Journal of Chromatography B (v.814, #2)

News Section (N1-N2).

OFC (CO1).

Glycyrrhizin (GLY) which has been widely used in traditional Chinese medicinal preparation possesses various pharmacological effects. In order to investigate the pharmacokinetic behavior of GLY in human after oral administration of GLY or licorice root, a liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of GLY and its major metabolite glycyrrhetic acid (GA) in human plasma. The method involved a solid phase extraction of GLY, GA, and α-hederin, the internal standard (IS), from plasma with Waters Oasis MCX solid phase extraction (SPE) cartridges (30 mg) and a detection using a Micromass Quattro LC liquid chromatography/tandem mass spectrometry system with electrospray ionization source in positive ion mode. Separation of the analytes was achieved within 5 min on a SepaxHP CN analytical column with a mobile phase of acetonitrile:water (50:50, v:v) containing 0.1% formic acid and 5 mM ammonium acetate. Multiple reaction monitoring (MRM) was utilized for the detection monitoring 823 → 453 for GLY, 471 → 177 for GA and 752 → 456 for IS. The LC–MS/MS method was validated for specificity, sensitivity, accuracy, precision, and calibration function. The assay had a calibration range from 10 to 10,000 ng/mL and a lower limit of quantification of 10 ng/mL for both GLY and GA when 0.2 mL plasma was used for extraction. The percent coefficient of variation for accuracy and precision (inter-run and intra-run) for this method was less than 11.0% with a %Nominal ranging from 87.6 to 106.4% for GLY and 93.7 to 107.8% for GA. Stability of the analytes over sample processing (freeze/thaw, bench-top and long-term storage) and in the extracted samples was also tested and established.
Keywords: Glycyrrhizin; Glycyrrhetic acid; Metabolite; LC–MS/MS;

Optimised affinity purification of polyclonal antibodies from hyper immunised ovine serum using a synthetic Protein A adsorbent, MAbsorbent® A2P by Anthony R. Newcombe; Chrissie Cresswell; Susannah Davies; Keith Watson; Guy Harris; Kieran O’Donovan; Richard Francis (209-215).
This report describes the applicability of a synthetic chromatography adsorbent for large-scale purification of polyclonal immunoglobulin G from hyper immunised ovine serum. Under optimised conditions, MAbsorbent® A2P was shown to bind ∼27 mg mL−1 of ovine immunoglobulin from undiluted serum, with eluted IgG purities of >95%, minor levels of albumin (∼1%) and undetectable levels of leached ligand in the purified preparations. The results presented here indicate that the optimised affinity capture of immunoglobulin from ovine serum using MAbsorbent® A2P is a feasible alternative to Protein A chromatography or sodium sulphate precipitation for the initial capture of antibodies from undiluted serum.
Keywords: Mimetic; Protein A; Chromatography; Affinity purification; Antibody; Serum;

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry by Luis H. Migliorança; Rafael E. Barrientos-Astigarraga; B.S. Schug; H.H. Blume; Alberto S. Pereira; Gilberto De Nucci (217-223).
A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C18 analytical column (100 mm × 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL−1 (r 2  > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL−1), 7.1% (0.6 ng mL−1) and 6.8% (7.5 ng mL−1). The between-run accuracy was ± 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.
Keywords: Felodipine; Antihipertensive; Heathy volunteer; LC–MS/MS; Bioequivalence;

In vitro metabolic stability studies are performed routinely in drug discovery to determine the rate of metabolism as well as the metabolic fate of compounds. These studies are labor intensive, involving incubation of the compound with a biological matrix, sampling at various time points, stopping the reaction, and sample preparation for analysis. All of these steps involve manual pipetting in the conventional method. An automated method for in vitro metabolism studies is reported here. The method reduces the time and manual labor required and has other advantages, such as better reproducibility and unattended operation. This method utilizes an autosampler custom configured with cooling and incubation capabilities. The autosampler is programmed to directly inject incubation samples at set time points onto an online extraction column. The extracted sample then enters an analytical column for separation and ultimately the mass spectrometer for detection. The injection has the dual function of stopping the reaction and starting the analysis on the LC–MS. This method was used for the metabolic stability study of a prodrug in plasma and liver S9 fractions of five different species. The stability data from the automated method were similar to those obtained using the conventional method. The potential for this method to increase throughput of metabolic stability studies in drug discovery is demonstrated.
Keywords: Liquid chromatography; Prodrug; Stability; LC/MS analysis; Automated online method; Metabolic study; Online incubation; Online analysis;

