Journal of Chromatography B (v.963, #C)

Quantitative analyses of CTP-499 and five major metabolites by core-structure analysis by Xiaonan Tang; Gary Bridson; Jing Ke; Lijun Wu; Halil Erol; Phillip Graham; Chih Hsien Lin; Virginia Braman; Harry Zhao; Julie F. Liu; Zhongping (John) Lin; Changfu Cheng (1-9).
CTP-499 is a novel oral multi-subtype selective inhibitor of PDEs that is currently in clinical testing, in combination with angiotensin modulators, as a potentially first-in-class treatment for diabetic kidney disease. The compound was discovered and developed by using Concert's proprietary DCE Platform® in which deuterium was incorporated at select positions of 1-((S)-5-hydroxyhexyl)-3,7-dimethylxanthine (HDX). CTP-499 metabolizes to five major metabolites: C-21256, D-M2, D-M3, D-M4 and M5, of which all contains deuterium except M5. During in vivo metabolism, however, H/D exchange takes place. As a result, each analyte, except M5, has multiple molecular masses. To accurately quantify the analytes, we developed an LC–MS/MS method focusing on the core structures of the molecules, termed “core-structure analyses”. The core-structure analyses method was then validated under GLP guidance in dog, rat and rabbit plasma, with a sample volume of 50 μL. Results demonstrated that this approach accurately quantifies each of the six analytes despite partial exchange of deuterium with hydrogen atoms in the in vivo samples. The validation parameters included accuracy, precision, sensitivity, stability, dilution integrity, hemolysis, matrix effect, selectivity, and recovery. Acceptable intra-run and inter-run assay precision (%CV ≤ 5.5%) and accuracy (90.1–106.7%) were achieved over a linear range of 10–5000 ng/mL of each analyte. Various stability tests, including bench-top, freeze/thaw, stock solution, and long-term storage, were also performed. All stability results met acceptance criteria. The robustness of the methods was demonstrated by the incurred sample reproducibility (ISR) tests. After validation, the method was successfully used in support of multiple toxicological studies of CTP-499.
Keywords: CTP-499; Core-structure analysis; Diabetic kidney disease; Quantitative analysis; Deuterium substitution;

Hen egg white cystatin, an inhibitor of cysteine proteinase, may have wide applications for improving human health. However, its pricy cost associated with extraction and preparation has hurdled its further utilization. The objective was to develop an improved method to extract and purify cystatin from egg white. After removal of ovomucin, a fraction containing cystatin was obtained by cation exchange chromatography, and further purified by affinity chromatography using a cm-papain-Sepharose column. The prepared cystatin was then characterized by SDS-PAGE, Western-Blot, and LC–MS/MS, and its purity was determined by HPLC method instead of the conventional immunodiffusion method. The protein content of cystatin extract was 66.4 ± 2.3%. In comparison with the conventional method, the purity of cystatin was improved from 56.6 ± 1.7% to 93.3 ± 4.0%, and its yield was improved from 21.3 ± 1.2% to 33.6 ± 1.5%. Relative activities of cystatin to inhibit papain prepared by our method and the conventional method were determined to be 88 ± 7% and 91 ± 4% respectively, against a cystatin standard from Sigma. This suggested no significant loss of activity during the separation process.
Keywords: Cystatin; Hen egg white; Affinity chromatography; Quantification; HPLC; Mass spectrometry;

