Journal of Chromatography B (v.813, #1-2)

News Section (N1-N4).

Proteomics, the analysis of the protein complement of a cell or an organism, has grown rapidly as a subdiscipline of the life sciences. Mass spectrometry (MS) is one of the central detection techniques in proteome analysis, yet it has to rely on prior sample preparation steps that reduce the enormous complexity of the protein mixtures obtained from biological systems. For that reason, a number of so-called tagging (or labeling) strategies have been developed that target specific amino acid residues or post-translational modifications, enabling the enrichment of subfractions via affinity clean-up, resulting in the identification of an ever increasing number of proteins. In addition, the attachment of stable-isotope-labeled tags now allows the relative quantitation of protein levels of two samples, e.g. those representing different cell states, which is of great significance for drug discovery and molecular biology. Finally, tagging schemes also serve to facilitate interpretation of MS/MS spectra, therefore assisting in de novo elucidation of protein sequences and automated database searching. This review summarizes the different application fields for tagging strategies for today's MS-based proteome analysis. Advantages and drawbacks of the numerous strategies that have appeared in the literature in the last years are highlighted, and an outlook on emerging tagging techniques is given.
Keywords: Affinity tags; Isotopic labeling; Mass spectrometry; Proteomics;

Simultaneous determination of hypericin and hyperforin in human plasma with liquid chromatography–tandem mass spectrometry by Klaus-Dieter Riedel; Karin Rieger; Meret Martin-Facklam; Gerd Mikus; Walter E. Haefeli; Jürgen Burhenne (27-33).
A selective and sensitive method for the simultaneous determination of hypericin and hyperforin—the two main active ingredients of St. John's Wort (SJW) extract—in human plasma depending on liquid/liquid-extraction and LC/MS/MS detection has been developed, validated after specifying the stability of the photosensitive hypericin in plasma samples during light exposure and applied to samples of a patient. After extraction with ethyl acetate/n-hexane in the darkness, sample extracts were chromatographed isocratically within 6 min on a Kromasil RP-18 column. The analytes were detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source. The limit of quantification was 0.05 ng/mL for hypericin and 0.035 ng/mL for hyperforin. The accuracy of the method varied between 101.9 and 114.2% and the precision ranged from 4.7 to 15.4% (S.D., batch-to-batch) for both analytes. The method was linear at least between 0.05 and 10 ng/mL for hypericin and between 0.035 and 100 ng/mL for hyperforin. Using this method hypericin and hyperforin were determined successfully in a patient over seven days following discontinuation of exposure with therapeutic doses of St. John's Wort extract.
Keywords: Hypericin; Hyperforin; Stability to light;

Multiresidue confirmation of β-agonists in bovine retina and liver using LC-ES/MS/MS by Lee D. Williams; Mona I. Churchwell; Daniel R. Doerge (35-45).
Misuse of numerous β-agonist drugs for their growth promoting effects in livestock production requires significant regulatory enforcement activities worldwide. The proof of illegal drug use needed for regulatory action usually requires the high degree of specificity derived from mass spectrometric analysis of suspect tissues and body fluids. In this paper, we describe a multiresidue screening method for confirmation of nine β-agonist compounds in bovine liver and retina. A wide range of analyte structures was selected in order to demonstrate applicability to other chemically related β-agonists for which standards are not currently available. The class-specific method, which is based on mixed mode cation exchange/reverse phase solid phase extraction, reverse phase gradient LC separation using a cyanopropyl-silica phase, and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode, yields high analyte recoveries at the target level of 1 ppb (ng/g). In addition, acquisition of multiple MRM transitions for each analyte permits simultaneous confirmation of β-agonists at the level of 1 ppb in liver and retina by using intensity ratios between fragment ions and protonated molecules. Estimated values for the limit of quantification (LOQ) for individual β-agonists were 0.08–0.3 ppb in liver and 0.02–0.5 in retina; the estimated limits of confirmation, using accepted criteria from international regulatory agencies, were 0.25–0.8 ppb in liver and 0.1–1 ppb in retina. This method should be useful in supporting regulatory enforcement programs that monitor β-agonist misuse.
Keywords: β-Agonists; Mass spectrometry; Retina; Clenbuterol; Ractopamine;

Hexanal and heptanal in human blood have been regarded as potential biomarkers of lung cancer. Owing to their high volatilities and activities, it is difficult to accurately measure the two biomarkers. In the current work, headspace solid-phase microextraction (HS-SPME) with on-fiber derivatization technique was developed for quantitative analysis of hexanal and heptanal in human blood. In the proposed method, the two aldehydes in blood were headspace extracted by using a poly (dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber with O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine (PFBHA) at 60 °C for 8 min. The aldehyde oximes formed on the fiber were desorbed and analyzed by gas chromatography–mass spectrometry (GC–MS). The method validations including detection limit, recovery and precision were studied. It was found that the method provided low detection limits of 0.006 nM for hexanal and 0.005 nM for heptanal, recoveries from 89% to 95% and R.S.D. values less than 8.5%. The present method was applied to quantitative analysis of hexanal and heptanal in normal blood and lung cancer blood. Hexanal concentrations from 7.33 to 15.23 μM and heptanal concentrations from 2.47 to 9.23 μM were found in the lung cancer blood, while both hexanal and heptanal in the control blood were lower than 0.6 μM. This further demonstrated that hexanal and heptanal might be the biomarkers of lung cancer. The experimental results showed that GC–MS and HS-SPME with on-fiber derivatization is a simple, rapid, sensitive and solvent-free method for determination of in hexanal and heptanal human blood.
Keywords: Headspace solid-phase microextraction; On-fiber derivatization; Hexanal; Heptanal; Blood; Lung cancer;

