Journal of Chromatography B (v.929, #C)

Determination of crenolanib in human serum and cerebrospinal fluid by liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) by Michael S. Roberts; David C. Turner; Thandranese S. Owens; Abhijit Ramachandran; Cynthia Wetmore; Stacy L. Throm; Clinton F. Stewart (1-5).
A LC–ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50 μl of serum or CSF) were prepared for analysis using liquid–liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3 μm, 100 mm × 4.6 mm I.D.) in a column heater set at 50 °C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45 mL/min, and the retention time for both analyte and ISTD was less than 3.5 min. Samples were analyzed with an API-5500 LC–MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4 → 373.1 and m/z 448.2 → 374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5–1000 ng/mL for serum and 0.5–1000 ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib.
Keywords: Crenolanib (CP-868,596); Human serum; Liquid–liquid extraction (LLE); Liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS); Pharmacokinetic studies;

Silica gel column chromatography combined with high performance counter-current chromatography (HPCCC) was employed for the separation of potential anti-tumor compounds from a petroleum ether fraction of a crude extract of Zanthoxylum ailanthoides Sieb. & Zucc. This traditional Chinese medicine was recently found to display high inhibitory activity against A-549 human cancer cells in vitro and Lewis lung cancer in vivo. A 75% aqueous ethanol extract of the stem bark of Z. ailanthoides was fractionated with petroleum ether, ethyl acetate and n-butanol. In this paper, the petroleum ether fraction was pre-separated by silica gel column chromatography with a petroleum ether–ethyl acetate gradient. Two fractions were further separated and purified by HPCCC using n-hexane–ethyl acetate–methanol–water (3:1:2:1, v/v) and petroleum–ethyl acetate–methanol–water (8:6:7:7, v/v). Finally, coumarins and lignans including luvangetin, xanthyletin, hinokinin and asarinin were isolated and identified by MS, 1H and 13C NMR. In total, 56 mg of xanthyletin (1), 140 mg of hinokinin (2), 850 mg of luvangetin (3) and 74 mg of asarinin (4) were obtained from approximately 50 g of petroleum ether extract, in 96.0%, 94.0%, 99.0% and 94.0% purity, respectively, as determined by HPLC. The separation method proved to be efficient, especially for those minor components.
Keywords: Antitumor activity; Coumarins; Counter-current chromatography (CCC); Zanthoxylum ailanthoides Sieb. & Zucc.; Lignans;

The integration of refolding, reconstitution and two-phase partitioning in aqueous two-phase systems (ATPS) which is composed of polyethylene glycol (PEG) and sodium carbonate (Na2CO3) was employed as a novel method for recovery and purification of recombinant Pseudomonas fluorescens proline dehydrogenase (ProDH). To obtain an optimal condition, the influence of different parameters, such as PEG molecular weight (MW), type and concentration of salt, pH, and NaCl addition on the partitioning features of target enzyme was also investigated. Combining the refolding, reconstitution and two-phase partitioning in an optimized ATPS of 14% (w/w) PEG-1000 and 12% (w/w) Na2CO3 at pH 8.0 resulted in a yield of 61.5%, purification factor of 27.0, recovery of 430.7% and specific activity of 600.0 U/mg. The recombinant P. fluorescens enzyme was preferentially partitioned into the top PEG-rich phase. NaCl addition decreased greatly the partition coefficient and recovery of ProDH. In addition, the resulting protein pattern by SDS-PAGE demonstrated the adequacy of presented procedure for enzyme recovery. Overall, our data confirmed that the PEG-1000/Na2CO3 aqueous two-phase partitioning combined with refolding and reconstitution can be used as an efficient integrated process for recovery and purification of recombinant ProDH from inclusion bodies in only one step.
Keywords: Aqueous two-phase systems (ATPS); Proline dehydrogenase (ProDH); Pseudomonas fluorescens; Process integration; Purification; Recovery;

