Journal of Chromatography B (v.828, #1-2)
Full-title Page - prelim iii (iii).
Publisher's note (1).
High-performance liquid chromatographic determination of vertilmicin in rat plasma using sensitive fluorometric derivatization by Zhen Liu; Yunfei Sha; Taomin Huang; Bei Yang; Geng-Li Duan (2-8).
A sensitive and reliable high-performance liquid chromatographic method was developed for the determination of vertilmicin in rat plasma. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C18 reversed-phase chromatography allowed the fluorimetric detection of vertilmicin. Optimal conditions for the derivatization of vertilmicin are described. The limit of quantification was 0.02 mg/L. The pharmacokinetics of vertilmicin was studied in 24 rats following intramuscular injection (i.m.) of different doses (4, 8, 16, 32 mg/kg of body weight). The pharmacokinetic parameter values were estimated by use of 3P97 program. In this study, we assessed the dose proportionality of vertilmicin after single intramuscular injection doses and obtained new information on the pharmacokinetics of the compound.
Keywords: Vertilmicin; Aminoglycosides; Fluorescene; High-performance liquid chromatography; 9-Fluorenylmethyl chloroformate; Pharmacokinetics;
Metabolic fingerprinting of rat urine by LC/MS by Helena Idborg; Leila Zamani; Per-Olof Edlund; Ina Schuppe-Koistinen; Sven P. Jacobsson (9-13).
Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography–electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C18 column and a ZIC®-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.
Keywords: High-throughput profiling; Metabonomics; Metabolomics; Metabolite profiling; Metabolic profiling; Metabolic fingerprints; Biomarker; Biological fluids; Hydrophilic interaction; HILIC; LC/MS; ZIC®-HILIC column;
Metabolic fingerprinting of rat urine by LC/MS by Helena Idborg; Leila Zamani; Per-Olof Edlund; Ina Schuppe-Koistinen; Sven P. Jacobsson (14-20).
Metabolic fingerprinting of biofluids like urine is a useful technique for detecting differences between individuals. With this approach, it might be possible to classify samples according to their biological relevance. In Part 1 of this work a method for the comprehensive screening of metabolites was described [H. Idborg, L. Zamani, P.-O. Edlund, I. Schuppe-Koistinen, S.P. Jacobsson, Part 1, J. Chromatogr. B 828 (2005) 9], using two different liquid chromatography (LC) column set-ups and detection by electrospray ionization mass spectrometry (ESI-MS). Data pretreatment of the resulting data described in [H. Idborg, L. Zamani, P.-O. Edlund, I. Schuppe-Koistinen, S.P. Jacobsson, Part 1, J. Chromatogr. B 828 (2005) 9] is needed to reduce the complexity of the data and to obtain useful metabolic fingerprints. Three different approaches, i.e., reduced dimensionality (RD), MarkerLynx™, and MS Resolver™, were compared for the extraction of information. The pretreated data were then subjected to multivariate data analysis by partial least squares discriminant analysis (PLS-DA) for classification. By combining two different chromatographic procedures and data analysis, the detection of metabolites was enhanced as well as the finding of metabolic fingerprints that govern classification. Additional potential biomarkers or xenobiotic metabolites were detected in the fraction containing highly polar compounds that are normally discarded when using reversed-phase liquid chromatography.
Keywords: High-throughput profiling; Metabonomics; Metabolomics; Metabolite profiling; Metabolic profiling; Metabolic fingerprints; Biomarker; Biological fluids; Hydrophilic interaction; HILIC; LC/MS; ZIC-HILIC column; Data pre-treatment; Classification; Chemometrics;
Quantitative analysis of androst-4-ene-3,6,17-trione and metabolites in human urine after the administration of a food supplement by liquid chromatography/ion trap-mass spectrometry by K. Deventer; P. Van Eenoo; P. Mikulčíková; W. Van Thuyne; F.T. Delbeke (21-26).
6-OXO®, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6α-ol-3,17-dione and androst-4-ene-6α,17β-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid–liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6α-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6α,17β-diol-3-one in the range of 5–1000 ng/mL. Using this method androst-4-ene-6α-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6α,17β-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.
