Journal of Chromatography B (v.855, #2)

The disposition into hair of new designer drugs; methylone, MBDB and methcathinone by Ruri Kikura-Hanajiri; Maiko Kawamura; Kazuhiro Saisho; Yukio Kodama; Yukihiro Goda (121-126).
The disposition into hair of methylone and other new designer drugs, methcathinone and MBDB, was studied with the animal model. Moreover, the incorporation rates of these drugs were compared with those of their related eight compounds previously studied in order to evaluate their incorporation tendency into hair and the usefulness of hair specimens for the retrospective confirmation of the use of these drugs. When the ratio of hair concentration to AUC in plasma ([Hair]/AUC) was represented as an index of the incorporation rate of drugs into hair, the [Hair]/AUC of methylone was 14 times higher than that of methcathinone. It might support earlier findings that the methylenedioxy group on the benzene ring leads to considerably higher incorporation rates. However, [Hair]/AUC of methylone was five-sevenths times lower in comparison with that of MDMA. This suggested that the beta-carbonyl group leads to lower incorporation rates. Although methylone has both groups in its structure, the positive effect of the methylenedioxy group may be stronger than the negative effect of the beta-carbonyl group. On the other hand, the [Hair]/AUC of MBDB, which has methylenedioxyphenyl-2-butanamine structure, was higher than that of MDMA while that of methcathinone, having beta-ketone in its structure, was extremely low. In conclusion, as with MA and MDMA, the incorporation tendency of methylone and MBDB (except for methcathinone) into hair is relatively high, and a hair sample would be a good specimen for the confirmation of retrospective use of these drugs.
Keywords: Methylone; Hair analysis; Drug disposition into hair; GC–MS;

Proof of a 1-(3-chlorophenyl)piperazine (mCPP) intake—Use as adulterant of cocaine resulting in drug–drug interactions? by Roland F. Staack; Liane D. Paul; Dagmar Schmid; Gabriele Roider; Burkhard Rolf (127-133).
Since 2005, increasing numbers of seizures of the designer drug of abuse 1-(3-chlorophenyl)piperazine (mCPP) have been reported. This paper describes the unequivocal proof of a mCPP intake. Differentiation from the intake of its precursor drugs trazodone and nefazodone was performed by a systematic toxicological analysis (STA) procedure using full-scan GC–MS after acid hydrolysis, liquid–liquid extraction and microwave-assisted acetylation. The found mCPP/hydroxy-mCPP ratio indicated altered metabolism of this cytochrome (CYP) 2D6 catalyzed reaction compared to published studies using the same procedure. Although this might be ascribed to a poor metabolizer (PM) phenotype, genotyping revealed no PM genotype but indications for an intermediate metabolizer genotype. However, a PM phenotype could also be caused by drug–drug interactions with CYP2D6 inhibitors or substrates such as the co-consumed cocaine and diltiazem and/or diltiazem metabolites, respectively. In conclusion, the presented data indicate a possible relevance of CYP2D6 polymorphism and/or drug interactions to mCPP toxicokinetics, which is important for risk assessment of this new designer drug of abuse, in particular if it is used as adulterant of CYP2D6 substrates such as cocaine.
Keywords: 1-(3-Chlorophenyl)piperazine; mCPP; Drug–drug interaction; Drug metabolism; GC–MS;

Hydrolysis and transesterification reactions of candesartan cilexetil observed during the solid phase extraction procedure by Nerea Ferreirós; Sebastian Dresen; Rosa María Alonso; Wolfgang Weinmann (134-138).
Candesartan cilexetil is an angiotensin receptor antagonist widely used in the treatment of high blood pressure. This prodrug is metabolised into candesartan, which blocks the receptors AT1 for angiotensin II decreasing the blood pressure levels. During the development of a solid phase extraction procedure for the chromatographic determination of eight antihypertensive compounds, lack of linearity and reproducibility was observed only for candesartan cilexetil. Due to this fact, a stability study for this prodrug was performed. It showed that the lack of linearity and reproducibility was based on hydrolysis and transesterification processes which occurred during the drying step after elution with methanol into glass tubes. These phenomena could be reproduced artificially under basic conditions, which demonstrated the presence of basic residues in glass tubes. The study of this potential hydrolysis and transesterification reactions is very important to assure that labile drugs containing ester groups remain unaffected.
Keywords: Candesartan cilexetil; Hydrolysis; Transesterification; Stability;

