Chromatographia (v.70, #9-10)

65th Birthday of Professor Maria Dolores (Lola) Luque de Castro by C. Cámara; J. L. Luque-García (1303-1303).

A Review of Microdialysis Sampling Systems by Nelson Torto (1305-1309).
The most important aspects of microdialysis are a theoretical understanding of the process, the microdialysis membrane and the design of the microdialysis probe including the inner cannula dimensions. Several efforts have been made to theoretically account for the processes that take place during microdialysis. These have been employed to develop optimal sampling conditions so as to increase the applicability of the technique for in situ sampling and as a sample clean-up technique prior to chromatography. On the occasion of Prof. Lo Gorton’s 60th birthday, this review highlights the challenge presented by low analyte recoveries that is the major bottleneck in the wider use of microdialysis. The discussion concludes by considering how to increase analyte recovery through a multiple probe approach or by an increase in recovery in the light of the advantages of nanotechnology. Both approaches could impact on the use of microdialysis as a sampling and sample clean-up technique for liquid chromatography.
Keywords: Microdialysis sampling system; Inner cannula dimensions; Review

This study explored feasibility of utilizing sodium phosphate and mixtures of sodium phosphate and sodium perchlorate salts in mobile phases as UV transparent alternatives to the ammonium formate salts commonly used in LC–MS mobile phases. Chromatography experiments were run at pH 3.5 in 25% acetonitrile mobile phase, using several model cationic analytes to evaluate cation retention on two different C18 columns as the type or amount of salt was varied. For both columns, phosphate consistently showed less cation retention than formate. In other respects, the two columns showed very different behavior. The study suggests that it is feasible to use UV transparent mobile phase additives to provide comparable cation retention of formate mobile phases, but that the exact composition needed for optimal retention agreement is column dependent.
Keywords: Cation retention; Chaotropic effect; Formate buffer; Ionic strength; Peak assignment

Screening and Identification of Permeable Components of Radix et Rhizoma Rhei Extract by Use of Immobilized Artificial Membrane Chromatography by Wenping Zhang; Jin Sun; Yongjun Wang; Xiaohong Liu; Yinghua Sun; Rong Lu; Zhonggui He (1321-1326).
The objective of this work was to screen and identify permeable components of Radix et Rhizoma Rhei extract by use of immobilized artificial membrane chromatography (IAMC) then to investigate the intestinal absorption behavior of these components and the mechanism of absorption at three concentrations (0.3, 0.6, and 1.5 mg mL−1) by use of the in situ rat intestinal absorption model. The percentage absorption of permeable components was then correlated with their retention behavior by using mobile phases of three different pH (5.0, 6.0, and 7.0) containing buffers of two different ionic strength (10 and 50 mmol L−1). There was better correlation between percentage intestinal absorption and IAMC retention at pH 6.0 (r 2 = 0.82) than at pH 5.0 (r 2 = 0.64) or 7.0 (r 2 = 0.74). The correlation coefficients suggest that most of the permeable components of Radix et Rhizoma Rhei were mainly absorbed by a passive diffusion mechanism. The four main permeable components were identified as rhein, aloe-emodin, chrysophanol, and emodin by comparison of chromatographic and spectrometric behavior with those of reference standards. IAMC seems useful as a promising rapid screening technique for identification of bioactive components in complex systems, e.g. traditional Chinese medicines.
Keywords: Column liquid chromatography; Immobilized artificial membrane chromatography; Intestinal absorption; Permeable components; Traditional Chinese medicine; Screening

Comparative LC Enantioseparation of Novel PPAR Agonists on Cellulose- and Amylose-Based Chiral Stationary Phases by Luca Piemontese; Salvatore Faliti; Giuseppe Carbonara; Antonio Laghezza; Paolo Tortorella; Fulvio Loiodice (1327-1333).
The LC enantiomeric separation of several dual PPARα/γ agonists on the commercially available Chiralcel OD and Chiralpak AD columns has been evaluated in normal phase mode using a mobile phase consisting in a mixture of n-hexane, 2-propanol and trifluoroacetic acid at constant volume ratio. Most compounds were separated as underivatized acids without requiring time consuming analysis. Some complementary selectivity was evidenced on the two investigated chiral stationary phases related to the different accessibility of the active sites of the helical cavities. Additional information on the chiral recognition mechanism were deduced from the chromatographic behaviour of some selected methyl esters.
Keywords: Column liquid chromatography; Chiral recognition mechanism; Enantiomeric resolution; Polysaccharide-based chiral stationary phases; PPAR agonists

