Journal of Chromatography B (v.928, #C)

Quantitative analysis of steroid hormones in human hair using a column-switching LC–APCI–MS/MS assay by Wei Gao; Tobias Stalder; Paul Foley; Manfred Rauh; Huihua Deng; Clemens Kirschbaum (1-8).
The analysis of steroid hormones in hair is increasingly used in the field of stress-related research to obtain a retrospective index of integrated long-term hormone secretion. Here, most laboratories have so far relied on immunochemical assays originally developed for salivary analyses. Although these assays are fast and easy to perform, they have a reduced reliability and specificity due to cross-reactivity with other substances and are limited to the detection of one hormone at a time. Here, we report the development of a LC–MS/MS-based method for simultaneous identification of endogenous concentrations of seven steroid hormones (cortisol, cortisone, testosterone, progesterone, corticosterone, dehydroepiandrosterone (DHEA) and androstenedione) in human hair. Hair samples were washed with isopropanol and steroid hormones were extracted from 10 mg whole, nonpulverized hair by methanol incubation. A column switching strategy for on-line solid phase extraction (SPE) was applied, followed by analyte detection on an AB Sciex API 5000 QTrap mass spectrometer. Results indicated linearity of the method for all steroids over ranges of 0.09–90 pg/mg (0.9–900 pg/mg for DHEA) with correlation coefficients ranging between 0.9995 and 0.9999. Intra- and inter-assay coefficients of variation were between 3.7 and 9.1%. The limits of quantification (LOQ) were below (or equal to) 0.1 pg/mg for all steroids, except of DHEA for which the LOQ was 0.9 pg/mg. An analysis of 30 natural hair samples (15 men/15 women) using this method confirmed that all steroid hormones could be quantified at endogenous levels in each individual. In addition, the use of whole hair samples and on-line SPE resulted in a significant reduction in sample throughput times, increasing the applicability of this method for research questions where a larger number of samples needs to be processed.
Keywords: Steroid hormone; Hair; Mass spectrometry; Chromatography, On-line solid phase extraction;

Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay by Wlodzimierz Tszyrsznic; Agnieszka Borowiec; Emilia Pawlowska; Radoslaw Jazwiec; Dorota Zochowska; Irena Bartlomiejczyk; Jolanta Zegarska; Leszek Paczek; Michal Dadlez (9-15).
Therapeutic drug monitoring of immunosuppressive agents is a critical and essential part of patient therapy after organ transplantation. We have developed high-throughput, robust, and rapid liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) methods with common pretreatment procedures for simultaneous quantification of four immunosuppressive agents (everolimus, sirolimus, tacrolimus, and cyclosporin A) in whole blood and one immunosuppressant (mycophenolic acid) in plasma. The new approach used in this work is based on improved sample preparation procedures allowing the analysis of five immunosuppressive drugs. Whole blood was prepared by transferring 100 μL of blood into a 1.5-mL silanized conical test tube. Zinc sulfate solution (150 μL), containing deuterated internal standards, was added to perform hemolysis. The samples were vortexing for 10 s, followed by the addition of 250 μL acetonitrile, containing internal standard for cyclosporin A, to precipitate proteins. The mixture was vortexed for 1 min and centrifuged for 2 min at 14,000 rpm. The whole supernatant was transferred to a vial. To prepare blood plasma, the hemolysis step involving the addition of zinc sulfate was omitted and, instead of acetonitrile, methanol was used as the solvent for the internal standard (mycophenolic acid-d3). The volumes of chemicals used in this procedure were the same as those used in the procedure for immunosuppressants in whole blood. The basic validation parameters for the analytical methods were limits of detection (0.5 ng/mL for everolimus, sirolimus and tacrolimus, 25 ng/mL for cyclosporin A and 100 ng/mL for mycophenolic acid), precision (<15%), recovery (>84%), repeatability and reproducibility. Possible mutual ion suppression was eliminated in the presence of internal standards. The method developed for the quantitation of immunosuppressants in whole blood was used to analyze 276 patient samples containing tacrolimus and 55 samples containing cyclosporin A. The results from LC/MS/MS were compared to those obtained from immunoassays of the same samples. Immunoassays significantly overestimated the concentrations of immunosuppressants.
Keywords: Immunosuppressants; Therapeutic drug monitoring; Liquid chromatography–tandem mass spectrometry;