Organophosphate and pyrethroid insecticides and phenoxyacetic acid herbicides represent important classes of pesticides applied in commercial and residential settings. Interest in assessing the extent of human exposure to these pesticides exists because of their widespread use and their potential adverse health effects. An analytical method for measuring 12 biomarkers of several of these pesticides in urine has been developed. The target analytes were extracted from one milliliter of urine by a semi-automated solid phase extraction technique, separated from each other and from other urinary biomolecules by reversed-phase high performance liquid chromatography, and detected using tandem mass spectrometry with isotope dilution quantitation. This method can be used to measure all the target analytes in one injection with similar repeatability and detection limits of previous methods which required more than one injection. Each step of the procedure was optimized to produce a robust, reproducible, accurate, precise and efficient method. The required selectivity and sensitivity for trace-level analysis (e.g., limits of detection below 0.5 ng/mL) was achieved using a narrow diameter analytical column, higher than unit mass resolution for certain analytes, and stable isotope labeled internal standards. The method was applied to the analysis of 55 samples collected from adult anonymous donors with no known exposure to the target pesticides. This efficient and cost-effective method is adequate to handle the large number of samples required for national biomonitoring surveys.
Keywords: Biomonitoring; Herbicides; LC–MS; Organophosphate insecticides; Pesticide metabolites; Pyrethroids;

► PMME was developed for the determination of N-methylcarbamate pesticides coupled with HPLC–DAD. ► A poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith was selected as the extraction medium for PMME. ► The method was applied to the determination of N-methylcarbamate pesticides in vegetable samples.A simple, rapid and sensitive method for simultaneous determination of three N-methylcarbamate pesticides (carbaryl, pirimicarb, and isoprocarb) in vegetables was developed by coupling polymer monolith microextraction (PMME) to high-performance liquid chromatography (HPLC). A poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith was selected as the extraction medium for PMME. To achieve optimum extraction performance, several parameters were investigated, including desorption solvent, desorption flow rate, sample flow rate, sample volume, sample pH values, inorganic salt and organic solvent content of the sample solution. Under the optimum experimental conditions, the method provides an acceptable linearity (5–5000 μg/kg), low limits of detection (0.36–2.6 μg/kg), good precision (intra-day relative standard deviations < 2.53%, inter-day relative standard deviations <6.36%). Finally, the developed method was successfully applied to the determination of N-methylcarbamate pesticides in vegetables, and the trueness was evaluated by recovery experiments. The obtained relative recoveries were in the range of 70.4–98.5%. This PMME method integrates sample extraction, purification, and preconcentration of analytes into one single step and it also has several advantages such as solvent-free extraction, small sample volume, high enrichment, convenience, and flexibility operation.
Keywords: Poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction; High performance liquid chromatography; N-methylcarbamate pesticides; Vegetables;

A selective and sensitive ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–MS) method was developed for the simultaneous determination of ten biogenic amines (tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, adrenaline, dopamine and spermine) in a thymopolypeptides injection from the Chinese market for the first time. Biogenic amines (BAs) were pre-column derivatised by dansyl chloride after direct sample dilution. Dansylated amines were separated on an ACQUITY UPLC BEH Shield RP18 column (2.1 mm × 150 mm I.D., 1.7 μm) using a gradient elution. Quantification was done by monitoring fragment ions of each derivative under the MS mode of multiple reaction monitoring (MRM). A satisfactory result of method validation was obtained. The linearity ranged from 0.32 to 1182.9 μg/L and the correlation coefficients (r) for all amines were above 0.99. The LOD ranged from 0.08 μg/L for 2-phenylethylamine and tyramine to 8.00 μg/L for adrenaline; the LOQ ranged from 0.32 μg/L for 2-phenylethylamine to 12.12 μg/L for dopamine. The recovery ranged from 75.8 to 110.3% after spiking standard solutions of BAs to a sample at three levels. The intra and inter-day precision RSD were 0.78–8.85% and 1.39–9.93% respectively. Eighty-four injections were analyzed by this method. Nine biogenic amines were found in them except adrenaline. Moreover, the relationship between the result of test for depressor substances and the content of BAs was statistically analyzed.
Keywords: Biogenic amines; UPLC–MS; Thymopolypeptides injection; Multiple reaction monitoring (MRM); Test for depressor substances;