Enantioselective Analysis of Fluoxetine and Norfluoxetine by LC in Culture Medium for Application in Biotransformation Studies Employing Fungi by Keyller Bastos Borges; Laura Tiemi Okano; Mônica Tallarico Pupo; Pierina Sueli Bonato (1335-1342).
A liquid chromatography method is described for the analysis of fluoxetine and norfluoxetine enantiomers in fungi cultures. The analytes were separated simultaneously by LC employing a serial system. The resolution was performed using a mobile phase of ethanol: 15 mM ammonium acetate buffer solution, pH 5.9: acetonitrile (77.5:17.5:5, v/v/v). UV detection was at 227 nm. Hexane: isoamyl alcohol (98:2, v/v) was used as extractor solvent. The calibration curves were linear over the concentration range of 12.5–3,750 ng mL−1 (r ≥ 0.996). The values for intra- and inter-day precision and accuracy were ≤10% for all analytes. The validated method was used to evaluate fluoxetine biotransformation to its mammalian metabolite, norfluoxetine, by selected endophytic fungi. Although the desired biotransformation was not observed in the conditions used here, the method could be used to evaluate the biotransformation of fluoxetine by other fungi or to be extended to other matrices with adequate procedures for sample preparation.
Keywords: Column liquid chromatography; Enantioseparation; Fluoxetine and norfluoxetine; Biotransformation; Endophytic fungi

A chemometrical approach was applied to develop a reversed-phase liquid chromatographic method for simultaneous determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form. According to contemporary literature, no method was developed for simultaneous determination of carbamazepine and these impurities by chemometrical approach. The fractional factorial design was used for selection of variables significantly influencing the chromatographic separation of the investigated substances. The investigated variables were: temperature of the column, the percentage of organic modifier, the acetate buffer concentration and pH of water phase. The first three variables were proved to be significant and were optimized by face centered, central composite design. Investigation was performed using C18 XBridge Shield analytical column (50 mm × 4.6 mm i.d., particle size 3.5 µm). The optimal conditions for the separation were established with the mobile phase composition of methanol–10 mM acetate buffer (pH adjusted to 2.21 with glacial acetic acid) (50:50, v/v) at a flow rate of 1.5 mL min−1, 25 °C column temperature and detection at 260 nm. Total analysis time was shortened to about 8 min. Finally, the method was successfully validated and subsequently applied to the analysis of commercially available carbamazepine tablets.
Keywords: Column liquid chromatography; Fractional factorial design; Central composite design; Carbamazepine; Iminostilbene and iminodibenzyl

Several products marketed as natural drugs for enhancement of anti-diabetic function have been analyzed and found to contain synthetic hypoglycemic drugs. In the work discussed in this paper, LC-MS analysis ten such drugs [gliquidone (GLQ), glipizide (GLZ), gliclazide (GLC), glibenclamide (GLB), glimepiride (GLM), rosiglitazone (RGL), repaglinide (RPG), metformin (DMBG), phenformin (DBI), and tolbutamide (TOL)] has been improved. Quantification was by use of multiple reaction monitoring mode. The intra-day and inter-day precision of the method ranged from 2.13 to 5.55% and from 3.78 to 8.14%, respectively. LOQ was 1, 1, 1.2, 2, 3, 3, 5, 5, 2, and 1 μg L−1 for GLQ, GLZ, GLC, GLB, GLM, RGL, RPG, DMBG, DBI, and TOL, respectively. The structures of the compounds were identified by collision-induced dissociation mass spectral analysis. The results showed that a variety of synthetic drugs had been illegally added to anti-diabetic herbal products. A surprising result was that some of the adulterants were added into the capsule shell instead of capsule contents. LC-MS–MS is a powerful tool, and the method has wide applicability.
Keywords: Column liquid chromatography-mass spectrometry; Anti-diabetic natural products; Adulterants; Synthetic hypoglycemic drugs; Detection

Fructus Gleditsiae abnormalis and Fructus Gleditsiae sinensis are widely used in traditional Chinese medicine. As different developmental stages of fruits of the same plant Gleditsia sinensis Lam., whether Fructus Gleditsiae sinensis can be used as the substitute of Fructus Gleditsiae abnormalis has long been debated. To compare the differences of the content of main active saponins between the two fruits, we established a new LC-ELSD method for simultaneous determination of four major saponins present in two fruits. After fully validated, the method was successfully applied to quantitatively analyze the four bioactive saponins of twenty samples, 11 of Fructus Gleditsiae abnormalis and 9 of Fructus Gleditsiae sinensis obtained from different locations. It was shown that there were no significant differences in saponins contents and LC profiles between the two fruits. Moreover, the developed method can be used for quality control of Fructus Gleditsiae abnormalis and Fructus Gleditsiae sinensis.
Keywords: Column liquid Chromatography; Evaporative light scattering detection; Quantitative analysis; Saponins; Fructus Gleditsiae abnormalis ; Fructus Gleditsiae sinensis