Keywords: Androst-4-ene-3,6,17-trione; Doping; Urine; Androst-4-ene-6α-ol-3,17-dione;
Quantification of betamethasone in human plasma by liquid chromatography–tandem mass spectrometry using atmospheric pressure photoionization in negative mode by Alberto dos Santos Pereira; Lina S.O.B. Oliveira; Gustavo D. Mendes; Jorge J. Gabbai; Gilberto De Nucci (27-32).
Betamethasone is a synthetic corticosteroid designed to exert a marked glucocorticoid activity. As the free alcohol, betamethasone finds widespread clinical applications related to its anti-inflammatory and immunosuppressant activity.In the present study, a fast, sensitive, robust method was developed for the determination and quantification of betamethasone in human plasma by liquid chromatography coupled with tandem mass spectrometry, using photospray ionization in negative mode.Betamethasone was extracted from 0.5 ml human plasma by liquid–liquid extraction (LLE) using chloramphenicol as internal standard. The method has a chromatographic run of 2.5 min using a C18 analytical column (100 mm × 2.1 mm i.d.) and the linear calibration curve over the range was linear from 0.05 to 50 ng ml−1 (r 2 > 0.993). The between-run precision, based on the relative standard deviation replicate quality controls was 94.1% (0.15 ng ml−1), 90.7% (4.0 ng ml−1) and 97.2% (40 ng ml−1). The between-run accuracy for the above-mentioned concentrations was 11.9, 9.0 and 9.8%, respectively. The method herein described was employed in a bioequivalence study of two formulations of dexchlorpheniramine/betamethasone 2 mg/0.25 mg tablets.
Keywords: Betamethasone; Corticosteroid; Photospray; Human plasma;
Application of microemulsion thin layer chromatography for the fingerprinting of licorice (Glycyrrhiza spp.) by Shufen Cui; Boqiang Fu; Frank Sen-Chun Lee; Xiaoru Wang (33-40).
Microemulsion thin layer chromatography (ME-TLC) has been developed for the fingerprinting of aqueous extract of licorice (Glycyrrhiza spp.). The separation conditions and operational processes of the method have been optimized, and its chromatographic characteristics compared with conventional TLC. The ME-TLC system is easier to operate, and with higher resolution and better reproducibility than the conventional TLC. The separation mechanism and retention behavior of ME-TLC are found to differ significantly from conventional TLC. The technique has been applied to the analysis of different licorice species including G. uralensis, G. glabra and G. inflata; and to monitor the dynamic accumulation of active ingredients in licorice plant harvested at different times during its growing cycle in a Good Agriculture Practice (GAP) research farm. Results show that without post-chromatographic derivatization, the ME-TLC fingerprinting images of different species appear as clear, well resolved bands and with strong intensities to reveal distinctively different compositional features of the samples. The technique has also been applied successfully to monitor the dynamic accumulation of active components in licorice plant as a function of growing time in an experimental licorice farm. The study demonstrates the potential of ME-TLC technique as a rapid fingerprinting tool for the authentication and quality assessment of licorice as well as other herbs.
Keywords: Microemulsion thin layer chromatography; Licorice; Characteristic; Fingerprint;
Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate dimethyl benzoylphenylurea (BPU) and its five metabolites in human plasma and urine for clinical pharmacology studies by Michelle A. Rudek; Ming Zhao; Ping He; Yelena Zabelina; Runyan Jin; Wells A. Messersmith; Antonio C. Wolff; Sharyn D. Baker (41-54).
A method has been developed for the quantitation of N-[4-(5-bromo-2-pyrimidinyloxy)-3-methylphenyl]-N′-(2-dimethylamino-benzoyl)urea (BPU) and its metabolites in human plasma and urine. BPU and metabolites were separated on a C18 column with acetonitrile–water mobile phase containing 0.1% formic acid using isocratic flow for 5 min. The analytes were monitored by tandem mass spectrometry. Calibration curves were generated over the range of 2.5–500 ng/mL for BPU, mmBPU, and aminoBPU in plasma; and 0.1–20, 0.1–20, 0.5–100, 10–2000, 1–200, and 3–600 ng/mL for BPU, mmBPU, aminoBPU, G280, G308, and G322 in urine, respectively. The method has been successfully applied to study the pharmacokinetics of BPU.
Keywords: Dimethyl benzoylphenylurea (BPU); LC/MS/MS; Pharmacokinetics;
Determination of schizandrin in rat plasma by high-performance liquid chromatography–mass spectrometry and its application in rat pharmacokinetic studies by Meijuan Xu; Guangji Wang; Haitang Xie; Rui Wang; Wei Wang; Xiaoyu Li; Hao Li; Danni Zhu; Lei Yue (55-61).
A sensitive liquid chromatography–mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C18 column with the mobile phase of acetonitrile–sodium acetate (10 μmol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005–2.000 μg/mL) (r = 0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 μL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 μg/mL. Intra- and inter-day relative standard deviations were 5.95–12.93% and 3.87–14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.
Keywords: Schizandrin; Plasma; LC/MS; Solid phase extraction (SPE); Pharmacokinetics;
Biocompatible in-tube solid-phase microextraction coupled to HPLC for the determination of angiotensin II receptor antagonists in human plasma and urine by Jing Nie; Min Zhang; Yi Fan; Yi Wen; Bingren Xiang; Yu-Qi Feng (62-69).
A poly (methacrylic acid-ethylene glycol dimethacrylate, MAA-EGDMA) monolithic capillary was used for the in-tube solid-phase microextraction (in-tube SPME) of several angiotensin II receptor antagonists (ARA-IIs) from human plasma and urine. Under the optimized extraction condition, the protein component of the biological sample was flushed through the monolithic capillary, while the analytes were successfully trapped. Coupled to HPLC with fluorescence detection, this on-line in-tube SPME method was successfully applied for the determination of candesartan, losartan, irbesartan, valsartan, telmisartan, and their detection limits were found to be 0.1–15.3 ng/mL and 0.1–15.2 ng/mL in human plasma and urine, respectively. The method was linear over the range of 0.5–200 ng/mL for telmisartan, 5–2000 ng/mL for candesartan and irbesartan, 10–2000 ng/mL for valsartan, and 50–5000 ng/mL for losartan with correlation coefficients being above 0.9985 in plasma sample and above 0.9994 in urine sample. The method reproducibility was evaluated at three concentration levels, resulting in the R.S.D. <7%. The poly (MAA-EGDMA) monolithic capillary was demonstrated to be robust and biocompatible by using direct injections of biological samples.
Keywords: Poly (methacrylic acid-ethylene glycol dimethacrylate); Monolithic capillary; In-tube SPME; Liquid chromatography; Angiotensin II receptor antagonists; Candesartan; Losartan; Irbesartan; Valsartan; Telmisartan;
Determination of glabridin in human plasma by solid-phase extraction and LC–MS/MS by Fumiki Aoki; Kaku Nakagawa; Akiyoshi Tanaka; Kazue Matsuzaki; Naoki Arai; Tatsumasa Mae (70-74).
Glabridin is a major flavonoid included specifically in licorice (Glycyrrhiza glabra L.), and has various physiological activities including antioxidant and anti-inflammatory effects. We have developed and validated an analytical method for determination of glabridin in human plasma by solid-phase extraction (SPE) and LC–MS/MS. Glabridin was extracted from plasma by SPE using a C8 cartridge and analyzed by LC–MS/MS using mefenamic acid as an internal standard (IS). The analyte were separated by a C18 column on LC, and monitored with a fragment ion of m/z 201 formed from a molecular ion of m/z 323 for glabridin and that of m/z 196 from m/z 240 for IS during negative ion mode with tandem MS detection. The lower limit of quantitation (LLOQ) of glabridin was 0.1 ng/mL in plasma, corresponding to 1.25 pg injected on-column. The calibration curves exhibited excellent linearity (r > 0.997) between 0.1 and 50 ng/mL. Precision and accuracy were <17 and <±7% at LLOQ, and <11 and <±5% at other concentrations. Glabridin was recovered >90%, and was stable when kept at 10 °C for 72 h, at −20 °C until 12 weeks, and after three freeze-thaw cycles. This is the first report on determination of glabridin in body fluids by the selective, sensitive, and reproducible method.
