Journal of Chromatography B (v.852, #1-2)

Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg by Rodolfo Valdés; Yenisley Medina; William Ferro; Biunayki Reyes; Déborah Geada; José Montero; Tatiana Alvarez; Alberto Leyva; Leonardo Gómez; Sigifredo Padilla; Leonardo Pacín; Alejandro Figueroa; Andrés Tamayo; Lorely Milá; Yurisleydi Aldama; Galina Moya; Jorge Reonde; María del Carmen Abrahantes (1-7).
This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31 mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43 mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23 mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3 ng IgG/μg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27 mg/mL. In the case of the ligand density of 2.23 mg/mL the IgG leached value was high (2.90 ng IgG/μg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43 mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose.
Keywords: Plant-derived antibody; Plantibody; Monoclonal antibody; Hepatitis B virus surface antigen; Immunoaffinity; Immunopurification;

Determination of ginsenoside Rd in dog plasma by liquid chromatography–mass spectrometry after solid-phase extraction and its application in dog pharmacokinetics studies by Wei Wang; Guang-Ji Wang; Hai-Tang Xie; Jian-Guo Sun; Shuai Zhao; Xi-ling Jiang; Hao Li; Hua Lv; Mei-Juan Xu; Rui Wang (8-14).
A sensitive liquid chromatography–mass spectrometric (LC/MS) method for the quantification of ginsenoside Rd in dog plasma was developed and validated after solid-phase extraction (SPE).Chromatographic separation was achieved on a reversed-phase Cromosil C18 column with the mobile phase of acetonitrile–ammonium chloride (500 μmol/L) and step gradient elution resulted in a total run time of about 5.5 min. The analytes were detected by using an electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005–2.500 μg/mL) (r  = 0.9998). Lower limit of quantification (LLOQ) was 5 ng/mL by using 500 μL plasma sample. Average recoveries ranged from 70.71 to 75.89% in plasma at the concentrations of 0.010, 0.100 and 2.500 μg/mL. Intra- and inter-day relative standard deviations were 8.49–11.71 and 5.71–16.48%, respectively. This method was successfully applied to the pharmacokinetic studies on dogs. The absolute bioavailability of Rd in dogs was 0.26%.
Keywords: Ginsenoside Rd; LC/MS; Solid-phase extraction (SPE); Pharmacokinetics; Bioavailability;

High throughput method for the determination of organochlorine pesticides and polychlorinated biphenyls in human serum by Fernando Goñi; Raul López; Arsenio Etxeandia; Esmeralda Millán; Pilar Amiano (15-21).
An improved method for the determination of selected organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum was developed. The method requires low volume of serum (500 μl) and 48–96 samples per day can be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Different denaturation, elution and clean-up conditions were tested. Quantification was carried out by gas chromatography equipped with electron capture detector (GC-ECD) or mass spectrometer (GC–MS). Recoveries of PCB congeners 28, 52, 101, 118, 138, 153 and 180 and OCPs HCB, β-HCH, p,p′-DDE and p,p′-DDT at two spiking levels (n  = 8) varied from 57 to 120%, and intra-day relative standard deviation from 1 to 11%, both depending on spiking level and compound. Inter-day relative standard deviation was <15% in all cases. Limit of quantification (LOQ) for these PCBs ranged from 0.08 to 0.13 ng/ml and for these OCPs from 0.16 to 0.40 ng/ml. The optimized method was applied to the analysis of 1000 serum samples from different places of Spain.
Keywords: Organochlorine pesticides; Polychlorinated biphenyls (PCBs); 96-Well solid-phase disk extraction plate; Gas chromatography; Human serum;

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses by Erin Chambers; Diane M. Wagrowski-Diehl; Ziling Lu; Jeffrey R. Mazzeo (22-34).
A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid–liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC® technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC® technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC® technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.
Keywords: Matrix effects; Quantitative bioanalysis; Mass spectrometry; Phospholipids; UPLC; HPLC; Sample preparation;

A sensitive and selective analytical method based on liquid chromatography–triple–quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r  = 0.9998 for plasma sample and r  = 0.9999 for urine sample) were obtained in the range of 1.0–20,000 ng ml−1 for mildronate. The detection limit was 1 ng ml−1. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC–MS–MS analysis of mildronate in plasma and urine.
Keywords: Mildronate; LC–MS–MS;

The chloroform extract of mango ginger (Curcuma amada Roxb.) rhizome was subjected to antioxidant activity-guided purification by repeated silica gel column chromatography to obtain a pure antioxidant compound. The structure was deduced by analyzing UV, IR, liquid chromatography–mass spectrometry (LC–MS) and two-dimensional heteronuclear multiple quantum coherence transfer spectroscopy (2D-HMQCT) NMR spectral data, and named it as “Amadannulen”, a novel compound. It exhibited DPPH radical scavenging activity, super oxide radical scavenging activity, lipid peroxidation inhibitory activity and metal chelating activity. Amadannulen also showed antibacterial activity against both Gram-positive and Gram-negative bacteria tested. It also exhibited bactericidal activity against M. luteus, B. cereus and B. subtilis.
Keywords: Curcuma amada; Mango ginger; Rhizome; Antioxidant activity; Antibacterial activity; Amadannulen;

The purpose of the present study was to develop a reverse-phase high performance liquid chromatographic (HPLC) assay for quantifying 5-aminolevulinic acid (ALA). The assay was applied to study the skin permeation of ALA and the influence of a novel skin penetration enhancement technology. Separation was achieved utilizing a Phenomenex Jupiter C18 column following fluorescence derivatization with fluorescamine. The assay was linear (r 2  > 0.99) with a minimum limit of quantitation of 400 ng/mL. The inter- and intraday variation was 1.6 and 0.9% at the lower end of the linear range and 1.5 and 1.9% at the upper end, respectively. The HPLC assay and fluorescence derivatization procedure is sensitive, simple, rapid, accurate and reproducible and offers advantages with regard to stability of ALA in comparison to other fluorescence derivatization methods. Results from the preliminary skin permeation study demonstrated substantial skin penetration of ALA only when applied with Dermaportation as a skin penetration enhancement device.
Keywords: HPLC; Penetration enhancement; Transdermal drug delivery; Electroporation;

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-μm column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 μL rat plasma was 0.05 μg/mL. The calibration curve was linear over a concentration range of 0.05–20 μg/mL. The mean recoveries were 80.6 ± 5.7, 83.4 ± 1.6 and 77.1 ± 3.4% with quality control (QC) level of 0.05, 1 and 20 μg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4 h incubation at room temperature, one month storage at −80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 °C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.
Keywords: Dibenzoylmethane; HPLC; Pharmacokinetics;

This article describes the fabrication of a rigid magnetic monodisperse bead (M-PGMA-TRI, 4.92 μm) with polyglycidyl methacrylate (PGMA) cross-linked by trimethylolpropane trimethacrylate (TRI). This was realized by adding a proper amount (2%, w/w) of TRI after 3 h of the dispersion-polymerization reaction with the monomer of GMA. The mono-sized microspheres were further processed to introduce magnetic granules by sulfonation and penetration-deposition approaches. The monodisperse bead (M-PGMA) without TRI addition was also fabricated for comparison. The morphology, size and magnetic characteristics of the microspheres were extensively characterized. The M-PGMA-TRI microspheres were nonporous, of smooth surface and superparamagnetic with a saturation magnetization of 13.0 emu/g. Recycled use of the material for protein adsorption exhibited stability of the magnetic properties of the M-PGMA-TRI, as compared to the significant loss of the saturation magnetization of the M-PGMA. The chemical stability of the M-PGMA-TRI was also confirmed by examining its protein adsorption and magnetic properties after incubation in various solutions such as acidic buffer (pH 2.2) for 24 h. The adsorption capacity of γ-globulin reached 287.2 mg/g and kept stable in the repeated adsorption/desorption/regeneration cycles. The results indicated that the introduction of 2% TRI was promising for producing rigid magnetic mono-sized microspheres for protein adsorption.
Keywords: Dispersion-polymerization; Penetration-deposition; Monodisperse; Magnetic polymer microsphere; Protein adsorption;

Two semi-automated, relatively high throughput methods using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) were developed for the simultaneous determination of ethinyl estradiol (EE) in combination with either 19-norethindrone (NE) or levonorgestrel (LN) in human plasma. Using 300 μL plasma, the methods were validated over the concentration ranges of 0.01–2 ng/mL and 0.1–20 ng/mL for EE and NE (or LN), respectively. The existing methods for the determination of the oral contraceptives in human plasma require large volumes of plasma (≥500 μL), and sample extraction is labor-intensive. The LC run time is at least 6 min, enabling analysis of only about 100 samples a day. In the present work the throughput was greatly improved by employing a semi-automated sample preparation process involving liquid–liquid extraction and derivatization with dansyl chloride followed by UPLC separation on a small particle size column achieving a run time of 2.7 min. The validation and actual sample analysis results show that both methods are rugged, precise, accurate, and well suitable to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in a day.
Keywords: Ethinyl estradiol; 19-Norethindrone; Levonorgestrel; Oral contraceptives; Ultra performance liquid chromatography; Mass spectrometry; Quantitative; Human plasma;

BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D5-stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert-butyl ether. The liquid–liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m/z 487 > 314 and 473 > 300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within ±5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at −30 °C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.
Keywords: BMS-068645; BMS-068645-acid; Liquid chromatography-tandem mass spectrometry; Diisopropyl fluorophosphate; DFP; Adenosine 2A agonist; Esterase inhibitor;

1,5-Dicaffeoylquinic acid (1,5-DCQA), a potent HIV-1 integrase inhibitor, is undergoing an evaluation as a promising novel HIV therapeutic agent. Here, we report a simple, rapid and robust LC–MS/MS method for simultaneous determination of 1,5-DCQA and its two active metabolites, 1-caffeoyl-5-feruoylquinic acid (1,5-CFQA) and 1,5-O-diferuoylquinic acid (1,5-DFQA) in human plasma. The quantitation of the target compounds was determined by selected reaction monitoring (SRM) mode using electrospray ionization (ESI). Good linearity was obtained in the 3–500 ng/ml range for each analyte and the analytical method was validated in terms of specificity, precision, accuracy, recovery, stability and matrix effect. These assays gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between −8.9 and 0.9%. In addition, the specificity, extraction recovery, stability and matrix effect were satisfactory too. Using the measured plasma concentrations of 1,5-DCQA and its active metabolites in five healthy volunteers, pharmacokinetic profiles of 1,5-DCQA and its active metabolites were evaluated, which supported the clinical pharmacokinetic studies successfully. Due to its high sensitivity, specificity and simplicity, the method could be used for pharmacokinetic studies of both 1,5-DCQA and its active metabolite, and for routine monitoring of their levels in human plasma.
Keywords: LC–MS–MS; 1,5-Dicaffeoylquinic acid; 1-Caffeoyl-5-feruoylquinic acid; 1,5-O-Diferuoylquinic acid; Pharmacokinetics;

Ultra-performance liquid chromatography/tandem mass spectrometric determination of diastereomers of SCH 503034 in monkey plasma by Ganfeng Wang; Yunsheng Hsieh; Kuo-Chi Cheng; Richard A. Morrison; Srikanth Venkatraman; F. George Njoroge; Larry Heimark; Walter A. Korfmacher (92-100).
This paper describes the development and qualification of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of diastereomers of SCH 503034 in monkey plasma. The analytical method involves direct protein precipitation with a mixture of methanol/acetonitrile (10/90) containing an internal standard, followed by separation of the stereoisomers on an Acquity UPLC™ C18 column and detected by selected reaction monitoring (SRM) in positive ionization mode using atmospheric pressure chemical ionization (APCI). The effects of ion-pairing agents on separation and ionization efficiency were investigated. The two diastereomers were well separated (R  = 1.3) with a runtime of 5 min under an isocratic condition. The method was qualified. The linear concentration range was 1–2500 ng/ml for the both stereoisomers. Inter-assay mean bias and relative standard deviation (R.S.D.) were in the range of −1.2% to 3.6% and 2.8–10%, respectively. Intra-assay mean bias and R.S.D. were in the range of −1.3% to 5.5% and 2.3–7.8%, respectively. Recoveries of the stereoisomers at concentration levels of 2.5, 50 and 1000 ng/ml were 87.2–90.0%, 89.1–90.4% and 92.3–94.3%, respectively. The LLOQ for this assay was 1 ng/ml. No matrix interferences were observed in six different sources of blank monkey plasma.
Keywords: UPLC; LC/MS/MS; SCH 503034; Diastereomers; Plasma;

