Journal of Chromatography B (v.788, #2)

News Section (N1-N2).

A sensitive and specific method using reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (LC–ESI-MS) has been developed for the quantitative determination of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DMF) and 3-hydroxyflunitrazepam (3-OHF) in biological fluids. After the addition of deuterium labelled standards of F,7-AF and N-DMF, the drugs were isolated from urine or plasma by automated solid-phase extraction, then chromatographed in an isocratic elution mode with a salt-free eluent. The quantification was performed using selected ion monitoring of protonated molecular ions (M+H+). Experiments were carried out to improve the extraction recovery (81–100%) and the sensitivity (limit of detection 0.025 ng/ml for F and 7-AF, 0.040 ng/ml for N-DMF and 0.200 ng/ml for 3-OHF). The method was applied to the determination of F and metabolites in drug addicts including withdrawal urine samples and in one date-rape plasma and urine sample.
Keywords: Flunitrazepam; 7-Aminoflunitrazepam; N-Desmethylflunitrazepam; 3-Hydroxyflunitrazepam;

A simple, sensitive, selective and reproducible method based on anion-exchange liquid chromatography with post-column derivatisation was developed for the determination of eflornithine (2-difluoromethyl-dl-ornithine; DFMO) in human plasma and cerebrospinal fluid. The 1-alkylthio-2-alkyl-isoindoles fluorescent derivative of the drug was separated from the internal standard (MDL 77246A) on an anion-exchange column (PRP-X300, 250×2.1 mm, 7-μm particle size: Hamilton, USA), with retention times of 6.9 and 10.7 min, respectively. Fluorescence detection was set at 430/340 nm (emission/excitation wavelength). The elution solvent consisted of a solution of 30 mM potassium dihydrogen phosphate buffer (pH 2.2) and acetonitrile (50:50, v/v), running through the column at a flow-rate of 0.3 ml/min. The chromatographic analysis was operated at 37 °C. Sample preparation for either plasma or CSF (100 μl) was done by single-step protein precipitation with 20% trichloroacetic acid after incubation at 4 °C for 1 h. Calibration curves for plasma (100, 200, 400, 600, 800 and 1200 nmol/100 μl, and 10, 20, 40, 80, 120 and 160 nmol/100 μl for the high and low concentration range curves, respectively) and CSF (1, 2, 4, 8, 16, 32 nmol/100 μl) were all linear with correlation coefficients better than 0.999. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) at high concentration range was below 15%, whereas at low concentration range was below 20% (% 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 ±15 and ±20% at high and low concentration range, respectively. The limit of quantification was accepted as 0.1 nmol using 100-μl samples. The mean recovery for DFMO and the internal standard were greater than 95%. The method was free from interference from commonly used drugs including antimalarials and antihelminthics. The method appears to be robust and has been applied to a pharmacokinetic study of DFMO in patients with African trypanosomiasis following oral doses of Ornidyl® (Aventis Pharma, Frankfurt, Germany) at 500 mg/kg body weight (125 mg q.i.d.) for 14 days.
Keywords: 2-Difluoromethylornithine; Eflornithine;

To make molecularly imprinted polymer (MIP) solid-phase extraction (SPE) capable of direct clean-up of aqueous biological samples, an anti-quercetin MIP with evident hydrophobic matrix was synthesized using acrylamide (AA) as the functional monomer and 2,2-bis(hydroxymethyl)butanol trimethacrylate (TRIM) as the crosslinker. The affinity and selectivity were evaluated by liquid chromatography, and the binding sites and the dissolution constants were measured by frontal chromatography. Compared with the AA–co-ethyleneglycol dimethacrylate (EDMA) MIP, the anti-quercetin AA–co-TRIM MIP exhibited stronger binding and possessed improved column efficacy. A linear plot of the peak area versus sample size (in the range of 0.4–2.2 μg) was obtained, which made it promising for the MIP columns to be directly used for analysis. Before MIP-SPE of the sample of plasma, several washing solvents were tested and it was shown that the careful choice of the right washing solvent is the key step to successful sample extraction. The anti-quercetin AA–co-TRIM polymer selectively extracted quercetin, the effective component in the plasma of rats fed the hydrolyzed extract of Gingko biloba L. The recovery (67%) for MIP-SPE was calculated using spiked plasma. The results of the present work showed that the properties of MIP could be improved by modifying the polymerization and that MIP-SPE could be used for direct clean-up of biological samples for the analysis of functional components in vivo originating from an extract of medicinal herbs.
Keywords: 2,2-Bis(hydroxymethyl)butanoltrimethacrylate; Quercetin;