Discrimination of Type 2 diabetic patients from healthy controls by using metabonomics method based on their serum fatty acid profiles by Jun Yang; Guowang Xu; Qunfa Hong; Hartmut M. Liebich; Katja Lutz; R.-M. Schmülling; Hans Günther Wahl (53-58).
Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields, including work in cardiovascular research and drug toxicology. In this study, metabonomics method was employed to the diagnosis of Type 2 diabetes mellitus (DM2) based on serum lipid metabolites. The results suggested that serum fatty acid profiles determined by capillary gas chromatography combined with pattern recognition analysis of the data might provide an effective approach to the discrimination of Type 2 diabetic patients from healthy controls. And the applications of pattern recognition methods have improved the sensitivity and specificity greatly.
Keywords: Pattern recognition; Diabetes mellitus; Fatty acid profiles; Linear discriminant analysis; Artificial neural networks; Metabonomics;

Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases by Jun Yang; Guowang Xu; Yufang Zheng; Hongwei Kong; Tao Pang; Shen Lv; Qing Yang (59-65).
Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields. Current metabonomics practice has relied on mass spectrometry (MS), gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR) to analyze metabolites. In this study, a novel approach of using high-performance liquid chromatography (HPLC) in conjunction with developed software was employed. Using the principal components analysis method (PCA), all (113) peaks of urinary metabolites with a cis-diol structure from patients with hepatitis and hepatocirrhosis were compared to those from liver cancer patients. The results showed that the metabonomics-PCA method might be useful to differentiate between patients with hepatocirrhosis and hepatitis from patients with liver cancer while lowering false-positive rate. These findings also suggest that a subset of the urinary nucleosides identified with metabonomics correlate better with cancer diagnosis than the traditional single tumor marker alpha-fetoprotein (AFP).
Keywords: Metabonomics; HPLC; Nucleosides; Methodological studies; Molecular diagnosis and prognosis;

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C18, have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH2 (TAPP), H-Tyr-(D)Ala-Phe-NH2 (TAP), H-Phe-Phe-NH2 (PP) and H-Phe-NH2 (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H2O as BGE, operating at 20 °C and 20 kV high voltage. For the C18 stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 °C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.
Keywords: Capillary electrochromatography; Peptides; Experimental design;

We developed a method for determining ebastine, a new generation of antihistamines, and its three metabolites (hydroxyebastine, carebastine and desalkylebastine) in plasma simultaneously using LC/MS/MS. Four compounds and terfenadine, an internal standard, were extracted from plasma using a mixture of diethylether and dichloromethane in the presence of 1 M HCl. After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:5 mM ammonium acetate, 50:50, v/v) and injected onto a reversed-phase C18 column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 470.7 → 167.1, 486.7 → 167.1, 500.6 → 167.1, 268.4 → 167.1 and 472.7 → 436.0 for ebastine, hydroxyebastine, carebastine, desalkylebastine and terfenadine, respectively. The coefficient of variation of the assay precision was less than 12.5%, and the accuracy exceeded 88%. The limit of detection was 0.5 ng/ml for desalkylebastine; 0.2 ng/ml for ebastine, hydroxyebastine and carebastine, respectively. This method was used to measure the plasma concentration of ebastine and its three metabolites from healthy subjects after a single 20 mg oral dose of ebastine. This analytic method is a very simple, sensitive, and accurate to determine the pharmacokinetic profiles of ebastine including its metabolites.
Keywords: Ebastine; Desalkylebastine; Hydroxyebastine; Carebastine; LC/MS/MS;

Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography by Ming Zhao; Michelle A. Rudek; Ping He; Carol Hartke; Steve Gore; Michael A. Carducci; Sharyn D. Baker (81-88).
5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80™ C18 column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 ± 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9 → 113.0 for 5AC and m/z+ 242.0 → 126.0 for 5-methyl-2′-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.
Keywords: 5-Azacytidine; LC/MS/MS; Pharmacokinetics;

A derivatization–extraction method that avoids tedious preconcentration steps is established in order to determine amino acids accurately at nanogram levels. The method involves conversion of the analytes of concern to N(O,S)-ethoxycarbonyl amino acid ethyl esters and subsequent extraction by single-drop microextraction (SDME) followed by GC analysis. The reaction proceeds smoothly and rapidly under ultrasonication which removes the bubbles from the bulk solution. Precision is acceptable and 12 non-hydrolyzed amino acids can be determined in urine in this manner. As long as the extraction conditions are consistently applied, quantitative analysis can be performed accurately. The limits of detection were satisfactory in the range 0.010–0.025 μg/ml for GC–FID and 0.26–68 ng/ml for GC–MS(SIM) with 1 ml sample volume.
Keywords: Amino acids; Ethylchloroformate; Derivatization; Urine; Single-drop microextraction; GC;

Determination of MK-0767 enantiomers in human plasma by normal phase LC–MS/MS by Kerri X. Yan; Hengchang Song; Man-Wai Lo (95-102).
A sensitive and selective analytical method for the enantioselective determination of MK-0767, a dual peroxisome proliferator-activated receptor (PPAR) α/γ agonist, in human plasma has been developed and validated. The chromatography is based on normal-phase chiral separation on a Kromasil, 5 μm, CHI-DMB 250 mm × 4.6 mm column. The detection involves the direct introduction of the normal phase eluent into MS/MS without the addition of a post-column reagent. Atmospheric pressure chemical ionization (APcI) mode was selected as the ion source in this method. With proper sample handling and processing procedures, ex vivo interconversion of the enantiomers was kept to minimum during sample collection, preparation and short term storage of frozen human plasma samples. The method was successfully utilized to determine the concentrations of MK-0767 enantiomers in human plasma to support pharmacokinetic investigation in man.
Keywords: Chiral separation; MK-0767 (KRP-297); LC–MS/MS; Liquid–liquid extraction; Normal phase; Dual PPAR; Diabetes; Thiazolidinediones;

Quantification of residual EDU (N-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) hydrolyzed urea derivative) and other residual by LC–MS/MS by Q. Paula Lei; David H. Lamb; Anthony G. Shannon; Xinxing Cai; Ronald K. Heller; Michael Huang; Earl Zablackis; Robert Ryall; Patricia Cash (103-112).
An LC–MS/MS method for determination of the break down product of N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) urea derivative, EDU, has been developed and validated for monitoring the residual coupling reagents. Results indicate that the method exhibits suitable specificity, sensitivity, precision, linearity and accuracy for quantification of residual EDU in the presence of meningococcal polysaccharide-diphtheria toxoid conjugate vaccine and other vaccine matrix compounds. The assay has been validated for a detection range of 10–100 ng/mL and then successfully transferred to quality control (QC) lab. This same method has also been applied to the determination of residual diaminohexane (DAH) in the presence of EDU. LC–MS/MS has proven to be useful as a quick and sensitive approach for simultaneous determination of multiple residual compounds in glycoconjugate vaccine samples.
Keywords: LC–MS; Quantification; Validation; Assay transfer; Polysaccharide protein conjugate vaccine; Residual material;

Two step procedure for purification of enzymatically active prostate-specific antigen from seminal plasma by B. Bindukumar; Elzbieta Kawinski; Craig Cherrin; Leah M. Gambino; Madhavan P.N. Nair; Stanley A. Schwartz; Kailash C. Chadha (113-120).
The role of prostate-specific antigen (PSA) during the onset of prostate cancer and subsequent tumor growth and metastasis is not well understood. We have developed a simple two step procedure, based on principles of hydrophobic charge-induction chromatography and molecular size chromatography to provide pure free-PSA (f-PSA) preparation that is free from all other known PSA complexes as well as human kallikrein 2 (hK2). The overall recovery of f-PSA is 72%. The isolated f-PSA consists of three known isoforms that corresponds to pI of 6.2, 6.4 and 7.2. f-PSA is enzymatically active and its enzymatic activity can be effectively neutralized by a serine protease inhibitor.
Keywords: Prostate cancer; Prostate-specific antigen; PSA; Serine protease; Thiophilic-interaction chromatography; PSA purification;

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC–ESI mass spectrometry by Kyung-Hwan Yoon; So-Young Lee; Won Kim; Jong-Sei Park; Hie-Joon Kim (121-127).
A simple, fast and sensitive high-performance liquid chromatography (HPLC)–mass spectrometric (MS) method has been developed for simultaneous determination of amoxicillin and clavulanic acid in human plasma using terbutaline as internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C8 reversed-phase column with formic acid–water–acetonirile (2:1000:100) and detected using electrospray ionization (ESI) mass spectrometry in negative selected ion monitoring (SIM) mode. The method was validated and successfully applied to analysis of amoxicillin and clavulanic acid in clinical studies. The limit of quantitation, 0.12 μg/ml for amoxicillin and 0.062 μg/ml for clavulanic acid, was five times lower than that of the published HPLC–UV method.
Keywords: Amoxicillin; Clavulanic acid; Electrospray ionization LC–MS;

The need for on-line sample preparation for high-throughput applications in bioanalysis has increased during the past decade. In this paper a robust and on-line sample preparation technique, micro extraction in packed syringe (MEPS) has been developed and validated. The method is a miniaturized, fully automated, solid-phase extraction (SPE) technique that can be connected on-line to GC or LC without any modification of the chromatographs. The performance of MEPS as sample preparation method is illustrated by the determination of local anaesthetics in human plasma samples on-line with high performance liquid chromatography (HPLC) and tandem mass spectrometry. The sampling sorbent was 1 mg silica based benzenesulphonic acid cation exchanger that was inserted in a 250 μl syringe. Ropicavine and two of its metabolites (PPX and 3-OH-ropivacine), lidocaine and bupivacine were used as model substances. The accuracy values of quality control samples (QC) were between 95% and 109%, and precision (relative standard deviation, R.S.D.) had a maximum deviation of 9% for the analytes.
Keywords: Microextraction in packed syringe; Sample preparation; Cation exchange sorbent; Local anaesthetics; Fully automated; High throughput; HPLC/MS/MS;

A new molecularly imprinted polymer for the selective extraction of naproxen from urine samples by solid-phase extraction by Ester Caro; Rosa M. Marcé; Peter A.G. Cormack; David C. Sherrington; Francesc Borrull (137-143).
A non-covalent molecularly imprinted polymer (MIP) was synthesised using naproxen (a non-steroidal, anti-inflammatory drug (NSAID)) as a template molecule. The MIP was chromatographically evaluated to confirm the imprinting effect, and was then applied as a selective sorbent in solid-phase extraction (SPE) to selectively extract naproxen. After this study, the MIP was used to extract naproxen from urine samples; it was demonstrated that by applying a selective washing step with acetonitrile (ACN) the compounds in the sample that were structurally related to naproxen could be eliminated.
Keywords: Molecularly imprinted polymer; Solid-phase extraction; Naproxen; Human urine samples;

Determination of linezolid in plasma and bronchoalveolar lavage by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method by Jérôme Toutain; Emmanuel Boselli; Sarah Djabarouti; Bernard Allaouchiche; Fabien Xuereb; Jean-Marc Bernadou; Boubakar Ba; Marie-Claude Saux; Dominique Breilh (145-150).
The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic assay for the determination of linezolid in human plasma, and bronchoalveolar lavage. The sample extraction was based on a fully automated solid-phase extraction with an OASIS HLB cartridge. The method used ultraviolet detection set at a wavelength of 254 nm and a separation with a Zorbax Eclipse XDB C8 column. The assay has been found linear over the concentration range 0.02–30 μg/ml and 0.04–30 μg/ml for linezolid, respectively, in plasma and bronchoalveolar lavage. It provided good validation data for accuracy and precision (CV <4.64% and 5.08%, accuracy in the range 96.93–102.67% and 97.33–105.67%, respectively, for intra- and inter-day). The assay will be applied to determine the penetration of linezolid in human bronchoalveolar lavage during pharmacokinetic steady-state.
Keywords: Linezolid; Oxazolidinones; Pharmacokinetics;

