Journal of Chromatography B (v.874, #1-2)

Identification and significance of sterols in MSW landfill leachate by Caixiang Zhang; Yanxin Wang; Shihua Qi (1-6).
The sterol content of leachate from two different landfills (labeled as landfill J and landfill R, respectively) at Wuhan, central China was examined by GC/MS. About 20 types of sterols were identified according to their mass spectra of TMS (trimethylsilyl derivates) ethers and their eluting orders. Three types of indices of sterols, namely the ratio of 5β/(5β + 5α) stanol, the ratio of coprostanol/epicoprostanol and the ratio of coprostanol/cholesterol, were used to assess and cross-validate sterol sources. The results showed that landfill R suffered faecal pollution while there are complex sterol sources in landfill J. The ratios of cholesterol/(chloesterol + cholestanol) were 0.24 in landfill R and 0.32 in landfill J, indicating cholesterol reduction in both landfills. C29 sterols consisted of 58% of total sterols in landfill J leachate. The sources for the landfill leachate included not only allochthonous domestic wastes, but biodegradation products of autochthonous wastes in the landfills.
Keywords: Sterols; Landfill leachate; Biomarkers; GC/MS;

Cyadox (CYX) is an antimicrobial growth-promoter of the quinoxalines. It is highly effective on improving growth and feed conversion of chicken with little toxicity. For food safety concerns, HPLC-UV methods were developed for the sequential determination of CYX and its major metabolites, 1,4-bisdesoxycyadox (BDCYX) and quinoxaline-2-carboxylic acid (QCA), in plasma, muscle, liver, kidney and fat of chicken. For CYX and BDCYX, samples were subjected to a deproteinisation, a degrease and a liquid–liquid extraction. For QCA, samples were subjected to an alkali hydrolysis, a liquid–liquid extraction and a cation exchange column (AG MP-50 resin) clean-up. Analysis was performed on a RP-C18 column by UV detection with a gradient program of wavelength. Gradient elution was performed at a flow of 1 mL/min. The limits of quantification for CYX, BDCYX and QCA in plasma and tissues were 0.025 μg/g, and 0.002 μg/g for QCA in muscle. The recoveries of three compounds in plasma and tissues were 70–87% with inter-day relative standard deviation (R.S.D.) less than 10%. An animal experiment was performed to show the applicability of the present methods in real biological samples, which demonstrated a satisfactory applicability since all compounds could be detected nearly in all tissues. The present methods were highly sensitive and accurate, and could therefore be useful in pharmacokinetic and residue studies for cyadox in chicken. The developed methods will be further applied in the residue screening of cyadox and its metabolites in chicken.
Keywords: Cyadox; Bisdesoxycyadox; Quinoxaline-2-carboxylic acid; HPLC-UV; Plasma; Tissues;

An automated SPE/LC/MS/MS method for the analysis of cocaine and metabolites in whole blood by Eshwar Jagerdeo; Madeline A. Montgomery; Marc A. LeBeau; Martin Sibum (15-20).
As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50 mm × 3.00 mm i.d., 5 μm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-μL sample injection, linearity was analyte dependent but generally fell between 8 and 500 ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16 ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47 ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and %precision as <9% for all components at each QC level.
Keywords: SPE; LC/MS; Cocaine; Cocaine metabolites; Whole blood;

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide–monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels.
Keywords: Affinity chromatography; Ligand; Peptide; Plasmid DNA purification; Single step purification;

A method for the simultaneous analysis of asymmetric dimethylarginine, symmetric dimethylarginine, monomethylarginine and arginine in human plasma and urine, with short analysis time and isotopic internal standardisation for each analyte is described. The method requires neither sample derivatisation nor the need for chromatographic separation of analytes. The method described shows good precision and accuracy and is suited for both research purposes and implementation in the busy, routine clinical laboratory. In addition the synthesis and utilisation of isotopically labelled symmetric dimethylarginine and monomethylarginine is described for the first time, avoiding the use of surrogates such as homoarginine for internal standardisation.
Keywords: Asymmetric dimethylarginine; Symmetric dimethylarginine; Monomethylarginine; Arginine; Mass spectrometry;

An automated, highly sensitive LC-MS/MS assay for the quantification of the opiate antagonist naltrexone and its major metabolite 6β-naltrexol in dog and human plasma by Claudia Clavijo; Jamie Bendrick-Peart; Yan Ling Zhang; Gillian Johnson; Antje Gasparic; Uwe Christians (33-41).
To support animal studies and clinical pharmacokinetic trials, we developed and validated an automated, specific and highly sensitive LC-MS/MS method for the quantification of naltrexone and 6β-naltrexol in the same run. In human plasma, the assay had a lower limit of quantitation of only 5 pg/mL. This was of critical importance to follow naltrexone pharmacokinetics during its terminal elimination phase. The assay had the following key performance characteristics for naltrexone in human plasma: range of reliable quantification: 0.005–100 ng/mL (r 2  > 0.99), inter-day accuracy (0.03 ng/mL): 103.7% and inter-day precision: 10.1%. There were no ion suppression, matrix interferences or carry-over.
Keywords: Naltrexone; 6β-Naltrexol; LC-MS/MS; Column switching; Dog plasma; Human plasma;

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma by Chadi Abbara; Isabelle Bardot; Annie Cailleux; Guy Lallement; Anne Le Bouil; Alain Turcant; Pascal Clair; Bertrand Diquet (42-50).
A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100 μl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra® MS C8 column (100 mm × 2.1 mm, i.d. 3.5 μm), with a quick stepwise gradient using a formate buffer (pH 3, 2 mM) and acetonitrile at a flow rate of 0.2 ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1–500 ng/ml for diazepam, 0.25–50 ng/ml for atropine and 5–1000 ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within ±15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone–atropine–pralidoxime combination, in healthy subjects.
Keywords: Avizafone; Diazepam; Atropine; Pralidoxime; Liquid chromatography/tandem mass spectrometry; Pharmacokinetics; Organophosphate agents;

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson’s disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP+), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP+ in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2 M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP+ in brain extracts with 0.4 M perchloric acid (HClO4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP+ using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP+ established the limits of detection (LOD) (S/N = 3) at 0.34 pg/mg tissue and 0.007 pg/mg tissue (5 μl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D. < 6%), and sensitive over a range of 0.001–1 ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP+ concentrations in vivo. Two reference agents, R-(−) Deprenyl and MK-801, known to alter the concentration of MPP+ in MPTP treated mice were evaluated.
Keywords: 1-Methyl-4-phenyl pyridinium; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Liquid chromatography; Tandem mass spectrometry; Mouse striatal tissue sample preparation;

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI–MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100 μL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2 M ZnSO4, and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1 × 12.5 mm column (at 60 °C), and washed with water–acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02 mM sodium acetate, to remove poorly retained components. After washing, water–MeOH (with 0.02% glacial acetic acid and 0.02 mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1–200 ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.
Keywords: Voclosporin; ISA247; Therapeutic drug monitoring; LC/MS/MS; Immunosuppressant; Cyclosporine A;

A SEC-HPLC-ICP MS hyphenated technique for identification of sulfur-containing arsenic metabolites in biological samples by Badal Kumar Mandal; Kazuo T. Suzuki; Kazunori Anzai; Kentaro Yamaguchi; Yoshihisa Sei (64-76).
The present study describes the synthesis and characterization of thioarsenicals using electro-spray ionization-MS and time of flight-MS. Separation of thioarsenicals was found to be better by size-exclusion column compared to anion exchange column coupled with HPLC-inductively coupled argon plasma mass spectrometer (ICP MS). Although four thioarsenicals were confirmed as dimethylthioarsinous acid (m/z  = 138), methylmonothioarsonous acid (m/z  = 122), dimethyldithioarsinic acid (m/z  = 170) and methyltrithioarsonic acid (m/z  = 188), it is noted that HPLC-ICP MS alone were not sufficient for their identification. Also, none of them was stable with time. This is the first report detailing the synthesis and identification of methyltrithioarsonic acid. Both dimethyldithioarsinic acid and dimethylthioarsinous acid were detected in human nail samples while dimethyldithioarsinic acid was found in urine samples. So, the above technique could be applicable to the identification of sulfur-containing biomolecules in the biological samples.
Keywords: Dimethyldithioarsinous acid; Dimethyldithioarsinic acid; Methylmonothioarsonous acid; Methyltrithioarsonic acid and biomolecules;

An in vivo microdialysis sampling method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for continuous simultaneous monitoring of unbound baicalin in rat blood and brain. Microdialysis probes were inserted into the jugular vein and brain cerebrospinal fluid (CSF) of Sprague-Dawley rats then, following administration of baicalin at doses of 24 mg/kg via the candal vein, samples were collected every 20 min and injected directly into the UPLC-MS/MS system. In vitro recoveries of the probes were 19.26% and 18.38%, while in vivo recoveries of the probes were 15.0% and 17.52% for blood and brain, respectively. This improved method offers a rapid quantitative procedure for the determination of baicalin with a retention time of only 1.6 min. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) based on a signal-to-noise ratio of 5 were 2.37 and 0.1 ng/ml for anticoagulant citrate dextrose (ACD) solution, and 1.185 and 0.3 ng/ml for artificial cerebrospinal fluid (aCSF), respectively. The pharmacokinetics results indicated that baicalin could pass through the blood–brain barrier (BBB) and was detectable in brain dialysate. These in vivo microdialysis-based measurements provide a technique for simple sampling and rapid sensitive analysis of unbound baicalin in rat blood and CSF and for further application in pharmacokinetic studies.
Keywords: Baicalin; Microdialysis; UPLC-MS/MS; Pharmacokinetics;

Quantification of sunitinib in human plasma by high-performance liquid chromatography–tandem mass spectrometry by Patton Minkin; Ming Zhao; Zhaoyuan Chen; Jan Ouwerkerk; Hans Gelderblom; Sharyn D. Baker (84-88).
A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.2 mL of plasma with 4.0 mL tert-butyl-methyl-ether extraction solution containing 25 ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50 mm × 2.1 mm i.d., 3.5 μm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150 mL/min for 3 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2–500 ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were ≤10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.
Keywords: Sunitinib; LC/MS/MS; Pharmacokinetics;

Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly by Maria Candida M. Mellado; Cristina Peixoto; Pedro E. Cruz; Manuel J.T. Carrondo; Paula M. Alves (89-94).
Rotavirus VP7 is a glycoprotein that forms the viral capsid outerlayer and is essential to the correct assembly of triple-layered rotavirus-like particles (RLPs). In this work, a novel purification strategy was designed to allow obtaining highly pure monomeric VP7 required for the RLPs in vitro assembly. VP7 production kinetics in baculovirus–insect cells at cell concentration at infection (CCI) of 1 × 106  cells mL−1 was compared in terms of VP7/glycoprotein 64 (gp64) ratio at different multiplicity of infection (MOI). The best productivity was achieved at MOI of 0.1 plaque forming unit (pfu) cell−1 and time of harvest of 80 h post-infection. After preliminary clarification steps, the proteins eluted from Concanavalin A were concentrated and loaded onto size exclusion chromatography. The polishing step was anion exchange chromatography with Mono Q. The high resolution of this column resulted in separation of monomers from dimers of VP7. Overall, the purification protocol yielded high level of purity (>90%). Purified VP7 was characterized by MALDI-TOF mass spectrometry and SDS-capillary gel electrophoresis. The M W and apparent M W were determined as 31.6 and 39 kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.
Keywords: Rotavirus VP7; Purification; VP7 purity; Baculovirus gp64; Contaminants;

A high accuracy method for the quantification of malachite green (MG) and leucomalachite green (LMG) in salmon is described. Analytical challenges including the effects of analyte instability and matrix suppression were minimised by the use of exact matching isotope dilution mass spectrometry. The developed method included overnight extraction in acidified acetonitrile/ammonium acetate buffer and analysis by LC–MS/MS utilising isotopic internal standards. This method was used to determine the level of MG and LMG in a sample of salmon used in an international inter-comparison organised by the Comité Consultatif pour la Quantité de Matière (CCQM). The sum of MG and LMG was found to be 9.32 ± 0.98 ng g−1 at the 95% confidence interval (relative expanded uncertainty 10.5% (k  = 2)). This encompassed the mean and median of the CCQM inter-comparison.
Keywords: Malachite green; Leucomalachite green; Exact matching isotope dilution mass spectrometry;