Prion protein (PrPc) interacts with histone H3 confirmed by affinity chromatography by Hanning Cai; Ying Xie; Lingyin Hu; Jingjing Fan; Renqiang Li (40-44).
The histones including H2a, H2b, H3 and H4 purified from pig liver tissue were immobilized onto Sepharose 4B to create a histone-Sepharose column. During chromatography of cow milk casein by histone-Sepharose column, two isoforms of prion protein (PrPc) with 34 and 30 kDa molecular mass corresponding to diglycosylated and monoglycosylated PrPc respectively were found to be captured by histone ligands. To further verify the interaction between histones and PrPc, the PrPc-Sepharose column was prepared and used to separate the histones. Two chromatography processes and SDS-PAGE demonstrated that only H3 in the histones was found to interact with PrPc. This study suggested H3 could be the target molecule of PrPC in nuclei, which might be useful for understanding the prion disease.
Keywords: Prion protein; Histone H3; Interaction; Affinity chromatography;

Determination of acyl-CoA esters and acyl-CoA synthetase activity in mouse brain areas by liquid chromatography–electrospray ionization-tandem mass spectrometry by Fumiyo Kasuya; Teiichi Masuyama; Taku Yamashita; Kazuo Nakamoto; Shougo Tokuyama; Hiromi Kawakami (45-50).
The acyl-CoA levels and the acyl-CoA synthetase activities in 7 areas of mouse brain were determined by liquid chromatography–electrospray ionization-tandem mass spectrometry. Twenty-one acyl-CoA esters of C2:0, C4:0, C6:0, C8:0, C10:0, C12:0, C14:1, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0, C20:4, C20:5, C22:0, C22:5, C22:6 and C24:0 were detected in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, hypothalamus, midbrain and medulla oblongata. The brain areas contained primarily the acyl-CoAs of the C16:0, C18:0, C18:1, C20:4 and C22:6 species. The relative abundances of the acyl-CoAs of C16:0, C18:0 and C18:1 were considerably higher than those of C20:4 and C22:6. The levels of medium-chain acyl-CoAs were only 1.2% that of the long-chain acyl-CoAs. The differences in the acyl-CoA synthetase activities in each area of mouse brain were less dramatic. The order of the acyl-CoA synthetase activities for fatty acids of different chain lengths was palmitic acid > arachidonic acid > docosahexaenoic acid > octanoic acid. The analytical method proved to be very useful for the analysis of the acyl-CoA profile of tissues. Our results have important implications for understanding the regulation of acyl-CoA synthetase activity and long-chain fatty acid turnover in the phospholipids in the brain.
Keywords: Acyl-CoAs; Acyl-CoA synthetase; Brain; PUFA; LC–ESI-MS/MS;