Microwave-assisted derivatization of 2,5-hexanedione in urine: evaluation using GC–MS and GC–ECD by Steffen Strassnig; Marion Gfrerer; Ernst P. Lankmayr (151-158).
2,5-Hexanedione, the main metabolite of n-hexane, can be responsible for axonal degeneration symptoms via formation of pyrrol-adducts with several amino acids. In order to make it amenable to gas chromatographic analysis, a protocol including microwave assisted derivatization is presented and compared to state-of-the-art technique of urine analysis. The applied methodology includes derivatization with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine, extraction of the oximes and final analysis using either GC–MS or GC–μECD. Furthermore, the mass spectra of derivatized 2,5-hexanedione and 5-hydroxy-2-hexanone as well as preliminary excretion kinetics are provided. Orthogonal regression methodology demonstrated superior sensitivity for the microwave heating. Limits of detection were calculated to be approximately 20 ng mL−1 with both MS and electron capture detection, the decompositon of excess derivatizing agent using sulfuric acid, following the reaction is beneficial. A matrix effect caused by urine was not observed, a calibration in aqueous matrix ensures accurate results therefore. Microwave heating yields excellent results regarding recovery, sensitivity and the time needed for sample preparation, furthermore, it is demonstrated that both mass selective as well as electron capture detection are of comparable suitability for this task.
Keywords: 2,5-Hexanedione; Microwave-assisted derivatization; Urine; GC–MS; GC–μECD;

Since we recently noticed poor recoveries of unsaturated fatty acids (UFA) when the parent lipids were first separated on TLC plates, we investigated the source of this error by examining several variables, including the brand of TLC plate, nature of the lipid, and conditions of methylation. Of the five commercial brands of plates used, two (Baker and Whatman) showed loss of UFA, and three (Alltech Hardlayer, Alltech Softlayer, and Merck) did not. This loss occurred in both neutral and phospholipids, did not affect saturated acids, and was independent of the methylation reagent used. No loss occurred, however, if the lipids were eluted from the silica gel before methylation, indicating that the loss is due to oxidation of UFA in presence of certain brands of silica gel. These results show that some brands of TLC plates may be unsuitable for lipid analysis, if the aim is to determine the fatty acid composition by GC using direct methylation.
Keywords: Thin-layer chromatography; Commercial silica gel plates; Unsaturated fatty acids; Phospholipids; Cholesteryl esters; Free fatty acids; Methylation; Fatty acid methyl esters; Gas liquid chromatography; Oxidative degradation;

A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase® digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris–Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 × 108 and 1.57 × 109 viral particles/mL, respectively. Linearity was between 3.0 × 109 and 1.0 × 1011 viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 × 10−6. The assay precision was < 5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the β-galactosidase gene.
Keywords: Retroviral vector; Vesicular stomatitis virus-G glycoprotein;

A sensitive and specific high performance liquid chromatographic method for quantitation of topiramate in human serum was developed using HPLC with fluorescence labeling reagent. Topiramate was extracted from human serum by dichloromethane and derivatized by reaction with 9-fluorenylmethyl chloroformate (FMOC-Cl) in the presence of borate buffer. Analysis was performed on a CN column with sodium phosphate buffer (pH 2.2) containing 1 ml/l triethylamine and methanol (52:48 (v/v)) as mobile phase. Amantadine was used as internal standard. The standard curve was linear over the range 20–5000 ng/ml of topiramate in human serum. The mean intra-day precision was from 10.5% (low concentration) to 1.2% (high concentration) and the within-day precision from 1.5 to 12.5% determined on spiked samples. The accuracy of the method was 96.5–107.5% (intra-day) and 98.4–105% (inter-day). The limit of quantification was 20 ng/ml of serum. This method was used in a bioequivalence study after administration of 2 × 25 mg topiramate in 24 healthy volunteers.
Keywords: Reverse phase chromatography; HPLC; Topiramate; Serum; Bioequivalence study;

We present a method based on electrospray liquid chromatography tandem mass spectrometry (LC–MS/MS) for determining in muscle and eggs the following nine coccidiostats: halofuginone, diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, monensin, lasalocid, narasin, salinomycin, and maduramicin. Dinitrocarbanilide-d8, nigericin, and diclazuril-bis were used as internal standards. The method uses extraction in acetonitrile followed by a clean-up on an SiOH solid-phase extraction column. High-performance liquid chromatography (HPLC) separation was performed on a Purospher® C18 column (125 mm × 3 mm i.d.) protected by a guard column, the mobile phase being a water–acetonitrile gradient (each gradient component containing 0.1% formic acid) at a flow rate of 1 ml min−1. For unequivocal identification of each analyte, two ions were detected and chosen for multiple reaction monitoring (MRM). Validation was carried out on spiked muscle and egg samples. The method described meets all the criteria of Decision 2002/657/EC and is easy to use in routine analysis. Validation results are presented with the measured CCα and CCβ values. This whole method allows extraction and analysis of up to 24 samples per day.
Keywords: Coccidiostats; Polyether ionophores; Residues; Meat; Eggs; Mass spectrometry;

The performance of monolithic HPLC columns Chromolith™ (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma–Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases – for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) – was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0–5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0 ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.
Keywords: Monolithic columns; Ketoprofen; Estrogel;

A method for quantification of total faecal sterols and bile acids (BAs) in human stool by using gas chromatography–mass spectrometry–single ion monitoring (GC–MS–SIM) is described. Cholesterol, coprostanol, coprostanone, cholestanol, iso-lithocholic acid (iso-LCA), lithocholic acid (LCA), iso-deoxycholic acid (iso-DCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), cholic acid (CA), and 12-oxo-deoxycholic acid (12-oxo-DCA) in faeces of 86 healthy subjects were determined. The sample preparation for sterol analysis requires hydrolysis and liquid extraction from matrix, but no derivatisation. The GC-flame ionisation detection (FID) and total ion current (TIC) in GC–MS were not sufficient for sterol and BA determination, whereas selectivity and specificity of the GC–MS–SIM ensured the analysis of sterols and BAs in faeces.
Keywords: Sterols; Bile acids;

