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Journal of Pharmaceutical and Biomedical Analysis (v.44, #4)

Editorial Board (pp. co2).

Analytical methodologies for the determination of omeprazole: An overview by M. Espinosa Bosch; A.J. Ruiz Sánchez; F. Sánchez Rojas; C. Bosch Ojeda (pp. 831-844).
Omeprazole, a gastric acid pump inhibitor, dose-dependently controls gastric acid secretion; the drug has greater antisecretory activity than histamine H2-receptor antagonists.Omeprazole has been determined in formulations and biological fluids by a variety of methods such as spectrophotometry, high-performance liquid chromatography with ultraviolet detection and liquid chromatography coupled with tandem mass spectrometry. The overview includes the most relevant analytical methodologies used in its determination since the origin still today.

Keywords: Omeprazole; Pharmaceutical analysis; Review; Analytical methodologies

The application of microbore UPLC/oa-TOF-MS and1H NMR spectroscopy to the metabonomic analysis of rat urine following the intravenous administration of pravastatin by E.M. Lenz; R.E. Williams; J. Sidaway; B.W. Smith; R.S. Plumb; K.A. Johnson; P. Rainville; J. Shockcor; C.L. Stumpf; J.H. Granger; I.D. Wilson (pp. 845-852).
The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)–oa-TOF-MS and, independently, by1H NMR spectroscopy. UPLC was performed using a 1mm microbore column packed with 1.7μm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC–MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic “trajectory” as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC–MS and NMR) were found to be complementary whilst the use of a 1mm i.d.×100mm column reduced the amount of sample required for analysis to 2μL, compared with 10μL for a 2.1mm i.d.×100mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.

Keywords: LC–MS; 1; H NMR spectroscopy; Metabonomics; UPLC; TOF-MS; Microbore columns; Pravastatin; Biomarkers; Hepatotoxicity

Quantitative determination of 1-deoxynojirimycin in mulberry leaves using liquid chromatography–tandem mass spectrometry by Nitra Nuengchamnong; Kornkanok Ingkaninan; Wiroje Kaewruang; Sathaporn Wongareonwanakij; Bhinai Hongthongdaeng (pp. 853-858).
A novel HPLC–MS/MS method was developed for the quantitative determination of 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor present in mulberry leaves ( Morus alba L.). DNJ was isolated from the mulberry leave extract on a TSKgel Amide-80 column using a mixture of 0.1% formic acid and acetonitrile as a mobile phase at a flow rate of 0.6ml/min. A triple quadrupole mass spectrometry using electrospray ionization source in a positive ion mode under multiple reaction monitoring with the [ M+H]+ ions, m/ z 164.4/109.9 was used. The detection limit (S/N=3) was 75pg and quantitation limit (S/N=10) was 100pg. The comparison of mulberry leaves of different ages showed that the DNJ level was higher in mulberry shoots than young and mature leaves.

Keywords: 1-Deoxynojirimycin; Mulberry leaves; Mulberry shoots; LC–MS/MS; MRM

Characterization of acid–base properties of unstable drugs using a continuous-flow system with UV–vis spectrophotometric detection by Anna Argemí; Javier Saurina (pp. 859-866).
In this paper, we propose a continuous-flow system for the study of the acid–base characteristics of unstable drugs. 5-Azacytidine has been selected as a first model of unstable compound, which progressively decomposes in aqueous solutions. Besides, other compounds undergoing hydrolysis and oxidation side reactions have been also analyzed to explore the performance of the method. In comparison with conventional batch titrations, the drug decomposition can be minimized by the continuous renewal of the analyte solution. The composition of the buffer mixture is varied on-line during the process from successive changes in the flow rates of acid and basic stock solutions. As a result, the pH value of the test solution is varied in a controlled manner in the range of 1–13. Multivariate curve resolution based on alternating least squares has been used to extract relevant information concerning the acid–base properties of analytes. Results from the continuous-flow system have been compared with those obtained, using batch spectrophotometric titrations, and in the case of fast degradations, the performance of the proposed procedure has been superior.