Orally administered racecadotril is rapidly hydrolyzed to the more potent enkephalinase inhibitor thiorphan in vivo. A sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated to quantify thiorphan in human plasma using lisinopril as the internal standard. After a simple protein precipitation with methanol, the post-treatment samples were analyzed on a CN column interfaced with a tripe-quadruple tandem mass spectrometer using negative electrospray ionization. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy, and precision of measurements. The assay was linear over the concentration range 9.38–600 ng/mL using a 5 μL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.9985 to 0.9995. The intra- and inter-day precisions over the entire concentration were not more than 6.33%. Methanol and water (35:65, v/v) is used as the isocratic mobile phase, with 0.1% of formic acid in water. The method was successfully applied for pharmacokinetic study after a single oral administration of 200 mg racecadotril to 20 healthy volunteers.
Keywords: Racecadotril; Liquid chromatography/tandem mass spectrometry; Thiorphan;

A SPE-HPLC method was developed and validated for the simultaneous determination of flavonols, isoquercitrin (1), hibifolin (2), myricetin (3), quercetin-3′-O-d-glucoside (4) and quercetin (5) in rat plasma and urine after oral administration of the total flavonoids from Abelmoschus manihot (TFA). The astragalin (6) and kaempferol (7) were used as internal standards (IS). Plasma and urine samples were pretreated by solid-phase extraction using Winchem™ C18 reversed-phase cartridges. Analysis of the plasma and urinary extract was performed on YMC-Pack ODS-A C18 and Thermo ODS-2HYEPRSIL C18 reversed-phase column, respectively and a mobile phase of acetonitrile–0.1% phosphoric acid was employed. HPLC analysis was conducted with different elution gradients. The flow rate was 1.0 mL/min and the detection wavelength was set at 370 nm. Calibration ranges in plasma for flavonols 25 were at 0.011–2.220, 0.014–2.856, 0.022–4.320, and 0.028–5.600 μg/mL, respectively. In urine calibration ranges for flavonols 1, 2, 4 and 5 were at 2.00–16.00, 8.56–102.72, 2.70–21.60, and 3.00–24.00 μg/mL, respectively. The RSD of intra- and inter-day was less than 5.40% and 4.89% in plasma, and less than 3.96% and 6.85% in urine for all the analyses. A preliminary experiment to investigate the plasma concentration and urinary excretion of the flavonols after oral administration of TFA to rats demonstrated that the present method was suitable for determining the flavonols in rat plasma and urine.
Keywords: Abelmoschus manihot; Flavonols; Plasma; Urine; SPE-HPLC;

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the estimation of rivastigmine in human plasma by Jignesh Bhatt; Gunta Subbaiah; Sandeep Kambli; Bhavin Shah; Samita Nigam; Mehul Patel; Ashish Saxena; Ashok Baliga; Hetal Parekh; Gunvat Yadav (115-121).
A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the estimation of rivastigmine in human plasma. Rivastigmine was extracted from human plasma by using solid-phase extraction technique. Zolpidem was used as the internal standard. A Betabasic-8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 251.20 → 206.10, 86.20 for rivastigmine and m/z 308.10 → 235.10 for zolpidem. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.2–20.0 ng/ml with a correlation coefficient ≥0.9988. The intra-run and inter-run precision and accuracy were within 10.0%. The overall recoveries for rivastigmine and zolpidem were 86.28% and 87.57%, respectively. The total run time was 2.0 min. The developed method was applied for the determination of the pharmacokinetic parameters of rivastigmine following a single oral administration of a 3 mg rivastigmine capsule in 20 healthy male volunteers.
Keywords: Rivastigmine; LC–MS/MS; Human plasma;

A rapid and sensitive liquid chromatographic/tandem mass spectrometric method for determination of misoprostol acid, the active metabolite of misoprostol, was developed and validated. Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a C18 column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 367–249 and 296–269 was performed to quantify misoprostol acid and the internal standard hydrochlorothiazide, respectively. The method was linear in the concentration range of 10.0–3000 pg mL−1 using 200 μL plasma. The lower limit of quantification was 10.0 pg mL−1. The intra- and inter-day relative standard deviation over the entire concentration range was less than 8.3%. Accuracy determined at three concentrations (25.0, 200 and 2700 pg mL−1 for misoprostol acid) ranged from −0.5 to 1.2% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method was successfully used in a pharmacokinetic study of misoprostol in human plasma after an oral administration of 0.6 mg misoprostol.
Keywords: Liquid chromatography–tandem mass spectrometry; Misoprostol; Misoprostol acid; Pharmacokinetics;

In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography–tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytochrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis–Menten constant (K m) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0–2000 ng/ml for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng/ml. The limit of detection (LOD) for the tested analytes was 0.48 ng/ml or better based on signal-to-noise ratio >3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC50 values) measured by this method demonstrated high precision and are consistent with the literature values.
Keywords: Acetaminophen; 4-Hydroxytolbutamide; 4′-Hydroxymephenytoin; Dextrorphan; 1′-Hydroxymidazolam; 3-Methyoxymorphinan; 6-Hydroxychlorzoxazone;

A simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans, phyllanthin (1), hypophyllanthin (2), phyltetralin (3) and niranthin (4) from Phyllanthus niruri L. in plasma. The method recorded limits of detection for 1, 2, 3 and 4 as 1.22, 6.02, 0.61 and 1.22 ng/ml, respectively, at a signal-to-noise ratio of 5:1 whereas their limits of quantification were 4.88, 24.41, 4.88 and 9.76 ng/ml, respectively, at a signal-to-noise ratio of 12:1. These values were comparable to those of other sensitive methods such as gas chromatography–mass spectrometry (GC–MS), high-performance liquid chromatography–MS (HPLC–MS) and HPLC–electrochemical detection (HPLC–ECD) for the analysis of plasma lignans. A further advantage over known methods was its simple protocol for sample preparation. The within-day and between-day accuracies for the analysis of the four lignans were between 87.69 and 110.07% with precision values below 10.51%. Their mean recoveries from extraction were between 91.39 and 114.67%. The method was successfully applied in the pharmacokinetic study of lignans in rats. Following intravenous administration, the lignans were eliminated slowly from the body with a mean clearance of 0.04, 0.01, 0.03 and 0.02 l/kg h and a mean half-life of 3.56, 3.87, 3.35 and 4.40 h for 1, 2, 3 and 4, respectively. Their peak plasma concentration upon oral administration was 0.18, 0.56, 0.12 and 0.62 μg/ml, respectively, after 1 h. However, their absorption was incomplete with a calculated absolute oral bioavailability of 0.62, 1.52, 4.01 and 2.66% for 1, 2, 3 and 4, respectively.
Keywords: Phyllanthus niruri L.; Euphorbiaceae; Lignans; HPLC–fluorescence detection; Pharmacokinetic study; Absolute oral bioavailability;

A simple, rapid and accurate method has been developed for effective separation and simultaneous determination of lomefloxacin, gatifloxacin, enoxacin, ciprofloxacin, ofloxacin, enrofloxacin and pefloxacin residues in porcine tissue by capillary electrophoresis with diode-array detector. The separation conditions were investigated and optimized. The sample was extracted with acetonitrile, and a mixture consisted of 25 mM NaH2PO4, 25 mM Na2B4O7 and 25 mM H3BO3 (pH 9.0) was used as a running buffer. A linear relationship between concentration and peak area for each compound was obtained in the concentration range of 0.5–100 mg/L with a correlation coefficient greater than 0.9994. For analysis of porcine tissue, the detection limits of lomefloxacin, gatifloxacin, enoxacin, ciprofloxacin, ofloxacin, enrofloxacin and pefloxacin were 0.013, 0.012, 0.023, 0.040, 0.037, 0.035 and 0.034 mg/kg, respectively. The recoveries are in the range of 72–93%. The intra-day precision is less than 5%, and the inter-day precision is less than 10%. The proposed method has high resolution, speed and the extremely small sample volume required. It can permit to confirm the presence of the studied seven fluoroquinolones in porcine tissue at the required maximum residue limit (MRL) level.
Keywords: Capillary electrophoresis; Simultaneous determination; Seven fluoroquinolones; Porcine tissues;

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5–250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n  = 5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.
Keywords: Stereospecific assay; Active metabolite; Enantioselective assay; Stereoselective pharmacokinetics; Stereoselective metabolism; Tramadol;

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection by Jonatan R. Catai; Javier Sastre Toraño; Peter M.J.M. Jongen; Gerhardus J. de Jong; Govert W. Somsen (160-166).
The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB–PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE–mass spectrometry (MS) of this sample, using a PB–PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE–UV and CE–MS indicated the presence of both deamidation and oxidation products.
Keywords: Capillary electrophoresis; Mass spectrometry; Recombinant human growth hormone; Biopharmaceutical; Protein stability; Bilayer coating;

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.
Keywords: Aqueous micellar two-phase system; Hydrophobically modified ethylene oxide; Electrostatic interaction; Micellar aggregate; Cytochrome P450 BM-3;

An optimized analytical method of fluconazole in human plasma by high-performance liquid chromatography with ultraviolet detection and its application to a bioequivalence study by Sung-Su Kim; Ho-Taek Im; Il-Mo Kang; Hyun-Su Lee; Heon-Woo Lee; Sung-Hee Cho; Jong-Bin Kim; Kyung-Tae Lee (174-179).
A sensitive and accurate HPLC-UV method for the quantification of fluconazole (FLA) level in human plasma has been developed. The sample was prepared by one-step liquid–liquid extraction (LLE) of FLA from plasma using dichloromethane. Phenacetin was used as the internal standard. The chromatographic retention times of FLA and phenacetin were 4.6 and 8.3 min, respectively. The lower limit of quantitation (LLOQ) was 0.05 μg/mL, and no interferences were detected in the chromatograms. The devised HPLC-UV method was validated by evaluating its intra- and inter-day precisions and accuracies in a linear concentration range between 0.05 and 10.00 μg/mL. The devised method was successfully applied to a bioequivalence studies involving the oral administration of a single 150 mg FLA tablet and 3 × 50 mg FLA capsules in healthy Korean male volunteers.
Keywords: Fluconazole; High performance liquid chromatography (HPLC); Validation; Bioequivalence; Human plasma;

A sensitive method for the quantification of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry is described. The sample preparation includes a liquid–liquid extraction with methyl tert-butylmethyl ether after addition of 2 M sodium hydroxide. Ethylmethylglycinexylidide (EMGX) is used as an internal standard. For chromatographic separation, an ODS Hypersil column was used. Isocratic elution was achieved with 0.01 M ammonium acetate and acetonitrile as mobile phases. Good linearity was observed in the range of 2.5–1000 ng ml−1 for lidocaine in both dog and horse plasma. For MEGX, linear calibration curves were obtained in the range of 5–1000 ng ml−1 and 20–1000 ng ml−1 for dog and horse plasma, respectively. In dog and horse plasma good linearity was observed in the range of 200–1500 ng ml−1 for GX. The limit of quantification (LOQ) in dog plasma for lidocaine, MEGX and GX was set at 2.5 ng ml−1, 20 ng ml−1 and 200 ng ml−1, respectively. For horse plasma a limit of quantification of 2.5 ng ml−1, 5 ng ml−1 and 200 ng ml−1 was achieved for lidocaine, MEGX and GX, respectively. In dog plasma, the limit of detection (LOD) was found to be 0.8 ng ml−1, 2.3 ng ml−1 and 55 ng ml−1 for lidocaine, MEGX and GX, respectively. In horse plasma the LOD's found for lidocaine, MEGX and GX, were 1.1 ng ml−1, 0.5 ng ml−1 and 13 ng ml−1, respectively. The method was shown to be of use in pharmacokinetic studies after application of a transdermal patch in dogs and after an intravenous infusion in horses.
Keywords: Lidocaine; Glycinexylidide; Monoethylglycinexylidide; Liquid chromatography/electrospray ionization tandem mass spectrometry; Quantification; Validation; Horse plasma; Dog plasma;

A general, robust method for the quality control of intact proteins using LC–ESI-MS by Gustav Sundqvist; Maria Stenvall; Helena Berglund; Jenny Ottosson; Harry Brumer (188-194).
A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.
Keywords: Electrospray ionization; Liquid chromatography; Mass spectrometry; Protein analysis; Quality control; Reduction; Alkylation; Column regeneration; Glycoprotein; Xyloglucan endo-transglycosylase (XET);

