SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP) for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05) at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively.     

Conditions for solubilization of Tamm-Horsfall protein/uromodulin in human urine and establishment of a sensitive and accurate enzyme-linked immunosorbent assay (ELISA) method

Tamm-Horsfall protein (THP), also referred to as uromodulin, is a major glycoprotein produced by kidney cells. The level of THP in urine is an indicator of renal function. THP levels are difficult to quantify accurately, because the protein forms aggregates in the pH and ion concentrations found in urine. Here, we describe conditions by which THP can be solubilized and kept soluble during measurement using Triton X-100, EDTA, and alkaline pH. Establishing conditions for precise quantification of THP by enzyme-linked immunosorbent assay (ELISA) will aid in studying the functions of THP.     

A luminescent oxygen channeling immunoassay for the determination of insulin in human plasma

The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 microL. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R(2) = 0.98). This method is convenient and represents an alternative to ELISA.     

Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine

Sensitive assays for the determination of cyclobenzaprine (I) in human plasma and urine were developed utilizing high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) and ultraviolet (UV) absorbance detections. These two analytical techniques were evaluated for reliability and sensitivity, and applied to support pharmacokinetic studies. Both methods employed a liquid-liquid extraction of the compound from basified biological sample. The organic extract was evaporated to dryness ,the residue was reconstituted in the mobile phase and injected onto the HPLC system. The HPLC assay with MS-MS detection was performed on a PE Sciex API III tandem mass spectrometer using the heated nebulizer interface. Multiple reaction monitoring using the parent → daughter ion combinations of m/z 276 → 215 and 296 → 208 was used to quantitate I and internal standard (II), respectively. The HPLC-MS-MS and HPLC-UV assays were validated in human plasma in the concentration range 0.1–50 ng/ml and 0.5–50 ng/ml, respectively. In urine, both methods were validatedin the concentration range 10–1000 ng/ml. The precision of the assays, as expressed as coefficients of variation (C.V.) was less than 10% over the entire concentration range, with adequate assay specificity and accuracy. In addition to better sensitivity, the HPLC-MS-MS assay was more efficient and allowed analysis of more biological fluid samples in a single working day than the HPLC-UV method.     

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50-5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.     

Simple and sensitive liquid chromatography-tandem mass spectrometry methods for quantification of paraquat in plasma and urine: application to experimental and clinical toxicological studies

Simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for quantification of paraquat (PQ) in plasma and urine. Plasma and urine sample preparation were carried out by one-step protein precipitation using cold acetonitrile (-20 to -10 °C). After centrifugation, an aliquot of 10 μL of supernatant was injected into a Kinetex? hydrophilic interaction chromatography (HILIC) column with a KrudKatcher? Ultra in-line filter. The chromatographic separation was achieved using the mobile phase mixture of 250 mM ammonium formate (with 0.8% aqueous formic acid) in water and acetonitrile at a flow rate of 0.3 mL/min. Detection was performed using an API2000 triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear over the concentration range of 10-5000 ng/mL, with an LLOQ of 10 ng/mL. The inter- and intra-day precision (% R.S.D.) were <8.5% and 6.4% for plasma and urine, respectively with the accuracies (%) within the range of 95.1-102.8%. PQ in plasma and urine samples was stable when stored at -70 °C for three freeze-thaw cycles. The methods were successfully applied to determine PQ concentration in rat and human samples.     