Keywords: Unstable drugs; 5-Azacytidine; Acid–base characterization; Continuous flow titration; Chemometric analysis

Micelle-mediated extraction and cloud point preconcentration for the analysis of aesculin and aesculetin in Cortex fraxini by HPLC by Zhihong Shi; Xiaomin Zhu; Hongyi Zhang (pp. 867-873).
In this paper, a micelle-mediated extraction and cloud point preconcentration method was developed for the determination of less hydrophobic compounds aesculin and aesculetin in Cortex fraxini by HPLC. Non-ionic surfactant oligoethylene glycol monoalkyl ether (Genapol X-080) was employed as the extraction solvent. Various experimental conditions were investigated to optimize the extraction process. Under optimum conditions, i.e. 5% Genapol X-080 (w/v), pH 1.0, liquid/solid ratio of 400:1 (ml/g), ultrasonic-assisted extraction for 30min, the extraction yield reached the highest value. For the preconcentration of aesculin and aesculetin by cloud point extraction (CPE), the solution was incubated in a thermostatic water bath at 55°C for 30min, and 20% NaCl (w/v) was added to the solution to facilitate the phase separation and increase the preconcentration factor during the CPE process. Compared with methanol, which was used in Chinese Pharmacopoeia (2005 edition) for the extraction of C. fraxini, the extraction efficiency of 5% Genapol X-080 reached higher value.

Keywords: Micelle-mediated extraction; Cloud point preconcentration; Genapol X-080; Cortex fraxini; Aesculin; Aesculetin

Determination of surface-adsorbed excipients of various types on drug particles prepared by antisolvent precipitation using HPLC with evaporative light scattering detection by Anne Zimmermann; Michiel Ringkjøbing Elema; Tue Hansen; Anette Müllertz; Lars Hovgaard (pp. 874-880).
A common challenge in the development of new drug substances is poor dissolution characteristics related to low aqueous solubility. One approach to overcome this problem is antisolvent precipitation in the presence of polymers or surfactants, which may enhance the dissolution rate through reduced particle size and increased wettability. In this study, a simple method based on size exclusion chromatography (SEC) with evaporative light scattering detection (ELSD) was developed for the determination of polymers and surfactants adsorbed to drug particles prepared by antisolvent precipitation of the poorly water-soluble model drug Lu 28–179. Detection of many polymeric excipients and surfactants is problematic due to the lack of UV-absorbing chromophores, but ELSD proved successful for the direct determination of the investigated compounds. A mixed mode column was used to effectively separate each of the excipient structures from the drug. The mobile phase comprised acetonitrile–ammonium formate (20mM; pH 6.5) (50:50, v/v) at a flow-rate of 0.6ml/min. Qualification studies showed that the method was adequately sensitive and precise with limits of detection between 0.72 and 4.32μg/ml. Linearity of the calibration curves was achieved by log–log modelling. The method was applied for determination of nine polymeric excipients and surfactants adsorbed to particles of the model drug. The extent of excipient adsorption varied between 0.07 and 1.39% (w/w) of the total particle weight.

Keywords: Poorly soluble drugs; Antisolvent precipitation; Surfactants; Polymers; Size exclusion chromatography (SEC); Evaporative light scattering detection (ELSD)

Separation and determination of flavonoids in Lamiophlomis rotata by capillary electrophoresis using borate as electrolyte by Mina Luo; Huawei Lu; Huaqiao Ma; Liang Zhao; Xia Liu; Shengxiang Jiang (pp. 881-886).
A capillary electrophoresis method was developed for the simultaneous determination of five flavonoids such as luteolin-7- O-glucoside, isorhamnetin, apigenin, luteolin and quercetin in Lamiophlomis rotata (Benth.) Kudo. Optimal conditions were obtained at pH 9.0 with 30mM borate as buffer containing 8% (v/v) acetonitrile, 20kV as driving voltage and 210nm as detection wavelength. The association constant K and the change in Gibbs free energy (Δ G) of the interaction of flavonoids with borate anion ion (a typical ion-dipole or ion-induced dipole interaction) were calculated for the quantitative evaluation and characterization of the interaction. The described method was successfully applied for the rapid and efficient quality control by quantifying flavonoids in L. rotata. Repeatability tests showed that the R.S.D.s of both intra- and inter-day migration times and peak areas were less than 5%. The LOD of the five flavonoids was less than 3.75mg/l. Recovery results ranged from 94.2% to 105.1%.