Free glycans derived from glycoproteins present in human sera by Kinya Iwatsuka; Sakie Watanabe; Mitsuhiro Kinoshita; Kazuya Kamisue; Keita Yamada; Takao Hayakawa; Tadashi Suzuki; Kazuaki Kakehi (16-21).
During the course of studies on the analysis of O-glycans in biological samples, we found that significant amount of free glycans are present in normal human serum samples. The most abundant free glycan was disialo-biantennary glycan typically observed in transferrin which is one of the abundant glycoproteins found in sera. Minor glycans were also considered to be mainly due to transferrin, but some glycans were derived from mucin-type O-glycans, although the amount was quite minute. However, high mannose-type glycans could not be detected at all. Although there have been many reports on the presence of intracellular “free” N-glycans (mainly derived from high mannose-type glycans) generated either from lipid-linked oligosaccharides or from misfolded glycoproteins through endoplasmic-reticulum associated protein degradation pathway, there is little information on the presence of free glycans in extracellular matrix and biological fluids such as serum. This report is the first one which demonstrates the presence of free glycans due to glycoproteins in sera.
Keywords: Free glycans; Serum; Transferrin; HPLC; ESI-TOF-MS;

There is a growing need both clinically and experimentally to improve the determination of the blood levels of multiple chemical constituents in herbal medicines. The conventional multiple reaction monitoring (cMRM), however, is not well suited for multi-component determination and could not provide qualitative information for identity confirmation. Here we apply a dynamic triggered MRM (DtMRM) algorithm for the quantification of 20 constituents in an herbal prescription Bu-Zhong-Yi-Qi-Tang (BZYQT) in rat plasma. Dynamic MRM (DMRM) dramatically reduced the number of concurrent MRM transitions that are monitored during each MS scan. This advantage has been enhanced with the addition of triggered MRM (tMRM) for simultaneous confirmation, which maximizes the dwell time in the primary MRM quantitation phase, and also acquires sufficient MRM data to create a composite product ion spectrum. By allowing optimized collision energy for each product ion and maximizing dwell times, tMRM is significantly more sensitive and reliable than conventional product ion scanning. The DtMRM approach provides much higher sensitivity and reproducibility than cMRM.
Keywords: Herbal medicines; Multi-component determination; Dynamic triggered multiple reaction monitoring; Triple quadrupole tandem mass spectrometer;

Applicability of LC–MS/MS to optimize derivatization of topiramate with FMOC-Cl using reacted/intact drug ratio by Bahareh Mohammadi; Esmail Tammari; Sajad Fakhri; Gholamreza Bahrami (32-36).
Topiramate is an antiepileptic agent, which is structurally different from the other anticonvulsants. The drug has no UV–Vis absorption or emits fluorescence. Thus for its analysis using high performance liquid chromatography (HPLC) with conventional UV or fluorescence detectors, the drug should be derivatized with a suitable reagent. In previous study using fluorenylmethyl chloroformate (FMOC-Cl) and HPLC coupled with fluorescence detector, we reported an analytical method for derivatization and analysis of the drug in human serum. In this method, several factors including time and temperature of the reaction, pH and concentration of the used buffer, ratio of organic phase in the medium and removal of the reagent excess by glycine should be optimized to obtain maximum yield of the product. In HPLC coupled with fluorescence detector, there is not any signal from intact topiramate and only the final product (FMOC-topiramate) is appeared. Thus to optimize the reaction conditions for obtaining the highest derived yield, intensity of the final product peak is considered as a criteria for progression of the reaction. In LC–MS/MS system however, both free and reacted topiramate are visible in observed spectra. In the present study reaction of the drug with FMOC-Cl was re-optimized using LC–MS/MS technique on the basis of reacted/free topiramate ratio as the new and more accurate index. The results showed that, ratio of organic/aqueous phase has a dominant effect on the reaction, the most efficient temperature is 70 °C and the reaction is reversed following addition of the glycine.
Keywords: Topiramate; Derivatization; FMOC-Cl; LC–MS/MS; Optimization;