A new LC–MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood by Tereza Moore; Anthony Le; Anna-Kaisa Niemi; Tony Kwan; Krinstina Cusmano-Ozog; Gregory M. Enns; Tina M. Cowan (51-55).
Reduced levels of glutathione (γ-glutamylcysteinylglycine, GSH) and the ratio of GSH to glutathione disulfide (GSSG) can serve as important indicators of oxidative stress and disease risk. Measured concentrations of GSH and GSSG vary widely between laboratories, largely due to the instability of GSH during sample handling and variables arising from different analytical methods. We have developed a simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for measuring whole blood GSH and GSSG that minimizes preanalytic and analytic variability, reliably eliminates interference from ion suppression, and can easily be implemented in clinical laboratories. Samples were deproteinized with sulfosalicylic acid (SSA) and derivatized with N-ethylmaleimide (NEM) in a single preparative step, and the resulting supernatants combined with stable-isotope internal standards (GSH-13C, 15N-NEM and GSSG-13C,15N), subjected to chromatographic separation using a Hypercarb column, and analyzed by MS/MS in the positive-ion mode. Results showed excellent linearity for both GSH and GSSG over the ranges of physiologic normal, with inter- and intra-assay CV's of 3.1–4.3% and accuracy between 95% and 101%. The lower limits of detection (LLOD) were 0.4 μM for GSH and 0.1 μM for GSSG and the lower limits of quantitation (LLOQ) were 1.5 μM for GSH and 0.1 μM for GSSG. Derivatized samples are stable for at least 3 years when stored at −80 °C, and underivatized samples for at least 24 h at either 4 °C or room temperature. Reference intervals were determined for 59 control samples, and were (mean ± SD): GSH 900 ± 140 μM; GSSG 1.17 ± 0.43 μM; GSH/GSSG 880 ± 370.
Keywords: Glutathione; Glutathione disulfide; Tandem mass spectrometry; Oxidative stress; Clinical testing;

Metabolite profiling of astilbin in rat sera using UPLC/MSE and impact of its metabolites on immunosuppressive activity by Jiayu Tang; Jianming Guo; Jingjing Fan; Cheng Qian; Feifei Tao; Xiang Zhou; Xuefeng Wu; Yang Sun; Jianxin Li; Yan Shen; Qiang Xu (56-62).
Astilbin, a natural flavonoid, has been shown to have a selective immunosuppressive activity on activated T lymphocytes. In our previous study, 3′-O-methylastilbin was identified as an active metabolite in vivo. However, more comprehensive information regarding the kinetics and metabolic characteristics of astilbin is yet unknown. Here, we isolated and identified 4 metabolites after incubating astilbin with rat liver microsomal/cytosolic fractions. Besides 3′-O-methylastilbin, 4′-O-methylastilbin was firstly identified and detected in the rat plasma after either oral or intravenous administration of astilbin. And phenotypic differences in the metabolic profile were observed between the two administration routes when using UPLC/MSE to measure the metabolites in the plasma. Moreover, 4′-O-methylastilbin decreased serum transaminases elevation in mice with concanavalin A-induced liver injury and reduced the mRNA expression of TNF-alpha and IFN-gamma in primed lymph cells upon antigen restimulation. The immunosuppressive activity of 4′-O-methylastilbin appears weaker than astilbin and 3′-O-methylastilbin. Taken together, the characterization of the comprehensive metabolic profile of astilbin confirmed 3′-O-methylastilbin as the major active form of astilbin metabolites, revealed 4′-O-methylastilbin as a minor active form, and helped us to evaluate the route of astilbin administration, which is beneficial for the treatment of human immune diseases.
Keywords: Astilbin; Metabolite; UPLC/MSE; Rat; Immunosuppression;

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) has been developed and validated for simultaneous quantification of the three main bioactive compounds, i.e., ligustilide, dehydrocostuslactone and α-cyperone in dog plasma after oral administration of the essential oil of Xiang–Fu–Si–Wu Formula (XEO). Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL min−1, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 190.5 → 90.9 for ligustilide, 231.1 → 185.1 for dehydrocostuslactone, 219.2 → 123.0 for α-cyperone and 748.5 → 158.1 for IS. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 5.12 ng/mL for ligustilide, 1.06 ng/mL for dehydrocostuslactone and 1.89 ng/mL for α-cyperone, respectively, and the calibration curves obtained were linear (r  > 0.99) over the concentration range approximately 1–1000 ng/mL. The intra- and inter-day precision was less than 15% and the accuracy was within ±9.2%. After validation, this method was successfully applied to a pharmacokinetic study where dogs were orally given 0.3 g/kg XEO, equivalent to 183.6 mg/kg of ligustilide, 5.0 mg/kg of dehydrocostuslactone and 26.2 mg/kg of α-cyperone, respectively.
Keywords: Ligustilide; Dehydrocostuslactone; α-Cyperone; UPLC–MS/MS; Pharmacokinetics; Dog plasma;