A liquid chromatographic method was developed to evaluate the quality of Petasites tricholobus through a simultaneous determination of four major active bakkenolides. The wavelengths at 265 and 235 nm were chosen to determine four bakkenolides: bakkenolide-B, bakkenolide-D, bakkenolide-IIIa and bakkenolide-IVa. The recovery of the method was in the range of 98.6 to 103.1%, and all the bakkenolides showed good linearity (r 2 > 0.999) within test ranges. The developed method was applied to the determination of four bakkenolides in the collected herb samples. The results showed that the content of bakkenolides in rhizome was higher than in other parts of the plant and the older the rhizome, the higher was the bakkenolide content. This simple, rapid, low-cost and reliable LC-VWD method is suitable for routine quantitative analysis and quality control of petasites species plants.
Keywords: Column liquid chromatography; Variable wavelength detection; Quality evaluation; Bakkenolides; Petasites tricholobus

A qualitative method, involving supported liquid–liquid extraction (SLE) and ultra high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS–MS), was developed for the rapid tentative identification of various drugs of abuse in urine. In this study, 28 drugs and metabolites were covered by the screening procedure. Before analysis, urine samples were extracted by SLE and good extraction recoveries were obtained for most investigated compounds. The UHPLC strategy was then selected for the rapid separation of amphetamines, cocaine, opiates and related compounds in urine. Using columns packed with sub-2 µm particles, analysis time was reduced down to 2 min, while maintaining acceptable performance. Finally, the detection was by tandem MS operating in the single reaction monitoring (SRM) mode. The most intense transition was selected for the different drugs and SRM dwell times set at 5 ms, to maintain sufficient data points across the narrow UHPLC peaks. The tentative identification of the drugs of interest, including amphetamines, opiates and cocaine, was based on both, retention times and mass spectrometry information. With the proposed method, limits of detection were estimated at about 1 ng mL−1 and the applicability was assessed by successfully analyzing several samples of drug abusers. Finally, this study demonstrates the potential of UHPLC coupled to tandem MS for the rapid screening of drugs of abuse in urine.
Keywords: Supported liquid–liquid extraction; Ultra high pressure liquid chromatography; Tandem mass spectrometry; Drugs of abuse in urine

A liquid chromatographic-tandem mass spectrometric method for the simultaneous determination of anabolic androgenic steroids and their esters in hair has been developed. The hair sample was treated with methanol to extract the esters, followed by alkaline digestion for optimum recovery of the anabolic androgenic steroids. After liquid–liquid extractions, the extract was dried, redissolved and analyzed by multiple reaction monitoring with a quadrupole mass spectrometer. The lower limits of detection ranged from 0.001 to 0.020 ng mg−1 for the 21 analytes. The applicability of the method was demonstrated using guinea pig hair samples gained from controlled experiments.
Keywords: Column liquid chromatography–tandem mass spectrometry; Anabolic androgenic steroids; Testosterone ester in hair

Validity Assessment for the Results of Three Inflammatory Markers in Exhaled Breath Condensate: A Pilot Study by Luis M. Gonzalez-Reche; Dirk Schaefer; Thomas Göen; Thomas Kraus (1387-1392).
This study was initiated to assess the validity of eicosanoid determination in exhaled breath condensate by immunoassays. The results were compared to those obtained from liquid chromatographic methods with mass spectrometric detection and with the theoretical spiked amount of condensate. Therefore, spiked exhaled breath condensate was prepared achieving samples for comparison of identical aliquots. The three most often analysed inflammatory markers in the literature, i.e. leukotriene B4, prostaglandin E2, and 8-isoprostane-prostaglandin F were selected for comparison. Three concentrations of spiked and non-spiked breath condensate matrix were examined. There was an up to 800% overestimation of the eicosanoid quantities revealed by the immunoassay kits used in this study compared to the liquid chromatographic method with mass spectrometric detection which was in line with the nominal amount spiked. Full validation of immunoassay for the determination of inflammatory markers should be performed before further interpretations and consequences will be taken from the absolute values quantified by enzyme-immunoassays. For improving the accuracy of enzyme linked immunoassay (ELISA) results a collaborative work applying ELISA and LC–MS–MS should be performed in order to spotlight the advantages of the ELISA-method.
Keywords: Column liquid chromatography–mass spectrometry; Enzyme linked immunoassay; Method comparison; Exhaled breath condensate