The present work describes a sensitive and specific automated immunoaffinity in-tube solid-phase microextraction coupled with liquid chromatography with fluorescence detection (LC-FD) method for the determination of interferon α2a in plasma samples for therapeutic drug monitoring. To this end, the intrinsic selectivity of the monoclonal antibodies has been combined with in-tube solid phase microextraction by immobilization of these antibodies into the fused silica capillary. The in-tube SPME variables, such as plasma sample volume, draw/eject volume, number of draw-eject cycles, as well as desorption procedure have been optimized, in order to improve the sensitivity of the proposed method. Analyses of interferon α2a in plasma samples were carried out within 12 min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range 0.006–3.0 MIU mL−1, with a correlation coefficient of 0.998. The inter-day precision of the method had a coefficient of variation lower than 6.2%. The proposed automated method has adequate analytical sensitivity and selectivity for the determination of interferon α2a in plasma samples for therapeutic drug monitoring. The proposed method was successfully applied to the plasmas samples analyses from patients in therapy with interferon α-2a, demonstrating a rare application of in-tube SPME for biopharmaceutical (protein) analyses.
Keywords: In-tube SPME; Interferon; Immunoaffinity; Liquid chromatography; Fluorescence detection;

A rapid and selective ultra high performance liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of four major ingredients in Cortex Dictamni extract, including limonin, dictamnine, obacunone and fraxinellone in rats plasma. Nimodipine was used as the internal standard. Following extraction by methyl tert-butyl ether, the analytes were separated on a Thermo Syncronis C18 column by a gradient elution within a runtime of 9 min. The mobile phase consisted of A (methanol) and B (2 mmol/L ammonium acetate in water). The detection was accomplished by using positive ion electrospray ionization in multiple reaction monitoring mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.998. The lower limits of quantification were 9.18 ng/mL for limonin, 12.0 ng/mL for dictamnine, 16.05 ng/mL for obacunone and 4.59 ng/mL for fraxinellone. The intra- and inter-day precision (RSD%) was within 10% and the accuracy (RE%) ranged from −12.9% to 9.7%. This rapid and sensitive method was fully validated and successfully applied to the pharmacokinetic study of limonin, dictamnine, obacunone and fraxinellone in the rat plasma after oral administration of Cortex Dictamni extract.
Keywords: Cortex Dictamni; Limonoids; Furoquinoline alkaloids; Quantitative analysis; Pharmacokinetics; UHPLC–MS/MS;

A selective, sensitive and accurate high-performance liquid chromatographic- tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of iloperidone and its two active metabolites, P88 and P95, in human plasma has been first developed and validated. The analytes and internal standard (IS), pioglitazone hydrochloride, were extracted from human plasma via liquid–liquid extraction with ethyl acetate and separated on a CAPCELL PAK C18 MG IIIcolumn (150 mm × 2.0 mm, 5 μm) set at 40 °C. The mobile phase was acetonitrile: 5 mM ammonium formate containing 0.3% formic acid (pH 4.8) (25:75, v/v), with a flow rate of 0.35 mL/min. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transitions m/z 427.2 → 261.2 for iloperidone, m/z 429.1 → 261.1 for P88 and P95, and m/z 357.1 → 133.7 for the I.S. (pioglitazone hydrochloride). The method was validated to be linear over the concentration range of 10–10,000 pg/mL for iloperidone and P88, 50–15,000 pg/mL for P95. The mean recoveries were more than 78.88%, and the intra- and inter-day precisions were less than 10.24% and accuracy was −5.78 to 5.40%, which indicated that the quantitative method was reliable and accurate. The validated method has been successfully applied to a human pharmacokinetic study of iloperidone and two active metabolites, P88 and P95, after oral administration of 4 mg iloperidone tablets in 12 healthy Chinese volunteers.
Keywords: Iloperidone; Metabolites; LC–MS/MS; Pharmacokinetic;