Nicotine is rapidly and extensively metabolized in humans. We present an analytical method to simultaneously quantify nicotine, cotinine, norcotinine, and trans-3′-hydroxycotinine in human oral fluid. Solid phase extraction (SPE) and GC/MS/EI with selected ion monitoring (SIM) were utilized. Linearity ranged from 5 to 1000 ng/mL of oral fluid; correlation coefficients for calibration curves were >0.99. Recoveries were 90–115% nicotine, 76–117% cotinine, 88–101% norcotinine, and 67–77% trans-3′-hydroxycotinine. Intra-assay precision and accuracy ranged from 1.6 to 5.7% and 1.6 to 17.8%, respectively. Inter-assay precision and accuracy ranged from 4.3 to 10.2% and 0 to 12.8%, respectively. Suitable precision and accuracy were achieved for the simultaneous determination of nicotine and three metabolites in the oral fluid of smokers. This assay is applicable to pharmacokinetic studies of nicotine, cotinine, and trans-3′-hydroxycotinine from tobacco smokers and can be utilized for routine monitoring of tobacco smoke exposure. 3-Hydroxycotinine requires additional investigation to determine its usefulness as a biomarker for tobacco smoke exposure.
Keywords: Nicotine; Cotinine; trans-3′-Hydroxycotinine; Norcotinine; Oral fluid;

An assay using high performance liquid chromatography (HPLC)–electrospray ionization–tandem mass spectrometry (ESI–MS–MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 μl samples of rat serum. Deuterated (d3) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins; acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were—morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%).
Keywords: Morphine; Morphine-3-glucuronide; Oxycodone; Noroxycodone; High performance liquid chromatography; Electrospray ionization; Tandem mass spectrometry;

Application of gas chromatography-mass spectrometry for the determination of urinary ethylenethiourea in humans by Silvia Fustinoni; Laura Campo; Claudio Colosio; Sarah Birindelli; Vito Foà (251-258).
Ethylenethiourea (ETU) is a major metabolite of ethylenebisdithiocarbamate pesticides: a sensitive and specific assay for its determination in human urine is proposed below. ETU is extracted on a diatomaceous earth column using dichloromethane and derivatized with the mixture of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide and tert-butyldimethyilsilyl chloride. The derivative is analyzed using GC/MS in the EI/SIM mode. The whole procedure is carried out in the presence of ethylenethiourea-d4 as internal standard. The analytical features of the method are: high specificity, >90% recovery, range of linearity 0–200 μg/L, within- and between-run precision as coefficient of variation, <17 and <20%, respectively, limit of quantification 2 μg/L. In specimens stored in the dark at −20 °C ETU is stable for at least 6 months. The procedure was successfully applied to the biological monitoring of vineyard workers exposed to EBDTC and of a matched group of subjects from the general population.
Keywords: Urinary ethylenethiourea; Biological monitoring; Occupational exposure;

Determination of Rhodamine 123 in cell lysate by HPLC with visible wavelength detection by Tahira Iqbal; Minori Kinjo; Thomas C. Dowling (259-262).
Rhodamine 123 (R123) is widely used to quantify P-glycoprotein (P-GP) functional efflux activity in vitro. We developed a rapid and specific high-performance liquid chromatography (HPLC) method to quantify Rhodamine 123 for use in experimental cell culture studies. The R123 standards (2.5–250 ng/mL) and quality controls (QCs) (5, 75, 200 ng/mL) were prepared in cell lysis buffer consisting of 0.75% Triton 100X and 0.2% sodium chloride. The mobile phase consisted of acetonitrile, 1.5 mM tetrabutyl ammonium bromide in 20 mM sodium acetate buffer (pH 4.0) (50:20:30) delivered at a rate of 1.0 mL/min. Samples (50 μL) were injected onto a C18 reversed-phase HPLC column with detection at 500 nm. Analyte retention times were 1.4 and 4.3 min for R123 and internal standard (R6G), respectively. Intra- and inter-day coefficients of variation were ≤4.2%. Samples were stable for at least three freeze-thaw cycles at room temperature for 24 and 48 h. This method was used to evaluate the functional activity of P-glycoprotein in renal tubule cell models including human kidney (HK-2), Madin–Darby canine kidney (MDCK) and multi-drug resistance gene-transfected MDCK cells (MDR1-MDCK).
Keywords: P-glycoprotein; Rhodamine 123; High-performance liquid chromatography (HPLC);