The coverage of abrasive (fused alumina) by resol, used in manufacturing of grinding tools, was estimated by means of inverse gas chromatography (IGC). γ S D and χ 12 parameters were used to describe the wettability of abrasive by resol. γ S D shows activity of the surface of fused alumina and it was found that it is correlated with the wettability of fused alumina by resol. By using χ 12 it was possible to calculate the degree of the coverage (DC parameter) of abrasive by resol. This method was used as an alternative for the Washburn sorption method. IGC was found to be more accurate and informative method. The results show that IGC can be applied to quantitatively determine the degree of coverage the abrasive material, fused alumina, by resol.
Keywords: Gas chromatography; Inverse gas chromatography; Degree of the coverage; Abrasive; Resol

An analytical procedure was developed and validated for the determination of atenolol in human plasma. Atenolol and metoprolol (internal standard) were extracted from human plasma with a mixture of chloroform and butanol at basic pH. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and analyzed by GC–MS. Calibration curves were linear over the concentration range 15–250 ng mL−1. Intra- and inter-day precision values for atenolol in human plasma were less than 7.4, and accuracy (relative error) was better than 6.4%. Recovery of atenolol from human plasma averaged 90.46%. The limits of detection (LOD) and quantitation (LOQ) of atenolol were 5.0 and 15 ng mL−1. This method was successfully applied to six patients with hypertension who had been given an oral tablet of 50 mg atenolol.
Keywords: Gas chromatography–mass spectrometry; Atenolol in plasma; Pharmacokinetics

Improved Method for the Determination of Cyanuric Acid in Animal Feed by GC–MS by Hubert P. O. Tang; Shirley S. L. Lai; Ashley Y. H. Lai; W. O. Lee (1405-1410).
A specific and sensitive analytical method for the quantitative determination of cyanuric acid in animal feed was developed. Sample preparation involved the diethylamine/acetonitrile/water extraction of feed using sonication and shaking. The extract was subjected to clean-up by dual solid phase extraction using mixed mode anionic and cationic extraction cartridges. After removal of clean-up solvent, cyanuric acid was converted to a tert-butyldimethylsilyl derivative and was determined by gas chromatography–mass spectrometry in the selected ion monitoring mode. 13C 3 15 N3-cyanuric acid was employed as the internal standard. The calibration curve was found to be linear up to 4 mg kg−1. LOD and LOQ were determined to be 0.06 to 0.4 mg kg−1 for fish and chicken feed. The mean recovery of cyanuric acid was 96 to 98% with relative pooled standard deviation of 1.8–7.4% in the range of 0.5 to 100 mg kg−1 for fish and chicken feed. The validated method was applicable for analysing cyanuric acid in animal feed.
Keywords: Gas chromatography–mass spectrometry; Solid phase extraction; Cyanuric acid in animal feed

Stability-Indicating LC-PDA Method for Determination of Idebenone in Nanoparticles Based on Chitosan and N-Carboxymethylchitosan by Clarissa M. de Amorim; Daisy J. A. Netz; Angelica G. Couto; Rilton A. de Freitas; Tania M. B. Bresolin (1411-1415).
A forced degradation study of idebenone was conducted under conditions of UV irradiation, acid, basic and oxidative hydrolysis and in order to develop an isocratic stability-indicating LC-UV method for drug quantification in chitosan and N-carboxymethylchitosan nanoparticles obtained by spray drying. The drug was more labile to alkaline treatment than under the other forced degradation conditions. Idebenone and its degradation products were optimally resolved (resolution >4) on a Luna Phenomenex C18 column with mobile phase composed by methanol:water: (80:20% v/v) at a flow rate of 1.0 mL min−1, at 30 °C, using wavelength of 279 nm for drug detection. The method was linear, over a drug concentration range of 2 to 10 μg mL−1. The RSD% value of intra- and inter-day precision studies was <1.5. The method showed excellent recoveries (99.4 to 101.1%). The LOD and LOQ values were found to be 0.18 and 0.59 μg mL−1, respectively. In conclusion this method can be used as a rapid and accurate assay of idebenone in the nanoparticles during stability tests.
Keywords: Column liquid chromatography; UV-Stability-indicating assay; Chitosan nanoparticles; N-Carboxymethylchitosan nanoparticles; Idebenone

A sensitive and rapid LC–MS–MS method was developed for the simultaneous determination of ebastine and carebastine in human plasma. Solid-phase extraction was used to isolate the compounds from the biological matrix followed by separation on a Symmetry C18 column under isocratic conditions. The mobile phase was 10 mM ammonium formate in water/acetonitrile (40:60, v/v). Detection was carried out using a triple-quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The method was fully validated over the concentration range of 0.1–10 ng mL−1 for ebastine and 0.2–200 ng mL−1 for carebastine in human plasma, respectively. The lower limit of quantification (LLOQ) was 0.1 ng mL−1 for ebastine and 0.2 ng mL−1 for carebastine. For ebastine and carebastine inter- and intra-day precision (CV%) and accuracy values were all within ±15% and 85–115%, respectively. The extraction recovery was on average 60.0% for ebastine and 60.3% for carebastine.
Keywords: Column liquid chromatography–tandem mass spectrometry; Ebastine; Carebastine; Human plasma