Analytical methodologies based on liquid or gas chromatography coupled to tandem mass spectrometry for the simultaneous determination of two or more endogenous steroid hormones in human and animal plasma and serum has received increased attention the last few years. Especially in the clinical setting steroid profiling is of major importance in disease diagnostics. This paper discusses recent findings in such multi-steroid hormone procedures published from 2001 to 2012. The aim was to elucidate possible relationships between chosen analytical technique and the obtained analyte sensitivity for endogenous steroid hormones. By evaluating the success, at which the currently applied techniques have been utilized, more general knowledge on the field is provided. Furthermore the evaluation provides directions in which future studies may be interesting to conduct.
Keywords: Steroid hormones; Plasma; Serum; Liquid chromatography; Gas chromatography; Tandem mass spectrometry;

The alkaloids of Piper longum L. (PLA) improved motor dysfunction and dopamine depletion in a rat model of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. A rapid, accurate, simple, and high-performance liquid chromatography–mass spectrometry method was developed and fully validated to simultaneously detect three P. longum L. antiparkinsonian alkaloids (piperine (PPR), piperlonguminine (PPL), and Δα,β-dihydropiperlonguminine (DPPL)) in rat plasma, heart, liver, spleen, lung, kidney, and brain tissues. Rat plasma and tissue homogenates were pretreated with methanol/acetonitrile (1:1, v/v) using a simple protein precipitation method. Chromatographic separation was achieved on a Phenomenex Gemini C18 column (50 mm × 2.00 mm, 5 μm) with a gradient mobile phase containing 0.1% (v/v) formic acid in water or acetonitrile. The elution was pumped at a flow rate of 0.4 ml/min, and the injection volume was 10 μl with a total running time of 4 min. The analysis was performed by selected reaction monitoring of the transitions m/z 285.9 → 201.1, m/z 274.3 → 209.9, and m/z 276.2 → 134.9 for PPR, PPL, and DPPL, respectively. All three analytes showed good linearity (R  > 0.995) in plasma and tissue homogenates, and the lower limit of quantification was 0.20 ng/ml. The distribution of PPR, PPL and DPPL in all 7 tissues was examined. The highest concentrations for PPR and PPL were observed in the liver, while the highest DPPL concentration was observed in the kidney. Following oral administration, the highest levels of PPR, PPL and DPPL in different tissues were found at approximately 2 h. PPR, PPL and DPPL could cross the blood–brain barrier. The present study provides evidences for that PPR, PPL and DPPL may play roles in improving motor dysfunction and dopamine depletion.
Keywords: Piper longum L.; Alkaloid; Antiparkinsonian; HPLC–ESI-MS/MS; Tissue distribution;

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification by Marcus O.D. Sjödin; Magnus Wetterhall; Kim Kultima; Konstantin Artemenko (83-92).
The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55–1.16, r 2: 0.61–0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR > iTRAQ QStar > LF LTQ-FTICR > DML QStar > LF QStar.
Keywords: Relative quantification; Proteomics; Mass spectrometry; Stable isotope labeling; Label free;

An HPLC-UV method for the simultaneous quantification of vemurafenib and erlotinib in plasma from cancer patients by Yi Zhen; Audrey Thomas-Schoemann; Lilia Sakji; Pascaline Boudou-Rouquette; Nicolas Dupin; Laurent Mortier; Michel Vidal; Francois Goldwasser; Benoit Blanchet (93-97).
Vemurafenib and erlotinib are two oral kinase inhibitors approved for the treatment of metastatic melanoma and advanced non-small cell lung cancer, respectively. In contrast with erlotinib, the single published method for analysis of vemurafenib in human plasma is based on mass spectrometry. The purpose of the present study was to develop an HPLC-UV method to simultaneously quantify these two drugs in plasma. Following liquid–liquid extraction, vemurafenib, erlotinib and sorafenib (internal standard) were separated isocratically on a C8 Xterra® MS using a mobile phase of glycine buffer (pH 9.0, 100 mM)/acetonitrile (45:55, v/v). Samples were eluted at a flow rate of 0.9 mL/min throughout the 12-min run. Dual UV wavelength mode was used, with vemurafenib and sorafenib monitored at 249 nm, and erlotinib at 331 nm. The calibration was linear in the range 1.25–100 mg/L and 50–4000 μg/L for vemurafenib and erlotinib, respectively. Inter- and intra-day precision was less than 6.7% and 6.6% for vemurafenib and erlotinib, respectively. This analytical method was successfully applied to assess the steady state plasma exposure of these drugs in cancer patients. This accurate method can be used in routine clinical practice to monitor vemurafenib or erlotinib concentrations in plasma from cancer patients.
Keywords: Vemurafenib; Erlotinib; Liquid chromatography; UV detection; Cancer patients; Therapeutic drug monitoring;