Simultaneous determination of the antiretroviral agents: amprenavir, lopinavir, ritonavir, saquinavir and efavirenz in human peripheral blood mononuclear cells by high-performance liquid chromatography–mass spectrometry by Agnès Rouzes; Karine Berthoin; Fabien Xuereb; Sarah Djabarouti; Isabelle Pellegrin; Jean Luc Pellegrin; Anne Cécile Coupet; Sylvie Augagneur; Hélène Budzinski; Marie Claude Saux; Dominique Breilh (209-216).
A selective and accurate assay for the simultaneous quantitation of four protease inhibitors (PIs) (amprenavir (APV), lopinavir (LPV), ritonavir (RTV) and saquinavir (SQV)) and a non-nucleoside reverse transcriptase inhibitor (NNRTI) (efavirenz, EFV) in human peripheral blood mononuclear cells using high-performance liquid chromatography–mass chromatography (LC/MS) has been developed and validated. After liquid–liquid extraction, the antiretroviral agents were separated within 15 min. The calibration curves of each drug showed a good linearity in a range of concentration between 2 and 200 ng/3 × 106 cells for amprenavir, lopinavir, efavirenz, 1.60 and 128 ng/3 × 106 cells for ritonavir and saquinavir. Mean intra- and inter-assay coefficients of variation over the ranges of the standard curves were less than 15% and mean extraction recoveries ranged 88.7–112.1%. The limits of quantification were 2 ng/3 × 106 cells for amprenavir, lopinavir, efavirenz, 1 ng/3 × 106 cells for ritonavir and 1.6 ng/3 × 106 cells for saquinavir. This novel LC/MS assay, which provides an excellent method for simultaneous intra-cellular determination of amprenavir, lopinavir, ritonavir, saquinavir and efavirenz in human peripheral blood mononuclear cells, could be successfully applied for therapeutic drug monitoring and pharmacokinetic studies.
Keywords: Intra-cellular quantitation; Protease inhibitors (PIs); Non-nucleoside reverse transcriptase inhibitor (NNRTI); LC/MS;

In order to establish a fast screening method for the determination of the CYP2D6 metabolic phenotype a sensitive LC–MS/MS assay to quantify dextromethorphan (DEX) and its O-demethylated metabolite dextrorphan (DOR) in human saliva was developed with limits of quantitation of 1 pmol/ml. Saliva was provided by 170 medical students 2 h after oral ingestion of 30 mg (81 μmol) dextromethorphan hydrobromide. Individual ratios of the concentrations DEX/DOR (metabolic ratio, MRDEX/DOR) varied more than 25,000-fold (0.03–780). Two groups comprising 156 ‘Extensive’ and 14 ‘Poor Metabolizers’ were clearly distinguished. For the investigation of individual differences in N-demethylation and glucuronidation, four additional metabolites of DEX, 3-methoxymorphinan (MOM), 3-hydroxymorphinan (HOM), and the two O-glucuronides (DORGlu and HOMGlu) were measured by LC–MS/MS analysis of 6-h urine of 24 volunteers. The N-demethylation reactions DEX-to-MOM and DOR-to-HOM defined by the respective MR were significantly correlated. The same holds for the glucuronidation pathways (MRDOR/DORGlu versus MRHOM/HOMGlu). The three poor CYP2D6 metabolizers excreted relatively high amounts of the parent compound DEX (up to 7 μmol), but only low amounts of glucuronides (DORGlu: 0.4–1.0 μmol; HOMGlu: 0.2–0.7 μmol). For the 21 ‘Extensive Metabolizers’, the two glucuronides were the most abundant, with relatively little interindividual variation (DORGlu: 10–44 μmol; HOMGlu: 5–17 μmol). For the excretion of the glucuronides, two normal distributions provided the best fit, indicating that the determination of the glucuronides alone could allow assignment of the CYP2D6 metabolic phenotype.
Keywords: Dextromethorphan; Metabolism; Phenotyping; CYP2D6; Monooxygenase; Demethylation; Glucuronosyltransferase; Individual; Statistics; Models;

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method has been developed and validated for the identification and quantification of zolmitriptan in human plasma. After the addition of the internal standard (IS) and 1.0 M sodium hydroxide solution, plasma samples were extracted with methylene chloride:ethyl acetate mixture (20:80, v/v). The organic layer was evaporated under a stream of nitrogen at 40 °C. The residue was reconstituted with 100 μl mobile phase. The compounds were separated on a prepacked Lichrospher CN (5 μm, 150 mm × 2.0 mm) column using a mixture of methanol:water (10 mM NH4AC, pH 4.0) = 78:22 as mobile phase. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.30–16.0 ng/ml with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (R.S.D.%) were lower than 15% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.30 ng/ml. The proposed method enables the unambiguous identification and quantification of zolmitriptan for pharmacokinetic, bioavailability or bioequivalence studies.
Keywords: Zolmitriptan; HPLC–ESI–MS; Human plasma; Pharmacokinetics;

On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the α3β4- and α4β2-nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases by Ruin Moaddel; Krzysztof Jozwiak; Rika Yamaguchi; Christopher Cobello; Kevin Whittington; Tarun K. Sarkar; Sankar Basak; Irving W. Wainer (235-240).
Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The α3β4 nAChR and α4β2 nAChR subtypes were used to prepare the chromatographic columns and [3H] epibatidine dihydrochloride ([3H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (K d  > 4700 nM for α3β4). The displacement of [3H] EB by each of the test compounds and internal controls was calculated and expressed as Δml. Functional studies were then conducted using a stably transfected cell line that expresses the α3β4 nAChR and EC50 values were determined for the test compounds and the internal controls. A comparison of the Δml and EC50 values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the α3β4 nAChR. A similar comparison could not be made for the α4β2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.
Keywords: On-line screening; Immobilized nicotinic receptors; Nicotinic agonists;