A sensitive and selective method based on liquid chromatography-tandem mass spectrometry (LC–MS/MS) has been developed for the quantitative determination of loperamide in human plasma.Automated solid-phase extraction (SPE) on disposable extraction cartridges (DEC) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC–MS/MS system. After conditioning, the plasma sample is loaded on the DEC filled with endcapped ethyl silica (C2EC) and washed twice with water. The analytes are therefore eluted by dispensing methanol. The eluate is then collected and added with ammonium acetate solution in order to inject an aliquot of this final extract in the LC–MS/MS system.On-line LC–MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of loperamide. The separation is obtained on a octadecylsilica based stationary phase using a mobile phase consisting in a mixture of methanol and 5 mM ammonium acetate solution (25:75, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/z 477 → 266 and 316 → 270 for loperamide and clonazepam, respectively. The most appropriate regression model of the response function as well as the limit of quantitation were first selected during the pre-validation step. These latter criteria were then assessed during the formal validation step. The limit of quantitation (LOQ) was around 50 pg/ml for loperamide. The method was also validated with respect to recovery, precision, trueness, accuracy and linearity.
Keywords: Loperamide; Pharmacokinetics; Validation;

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography by H.M. Liebich; S. Müller-Hagedorn; M. Bacher; H.-G. Scheel-Walter; X. Lu; A. Frickenschmidt; B. Kammerer; K.-R. Kim; H. Gérard (275-283).
Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b 0 + b 1 (1/x) with the regression coefficients b 0 and b 1, the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1–2 months, when they reach their absolute maximum for all age periods, and then decrease.
Keywords: Nucleosides; Age dependence;

A new developed gas chromatographic–high resolution mass spectrometric method for the sensitive simultaneous determination of trans-chrysanthemumdicarboxylic acid, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine is presented. These metabolites are biomarkers for an exposure to pyrethrum, allethrin, resmethrin, phenothrin, tetramethrin, cyfluthrin, cypermethrin, deltamethrin or permethrin. Therefore, with the help of this method for the first time a complete assessment of exposure to pyrethroid and pyrethrin insecticides is possible. After acid hydrolysis and extraction with tert-butyl-methyl-ether the residue is derivatized with 1,1,1,3,3,3-hexafluoroisopropanol and analyzed by GC/HRMS in electron impact mode (detection limits < 0.1 μg/l) as well as in negative chemical ionization mode (detection limit < 0.05 μg/l urine).
Keywords: Biomonitoring; Pyrethroids; Pyrethrum; Urine; GC/HRMS; Derivatization;

A sensitive and reliable method for the determination of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in human plasma and cerebrospinal fluid (CSF) has been developed. The method is based on capillary liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using deuterium-labeled GABA (γ-aminobutyric acid-2,2-D2, GABA-d2) as internal standard. Pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) was deployed, allowing both effective in-line pre-concentration and sensitive tandem MS detection of the analyte. An extraction column (10 mm × 0.25 mm, 7 μm, C18) was used for preconcentrating and stacking the sample. Separation was carried out on an analytical column (50 mm × 0.25 mm, 5 μm, C18). Characteristic precursor-to-product ion transitions, m/z 267 → 249 (for NBD-GABA) and m/z 269 → 251 (for NBD-GABA-d2) were monitered for the quantification. A linear calibration curve from 10 to 250 ng/mL GABA with an r 2 value of 0.9994 was obtained. Detection limit was estimated to be 5.00 ng/mL GABA (S/N = 3). Human plasma and CSF samples were analyzed. The concentrations of GABA were found to be 98.6 ± 33.9 ng/mL (mean ± S.D., n  = 12), and 44.3 ± 10.0 ng/mL (n  = 6) in plasma and CSF, respectively.
Keywords: γ-Aminobutyric acid; GABA; Capillary liquid chromatography/tandem mass spectrometry; Cerebral spinal fluid; Plasma;