The long-term precision of three retention parameters, the absolute retention time (RT), the relative retention time related to dibenzepin (RRT), and the internal retention index based on the alkylfluoroaniline series (RI), were studied with 14 basic drugs on HP-5 and DB-17 columns with and without the use of the retention time locking option (RTL). Using the constant flow mode in all experiments, the RTL method was found to produce superior precision with all three retention parameters compared to the non-RTL method on each column. The results showed that RTL offers a significant advantage within a single instrument method, not only between methods, with CV<0.1% by RRT. Consequently, a dual-column gas chromatographic procedure with nitrogen–phosphorus detection was described for comprehensive screening for basic drugs in 1-ml whole blood samples. The method consisted of one-step liquid–liquid extraction with butyl acetate, identification using RRT in the RTL mode, and quantification based on single point calibration. The method allowed reliable screening and quantification of 124 basic drugs at therapeutic and toxic concentration levels in autopsy blood.

Generic solid phase extraction–liquid chromatography–tandem mass spectrometry method for fast determination of drugs in biological fluids by Anniek Schellen; Bert Ooms; Dick van de Lagemaat; Rob Vreeken; William D van Dongen (251-259).
A generic method was developed for the fast determination of a wide range of drugs in serum or plasma. The methodology comprises generic solid-phase extraction, on-line coupled to gradient HPLC with tandem mass spectrometric detection (SPE–LC–MS/MS). The individual components of the SPE–LC–MS/MS system were optimized in an integrated approach to maximize the application range and minimize the method development time. The optimized generic SPE–LC–MS/MS protocol was evaluated for 11 drugs with different physicochemical properties. Good quantification for 10 out of 11 of the pharmaceuticals in serum or plasma could be readily achieved. The quantitative assays gave recoveries better than 95%, lower quantification limits of 0.2–2.0 ng/ml, acceptable precision and accuracy and good linearity over 2–4 orders of magnitude. Carry-over was determined to be in the range of 0.02–0.10%, without optimization.

Dextromethorphan is an effective and safe antitussive, but has liabilities with respect to its abuse potential at doses above the therapeutic dose. At these higher doses, people report phencyclidine-like effects from the drug. A number of animal models have suggested that dextrorphan, an active metabolite of dextromethorphan, is responsible for the abuse liability of the parent compound when dextromethorphan is taken at high doses. Full pharmacokinetic profiles in single animals have not been demonstrated in these studies due to a lack of analytical sensitivity and/or selectivity for dextromethorphan and its metabolites. We have developed a low-cost liquid chromatographic method capable of characterizing the concentration–time profile for dextromethorphan and dextrorphan for 8 h in rats following an 18 mg/kg i.p. dose of dextromethorphan. Limits of quantitation (S/N=10) in 100 μL of serum were 0.25, 0.19, 0.27, and 0.22 nmol/mL for 3-hydroxymorphinan, dextrorphan, 3-methoxymorphinan, and dextromethorphan, respectively. Inter-day precision was better than 11% across the dynamic range of the method.
Keywords: Dextromethorphan; 3-Hydroxymorphinan; Dextrorphan; 3-Methoxymorphinan;

Determination of catechins in human plasma was carried out by high-performance liquid chromatography with electrochemical detection using a microbore octadecylsilica column. Peak heights for catechins were found to be linearly related to the amount of each catechin injected, from 2 pmol/ml to 2 nmol/ml (r>0.999). Conjugated-form catechins in plasma were hydrolyzed enzymatically using β-glucuronidase and sulfatase. Catechins in plasma and the hydrolyzed solution were extracted with ethyl acetate and determined by the present method. The time courses of concentrations of catechins in human plasma showed maxima at 1–2 h after ingestion of 340 ml of commercial canned green tea.
Keywords: Catechins;