Rapid determination of nerve agent biomarkers at low-ppb levels in urine samples was achieved by direct derivatization and sample analysis using gas chromatography–tandem mass spectrometry. The studied biomarkers were alkylphosphonic acids (APAs), as they are specific hydrolysis products of organophosphorus nerve agents that can be used to verify nerve agent exposure. The sample preparation technique employed involves rapid direct derivatization (5 min) of acidified urine samples (25 μL) using a highly fluorinated phenyldiazomethane reagent [1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene]. The derivatization conditions were optimized using statistical experimental design and multivariate data analysis. The APA derivatives were analyzed by GC–MS and MS/MS using negative ion chemical ionization. The selectivity and sensitivity of analyses performed by low and high resolution single ion monitoring MS-mode were compared with those performed by multiple reaction monitoring MS/MS-mode. The MS/MS technique offered the greatest sensitivity and selectivity of the tested mass spectrometric techniques, with limits of detection ranging from 0.5 to 1 ng APAs/mL of urine. The method's robustness was evaluated using urine samples from the OPCW 2nd biomedical confidence building exercise and all APAs present in the samples were conclusively identified. The method thus offers excellent performance and is viable for the simultaneous trace determination of a wide range of nerve agent markers.
Keywords: Chemical warfare agents; Organophosphorus nerve agents; Alkylphosphonic acids; Biomedical samples; Direct derivatization;

The aqueous two-phase system (ATPS) with polyethylene glycol (PEG) and hydroxypropyl starch (HPS) was evaluated for the extraction of human immunoglobulin G (IgG) from human serum albumin (HSA). The partitions of IgG in PEG/sulphate, PEG/phosphate, PEG/Dextran and PEG/HPS ATPSs were compared, and the results indicated that PEG/HPS system was most suitable for IgG extraction. The effects of the concentrations of PEG, HPS and NaCl addition and pH on the partition of IgG and HSA in PEG/HPS ATPS were investigated. It was found that with 15% NaCl addition at pH 8.0 IgG could be largely recovered in the top PEG-rich phase and most of HSA kept in the bottom phase. In addition, the response surface methodology (RSM) was used to optimize the extraction of IgG from the protein mixture of IgG and HSA. The optimal conditions were obtained as 12% (w/w) PEG 4000, 18% (w/w) HPS and 10% (w/w) NaCl at pH 8.0. The extraction yield of IgG in the top phase was 99.2% and the purification factor could reach 5.28. The back extractions of IgG into a phosphate-rich bottom phase were also studied. The total purification factor was 5.73 with the yield of 84.0%. The results indicated that PEG/HPS ATPS might be a promising alternative for the primary recovery of IgG from the complicated feedstock.
Keywords: Aqueous two-phase system; Hydroxypropyl starch; Human immunoglobulin G; Purification; Response surface methodology;