Keywords: Flavonoid; Capillary electrophoresis; Lamiophlomis rotata; Association constant

Use of liquid chromatography–mass spectrometry and a chemical cleavage reaction for the structure elucidation of a new sildenafil analogue detected as an adulterant in an herbal dietary supplement by John C. Reepmeyer; Jeffrey T. Woodruff (pp. 887-893).
An herbal dietary supplement, marketed as a natural product for the enhancement of sexual function, was analyzed by HPLC with photodiode array and mass spectral detection and found to contain a compound related to the synthetic phosphodiesterase-5 (PDE-5) inhibitors. Based on UV spectra, mass spectra and direct infusion MS n, the structure of the compound was tentatively identified as a sildenafil analogue in which the sulfonyl group had been replaced with an acetyl group. This new analogue is similar to acetildenafil, a previously reported sildenafil analogue, but differs in that it contains an N-methyl group where acetildenafil contains an N-ethyl group. The structure of the unknown was unequivocally established by chemical cleavage of the phenacylamine group of the molecule to generate N-methylpiperazine; other cleavage products matched those generated from acetildenafil. Since the new compound has one less CH2 group than acetildenafil, it was named nor-acetildenafil.

Keywords: Sildenafil analogue; Acetildenafil; Nor-acetildenafil; Erectile dysfunction; Phosphodiesterase-5 inhibitor; Dietary supplement; Liquid chromatography–mass spectrometry (LC–MS)

Column selection for pharmaceutical analyses based on a column classification using four test parameters by Kristóf Kóczián; Erik Haghedooren; Sanja Dragovic; Béla Noszál; Jos Hoogmartens; Erwin Adams (pp. 894-905).
This paper focuses on the usability of a previously developed column classification system, applied to pharmaceutical analyses. The separation of two drugs from their respective related substances was investigated on 65 new reversed-phase liquid chromatographic columns. The chromatographic procedure for fluoxetine hydrochloride was performed according to the method prescribed in the European Pharmacopoeia monograph while the separation of gemcitabine hydrochloride was carried out according to the United States Pharmacopeia monograph. It was shown that the column ranking system is a helpful tool in the selection of a suitable column.

Keywords: Chromatographic tests; Column ranking; Fluoxetine; Gemcitabine

Is HPLC assay for drug substance a useful quality control attribute? by Jeffrey D. Hofer; Bernard A. Olsen; Eugene C. Rickard (pp. 906-913).
HPLC is a generally accepted method for assay of drug substances. However, recent claims cast doubts on the utility of HPLC assay methods for characterizing quality [S. Görög, J. Pharm. Biomed. Anal. 36 (2005) 931–937]. This study examines the utility of the traditional drug substance HPLC assay as a quality control parameter. HPLC assay data from more than 100 batches for each of eight drug substances were compared to results from a mass balance approach (100−impurities%). Estimates of the variability of HPLC assays from our data and from the literature ranged from 0.6 to 1.1% R.S.D. This variability is an appreciable portion of a typical acceptance range (e.g., 98.0–102.0%) and frequently exceeds the variability of the manufacturing process. Therefore, the results of the HPLC assay are questionable at best to determine the acceptability of the drug substance batch. The high variability also can generate a significant percentage of false out-of-specification (OOS) results, even when the “true” purity is 99.0–100.0%. Each false OOS leads to inefficiencies because of unwarranted investigations for a root cause and/or implementation of countermeasures for a problem that does not exist. Lastly, low precision makes it nearly impossible to detect significant changes in the process mean and/or degradation during a stability study. The use of a mass balance approach for assay retains essentially the same average results as the HPLC assay but gives standard deviations that are up to 10 times less. Monitoring the assay by mass balance allows for more precise process and stability monitoring and facilitates more rapid and accurate identification of process changes.

Keywords: HPLC assay; Drug substance; Assay variability; Out-of-specification; Precision

Pharmaceutical analysis of synthetic lipid A-based vaccine adjuvants in poly (d,l-lactic- co-glycolic acid) nanoparticle formulations by Samar Hamdy; Azita Haddadi; Vishwa Somayaji; David Ruan; John Samuel (pp. 914-923).
The present study had two main objectives. First, was to compare the immune stimulatory effect of two synthetic lipid A analogues (7-acyl lipid A and pentaerythritol-based lipid A (PET lipid A)) on maturation/stimulation of bone marrow derived dendritic cells (DCs). Our second objective was to develop a liquid chromatography/mass spectrometry (LC-MS) method for the quantitative analysis of lipid A-based vaccine adjuvants. Treatment of immature DCs with 7-acyl lipid A and PET lipid A up regulated the surface expression of CD86 and CD40 molecules, and also induced similar profile of pro-inflammatory cytokine secretion. LC-MS analyses were performed using a Waters Micromass ZQ 4000 spectrometer, coupled to a Waters 2795 separations module with an autosampler. Calibration curves with R2>0.999 were constructed over the concentration range of 1.25–20μg/ml for the solution of 7-acyl lipid A and PET lipid A. The method was tested in a 3 day validation protocol. The accuracy of the assay at different concentrations tested ranged from 89 to 108% and from 92 to 107% for 7-acyl lipid A and PET lipid A, respectively. The limit of quantification for both 7-acyl lipid A and PET lipid A was 1.25μg/ml (signal/noise (S/N)) ratio >15:1. The sensitivity of the method (the limit of detection) was 0.35 and 0.15ng for 7-acyl lipid A and PET lipid A, respectively (S/N ratio between 4:1 or 3:1). As a preliminary application, this method has been successfully applied to the determination of 7-acyl lipid A and PET lipid A content in poly (d,l-lactic- co-glycolic acid) nanoparticles (PLGA-NP).