Dexmedetomidine (Dex) is a lipophilic imidazole derivative used primarily for the sedation and anxiolysis of adults in the intensive care setting. Dex is being used more frequently in the pediatric intensive care unit. This report describes a selective and highly sensitive assay for Dex in pediatric plasma employing liquid chromatography–tandem mass spectrometry (LC–MS/MS). Dex was extracted from 200 μL of plasma by solid-phase extraction (SPE). High performance liquid chromatography (HPLC) separation was conducted on an YMC ODS-AQ C18 column with a flow rate of 0.3 mL/min using a mobile phase comprised of 5 mM ammonium acetate buffer/0.03% formic acid in the solvent mixture of methanol/acetonitrile/water (20:20:60, v/v/v). The intra-day precision (coefficient of variation, % CV) and accuracy for quality control samples, ranged from 1.04 to 6.84% and 90.2 to 100.8%, respectively. The inter-day precision and accuracy ranged from 4.08 to 5.37% and 92.7 to 98.6%, respectively. Stability studies showed that Dex was stable during both the assay procedure and storage. The overall recovery was 76.6–78.3% for Dex in plasma. The analytical method showed excellent sensitivity using a small sample volume (200 μL) with a lower limit of quantitation of 5 pg/mL. This method is robust and has been successfully employed in a pharmacokinetic study of Dex in infants postoperative from cardiac surgery.
Keywords: Dexmedetomidine; LC–MS/MS; Pediatric; Pharmacokinetics;

Liquid chromatography–tandem mass spectrometry assay for the quantitation of β-dihydroartemisinin in rat plasma by Jie Xing; Hong-Xia Yan; Rui-Li Wang; Li-Feng Zhang; Shu-Qiu Zhang (202-207).
Dihydroartemisinin (DHA) is a sesquiterpene used in the world as an antimalarial. To evaluate the pharmacokinetics of dihydroartemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC–MS/MS) method was developed and validated for the quantitation of dihydroartemisinin in rat plasma. For detection, a Sciex API 4000 LC–MS/MS with a TurboIonSpray ionization (ESI) inlet in the positive ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pre-treated by a simple liquid–liquid extraction with diethyl ether. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.2–100.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.2 ng/mL for β-dihydroartemisinin in 100 μL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of β-dihydroartemisinin in rats after oral administration.
Keywords: Dihydroartemisinin; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics; Rat plasma;

An isocratic and sensitive HPLC assay was developed allowing the determination of the new anticancer drug nilotinib (AMN107) in human plasma, urine, culture medium and cell samples. After protein precipitation with perchloric acid, AMN107 underwent an online enrichment using a Zirchrom-PBD precolumn, was separated on a Macherey-Nagel C18-HD column and finally quantified by UV-detection at 258 nm. The total run time is 25 min. The assay demonstrates linearity within a concentration range of 0.005–5.0 μg/ml in plasma (r 2  = 0.9998) and 0.1–10.0 μg/ml in urine (r 2  = 0.9913). The intra-day precision expressed as coefficients of variation ranged depending on the spiked concentration between 1.27–9.23% in plasma and 1.77–3.29% in urine, respectively. The coefficients of variation of inter-day precision was lower than 10%. Limit of detection was 0.002 μg/ml in plasma and 0.01 μg/ml in urine. The described method is stable, simple, economic and is routinely used for in vivo and in vitro pharmacokinetic studies of AMN107.
Keywords: Nilotinib; AMN107; Imatinb; Chronic myelogenous leukaemia; Tyrosine kinase; BCR-ABL mutant; HPLC; Intracellular concentration;

A rapid, sensitive, and specific LC/MS/MS-based method was developed for determining the concentration of DMXAA in human and mouse plasma. Sample preparation involved a single protein precipitation step using acetonitrile. Separation of DMXAA and 6-isopropoxy-9-oxoxanthene-2-carboxylic acid, the internal standard, was achieved on a Waters X-Terra™ C18 (50 mm × 2.1 mm i.d., 3.5 μm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (55:45, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 5–3000 ng/mL. The values for precision and accuracy were <9.6%, except at the LLOQ (5 ng/mL) level, which was within 16.8%. Recovery of DMXAA in mouse plasma was >65%. DMXAA was stable through 2 freeze/thaw cycles, to 2 h in mouse plasma or 50% acetonitrile, and on the autosampler to 5.1 h. This method was subsequently used to measure concentrations of DMXAA in mice following intraperitoneal administration.
Keywords: DMXAA; LC/MS/MS; Pharmacokinetics;

Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection by Stéphanie Chhun; Elisabeth Rey; Agnes Tran; Olivier Lortholary; Gérard Pons; Vincent Jullien (223-228).
A sensitive and selective high-performance liquid chromatographic (HPLC) method with ultra-violet detection has been developed and validated for the simultaneous determination of posaconazole and voriconazole, two systemic anti-fungal agents. An internal standard diazepam was added to 100 μL of human plasma followed by 3 mL of hexane–methylene chloride (70:30, v/v). The organic layer was evaporated to dryness and the residue was reconstituted with 100 μL of mobile phase before being injected in the chromatographic system. The compounds were separated on a C8 column using sodium potassium phosphate buffer (0.04 M, pH 6.0): acetonitrile:ultrapure water (45:52.5:2.5, v/v/v) as mobile phase. All compounds were detected at a wavelength of 255 nm. The assay was linear and validated over the range 0.2–10.0 mg/L for voriconazole and 0.05–10.0 mg/L for posaconazole. The biases were comprised between −3 and 5% for voriconazole and −2 and 8% for posaconazole. The intra- and inter-day precisions of the method were lower than 8% for the routine quality control (QC). The mean recovery was 98% for voriconazole and 108% for posaconazole. This method provides a useful tool for therapeutic drug monitoring.
Keywords: Posaconazole; Voriconazole; Liquid chromatography; UV detection;

Improved synthesis methods of standards used for quantitative determination of total isothiocyanates from broccoli in human urine by Mette Kristensen; Kirstine S. Krogholm; Hanne Frederiksen; Fritz Duus; Claus Cornett; Susanne H. Bügel; Salka E. Rasmussen (229-234).
A well-known method for quantification of isothiocyanates (ITCs) and their metabolites is the condensation reaction with 1,2-benzenedithiole to produce 1,3-benzodithiole-2-thione, which can be quantified by high-performance liquid chromatography. Standards of an ITC metabolite and 1,3-benzodithiole-2-thione are required for this assay but are not commercially available. In the present study, we report on an improved synthesis of the ITC metabolite N-acetyl-S-(N-4-methylsulfinylbutylthiocarbamoyl)-l-cysteine and 1,3-benzodithiole-2-thione. The standards were used to quantify the urinary excretion of ITCs from 10 healthy subjects who consumed 350 g broccoli. The excretion was investigated throughout 48 h showing a cumulative urinary ITC excretion of 49.1 ± 25.2% of the dose.
Keywords: Isothiocyantes; Biomarker; Synthesis; 1,3-Benzodithiole-2-thione; N-acetyl-S-(N-4-methylsulfinylbutylthiocarbamoyl)-l-cysteine;

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry by Douglas B. Mawhinney; Elizabeth I. Hamelin; Rheaclare Fraser; Sathya S. Silva; Antonis J. Pavlopoulos; Robert J. Kobelski (235-243).
A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k′ = 3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (−0.5 to +3.4%) and precise (coefficients of variation of 2.1–3.6%) quantitative results over the clinically relevant urine concentration range of 1–200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.
Keywords: Hydrophilic interaction chromatography; HILIC chromatography; LC/MS/MS; Organophosphorus nerve agents; Nerve agent metabolites; Chemical warfare agent; Chemical terrorism;

The partitioning of bovine trypsin and α-chymotrypsin – proteases of similar physico-chemical properties – in different polyethyleneglycol/sodium citrate aqueous two-phase systems was investigated. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length, temperature and the presence of an inorganic salt on the protein partition coefficient were analysed. Both a decrease in PEG molecular weight and an increase in pH led to a higher partition coefficient for both enzymes. Aqueous two-phase systems formed by PEG of molecular weight 3350 and citrate pH 5.2 showed the best separation capability which was enhanced in presence of sodium chloride 3%. The transfer of both proteins to the top phase was associated with negative enthalpic and entropic changes.
Keywords: Trypsin; α-chymotrypsin; Pancreatic proteases; Aqueous two-phase systems;

Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function by Bernhard Antes; Sabine Amon; Andreas Rizzi; Susi Wiederkum; Manuela Kainer; Oliver Szolar; Markus Fido; Ralf Kircheis; Andreas Nechansky (250-256).
During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/κ) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC–MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants – clipped and unclipped – elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.
Keywords: IEF; mAb; Charge heterogeneity; Lewis-Y; Lysine clipping;

Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): Up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer by Tatsuhiko Kakisaka; Tadashi Kondo; Tetsuya Okano; Kiyonaga Fujii; Kazufumi Honda; Mitsufumi Endo; Akihiko Tsuchida; Tatsuya Aoki; Takao Itoi; Fuminori Moriyasu; Tesshi Yamada; Harubumi Kato; Toshihide Nishimura; Satoru Todo; Setsuo Hirohashi (257-267).
We investigated the aberrant expression of plasma proteins in patients with pancreatic cancer. High-abundance plasma proteins (albumin, transferrin, haptoglobin, alpha-1-antitrypsin, IgG and IgA) were depleted by use of an immuno-affinity column, and low-abundance ones were separated into five fractions by anion-exchange chromatography. The fractionated plasma proteins were subjected to 2D-DIGE with highly sensitive fluorescent dyes. The quantitative protein expression profiles obtained by 2D-DIGE were compared between two plasma protein mixtures: one from five non-cancer bearing healthy donors and the other from five patients with pancreatic cancer. Among 1200 protein spots, we found that 33 protein spots were differently expressed between the two mixtures; 27 of these were up-regulated and six were down-regulated in cancer. Mass spectrometry and database searching allowed the identification of the proteins corresponding to the gel spots. Up-regulation of leucine-rich alpha-2-glycoprotein (LRG), which has not previously been implicated in pancreatic cancer, was observed. Western blotting with an anti-LRG antibody validated the up-regulation of LRG in an independent series of plasma samples from healthy controls, patients with chronic pancreatitis, and patients with pancreatic cancer. Our results demonstrate the application of a combination of multi-dimensional liquid chromatography with 2D-DIGE for plasma proteomics and suggest the clinical utility of LRG plasma level measurement.
Keywords: Plasma proteomics; Pancreatic cancer; Two-dimensional difference gel electrophoresis (2D-DIGE); Leucine-rich alpha-2-glycoprotein;

It was demonstrated that a shotgun approach can be utilized for the characterization of phospholipids (PLs) extracted from mouse liver and brain by using nanoflow reversed phase liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). In this study, a dual scan method was introduced for the high throughput analysis of complex PL mixtures. Two consecutive LC–ESI-MS–MS runs were made in positive ion mode (for phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)) first followed by analysis in negative ion mode (for phosphatidylserine (PSs) and phosphatidylinositol (PIs)) using the same binary gradient elution with and without adding formic acid, respectively. The separation of the PLs was carried out using a home made pull tip capillary column (C18) with an end frit. The MS analysis of the eluted PL molecules was performed with a precursor scan followed by a data dependent MS–MS scan. The developed dual scan method was tested with the extracts of PCs and PIs mixtures from soybean, PEs from Escherichia coli, and PSs from bovine brain. It was further applied for the characterization of intact PL samples that were extracted from both mouse liver and mouse brain in the laboratory, and resulted in the identification of 90 and 80 PL species, respectively.
Keywords: Lipidomics; Phospholipids; Nanoflow liquid chromatography–tandem mass spectrometry; LC–MS–MS; Electrospray ionization; Mouse liver; Mouse brain;

An analytical method was developed for the determination of potassium in vitreous humor by low pressure ion chromatography (LPIC). Experimental conditions for LPIC analysis were optimized. High sensitivity and selectivity were obtained using this method. The LOD and LOQ were 1 and 2 mmol l−1, respectively. The linearity was demonstrated from 2 to 20 mmol l−1. The intra- and inter-day precision (CV) based on three concentrations was less than 5.0%. It was a simple and fast method to measure potassium and was suitable for evaluating the postmortem interval (PMI) in relatively well-preserved bodies. Sixty-two samples from medical-legal autopsies with known PMI were analyzed. A linear correlation equation for potassium concentration in the vitreous humor and PMI was established: [K+] = 0.1702PMI + 5.5678, r  = 0.8692.
Keywords: Potassium; Low pressure ion chromatography; Forensic science; Postmortem interval;