Analysis of interaction property of calycosin-7-O-β-d-glucoside with human gut microbiota by Wei Zhang; Shu Jiang; Dawei Qian; Er-xin Shang; Jin-ao Duan (16-23).
Calycosin-7-O-β-d-glucoside as the major isoflavonoids in Astragali Radix has been investigated intensively and has been reported to possess a wide range of pharmacological properties. However, the route and metabolites of calycosin-7-O-β-d-glucoside by human intestinal bacteria are not well understood and its metabolites may accumulate to exert physiological effects. Therefore, the objective of this study was to screen the ability of the bacteria to metabolize calycosin-7-O-β-d-glucoside and assess the effect of this compound on the intestinal bacteria. Finally, five strains including Bacteroides sp.13, and sp.58, Clostridium sp.21-2, Veillonella sp.43-1, and Bacillus sp.46 were isolated from human intestinal bacteria and were studied their abilities to convert calycosin-7-O-β-d-glucoside. A total of six metabolites were identified in human incubated solution by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF-MS). The results indicated that hydrolysis, demethylation, dehydroxylation and acetylation were the major metabolism of calycosin-7-O-β-d-glucoside. On the other hand, different strains of intestinal bacteria have varying degrees of growth sensitivity to calycosin-7-O-β-d-glucoside. Growth of certain pathogenic bacteria such as Enterobacter, Enterococcus, Clostridium and Bacteroides was significantly repressed by calycosin-7-O-β-d-glucoside, while commensal probiotics such as Lactobacillus, Bifidobacterium were less severely affected. This indicates that calycosin-7-O-β-d-glucoside exert significant effects on the intestinal environment by modulation of the intestinal bacterial population. Our observation provided further evidence for the importance of intestinal bacteria in the metabolism, absorption and potential activity of calycosin-7-O-β-d-glucoside in human health and diseases.
Keywords: Calycosin-7-O-β-d-glucoside; Fecal bacteria; UPLC-Q-TOF/MS; Metabolism; Modulation;

Development and validation a liquid chromatography mass spectrometry for determination of solasodine in rat plasma and its application to a pharmacokinetic study by Jianshe Ma; Xitao Ding; Chengxiang Sun; Chongliang Lin; Xinxin An; Guanyang Lin; Xuezhi Yang; Xianqin Wang (24-28).
Solasodine is a poisonous alkaloid chemical compound that occurs in plants of the Solanaceae family. A simple and selective liquid chromatography mass spectrometry method for determination of solasodine in rat plasma was developed and validated over the range of 3–1000 ng/mL. Chromatographic separation was achieved on a C18 (2.1 mm × 50 mm, 3.5 μm) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. The flow rate was set at 0.4 mL/min. After addition of midazolam as internal standard (IS), liquid–liquid extraction by ethyl acetate was used as sample preparation. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification with target ions m/z 414 for solasodine and m/z 326 for IS. Mean recoveries of solasodine in rat plasma were in the range of 87.6–94.1%. Matrix effects for solasodine were between 94.9% and 102.3%. Coefficient of variation of intra-day and inter-day precision were both <13%. The accuracy of the method ranged from 94.4% to 105.3%. The method was successfully applied to a pharmacokinetic study of solasodine after oral administration of 20 mg/kg in rats.
Keywords: Solasodine; LC–MS; Pharmacokinetics; Rat plasma;