The applicability of capillary electrophoresis (CE) with UV and mass spectrometric (MS) detection for the determination of dopamine and methoxycatecholamines in urine was evaluated in comparison with the liquid chromatography–electrochemical detection (LC–EC) method widely used in catecholamine analysis. The catecholamines in urine were deconjugated with acid or enzyme hydrolysis, purified by cation exchange (CEX) or solid-phase extraction (SPE) with a copolymer of N-divinylpyrrolidone and divinylbenzene and analyzed by LC–EC, CE–UV, and CE–MS. Acid hydrolysis was more effective in the deconjugation than enzymatic hydrolysis with Helix pomatia. However, the recoveries of HMBA, DA and NMN from spiked samples were less than 30% after acid hydrolysis and SPE purification. The CEX purification was more efficient than SPE in removing matrix compounds from the urine samples. The limits of detection were lower in LC–EC analysis than in CE–UV or CE–MS. Many factors in the analytical procedure caused deviations in the concentrations measured for urinary dopamine and methoxycatecholamines. The recovery of HMBA, which was used as the internal standard, was poor after acid hydrolysis and SPE purification. The purification methods were validated in conjunction with the analytical methods and therefore cross analysis was unsuccessful. The LC–EC method was the most sensitive, but CE–UV and CE–MS were sensitive enough for the determination of dopamine and methoxycatecholamines even in healthy patient urine. The EC and MS detections were superior to the UV detection in specificity since, after acid hydrolysis, some matrix compounds were migrating close to I.S., DA and 3MT.
Keywords: Dopamine; Methoxycatecholamines;

Rapid determination of 5-fluorouracil in plasma using capillary electrophoresis by Hao-jie Lu; Yin-long Guo; Hong Zhang; Qing-yu Ou (291-296).
A rapid, simple and sensitive capillary electrophoresis (CE) method used for the determination of 5-fluorouracil in rabbit plasma is described in the present paper. In this method, samples were simply pretreated by a solvent extraction procedure prior to injection. With a running buffer composed of 30 mM Tris–H3PO4 (pH 7.0) and 5% isopropanol, 5-fluorouracil was easily separated from the external standard α-phenethylol as well as other substances existed in the plasma. A linearity of 5-fluorouracil was determined in the range from 0.17 to 42.50 μg/ml with a correlation coefficient of 0.999. A limit of quantitation (LOQ) corresponding to signal-to-noise ratio of 10 was obtained (LOQ=0.08 μg/ml). The method was successfully used for determining the 5-fluorouracil in real plasma samples from rabbits.
Keywords: 5-Fluorouracil;

Rimadyl® (carprofen) was administered orally to the racing greyhound at a dose of 2.2 mg kg−1. Following both alkaline and enzymatic hydrolysis, postadministration urine samples were extracted by mixed mode solid-phase extraction (SPE) cartridges to identify target analyte(s) for forensic screening and confirmatory analysis methods. The acidic isolates were derivatised as trimethylsilyl ethers (TMS) and analysed by gas chromatography–mass spectrometry (GC–MS). Carprofen and five phase I metabolites were identified. Positive ion electron ionisation (EI+) mass spectra of the TMS derivatives of carprofen and its metabolites show a diagnostic base peak at M+⋅ −117 corresponding to the loss of COO–Si–(CH3)3 group as a radical. GC–MS with positive ion ammonia chemical ionisation (CI+) of the compounds provided both derivatised molecular mass and some structural information. Deutromethylation-TMS derivatisation was used to distinguish between aromatic and aliphatic oxidations of carprofen. The drug is rapidly absorbed, extensively metabolised and excreted as phase II conjugates in urine. Carprofen, three aromatic hydroxy and a minor N-hydroxy metabolite were detected for up to 48 h. For samples collected between 2 and 8 h after administration, the concentration of total carprofen ranged between 200 and 490 ng ml−1. The major metabolite, α-hydroxycarprofen was detected for over 72 h and therefore can also be used as a marker for the forensic screening of carprofen in greyhound urine.
Keywords: Carprofen;

The validation of a liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the determination of the selective cyclooxygenase-2 inhibitor etoricoxib in human plasma with phenazone as internal standard is described. The plasma samples were extracted by solid-phase extraction using polymer-based cartridges. Chromatography was carried out on a short, narrow bore RP C18 column (30×2 mm). Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2→280.2 and m/z 189.0→104.1. The analytical method was validated over the concentration range 0.2–200 ng/ml. The limit of quantification was 0.2 ng/ml. The method is applicable to pharmacokinetic studies in humans.
Keywords: Etoricoxib;

An automated online sample extraction method for rat plasma was developed and validated for the quantification of (R)- and (S)-propranolol following the intravenous administration of either the racemate or the individual enantiomers at 5 mg/kg. A dual-column extraction system coupled to a chiral stationary phase (CSP) was used in conjunction with liquid chromatography–tandem mass spectrometry. In this method, two Oasis HLB extraction columns (50×1.0 mm) in parallel were used for online plasma sample purification and teicoplanin CSP (Chirobiotic T) was used for the enantiomeric separation. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for re-conditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a relatively shorter run time and higher throughput. The lower limit of detection was 0.5 ng/ml and the lower limit of quantification was 2 ng/ml for each enantiomer using 25 μl of rat plasma. The method was validated with a linear calibration curve between 2 and 2000 ng/ml for (R)- and (S)-propranolol, respectively. The intra- and inter-day precision (C.V.) was no more than 7.6% and the accuracy of the assay was between 92 and 103%. The teicoplanin CSP proved to be rugged with excellent reproducibility of chromatographic parameters.
Keywords: Teicoplanin; Propranolol;

In this work, monolithic columns were used as a fast separation tool for multiple-component quantitative liquid chromatography–tandem mass spectrometry (LC–MS–MS) assays of drug candidates in biological fluids. A considerably reduced runtime was achieved while maintaining good chromatographic separations. This significantly improved separation speed demanded higher throughput on sample extraction. To this end, monolithic separations were coupled on-line with high-flow extraction, which allowed for the fast extraction and separation of samples containing multiple analytes. An evaluation of this system was performed using a mixture of fenfluramine, temazepam, oxazepam, and tamoxifen in plasma. A total cycle time of 1.2 min was achieved which included both sample extraction and subsequent monolithic column separation via column switching. A total of over 400 plasma samples were analyzed in less than 10 h. The sensitivity and responses were reproducible throughout the run. The system has been routinely used in the authors’ laboratory for high-throughput quantitation of compounds in biological fluids in support of drug discovery programs. The assay for samples from a 9-in-1 dog pharmacokinetic study is shown as an example to demonstrate the capability of this system.

Therapeutic drug monitoring of antiretroviral drugs has become more and more important. Therefore, a highly specific method is presented, which is capable of quantifying the different proteinase inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz, nelfinavir). The antiretroviral agents were separated and detected using LC–MS and atmospheric pressure chemical ionization. After solid-phase extraction, the antiretrovirals were separated within 21 min using gradient elution. The calibration range of each drug was linear including the expected minimum and maximum concentrations measured in plasma after the administration of the different drugs. All within-day and between-day coefficients of variation were below 10% and the recovery rates were between 34.8 and 124%. The respective quantification limits were 1 μg/l (indinavir), 10 μg/l (amprenavir, efavirenz), 50 μg/l (saquinavir), 90 μg/l (nelfinavir), 200 μg/l (nevirapine, ritonavir) and 250 μg/l (lopinavir).
Keywords: Indinavir; Amprenavir; Efavirenz; Saquinavir; Nelfinavir; Nevirapine; Ritonavir; Lopinavir;

Determination of residues of malachite green in aquatic animals by Aldert A Bergwerff; Peter Scherpenisse (351-359).
Residues of malachite green (MG) were extracted from homogenized animal tissues with a mixture of McIlvaine buffer (pH 3.0)–acetonitrile, and purified over an aromatic sulfonic acid solid-phase extraction column followed by HPLC or LC–ESI-MS–MS analysis. Ascorbic acid and N,N,N′,N′-tetramethyl-1,4-phenylenediamine dihydrochloride were added to reduce de-methylation of the dye. Responses were recorded at 620 nm (HPLC) or by multiple-reaction-monitoring (LC–MS–MS) after post-column oxidation using PbO2. MG and its primary metabolite leuco-malachite green (LMG) were successfully determined at 2.5–2000 μg/kg in catfish, eel, rainbow trout, salmon, tropical prawns and turbot, with a limit of detection at 1 μg/kg (HPLC) and 0.2 μg/kg (LC–MS–MS) for both MG and LMG. Recoveries for LMG were between 86±15% (prawn) and 105±14% (eel). Freeze–thawing cycles, and storage at 4 °C and −20 °C affected the recovery of both MG and LMG. Analyses of eel, trout and (processed) salmon field samples collected at local retailers, fish-market and -shops demonstrated trace levels of MG-residues.
Keywords: Malachite green;

Selective, sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of alfuzosin in human plasma by J.L Wiesner; F.C.W Sutherland; G.H van Essen; H.K.L Hundt; K.J Swart; A.F Hundt (361-368).
A selective, sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of alfuzosin in plasma was developed. A PE Sciex API 2000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray with positive ionisation was used. Using prazosin as an internal standard, liquid–liquid extraction was followed by C18 reversed-phase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosin was 82.9% with a lower limit of quantification set at 0.298 ng/ml, the calibration range being between 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS–MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of alfuzosin in human plasma than has previously been described. The assay method was used to quantify alfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steady state.
Keywords: Alfuzosin;

Glutathione (GSH) is an important component of antioxidant defenses in airway surface liquid (ASL), a thin layer (10–30 μm) of liquid covering the epithelial cells lining the airways of the lung. Decreased levels of ASL GSH have been reported in cystic fibrosis (CF), potentially contributing to the severe oxidative stress seen in this disease. To help investigate the role of GSH in ASL, we developed a technique suitable for analysis of GSH and its oxidized form (GSSG) in microliter samples using capillary sampling followed by capillary zone electrophoresis (CZE) analysis with conductivity detection. CZE was carried out in 100 mM CHES and 40 mM lithium hydroxide with 5 mM spermine at pH 9.1 under an applied electric field of −416 V cm−1. To prevent any autooxidation of GSH during sample manipulations, the samples were treated with N-ethylmaleimide (50 mM) to alkylate free thiol (–SH). Under these conditions, GSH and GSSG were cleanly separated without interference from common anions (e.g. Cl, PO4 3−, HCO3 , etc.) and the limit of detection for ASL analysis was 11 μM for GSH and 8 μM for GSSG (S/N=3). GSH and GSSG were also measured in rat plasma. Baseline values of 897±210 μM (GSH) and 215±61 μM (GSSG) were obtained for rat ASL (n=8), whereas 12.4±2.7 μM (GSH) and 14.8±6.7 μM (GSSG) were obtained for rat plasma (n=5).
Keywords: Glutathione;

A selective, accurate, and reproducible LC–MS–MS assay was developed for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma samples. The method involved automated solid-phase extraction of atazanavir and a stable isotope analog internal standard (I.S.) using Oasis HLB 10 mg 96-well SPE plates. A portion of the reconstituted sample residue was injected onto a C18 HDO analytical column which was configured with a triple quad mass spectrometer for analyte determination by positive ion electrospray. The assay was linear from 1.00 to 1000 ng/ml with a lower limit of quantitation of 1.00 ng/ml. The inter- and intra-day coefficients of variation (C.V.) for the assay were <4%, and the accuracy was 99–102%. Atazanavir was stable in human plasma for at least 109 h at room temperature and for at least 1 year at −20 °C.
Keywords: Atazanavir;

A high-performance liquid chromatographic method for the determination of dehydrotumulosic acid in plasma of rats having been administrated orally with the traditional Chinese medicinal preparation Ling-Gui-Zhu-Gan decoction was developed. Plasma samples taken from rats were acidified with hydrochloric acid and extracted with ethyl acetate. Separation of the main effective constituent dehydrotumulosic acid was accomplished on a C18 stationary phase and a mobile phase of methanol–acetonitrile–2% glacial acetic acid (13:12:10, v/v), with a UV detector setting at 242 nm. After validation, the method was used for preliminary investigation of the pharmacokinetic profiles of dehydrotumulosic acid administrated in Ling-Gui-Zhu-Gan decoction.
Keywords: Dehydrotumulosic acid;

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed to detect perchlorate in tissues of male and female rats, both pregnant and lactating (including milk) after administration of perchlorate. Supernatants of ethanol precipitated rat fluids and tissues were evaporated to dryness under nitrogen and reconstituted in deionized water. Reconstituted samples were injected into HPLC system coupled with conductivity detection. Isocratic separation of perchlorate was achieved using an anion-exchange column with sodium hydroxide as mobile phase and a conductivity detector. In this method, perchlorate showed a linear response range from 5 to 100 ng/ml. The lower detection limits for perchlorate in fluids and tissues of rats were 3–6 ng/ml and 0.007–0.7 mg/kg, respectively. The described method has the unique advantage over the existing methods of determining low traces of perchlorate in different biological matrices without complex sample preparation.
Keywords: Perchlorate;

A highly sensitive high-performance liquid chromatography (HPLC) method for the determination of urinary N-acetylneuraminic acid (NeuAc) using 3-[(1-{[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl}pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent was developed. The labeling reaction was carried out at 30 °C for 30 min in the presence of pyridine. The derivative was monitored at Ex 314 nm and Em 388 nm. The detection limit of NeuAc was about 48 fmol per injection. The relative standard deviations of within-day and between-day precisions were 2.6–3.3 and 1.7–3.3%, respectively. Urine diluted 10 times with distilled water was analyzed by employing the standard-addition method. The concentrations were 8–89 nmol/mg creatinine (30±28 nmol/mg creatinine, n=9).
Keywords: N-Acetylneuraminic acid;

Determination of three phthalate metabolites in human urine using on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry by Kayoko Kato; Sachiko Shoda; Masakazu Takahashi; Natsuko Doi; Yoshihiro Yoshimura; Hiroyuki Nakazawa (407-411).
An on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (on-line SPE–HPLC–MS/MS) method was developed for the analysis of metabolites of three phthalate esters in human urine at the low nanogram per milliliter level. The recoveries were above 84.3% and relative standard deviations varied from 0.8 to 4.8%. The compounds along with their deuterated internal standards were detected in the negative ion mode by selective reaction monitoring and the accuracy of the method was improved by isotope dilution. Monobutyl phthalate was detected with median level of 22.5 ng/ml. The median levels for monobenzyl phthalate and monoethylhexyl phthalate were less than the limit of quantitation (LOQ). The on-line SPE–HPLC–MS/MS method allowed the possibility of determining these metabolites within a short time, with increased sensitivity and by using decreased amounts of sample and solvent.
Keywords: Phthalate monoester;

gamma Aminobutyric acid (GABA) was determined by precolumn derivatization with 2-hydroxynaphthaldehyde and elution was made using Phenomenex C18, 5 μm column with methanol: water (62:38 v/v) and UV detection at 330 nm. In a mixture containing glycine, l-lysine and tyramine GABA separated completely. A number of amines and amino acids tested did not affect the response of GABA. A linear calibration curve was obtained for GABA in the range of 1.2–28.0 μg/ml with detection limit of 2.8 ng/injection (5 μl). The method was used for the determination of GABA in cerebral spinal fluid (CSF) samples and gave results of 19.0 to 22.4 μg/m1 with coefficient of variation 2.4%
Keywords: γ-Aminobutyric acid;