A rapid and sensitive LC-UV method was developed and validated for the determination of meropenem, in human plasma and urine. Meropenem retention time was 4.8 min. Method development was based on comparative analysis of different extraction methods published as well as careful study of meropenem stability in biological samples under different conditions. Best results in plasma sample preparation were obtained from protein precipitation with methanol. LOQ was 0.1 µg mL−1 for plasma and 1 µg mL−1 for urine samples. Meropenem in plasma has low stability at room temperature (<20% of original content after 12 h), but had acceptable stability when the whole analysis procedure was designed to minimize the exposure of meropenem-containing samples and solutions to temperatures higher than 4 °C. The developed method was applied to a human pharmacokinetic study in patients with acute peritonitis.
Keywords: Column liquid chromatography; UV detection; Pharmacokinetic study in human plasma and urine; Meropenem

A simple and novel LC method has been developed for determination of isepamicin (ISP) in rat plasma, an aminoglycoside antibiotic agent. After protein precipitation and clean-up procedure to remove lipophilic contaminants, ISP is derivatized by pre-column with 9-fluorenylmethyl chloroformate for fluorescence detection. Chromatographic separations are achieved using a C18 column and mobile phase consisting of water and acetonitrile (68/32, v/v). Amikacin was used as an internal standard. The calibration curve was linear over a concentration range of 0.625–15 μg mL−1. The limit of quantification was 0.45 μg mL−1. The intra- and inter-day variabilities of ISP were both less than 5%. Both derivatives were stable for at least a week at ambient condition. This assay procedure should have useful application in therapeutic drug monitoring of ISP. The limit of detection was 0.10 μg mL−1. The specificity, assay linearity, low level assay linearity and assay repeatability were also investigated. The established method provides a reliable bioanalytical method to carry out isepamicin pharmacokinetics in rat plasma.
Keywords: Column liquid chromatography; Pharmacokinetic study; 9-Fluorenylmethyl chloroformate; Isepamicin

A simple, rapid, and reproducible reversed-phase LC method with UV detection at 215 nm has been developed for analysis of SP-8203 in rat samples. A C18 column was used with 3,000:1,050 (v/v) 0.01 m K2HPO4 buffer (pH 3)–acetonitrile as mobile phase at a flow rate of 1.7 mL min−1 at 50 °C. Samples were extracted with dichloromethane containing ondansetron (internal standard). Detection limits for SP-8203 in plasma, urine, and gastrointestinal tract samples were 0.05, 0.5, and 10 μg mL−1, respectively. The method was suitable for pharmacokinetic study of SP-8203 in rats after intravenous administration.
Keywords: Column liquid chromatography; SP-8203; Rat plasma, urine, and gastrointestinal tract

Simultaneous Determination of Andrographolide and Dehydroandrographolide in Chicken Plasma for Application to Pharmacokinetic Studies by Kaiyong Liu; Limin He; Hai Gao; Xianhui Huang; Zhigang Jiang; Zhenling Zeng (1441-1445).
A simple, suitable reverse phase liquid chromatographic method was developed for simultaneous determination of andrographolide (1) and dehydroandrographolide (2) in chicken plasma after orally administrating the ultra-fine powder of Andrographis paniculata. Plasma samples were extracted with ethyl acetate. Analysis of the extract was performed on a reversed-phase C18 column with gradient eluent composed of acetonitrile and 0.5% acetic acid. The flow rate was kept at 1 mL min−1 and the detection wavelength was set at 225 and 255 nm for 1 and 2, respectively. All calibration curves showed good linear regression (R ≥ 0.9991). The good precision and recoveries with intra-day and inter-day were 3.2–8.7% and 91.1–98.4%, respectively. The limit of detection was 0.016 µg mL−1 and the limit of quantitation was 0.040 µg mL−1 for the target analytes. This validated method has been successfully applied in the pharmacokinetics study of 1 and 2 after orally administrating the Andrographis paniculata ultra-fine powder to chicken.
Keywords: Column liquid chromatography; Validated method; Andrographis paniculata

A novel method for separation and on-line characterization of flavonoids from Asparagus officinalis by medium-pressure liquid chromatography coupled to electrospray ionization multi-stage mass spectrometry (MPLC-ESI-MSn) was successfully established. The hyphenation between MPLC and ESI-MSn was designed to keep the split ratio exactly in the range from 1:100 to 1:300. The separation procedure was guided by the chromatogram of ion current of MSn and the structures of compounds were characterized by fragments information at the same time. Consequently, it was proved that MPLC coupled with ESI-MSn was an effective method for separation of compounds from multi-component mixtures with high purity and desired amounts and simultaneous elucidation of chemical structures.
Keywords: Column liquid chromotography; Medium-pressure liquid chromatography; Mass spectrometry; Flavonoids; Asparagus officinalis

LC Determination of Five Flavonoid Aglycones in the Tibetan Medicinal Plant Oxytropis falcata Bunge by Huan Yang; Jun Chen; Hao Cai; Huiqin Xu; Li Tong; Baochang Cai (1451-1454).
A simple, efficient and accurate liquid chromatographic method was established to determine five flavonoid aglycones, 7-hydroxy flavonone, pinocembrin, 2′,4′-dihydroxy chalcone, 2′-hydroxy-4′-methoxy chalcone and pinostrobin in the whole plant powder of Oxytropis falcata Bunge. These five compounds were separated on an Agilent Zorbax Eclipse XDB-C8 column (150 × 4.6 mm, 5 μm). Mobile phases were composed of water containing 0.1% v/v formic acid and acetonitrile using gradient elution. The established method was validated for linearity, accuracy, precision, limit of detection and quantitation, repeatability and stability.
Keywords: Column liquid chromatography; Flavonoid aglycones; Oxytropis falcata Bunge

An LC–DAD method has been developed in order to evaluate qualitatively and quantitatively quaternary aporphine alkaloids, flavonoid glycosides and styrylpyrones, which are the main secondary metabolites of leaves from C. mandioccana. The chromatographic method was validated considering both internal and external standard quantification methods and showed good performances in terms of selectivity, linearity, precision (repeatability and intermediate precision), limit of detection, limit of quantification, accuracy and stability.
Keywords: Column liquid chromatography–diode array detection; Fingerprint analysis; Quaternary aporphine alkaloids; Flavonoid glycosides; Styrylpyrones; Cryptocarya mandioccana

Determination of Picoxystrobin and Pyraclostrobin by MEKC with On-Line Analyte Concentration by Cabrini F. de Souza; Alessandra L. M. C. da Cunha; Ricardo Queiroz Aucélio (1461-1466).
Micellar electrokinetic capillary chromatography was used for the determination of picoxystrobin and pyraclostrobin. The background electrolyte consisted of borate buffer (40 mmol L−1 pH 8.5), SDS (30 mmol L−1) and acetonitrile (15% in volume). Runs were made at 25 °C with 25 kV applied potential. The developed method was applied to analyte fortified urine samples. On-line analyte concentration, combined with a capillary of a longer optical path length, allowed limits of quantification of 8.6 × 10−8 mol L−1 for picoxystrobin and 1.8 × 10−7 mol L−1 for pyraclostrobin.
Keywords: Micellar electrokinetic capillary chromatography; On-line analyte concentration; Picoxystrobin; Pyraclostrobin

A micellar electrokinetic chromatography method was developed for the simultaneous determination of dopamine, epinephrine and 5-hydroxytryptamine. Several experimental parameters such as surfactant type and concentration, buffer concentration and pH, type and concentration of organic modifier were evaluated for the analysis of the studied compounds. Among the investigated separation conditions, the composition of micelles, pH and the methanol concentration were the critical parameters. Dopamine, epinephrine and 5-hydroxytryptamine were separated and determined successfully within 7.5 min in Toad venom and Common yam rhizome under the optimum conditions.
Keywords: Micellar electrokinetic chromatography; Toad venom; Common yam rhizome; Dopamine, epinephrine and 5-hydroxytryptamine

Optimization and Validation of a Capillary MEKC Method for Determination of Proteins in Urine by Nina Kočevar Glavač; Rade Injac; Samo Kreft (1473-1478).
Proteinuria, i.e. increased excretion of proteins in urine, is a common sign indicating renal or urinary tract diseases. In this study, a fast and simple procedure for urine sample preparation and capillary micellar electrokinetic chromatographic analysis is presented, without any sample pretreatment prior to the analysis. The developed MEKC method was employed for simultaneous determination of albumin (ALB), haemoglobin (HGB), and myoglobin (MYO) in human urine samples obtained from patients with diagnosed proteinuria. Optimum conditions for detection and separation of ALB, HGB, and MYO are 50 mmol L−1 borate buffer containing 20 mmol L−1 SDS (pH 9.3), injection 40 mbar × 20 s, voltage 25 kV, temperature 30 °C, and detection wavelength 200 nm. The method was shown to be specific, accurate, linear (correlation coefficients r 2 > 0.99), and precise (RSD below 3.75 and 7.23% for migration time and peak area, respectively). Multi-variable-at-a-time (MVAT) approach for robustness testing shows no significant variations in accuracy, specificity, and precision as RSD values were lower than 5 and 10% for migration time and peak area, respectively. The presented method is applicable for routine analyses of urine samples as a screening method for patients with excess ALB, HGB, and MYO.
Keywords: Micellar electrokinetic chromatography; Optimization and validation; Albumin, haemoglobin and myoglobin in urine