A rapid, sensitive and reliable method was developed to quantitate omeprazole in human plasma using liquid chromatography–tandem mass spectrometry. The assay is based on protein precipitation with acetonitrile and reversed-phase liquid chromatography performed on an octadecylsilica column (55 mm × 2 mm, 3 μm particles), the mobile phase consisted of methanol–10 mM ammonium acetate (60:40, v/v). Omeprazole and flunitrazepam, the internal standard, elute at 0.80 ± 0.10 min with a total run time 1.35 min. Quantification was through positive ion mode and selected reaction monitoring mode at m/z 346.1 → 197.9 for omeprazole and m/z 314.0 → 268.0 for flunitrazepam, respectively. The lower limit of quantitation was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 12%. The assay was applied to the analysis of samples from a pharmacokinetic study.
Keywords: Omeprazole; Protein precipitation; Liquid chromatography; Mass spectrometry;

Studies on endotoxin removal mechanism of adsorbents with amino acid ligands by Zhong Wei; Wei Huang; Jihong Li; Guanghui Hou; Jie Fang; Zhi Yuan (288-292).
In this paper, a series of adsorbents with different amino acid ligands for endotoxin removal were prepared and endotoxin adsorption capacities (EAC) in aqueous solution were studied using an affinity column. The results showed that the property and structure of amino acid ligands have great influence on EAC. As the increasing of isoelectric point and polarity of amino acids ligands, EACs of the adsorbents increased. In addition, computer simulation method was employed to a further investigation on the interaction between endotoxins and ligands. Based on the results, some adsorbents were applied to remove endotoxin from endotoxemia rabbit's serum. Similar adsorption results were observed and the removal efficiency of adsorbents with Arg, Ser ligands is up to 78%.
Keywords: Endotoxin; Amino acids; Affinity adsorbents; Computer simulation;

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma by Puran Singhal; Ashwani Gaur; Vanita Behl; Anirudh Gautam; Brijesh Varshney; Jyoti Paliwal; Vijay Batra (293-299).
A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC–MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3 → 247.2 and m/z 409.1 → 205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0–489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0–489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.
Keywords: Antimalarial; Chloroquine; Liquid chromatography/tandem mass spectrometry; Pharmacokinetic study; Plasma;

Stable isotope dilution analysis of salicylic acid and hydroquinone in human skin samples by gas chromatography with mass spectrometric detection by Anja Judefeind; Peet Jansen van Rensburg; Stephan Langelaar; Jeanetta du Plessis (300-307).
A sensitive and accurate gas chromatographic–mass spectrometric (GC–MS) method has been developed for the quantitative determination of salicylic acid (SA) and hydroquinone (HQ) from human skin samples and cosmetic emulsions. Deuterium labeled SA-d6 and HQ-d6 were used as internal standards (IS). The samples were extracted with methanol, dried under nitrogen and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS). Quantification was performed in SIM mode with a limit of quantification (LOQ) of 50 ng ml−1 for SA and 10 ng ml−1 for HQ. The inter-day variation (R.S.D.) was less than 5% and the accuracy was better than 13.3% for both compounds. The recoveries from the different matrices ranged between 93.1 and 103.3% for SA, and 97.3 and 100.8% for HQ.
Keywords: Hydroquinone; Salicylic acid; GC–MS; BSTFA; Skin penetration; Emulsion;

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: Application to pharmacokinetic studies by Marlice A. Sípoli Marques; Alcenir de Souza Soares; Olivia Woyames Pinto; Pedro Tupinambá Werneck Barroso; Douglas Pereira Pinto; Milton Ferreira-Filho; Eduardo Werneck-Barroso (308-316).
A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC–MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water–acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C18 (100 mm × 2.0 mm i.d., 3 μm) column connected to a guard column MS-pursuit (0.20 mm × 0.20 mm i.d., 5 μm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r 2 values of 0.995 (CV = 0.24%, n  = 10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C max (1667.25 ng/mL) and T max (3.89 h).
Keywords: Metformin; Liquid chromatography; Mass spectrometry; Plasma; Pharmacokinetic;

Determination of ximelagatran, melagatran and two intermediary metabolites in plasma by mixed-mode solid phase extraction and LC–MS/MS by Kristina Dunér; Jonas Bäckström; Niklas Magnell; Henrik Svennberg; Martin Ahnoff; Ulrika Logren (317-324).
An analytical method was developed for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the two intermediate metabolites, OH-melagatran and ethyl-melagatran in human plasma. Extraction of plasma was carried out on a mixed mode bonded sorbent material (C8/SO3 ). All four analytes, including their isotope-labelled internal standards, were eluted at high ionic strength with a mixture of 50% methanol and 50% buffer (0.25 M ammonium acetate and 0.05 M formic acid, pH 5.3) with an extraction recovery above 80%. The extracts were demonstrated to be clean in terms of a low concentration of albumin and lysoPC. The sample extraction was fully automated and performed in 96-well plates using a Tecan Genesis pipetting robot. Analysis of the extracts were performed with liquid chromatography followed by positive electrospray ionization mass spectrometry. The low organic content and the low pH of the extracts allowed for, after dilution 1:3 with buffer, direct injection onto the LC-column. The four analytes were separated on a C18 analytical LC-column using gradient elution with the acetonitrile concentration varying from 10 to 30% (v/v) and the ammonium acetate and acetic acid concentration kept constant at 10 and 5 mmol/L, respectively, at a flow rate of 0.75 mL/min. Linearity was achieved over the calibrated range 0.010–4.0 μmol/L with accuracy and relative standard deviation in the range 96.9–101.2% and 6.6–17.1%, respectively at LLOQ, and in the range 94.7–102.6% and 2.7–6.8%, respectively at concentrations above 3 × LLOQ. The method replaces a manual method, and displays the advantages of having a fully automated sample clean-up, no evaporation/reconstitution step, high recovery, and complete LC-separation of all four analytes.
Keywords: Thrombin inhibitor; Mixed-mode solid phase extraction;

A sensitive and reproducible method was developed for the separation and determination of three water-soluble components—protocatechuic aldehyde (PAH), β-(3,4-dihydroxyphenyl) lactic acid (DSS) and protocatechuic acid (PA) in medicine plant Salvia miltiorrhiza Bunge and two related traditional medicinal preparations using flow injection-capillary electrophoresis system. This analysis was carried out by using an unmodified fused-silica capillary (28.4 cm × 75 μm i.d. × 375 μm o.d., 25 cm effective separation length) and direct ultraviolet detection at 214 nm, 7.0 kV applied voltage. With boric acid (200 mM) adjusted to pH 7.8 as a background electrolyte. The separation was achieved in 9 min. The sample throughput rate could reach up to 15 h−1. Calibration curves showed good linearity with correlation coefficients (r) more than 0.9986. The repeatability (defined as R.S.D.) were 0.20%, 0.46%, 0.47% with migration time evaluation and 0.62%, 3.66%, 1.50% with peak area evaluation for PAH, DSS and PA, respectively. The limits of detection (S/N = 3) were 0.36 μg/mL, 0.84 μg/mL, and 0.73 μg/mL for PAH, DSS, and PA, respectively. The mean recoveries of PAH, DSS and PA were 103.2%, 98.1% and 100.5%, respectively. This method has been applied successfully to monitor these three components in Salvia miltiorrhiza Bunge and its two traditional medicinal preparations.
Keywords: Protocatechuic aldehyde; β-(3,4-Dihydroxyphenyl) lactic acid; Protocatechuic acid; Nicotinic acid; Salvia miltiorrhiza Bunge; Traditional medicinal preparation; Flow injection-capillary electrophoresis;

Determination of the volume of sweat accumulated in a sweat-patch using sodium and potassium as internal reference by Brice M.R. Appenzeller; Claude Schummer; Sophie Boura Rodrigues; Robert Wennig (333-337).
In the present work, we assessed the suitability of sodium and potassium physiologically present in sweat, as internal reference allowing to re-calculate the corresponding volume of sweat collected on a PharmChek™ Patch. A method using capillary electrophoresis with indirect ultra-violet detection was developed for the determination of sodium and potassium in sweat. The concentrations determined in specimens collected from 12 females and 10 males, using a home-made system composed of polypropylene copolymer bag, were 1039 ± 89 mg/L and 711 ± 45 mg/L for sodium, and 489 ± 293 mg/L and 474 ± 196 mg/L for potassium, respectively. In parallel, for seven females and eight males, the comparison of the volume of sweat collected in the same way to the re-calculated volume of sweat accumulated in a patch using sodium as internal standard, gave an average agreement of 98.4 ± 15.0%. Results demonstrated the usefulness of sodium as internal standard to determine the volume of sweat accumulated in a patch, and confirm the suitability of PharmChek™ patch for the collection and determination of cations in sweat.
Keywords: Sweat patch; Sodium; Potassium; Internal reference; Capillary electrophoresis;

In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS.The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC® MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 °C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001–2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation—%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%.A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC–MS that involves derivatisation.
Keywords: Cocaine and metabolites; Forensic samples; LC/MS/MS;

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte®. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm × 2.1 mm, 3 μm) with a 10 mm × 2 mm, 5 μm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.
Keywords: Cyclosporine; Lymphocytes; Mass spectrometry; Intracellular; Metabolites; T-cells;

Biopartitioning micellar chromatography: An alternative high-throughput method for assessing the ecotoxicity of anilines and phenols by J.M. Bermúdez-Saldaña; L. Escuder-Gilabert; M.J. Medina-Hernández; R.M. Villanueva-Camañas; S. Sagrado (353-361).
An investigation of the use of the chromatographic retention (log k) as an in vitro approach for modelling the toxicity to Fathead Minnows of anilines and phenols is developed. A data set of 65 compounds with available experimental toxicity data was used. Log k data at three pH values were used for the compounds classification and two groups or ‘MODEs’ were identified. For one ‘MODE’ a quantitative retention-activity relationship (QRAR) model was calculated. Finally, it was used to estimate the toxicity to Fathead minnows of anilines and phenols for which experimental data are not available. These estimations were compared to those obtained from another toxicity (to Tetrahymena pyriformis) data set and those estimated from a U.S. EPA QSAR approach (ECOSAR software) to decide on the toxicity level according to the Directive 3/21/EEC.
Keywords: Biopartitioning micellar chromatography; Ecotoxicity; Quantitative retention-activity relationships; Phenols; Anilines;

Screening of rifamycin producing marine sponge bacteria by LC–MS–MS by Amitha K. Hewavitharana; P. Nicholas Shaw; Tae Kyung Kim; John A. Fuerst (362-366).
HPLC–MS–MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.
Keywords: HPLC–MS–MS; HPLC–MS; Tandem mass spectrometry; Salinispora; Rifamycin;

Alterations in the molecular species of plasmalogen phospholipids and glycolipids due to peroxisomal dysfunction in Chinese hamster ovary-mutant Z65 cells by FABMS method by Makiko Saito; Makoto Horikawa; Yuriko Iwamori; Yoichi Sakakihara; Masashi Mizuguchi; Takashi Igarashi; Yukio Fujiki; Masao Iwamori (367-373).
Changes in the molecular species of lipids associated with Pex2 gene-mutation were investigated to elucidate the pathogeneses of peroxisome biogenesis disorders. Although no differences were observed in the concentrations of cholesterol and phosphatidyl choline between mutated Z65 and control CHO-K1 cells, the amounts of cholesterol esters and glycolipids in Z65 cells were twice those in CHO-K1 cells, but phosphatidyl ethanolamine (PE), particularly 1-O-octadec-1′-enyl-2-oleoyl PE, was absent in Z65 cells by FABMS. Enhanced synthesis of glycolipids in Z65 cells was associated with an abundance of lignoceric acid-containing ones, suggesting a role of glycolipids in the retention of longer saturated fatty acids.
Keywords: Peroxisome biogenesis disorder; Pex2-mutation; Ganglioside GM3; Lignoceric acid; Cholesterol esters; Negative ion FABMS;

A high throughput and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the estimation of bisoprolol in human plasma using multiplexing technique by Jignesh Bhatt; Gunta Subbaiah; Sandeep Kambli; Bhavin Shah; Mehul Patel; Ashish Saxena; Ashok Baliga; Samita Nigam; Hetal Parekh; Gunvat Yadav (374-381).
A high throughput and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the estimation of bisoprolol in human plasma using multiplexing technique (two HPLC units connected to one MS). Bisoprolol was extracted from human plasma using solid-phase extraction technique using metoprolol as internal standard. A Betabasic 8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 326.2 → 116.1 for bisoprolol and m/z 268.2 → 191.0 for metoprolol. The method involves a simple multiplexing, rapid solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.5–70.0 ng/mL with correlation coefficient ≥0.9991. The precision and accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for bisoprolol and metoprolol were 93.89% and 77.65%, respectively. Total MS run time was 0.90 min only. The developed method was applied for the determination of pharmacokinetic parameters of bisoprolol following a single oral administration of a 10 mg bisoprolol tablet in 18 healthy male volunteers.
Keywords: Bisoprolol; LC–MS/MS; Human plasma; β-Blockers; Multiplexing;

We developed and validated a simple high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection system for determining penciclovir (active metabolite of famciclovir) levels in human plasma using acyclovir as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 254.00 > 152.09 for penciclovir and m/z 226.00 > 152.09 for IS. The analytes were chromatographed on a Capcellpak MGII C18 reversed-phase chromatographic column by isocratic elution using 30% methanol and 70% Milli-Q water containing 10 mM ammonium formate (adjusted to pH 3.1 with formic acid). Results were linear over the studied range (0.05–10 μg/ml) with r 2  = 0.9999, and the total analysis time for each run was 2 min. Intra- and inter-assay precisions were 2.3–7.8 and 3.7–7.5%, respectively, and intra- and inter-assay relative errors (RE) were 2.0–8.4 and 1.9–9.1%, respectively. The lower limit of quantification (LLOQ) was 0.05 μg/ml. At this concentration mean intra- and inter-assay precisions were 7.8 and 7.5%, respectively, and mean intra- and inter-assay relative errors were 2.2 and 9.1%, respectively. Penciclovir was found to be stable in plasma samples under short-, long-term storage and processing conditions. The developed assay was successfully applied to a bioequivalence study of penciclovir administered as a single oral dose (500 mg as famciclovir) to healthy volunteers.
Keywords: Penciclovir; Famciclovir; Acyclovir; LC–MS/MS; Human plasma; Bioequivalence study;

Enantioselective determination of azelnidipine in human plasma using liquid chromatography–tandem mass spectrometry by Kiyoshi Kawabata; Naozumi Samata; Yoko Urasaki; Ichiro Fukazawa; Naoki Uchida; Eiji Uchida; Hajime Yasuhara (389-397).
A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [2H6]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing α1-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583 → 167 for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589 → 167 for [2H6]-azelnidipine. The standard curve was linear over the studied range (0.05–20 ng/mL), with r 2  > 0.997 using weighted (1/x 2) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2–8.2% and 2.4–5.8% for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2–117.0% for (R)-(−)-azelnidipine and 100.0–107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine were 71.4–79.7% and 71.7–84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.
Keywords: Azelnidipine; Enantiomer separation; Quantitative analysis; LC/APCI–MS/MS;

Safflomide (N-caffeoyltryptamine) is a compound belonging to a group of phenylpropanoid amides found in plants. In this study, safflomide was chemically synthesized and confirmed by LC–MS, LC–MS/MS and NMR spectroscopic methods, and a high-performance liquid chromatography (HPLC) method was developed for quantifying safflomide in biological samples. The synthesis was simple, and the yield of safflomide was greater than 50%. Using the synthesized safflomide as a standard, HPLC separation was performed on a Nova-Pak C18 column using an isocratic buffer, and the separation was detected using a coulometric electrochemical detector. The detection of safflomide yielded an excellent peak resolution at the retention time of 21 min, and the lower limit of the detection was as little as 100 fmol. Using this HPLC method, the plasma concentrations of safflomide were determined in mouse blood, collected at 5, 10, 15, 20, 25, 30, and 35 min following its oral administrations (1 and 3 mg/30 g body weight). This HPLC method standardized with safflomide is the first reported method able to quantify the compound in standard and plasma samples with good detection limit and consistent reproducibility.
Keywords: N-Caffeoyltryptamine; Safflomide; HPLC; Oral administration; Mouse plasma; Bioavailability;

A highly selective, sensitive and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid–liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C-18 column (250 mm × 4.6 mm, 5 μm, Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M triethylamine + 0.020 M orthophosphoric acid (50/50, v/v). The analytes were detected at 225 nm. The assay exhibited a linear range of 5–600 ng/mL for tadalafil in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL. The within- and between batch precision (expressed as coefficient of variation, C.V.) did not exceed 10.3% and the accuracy was within −7.6% deviation of the nominal concentration. The recovery of tadalafil from plasma was greater than 66.1%. Stability of tadalafil in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and 30 days storage in a freezer. This validated method is applied for the clinical study of the tadalafil in human volunteers.
Keywords: Tadalafil; Liquid–liquid extraction; HPLC–UV; Plasma; Quantitation;

High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)–dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.
Keywords: Aqueous two-phase systems; Metal affinity partitioning; Granulocyte-colony stimulating factor; Inclusion bodies; Protein refolding;

Determination of aciclovir and ganciclovir in human plasma by liquid chromatography–spectrofluorimetric detection and stability studies in blood samples by N. Perrottet; A. Beguin; P. Meylan; M. Pascual; O. Manuel; T. Buclin; J. Biollaz; L.A. Decosterd (420-429).
A sensitive HPLC method has been developed for the assay of aciclovir and ganciclovir in human plasma, by HPLC coupled with spectrofluorimetric detection. Plasma (1000 μl), with 9-ethyl-guanine added as internal standard, is submitted to protein precipitation with trichloroacetic acid solution 20%. The supernatant, evaporated to dryness at 37 °C, is reconstituted in 100 μl of a solution of sodium heptanosulfonate 0.4% adjusted with acetic acid to pH 2.60 and a 30 μl volume is then injected onto a Nucleosil 100–5 μm C18 column. Aciclovir and ganciclovir are analysed by spectrofluorimetric detection set at 260 nm (excitation) and 380 nm (emission) using a gradient elution program with solvents constituted of acetonitrile and a solution of sodium heptanosulfonate 0.4% adjusted to pH 2.60. The calibration curves are linear between 0.1 and 10 μg/ml. The mean absolute recovery of aciclovir and ganciclovir are 99.2 ± 2.5 and 100.3 ± 2.5%, respectively. The method is precise (with mean inter-day C.V.s within 1.0–1.6% for aciclovir and 1.2–3.5% for ganciclovir), and accurate (range of inter-day deviations −1.6 to +1.6% for aciclovir and −0.4 to −1.4% for ganciclovir). The method has been applied in stability studies of ganciclovir in patients’ blood samples, demonstrating its good stability in plasma at −20 °C and at room temperature. The distribution of ganciclovir and aciclovir in plasma and red blood cells was also investigated in vitro in spiking experiments with whole blood, which showed an initial drop of ganciclovir and aciclovir levels in plasma (about −25%) due to the cellular uptake of aciclovir and ganciclovir by red blood cells. The method has been validated and is currently applied in a clinical study assessing the ganciclovir plasma concentration variability after administration of valganciclovir in a population of solid organ transplant patients.
Keywords: Aciclovir; Ganciclovir; Stability; HPLC;

Determination of muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry by Kenji Tsujikawa; Kenji Kuwayama; Hajime Miyaguchi; Tatsuyuki Kanamori; Yuko Iwata; Hiroyuki Inoue; Takemi Yoshida; Tohru Kishi (430-435).
A reliable analytical method was developed for the quantification and identification of muscimol (MUS) and ibotenic acid (IBO), the toxic constituents of Amanita muscaria and Amanita pantherina. MUS and IBO were extracted from mushrooms by aqueous methanol and derivatized with dansyl chloride (DNS-Cl). After extraction with ethyl acetate and evaporation of the solvent, the residue was ethylated with 1.25 M hydrogen chloride in ethanol. The resulting derivatives were quantified by high-performance liquid chromatography with UV detection and identified by liquid chromatography electrospray ionization tandem mass spectrometry. Calibration curves were linear in the range of 25–2500 ppm for MUS and 40–2500 ppm for IBO, respectively. This method was successfully applied to identify and quantify MUS and IBO in Aminita mushrooms naturally grown and circulated in the drug market.
Keywords: Amanita muscaria; Amanita pantherina; Muscimol; Ibotenic acid; Dansylation; HPLC; LC/MS;

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of valsartan and hydrochlorothiazide in human plasma by Hao Li; Yingwu Wang; Yao Jiang; Yunbiao Tang; Jiang Wang; Limei Zhao; Jingkai Gu (436-442).
A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of valsartan and hydrochlorothiazide in human plasma. After a simple protein precipitation using acetonitrile, the analytes were separated on a Zorbax SB-Aq C18 column using acetonitrile −10 mM ammonium acetate (60:40, v/v, pH 4.5) as mobile phase at a flow rate of 1.2 mL/min. Valsartan and hydrochlorothiazide were eluted at 2.08 min and 1.50 min, respectively, ionized using ESI source, and then detected by multiple reaction monitoring (MRM) mode. The precursor to product ion transitions of m/z 434.2–350.2 and m/z 295.9–268.9 were used to quantify valsartan and hydrochlorothiazide, respectively. The method was linear in the concentration range of 4–3600 ng/mL for valsartan and 1–900 ng/mL for hydrochlorothiazide. The method was successfully employed in a pharmacokinetic study after an oral administration of a dispersible tablet containing 80 mg valsartan and 12.5 mg hydrochlorothiazide to each of the 20 healthy volunteers.
Keywords: Valsartan; Hydrochlorothiazide; LC/MS/MS; Pharmacokinetics;

An automated solid-phase extraction procedure combined with the gas chromatography–mass spectrometry (GC–MS) methodology, without derivatization, has been developed for the determination of ketamine (K), norketamine (NK), and dehydronorketamine (DHNK) in urine. The analytical approach is simple and rapid, yet reliable, achieving good linearity (r 2  > 0.999 over the concentration range of 30 to 1000 ng/mL), sensitivity (limits of quantification = 15, 10, and 20 ng/mL for K, NK, and DHNK, respectively), accuracy (90–104%), and precision (RSD < 8.1%) for all analytes. Two hundred and six urine specimens collected from suspected drug users were analyzed by this protocol and also screened by Neogen ELISA method to evaluate the efficiency as well as the compatibility of these two methods. Neogen ELISA showed high efficiency (98.1%), high sensitivity (90.9%), high specificity (98.9%), low false-positive rate (1.1%), and moderate false-negative rate (9.1%), adopting 10 ng/mL K as the cutoff. Neogen ELISA screening followed by GC–MS analysis appeared to be a good screening-confirmation test scheme for the analysis of K in urine. Twenty of the 22 positive urine specimens contained all three analytes simultaneously, with DHNK showing the highest and K the lowest concentrations.
Keywords: Ketamine; Norketamine; Dehydronorketamine; GC–MS; ELISA;

We describe the development and validation of a method for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3-hydroxy-4-methoxymethamphetamine (HMMA), 3-hydroxy-4-methoxyamphetamine (HMA), 3,4-methylenedioxyethylamphetamine (MDEA), methamphetamine (MAMP) and amphetamine (AMP) in sweat. Drugs were eluted from PharmChek™ sweat patches with sodium acetate buffer, extracted with disk solid phase extraction and analyzed using GC/MS-EI with selected ion monitoring. Limits of quantification (LOQ) for MDMA, MDEA, MAMP and AMP were 2.5 ng/patch, and 5 ng/patch for MDA, HMA and HMMA. This fully validated procedure was more sensitive than previously published analytical methods and permitted the simultaneous analysis of multiple amphetamine analogs in human sweat.
Keywords: MDMA; Amphetamine; Methamphetamine; Sweat patches; GC/MS;

Tetrahydrocannabinol (THC) is an important psychoactive ingredient in marijuana, which is the most widely used illegal recreational drug in the USA. Since it is generally smoked, the constituents of the plant material, as well as THC may be present in oral fluid specimens collected for the purposes of drug testing. We present an analytical procedure for the simultaneous determination of the pyrolytic precursor Δ9-tetrahydrocannabinolic acid A, tetrahydrocannabinol, cannabinol and cannabidiol in human oral fluid specimens using gas chromatography mass spectrometry (GC/MS). Solid phase extraction and GC/MS/EI with selected ion monitoring were used, and the linearity of the method ranged from 0–16 ng/mL of neat oral fluid. The recovery of the cannabinoids from the collection pad into the transportation buffer was greater than 70% for all cannabinoids tested at 4 ng/mL, and the intra- and inter-day precision was less than 10.3 and 15.2% for all analytes. The stability of the drugs in oral fluid and of the extracted derivatives was investigated. The procedure was applied to oral fluid specimens taken from habitual marijuana smokers. We have previously reported the presence of the metabolite 11-nor-Δ9-tetra-hydrocannabinol-9-carboxylic acid in oral fluid, but this is the first report of the plant constituent 2-carboxy-THC being detected in saliva.
Keywords: Oral fluid; Tetrahydrocannabinol; Cannabinol; Cannabidiol; 2-Carboxy-THC;

Determination of N-acetyl retigabine in dog plasma by LC/MS/MS following off-line μElution 96-well solid phase extraction by Wei Bu; Mai Nguyen; Christine Xu; Chin-Chung Lin; Li-Tain Yeh; Virginia Borges (465-472).
A high throughput off-line μElution 96-well solid phase extraction (SPE) followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for the determination of N-acetyl retigabine in dog plasma has been developed and validated. The method involves the use of μElution 96-well SPE for the simultaneous extraction of N-acetyl retigabine and rapid removal of its N-glucuronide metabolite that has shown to be problematic due to its instability using other clean-up methods. The μElution SPE technology eliminates the need for post-extraction solvent evaporation and greatly reduces sample preparation time consequently improving assay efficiency.
Keywords: N-Acetyl retigabine; μElution SPE; LC/MS/MS; Dog plasma;

Isoimperatorin is one of the major furanocoumarins isolated from the dried root of Angelica dahuricae Benth.et Hook. The aim of the present study is to develop a procedure based on gas chromatography–mass spectrometry (GC–MS) to describe the analysis of isoimperatorin in rat plasma and tissue. The method was set up and adapted for the analysis of small biological samples taken from rats. Biological samples were extracted by liquid–liquid extraction. Extracted compounds were acetic ether/light petroleum (1:2). They were separated by GC on a DB-5MS analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.027–5.32 μg/mL (r  > 0.99) for plasma samples and 0.108–21.28 μg/g (r  > 0.99) for the tissue samples. The limit of detection (LOD) was 1.0 ng/mL or 1.0 ng/g (three times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.81–5.22% and 4.72–6.52%, respectively. The method recoveries for all samples were >80%. The main pharmacokinetic parameters obtained were T max  = (1.06 ± 0.12) h, C max  = (0.72 ± 0.14) μg/mL, AUC = (2.11 ± 0.29) h μg/mL and K a  = (1.76 ± 0.13)/h. The concentrations of isoimperatorin in rat liver, heart, cerebellum and cerebrum were higher than those in other organs. The results presented here clearly indicate that this proposed method could be applicable to investigate the pharmacokinetic and tissue distribution of isoimperatorin in rats after administration.
Keywords: Isoimperatorin; GC/MS; Pharmacokinetics; Tissue distribution; Angelica dahuricae;

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to pharmacokinetic study of nalmefene in humans by Ping Li; Xiaoyan Chen; Xiaojian Dai; Aidong Wen; Yifan Zhang; Dafang Zhong (479-484).
A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for quantification of nalmefene in human plasma. An aliquot of 200 μL plasma sample was simply precipitated by 400 μL methanol. Separation of nalmefene and the internal standard hydromorphone from the interferences was achieved on a C18 column followed by MS/MS detection. The analytes were monitored in the positive ionization mode with a TurboIonspray source. The method had a total chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 10–5000 pg/mL. The lower limit of quantification (LLOQ) was 10 pg/mL. The intra- and inter-day precision was less than 10.1% determined from QC samples at concentrations of 30, 300 and 4500 pg/mL, and the accuracy was within ±3.4%. As the method was more sensitive (10 times higher) than those reported previously, we investigated the pharmacokinetics of nalmefene in healthy volunteers after a single intravenous injection of low dose (30 μg) of nalmefene hydrochloride for the first time.
Keywords: Nalmefene; LC/MS/MS; Pharmacokinetics;

Liquid chromatography–mass spectrometry assay for quantification of Gluten Exorphin B5 in cerebrospinal fluid by Giuseppe Fanciulli; Emanuela Azara; Troy D. Wood; Giuseppe Delitala; Mauro Marchetti (485-490).
A sensitive, precise and accurate method for the quantification of the alimentary opioid peptide Gluten Exorphin B5 (GE-B5, Tyr-Gly-Gly-Trp-Leu) in cerebrospinal fluid (CSF) was developed using liquid chromatography–mass spectrometry (LC–MS). Aliquots (10 μL) of sheep CSF were injected into a LC–MS instrument equipped with a reversed-phase C12 column at a flow rate of 250 μL/min. The mobile phase consisted of Eluent A water with 0.01% acetic acid as an ion-pairing reagent, and Eluent B acetonitrile. The LC–MS system was programmed to divert column flow to waste for 3.5 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. DADLE (Tyr-D-Ala-Gly-Phe-D-Leu) was used as Internal Standard. No significant interfering peaks were detected at the retention times of GE-B5 in CSF blanks. The calibration curves were linear in the range of 0.39–78.00 ng/mL. The lower limit of detection and the lower limit of quantitation values for GE-B5 in CSF were established at 0.30 and 0.78 ng/mL, respectively. The intra-day and inter-day precision values were <12% relative standard deviation. The intra-day and inter-day accuracy were 99.46–100.86% and 98.95–100.02%, respectively. Recovery of GE-B5 in CSF samples was greater than 80%. Stability studies indicate that GE-B5 in CSF undergoes significant degradation (>55% after 600 min), which is reduced by the addition of protease inhibitors. This is the first reported method for the quantification of GE-B5 in CSF.
Keywords: Cerebrospinal fluid; Gluten Exorphin B5; Liquid chromatography–mass spectrometry;

Direct detection and quantification of 19-norandrosterone sulfate in human urine by liquid chromatography-linear ion trap mass spectrometry by Emmanuel Strahm; Christophe Saudan; Pierre-Edouard Sottas; Patrice Mangin; Martial Saugy (491-496).
19-Norandrosterone sulfate (19-NAS) is the sulfoconjugated form of 19-norandrosterone (19-NA), the major metabolite of the steroid nandrolone. A sensitive and accurate liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the direct measurement of 19-NAS in human urine samples. The method involved a quaternary amine SPE protocol and subsequently injection of the extract onto an analytical column (Uptisphere® ODB, 150 mm × 3.0 mm, 5 μm) for chromatographic separation and mass spectrometry detection in negative electrospray ionisation mode. The sulfoconjugate of 19-NA was identified in urine by comparison of mass spectra and retention time with a reference substance. The limit of detection (LOD) and lowest limit of quantification (LLOQ) of 19-NAS were of 40 pg/mL and 200 pg/mL, respectively. For a nominal concentration of 2 ng/mL, recovery (94%), intra-day precision (2.7%), intra-assay precision (6.6%) and inter-assay precision (14.3%) were determined. Finally, this analytical method was applied for quantifying the concentration of 19-NAS in doping samples, using calibration curves (0.2–20 ng/mL) and the standard-addition method. The results show the feasibility of applying this LC-MS/MS assay as a complementary tool to detect misuse of nandrolone or nandrolone precursors.
Keywords: Nandrolone; 19-Norandrosterone sulfate; Liquid chromatography-mass spectrometry; Doping control;

A robust and validated liquid–liquid extraction LC-MS/MS method was developed for population pharmacokinetic analysis and therapeutic drug monitoring of risperidone and the enantiomers of its major active metabolite (+)-and (−)9-hydroxyrisperidone in pediatric patients. The method was rapid, sensitive and used a low sample amount (200 μL), which is very desirable for the pediatric population. The assay was validated from 0.2 to 50 ng/mL in plasma for all analytes. LLOQ for all analytes was 0.2 ng/mL. The extracts were analyzed by normal phase LC-MS/MS. The sample run time was 8 min. Intra- and interday precision for all analytes was ≤6%; method accuracy was between 89 and 99%. Additional experiments were performed to analyze matrix effects and identify a proper internal standard for each analyte. The validated method was used to study risperidone and its enantiomer metabolites in plasma as part of a population pharmacokinetic study in pediatric patients with pervasive developmental disorder (PDD).
Keywords: Risperidone; 9-Hydroxyrisperidone enantiomers; LC-MS/MS method; Matrix effects;

A hydrophilic interaction chromatography (HILIC)/mass spectrometric assay was developed for the determination of zanamivir, a neuraminidase inhibitor used to treat influenza, in rat and monkey plasma. An organic solvent with hydrophilic properties, methanol, was used to precipitate proteins in plasma to assure the highly polar zanamivir of staying in solution. Chromatographic separation was obtained using a HILIC silica column with multiple reaction monitoring turboionspray positive ion detection. The stable label of zanamivir, [13C1 15N2] GR121167C, was used as the internal standard. The assay was validated for the determination of zanamivir in rat and monkey plasma. The lower and upper limits of quantitation were 2 and 10000 ng/mL, using 0.05 mL plasma aliquot, respectively. The signal to noise ratio of a typical 2 ng/mL was ∼5:1. The inter-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 6 to 10% and −6.5 to 0.2%, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in monkey plasma, derived from the analysis of validation samples at five concentrations, ranged from 2 to 8% and −2.3 to 2.1%, respectively. Zanamivir was found to be stable for at least 5 days at approximately −80 °C and at room temperature in plasma. This assay incorporates a simple protein precipitation with methanol and hydrophilic interaction chromatography which is sensitive, accurate, precise, and is being used to support oral formulation and toxicokinetic studies in rat and monkey, respectively.
Keywords: Zanamivir; Neuraminidase inhibitor; LC–MS/MS; Hydrophilic interaction chromatography (HILIC);

Micro-thermal field-flow fractionation of bacteria by Josef Janča; Věra Kašpárková; Věra Halabalová; Lubomír Šimek; Jan Růžička; Eva Barošová (512-518).
The retention of Staphylococcus epidermidis bacteria cells, achieved with the use of micro-thermal field-flow fractionation and described in this paper, represents the first experimental proof that the separation and characterization of the bio-macromolecules and biological particles is possible by exploiting Ludwig–Soret effect of thermal diffusion. The experiments were carried out under gentle experimental conditions preventing the denaturation of the bacteria. Lift forces, appearing at high linear velocities of the carrier liquid, generated the focusing mechanism of the retention which resulted in high-speed and high-performance separation performed in less than 10 min.
Keywords: Micro-thermal field-flow fractionation; Bacteria; Staphylococcus epidermidis;

Determination of fluoxetine, norfluoxetine and their enantiomers in rat plasma and brain samples by liquid chromatography with fluorescence detection by Nora Unceta; Sergio Barrondo; Iñigo Ruiz de Azúa; Alberto Gómez-Caballero; M. Aranzazu Goicolea; Joan Sallés; Ramón J. Barrio (519-528).
Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection (λ exc  = 227 nm, λ em  = 305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5–1000 ng ml−1 and 100–40,000 ng g−1 for doped samples, with detection limits of 3.2 and 2.1 ng ml−1 in plasma and 31.5 and 26.1 ng g−1 in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD (λ exc  = 224 nm, λ em  = 336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2–1000 ng ml−1 and 20–7000 ng g−1 with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml−1 in plasma and between 3.0 and 8.2 ng g−1 in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV < 9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.
Keywords: Fluoxetine and norfluoxetine enantiomers; HPLC-FD; Chiral derivatization; Chronic treatment with fluoxetine; Rat plasma and brain levels;

A liquid chromatographic (LC)–tandem mass spectrometry (MS/MS) method was developed for simultaneous determination of stilbenes, diethylstilbestrol (DES), hexestrol (HEX), and dienoestrol (DEN) in animal tissue. Sample clean-up and analyte enrichment was performed by automated solid-phase extraction (ASPE) with a silica gel cartridge. Detection capabilities (CCβ) related to the transition products of lowest abundance for the method were 0.04–0.45 ng g−1 in tissue and were achieved using atmospheric pressure chemical ionization (APCI) in negative mode. The use of an internal standard in combination with the simplified sample preparation led to a sensitive and reliable analytical method. The recovery level of the method was 84–108% for DES and DEN between 0.5 and 5 ng g−1, and 59–87% for HEX between 0.25 and 2.5 ng g−1.
Keywords: Liquid chromatographic–tandem mass spectrometry; Stilbenes; Residues; Animal tissue;

Nifedipine (NIF), a calcium channel antagonist, is metabolized primarily by cytochrome P450 (CYP3A4) to dehydronifedipine (DNIF). As such, NIF is often used as a probe drug for determining CYP3A4 activity in human studies. A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine NIF and DNIF in human plasma using nitrendipine as the internal standard (IS). After extraction of the plasma samples by ether-n-hexane (3:1, v/v), NIF, DNIF and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm × 2.1 mm, i.d., 3 μm). The method had a chromatographic running time of approximately 2.5 min and linear calibration curves over the concentrations of 0.5–100 ng/mL for NIF and DNIF. The recoveries of the one-step liquid extraction method were 81.3–89.1% for NIF and 71.6–80.4% for DNIF. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for both analytes. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL. The validated LC/MS/MS method has been successfully used to study pharmacokinetic interactions of NIF with the herbal antidepressant St. John's wort in healthy volunteers. These results indicated that the developed LC/MS/MS method was efficient with a significantly shorter running time (2.5 min) for NIF and DNIF compared to those methods previously reported in the literature. The presented LC/MS/MS method had acceptable accuracy, precision and sensitivity and was used in a clinical pharmacokinetic interaction study of NIF with St. John's wort, a known herbal inducer of CYP3A4. St. John's wort was shown to induce NIF metabolism with increased plasma concentrations of DNIF.
Keywords: Nifedipine; Dehydronifedipine; Pharmacokinetics; Liquid chromatography/tandem mass spectrometry; St. John's wort;

A method for the simultaneous determination of cyclophosphamide (CP), doxorubicin (dox), and doxorubicinol (dol) was developed and validated to analyze 400 μL of plasma from patients receiving chemotherapeutic treatment with CP and dox. Final calibration ranges for the analytes were 0.440–60.0 μg/mL for cyclophosphamide, 7.20–984 ng/mL for dox and 3.04–104 ng/mL for dol. The samples were prepared using solid phase extraction and analyzed using a gradient separation over a Waters Symmetry® C18, 2.1 by 30 mm (Milford, MA) column. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer.
Keywords: Anthracyclines; Pharmacokinetics; Obesity; Assay;

A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of aminothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients (r) > 0.99. The intra- and inter-day precision and accuracy were within ±10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of aminothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease.
Keywords: Aminothiols; Homocysteine; Cysteine; Cysteinylglycine; Glutathione; Oxidative stress;

Separation and mass spectrometric characterization of covalently bound skin ceramides using LC/APCI-MS and Nano-ESI-MS/MS by Hany Farwanah; Barbara Pierstorff; Christian E.H. Schmelzer; Klaus Raith; Reinhard H.H. Neubert; Thomas Kolter; Konrad Sandhoff (562-570).
Ceramides covalently bound to keratinocytes are essential for the barrier function of the skin, which can be disturbed in diseases, such as psoriasis and atopic dermatitis. These ceramides of the classes ω-hydroxyacyl-sphingosine and ω-hydroxyacyl-6-hydroxysphingosine contain an ω-hydroxy fatty acid. For their separation and identification, a new analytical approach based on normal phase liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry and tandem nano-electrospray mass spectrometry, respectively, is presented here. Tandem mass spectrometry provided structural information about the sphingoid base as well as the fatty acid moieties. The chain lengths of the bases ranged from C12 to C22, the chain lengths of the fatty acids varied between C28 and C36. In total, 67 ceramide species have been identified in human skin. The analytical methods presented in this work can be helpful for investigating alterations in the ceramide composition of the skin as seen in psoriasis, atopic dermatitis, and diseases with impaired epidermal barrier function.
Keywords: Skin; Covalently bound ceramides; LC/APCI-MS; Nano-ESI-MS/MS;

HPLC determination of midazolam and its three hydroxy metabolites in perfusion medium and plasma from rats by Jan Juřica; Miroslav Dostálek; Jiří Konečný; Zdeněk Glatz; Eva Hadašová; Josef Tomandl (571-577).
A new, simple, rapid, sensitive, and repeatable isocratic reverse-phase HPLC method was developed and validated for simultaneous determination of midazolam and its main three hydroxylated metabolites, i.e. 1′-hydroxymidazolam, 4-hydroxymidazolam, and 1′,4-dihydroxymidazolam in rat liver perfusate and also plasma. Diazepam was used as an internal standard to ensure precision and accuracy of this method. Analytes were extracted from alkalinized samples into diethyl ether using single-step liquid–liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and sodium acetate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 7.9 and 19.6 μg/L for midazolam and its hydroxy metabolites. The overall recovery for the analytes exceeded 92%, for concentrations twice the limits of detection. The intra- and inter-day precision at three different concentrations never exceeded 8 and 11% variation, respectively. This method is applicable for modeling and description of possible pharmacological interactions on rat (CYP3A1/2) or human (CYP3A4/5) cytochrome P450 enzymes.
Keywords: Cytochrome P450; Midazolam; 1′-Hydroxymidazolam; 4-Hydroxymidazolam; 1′,4-Dihydroxymidazolam; Rat; Perfusion; Plasma; HPLC;

The methylenedioxy-derivatives of amphetamine represent the largest group of designer drugs. The 4-methyl (DOM), -ethyl (DOET) and -propyl (DOPR) derivatives of 2,5-dimethoxy-amphetamine (2,5-DMA) were found to possess quite similar serotonin receptor affinities [R.A. Glennon, D.L. Doot, R. Young, Pharmacol. Biochem. Behav. 14 (1981) 287.]. This paper describes a method to screen for and quantify DOM, DOET and DOPR in urine samples, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validated according to international guidelines. Data for accuracy and precision were within required limits. Calibration curves were generated ranging from 10 to 1000 ng/mL and correlation coefficients always exceeded 0.996.
Keywords: Urine; 2,5-Dimethoxy-4-methylamphetamine (DOM); 2,5-Dimethoxy-4-ethylamphetamine (DOET); 2,5-Dimethoxy-4-propylamphetamine (DOPR); Capillary electrophoresis-mass spectrometry (CE-MS);

A sensitive bioanalytical method for the measurement of two major circulating metabolites of duloxetine [4-hydroxy duloxetine glucuronide (LY550408) and 5-hydroxy-6-methoxy duloxetine sulfate (LY581920)] in plasma is reported. This method produced acceptable precision and accuracy over the validation range of 1–1000 ng/mL. Several issues had to be addressed in order to develop an LC/MS/MS assay for these metabolites. First, 4-hydroxy duloxetine glucuronide required chromatographic resolution from the 5-, and 6-hydroxy duloxetine glucuronide isomers. Second, the glucuronide conjugate is readily ionized under positive ESI conditions, while the sulfate conjugate required negative ESI conditions to obtain adequate sensitivity. Finally, the chromatographic conditions needed to separate the glucuronide isomers were not suitable for the analysis of the sulfate conjugate. The present method addressed these challenges, and was successfully applied to multiple human pharmacokinetic studies in which subjects received oral doses of duloxetine hydrochloride.
Keywords: Duloxetine metabolites; Validation; Plasma assay;

Ultra sensitive determination of limaprost, a prostaglandin E1 analogue, in human plasma using on-line two-dimensional reversed-phase liquid chromatography–tandem mass spectrometry by Junji Komaba; Yoriko Masuda; Yoshitaka Hashimoto; Sachiko Nago; Mayumi Takamoto; Kimio Shibakawa; Susumu Nakade; Yasuyuki Miyata (590-597).
A highly sensitive and selective method has been developed and validated to determine limaprost, a prostaglandin (PG) E1 analogue, in human plasma by on-line two-dimensional reversed-phase liquid chromatography–tandem mass spectrometry (2D-LC/MS/MS) due to the lack of efficient methods to determine very low levels of limaprost in plasma. Limaprost and its deuterium derivatives, used as internal standard, were extracted by protein precipitation and following three-step solid phase extractions. After extraction procedure, samples were analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system consists of Phenyl column at first dimension and ODS at second dimension with a trapping column placed between the separation columns. The linear dynamic range of this method was 0.1–10 pg/ml with 3 ml of plasma (r  > 0.9987). Acceptable precision and accuracy were obtained over the calibration curve ranges. The assay has been successfully used in analyses of human plasma samples to support clinical pharmacokinetics studies.
Keywords: Limaprost; OP-1206; Prostaglandin; Two-dimensional;

Analysis of interaction property of bioactive components in Danggui Buxue Decoction with protein by microdialysis coupled with HPLC–DAD–MS by Xiao-Dong Wen; Lian-Wen Qi; Jun Chen; Yue Song; Ling Yi; Xiao-Wei Yang; Ping Li (598-604).
Interaction of a commonly used combined prescription of Danggui Buxue Decoction (CPDBD) with protein was studied by microdialysis coupled with HPLC–DAD–MS. Nine compounds in CPDBD were unequivocally identified by comparing with their t R, MS data and UV spectra with those of reference compounds, and simultaneously quantified. Microdialysis recoveries and binding degrees of 20 compounds in CPDBD with bovine serum albumin (BSA) were determined. Recoveries of microdialysis sampling ranged from 66.9 to 91.5% with RSD below 3.0%, and the binding degrees of those to BSA ranged from 6.3 to 59.8% (0.3 mM BSA) and from 6.9 to 86.6% (0.6 mM BSA). The results were determined at pH 7.4 and the influence of different pH value was investigated. Furthermore, the binding degrees of eight reference compounds were determined separately under the same conditions, indicating a significant effect of the interaction of compounds with each other on their binding degrees to BSA. By comparing their binding degrees with BSA with those of proven active compounds in CPDBD, i.e. chlorogenic acid (3), ferulic acid (6), ononin (12) and calycosin (16), other five compounds were found to possess potential activities, which were tentatively identified as calycosin-7-O-β-d-glucoside-6-O-malonate (9), senkyunolide I or H (10), formononetin-7-O-β-d-glucoside-6-O-malonate (17), and two unknown compounds.
Keywords: Protein; Microdialysis; HPLC–DAD–MS; Bioactive components; Combined prescription of Danggui Buxue Decoction;

NC100692 is under development as a diagnostic radiopharmaceutical for targeting angiogenesis associated with diseases, such as cancer and endometriosis. NC100692 consists of a cyclic RGD-containing peptide with an ethylene glycol chain linked to the C-terminal amino acid and a 99mTc-binding chelator linked to the N-terminal amino acid. The present report describes a method for quantification of NC100692 in human citrated plasma. The method is based on solid-phase extraction followed by reversed-phase liquid chromatography using a gradient of water and acetonitrile with 0.1% formic acid. The chromatographic system was coupled on-line with an electrospray mass spectrometer. The analyses were performed by selective ion monitoring of the [M  + 2H]2+ and the [M  + 3H]3+ ions of NC100692 and the internal standard, which was identical to NC100692 except for containing twice the length of the ethyleneglycol chain. The limit of quantification of the method was 0.5 ng NC100692/ml plasma. The calibration curve ranged from 0.5 to 250 ng NC100692/ml plasma and was fitted to a quadratic equation with a weighing factor of 1/y and found to be highly reproducible. The total precision of the method, expressed as the relative standard error of the mean, was 11.1, 10.8 and 9.7% for the low, medium and high control samples, respectively. The accuracy of the method was 103.4, 111.1 and 107.5% for the low, medium and high control samples, respectively. NC100692 was stable in human plasma during at least 3 freeze/thaw cycles, during 48 h on dry ice and at least 8 weeks when stored in a −20 °C freezer.
Keywords: NC100692; Human plasma; LC–MS; Ion-trap; Imaging; Angiogenesis;