An analytical method for simultaneous determination of benazepril and its active metabolite, benazeprilat, in human plasma by high-performance liquid chromatography/electrospray-mass spectrometry was developed and validated. Rutaecarpine was selected as the internal standard. The separation was achieved on a C18 column with acetonitrile and aqueous solution (0.1% formic acid) as mobile phase with a gradient mode. The quantification of target compounds was using a selective ionization recording at m/z 425.5 for benazepril, m/z 397.5 for benzeprilat and m/z 288.3 for rutaecarpine. The correlation coefficients of the calibration curves were better than 0.992 (n  = 6), in the range of 6.67–666.67 ng/ml for benazepril and benazeprilat. The inter- and intra-day accuracy, precision, linear range had been investigated in detail. The method can be used to assess the bioavailability and pharmacokinetics of the drug.
Keywords: Benazepril; Benazeprilat; Rutaecarpine; HPLC–ESI-MS;

Micropipette-tip solid phase extraction (SPE) systems are common in proteomic analyses for desalting and concentrating samples for mass spectrometry, removing interferences, and increasing sensitivity. These systems are inexpensive, disposable, and highly efficient. Here, we show micropipette-tip solid phase extraction is a direct sample preparation method for 14C-accelerator mass spectrometry (AMS), removing salts or reagent from labeled macromolecules. We compared loading, recovery and desalting efficiency in commercially available SPE micro-tips using 14C-labeled peptides and proteins, AMS, and alpha spectrometry ion energy loss quantitation. The polypropylene in the tips was nearly 14C-free and simultaneously provided low-background carrier for AMS. The silica material did not interfere with the analysis. Alpha spectrometry provided an absolute measurement of desalting efficiency.
Keywords: Solid phase extraction; Quantitation; Recovery; Peptides; Proteins; Accelerator mass spectrometry;

Racemic methadone (MET) is administered to heroin users undergoing methadone maintenance therapy (MMT) in Australia. The enantiomers of methadone possess different pharmacological effects, and the enantioselective metabolism of methadone to its two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) has been demonstrated. Therefore, a stereoselective method capable of quantifying methadone, EDDP and EMDP in biological samples could be of benefit in the monitoring of MMT patients. In particular, the analysis of hair samples would provide a means by which long-term monitoring of MMT patients could be achieved. To date, no HPLC method has been published for the simultaneous separation of the six enantiomers. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the chiral analysis of methadone, EDDP and EMDP was developed using an α-glycoprotein (AGP) stationary phase. The method development involved the utilisation of factorial analysis experimental designs and the application of artificial neural networks (ANNs) to model the chromatographic response surfaces. The optimal conditions were determined to be 20 mM acetic acid: isopropanol (93:7, pH 7.4), with a flow rate of 0.9 mL/min. The method was validated and subsequently applied to the analysis of 20 hair samples collected from MMT patients.
Keywords: Methadone; EDDP; EMDP; Chiral; α-Glycoprotein; AGP; LC–MS; Artificial neural networks; Hair;

A sensitive reversed phase HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of the hydrophobic surfactant protein B (SP-B) in human bronchoalveolar lavage fluid. Samples were extracted two times with CHCl3:MeOH:HCl (2:3:0.005N) solution in a ratio of 1:2 by volume. The extract of the lower phase was separated on a C4 butyl silica gel column with an isocratic elution using a mobile phase, consisting of 97% methanol, 2.75% chloroform and 0.25% 0.1 M trifluoroacetic acid (by volume), at a flow rate of 1 ml/min. SP-B was detected by ELSD and quantified by comparison to an external standard. The duration of a run was 7 min, the quantification limit 30 ng and the limit of detection was at about 15 ng of SP-B. This method is suitable for the rapid routine quantification of SP-B in human bronchoalveolar lavage fluid samples.
Keywords: Surfactant protein B; Lipid extraction;