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography–mass spectrometry by Fan Pu; Andrew R. McKinney; Allen M. Stenhouse; Craig J. Suann; Malcolm D. McLeod (241-246).
A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS2 were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.
Keywords: Boldenone sulfate; Boldenone; Glucuronide;

Direct injection, column switching–liquid chromatographic technique for the estimation of rabeprazole in bioequivalence study by Sonu Sundd Singh; Manish Jain; Hiten Shah; Sapna Gupta; Purav Thakker; Ruchy Shah; Braj Bhushan Lohray (247-254).
A rapid, simple and sensitive high-performance liquid chromatography–ultra violet (HPLC–UV) method with column switching between sample pre-treatment column and analytical column was developed for the quantitation of rabeprazole in human plasma; on a Bio-Sample Analysis system (Co-sense® for BA) from Shimadzu Corporation, Kyoto, Japan. Zaleplon was used as an internal standard. The method was validated as per USFDA guidelines for the concentration range of 20.0–1200.0 ng/mL and the correlation coefficient were found to be better than 0.999. Recovery of rabeprazole as well as the internal standard from human plasma was more than 90.0%. Rabeprazole was stable in human plasma for 4 months at −70 ± 5 °C and for 20.0 h at ambient temperature. In the auto sampler, the drug was stable for 24.0 h at 4 °C. The method was specific as there were no interfering peaks in the human plasma eluting at the retention times of the rabeprazole and the internal standard. The frozen plasma samples containing rabeprazole were stable to three freeze thaw cycles. The bioanalytical method was rugged in terms of inter- and intra-day accuracy and precision. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of 20 mg rabeprazole tablet of German Remedies Ltd. (A division of Cadila Healthcare Ltd.), India versus Pariet tablet of Eisai Ltd. & Janssen-Cilag Ltd., Japan in male human subjects.
Keywords: Rabeprazole; Human plasma; Validation and bioequivalence;

Method for a direct determination of 8-hydroxy-2′-deoxyguanosine (8OHdG) in untreated urine samples by capillary electrophoresis with optical detection was developed. Optimisation of conditions resulted in a significant lowering of the limit of detection (LOD) by a factor of 400 as compared to our previous study. Optimum separation of 8OHdG from other urine components was achieved using the separation electrolyte containing 80 mM 2-(cyclohexylamino)ethanesulfonic acid, 9 mM LiOH (pH 8.6), and 0.1 mM cetyltrimethylammonium bromide ensuring the electro-osmotic flow inversion. In the model aqueous samples, these conditions allow separating 8OHdG and 2′-deoxyguanosine (dG) from other nucleosides/nucleotides including 2′-deoxycitidine 5′-monophosphate (dCMP), thymidine 5′-monophosphate (TMP), adenosine (A), and thymidine (T). On the other hand, 2′-deoxyadenosine 5′-monophosphate (dAMP) and 2′-deoxyguanosine 5′-monophosphate (dGMP) migrate together, and guanosine (G), 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC) are transported as neutral species with the electro-osmotic flow. In the spiked urine samples, 8OHdG and dG are well separated from each other and from other urine components and exhibit a linear calibration over the concentration range of 0.1–2.0 μM for 8OHdG (LOD = 42 nM) and 0.2–5.0 μM for dG (LOD = 86 nM), but urine metabolites interfere with the determination of dCMP, TMP, A and T. Method is applicable to untreated urine samples with slightly enhanced levels of 8OHdG compared to that found in healthy individuals.
Keywords: Capillary electrophoresis; 8-Hydroxy-2′-deoxyguanosine; Detection limit; Untreated urine;

A liquid chromatography–mass spectrometry method is described for the determination of tetramethylpyrazine (TMP) and its active metabolite, 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) in dog plasma. This method involves a plasma clean-up step using protein precipitation procedure followed by LC separation and positive electrospray ionization mass spectrometry detection (ESI-MS). Chromatographic separation of the analytes was achieved on a C18 column using a mobile phase of methanol, water and acetic acid (50:50:0.6, v/v/v) at a flow rate of 1.0 ml/min. Selected ion monitoring (SIM) mode was used for analyte quantitation at m/z 137.2 for TMP, m/z 153.2 for HTMP and m/z 195.2 for caffeine. The linearity was obtained over the concentration ranges of 20–6000 ng/ml for TMP and 20–4000 ng/ml for HTMP and the lower limit of quantitation was 20 ng/ml for both analytes. For each level of QC samples, both inter- and intra-day precisions (R.S.D.) were ≤7.4% for TMP and ≤6.0% for HTMP, and accuracy (R.E.) was ±6.0% for TMP and ≤3.5% for HTMP. The proposed LC–MS method was successfully applied to the pharmacokinetic studies of a TMP formulation preparation after oral administration to beagle dogs.
Keywords: Tetramethylpyrazine; Metabolite; Liquid chromatography–mass spectrometry; Dog plasma;

The separation of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether (PBDE) congeners was evaluated on four capillary columns: 60 m × 0.25 mm i.d., 0.25 μm film thickness RTX-5MS and DB-XLB capillary columns, and 60 m × 0.18 mm i.d., 0.25 μm film thickness DB-XLB and DB-5MS capillary columns. Based on performance, capacity, and cost, the RTX-5MS (60 m × 0.25 mm i.d., 0.25 μm thickness) and the DB-XLB (60 m × 0.25 mm i.d., 0.25 μm film thickness) were selected for the analysis of human serum extracts by using gas chromatography/electron-capture detection. In contrast to previous studies, the oven temperature program affords the separation of congeners that are not separated by using other combinations of capillary columns, most notably PBDE-47 and PCB 180. In addition, the method enables determination of OCPs, PCBs, and PBDEs prevalent in a single extract of serum, which can lead to considerable time savings in the analysis of large number of samples collected for epidemiologic studies.
Keywords: Polychlorinated biphenyls (PCBs); Organochlorine pesticides; Polybrominated diphenyl ethers (PBDEs); Persistent organic pollutants (POPs); Capillary column; GC-ECD;

Determination of total mycophenolic acid and its glucuronide metabolite using liquid chromatography with ultraviolet detection and unbound mycophenolic acid using tandem mass spectrometry by Chirag G. Patel; Anisha E. Mendonza; Fatemeh Akhlaghi; Oneeb Majid; Andrew K. Trull; Terry Lee; David W. Holt (287-294).
Two simple, sensitive and reproducible methods for determination of total mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) as well as unbound MPA (fMPA) was developed by the use of HPLC-UV and LC–MS/MS methods, respectively. For the total MPA/MPAG method, the analytes were extracted using Isolute C2 solid-phase extraction (SPE) cartridges and analyzed at 254 nm over a Zorbax Rx C8 column (150 mm × 4.6 mm, 5 μm). The mobile phase was a gradient mixture of methanol and water (containing 0.1% (v/v) phosphoric acid). The total run time was 18 min and the extraction recovery was 77% for MPA and 84% for MPAG. The method was precise and accurate with a lower limit of quantification (LLOQ) of 0.5 mg/l for MPA and 5.0 mg/l for MPAG. For the fMPA method, plasma was subjected to ultrafiltration followed by SPE using C18 cartridges. Analytical column was the same as the HPLC-UV method and the mobile phase was a gradient composition of methanol:0.05% formic acid with a flow rate of 0.6 ml/min for the first 3 min and 0.7 ml for the last 4 min. The chromatographic method separated MPA from its metabolites MPAG and Acyl-MPAG. Mass transitions in negative ionization mode for MPA and the internal standard, indomethacin were m/z: 319 → 190.9 and m/z: 356 → 312.2, respectively. The assay was linear in the concentration range of 1–1000 μg/l for fMPA with a LLOQ of 1 μg/l and an accuracy of >95%. The two methods reported have an adequate degree of robustness and dynamic concentration range for the measurement of MPA, MPAG and fMPA for therapeutic drug monitoring purposes or pharmacokinetics investigations.
Keywords: MPA; MPAG; HPLC-UV; LC–MS/MS; Unbound concentration;

Humic substances are the most important soil components affecting the behaviour and performances of herbicides in the soil–water–organism system. In this paper, a chromatographic approach was used for analysis of anticoagulant rodenticide–humic acid adsorption mechanisms. Using an equilibrium perturbation method, it was clearly shown that: (i) humic acid can be adsorbed on the C18 stationary phase, and (ii) all the rodenticides can be adsorbed on the humic acid adsorbed on the C18 stationary phase. This approach allowed the determination of the adsorption constant values between the anticoagulant rodenticides and humic acid as well as the corresponding thermodynamic data of this adsorption mechanism. The role of humic acid on the toxicity of these rodenticides on human keratinocytes was also clearly described in relation to these physico-chemical data.
Keywords: Rodenticide; Humic acid; Keratinocytes;

Urinary excretion of lignans after administration of isolated plant lignans to rats: the effect of single dose and ten-day exposures by Annika I. Smeds; Niina M. Saarinen; Teija T. Hurmerinta; Pauliina E. Penttinen; Rainer E. Sjöholm; Sari I. Mäkelä (303-312).
The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography–tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.
Keywords: 7-Hydroxymatairesinol; Matairesinol; Secoisolariciresinol; Lariciresinol; Cyclolariciresinol; Enterolactone; 7-Hydroxyenterolactone; Enterodiol; Plant lignans; Mammalian lignans; HPLC–MS/MS;

Fluorotelomer alcohols (FTOHs) constitute an important group of compounds among the perfluoroalkyl substances (PFAS). The PFAS have recently been a focus of many environmental and biological studies. This generated a strong need for analytical methods for analysis of PFAS at trace levels in various environmental and biological matrices. A quantitative analytical method for analysis of 8:2 FTOH in rat plasma and rat liver, kidney, and adipose tissue using GC–MS with electron impact (EI) ionization was developed and validated. Extraction of water-diluted plasma with methyl tert-butyl ether (MTBE) was used for rat plasma. The analysis of rat liver or kidney tissues required homogenization of tissue on ice, extraction with hexane, and clean up of the extract by silica (Si) normal-phase solid phase extraction (SPE). Similarly, the adipose tissue was dissolved in n-heptane and cleaned up by Si SPE. The methods were validated by performing spike recovery experiments for each type of matrix investigated and tested on authentic samples originating from 8:2 FTOH toxicological studies.
Keywords: Fluorotelomer alcohols; Biological tissues; GC–MS;

The study presented here shows that GC–MS with ion trap detection can be used for screening post mortem blood. The method described was used to simultaneously screen for unknowns, identify basic drugs present and semi-quantitate 14 drugs commonly encountered in coroner's toxicology (i.e. was used to determine whether the drugs were present in sub-therapeutic, therapeutic or greater than therapeutic amounts). The equipment used included a Varian Saturn 2000 GC–MS operating in full scan mode, a CP-3800 GC, a CP-8400 autosampler and Saturn GC–MS workstation Version 5.5 software. Post mortem blood samples were extracted using a standard liquid–liquid procedure; diethylether followed by back extraction into 0.1 M HCl. Standard curves for the 14 drugs which were semi-quantitated (amitriptyline, citalopram, clozapine, cocaine, cyclizine, diazepam, dihydrocodeine, dothiepin, methadone, mirtazapine, procyclidine, sertraline, tramadol, venlafaxine) were prepared covering the concentration range 0–1.0 ug/mL. The procedure is in routine use for coroners toxicology; semi-quantitation has been used (i) to speed-up the through put of cases where drugs are an incidental finding and (ii) for cases where the amount of sample submitted for analysis was too small to allow for screening, identification and quantitation on separate sample volumes.
Keywords: Post mortem blood; Ion trap GC–MS; Semi-quantitation;