Keywords: Glabridin; Licorice; Glycyrrhiza glabra L.; Flavonoid; Human plasma; Solid-phase extraction; LC–MS/MS; Determination; Validation;
Liquid chromatography–tandem mass spectrometry method for determination of phencynonate in rat blood and urine by Yuying Kou; Yanxia Xu; Ming Xue; Jinxiu Ruan; Zhenqing Zhang; Keliang Liu (75-79).
A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid–liquid extraction with ethyl ether–dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150 mm × 2.1 mm i.d., 3 μm). The mobile phase was composed of methanol–water (85:15, v/v), containing 0.5‰ formic acid, which was pumped at a flow-rate of 0.2 ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25 min, thiencynonate eluted at approximately 5.10 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1–100 ng/ml in rat blood and 1–500 ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57–79.49% in blood and 56.85–64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies.
Keywords: Phencynonate; Anticholinergic drug; Liquid chromatography–mass spectrometry; Rat blood; Quantification;
Liquid chromatography–electrospray mass spectrometry determination of carbamazepine, oxcarbazepine and eight of their metabolites in human plasma by Hélène Breton; Marylène Cociglio; Françoise Bressolle; Hélène Peyriere; Jean Pierre Blayac; Dominique Hillaire-Buys (80-90).
Carbamazepine (CBZ) and oxcarbazepine (OXCBZ) are both antiepileptic drugs, which are prescribed as first-line drugs for the treatment of partial and generalized tonic–clonic epileptic seizures. In this paper, a specific and sensitive liquid chromatography–electrospray ionization mass spectrometry method was described for the simultaneous determination of carbamazepine (CBZ), oxcarbazepine (OXCBZ) and eight of their metabolites [CBZ-10,11-epoxide (CBZ-EP), 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine (DiOH-CBZ), 10-hydroxy-10,11-dihydroCBZ (10-OH-CBZ), 2-hydroxycarbamazepine (2-OH-CBZ), 3-hydroxycarbamazepine (3-OH-CBZ), iminostilbene (IM), acridone (AO) and acridine (AI)] in human plasma. The work-up procedure involved a simple precipitation with acetone. Separation of the analytes was achieved within 50 min using a Zorbax eclipse XD8 C8 analytical column. The mobile phase consisted of a mixture of acetonitrile–formate buffer (2 mM, pH 3). Detection was performed using a quadrupole mass spectrometer fitted with an electrospray ion source. Mass spectrometric data were acquired in single ion recording mode at m/z 237 for CBZ, m/z 180 for CBZ-EP and AI, m/z 236 for OXCBZ, m/z 237 for 10-OH-CBZ, m/z 253 for 2-OH-CBZ, 3-OH-CBZ and DiOH-CBZ, m/z 196 for AO and m/z 194 for IM. For all analytes, the drug/internal standard peak height ratios were linked via a quadratic relationship to plasma concentrations. The extraction recovery averaged 90% for CBZ, 80% for OXCBZ and was 80–105% for the metabolites. The lower limit of quantitation was 0.5 mg/l for CBZ, 0.4 mg/l for OXCBZ and ranged from 0.02 to 0.3 mg/l for the metabolites. Precision ranged from 2 to 13% and accuracy was between 86 and 112%. This method was found suitable for the analysis of plasma samples collected during therapeutic drug monitoring of patients treated with CBZ or OXCBZ.
Keywords: Liquid chromatography–mass spectrometry; Carbamazepine; Oxcarbazepine; Metabolites; Validation;
Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography by Hao Liang; Qipeng Yuan; Qian Xiao (91-96).
Sulforaphane is an isothiocyanate that is present naturally in widely consumed Brassica oleracea vegetables and has been shown to block the formation of tumors. The contents of sulforaphane in five groups of B. oleracea seeds (broccoli, Brussels sprouts, cabbage, cauliflower and kale) were determined by RP-HPLC using linear gradient of acetonitrile in water. A new low-cost method to isolate and purify natural sulforaphane from B. oleracea seed meal was described in this work. Crude sulforaphane was first separated from B. oleracea seed meal by using immiscible solvent extraction with ethyl acetate, 10% ethanol and hexane, and the crude sulforaphane was used as raw materials to prepare high purity sulforaphane by low-pressure column chromatography of silica gel (200–300 mesh) with different eluents and elution modes. Compared with these different elution methods, the gradient elution was preferable to the isocratic elution for reducing the elution time and the eluent consumption and increasing the purity of sulforaphane product. The purity and recovery of sulforaphane were more than 90% in gradient elution.
Keywords: Sulforaphane; Purification; Low-pressure column chromatography (LPCC); Brassica oleracea seed;
Comparison of HPLC method and commercial ELISA assay for asymmetric dimethylarginine (ADMA) determination in human serum by Pirjo Valtonen; Jouni Karppi; Kristiina Nyyssönen; Veli-Pekka Valkonen; Toivo Halonen; Kari Punnonen (97-102).
The performance of a new ELISA assay kit (DLD Diagnostika GmbH, Hamburg, Germany) for the determination of asymmetric dimethylarginine (ADMA) was evaluated against a reversed phase HPLC method. ADMA concentrations of 55 serum samples were measured with both methods. The intra-assay CV for ADMA-ELISA was 19% (n = 10). Inter-assay CVs for ADMA-ELISA were 9% for kit control 1 (0.410 ± 0.037 μM) and 14% for kit control 2 (1.174 ± 0.165 μM). The intra- and inter-assay CVs for HPLC assay for ADMA were 2.5% (0.586 ± 0.015 μM) and 4.2% (0.664 ± 0.028 μM), respectively. There was no correlation between these two methods (R 2 = 0.0972). The effect of storage conditions of the samples on ADMA concentrations was investigated by HPLC. ADMA concentration was stable after four freezing and thawing cycles. Overall, the HPLC method offered better sensitivity, selectivity and, very importantly, simultaneous determination of ADMA, SDMA, l-homoarginine and l-arginine.
Keywords: ADMA; SDMA; l-Homoarginine; l-Arginine; ELISA; HPLC;
Development of a solid phase microextraction–gas chromatography method to determine N-hydroxymethyl-N-methylformamide and N-methylformamide in urine by Vagner Fernandes Knupp; Edna Maria Alvarez Leite; Zenilda de Lourdes Cardeal (103-107).
A headspace solid phase microextraction (SPME) method has been developed to determine metabolites of dimethylformamide, N-hydroxymethyl-N-methylformamide, and N-methylformamide (NMF) as NMF in urine by gas chromatography with nitrogen–phosphorus detector (GC-NPD). An SPME holder with a 65-μm PDMS/DVB fiber coating was used. Optimal desorption conditions were 250 °C for 1 min, adsorption at 80 °C for 15 min, and 3.00 mL of sample in the headspace vial. The method presented good resolution, repeatability, recovery, detection limit, ruggedness and response linearity.
Keywords: Dimethylformamide; N-Methylformamide; N-Hydroxymethyl-N-methylformamide; GC-SPME;
Determination and validation of a simple high-performance liquid chromatographic method for simultaneous assay of iprodione and vinclozolin in human urine by Giuseppe Carlucci; Dorina Di Pasquale; Fabrizio Ruggieri; Pietro Mazzeo (108-112).
A method based on solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed for the simultaneous determination of 3-(3,5-diclorophenyl)-5-ethenyl-5-methyl-2,4-oxazolidinedione (vinclozolin) and 3-(3,5-diclorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide (iprodione) in human urine. Urine samples containing vinclozolin and iprodione were collected by solid phase extraction using C18 cartridges. The chromatographic separation was achieved on a Spherisorb ODS2 (250 mm × 4.6 mm, 5 μm) column with an isocratic mobile phase of acetonitrile–water (60:40, v/v). Detection was UV absorbance at 220 nm. The calibration graphs were linear from 30 to 1000 ng/mL for the two fungicides. Intra- and inter-day R.S.D. did not exceed 2.9%. The quantitation limit was 50 ng/mL for vinclozolin and 30 ng/mL for iprodione, respectively.
Keywords: Pesticides; HPLC; Human urine; SPE;
Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy by Manuela Contin; Monica Balboni; Erica Callegati; Carmina Candela; Fiorenzo Albani; Roberto Riva; Agostino Baruzzi (113-117).