Keywords: Immunostimulatory adjuvant; Dendritic cells; Nanoparticles; Lipid A; Liquid chromatography/mass spectrometry

Development and full validation of a sensitive quantitative assay for the determination of clemastine in human plasma by liquid chromatography–tandem mass spectrometry by Zhiyong Xie; Qiongfeng Liao; Zuojun Li; Chenchen Zhu; Yuaner Zeng; Shikun Liu (pp. 924-930).
A sensitive high-performance liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of clemastine in human plasma. After having been extracted from plasma samples by ethyl acetate, clemastine and internal standard, diphenhydramine, were separated on a C18 column. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source. The method was linear in the concentration range of 5.0–1000.0pg/ml for clemastine. The intra- and inter-day precisions were within 13.4% and the deviations were between −1.1% and 5.6%. The fully validated LC/ESI-MS/MS method has been successfully applied to the preliminary pharmacokinetic study in healthy male Chinese volunteers.

Keywords: Clemastine; LC/MS/MS; Determination; Human plasma; Quantitative assay

Determination of berberine in human plasma by liquid chromatography–electrospray ionization–mass spectrometry by Wenyan Hua; Li Ding; Yan Chen; Bin Gong; Jianchang He; Guili Xu (pp. 931-937).
A liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) method for the determination of berberine in human plasma using chlorobenzylidine as the internal standard (IS) has been developed and validated. The plasma samples were prepared by LLE and the analytes were chromatographically separated on a Hanbon Lichrospher 5-C18 HPLC column under gradient elution with a mobile phase consisted of acetonitrile and 10mm ammonium acetate buffer containing 0.1% formic acid. Berberine was determined with electrospray ionisation–mass spectrometry (ESI–MS). LC–ESI–MS was performed in the selected-ion monitoring (SIM) mode using target ions at M+ m/ z 336.1 for berberine and M+ m/ z 464.1 for the IS. Calibration curve was linear over the range of 0.020–3.0ng/ml. The lower limit of quantification (LLOQ) was 0.020ng/ml. The intra- and inter-run variability values were less than 6.7 and 7.7%, respectively. The method has been successfully applied to determine the plasma concentration of berberine in healthy Chinese volunteers.

Keywords: Berberine; LC–ESI–MS; Pharmacokinetics

Validation and implementation of drug-dependent antibody assays in clinical trials for safety monitoring of patients dosed with roxifiban, an orally bioavailable glycoprotein IIb/IIIa antagonist by Yu Chen Barrett; William Ebling; Henry Pieniaszek; Jeffrey Billheimer; Dietmar Seiffert (pp. 938-946).
Thrombocytopenia exposes patients to increased bleeding risk. This serious adverse event was observed with a frequency of approximately 2% in early clinical trials with the potent, orally bioavailable glycoprotein (GP) IIb/IIIa receptor antagonist roxifiban. We previously reported that drug-dependent antibodies (DDAbs) to GP IIb/IIIa are the main cause of thrombocytopenia with roxifiban. Two ELISA assays for detection of free DDAbs (in citrate plasma) and total DDAbs (in EDTA plasma to elute platelet bound DDAbs) were developed and analytically validated. These tests served two purposes during the clinical development program, to pre-screen patients for pre-existing antibodies and monitor patients for increasing antibody titers as a surrogate for eminent thrombocytopenia. The free DDAb assay showed inter and intra-assay precision of 5–12 and 12–14% CV, respectively. The total DDAb assay showed a precision of 5–10 and 4–12% CV, respectively. Three cycles of freeze–thaw did not significantly alter DDAb values in citrate plasma, EDTA plasma or extraction solution. The clinical qualifications of the two assays were conducted in two phase II clinical trials in coronary arterial disease (CAD) patients dosed with roxifiban. Both assays have demonstrated clinical sensitivity of nearly 99–100% and clinical specificity of nearly 95%.