An enzyme-linked immunosorbent assay for rat prolactin

A sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for rat prolactin was developed using reagents from the National Institute of Arthritis, Diabetes, Digestive Diseases and Kidney. In this assay soluble prolactin and prolactin adsorbed to a solid-phase support compete for rabbit anti-prolactin antibody binding sites. Therefore, a high concentration of soluble prolactin in the sample will result in a low concentration of antibody immobilized to the adsorbed prolactin. The immobilized antibody-prolactin complex is detected and quantified using goat anti-rabbit immunoglobulin G covalently conjugated to the enzyme horseradish peroxidase. Assay parameters were optimized by investigating the concentration of reagents and the reaction kinetics in each of the assay steps. The assay can be performed in 24 h. A sensitivity range of 0.06 to 6 ng in the region of 90 to 10% binding was obtained. Near 50% binding (0.6 ng), the intraassay coefficient of variation (CV) was 4.2% and the interassay CV was 7.6%. The correlation between radioimmunoassay and the ELISA was 0.868. Selected applications of the assay are described. The assay should prove a useful alternative to the radioimmunoassay in those instances where steps involving the use of 125I become limiting, for example, iodination facility and gamma counter availability or prolonged reagent storage.     

GC-MS quantification of ketamine, norketamine, and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology

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

Validation of a sensitive LC-MS assay for quantification of glyburide and its metabolite 4-transhydroxy glyburide in plasma and urine: an OPRU Network study

Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 mL) or urine (0.1 mL). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was <15% whereas the accuracy (% deviation from expected) ranged from -10.1 to 14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of pyronaridine in human urine

A reliable method has been developed for the determination of pyronaridine in human urine using amodiaquine as an internal standard. Liquid-liquid extraction was used for sample preparation. Analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Chromatography was carried out using a Gemini 5 microm C18 3.0 mmx150 mm column using 2 mM perflurooctanoic acid and acetonitrile mixture as a mobile phase delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.1 and 8.1 min respectively, with a total run time of 14 min. The assay was linear over a range of 14.3-1425 ng/mL for pyronaridine (R2>or=0.992, weighted 1/Concentration). The analysis of quality control samples for pyronaridine at 28.5, 285, 684 and 1140 ng/mL demonstrated excellent precision with relative standard deviation of 5.1, 2.3, 3.9 and 9.2%, respectively (n=5). Recoveries at concentrations of 28.5, 285, 684 and 1140 ng/mL were all greater than 85%.This LC-MS method for the determination of pyronaridine in human urine has excellent specifications for sensitivity, reproducibility and accuracy and can reliably quantitate concentrations of pyronaridine in urine as low as 14.3 ng/mL. The method will be used to quantify pyronaridine in human urine for pharmacokinetic and drug safety studies.     

Biocompatible in-tube solid-phase microextraction coupled to HPLC for the determination of angiotensin II receptor antagonists in human plasma and urine

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 0.1-15.3ng/mL and 0.1-15.2ng/mL in human plasma and urine, respectively. The method was linear over the range of 0.5-200ng/mL for telmisartan, 5-2000ng/mL for candesartan and irbesartan, 10-2000ng/mL for valsartan, and 50-5000ng/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.     

Development and validation of a liquid chromatography and tandem mass spectrometry method for determination of roscovitine in plasma and urine samples utilizing on-line sample preparation

Roscovitine, a purine analogue that selectively inhibits cyclin-dependent kinases, has been considered as a potential anti-tumor drug. The determination of roscovitine in plasma and urine was performed using microextraction in packed syringe as on-line sample preparation method with liquid chromatography and tandem mass spectrometry. The sampling sorbent utilized was polystyrene polymer. 2H3-lidocaine was used as internal standard. The limit of detection for roscovitine was as low as 0.5 ng/mL and the lower limit of quantification was 1.0 ng/mL. The accuracy and precision values of quality control samples were between +/-15% and < or =11%, respectively. The calibration curve was obtained within the concentration range 0.5-2000 ng/mL in both plasma and urine. The regression correlation coefficients for plasma and urine samples were > or =0.999 for all runs. The present method is miniaturized and fully automated and can be used for pharmacokinetic and pharmacodynamic studies.     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Development and validation of an LC-MS method with electrospray ionization for quantitation of digoxin in human plasma and urine: application to a pharmacokinetic study