In this paper, p-toluenesulfonyl isocyanate has been reported as a novel derivatization reagent with strong nuclephilic reactivity for the hydroxyl compounds. The derivatization for the two pharmacologically active 3-hydroxyl metabolites, 3α-hydroxyl-7-methyl-norethynodrel and 3β-hydroxyl-7-methyl-norethynodrel by p-toluenesulfonyl isocyanate can be accomplished in 2 min under room temperature. The offline derivatization procedure introduced an easily ionizable sulfonylcarbamic ester moiety to the metabolites. This greatly improved the analyte's sensitivity in negative electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 100 pg/ml in plasma. Therefore, a sensitive high performance liquid chromatography-mass spectrometry (HPLC–MS) method for the analysis of the two stereo isomers was developed. The method had been validated to be accurate, precise, and sensitive, and can be used for the metabolism pharmacokinetic study of tibolone in human subjects.
Keywords: p-Toluenesulfonyl isocyanate; Derivatization;

A rapid, accurate and reproducible assay utilising high performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at −80 °C. Samples were reconstituted in 60% acetonitrile for LC–MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm × 150 mm i.d., 5 μm particle size, 300 Å pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y  = 0.01x  + 0.0045, r 2  = 0.959, r  = 0.979, p  < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 ± 7.9% (50 ng/ml) and 91.5 ± 3.72% (1 ng/ml), and for blow was 83.3 ± 6.8% (50 ng/ml) and 85.8 ± 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 ± 14.2% and in blow was 78.63 ± 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73–23 ng/ml, n  = 10) and blow (14.71–86.20 ng/ml, n  = 11) samples of captive bottlenose dolphins.
Keywords: Liquid chromatography; Mass spectrometry; Dolphin; Testosterone; Saliva; Blow exudate;

In this study, the effect of several factors that govern the partitioning behaviour of three model proteins, such as bovine serum albumin, lysozyme and trypsin was analysed in a two-phase system formed by maltodextrin and a copolymer of ethylene and propylene oxides. The protein partition coefficient (K r) showed to be very sensitive to temperature changes, protein molecular weight, pH medium and the lyotropic ion presence. The phase diagram obtained for these novel polymer–polymer two-phase systems shows two phases with high polymer concentrations. The maltodextrin is enriched in the bottom phase while the copolymer of ethylene and propylene oxides is found in the upper phase. Since this copolymer is thermoreactive, the upper phase can be removed and heated above the copolymer's cloud point resulting in the formation of a new two-phase system with a lower water phase, containing the target protein and an upper copolymer-rich phase. Our results show that systems formed by maltodextrin and a copolymer of ethylene and propylene oxides may be considered as an interesting alternative to be used in protein purification due to their low cost, and also because they offer a viable solution to problems of polymer removal and recycling.
Keywords: Ethylene oxide propylene oxide random copolymer; Maltodextrin; Temperature-induced two-phase system; Partitioning;

Determination of total phthalates in urine by isotope-dilution liquid chromatography–tandem mass spectrometry by Kayoko Kato; Manori J. Silva; Larry L. Needham; Antonia M. Calafat (355-360).
Diesters of 1,2-benzenedicarboxylic acid are a family of industrial compounds called “phthalates.” The physical and chemical properties of these diesters, and therefore their potential uses, depend on the structure of the dialkyl or alkyl/aryl side chain. The urinary concentrations of phthalate monoesters, which are metabolites, have been used as biomarkers of human exposure to specific phthalates. However, several phthalates, particularly those with side chains of eight or more carbon atoms, are complex mixtures of isomers. For these, the phthalate metabolites to be used as biomarkers of exposure have not been unequivocally identified. We developed a method for assessing total exposure to phthalates, including the isomeric mixtures of high molecular weight phthalates, by measuring the concentration of phthalic acid (PA) in human urine after acid hydrolysis of the phthalate metabolites to PA. The present method accurately assesses total exposure to phthalates without noticeable contamination from the ubiquitous phthalates in the environment, but it gives no information about the parent phthalate.
Keywords: Phthalic acid; Acid hydrolysis; Phthalates; Total exposure; Body burden;