A rapid and sensitive capillary electrophoresis-mass spectrometry with on-line pH-mediated stacking method for the simultaneous quantification of 19 amino acids in urine was established and applied for the analysis of the urinary amino acids of 20 bladder cancer patients and 20 healthy subjects. The two groups could be well classified by pattern recognition, which indicated that the two groups had different urinary amino acids profiles. Compared to those of healthy subjects, the relative concentrations of three glycogenic amino acids (methionine, cysteine and valine) decreased significantly in the urine of bladder cancer patients.
Keywords: Capillary electrophoresis-mass spectrometry; pH-Mediated stacking; Pattern recognition; Bladder cancer; Amino acid in urine

Polyunsaturated Fatty Acids in Dried Milk Samples: Validation of a Lipid Separation-Free Method by Daniela Gastaldi; Claudio Medana; Riccardo Aigotti; Valeria Giancotti; Claudio Baiocchi (1485-1489).
A method to determine polyunsaturated fatty acids (linoleic and α-linolenic acids and its long chain metabolite, DHA) in milk samples, avoiding the fat separation step, was developed and validated. The transesterification reaction was carried out directly on freeze-dried milk samples to produce fatty acid methyl esters. Separation, identification and quantification of analytes were performed by GC–MS. In these experimental conditions the matrix effect was negligible. Values for repeatability and intermediate precision demonstrate excellent method precision. To test the method applicability, goat’s, cow’s and human’s milk with different fat contents and essential fatty acids concentrations were examined.
Keywords: Gas chromatography–mass spectrometry; Fatty acids; Milk fat

A Simple and Sensitive Analytical Method for Determination of Naltrexone Level in Plasma by GC–MS by Rezaei Mehrdad; Abdi Khosrou; Dinarvand Rassoul; Vosough-Ghanabri Sanaz; Amini Mohsen (1491-1494).
A gas chromatography-mass spectrometry method for determination of naltrexone in plasma is presented. The method is based on pre-column derivatization of analyte to trimethylsilyl derivative of naltrexone. The analyte and internal standard were extracted from plasma by liquid–liquid extraction. Validation of the method has been studied in the concentration range 1–50 ng mL−1.
Keywords: Gas chromatography-mass spectrometry; Derivatization; Naltrexone in plasma

Determination of T-2 Toxin in Traditional Chinese Herbal Medicines by GC-ECD by Yan-Tao Yue; Xiao-Fei Zhang; Zhen Ou-Yang; Wei-Wei Gao; Jun Wu; Mei-Hua Yang (1495-1499).
Gas chromatography with electron capture detection has been applied for the determination of T-2 toxin (T-2) in traditional Chinese herbal medicines (TCHM). The method consists of extracting the sample with aqueous methanol followed by cleanup of the resulting extract with an immunoaffinity column. T-2 was determined as its heptafluorobutyl ester. The reaction temperature and time of derivatization were investigated to obtain the optimum conditions. Recoveries from different TCHMs, spiked with T-2 at levels ranging from 50 to 1,000 μg kg−1, were from 82.2 to 98.6%, with relative standard deviations of less than 7.5%. The limit of detection was 2.5 μg kg−1. Out of 32 commercially available TCHM samples analyzed, none were found to contain any detectable amount of T-2.
Keywords: Gas chromatography; Derivatization; T-2 toxin; Traditional Chinese herbal medicines

Investigation of Novel Peptide Chiral Selectors Prepared by Solid-Phase Synthesis with a tert-Butoxycarbonyl Amino Acid by Kaname Ohyama; Kana Oyamada; Naoya Kishikawa; Miyuki Arakawa; Yoshihito Ohba; Masahiro Kamino; Mitsuhiro Wada; Kenichiro Nakashima; Naotaka Kuroda (1501-1504).
A new class of chiral stationary phases (CSP) with peptide chiral selectors was prepared by solid-phase synthesis with a tert-butoxycarbonyl-L-amino acid on silica. The type of amino acid that is favorable for this class of CSP is discussed. Using the CSP with the phenylalanine peptide selector, the effect of peptide length on the enantioselectivity was investigated in normal-phase mode. The applicability of the CSP with a phenylalanine peptide to chiral ligand-exchange chromatography was also examined.
Keywords: Column liquid chromatography; Peptide chiral selectors; Normal-phase separations; Chiral ligand-exchange; Secondary structure