A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 μL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 μm Hydro-RP, 150 mm × 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1–20 μg/mL for lamotrigine, 2–40 μg/mL for monohydroxycarbamazepine and 10–120 μg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.
Keywords: Lamotrigine; Oxcarbazepine main active metabolite; Felbamate; HPLC analysis; Therapeutic drug monitoring;
Liquid chromatographic-tandem mass spectrometric method for the quantitation of sildenafil in human plasma by Yingwu Wang; Jiang Wang; Yimin Cui; J. Paul Fawcett; Jingkai Gu (118-121).
A method to determine sildenafil in human plasma involving liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed. Sildenafil and the internal standard (I.S.), diazepam, are extracted from human plasma with ether–dichloromethane (3:2, v/v) at basic pH and analyzed by reversed-phase high-performance liquid chromatography (HPLC) using methanol–10 mM ammonium acetate pH 7.0 (85:15, v/v) as the mobile phase. Detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was linear over the concentration range 0.125–40.0 ng/ml. Intra- and inter-day precision of the assay at four concentrations within this range were 2.5–8.0%. The method was used to evaluate plasma concentration–time profiles in healthy volunteers given an oral dose of 20 mg sildenafil as a combination tablet also containing apomorphine.
Adaptation of a high-performance liquid chromatographic method for quantitative determination of homocysteine in urine by Barbara Proksch; Simone Jelesnianski; Walter Oberrauch; Richard Fux; Christoph H. Gleiter (122-125).
A modification of the Bio-Rad® total homocysteine HPLC-test is presented in order to enable not only plasma homocysteine measurements but also the quantification of homocysteine in urine samples using the same principle of measurement. Coelution of the internal standard provided in the test kit with an endogenous compound in urine demands for an alternative analytical procedure. Therefore, we introduced 3-mercaptopropionic acid as a substitute for the internal standard. The analytical method validation was performed for the matrix of urine specimens. The applicability of this method was demonstrated in a clinical study with volunteers after homocysteine thiolactone hydrochloride loading.
Keywords: Homocysteine; Urine; HPLC;
Determination of telmisartan in human plasma by liquid chromatography–tandem mass spectrometry by Pengfei Li; Yingwu Wang; Yan Wang; Yunbiao Tang; J. Paul Fawcett; Yimin Cui; Jingkai Gu (126-129).
A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether–dichloromethane (60:40, v/v), and separated on a Zorbax extend C18 column using methanol–10 mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap™ LC–MS/MS system with an ESI interface. The assay was linear over the range 0.5–600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9–111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.
Keywords: Telmisartan; LC–MS/MS;
A homotetrameric agglutinin with antiproliferative and mitogenic activities from haricot beans by Jack Ho Wong; T.B. Ng (130-135).
A homotetrameric agglutinin with a molecular mass of 130 kDa was isolated from seeds of the haricot bean. The agglutinin was isolated using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 200. Haricot bean agglutinin was adsorbed on DEAE-cellulose and Affi-gel blue gel. The hemagglutinating activity of the agglutinin was stable up to 40 °C. It underwent a 40% decline when the temperature was raised to 50 °C and a complete loss when the temperature was further increased to 80 °C. The hemagglutinating activity exhibited a time-dependent loss in activity when the agglutinin was incubated at 100 °C for different durations. No activity was discernible when the agglutinin was left at 100 °C for 1 min. The activity also underwent a decline in the presence of 500 mM FeCl3 and CaCl2. Haricot bean agglutinin manifested a weaker mitogenic activity than concanavalin A toward mouse splenocytes. It exhibited antiproliferative activity toward the tumor cell lines M1 [leukemia], HepG2 [hepatoma] and L1210 [leukemia] cells.
Keywords: Haricot bean; Isolation; Agglutinin;
Erratum to “Wide concentration range investigation of recovery, precision and error structure in liquid chromatography” [J. Chromatogr. B 810 (2004) 111–118] by U. Schepers; J. Ermer; L. Preu; H. Wätzig (136).
Author Index (137-138).
Keyword Index (139-142).