A highly sensitive and specific LC-MS method was developed and validated for the quantification of digoxin in human plasma and urine using d5-dihydrodigoxin as internal standard (IS). The assay procedure involved extraction of digoxin and IS from human plasma with chloroform-isopropanol (95:5, v/v). Chromatogrphic separation was achieved on a Spherisorb ODS2 column using a gradient mobile phase with 5 mmol/L ammonium acetate in water with 1% acetic acid and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective [M+K](+) ions, m/z 819.4 for digoxin and m/z 826.4 for IS. The method was proved to be accurate and precise at linearity range of 0.12-19.60 ng/mL in plasma with a correlation coefficient (r(2)) of >or=0.9968 and 1.2-196.0 ng/mL in urine. The limit of quantification achieved with this method was 0.12 ng/mL in plasma and 1.2 ng/mL in urine. The intra- and inter-assay precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers following intravenous administration of digoxin.     

Bioassay for determination of fosmidomycin in plasma and urine: application for pharmacokinetic dose optimisation

A simple, sensitive, selective and reproducible method based on agar diffusion disk assay was developed for the determination of fosmidomycin and clindamycin in human plasma and urine. A disk diffusion technique was used, essentially as previously described but utilising the assay organism Enterobacter cloacae ATCC 23355 strain to seed the agar assay plates. Calibration curves were prepared from concentration response curves in plasma (0, 1, 2.5, 5, 7.5, 10, 25, 50 ng/microl) and urine (0, 10, 25, 50, 75, 100, 250 and 500 microg/microl) were all linear with correlation coefficients better than 0.990. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 5% (% coefficient of variations: %C.V.). Good accuracy was observed for both the intra-day or inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +/-5%). Limit of quantification (L.O.Q.) was accepted as 1 ng using 40-microl plasma or 7.5-microl urine sample. The mean recovery for fosmidomycin was greater than 99%. The method was free from interference from other commonly used antibiotics including clindamycin, carbenicillin, cephalothin, chloramphenicol, kanamycin, methicillin, penicillin, erythromycin, lincomycin, tetracycline and paromomycin. The method appears to be robust and has been applied to a pharmacokinetic study in plasma and urinary excretion of fosmidomycin in a patient with malaria following oral doses of clindamycin at 1200 mg given every 8 h for 7 days.     

Validated liquid chromatographic-ultraviolet method for the quantitation of tadalafil in human plasma using liquid-liquid extraction

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

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry: assay development, validation and application to a human pharmacokinetic study

Rasagiline is a highly potent, selective and irreversible second-generation monoamine oxidase inhibitor with selectivity for type B of the enzyme (MAO-B). The present studies aimed at developing and validating a rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of rasagiline in human plasma and urine. LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI(+)) and selected reaction monitoring (SRM). The assay for rasagiline was linear over the range of 0.01-40 ng/mL in plasma and 0.025-40 ng/mL in urine. It took 5.5 min to analyze a sample. The average recoveries in plasma and urine samples were both >85%. The RSD of precision and bias of accuracy were less than 15% and 10%, respectively, of their nominal values based on the intra- and inter-day analysis. The developed method was proved to be suitable for use in clinical pharmacokinetic study after single oral administration of 0.5, 1 and 2 mg rasagiline mesylate tablets in healthy Chinese volunteers.     

Simultaneous determination of four active alkaloids from a traditional Chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC-MS-MS

A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 microm, 100 x 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 microm, 20 x 4 mm). The method was validated with the concentration range 1-1000 ng/mL in plasma and 10-1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC-MS-MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

Simultaneous analysis of verapamil and norverapamil enantiomers in human plasma by high-performance liquid chromatography

An improved HPLC method for the simultaneous determination of the enantiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N-acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexane). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an α₁-acid glycoprotein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision and robustness of the method. The assay is sensitive and specific. Baseline separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precision and accuracy better than 15% at those levels. Detector response is linear in the range tested (3–200 ng/ml for V and 2–100 ng/ml for NV, single enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers.