Journal of Chromatography B (v.798, #2)

OFC:Update (OFC).

News Section (N1-N2).

Highly sensitive gas chromatographic determination of ethanol in human urine samples by Michael Zilly; Peter Langmann; Ulrike Lenker; Verena Satzinger; Diana Schirmer; Hartwig Klinker (179-186).
In order to evaluate recent alcohol consumption, a very sensitive and specific gas chromatographic method for ethanol determination in human urine samples was developed. The non-invasive method was performed without any pretreatment and carried out on a Stabilwax capillary column, 30  m×0.53  mm×1.0  μm film thickness. Helium was used as carrier gas with a constant inlet pressure of 27.72 kPa (0.277 bar) and a flame ionization detector (FID). Quantification was performed with the use of acetonitrile as an internal standard (IS). The calibration curve was linear throughout the concentration range from 0.5 to 500 mg/l. The calculated intra- and inter-day coefficients of variation were below 8%. A clear chromatographic separation of ethanol from methanol, acetone, 1-propanol and 2-propanol was achieved.
Keywords: Ethanol;

Measurement of carvedilol in plasma by high-performance liquid chromatography with electrochemical detection by Maiko Machida; Masato Watanabe; Shigeru Takechi; Shigeo Kakinoki; Akikazu Nomura (187-191).
Carvedilol is a β/α1-adrenoceptor blocker. A sensitive method for measuring plasma levels of carvedilol in human administrated low doses is needed since its plasma concentration is low. We measured carvedilol and carvedilol M21-aglycon using high-performance liquid chromatography (HPLC) with electrochmical detection. The amperometric detector was operated at 930 mV versus Ag/AgCl. Mean coefficients of variation (n=5) for carvedilol and M21-aglycon were 4.0 and 7.7% (intra) and 6.1 and 6.7% (inter), respectively. The lower limit of quantification for each analyte was 0.10 ng/ml (signal-to-noise ratio=3). This lower limit of quantification for carvedilol was sufficient for clinical use.
Keywords: Electrochemical detection; Carvedilol;

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis® hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 μm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.
Keywords: Steroids; Prednisolone; Cortisol;

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at −70 °C. The method has been applied to 150 patient samples as part of a medication adherence study.
Keywords: Metformin;

A simple high performance liquid chromatography (HPLC) method for the measurement of the new antidepressant mirtazapine and its N-demethyl metabolite, normirtazapine, in human plasma or serum during low dose mirtazapine therapy has been developed. A Waters Spherisorb S5 SCX column was used with ammonium perchlorate (50 mmol/l) in methanol/water (95 + 5 (v/v)), apparent pH 6.7, as eluent, and fluorescence detection. Only small volumes of sample (0.2 ml) and extraction solvent are used. An interference study found no significant co-elution with drug or metabolite, although paroxetine co-elutes with the internal standard. The recovery of mirtazapine and normirtazapine (mean±S.D.) was 79±2, and 64±3%, respectively. The LOD was estimated as 0.5 μg/l, LLOQ was 1 μg/l, with a linear response over the concentration range 4–1000 μg/l (both analytes). The analytes were stable in serum for at least 10 months when stored at −20 °C. Intra- and inter-day accuracy were in the range 91–107 and 93–103%, respectively. In clinical samples (n=14, median mirtazapine dose 45 mg per day, range 15–45 mg per day) the median (range) mirtazapine and normirtazapine concentrations were 26 (8–40) and 21 (8–32) μg/l, respectively.
Keywords: Mirtazapine; Normirtazapine;

The retention of 8-methoxypsoralen (8-MOP) on an immobilised hydroxypropyl-β-cyclodextrin (HP-β-CD) column was analysed in HPLC by the determination of its Langmuir distribution isotherm. A such method was used to confirm the potential drug complexing role of this cyclodextrin. The 8-MOP/HP-β-CD association constant (K) was equal to 29.5 and 18.7 M−1, respectively, at a temperature equal to 5 and 25 °C, respectively. These association constant values were used to determine the cytotoxicity profile of human keratinocyte cell line (HaCaT) in relation to the complex concentration. It was showed through these data that HP-β-CD had a cytoprotective since a reverse effect of HP-β-CD on 8-MOP cytotoxicity was observed.
Keywords: Association constant; 8-Methoxypsoralen; Hydroxypropyl-cyclodextrin;

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography–tandem mass spectrometry by Lutz Bräutigam; Maic Seegel; Irmgard Tegeder; Helmut Schmidt; Silke Meier; Maurizio Podda; Roland Kaufmann; Marcella Grundmann-Kollmann; Gerd Geisslinger (223-229).
The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid–liquid extraction with diisopropylether using 4,5′,8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30  mm×2.0  mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2→174.0 for 8-MOP and m/z 229.1→142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70  mm×2.0  mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2→174.0. The assays were validated over the concentration ranges of 0.5–50 ng/ml for plasma samples and 0.25–50 ng/ml for microdialysates, respectively.
Keywords: 8-Methoxypsoralen;

Nitric oxide (NO) is synthesized by NO synthase from l-arginine, which can be competitively blocked by endogenous inhibitors such as asymmetric dimethylarginine (ADMA), but not by symmetric dimethylarginine (SDMA). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH) to dimethylamine and citrulline. A growing number of published clinical studies documented a strong correlation between increased ADMA blood levels and cardiovascular morbidity and mortality. We present here a highly sensitive method for the determination of this compounds in plasma and urine by means of HPLC–MS. The sample preparation is very simple and comprises only protein precipitation and concentration in the case of plasma samples and dilution in the case of urine. The samples are derivatized automatically with orthophthaldialdehyde and 2-mercaptoethanol, are separated on a 250  mm×4  mm RP18 column by gradient elution with formate buffer/methanol and are detected by ESI-MS. The calibration functions are linear and cover the range from normal to pathologic concentration values of the analytes. The intra-day relative standard deviation (R.S.D.) of the assay for ADMA in plasma is 7.5% and the corresponding inter-day R.S.D. is 5.7%. In urine, these values for ADMA are 3.8 and 6.4%, respectively. All other analytes in plasma as well as in urine exhibit intra-day R.S.D. below 8%. The corresponding inter-day R.S.D. are all below 13%.
Keywords: Arginine; Asymmetric dimethylarginine; Symmetric dimethylarginine; Citrulline;

A simple and specific method based on gas chromatography–selected ion monitoring-mass spectrometry (GC–SIM-MS) for the analysis of in vivo metabolism of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) in rats is described. Three male rats were administered 20 mg/kg of 2C-T-2 by intra-peritoneal injection, and 24 h urine fractions were collected before and after the administration for analysis. After acidic hydrolysis of the urine samples, the metabolites were liquid–liquid extraction and analyzed by a quadruple mass spectrometer in the selected ion monitoring mode. The findings show that four metabolites of 2-(4-ethylthio-2,5-dimethoxyphenyl)-ethanol (M w: 242), 4-ethylthio-2,5-dimethoxyphenyl acetic acid (M w: 256), 1-acetoamino-2-(2-hydroxy-4-ethylthio-5-methoxyphenyl)-ethane (M w: 269) and 1-acetoamino-2-(2-methoxy-4-ethylthio-5-hydroxyphenyl)-ethane (M w: 269) are present and the metabolic pathway for 2C-T-2 in the rat is proposed.
Keywords: 2,5-Dimethoxy-4-ethylthiophenethylamine;

Development and validation of a gas chromatography-mass spectrometry assay for hair analysis of amphetamine, methamphetamine and methylenedioxy derivatives by Mitona Pujadas; Simona Pichini; Sandra Poudevida; Ester Menoyo; Piergiorgio Zuccaro; Magı́ Farré; Rafael de la Torre (249-255).
A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), 3,4-methylenedioxyethylamphetamine (MDE or MDEA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair. Hair samples were digested with 1 M sodium sulfide at 37 °C (by shaking for 3 h and was kept at room temperature overnight), and extracted with two sequential extraction procedures: liquid-liquid extraction with tert-butyl methyl ether and solid-phase extraction with Bond-Elut Certify columns. Extracted analytes were derivatised with N-methyl-bis(trifluoroacetamide), separated by a 5% phenylmethylsilicone column and determined by a mass spectrometer detector in selected ion monitoring mode. A good reproducibility (intra-assay R.S.D.=1.5–15.7%), accuracy (intra-assay error=2.0–11.7%) and sensitivity (LOD=0.03–0.08 ng/mg hair) were attained. The method was successfully applied to the analysis of the proximal (1 cm) hair segment to assess recent self-reported use in “ecstasy” consumers. Otherwise, further studies are needed to validate methodology developed in case of amphetamine consumption.
Keywords: Derivatization, GC; Amphetamine; Methamphetamine;

A rapid, selective and sensitive high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of triflusal and its major active metabolite, 2-hydroxy-4-trifluoromethyl benzoic acid (HTB), in rat and human plasma. HPLC analysis was carried out using a 5-μm particle size, C18-bonded silica column and acetonitrile–methanol–water (25:10:65, v/v/v) as the mobile phase and UV detection at 234 nm. Furosemide was used as the internal standard. The method involved extraction with an acetonitrile–chloroform mixture (60:40, v/v) and evaporation to dryness with nitrogen stream. The chromatograms showed good resolution and sensitivity and no interferences by plasma constituents. The mean absolute recovery for human plasma was 93.5±4.2% for triflusal and 98.5±3.1% for HTB. The lower limits of quantification of triflusal and HTB in human plasma were 20 and 100 ng/ml, respectively. The calibration curves in human plasma were linear over the concentration range 0.02–5.0 μg/ml for triflusal and 0.1–200.0 μg/ml for HTB with correlation coefficients greater than 0.999 and with inter- or intra-day coefficients of variation (CV) not exceeding 10.0%. This assay procedure was applied to the study of metabolite pharmacokinetics of triflusal and HTB in rat and human.
Keywords: Triflusal; 2-Hydroxy-4-trifluoromethyl benzoic acid;

Verapamil is a widely prescribed calcium antagonist, but suffers from extensive first pass metabolism. Despite its frequent use in drug metabolism a complete understanding of its metabolic pathway is still lacking. We thus investigated verapamil’s metabolism in cultures of primary rat hepatocytes and isolated metabolites from cell culture media by solid phase extraction (SPE). In detail, we investigated their structure in multiple liquid chromatography–mass spectrometry (LC–MS n ) experiments and found 25 phase I and 14 phase II metabolites. We showed many metabolites to be produced by oxidative dealkylation, and several yet unknown metabolites were identified that stem from hydroxylation and dealkylation reactions. Furthermore, we identified an array of glucuronides and, additionally, a glucoside. Finally, we investigated the enantioselective biotransformation of verapamil and found preferential metabolism of the S-enantiomers. In conclusion, this illustrates again the true complexity of verapamil’s disposition.
Keywords: Verapamil metabolites;

A direct injection high-performance liquid chromatographic (HPLC) method, with column-switching, for the determination of omeprazole enantiomers in human plasma is described. A restricted access media (RAM) of bovine serum albumin (BSA) octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the RAM column onto an amylose tris(3,5-dimethylphenylcarbamate) chiral column by the use of a column-switching valve and the enantioseparation was performed using acetonitrile–water (60:40 v/v) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40 mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. The assay was able to determine the cytochrome P450 2C19 phenotype of the subjects participating in this study.
Keywords: Enantiomer separation; Direct plasma injection; Omeprazole;

Glycosylation study of the major genetic variants of human α1-acid glycoprotein and of their pharmacokinetics in the rat by Françoise Hervé; Philippe d’Athis; Dominique Tremblay; Jean-Paul Tillement; Jérôme Barré (283-294).
Human α1-acid glycoprotein (AAG) is a mixture of at least two genetic variants, the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. AAG is also an extensively glycosylated protein which possesses five N-linked glycans exhibiting substantial heterogeneity in their structures. The first objective of this study was to investigate the glycosylation of the two major gene products of AAG, i.e. the A variant and a mixture of the F1 and S variants (F1*S). To this end, we combined a chromatographic method for the fractionation of the AAG variants with a lectin-binding assay to characterise the glycosylation of purified glycoproteins. Secondly, because the oligosaccharides can influence the disposition of AAG, a kinetic study of the AAG variants was carried out in the rat. After intravenous administration of whole human AAG, the separation and quantification of the AAG variants in plasma was performed by application of specific methods by isoelectric focusing and immunonephelometry. The binding studies carried out on a panel of lectins showed significant differences in the lectin-binding characteristics of the separated F1*S and A variants, accounting for differences in the degree of branching of their glycan chains and substitution with sialic acid and fucose. The plasma concentration-time profiles of the F1*S and A variants were biphasic, and only small differences were observed between the variants for their initial and terminal half-lives, clearance and distribution volume. This indicates that the structural differences between the two AAG gene products do not affect their pharmacokinetics in the rat. Specific drug transport roles have been previously demonstrated for the F1*S and A variants, calling for further investigations into their effects on the disposition of drugs they bind in plasma. The present study shows that such investigations are possible without being complicated by kinetic differences between these variants.
Keywords: Glycosylation; Pharmacokinetics; α1-Acid glycoprotein; Genetic variants;

A novel sensitive high-performance liquid chromatography-electrospray mass spectrometry method has been developed for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates, glycoursodeoxycholic acid (GDCA) and tauroursodeoxycholic acid (TDCA). The procedure involved a solid phase extraction of UDCA, GDCA, TDCA and the internal standard, 23-nordeoxycholic acid from human plasma on a C18 Bond Elut cartridge. Chromatography was performed by isocratic reverse phase separation with methanol/25 mM ammonium acetate (40/60, v/v) containing 0.05% acetic acid on a C18 column with embedded polar functional group. Detection was achieved using an LC-MS/MS system. The standard curve was linear over a working range of 10–3000 ng/ml for all analytes and gave an average correlation coefficient of 0.9992 or better during validation. The absolute recovery for UDCA, GDCA, TDCA and the internal standard was 87.3, 83.7, 79.5 and 95.8%, respectively. This method is simple, sensitive and suitable for pharmacokinetics, bioequivalence or clinical studies.
Keywords: Ursodeoxycholic acid;

Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol by Tomás Pérez-Ruiz; Carmen Martı́nez-Lozano; Virginia Tomás; Raquel Galera (303-308).
A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.
Keywords: Allopurinol; Oxypurinol;

In this study, a method for partly automated sample preparation and fully automated solid-phase extraction method for plasma, kidney and liver samples for various retinoids like all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, retinol and retinyl palmitate was established. Plasma, embryo-, kidney-and liver-homogenates were automatically mixed and extracted on multiple usage solid-phase (C2) extraction cartridges immediately before HPLC analysis. Automated cleaning, preconditioning and incorporation of the loaded cartridge to fully automated HPLC separation and quantification of the various retinoids in a single HPLC run was established. The recovery of the retinoids was generally between 80 and 90%. Intra-day repeatability was <11.7%. As little as 1.2 ng/ml could be quantified in lipid-mixture standard samples. This method allows a highly automated sample preparation and a fully automated solid-phase extraction with good selectivity for the study of endogenous retinoids and retinoids after nutritional supplementations and pharmacological applications in several biological samples.
Keywords: Retinoid;

Two different clean-up procedures for liquid chromatographic determination of ochratoxin A in urine by Ana-Marija Domijan; Maja Peraica; Marica Miletić-Medved; Ana Lucić; Radovan Fuchs (317-321).
This paper describes two different procedures for extraction of ochratoxin A (OTA) from urine samples: one using acidic chloroform–methanol mixture, followed by solid-phase extraction (SPE) clean-up and the other using commercial Chem Elut columns and a chloroform–formic acid mixture. The recovery of OTA using the procedure with silica gel columns was 82% with a R.S.D.<8.4% and the detection and quantitation limits were 0.5 and 1.5 ng OTA/ml, respectively. The recovery of OTA in the second procedure with urine samples purified only on commercial Chem Elut columns was 95% with R.S.D.<4.0%, and detection and quantitation limits 0.3 and 0.9 ng/ml, respectively. Both procedures of OTA extraction effectively eliminate interfering substances and give reliable and repeatable results. However, the procedure with Chem Elut columns gave higher recovery and lower detection and quantitation limits. It was successfully applied in determining OTA in human urine samples.
Keywords: Clean-up procedures; Ochratoxin A;