A highly sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) has been developed and validated for simultaneous quantification of seven components in rat plasma and five components in rat tissues after oral administration of the extracts of different combination Radix Angelicae Sinensis–Radix Paeoniae Alba herb couple and has been applied to compare the different pharmacokinetics and tissue distribution properties of these bioactive components. The extracts of Radix Angelicae Sinensis (RAS), Radix Paeoniae Alba (RPA) and Radix Angelicae Sinensis–Radix Paeoniae Alba herb couple (RRHC) were orally administrated to rats, respectively. The concentrations of ferulic acid, caffeic acid, vanillic acid, ligustilide, paeoniflorin, albiflorin and oxypaeoniflorin in rat plasma and the concentrations of ferulic acid, vanillic acid, paeoniflorin, albiflorin and oxypaeoniflorin in tissues were determined by UPLC–MS/MS. The plasma samples were pretreated by protein precipitation with methanol and the tissue samples were homogenated with water and pretreated by protein precipitation with methanol. Chromatographic separation was performed on a C18 column using 0.1% formic acid–acetonitrile as mobile phase for gradient elution. A triple quadrupole (TQ) tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating both in positive and negative ionization mode and operated by multiple-reaction monitoring (MRM) scanning. Noncompartmental pharmacokinetic parameters were calculated by DAS 2.0 program. The differences between each group were compared by SPSS 16.0 with Independent-Samples T-test. The pharmacokinetic parameters (such as C max, T max, T 1/2, AUC0–T , MRT0–T , V z/F or CLz/F) of all the detected components between the single herb (RAS or RPA) and herb pair (RRHP) showed significant differences (P  < 0.05). It indicated that the compatibility of RAS and RPA could alter the pharmacokinetics features of each component. Tissue distribution results showed that ferulic acid, vanillic acid, paeoniflorin, albiflorin and oxypaeoniflorin mostly distributed in liver and kidney both in herb couple and single herb distributed most in liver and kidney. Compared with single herb, RRHC could increase or decrease the concentrations of five components at different time points compared with the sing herb. The results indicated the method was successfully applied to the comparative study on pharmacokinetics and tissue distribution of different combination of RRHC in rats. The compatibility of two Chinese herbs could alter the pharmacokinetics and tissue distribution properties of major bio-active components in the single herb. The results might be helpful for further investigation of compatibility mechanism of RRHC.
Keywords: Pharmacokinetics; Tissue distribution; Radix Angelicae Sinensis; Radix Paeoniae Alba; Herb couple; UPLC–MS/MS;

Sedimentation field flow fractionation monitoring of in vitro enrichment in cancer stem cells by specific serum-free culture medium by Carole Mélin; Aurélie Perraud; Christophe Bounaix Morand du Puch; Elodie Loum; Stéphanie Giraud; Philippe Cardot; Marie-Odile Jauberteau; Christophe Lautrette; Serge Battu; Muriel Mathonnet (40-46).
The development of methods to enrich cell populations for cancer stem cells (CSC) is urgently needed to help understand tumor progression, therapeutic escape and to evaluate new drugs, in particular for colorectal cancer (CRC). In this work, we describe the in vitro use of OncoMiD for colon, a CRC-specific primary cell culture medium, to enrich CRC cell lines in CSC. Sedimentation field flow fractionation (SdFFF) was used to monitor the evolution of subpopulations composition. In these models, medium induced a loss of adherence properties associated with a balance between proliferation and apoptosis rates and, more important, an increased expression of relevant CSC markers, leading to specific SdFFF elution profile changes.
Keywords: Cell enrichment; Colorectal cancer; Defined medium; Cancer stem cells; Sedimentation field flow fractionation;

In this study, a headspace, solid-phase dynamic extraction method coupled to gas chromatography–tandem mass spectrometry (HS-SPDE-GC–MS/MS) method was developed for the simultaneous determination of four volatile compounds, namely, isoborneol, borneol, muscone and cinnamaldehyde, in rat plasma after oral administration of Shexiang Baoxin Pill (SBP) using naphthalene as an internal standard (IS). The target compounds were extracted using an SPDE needle device coated with a poly (dimethylsiloxane) (PDMS) phase. The detection was achieved by GC–MS/MS in multiple reaction monitoring (MRM) mode. The optimised mass transition ion pairs (m/z) for quantitation were 95.1/67.1 for isoborneol and borneol, 85.0/67.0 for muscone, 131.0/77.0 for cinnamaldehyde and 128.1/102.1 for the IS. The parameters that affect the extraction ratio, such as the pre-incubation time, extraction temperature, number of extraction cycles, desorption volume and pH, were also optimised. The method was thoroughly validated with respect to specificity, linearity, precision, accuracy, recovery and stability. A sufficiently sensitive HS-SPDE-GC–MS/MS method was first developed in this study to determine the pharmacokinetics of volatile compounds found in rat plasma following oral administration of SBP. The method developed uses a simple procedure for plasma sample preparation and could be a promising tool for the analysis of complex volatile samples, such as traditional Chinese medicine (TCM).
Keywords: Shexiang Baoxin Pill; Volatile compounds; HS-SPDE-GC–MS/MS; MRM; Pharmacokinetics;