Quantitative Determination of Busulfan in Human Plasma by UPLC by Quanyun Alan Xu; Reza Kazerooni; Jay K. Thapar; Borje D. Andersson; Timothy L. Madden (1505-1510).
A rapid and reliable UPLC method was developed and validated for the determination of busulfan in human plasma. After protein precipitation, derivatization, and liquid–liquid extraction, separation of derivatized busulfan was achieved on an Acquity BEH C18 column using a gradient mobile phase consisting of a trifluoroacetic acid aqueous solution (0.2%, v/v) and acetonitrile. The column temperature was maintained at 50 °C and UV detection was carried out at 254 nm. The complete analytical run time was 1.3 min, 7-fold faster than our previous LC methodology. Quantification was performed using external standardization and calibration curves were linear (r ≥ 0.999) over the dynamic range of 0.05–5.00 μg mL−1. Intra-day and inter-day coefficients of variation were ≤6.9 and 3.9%, respectively, across the range of concentrations. Accuracy of the analytical method expressed as the relative error percentage was better than 5.4%. LOD and LOQ were 0.013 and 0.025 μg mL−1, respectively. Data obtained using the UPLC method was compared to those obtained from our previously used LC method by Deming regression analysis. The UPLC method was accurate, sensitive, and greatly increased sample analysis throughput as compared to our previous LC methodology allowing for a 4-fold increase in the number of patients who could be monitored during transplant therapy.
Keywords: Ultra performance liquid chromatography; Human plasma; Bone marrow transplantation; Busulfan

Simple and Sensitive Determination of Metformin in Human Plasma Using an Ion-Pair LC Method by Qing-Feng Liu; Zhong-Dong Li; Xiao-Jin Shi; Zheng Jiao; Ming-Kang Zhong (1511-1514).
A simple, selective and sensitive ion-pair liquid chromatography method was described for the determination of metformin in human plasma. Plasma samples (200 μL) were deproteinated with 10% (v/v) perchloric acid followed by LC analysis using a Symmetry C18 column and a mobile phase of 29% acetonitrile—71% 2 mM sodium dodecyl sulfate (with 350 μL 0.1 M HCl) operated at a flow rate of 1.5 mL min−1. Metformin and the internal standard (sodium phenytoin) were detected at 232 nm and eluted at 6.8 and 7.9 min, respectively. Calibration curves were linear (r > 0.9990) between 9 and 2,000 ng mL−1. The quantitation limit was 9 ng mL−1, intra- and inter-day precision (CV) were 5.26% or less, and accuracy values were found to be within 5.95% of the nominal concentration.
Keywords: Ion-pair column liquid chromatography; Metformin analysis; Human plasma

A simple and sensitive liquid chromatographic method has been established for simultaneous analysis of four compounds (aloe-emodin, rhein, emodin, and chrysophanol) in Rhizoma Rhei-type preparations. The compounds were separated in less than 20 min on a C18 column with 70:30 methanol–0.2% aqueous acetic acid as mobile phase at a flow rate of 1 mL min−1. The method was validated for specificity, accuracy, precision, and limits of detection. Good linear regression data (r 2 > 0.9980) were obtained for all the calibration plots within the ranges tested. The method is an attractive alternative for evaluation of the quality of Rhizoma Rhei-type preparations.
Keywords: Column liquid chromatography; Traditional Chinese medicine; Rhizoma Rhei; Anthraquinones

Simultaneous LC Analysis of Food Dyes in Soft Drinks by Maja Serdar; Zorka Knežević (1519-1521).
Liquid chromatography with diode-array detection has been used for simultaneous analysis of eight water-soluble synthetic colorants (E102, E104, E110, E122, E124, E129, E131, and E133) in non-alcoholic beverages. The colors were separated in 15 min on a C18 reversed-phase column with a linear mobile phase gradient prepared from tetrabutylammonium hydrogen sulfate, methanol, and deionized water. The analytical characteristics of the separation were evaluated. Good linearity (r 2 = 0.9988–0.9999), adequate limits of quantification, and high recovery (from 96.3 to 98.5%) were achieved. The method was used for analysis of 57 samples of soft drinks. The experimental results showed the colorants were present in 34 of the samples, and confirmed the method is sensitive, rapid, precise, and suitable for routine analysis of synthetic organic dyes.
Keywords: Column liquid chromatography; Food dyes; Soft drinks

Chirality at the Nanoscale by Hassan Y. Aboul-Enein (1523-1523).