Keywords: Immunogenicity; Drug-dependent antibody assay; Anti-drug antibody assay; Validation; Roxifiban; Differential ELISA

Quantification of13C pyruvate and13C lactate in dog blood by reversed-phase liquid chromatography–electrospray ionization mass spectrometry after derivatization with 3-nitrophenylhydrazine by Steinar Uran; Kristin Eitrem Landmark; Gro Hjellum; Tore Skotland (pp. 947-954).
Injection of hyperpolarized13C-labelled pyruvate (13C pyruvate) is under evaluation as an agent for medical metabolic imaging by measuring formation of13C lactate using magnetic resonance spectroscopy of the13C nuclei. A quantitative method for analysis of these13C-labelled substances in dog blood was needed as part of the development of this agent and we here describe a liquid chromatography-mass spectrometry method for that purpose. Immediately after blood collection, the blood proteins were precipitated using methanol added internal standard ([U-13C]pyruvate and [U-13C]lactate). Prior to analysis, the compounds were derivatized using 3-nitrophenylhydrazine. Following separation on a Supelco Discovery HS C18 column,13C pyruvate and13C lactate were detected using negative electrospray ionization mass spectrometry. Calibration standards (4.5–4500μM13C pyruvate and 9–9000μM13C lactate) and added internal standard were used to make the calibration curves, which were fitted to a non-linear equation y= a+ bx+ cx2 and weighted with a weighting factor of 1/ y2. The analytical lower limit of quantification of13C pyruvate and13C lactate was 4.5 and 9μM, respectively. The total precision of the method was below 9.2% for13C pyruvate and below 5.8% for13C lactate. The accuracy of the method showed a relative error less than 2.4% for13C pyruvate and less than 6.3% for13C lactate. The recoveries were in the range 93–115% for13C pyruvate and 70–111% for13C lactate. Both substances were stable in protein-free supernatant when stored for up to 3 weeks in a −20°C freezer, during three freeze/thaw cycles, and when stored in an autosampler for at least 30h.

Keywords: LC–MS; 13; C pyruvate; 13; C lactate

Reliable HPLC method for therapeutic drug monitoring of frequently prescribed tricyclic and nontricyclic antidepressants by Wilson Roberto Malfará; Carlo Bertucci; Maria Eugênia Costa Queiroz; Sonia Ap. Dreossi Carvalho; Maria de Lourdes Pires Bianchi; Evandro José Cesarino; José Alexandre Crippa; Regina Helena Costa Queiroz (pp. 955-962).
A new high-performance liquid chromatography method is presented for the determination of 10 frequently prescribed tricyclic and nontricyclic antidepressants: imipramine, amitriptyline, clomipramine, fluoxetine, sertraline, paroxetine, citalopram, mirtazapine, moclobemide and duloxetine. The simple and accurate sample preparation step, consisted of liquid:liquid extraction with recoveries ranging between 72% and 86%, except for moclobemide (59%). Separation was obtained using a reverse phase Select B column under isocratic conditions with UV detection (230nm). The mobile phase consisted of 35% of a mixture of acetonitrile/methanol (92:8, v/v) and 65% of 0.25molL−1 sodium acetate buffer, pH 4.5. The standard curves were linear over a working range of 2.5–1000ngmL−1 for moclobemide, 5–2000ngmL−1 for citalopram, duloxetine, fluoxetine, 10–2000ngmL−1 for sertraline, imipramine, paroxetine, mirtazapine and clomipramine. The intra-assay and inter-assay precision and accuracy were studied at three concentrations (50, 200, and 500ngmL−1). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8%, and all inter-CVs were less than 10%. Limits of quantification were 2.5ngmL−1 for moclobemide, 5ngmL−1 for citalopram, duloxetine and amitriptyline, and 10ngmL−1 for mirtazapine, paroxetine, imipramine, fluoxetine, sertraline, and clomipramine. No interference of the drugs normally associated with antidepressants was observed. The method has been successfully applied to the analysis of real samples, for the drug monitoring of ten frequently prescribed tricyclic and non-tricyclic antidepressant drugs.