Simultaneous quantitative determination of microcystin-LR and its glutathione metabolites in rat liver by liquid chromatography–tandem mass spectrometry by Xiaochun Guo; Ping Xie; Jun Chen; Xun Tuo; Xuwei Deng; Shangchun Li; Dezhao Yu; Cheng Zeng (54-61).
The roles of glutathione (GSH) and cysteine (Cys) in the detoxification of Microcystin-LR (MC-LR) have recently become a popular area of research. However, lacking analysis methods for MC-LR-GSH and MC-LR-Cys (two main GSH pathway metabolites) in mammals, elucidation of the detoxification mechanism and metabolic pathway of MC-LR in mammals is difficult. In this study, a novel method for the simultaneous quantitative analysis of MC-LR, MC-LR-GSH and MC-LR-Cys in rat liver was developed and validated. The analytes were simultaneously extracted from rat liver using 3 M sodium chloride solution containing 0.01 M EDTA-Na2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges and determination by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS/MS). Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR, MC-LR-GSH and MC-LR-Cys were obtained at three concentrations (0.2, 1.0 and 2.5 μg g−1 dry weight (DW)) with values ranging from 97.7 ± 4.2 to 98.7 ± 5.1%, 70.1 ± 4.8 to 71.1 ± 4.1% and 79.8 ± 3.5 to 81.4 ± 4.0%, respectively. The relative standard deviations (RSDs) of these compounds at 0.2, 1.0 and 2.5 μg g−1 DW were between 4.3% and 6.9%. The limits of detection (LODs) were 0.005, 0.007 and 0.006 μg g−1 DW and the limits of quantification (LOQs) were 0.017, 0.023 and 0.020 μg g−1 DW for MC-LR, MC-LR-GSH and MC-LR-Cys, respectively. Furthermore, this method was successfully applied to both time- and dosage-effect studies of MC-LR, MC-LR-GSH and MC-LR-Cys in vivo.
Keywords: Microcystin-LR; Microcystin-LR-GSH; Microcystin-LR-Cys; LC–ESI–MS/MS; Quantitative determination; Rat liver;

A specific and sensitive high performance liquid chromatography coupled with tandem mass spectrometric (HPLC-MS/MS) method was developed and validated for the simultaneous determination of geniposidic acid and aucubin in rat plasma after oral administration of Du-zhong tea extract. The plasma samples were pretreated by protein precipitation with methanol and the chromatographic separation was performed on a Hypersil C18 column (4.6 mm × 250 mm, 5 μm), using a gradient mobile phase system of water-methanol (0.05% formic acid). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization source operating in the negative ionization mode. The linear range was 1–1000 ng/mL for geniposidic acid and 0.2–200 ng/mL for aucubin, respectively. The accuracy (relative error, R.E.%) were between −5.40 and 5.00%, while the intra-day and inter-day precisions were less than 7.95 and 7.87% for the two analytes, respectively. The method was fully validated for the sensitivity, selectivity, recovery, matrix effect and stability. Then this method was successfully applied to the pharmacokinetic study of geniposidic acid and aucubin after oral administration of Du-zhong tea extract to rats and the results indicated that this HPLC-MS/MS assay is a valuable method for the pharmacokinetic study of geniposidic acid and aucubin in rat plasma.
Keywords: Du-zhong tea; Geniposidic acid; Aucubin; HPLC-MS/MS; Rat plasma; Pharmacokinetics;