Detection of C-terminal peptide of proteins using isotope coding strategies by Samir Julka; Demetrius Dielman; Scott A. Young (101-110).
The determination of C-terminal peptide sequence is critical since the C-terminal peptide contains biologically relevant information and often undergoes post-translational processing. Another important application is in estimating purity of the biopharmaceuticals, especially for determining the presence of ragged processed ends and for N-terminally blocked polypeptides and proteins. In this paper, different isotope coding strategies in combination with reversed phase chromatography (RPC) coupled with electrospray ionization–mass spectrometry (ESI–MS) were evaluated to detect the C-terminal peptide from proteolytic digests. These were (i) O18 (ii) acylation and (iii) esterification based isotope coding strategies. Using reversed phase chromatography, the C-terminal peptide was resolved from other internal peptides. The isotope coding approaches specifically rendered a characteristic MS signature to the C-terminal peptide, thereby facilitating its detection. The unique MS signature, along with accurate mass data for the C-terminal peptide was found to be sufficient for its detection and identification. The advantages and limitations of the three approaches will be discussed.
Keywords: C-terminal detection; Isotope coding; O18; Acylation; Esterification; LC–ESI–MS;

Assessment of caspase-4 released free AFC by RP-HPLC and fluorescence detection by Sandra Koehn; Mike Trueck; Tobias G. Poehlmann; Ekkehard Schleussner; Udo R. Markert; Lydia Seyfarth (111-114).
A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40 pmol/mL to 3 nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time (∼7 min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50 μL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.
Keywords: Caspase; Caspase-4-activity; AFC; Ac-Leu-Glu-Val-Asp-AFC; RP-HPLC; Fluorescence;

An approach using microwave-assisted derivatization (MAD) following solid-phase extraction (SPE) combined with gas chromatography–mass spectrometry (GC–MS) was developed to determine amphetamines in urine samples. The parameters affecting the derivatization efficiency – including microwave power and irradiation time – were investigated. Besides, solvent is thought critically important to MAD. Derivatization performance was studied using various solvents and compared with the performance obtained without solvent. Derivatization efficiency was clearly found to be enhanced by the presence of solvent. The highest derivatization efficiencies were obtained in ethyl acetate (EA) under microwave power of 250 W for 1 min. Calibration curves for all amphetamines were linear over a range from 1 to 1000 ng/mL, with correlation coefficients above 0.9992. The intra-day and inter-day precision were less than 15%. The applicability of the method was tested by analyzing amphetamine-abusing subjects urine samples. Accordingly, the solvent-enhanced MAD-GC–MS method appears to be adequate for determining amphetamines in urine.
Keywords: Amphetamines; GC–MS; Microwave-assisted derivatization; Solvent; Urine;

3,4-Methylenedioxymethamphetamine (MDMA) is a psychoactive drug with abuse liability and neurotoxic potential. Mechanisms by which MDMA produces behavioral and neurotoxic effects have yet to be elucidated. By measuring concentrations of MDMA and its metabolites in relevant brain sites, it may be possible to gain insight into mechanisms underlying MDMA actions. For this purpose, an LC–MS assay with electrospray ionization was developed after homogenization of rat brain and enzymatic conjugate cleavage. The method was successfully validated with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect and its use should help to delineate the neurotoxic mechanism of action of MDMA.
Keywords: MDMA; Metabolites; LC–ESI-MS; Validation; Neurotoxicity; Brain; Rat;

Corrigendum to “LC–MS-based metabonomics analysis” [J. Chromatogr. B 866 (2008) 64] by Xin Lu; Xinjie Zhao; Changmin Bai; Chunxia Zhao; Guo Lu; Guowang Xu (125).