Natural phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated and purified from soybean degummed oil residues in this work. Crude PC and PE were first separated from degummed oil residues by extraction with 95% ethanol, and then the crude PC and PE were used as raw materials to prepare high purity PC and PE by using column chromatography of silica gel (100–200 mesh) with different eluents and elution modes. The high purity PC (content >90%) was obtained from the crude PC by using isocratic elution with methanol as eluent. Compared with the methods reported by using isocratic elution with mixed solvents as eluent or gradient elution, the procedure proposed exhibits low cost and industry potentialities because of some advantages, such as operation simplicity, cheap equipment and solvent to be recovered easily. The purity of the PE product prepared from the crude PE was more than 75%. The gradient elution was preferable to isocratic elution for reducing the elution time and eluent consumption when to prepare PE from the crude PE. The effects of loading amount and the flow-rate on separation efficiency were also investigated. For obtaining high separation efficiency, the loading amount should be less than 2.0 g crude PC or PE/100 g silica gel, and the flow-rate should be controlled under 4 ml/min for crude PC and 3 ml/min for crude PE, respectively.
Keywords: Phosphatidylcholine; Phosphatidylethanolamine;

Studies are described on the toxicological analysis of the piperazine-derived designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat urine using gas chromatography–mass spectrometry (GC–MS). The authors’ systematic toxicological analysis (STA) procedure using full-scan GC–MS after acid hydrolysis, liquid–liquid extraction and microwave-assisted acetylation allowed the detection of MeOPP and its metabolites 1-(4-hydroxy phenyl)piperazine and 4-hydroxyaniline in rat urine after administration of a single dose corresponding to doses commonly taken by drug users. Therefore, this procedure should also be suitable for detection of a MeOPP intake in human urine. However, the metabolites of MeOPP are not unique and can be produced from other drugs. Therefore, differentiation of use of this designer drug from use of the medicaments dropropizine, oxypertine or others, which are metabolized to the MeOPP isomer 1-(2-methoxyphenyl)piperazine, is discussed.
Keywords: Designer drug; 1-(4-Methoxyphenyl)piperazine; Dropropizine; Oxypertine;

Fast liquid chromatography–mass spectrometry glutathione measurement in whole blood: micromolar GSSG is a sample preparation artifact by Jean-Paul Steghens; Françoise Flourié; Khelifa Arab; Christian Collombel (343-349).
We describe a new, fast (6 min) and reliable method to measure reduced or oxidized glutathione (GSH) or (GSSG) in whole blood. The method is based on a LC/MS measurement in positive electrospray ionization mode after a chromatographic separation on a specific column which does not need any counter-ion in the mobile phase, improving the sensitivity of detection. A 50 μl sample of whole blood is sufficient for analysis. We demonstrate that the lack of an alkylating agent during the sample preparation brings out an underestimation of GSH and an artefactual production of GSSG, corresponding to 2–3% of GSH. The simultaneous use of N-ethyl-maleimide and a strong deproteinising acid prevents these two drawbacks. This efficient and new method of preparation and analysis lets us show that, unexpectedly, GSH is stable in whole blood for some hours and that deproteinised samples can be stored without GSH loss for at least three weeks at −20 or −80 °C. The reference interval, measured on 22 volunteers, on blood samples collected either with heparin or with EDTA, is 1310±118 μM for GSH and 0.62 μM for GSSG. The within-run precision of this method, with γ glutamyl-glutamic acid as an internal standard, evaluated in three successive series (n=30), lies between 2.1 and 4.8% for a GSH level at 580 or 1150 μM. The one step sample preparation we propose seems well suited for GSH routine measurements in hospital laboratories and avoids any underestimation of GSH, a now well accepted biomarker of oxidative stress.
Keywords: Artifacts; Glutathione;

Testing for kavain in human hair using gas chromatography–tandem mass spectrometry by M. Villain; V. Cirimele; A. Tracqui; F.X. Ricaut; B. Ludes; P. Kintz (351-354).
A sensitive, specific and reproducible method for the quantitative determination of kavain in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 50 mg) was incubated in 1 ml of methanol for 1 h, in an ultrasonic bath, in presence of 20 ng of methaqualone-d7 used as internal standard. The methanolic solution was evaporated to dryness, and the residue reconstitued by adding 30 μl of methanol. A 2 μl aliquot of the extract was injected onto the column (Optima5-MS capillary column, 5% phenyl–95% methylsiloxane, 30  m×0.25  mm i.d.×0.25 mm film thickness) of a Hewlett-Packard (Palo Alto, CA) gas chromatograph (5890). Kavain was detected by its parent ion at m/z 230 and daughter ions at m/z 111 and 202 through a Finnigan TSQ 700 MS/MS system. The assay was capable of detecting 30 pg/mg of kavain (limit of detection (LOD)). Linearity was observed for kavain concentrations ranging from 100 to 2000 pg/mg with a correlation coefficient of 0.998. Intra-day precision at 400 pg/mg was 13.7%. The analysis of a segment of hair, obtained from an occasional consumer, revealed the presence of kavain at the concentration of 418 pg/mg. A higher concentration (1708 pg/mg) was detected in the corresponding pubic hair.
Keywords: Kavain;

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of itraconazole and its active metabolite, hydroxyitraconazole, in human plasma. Prior to analysis, both compounds together with the internal standard were extracted from alkalinized plasma samples using a 3:2 (v/v) mixture of 2,2,4-trimethylpentane and dichloromethane. The mobile phase comprised 0.02 M potassium dihydrogen phosphate-acetonitrile (1:1, v/v) adjusted to pH 3.0. Analysis was run at flow-rate of 0.9 ml/min with excitation and emission wavelengths set at 260 and 365 nm, respectively. Itraconazole was found to adsorb on glass or plastic tubes, but could be circumvented by prior treating the tubes using 10% dichlorodimethylsilane in toluene. Moreover, rinsing the injector port with acetonitrile helped to overcome any carry-over effect. This problem was not encountered with hydroxyitraconazole. The method was sensitive with limit of quantification of 3 ng/ml for itraconazole and 6 ng/ml for hydroxyitraconazole. The calibration curve was linear over a concentration range of 2.8–720 ng/ml for itraconazole and 5.6–720 ng/ml for the hydroxy metabolite. Mean recovery value of the extraction procedure for both compounds was about 85%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 15%. Hence, the method is suitable for use in pharmacokinetic and bioavailability studies of itraconazole.
Keywords: Itraconazole; Hydroxyitraconazole;

Determination of cocaine, benzoylecgonine and cocaethylene in human hair by solid-phase microextraction and gas chromatography–mass spectrometry by Fernanda Crossi Pereira de Toledo; Mauricio Yonamine; Regina Lucia de Moraes Moreau; Ovandir Alves Silva (361-365).
The present work describes a highly precise and sensitive method developed to detect cocaine (COC), benzoylecgonine (BE, its main metabolite) and cocaethylene (CE, transesterification product of the coingestion of COC with ethanol) in human head hair samples. The method was based on an alkylchloroformate derivatization of benzoylecgonine and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography–mass spectrometry (GC–MS) was used to identify and quantify the analytes in selected ion monitoring mode (SIM). The limits of quantification and detection (LOQ and LOD) were: 0.1 ng/mg for COC and CE, and 0.5 ng/mg for BE. Good inter- and intra-assay precision was observed. The dynamic range of the assay was 0.1–50 ng/mg. The method is not time consuming and was shown to be easy to perform.
Keywords: Cocaine; Benzoylecgonine; Cocaethylene;