Voriconazole is a novel broad-spectrum antifungal agent. We developed an on-line LC–LC–MS–MS method for fully automated and direct analysis of voriconazole in raw human serum. After injection of human serum size-selective sample fractionation and analyte extraction was achieved using an extraction column (25 mm × 4 mm) packed with a restricted access material (RAM, LiChrospher® ADS C8, 25 μm). On-line transfer of voriconazole from the extraction column was followed by chromatography separation on a C18 column. Detection was done by ESI-MS–MS. The total analysis time was 13 min, managed by parallel extraction and chromatographic separation. This LC–MS assay was fully validated. The lower limit of quantification was 0.05 μg/ml. The automated inline extraction of voriconazole described here eliminates the need for difficult and time-consuming sample pre-treatment. Other advantages of the new method are that only a small quantity (5 μl) of serum is needed and that the method is very specific.
Keywords: Voriconazole; Column-switching; LC–LC–ESI-MS–MS; Serum; On-line sample preparation;

This paper details a validated liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI–MS/MS) method for the quantification of methadone, and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) and methadol in human meconium. Limits of detection (LOD) were determined to be 1.0 ng/g for methadone, EDDP and EMDP and 2.5 ng/g for methadol. The limits of quantitation (LOQ) for methadone, EDDP, EMDP were 5 and 25 ng/g for methadol. Linearity ranged from 5.0 to 500 ng/g. Following solid-phase extraction, no matrix effect was observed. This method proved to be suitable for the quantification of methadone, EDDP and EMDP and the semi-quantitation of methadol in meconium. Literature review revealed no other published LC–APCI–MS/MS method for the detection of methadone and its three main metabolites in meconium specimens.
Keywords: Methadone; Meconium; LC–APCI–MS/MS;

A technique for determination of drug–protein binding based on a membrane extraction technique termed “equilibrium sampling through membrane (ESTM)” is presented. It involves the establishment of an equilibrium between an aqueous buffer and either a blood plasma sample or a matched buffer, both containing the drug. Analysis of the aqueous buffer in the two cases gives the drug–protein binding. The principle bypasses some sources of systematic error found with common techniques for this measurement based on e.g. ultrafiltration, as it senses the equilibrium conditions without disturbing the sample. The technique is applied to some local anesthetic drugs as model substances and two alternative ways for the evaluation are presented. Results with these evaluation methods are compared with literature values for the drug–protein binding of these compounds. It is found that the drug–protein binding values obtained are lower than literature values, which is attributed to reduced systematic error.
Keywords: Drug–protein binding; Local anesthetics; Supported liquid membrane extraction; Equilibrium extraction; Equilibrium sampling through membrane (ESTM);

Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis by Hua He; Cheng Sun; Xiao-Rong Wang; Chuong Pham-Huy; Nassima Chikhi-Chorfi; Hervé Galons; Marc Thevenin; Jean-Roger Claude; Jean-Michel Warnet (385-391).
The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction.After addition of serum or urine samples, these two cartridges were washed with a water–methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95–102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6–7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.
Keywords: Polymeric extraction cartridges; Methadone; EDDP; Diazepam; Flunitrazepam; Nitrazepam; Oxazepam; Enantiomer separation;

Simultaneous determination of l- and d-lactic acid in plasma by capillary electrophoresis by Li Tan; Yu Wang; Xiaoquan Liu; Huangxian Ju; Jieshou Li (393-398).
A novel method for simultaneous determination of d- and l-lactic acids in plasma was presented by capillary electrophoresis with photodiode array detection at 195 nm. The separation was performed in an uncoated fused-silica capillary. The parameters influencing the resolution and the migration time of lactic acids were optimized. When 150 mM phosphate–Tris buffer (pH 7.0) consisting of 220 mM 2-hydroxypropyl-β-cyclodextrin and 0.2 mM tetradecyltrimethylammonium bromide was utilized as the running buffer, highly effective chiral separation of d- and l-lactic acids was achieved at about 42 min at an effective voltage of −25 kV. The resolution of lactic acid enantiomers was ≥1.25. The limits of detection of d- and l-lactic acids in standard solution without any pretreatment were 80 and 50 μM (S/N = 3), respectively. Sample pretreatment was preceded by protein-removal procedure with acetonitrile. With a pre-concentration procedure by 10 times, the limits of detection of d- and l-lactic acids were 20 and 15 μM (S/N = 10), respectively. The satisfactory analytical performance of the proposed method was validated.
Keywords: d-Lactic acid; l-Lactic acid; Capillary electrophoresis; Plasma; Simultaneous determination;