Determination of cephalomannine in rat plasma by gradient elution UPLC–MS/MS method by Xin-shuai Wang; Jia-chun Sun; Rui-na Yang; Jing Ren; Shuo Liang; Ming Sun; Ying-fei Wang; She-Gan Gao (70-74).
A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cephalomannine in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of perchloric acid–methanol (1:9, v/v) to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile–0.1% formic acid in water with gradient elution. The total run time was 2.0 min and the elution of cephalomannine was at 1.60 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 832.8 → 264.1 for cephalomannine and m/z 812.6 → 286.0 for 10-DAT (internal standard), respectively. The calibration curve was linear over the range of 10–2000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. Mean recovery of cephalomannine in plasma was in the range of 80.9–85.3%. Intra-day and inter-day precision were both <11.2%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0 mg/kg cephalomannine in rats.
Keywords: Cephalomannine; UPLC–MS/MS; Rat plasma; Pharmacokinetics;

A simple and reliable method for simultaneous determination of deoxynivalenol-3-glucoside and major type B trichothecenes (deoxynivalenol, nivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol and deepoxy-deoxynivalenol) in animal feed and raw materials has been developed and validated in this study. The method was based on an improved dispersive solid-phase extraction (DSPE) followed by analysis using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Also, matrix-matched calibration curve (R 2  > 0.99) was employed to minimize matrix effects and ensure accurate quantification. The recoveries during sample preparation process (including extraction and clean-up) ranged from 79.03% to 118.39%, with intra-day and inter-day relative standard deviation lower than 20% for all the analytes. The limit of quantification ranged from 5.0 μg/kg for deoxynivalenol to 13.6 μg/kg for fusarenon X. The validated method was successfully applied to the analysis of animal feed and corn. The pilot study showed that 37 out of 41 samples were contaminated with deoxynivalenol-3-glucoside at the levels of 6.0–121.0 μg/kg. Most of the type B trichothecenes were also found with the exception of fusarenon X, at the contaminated levels of 10.0–1382 μg/kg. To the best of our knowledge, this was the first scientific report on the co-occurrence of masked deoxynivalenol and type B trichothecenes in animal feed and raw materials.
Keywords: Mycotoxin; Deoxynivalenol-3-glucoside; Type B trichothecenes; Animal feed; LC-MS/MS; Simultaneous determination;

Development and validation of an LC–MS/MS method for determination of the L-type voltage-gated calcium channel/NMDA receptor antagonist NGP1-01 in mouse serum by Harini Jogiraju; Xiang Zhou; Ashta Lakshmi Prasad Gobburi; Kiran K. Pedada; Werner J. Geldenhuys; Cornelis J. Van der Schyf; Samuel D. Crish; David J. Anderson (83-89).
NGP1-01 (8-benzylamino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane) is a heterocyclic cage compound with multifunctional calcium channel blocking activity that has been demonstrated to be neuroprotective in several neurodegenerative models. A sensitive internal standard LC–MS/MS method was developed and validated to quantify NGP1-01 in mouse serum. The internal standard (IS) was 8-(2-phenylethylamino)-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane. Sample preparation involved a protein precipitation procedure by addition of acetonitrile. Chromatographic separation was carried out on a Phenomenex Kinetex phenyl-hexyl column (100 mm × 2.1 mm, 2.6 μm) employing a gradient (45% isocratic 3 min, 45–95% linear gradient 6 min, 95% isocratic 3 min) of an elution mobile phase of 5 mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5 mM ammonium acetate in 2% acetonitrile. Detection was achieved by a QTrap 5500 mass spectrometer (AB Sciex) employing electrospray ionization in the positive mode with multiple-reaction-monitoring (MRM) for NGP1-01 (m/z 266→91) and IS (m/z 280→105). The method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method had a linear range of at least 0.5–50 ng/mL with a correlation coefficient 0.999. The intra-assay and inter-assay precisions (%CV) were equal to or within the range of 1.0–4.3% and the accuracies (% relative error) equal to or within −2.5% to 3.4%. The analyte was stable for at least 2 months at −20 °C, for at least 8 h at room temperature and for at least three freeze–thaw cycles. The extraction recovery was 94.9 to 105.0%, with a %CV ≤ 9.5%. The technique was found to be free of any matrix effects as determined by experiments involving five different lots of mouse serum. Cross-talk interferences were not present. Two different gradient slope chromatography runs were done on dosed mouse serum samples to assess a possible positive error in peak area determination from in-source fragmentation of metabolites generating the same MRM transitions as the parent drug or IS. No such interference was found in the NGP1-01 peak, while a minor interference was identified in the IS peak. The optimized method was applied to the measurement of NGP1-01 in serum of dosed mice.
Keywords: NGP1-01; Pentacycloundecylamine; Neuroprotective agent; Multifunctional drug; Mouse serum; LC–MS/MS;