A sensitive analytical method for determination of doxycycline (DC) residues in chicken fat/fat and skin was developed. The extraction, in the presence of the internal standard (IS) minocycline (MINO), was carried out using solution of oxalic acid (pH 4.0) and ethyl acetate. The samples were cleaned up by solid phase extraction (SPE) procedure using, at first carboxylic acid and then polymeric Strata X cartridges. Chromatographic separation of DC by LC–UV was achieved on a Luna C8 analytical column and for LC–MS/MS analysis Luna C18 column was used. The presented procedures were evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCβ), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. The limit of detection (LOD) was 10 μg/kg for LC–UV and 1 μg/kg for LC–MS/MS method. The limit of quantitation (LOQ) was 15 and 2 μg/kg for LC–UV and LC–MS/MS, respectively. The absolute recovery for the LC–UV and relative recovery for the LC–MS/MS method at 300 μg/kg concentration level were 79%; 101% for fat and 82%; 99% for fat and skin, respectively. The developed liquid chromatography with ultraviolet detection (LC–UV) and liquid chromatography tandem mass spectrometry (LC–MS/MS) methods have been applied to quantitative determination of doxycycline (DC) in samples of chicken fat tissue obtained from animals treated with DC.
Keywords: Doxycycline; Fat; Validation; LC–UV; LC–MS/MS;

Small molecules that bind DNA with high specificity present a promising opportunity for application as chromatographic ligands for plasmid DNA (pDNA) purification. This research used the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as an immobilized ligand for the specific separation of supercoiled (sc) pDNA by affinity chromatography. The results showed that the protonated DAPP-Sepharose support has a great affinity for sc pDNA isoform, separating it from the less active open circular and linear isoforms. All pDNA isoforms were retained in the column using 10 mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22 M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55 M led to the elution of the sc isoform. The binding of pDNA to DAPP-Sepharose varies in function of pH, and the stability of the protonated DAPP–DNA complex decreases with increasing salt concentration.
Keywords: Small DNA ligands; DAPP; Intercalator; Affinity chromatography; Supercoiled plasmid DNA;

The development of an analytical method for urinary metabolites of the riot control agent 2-chlorobenzylidene malononitrile (CS) by James R. Riches; Robert W. Read; Robin M. Black; John M. Harrison; Dawn A. Shand; Eleanor V. Tomsett; Catherine R. Newsome; Nicola C. Bailey; Neil Roughley; Matthew R. Gravett; Sarah J. Stubbs; Richard R. McColm (125-130).
The analysis of biomedical samples such as urine and blood can provide evidence of exposure to chemicals for a range of applications including occupational exposure monitoring, detection of drugs of abuse, performance enhancement in sport and investigations of poisoning and incapacitation. This paper reports the development of an analytical method for two suspected urinary metabolites of the riot control agent 2-chlorobenzylidene malononitrile (CS): 2-chlorohippuric acid and 2-chlorobenzyl-N-acetylcysteine. 2-Chlorohippuric acid was identified in all 2 h post-exposure samples from a set of urine samples taken from army recruits exposed to low levels of thermally dispersed CS during training. 2-Chlorobenzyl-N-acetylcysteine, a metabolite known to be formed in the rat, was not identified in any of the samples. The lower limit of detection (LLOD) for 2-chlorohippuric acid and 2-chlorobenzyl-N-acetylcysteine was 1 ng/ml and 0.5 ng/ml in pooled urine from the pre-exposed subjects. 2-Chlorohippuric acid was rapidly excreted but was detectable in the urine of 17 of the 19 subjects tested 20 h after exposure.
Keywords: 2-Chlorobenzylidene malononitrile; CS; Tear gas; Riot control agents; LC/MS/MS; 2-Chlorohippuric acid;

Adsorption and separation of proteins by collagen fiber adsorbent by Juan Li; Xue-pin Liao; Qi-xian Zhang; Bi Shi (131-138).
The separation of proteins is a key step in biomedical and pharmaceutical industries. In the present investigation, the collagen fiber adsorbent (CFA) was exploited as column packing material to separate proteins. Bovine serum albumin (BSA), bovine hemoglobin (Hb) and lysozyme (LYS) that have different isoelectric points (pIs) were selected as model proteins to investigate the separation ability of CFA to proteins. In batch adsorption, the adsorption behaviors of these proteins on CFA under different pHs and ionic strengths indicated that the electrostatic interaction plays a predominant role in the adsorption of proteins on CFA. CFA exhibited high adsorption capacity to Hb and LYS. In column separation, the proteins were completely separated by adjusting pH and ionic strength of the eluent. The increase of flow rate could reduce the separation time with no influence on the recovery of protein in the experimental range. The protein recovery was higher than 90% even when the CFA column was re-used for 4 times in separation of BSA and LYS, and the retention time of BSA or LYS was almost constant during the repeated applications. In addition, as a practical application, LYS was successfully separated from chicken egg white powder by CFA column.
Keywords: Adsorption; Separation; Protein; Collagen fiber adsorbent; Electrostatic interaction;