Domperidone is currently used in Canada and Europe for the treatment of intestinal motility disorders as well as for its antiemetic properties. Recent drug metabolism studies have indicated that domperidone is a substrate of different subtypes of CYP3A family and consequently, the drug requires complete characterization of its metabolism for the identification of major drug–drug interactions. Therefore, the purpose of our studies was to develop a simple, sensitive and rapid HPLC assay for the determination of domperidone and its major metabolites. This assay had to be suitable for the conduct of in vitro drug metabolism study with human liver microsomes. Baseline resolution of internal standard, domperidone and three of its major metabolites was achieved in a run time of less than 15 min using an Ultrasphere ODS column (250 mm × 4.6 mm × 5 μM) and a mobile phase consisting of disodium citrate buffer (10 mM, pH 3.4):methanol:acetonitrile:trietylamine, 54.6:34.7:9.9:0.8 at a flow rate of 1.0 mL/min. Chromatographic separation was executed at room temperature. Quantification was performed by tandem fluorescence (excitation λ  = 282 nm and emission λ  = 328 nm) and ultraviolet detectors (λ  = 254 nm for the quantification of encainide, internal standard). Calibration curves were constructed and showed linearity in the range of 0.1–20 μmol/L and 10–250 μmol/L. Intra- and interday coefficients of variation were less than 8% and 11%, respectively. Mean accuracy was 100.5 ± 9.9% and limit of quantification was established at 0.06 μmol/L for domperidone and its metabolites. The assay allows estimation of enzymatic parameters (K m and V max) of domperidone for the formation of its various metabolites and sensitivity is sufficient for the conduct of inhibition studies with potent CYP3A inhibitors.
Keywords: HPLC; Domperidone; CYP3A4; Cytochrome P450; Metabolites; Drug metabolism;

A novel HPLC–UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05–2 μg/ml (r 2  = 0.9995). The lower limit of quantification (LOQ) was 0.05 μg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC0−t were 5.72 ± 2.42 and 1.35 ± 0.41 μg h/ml; t 1/2 were 4.6 ± 2.1 and 1.7 ± 0.6 h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.
Keywords: 1,2-[bis(1,2-Benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE); 2-Mercaptoethanol; Pre-column derivatization; High performance liquid chromatography (HPLC);

Multiresidue determination of quinolone antibacterials in eggs of laying hens by liquid chromatography with fluorescence detection by M.K. Hassouan; O. Ballesteros; J. Taoufiki; J.L. Vílchez; M. Cabrera-Aguilera; A. Navalón (625-630).
An analytical method for the simultaneous determination of seven quinolones (ciprofloxacin, enrofloxacin, danofloxacin, difloxacin, flumequine, oxolinic acid and sarafloxacin) in egg samples of laying hens was developed. Their use is totally prohibited in animals from which eggs are produced for human consumption. Protein precipitation was achieved by addition of acetonitrile and ammonia, removal of acetonitrile with dichloromethane, the quinolones remaining in the basic aqueous extract. The aqueous extract was analysed by liquid chromatography with fluorescence detection (LC–FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile–water (12:88, v/v) and using linear gradient elution. Norfloxacin was used as an internal standard. The limits of detection found were 4–12 ng g−1. These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in different tissues of eggs-producing animals.
Keywords: Quinolones; Eggs analysis; Liquid chromatography–fluorescence detection (LC–FD);

Quantitative analysis of Tenecteplase in rat plasma samples using LC–MS/MS as an alternative for ELISA by B.A.P. Buscher; H. Gerritsen; I. van Schöll; N.H.P. Cnubben; L.P. Brüll (631-634).
An LC–MS/MS method has been developed for the quantitative determination of a protein drug (Tenecteplase; M W 58,777 Da) in rat plasma. The protein was digested with trypsin without prior clean-up of the plasma sample, without the use of a label nor internal standard. A limited validation was performed to assess the linearity, the sensitivity and the specificity of the method. In addition, the developed method was applied to the quantitative analysis of Tenecteplase in rat plasma samples originating from a single-dose study in rats.
Keywords: Quantitative analysis; Proteins; Plasma; Digestion; Liquid chromatography; Mass spectrometry;

Gas chromatography–mass spectrometric assay for propofol in cerebrospinal fluid of traumatic brain patients by Mariska Y.M. Peeters; Hiltjo Kuiper; Ben Greijdanus; Joukje van der Naalt; Catherijne A.J. Knibbe; Donald R.A. Uges (635-639).
A sensitive gas chromatography–mass spectrometry method for measuring propofol in cerebrospinal fluid is described, validated and applied to four patients after traumatic brain injury. The limit of quantitation was 2 μg/L using a volume of 0.5 mL. The inter- and intra-assay coefficients of variation were 5.9 and 5.1%, respectively. The assay covers the linear concentration range of 2–50 μg/L, which is relevant in clinical pharmacokinetic studies when propofol is used in the Intensive Care Unit as a sedative agent or to lower the intracranial pressure.
Keywords: Propofol; CSF; GC–MS;

Determination of pindolol enantiomers in amniotic fluid and breast milk by high-performance liquid chromatography: Applications to pharmacokinetics in pregnant and lactating women by Paulo Vinicius Bernardes Gonçalves; Ricardo Carvalho Cavalli; Sergio Pereira da Cunha; Vera Lucia Lanchote (640-645).
A method for the determination of pindolol enantiomers in amniotic fluid and breast milk was developed, validated, and applied to the investigation of six pregnant women treated with rac-pindolol (10 mg/12 h). Biological samples were extracted with tert-methyl-butyl ether, and the pindolol enantiomers were resolved on a Chiralpak AD column. Amniotic fluid/plasma and milk/plasma concentrations ratios ranged from 0.4 to 4.5 and from 0.6 to 3.7, respectively, for (+)-R-pindolol and from 0.5 to 3.5 and from 1.1 to 2.8, respectively, for (−)-S-pindolol. Preliminary data suggest that amniotic fluid and breast milk are routes of fetal exposure to pindolol enantiomers.
Keywords: Pindolol; Enantiomers; Amniotic fluid; Breast milk; Pharmacokinetics; Pregnancy;

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 μmol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1–7%, and inter-assay CV was 2–12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.
Keywords: Chromatography; Amino acids; HPLC; Reverse phase; Fluorescence;

A highly sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed to determine meloxicam of low concentration in human plasma. After a simple sample preparation procedure by one-step protein precipitation with methanol, meloxicam and the internal standard piroxicam were chromatographed on a Zorbax SB C18 column. The mobile phase consisted of acetonitrile–water–formic acid (80:20:0.2, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via eletrospray ionization (ESI) source. The method had a lower limit of quantification of 0.10 ng/ml. The calibration curve was demonstrated to be linear over the concentration range of 0.10–50.0 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were within ±2.5%), precise (intra- and inter-day relative standard deviation (R.S.D.) <7%). The validated method was successfully applied to the determination of meloxicam in human plasma collected up to 180 h after a transdermal administration of 30 mg meloxicam for evaluation of the pharmacokinetics.
Keywords: Meloxicam; LC–MS–MS; Pharmacokinetics; Transdermal;

We developed a simple and sensitive high-performance liquid chromatography method on a biphenyl column to determine oxytetracycline (OTC) levels in rainbow trout serum. The assay used deproteination, filtration, and subsequent separation on a reverse-phase biphenyl column, with UV detection at 355 nm. OTC (7.8–7.9 min) was completely resolved from structurally similar riboflavin (10.4–10.5 min), a common feed supplement. Estimated limits of detection and quantitation of OTC were 0.01 and 0.04 μg/mL, respectively. The average recovery for OTC was 102% with a R.S.D. of 8.34%. Calibration standards were linear from 0.01 to 10 μg/mL.
Keywords: Oxytetracycline; HPLC; High-performance liquid chromatography; Riboflavin; Fish; Aquaculture; Rainbow trout;

Screening procedure for the analysis of distigmine bromide in serum by high-performance liquid chromatography–electrospray ionization mass spectrometry by Takeshi Saito; Fumiko Satoh; Kozo Tamura; Hiroyuki Otsuka; Shigeaki Inoue; Isotoshi Yamamoto; Sadaki Inokuchi (659-664).
A screening procedure was developed for the identification and quantification of distigmine bromide in serum samples by using liquid chromatography (LC)–electrospray ionization (ESI)-mass spectrometry (MS). In this method, distigmine bromide was analyzed in 0.5 mL serum by using pancuronium bromide as the internal standard, and gradient elution was performed using a reversed-phase column and a mixture of 10 mM-ammonium formate and methanol as the mobile phase. A highly sensitive assay could be performed with simple solid phase extraction using a cation exchange cartridge column by carrying out selected ion monitoring analysis in the positive ion detection mode. The procedure was validated in terms of linearity (0.997 <  r 2  < 0.999 for concentrations ranging from 5 to 250 ng/mL), extraction recovery (83.0% to 89.3%, n  = 5), and detection limit (S/N ratio, >3 at 2.5 ng/mL). The inter- and intra-day precisions (coefficient of variation; CV%) were <8.5% and <9.7%, respectively. The analytes were evaluated for stability and were found to be stable in serum for 1 week at 4 °C and 4 weeks at −30 °C, and successfully applied to in the analysis of two overdose cases. This method is sensitive and useful for the detection, quantification, and confirmation of distigmine bromide in serum.
Keywords: Distigmine bromide; Serum; Validation; LC–ESI-MS;

The pharmaceutical industry standard for bioanalysis is LC/MS/MS. There are, however, many instances where a single quadrupole detector could successfully be used to provide adequate sensitivity and selectivity for quantitation of drug substances in biological matrices. This paper presents one example of how a single quadrupole detector can be employed in a sensitive and selective analytical method for quantitation of carvedilol. A Synergi Hydro-RP (50 mm × 2 mm i.d.; 4 μm) column was used with acetonitile:water:formic acid mobile phase (32:68:0.01, v/v) at a flow rate of 200 μL/min into a single quadrupole mass spectrometer with an electrospray interface in the positive SIM mode. Using a 300 μL plasma aliquot and a liquid–liquid extraction procedure the limit of quantitation for the assay was 1 ng/mL. The assay utility was demonstrated in the analysis of carvedilol pharmacokinetic profiles in beagle dogs following oral carvedilol administration.
Keywords: Carvedilol; Liquid chromatography–mass spectrometry; Triple quadrupole; Single quadrupole;

A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C18 column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5–10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.
Keywords: Macrolide antibiotics; FMOC derivatives; Solid-phase extraction; UV and fluorescence detection; Pharmacokinetics;

A simple and sensitive HPLC method for the simultaneous analysis of free MPA and free MPAG was developed. Separation was achieved on a X-Terra RP18 column with acetonitrile–40 mM orthophosphoric acid as eluents using a gradient elution mode over 35 min at a flow rate of 1.5 ml/min. The assay was linear in the range 0.005 mg/L (LOQ) to 5 mg/L for free MPA and 0.05 mg/L (LOQ) to 200 mg/L for free MPAG. Isolation of free MPA and free MPAG was done by ultrafiltration and the ultrafiltrate was directly injected. A positive correlation between MPA free fractions and free MPAG concentrations was found. Likewise, free MPAG was related to total MPAG concentrations in the seven heart transplant patients.
Keywords: Mycophenolic acid; HPLC; Free fraction; Ultrafiltration;

Preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) was successfully applied to the isolation and purification of four triterpene saponins from Clematis mandshurica by one-step separation. The solvent system was composed of ethyl acetate–n-butanol–ethanol–0.05% TFA (5:10:2:20, v/v). The purities of all the saponins obtained were better than 97%. The structures of the compounds were elucidated by the means of spectroscopic methods.
Keywords: Clematis mandshurica Rupr.; High-speed counter-current chromatography; Triterpene saponins;

Detection of testosterone propionate administration in horse hair samples by S. Boyer; P. Garcia; M.A. Popot; V. Steiner; M. Lesieur (684-688).
A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100 mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 °C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 μL of acetonitrile and 30 μL of PFPA then incubating for 15 min at 60 °C. After evaporation, 30 μL of hexane was added and 2.5 μL was injected into the column (a bonded phase fused silica capillary column DB5MS, 30 m × 0.25 mm i.d. × 0.25 μm film thickness) of a Trace GC chromatograph. In order to improve the sensitivity of the method, damping gas flow has been optimized. Testosterone was identified in MS2 full scan mode on the Polaris Q instrument. The assay was capable of detecting less than 1 pg mg−1. The recovery was close to 90%. The analysis of tail and mane samples collected from a gelding horse having received a single dose of testosterone propionate (1 mg kg−1) showed the presence of testosterone in the range of 1–6 pg mg−1 in hair collected during 5 months after administration.
Keywords: Testosterone; Testosterone propionate; Horse hair; Gas chromatography–mass spectrometry; Ion trap mass spectrometry; Triple quadrupole mass spectrometer;