A selective, sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of levonorgestrel in plasma was developed. An Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, using atmospheric pressure photospray ionisation (APPI) in the positive mode. Using 17-α-methyltestosterone as internal standard (IS), liquid–liquid extraction was followed by reversed phase liquid chromatography using a phenyl–hexyl column and tandem mass spectrometric detection. The mean recovery for levonorgestrel and 17-α-methyltestosterone was 99.5 and 62.9%, respectively. The method was validated from 0.265 to 130 ng levonorgestrel/ml plasma with the lower limit of quantification (LLOQ) set at 0.265 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection, allowing for a rapid (extraction and chromatography) and selective method for the determination of levonorgestrel in human plasma. The assay method was used in a pharmacokinetic study to quantify levonorgestrel in human plasma samples generated after administrating a single oral dose of 1.5 mg levonorgestrel to healthy female volunteers for up to five half lives. The total chromatographic runtime of this method was 5.0 min per sample, allowing for analysis of a large number of samples per batch.
Keywords: Levonorgestrel; 17-α-Methyltestosterone; Liquid–liquid extraction; Plasma; Liquid chromatography; Mass spectrometry; Atmospheric pressure photospray ionisation (APPI);

A rapid, sensitive, and highly selective liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination of enalapril and its major active metabolite enalaprilat in human plasma. The analytes were extracted from plasma samples by liquid–liquid extraction, separated on a Zorbax Extend-C18 column, and detected by tandem mass spectrometry with a Turbo IonSpray ionization interface. The method has a lower limit of quantification (LLOQ) of 0.1 ng/ml for both enalapril and enalaprilat. The chromatographic run time was approximately 3.5 min. The standard calibration curves for both enalapril and enalaprilat were linear in the concentration ranges of 0.10–100.0 ng/ml in human plasma. The intra- and inter-run precisions, expressed as the relative standard deviation (R.S.D.), were less than 7.7 and 7.8%, determined from QC samples for enalapril and enalaprilat, and accuracy was within ±3.9 and ±2.7% in terms of relative error, respectively. The method was successfully applied for the evaluation of the pharmacokinetics of enalapril and enalaprilat in 20 volunteers after an oral dose of 10 mg enalapril maleate.
Keywords: Enalapril; Enalaprilat;

Enantioselective pharmacokinetics of lercanidipine in healthy volunteers by Valquiria Aparecida Polisel Jabor; Eduardo Barbosa Coelho; Vera Lucia Lanchote (343-346).
The enantioselective kinetic disposition of lercanidipine, a dihydropyridine type of third-generation calcium antagonist, was investigated in six healthy male volunteers following a single 20 mg racemic oral dose.Serial plasma samples were obtained from 0 to 24 h after drug administration. Lercanidipine enantiomers were analysed using a chiral LC–MS–MS method.The following differences (p  < 0.05, Wilcoxon test) between (S) and (R) enantiomers were found (median): C max 2.071 ng mL−1 versus 1.681 ng mL−1; AUC0–2412.352 ng h mL−1 versus 10.063 ng h mL−1 and Cl/f 732.16 L h−1 versus 1891.84 L h−1. The AUC0–∞ values for (S)-LER were 1.21-fold higher than those for (R)-LER.The pharmacokinetics of LER was enantioselective in healthy volunteers following a single dose of 20 mg of the unlabeled racemic drug.
Keywords: Lercanidipine; Enantiomers; Pharmacokinetics; Healthy volunteers; Calcium antagonists; Dihydropyridines;

Simultaneous determination of digoxin and permeability markers in rat in situ intestinal perfusion samples by RP-HPLC by Manthena V.S. Varma; Namita Kapoor; Mahua Sarkar; Ramesh Panchagnula (347-352).
A simple, sensitive and specific reversed-phase high performance liquid chromatographic (RP-HPLC) assay for simultaneous determination of digoxin and permeability markers, in samples obtained from intestinal in situ single-pass perfusion studies, was developed and validated. Chromatography was carried on C-18 column with mobile phase comprising of acetate buffer (pH 3.0), acetonitrile and methanol in the ratio of 50:25:25 (v/v/v), was pumped at a flow rate of 0.5 ml/min and UV detection was employed at 220 nm. The average retention times for phenolred, propranolol, frusemide and digoxin were 9.1, 10.7, 12.9 and 15.3 min, respectively. The calibration curves were linear (R 2  > 0.998) in the range for each analyte. The method is specific and sensitive with limit of quantification of 25 ng/ml for digoxin and frusemide and 10 ng/ml for phenolred and propranolol. The method is accurate and precise with recoveries of digoxin in the range of 95.2 and 103.2% and relative standard deviation (R.S.D.) <5%. We found that this method was simple and reliable in permeability determination and to estimate the contribution of P-glycoprotein in limiting intestinal absorption.
Keywords: Digoxin; Permeability markers; Rat in situ permeability studies; P-glycoprotein; RP-HPLC;

A global method is proposed for therapeutic drug monitoring of atazanavir, a novel protease inhibitor and of all other protease inhibitors (PI) and non nucleoside reverse transcriptase inhibitors (NNRTI) which are currently used to treat HIV patients. All drugs are extracted after a liquid–liquid extraction and separated on a C18 column with a binary gradient elution except lopinavir which is separated without this gradient. The absorbance is measured at 259 nm except for lopinavir (205 nm) and nevirapine (320 nm). This method is specific, accurate, precise (the intra-day and inter-day imprecision and inaccuracy are lower than 15%) and the limits of quantitation (0.40 mg/L for nevirapine, 0.10 mg/L for indinavir, 0.10 mg/l for M8, 0.05 mg/L for amprenavir, 0.10 mg/L for nelfinavir, 0.10 mg/L for saquinavir, 0.10 mg/L for ritonavir, 0.10 mg/L for efavirenz, 0.10 mg/L for atazanavir and 0.20 mg/L for lopinavir) are consistent with trough plasma concentrations allowing to use this method for therapeutic drug monitoring of PI and NNRTI.
Keywords: Indinavir; Atazanavir; Amprenavir; Nelfinavir; M8; Saquinavir; Ritonavir; Lopinavir; Nevirapine; Efavirenz;