Concentrations of nicotine and its metabolites in blood are indicative of patients’ current tobacco exposure, and their quantifications have been clinically applied to multiple assessments including demonstration of abstinence prior to heart–lung transplantation. For the purpose of transplant evaluation, the laboratory work up is extensive; thereby an assay with minimal sample volume is preferred. We developed and validated a rapid LC–MS/MS assay to simultaneously quantitate nicotine and its major metabolites, Cotinine and trans-3′-OH-cotinine (3-OH-Cot), in serum. 100 μL of serum was spiked with deuterated internal standards and extracted by Oasis HLB solid phase extraction cartridge. Nicotine and metabolites in the reconstituted serum extract were separated by Agilent Eclipse XDB-C8 3.5 μm 2.1 mm × 50 mm HPLC column within 4.7 min, and quantified by MS/MS with positive mode electrospray ionization and multiple reaction monitoring. Ion suppression was insignificant, and extraction efficiency was 79–110% at 50 ng/mL for all compounds. Limit of detection was 1.0 ng/mL for nicotine and 3-OH-Cot, and <0.5 ng/mL for Cotinine. Linearity ranges for nicotine, cotinine and 3-OH-Cot were 2–100, 2–1000, and 5–1000 ng/mL with recoveries of 86–115%. Within-day and twenty-day imprecision at nicotine/cotinine/3-OH-Cot levels of 22/150/90, 37/250/150, and 50/800/500 ng/mL were all 1.1–6.5%. The reconstituted serum extracts were stable for at least 7 days stored in the HPLC autosampler at 5 °C. Our method correlates well with alternative LC–MS/MS methods. We successfully developed and validated an LC–MS/MS assay to quantitate concentrations of nicotine and its metabolites in serum with minimal sample volume to assess tobacco exposure of heart–lung transplant patients.
Keywords: LC–MS/MS; Nicotine; Cotinine; Trans-3′-Hydroxycotinine; Transplant;

Validation of an LC–MS bioanalytical method for quantification of phytate levels in rat, dog and human plasma by Fernando Tur; Eva Tur; Irene Lentheric; Paula Mendoza; Maximo Encabo; Bernat Isern; Felix Grases; Ciriaco Maraschiello; Joan Perelló (146-154).
Myo-inositol hexakisphosphate (phytate, IP6) is a naturally occuring compound whose determination in biological matrices is chanllenging. Several benefitial properties have been attributed to IP6 in parallel with the development of suitable analytical methodologies for its analytical determination in urine and some tissues. However, there is a lack of appropriate tools for its determination in plasma samples. In this paper, a direct, sensitive and selective bioanalytical method for the determination of IP6 based on LC–MS is presented. It is the first method published to quantify IP6 in plasma matrices directly through its molecular weight, being consequently a highly specific methodology. The method has been validated in rat, dog and human plasma, according to the acceptance criteria laid down in the FDA guidance Bioanalytical Method Validation. Accuracy and precision were not greater than 15% at medium and high concentrations and not greater than 20% at the LLOQ concentration. The mean absolute recovery obtained ranged from 78.74 to 102.44%, 62.10 to 87.21% and 61.61 to 86.99% for rat, dog and human plasma respectively. The LLOQ was 500 ng mL−1 due to the presence of endogenous IP6 in blank plasma samples and the limit of detection was within the range 30–80 ng mL−1.
Keywords: Phytate; Myo-inositol hexaphosphate; Direct determination; LC–MS; Bioanalytical method; Biological matrices;