Keywords: Therapeutic drug monitoring; Tricyclic; Nontricyclic antidepressants; HPLC UV detection

Determination of rifampicin in human plasma and blood spots by high performance liquid chromatography with UV detection: A potential method for therapeutic drug monitoring by A.L. Allanson; M.M. Cotton; J.N.A. Tettey; A.C. Boyter (pp. 963-969).
A high performance liquid chromatography method has been developed that allows quantification of concentrations of rifampicin in human plasma and blood spots. Rifampicin and papaverine hydrochloride (internal standard) were extracted from plasma using a Strata-X-CW extraction cartridge. These analytes were also extracted into acetonitrile from blood spots dried onto a specimen collection card. The recovery of rifampicin from plasma and blood spots was 84.5% and 65.0%, respectively. Separation was achieved by HPLC on a Kromasil C18 column with a mobile phase composed of ammonium acetate (20mM, pH 4.0) and acetonitrile, delivered on a gradient programme. Optimum detection was at 334nm. The assay was linear over the concentration range of 0.5–20μg/ml. The limit of quantification was 0.5μg/ml in plasma; 1.5μg/ml in blood spots. Both intraday and interday precision data showed reproducibility (R.S.D.≤8.0, n=9). Stability studies showed rifampicin was stable in plasma for up to 9h after thawing; the samples were also stable for up to 9h after preparation. Five patient samples were analysed using the methods described. A correlation was found between the concentrations of RIF in plasma and blood spots ( r2=0.92). This method is proposed as a means of therapeutic drug monitoring of rifampicin in patients with tuberculosis.

Keywords: Rifampicin; HPLC; Dried blood spots; Therapeutic drug monitoring; Tuberculosis

A novel antibiotic bone assay by liquid chromatography/tandem mass spectrometry for quantitation of tigecycline in rat bone by Allena J. Ji; James P. Saunders; Nandan D. Wadgaonkar; Peter J. Petersen; Kenneth O’Leary; Williams E. McWilliams; Peter Amorusi; Mauricio Leal; Eric N. Fluhler (pp. 970-979).
Tigecycline (Tygacil®) is a first-in-class, broad spectrum antibiotic with activity against antibiotic-resistant organisms. In rats and humans, tigecycline readily distributes to bone tissue but its accuracy of quantitation via liquid chromatography/mass spectrometry (LC/MS/MS) is hindered by a low extraction recovery when using a conventional plasma extraction method. To overcome this issue, we have identified an effective extraction solvent for quantitation of tigecycline in rat bone. The current LC/MS/MS bone assay is novel, simple, and sensitive, and has a wide linear range of 50–10,000ng/g. The assay requires homogenization of the rat bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation of the supernatant with liquid chromatography, and detection of tigecycline with tandem mass spectrometry. The incurred pooled rat bone samples obtained from rats given 3mg/kg/day of [14C]-tigecycline and non-radio-labeled tigecycline were analyzed with the current method. The absolute extraction recovery of the bone assay for tigecycline was 77.1%. The intra-day accuracy ranged from 91.7 to 106% with precision (CV) of 1.9–10.7%, and inter-day accuracy ranged from 96.1 to 100% with a precision of 6.3–8.7%. In addition, biological activity was demonstrated for the tigecycline extracted from incurred rat bone. This bone assay provides an important analytical tool for the determination of drug concentrations (especially, antimicrobials) in rodent bone tissues and has served as the foundation of development and validation of a similar bone assay for tigecycline in human bone tissues.

Keywords: Tigecycline; Tygacil; GAR-936; Antibiotics; Bone assay; LC/MS/MS

The enhancement of isoflavones water solubility by complexation with modified cyclodextrins: A spectroscopic investigation with implications in the pharmaceutical analysis by R. Stancanelli; A. Mazzaglia; S. Tommasini; M.L. Calabrò; V. Villari; M. Guardo; P. Ficarra; R. Ficarra (pp. 980-984).
The improvement of isoflavones bioavailability by complexation with chemically modified cyclodextrins (CyDs) has been exploited to analyse the drug/macrocycle binding affinity by a conventional method with new useful measures. Genistein (Gen) and daidzein (Daidz) were investigated in aqueous medium and in presence an amount of (2-hydroxypropyl)-β-cyclodextrin (HP-β-CyD) at different host/guest molar ratios. The solubility in pure water, ∼3×10−6M for Gen and ∼10×10−6M for Daidz, was obtained by distributing the of guest molecule between water and the organic solvent. The stoichiometric ratios and stability constants describing the extent of formation of the complexes have been determined by phase-solubility UV–vis measurements and confirmed by circular dichroism data. These results have implications in the determination of the carrier's capacity for the complexation of the drug in water solution.