Comprehensive identification of active triterpenoid metabolites in frankincense using a coupling strategy by Yuanyan Liu; Zhenli Liu; Cheng Lu; Jianbei Li; Zhangchi Ning; Zhiqian Song; Chun Wang; Zhiyong Du; Xinmiao Lu; Siyu Zhao; Aiping Lu (90-98).
Frankincense resins are extensively used as natural remedies in regions ranging from North Africa to China. Triterpenoid metabolites from frankincense exhibit notable anti-inflammatory and anti-tumor properties. In the present paper, without the use of an isolation process, the fragmentation rules and NMR spectral characteristics of triterpenoid metabolites in frankincense are summarized through a coupling method using high performance liquid chromatography-diode array detection/electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI-MSn) combined with HPLC-nuclear magnetic resonance (NMR) experiments. Based on this groundwork, a coupling strategy for the comprehensive metabolic profiling of active triterpenoid metabolites from enriched fractions of frankincense was developed. The proposed strategy may serve as a method for the holistic screening of bioactive metabolites in complex TCM samples.
Keywords: Frankincense resins; Triterpenoid metabolites; Mass spectrometry; Nuclear magnetic resonance; Metabolic profiling;

Trapping of NAPQI, the intermediate toxic paracetamol metabolite, by aqueous sulfide (S2−) and analysis by GC–MS/MS by Arne Trettin; Sandor Batkai; Thomas Thum; Jens Jordan; Dimitrios Tsikas (99-105).
NAPQI, i.e., N-acetyl-p-benzoquinone imine, is considered the toxic metabolite of the widely used analgesic drug paracetamol (acetaminophen, APAP). Due to its high reactivity towards nucleophiles both in low- and high-molecular-mass biomolecules, NAPQI is hardly detectable in its native form. Upon conjugation with glutathione, NAPQI is finally excreted in the urine as the paracetamol mercapturic acid. Thus, determination of paracetamol mercapturate may provide a measure of in vivo NAPQI formation. In this work, we propose the use of Na2S in aqueous solution to trap NAPQI and to analyze the reaction product, i.e., 3-thio-paracetamol, together with paracetamol by GC–MS/MS in the electron-capture negative-ion chemical ionization mode after solvent extraction with ethyl acetate and derivatization with pentafluorobenzyl bromide. In mechanistic studies, we used newly synthesized N-acetyl-p-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI). In quantitative analyses, N-(4-hydroxyphenyl)-[2,3,5,6-2H4]acetamide (d4-APAP) was used as the internal standard both for NAPQI and APAP. 3-Thio-d3-paracetamol, prepared from d4-NAPQI and Na2S, may also be useful as an internal standard. We showed NAPQI in vitro formation from APAP by recombinant cyclooxygenase-1 as well as by dog liver homogenate. In vivo formation of NAPQI was demonstrated in mice given paracetamol intraperitoneally (about 150 mg/kg).
Keywords: Acetaminophen; Liver; Mice; NAPQI; Quantification; 3-Thio-paracetamol;