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC–MS/MS by Xijing Chen; Jing Jing; Weichao Ren; De-en Han; Ning Jing; Guangji Wang (140-144).
A liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol–water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI+). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127 → 110 for AICA, and 172 → 128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.
Keywords: Orazamide; AICA; Ion-pair extraction; LC–MS/MS;

Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies by Hui He; Ying Zhao; Xijing Chen; Yuanting Zheng; Xiaolan Wu; Rui Wang; Tingting Li; Qiangling Yu; Jing Jing; Le Ma; Weichao Ren; Deen Han; Guangji Wang (145-151).
A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid–liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 μm, 4.6 mm × 250 mm) and methanol/distilled water as the mobile phase (flow rate = 1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25–40 μg/mL for plasma sample and 1.0–500 μM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7 ± 7.86%, 96.8 ± 3.20% and 102.7 ± 9.72% recovery from 0.5, 10, and 40 μg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze–thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.
Keywords: trans-Polydatin; HPLC; Plasma concentration; Pharmacokinetics;

Quantitative gas chromatographic method for the analysis of cis-9, trans-11 and trans-10, cis-12 isomers of the conjugated linoleic acid in liver by A. Zabala; M.P. Portillo; V. Navarro; M.T. Macarulla; L.J.R. Barron; A. Fernández-Quintela (152-158).
A quantitative GC method for conjugated linoleic acid (CLA) isomers of physiological significance (cis-9, trans-11 CLA and trans-10, cis-12 CLA) as non-esterified fatty acids (NEFA) or triacylglycerols (TAG) was developed. Furthermore, the effect of the internal standard addition point (sample or fat extract) was studied. Response linearity, recovery and precision assays, detection and quantification limits were determined. Linearity was demonstrated over a range from 0.1 to 10 μg/mL. When CLA isomers were present as NEFA, the recovery significantly decreased (P  ≤ 0.05) from 76% to 27.1% (cis-9, trans-11 CLA) and 28.5% (trans-10, cis-12 CLA) when the standards were added to the fat extract or to the initial tissue, respectively. As an application, liver samples from hamsters fed a diet supplemented with both CLA isomers were analyzed. The CLA isomers in liver samples were detected with reasonable reproducibility.
Keywords: Quantitative method; GC; CLA analysis; Liver;

A novel derivatization method employing 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) to improve the mass spectrometric response for phenolic compounds in liquid chromatography electrospray ionization mass spectrometry (LC–ESI-MS) and tandem mass spectrometry (LC–ESI-MS/MS) is described. Several environmentally relevant compounds, including chloro-, aryl- and alkylphenols, steroidal estrogens, and hydroxy-polycyclic aromatic hydrocarbons (OHPAHs), were selected to evaluate this technique. A facile derivatization procedure employing DMISC results in dimethylimidazolesulfonyl (DMIS) derivatives that are stable in aqueous solution. These DMIS derivatives produced intense [M  + H]+ ions in positive-ion LC–ESI-MS. The product ion spectra of the [M  + H]+ ions of simple phenols were dominated by ions representing the DMIS and dimethylimidazole moieties, whereas product ion spectra of the DMIS derivatives of OHPAHs with three or more fused aromatic rings showed prominent ArO+ ions, the relative intensity of which increased with the number of rings. The DMIS derivatives of the selected phenolic compounds showed excellent chromatographic properties. To substantiate the utility of derivatization with DMISC, an analytical method employing enzyme hydrolysis, solid phase extraction, derivatization with DMISC, and analysis by LC–ESI-MS/MS with multiple reaction monitoring for determination in human urine of 1-hydroxypyrene, a widely used biomarker for the assessment of human exposure to PAHs, was developed and validated.
Keywords: Liquid chromatography electrospray ionization tandem mass spectrometry; Chemical derivatization of phenols; Dimethylimidazolesulfonyl derivatives; 1-Hydroxypyrene;

Isolation, characterization and chemobiological quantification of α-glucosidase enzyme inhibitory and free radical scavenging constituents from Derris scandens Benth by Sridhar A. Rao; Pullela V. Srinivas; Ashok K. Tiwari; Uma Maheswara S. Vanka; Rama V. Subba Rao; Krishna R. Dasari; Madhusudana J. Rao (166-172).
The hexane and chloroform extracts of Derris scandens have displayed potent α-glucosidase inhibitory and moderate free radical scavenging activities. Phytochemical investigation of the active extracts led to the isolation of three new prenylated isoflavones, isoscandinone, scandenin A and scandenin B in addition to scandenone, scandinone and 4′, 5′, 7-trihydroxybiprenylisoflavone as the main constituents, having α-glucosidase enzyme inhibitory and free radical scavenging properties. A reversed-phase HPLC method is developed to quantify these active principles in the plant material, which can serve as an effective quality control method for standardization of D. scandens.
Keywords: Derris scandens; Isoflavones; α-Glucosidase inhibitors; High-performance liquid chromatography; Chemobiological standardization;

An LC–MS/MS assay for the quantitative determination of a new antibacterial agent (AVE6971) has been developed and validated in human white blood cells (WBC). The assay involved a lysing procedure of white blood cells and ultra centrifugation of the extracts. Chromatography was performed on a Supelcosil ABZ+ C18 (2.1 mm × 50 mm, 5 μm) column using a mobile phase consisting of methanol/acetonitrile/10 mM ammonium formate mixture (10:30:60, v/v/v) at a flow rate of 0.2 ml/min. The linearity was within the range of 10–10000 ng/ml of extracts, corresponding to 0.5–500 ng of AVE6971 in WBC pellets tubes. The validated lower limit of quantification was 10 ng/ml. The inter- and intra-run coefficients of variation (CV) for the assay were <12.9% and the accuracy were from −9.0 to −1.2%. AVE6971 was stable in WBC for at least 1 month at −75 °C. This assay proved to be suitable for the determination of AVE6971 in WBC from clinical studies.
Keywords: Antibacterial agents; Leukocytes; Mass spectrometry; Liquid chromatography;

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. After a simple protein-precipitation using methanol, the post-treatment samples were separated on a CAPCELL UG120 column with a mobile phase of a mixture of methanol and water (35:65) containing 0.1% formic acid. The serial chiral analytes and internal standard (IS) were all detected by the use of selected reaction monitoring mode (SRM). The method of all serial chiral analytes developed was validated in rat plasma with a daily working range of 0.5–100 ng/ml with correlation coefficient, R 2  ≥ 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision and stability studies for all serial chiral analytes. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.
Keywords: Anticholinergic compounds; Serial chiral analytes; Liquid chromatography–tandem electrospray mass spectrometry; Rat plasma; Quantification;

Liquid chromatography coupled with electrospray tandem mass spectrometry (LC–MS/MS) was used to identify palmitoyl-lineloyl-glycerophosphatidylcholine oxidation products (PL(O1–6)PC). Structural and positional isomers of keto, hydroxy and/or epoxy, and hydroperoxide derivatives of PLPC were identified based on MS/MS data, namely product ions attributed to lyso-phosphatidylcholines, product ions formed by loss of nH2O and H2O2 from [MH]+ ions groups, and product ions involving the hydroxy groups, providing information about the position of these groups and of the double bonds along the carbon chain of lineloyl moiety.
Keywords: Phospholipids; Glycerophosphatidylcholine; Oxidation; LC–MS/MS;

Fast and sensitive liquid chromatography–tandem mass spectrometric assays for the determination of salinomycin in mouse plasma, liver, brain and small intestinal contents and in OptiMEM cell culture medium, were developed and validated using simple sample pre-treatment procedures. Tissue samples were homogenized with phosphate buffered saline or, for high levels in liver, with human plasma. After addition of monensin as the internal standard to plasma, homogenate or culture medium and acetonitrile extraction for tissue and plasma, the diluted medium or the supernatant was directly injected into the isocratic chromatographic system using a polar embedded reversed-phase column and formic acid in water–acetonitrile as the eluent. The eluate was completely led into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assays were successfully validated in the ranges 10–2000 ng/ml for OptiMEM cell culture medium, 1–2000 ng/ml for plasma and 3–2000 ng/g in liver brain and small intestinal contents. At the lowest levels, the intra-day precisions were ≤9%, inter-day precisions were ≤14% and accuracies were between 91 and 112%. The analytes were chemically stable under all relevant conditions and the assays were applied in different in vitro transport studies and in pharmacokinetic and tissue distribution studies with salinomycin in mice.
Keywords: Salinomycin; Monensin; LC/MS/MS; OptiMEM cell culture medium; Mouse tissues;

Simple and rapid reversed phase HPLC methods for individual as well as simultaneous analysis of paclitaxel and carboplatin with cremophorEL (CrEL) in an amphiphilic polymer matrix were developed. Different analytical performance parameters such as linearity, accuracy, precision, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to ICH guidelines. All the analytical methods were developed by reverse phase HPLC on C-18 column with a mobile phase comprising of water–acetonitrile run on isocratic mode for the analysis of carboplatin and gradient mode for individual analysis of paclitaxel and for simultaneous analysis of the two drugs at a flow rate of 1 ml/min at 227 nm. The proposed methods for independent analysis of the drugs elute out carboplatin in 4.3 min and paclitaxel in 10.5 min while in simultaneous analysis carboplatin shows R t at 4 min and paclitaxel at 18 min with a continuous run for 17 more minutes to elute out CrEL. These methods were found to be specific as none of the components of the media, i.e. polymer, CrEL and buffer interfered with the drug peaks. The linearity of the calibration curves for each analyte in the desired concentration range was found to be good (r 2  > 0.9995). The methods were accurate and precise with recoveries ranging from 98 to 101% for each drug and relative standard deviation (%RSD) <2%. Peaks corresponding to each of the drug showed positive value for the minimum peak purity index over the entire range of integrated chromatographic peak thus indicating the purity of the peaks. Stability analysis of the two drugs revealed that the drugs remain stable during the period of study.
Keywords: Reversed phase HPLC method; Paclitaxel; Carboplatin; CremophorEL;

This paper describes the development and validation of a method for the detection of raloxifene (Ral) and its two glucuronide metabolites, raloxifene-6-glucuronide (M1) and raloxifene-4′-glucuronide (M2), in human plasma samples. Both glucuronides were synthesized enzymatically, purified and used as authentic standards. The assay involves a simple solid phase extraction (SPE) procedure of 0.5 mL of human plasma and subsequent analysis by LC–MS–MS. The recoveries were higher than 71% and chromatographic separation of all the analytes was accomplished in less than 7 min. Linear ranges (r 2  > 0.99) were found from 0.200 to 340 μg/L, from 1.600 to 2720 μg/L and from 0.088 to 60.00 μg/L, for M1, M2 and Ral, respectively. The limits of detection achieved were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method presented was successfully applied to a genetic polymorphism study of 47 plasma samples from women taking Evista (raloxifene hydrochloride).
Keywords: Raloxifene hydrochloride; Raloxifene glucuronides; LC/MS/MS; Plasma samples;

Three phase liquid phase microextraction of phenylacetic acid and phenylpropionic acid from biological fluids by Shahab Shariati; Yadollah Yamini; Majid Darabi; Mohsen Amini (228-235).
Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0–3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 μl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3 M Na2SO4, pH 2.0–3.5; organic membrane: 95 μl of 1-hexanol; acceptor solution: 4.0 μl of 0.1 M NH3/NH4 + with pH 11.1; donor solution temperature: 45–50 °C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1–5000 μg/l with r 2  > 0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 μg/l (based on S/N = 3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.
Keywords: Three phase liquid phase microextraction; Phenylacetic acid; Phenylpropionic acid; High performance liquid chromatography;

Identification of a peptide from α-gliadin resistant to digestive enzymes: Implications for celiac disease by Gianfranco Mamone; Pasquale Ferranti; Mauro Rossi; Peter Roepstorff; Olga Fierro; Antonio Malorni; Francesco Addeo (236-241).
Current knowledge indicates that both innate and adaptive immune responses are involved in Celiac disease (CD) driven by different gliadin peptides. By studying a representative recombinant α-gliadin form, a further 25-mer peptide resistant to gastric, pancreatic, and human intestinal brush-border membrane enzymes was detected. This peptide latter encompasses the sequence 31–43 known to elicit the innate immune response in CD. The resistance of 25-mer, as well as that of the already described 33-mer related to the CD adaptive immune response, was confirmed on a standard flour wheat sample representative of the most widespread European varieties.
Keywords: Celiac disease; Gliadin; Brush-border membrane enzymes; Prolamin Working Group standard; Mass spectrometry;

Determination of ochratoxin A in dry-cured meat products by a HPLC–FLD quantitative method by Tania Toscani; Alessandra Moseriti; Arnaldo Dossena; Chiara Dall’Asta; Nicoletta Simoncini; Roberta Virgili (242-248).
A fast and sensitive method for the quantification of the mycotoxin ochratoxin A (OTA) in dry-cured meat products has been developed, which does not require a clean-up step, by HPLC with an alkaline mobile phase (pH 9.8). Validation procedures for specificity, trueness, ruggedness, stability, recovery and repeatability were performed. The decision limit (CCα) and the decision capability (CCβ) were calculated at 1.10 and 1.23 μg/kg, respectively. The procedure was applied to representative dehydration levels of dry-cured meat samples.
Keywords: Dry-cured meat products; Ochratoxin A; Validation;

In the present work, human male and female fetal cord blood samples were purified, selectively extracted and separated to examine a fraction of steroids ranging from polar estetrol to relatively non-polar progesterone using solid phase extraction based on C-18 tubes and β-cyclodextrin driven temperature dependent inclusion chromatography. Resulting UV diode array chromatographic patterns revealed the presence of 27 peaks. Chromatographic patterns of UV detected steroids were analyzed using principal components analysis which revealed differences between male/female and labour/not-in-labour clusters. Quantitative analysis of nine identified steroids including: estetrol, 17β-estradiol, estrone, estriol, cortisol, cortisone, progesterone, 20α-hydroxyprogesterone and 17α-hydroxyprogesterone were not significantly different between males and females. Significant differences between male and female fetuses were related to as yet unidentified compounds. Four peaks were significantly different with labour which corresponded with cortisol, cortisone and two unidentified compounds. This protocol may distinguish significant differences between clinical groups that are not readily identifiable using univariate measurements of single steroids or different low molecular mass biomarkers. Moreover, we have provided new evidence that despite the absence of testosterone there are number of steroids and low molecular mass compounds that differ between male and female fetuses.
Keywords: Human pregnancy; Fetal sex; Labour; Cord blood; Chromatography; Principle component analysis;

A selective, rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed for the quantitative determination of azithromycin in human plasma and its application in a pharmacokinetic study. With roxithromycin as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The analysis was carried out on an ACQUITY UPLC™ BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 μm) with gradient elution at flow rate of 0.35 mL/min. The mobile phase was 50 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 1–1000 ng/mL, with a lower limit of quantification of 1 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was −1.3% to 5.7% at all QC levels. The method was applicable to clinical pharmacokinetic study of azithromycin in healthy volunteers following oral administration.
Keywords: Azithromycin; UPLC–ESI-MS/MS; Human plasma; Pharmacokinetic;

3,4-Methylenedioxymethamphetamine (MDMA) is a recreational drug with neurotoxic potential. Pharmacokinetic data of MDMA and its metabolites may shed light on the mechanism of MDMA neurotoxicity. An LC–MS assay with electrospray ionization (ESI) is presented for quantifying MDMA and its metabolites 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA) in squirrel monkey plasma. The method involved enzymatic conjugate cleavage and protein precipitation. Separation was achieved within 14 min. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect. The present method should prove useful for acquiring pharmacokinetic and toxicokinetic data in squirrel monkeys.
Keywords: MDMA; Metabolites; LC–ESI-MS; Validation; Neurotoxicity; Plasma;

Determination of ornithine in human plasma by hydrophilic interaction chromatography–tandem mass spectrometry by Jens Martens-Lobenhoffer; Sylvia Postel; Uwe Tröger; Stefanie M. Bode-Böger (271-275).
The amino acid ornithine (Orn) acts as a vital part in the physiologically fundamental urea cycle. As such, it is a main intermediate in the catabolic breakdown as well as in the synthesis of arginine and is involved in many other metabolic pathways with potential clinical implications. We here describe a LC–MS-MS method for the detection of Orn in human plasma which is fast, easy and precise. The sample preparation comprises only protein precipitation and the addition of the isotopic labeled I.S. The analytes are separated by hydrophilic interaction chromatography (HILIC) in less than 4 min on a silica column with an isocratic mobile phase consisting of 0.1% trifluoroacetic acid in water and acetonitrile in the ratio of 25:75. Orn and its I.S. are detected and quantified by APCI tandem mass spectrometry. The calibration function is linear from 7.5 to 205 μmol/l and covers the range of concentrations found in patients undergoing different clinical interventions. The quantification results are independent with regard to the biological matrix analyzed. The intra-day and inter-day relative standard deviations are 1.1% and 3.5%, respectively. As an application of the described method in clinical investigations, we report arginine and ornithine plasma concentration results from an arginine supplementation study enrolling healthy volunteers and patients suffering from hypercholesterolemia. After oral dosing of 110 mg/kg arginine, ornithine plasma concentrations rose from 54 to 148 μmol/l after 2 h and were back to baseline after 24 h. However, arginine to ornithine ratios kept constant during the complete observation time.
Keywords: Ornithine; Arginine; HPLC; APCI; MS-MS; HILIC;

Determination of ellagic acid in oak leaves and in sheep ruminal fluid by ion-pair RP-HPLC by P. García del Moral; M.J. Arín; J.A. Resines; M.T. Díez (276-279).
An isocratic ion-pair high-performance liquid chromatography (IP-RP-HPLC) method with UV detection was developed to identify and quantify ellagic acid (EA). This phenolic compound is widely distributed in the plants and is often present in the diet of ruminants. The method was validated and validation parameters were: linearity range 5–100 mg/L; correlation coefficient, 0.9995; mean recoveries (99.94 and 101.07%) and detection limit 1.4 mg/L. Method was applied for the determination of ellagic acid in oak leaves and in ruminal fluid from to a vitro ruminal system. The proposed method proved to be rapid and accurate and can be successfully used in ruminant nutrition studies.
Keywords: Ellagic acid; Oak leaves; Ruminal fluid; Ion-pair reversed-phase liquid chromatography;

Capillary electrophoretic immunoassay for alpha-fetoprotein with chemiluminescence detection by Yan-Ming Liu; Hai-Bei Mu; Yan-Li Zheng; Cheng-Quan Wang; Yong-Hong Chen; Fu-Rong Li; Jun-Hua Wang; Jie-Ke Cheng (280-285).
A capillary electrophoretic immunoassay with chemiluminescence detection (CEIA–CL) using a non-competitive format for analyzing tumor marker alpha-fetoprotein (AFP) has been developed. In this method, antigen (Ag) AFP reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ab*–Ag complex produced in the solution are separated by CE in a separation capillary. Then they catalyze the reaction of enzyme substrate luminol and H2O2 in a reaction capillary following the separation capillary. Parameters affecting the CE separation and CL detection were investigated. Under the optimal conditions, the free Ab* and the Ab*–Ag complex were well separated within 4 min, the linear range and the detection limit (S/N = 3) for AFP were 5–500 ng/ml and 0.85 ng/ml (1.2 × 10−11  M), respectively. The proposed method has been applied satisfactorily in the analysis of human sera samples.
Keywords: Capillary electrophoresis; Enzyme immunoassay; Chemiluminescence detection; Alpha-fetoprotein;

Apigenin is a flavone and is being developed for treatment of cardiovascular disease. A sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the measurement of apigenin and luteolin levels in rat plasma is described. Analytes were separated on a separation by a Luna C18 (5 μm, 100 mm × 2.0 mm) column with acetonitrile:methanol:water (35:40:60, v/v/v) as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. Good linearity (R 2  > 0.9997) was observed for both analytes over the range of 2.5–5000 ng/mL in 0.1 mL of rat plasma. The overall accuracy of this method was 93–105% for apigenin and 95–112% for luteolin in rat plasma. Intra-assay and inter-assay variabilities were less than 11% in plasma. The lowest quantitation limit for both apigenin and luteolin was 2.5 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC/MS/MS method was demonstrated in a pilot pharmacokinetic study in rats following intravenous administration of apigenin. Metabolism of apigenin to luteolin in vivo was established.
Keywords: Apigenin; Metabolism; Luteolin; LC/MS/MS; Pharmacokinetics;

A rapid and sensitive ultra-performance liquid chromatography and mass spectrometry (UPLC/MS) method was developed to simultaneously quantify seven monohydroxyl testosterone metabolites (16α-, 2α-, 7α-, 6α-, 2β-, 6β-, and 16β-hydroxyl testosterones) in rat liver microsomes. The UPLC system used a short 1.7-μm particle size column coupled to a Sciex 4000 Q trap in multiple reaction monitor (MRM) mode. All hydroxyl testosterones were resolved within 2.5 min. A 4-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method in rat liver microsomes. This method is applicable to the measurement of the testosterone hydroxylase activity in biological matrices such as the liver microsome incubates.
Keywords: UPLC; LC/MS; Quantitation; Testosterone; Metabolites;