Keywords: (2-Hydroxypropyl)-β-cyclodextrin; Isoflavones; UV–vis spectroscopy; Circular dichroism; Binding constant; Water solubility measurements

Validation of rapid and simple LC–MS/MS method for determination of voriconazole in rat plasma by B.V. Araujo; D.J. Conrado; E.C. Palma; Teresa Dalla Costa (pp. 985-990).
A rapid, simple and sensitive LC–MS/MS analytical method was developed and validated for the determination of voriconazole (VRC) in rat plasma, using ketoconazole as internal standard (IS). Analysis was performed on a Shimadzu® HPLC system using a Shimadzu® C18 column and isocratic elution with acetonitrile–water–formic acid (60:40:0.05, v/v/v), at a flow of 1.0mL/min (split ratio 1:5), and a mass spectrometer Micromass®, equiped with a double quadrupole and an electrospray ionization interface, operated in a positive mode. Plasma samples were deproteinized with methanol (1:2) and 30μL of the supernatant was injected into the system. The retention times of VRC and IS were approximately 3.3 and 2.7min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 50–2500ng/mL with determination coefficient >0.98. The lower limit of quantification was 50ng/mL. The accuracy of the method was within 5%. Intra- and inter-day relative standard deviations were less or equal to 12.5 and 7.7%, respectively. The applicability of the LC–MS–MS method for pharmacokinetic studies was tested using plasma samples obtained after intravenous administration of VRC to male Wistar rats. The reported method provided the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of VRC in pre-clinical pharmacokinetic studies.

Keywords: Voriconazole; LC–MS/MS; Pharmacokinetics; Biologic fluid; Validation

Continuous wavelet and derivative transforms for the simultaneous quantitative analysis and dissolution test of levodopa–benserazide tablets by Erdal Dinç; Süha Kaya; Tanver Doganay; Dumitru Baleanu (pp. 991-995).
Simultaneous analyses and dissolution tests of levodopa–benserazide tablets were carried out by continuous wavelet transform (CWT) and classic derivative spectrophotometry (DS) without using any chemical separation step. The developed two spectrophotometric resolutions are based on the transformation of the original UV spectra. The original absorption spectra of levodopa and benserazide in the concentration range of 1–80μg/mL and 5–240μg/mL in USP simulated gastric juice were registered in the spectral range of 250–310nm, respectively. Various wavelet families and different spectrophotometric derivative orders were tested to find the optimal signal processing for obtaining desirable calibration graphs and reliable determinations of the investigated drugs. Under the optimized conditions of the methods, symlets wavelet family using a=128 with sixth order (SYM6–CWT) and the first derivative transform with Δ λ=10nm were identified as optimal signal processing methods for the determinations and dissolution tests. The calibration functions for each drug were obtained by measuring the values of the CWT and derivative amplitudes. The validation of the developed methods was confirmed by analyzing various synthetic mixtures of the investigated drugs. Mean recovery values were found between 99.1% and 104.7% for DS and 100% and 102.9% for CWT, respectively for determination of BEN and LEV in synthetic mixtures. Each developed approaches were successfully applied to the simultaneous determination and dissolution test of levodopa and benserazide in their commercial tablets and a good agreement was observed.

Keywords: Dissolution test; Levodopa; Benserazide; Continuous wavelet transform; Derivative transform

A rapid method for the simultaneous determination of 11 saponins in Panax notoginseng using ultra performance liquid chromatography by J. Guan; C.M. Lai; S.P. Li (pp. 996-1000).
A rapid ultra performance liquid chromatography coupled with photo diode array detection method (UPLC-PDA) was developed for the simultaneous determination of 11 saponins, namely notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, Rd and Rg3 in Panax notoginseng. The analysis was performed on Acquity UPLC system with Acquity UPLC BEH C18 column and gradient elution of water and acetonitrile in 12min. The high correlation coefficient ( r2>0.9968) values indicated good correlations between the investigated compounds’ concentrations and their peak areas within the test ranges. The LOQ and LOD were lower to 0.2–2.4 and 0.1–1.8ng on column, respectively. The overall intra- and inter-day variations (R.S.D.) of 11 saponins were lower than 3.1%. The developed method was successfully used for the analysis of saponins in P. notoginseng with overall recovery of 93.0–101.6% for the analytes. The results show that UPLC is a powerful tool for analysis of components in Chinese medicines.