Urine homogentisic acid and tyrosine: Simultaneous analysis by liquid chromatography tandem mass spectrometry by A.T. Hughes; A.M. Milan; P. Christensen; G. Ross; A.S. Davison; J.A. Gallagher; J.J. Dutton; L.R. Ranganath (106-112).
Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid (HGA) which although excreted in gram quantities in the urine, is deposited as an ochronotic pigment in connective tissues, especially cartilage. Ochronosis leads to a severe, early-onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, Nitisinone, an inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of HGA, resulting in accumulation of the upstream pre-cursor, tyrosine. Using reverse phase LC–MS/MS, a method has been developed to simultaneously quantify urinary HGA and tyrosine. Using matrix-matched calibration standards, two product ion transitions were identified for each compound and their appropriate isotopically labelled internal standards. Validation was performed across the AKU and post-treatment concentrations expected. Intrabatch accuracy for acidified urine was 96–109% for tyrosine and 94–107% for HGA; interbatch accuracy (n  = 20 across ten assays) was 95–110% for tyrosine and 91–109% for HGA. Precision, both intra- and interbatch was <10% for tyrosine and <5% for HGA. Matrix effects observed with acidified urine (12% decrease, CV 5.6%) were normalised by the internal standard. Tyrosine and HGA were proved stable under various storage conditions and no carryover, was observed. Overall the method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of patients with AKU, pre and post-nitisinone therapy
Keywords: Alkaptonuria; Homogentisic acid; Tyrosine; Liquid chromatography; Tandem mass spectrometry;

A rapid and sensitive liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for the simultaneous determination of microcystin-RR (MC-RR) and its glutathione and cysteine conjugates (MC-RR-GSH and MC-RR-Cys, respectively) in fish plasma and bile. The analytes were extracted using methanol, followed by an Oasis mixed-mode cation-exchange polymeric sorbent. The separation was performed on a reversed-phase Waters XBridge C18 column with the gradient mobile phase, consisting of water and acetonitrile (both acidified with 0.5‰ formic acid). Mean recoveries of MC-RR, MC-RR-GSH and MC-RR-Cys ranged from 80.7 to 93.7%, 81.1 to 93.1% and 80.3 to 93.2%, respectively, at three concentrations (0.2, 1.0 and 5.0 μg mL−1). Limits of detection (LODs) for MC-RR, MC-RR-GSH and MC-RR-Cys were 6, 12 and 9 ng mL−1, respectively. Limits of quantification (LOQs) were 15, 30 and 22.5 ng mL−1 for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. This method makes it feasible for the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in limited and complex biological fluid samples (such as plasma and bile, typically 50 μL), which were previously excluded or difficult to study due to the relatively large sample volumes.
Keywords: Microcystin-RR; LC–MS/MS; Metabolites; Quantification;

Quantification of levornidazole and its metabolites in human plasma and urine by ultra-performance liquid chromatography–mass spectrometry by Yuran Cao; Miao Zhao; Xiaojie Wu; Beining Guo; Yuancheng Chen; Jicheng Yu; Guoying Cao; Jing Zhang; Yaoguo Shi; Yingyuan Zhang (119-127).
We developed and validated an ultra-performance liquid chromatographic (UPLC) method coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry for simultaneous determination of levornidazole and its first-pass metabolites, l-chloro-3-(2-hydroxymethyl-5-nitro-l-imidazolyl)-2-propanol (Ml), 2-methyl-5-nitroimidazole (M2) and 3-(2-methyl-5-nitro-1-imidazolyl)-1,2-propanediol (M4), in human plasma and urine. The biological samples were pretreated by protein precipitation and liquid–liquid extraction and analyzed using an ACQUITY UPLC CSH C18 column (2.1 × 50 mm, 1.7 μm) and a QTRAP mass spectrometer in multiple reaction monitoring mode via APCI. Acetonitrile and 0.1% formic acid in water was used as the mobile phase in gradient elution at a flow rate of 0.6 mL/min. The lower limit of quantification of this method was 0.0100, 0.00500, 0.0200 and 0.00250 μg/mL for levornidazole, M1, M2 and M4, respectively. The linear calibration curves were obtained for levornidazole, M1, M2, and M4 over the range of 0.0100–5.00, 0.00500–2.50, 0.0200–10.0 and 0.00250–1.25 μg/mL, respectively. The intra- and inter-batch precision was less than 12.2% in plasma and less than 10.8% in urine. The intra- and inter-batch accuracy was 87.8–105.7% in plasma and 92.8–109.2% in urine. The mean recovery of levornidazole, M1, M2 and M4 was 91.1–105.1%, 95.8–103.8%, 87.8–96.8%, 96.8–100.6% from plasma and 96.0–100.9%, 96.9–107.9%, 95.1–102.7%, 103.7–105.9% from urine respectively. This method was validated under various conditions, including room temperature, freeze–thaw cycles, long-term storage at −40 ± 5 °C, after pretreatment in the autosampler (at 10 °C), and 10- and 100-fold dilution. This newly established analytical method was successfully applied in a pharmacokinetic study following single intravenous infusion of levornidazole in 24 healthy Chinese subjects.
Keywords: Levornidazole; Metabolite; LC–MS/MS; Pharmacokinetics;