Keywords: Ultra performance liquid chromatography; Panax notoginseng; Ginsenoside; Notoginsenoside; Pressurized liquid extraction

A validated liquid chromatography method for the simultaneous determination of vitamins A and E in human plasma by Danuta Siluk; Regina V. Oliveira; Maria Esther-Rodriguez-Rosas; Shari Ling; Angelo Bos; Luigi Ferrucci; Irving W. Wainer (pp. 1001-1007).
A fast (15min) and simple HPLC method for determination of all- trans-retinol and α-, γ- and δ-tocopherols in human plasma has been developed. The assay utilized 200μl of plasma to which 20μl of internal standard solution (retinol acetate) was added followed by 200μl of water, 400μl of ethanol and 800μl of hexane. The hexane layer was collected, evaporated, the residue dissolved in 200μl of methanol and analyzed on a Zorbax Eclipse XDB-C18 column using a step gradient with a polar organic mobile phase composed of acetonitrile and methanol and variable wavelength fluorescence detection. The quantification limits for all- trans-retinol and γ-tocopherol were 20ng/ml and for α- and δ-tocopherols 500ng/ml and 10ng/ml, respectively. The procedure was validated and applied to the analysis of plasma samples from the Baltimore Longitudinal Study of Aging.

Keywords: Abbreviations; RP-HPLC; reversed-phase high performance liquid chromatography; Eqn.; equation; RE; relative error; BLSA; Baltimore Longitudinal Study of Aging; NHANES III; The Third National Health and Nutrition Examination SurveyVitamins; All-; trans; -retinol; α-Tocopherol; γ-Tocopherol; δ-Tocopherol; HPLC; Fluorescence detection

Development and validation of a rapid HPLC method for the determination of oseltamivir phosphate in Tamiflu® and generic versions by J. Joseph-Charles; C. Geneste; E. Laborde-Kummer; R. Gheyouche; H. Boudis; J.-P. Dubost (pp. 1008-1013).
Oseltamivir phosphate (OP) is an antiviral drug that is used in the treatment and prophylaxis of both influenza A and influenza B. It is effective against all known influenza viruses than can infect humans, including pandemic influenza viruses and may be the most appropriate antiviral option against avian influenza caused by H5N1 virus. Tamiflu®, the registered trademark used under exclusive license by Roche laboratories with OP as active pharmaceutical ingredient, is considered the best treatment for the bird flu disease.A simple, selective, linear, accurate and precise HPLC method was developed and validated for rapid assay of OP aimed to the quality control of Tamiflu® capsules and generic versions. Isocratic elution at a flow rate of 1.2mL/min was employed on a Zorbax CN column (150mm×4.6mm; 5μm) at ambient temperature. The mobile phase consisted of methanol and 0.04M formic acid pH 3.0 (50:50, v/v). The UV detection wavelength was 226nm and 20μL of sample was injected. Sotalol hydrochloride was used as the internal standard (IS). The retention times for OP and IS were 3.40 and 2.25min, respectively. The method was successfully applied to commercial pharmaceuticals, Tamiflu® and generic versions. The proposed method could be applicable for routine analysis of OP and monitoring of the quality of marketed drugs as possibly counterfeit Tamiflu®.

Keywords: Oseltamivir phosphate; Tamiflu; ®; Bird flu; RPLC–UV; Generic versions; Counterfeit

Determination of ochratoxin A in human urine by solid-phase microextraction coupled with liquid chromatography-fluorescence detection by Rosa Vatinno; Antonella Aresta; Carlo G. Zambonin; Francesco Palmisano (pp. 1014-1018).
A new method for the determination of ochratoxin A (OTA) in human urine samples has been developed using solid-phase microextraction (SPME) interfaced with liquid chromatography-fluorescence detection (LC-FD). This method is simpler and cheaper compared to the most widely adopted clean-up procedures for OTA extraction from urine (usually based on immunoaffinity columns).Briefly, urine samples, diluted 1:5 with phosphate buffer (10mM, pH 3), were partitioned against chloroform and the aqueous phase extracted by a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber. The fiber was then “statically” desorbed, through a SPME interface, into a LC system operating in isocratic conditions.The linear range investigated in urine was 0.01–1ng/ml. Within-day R.S.D.% in urine spiked at 0.1 and 1ng/ml were 3.9 and 1.9, respectively, whereas the between-days R.S.D.% were 5.5 and 3.0, respectively.The limits of detection (LOD) and quantitation (LOQ) calculated at a signal-to-noise ratio of 3 and 10 (noise calculated peak to peak on a blank chromatogram at the OTA retention time) were 0.01 and 0.05ng/ml, respectively.

Keywords: Mycotoxins; SPME; LC-FD; Ochratoxin A; Urine

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