Quantification of phylloquinone and menaquinones in feces, serum, and food by high-performance liquid chromatography–mass spectrometry by J. Philip Karl; Xueyan Fu; Gregory G. Dolnikowski; Edward Saltzman; Sarah L. Booth (128-133).
Vitamin K, comprising phylloquinone (PK) and menaquinones (MKn), is a family of vitamers found in multiple biological and environmental matrices. Advancing emerging evidence for novel and distinct physiologic roles of these vitamers in human health and disease necessitates sensitive and selective methods for quantifying PK and MKn in these matrices. We developed a novel method employing high-performance liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization (LC–APCI–MS) for simultaneous quantification of 11 vitamin K vitamers that can be applied in feces, serum and food. Minimal detectable concentrations of vitamin K vitamers ranged from 1 pmol/g to 30 pmol/g. Limits of quantification ranged from 5 pmol/g to 90 pmol/g. Inter-assay and intra-assay variations were <17% and <8%, respectively, in food, and <12% and <8%, respectively, in feces. Recovery exceeded 80% for all vitamers in both food and feces. The method successfully quantified PK and MKn concentrations in rat chow, feces and serum. In summary, this LC–APCI–MS method provides a sensitive and selective tool for quantifying vitamin K vitamers in feces, serum and food. This method can be applied in human and animal studies examining the role of vitamin K vitamers derived from the diet and gut bacteria synthesis in health and disease.
Keywords: High-performance liquid chromatography; Mass spectrometry; Solid-phase extraction; Microbiota; Bacteria; Vitamin K;

Fast on-site diagnosis of influenza A virus by Palm PCR and portable capillary electrophoresis by Seoyeon Lim; He Nan; Min-Jun Lee; Seong Ho Kang (134-139).
A method combining Palm polymerase chain reaction (PCR) and portable capillary electrophoresis (CE) was developed for rapid on-site analysis of influenza A (H1N1) virus. The portable CE system was suitable for rapid diagnosis which was able to detect a sample in ∼4 min after sample loading, while the ‘Palm PCR’ system allowed for high-speed nucleic acid amplification in ∼16 min. The analysis time from DNA sample to analysis of amplified target DNA molecule was only ∼20 min, which was significantly less than slab gel electrophoresis with other commercially available PCR machine. When the 100-bp DNA ladder was separated, the relative standard deviation values (n  = 5) for the migration times and peak areas of the 100 and 200-bp DNA molecules were 0.26 and 8.9%. The detection limits were 6.3 and 7.2 pg/μL, respectively. The combined method was also able to identify two influenza A-associated genes (the HA and NP genes of the novel H1N1 influenza). CE separation was achieved with a sieving matrix of 1% poly(vinylpyrrolidone) (M r  = 1,300,000) in 1× TBE buffer (pH 8.45). The combined Palm PCR-portable CE system should provide an improved, fast on-site molecular genetic diagnostic method.
Keywords: H1N1 influenza; Fast diagnosis; Portable CE; Palm PCR; On-site analysis;