Berbamine (BBM), a natural compound from Chinese herb Berberis amurensis, has recently received a great deal of attention due to its anti-leukemia activity. In this study, a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of BBM in rat plasma was developed for the first time. Caffeine was used as an internal standard. Chromatographic separation was performed on an ODS column with gradient elution using methanol-1% formic acid as mobile phase at a flow rate of 0.3 mL/min. Quantification was through tandem mass spectrometry with positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) at m/z 305.2 → 566.3 and 195.1 → 138.0 for BBM and IS, respectively. The lower limit of quantification was 1 ng/mL with a linear range of 1–1000 ng/mL. The intra- and inter-day assay precision (RSD) ranged from 2.0–6.4% to 2.5–5.5%, respectively, and the intra- and inter-day assay accuracy (RE) was between −5.8–6.0% and −6.5–1.4%, respectively. The validated method was successfully applied to the preclinical pharmacokinetic studies of BBM in rats. The elimination half-lives (t 1/2) were (472.4 ± 66.1), (509.6 ± 97.0) and (486.2 ± 94.6) min after single intravenous administration of 2, 4 and 8 mg/kg BBM, respectively. The area under the plasma concentration versus time curve (AUC0–24 h) and initial plasma concentration (C 0) were linearly related to dose.
Keywords: Berbamine; LC–MS/MS; Pharmacokinetics; Rat plasma;

Evaluation of oxidation and glyco-oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylserine by LC–MS/MS by Elisabete Maciel; Renata Faria; Deolinda Santinha; M. Rosário M. Domingues; Pedro Domingues (76-83).
Phosphatidylserine (PS) is an aminophospholipid found mainly in the plasma membranes of all mammalians cells, playing important roles in biological processes such as apoptosis and cell signaling. Due to the presence of a free amine group, under hyperglycemic conditions, PS can undergo glycation reaction, which may increase the susceptibility to oxidation. However, far too little attention has been paid to glycation and oxidation of PS. In this work we studied the oxidation, glycation and glyco-oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-l-serine (PAPS). PAPS and glycated PAPS were oxidized through a Fenton reaction and the oxidation products were monitored by ESI-MS in negative mode. Also, we developed a new sensitive liquid chromatography method coupled with tandem mass spectrometry (LC–MS/MS) to provide a complete profile of oxidized and glyco-oxidized PS. We were able to separate and identify several oxidation products of PAPS and glycated PAPS with modifications in unsaturated fatty acyl chain as long chain oxidation product (hydroxy and mono to tetra-hydroperoxy derivatives), and short chain products with a shortened fatty acyl chain with C5 and C8 length and aldehyde or carboxylic terminal. We have also observed oxidation products arising from structural changes in the serine polar head, which lead to oxidation products with an acetic acid terminal (glycerophosphoacetic acid derivatives) and lysoPS species. Oxidation of glycated PAPS gave rise to several products involving oxidative cleavages of the glucose moiety, mainly between C1 and C2 of the sugar unit. These oxidation products with different polar head groups have shown distinct neutral loss fragmentation patterns. Simultaneous oxidative modifications of the polar head and the fatty acyl chains were also observed. The findings from this study contribute to an ongoing effort to detect PS oxidation and glyco-oxidation products in biological systems.
Keywords: Phosphatidylserine; Aminophospholipid; Oxidative stress; Glycation; Mass spectrometry;

Here, we describe the development and application of a liquid chromatography–tandem mass spectrometry method with positive electrospray ionisation and scheduled multiple reaction monitoring algorithm (s-MRM) to analyse synthetic cannabinoids (SC) combined with new designer drugs (NDD) in human serum. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column and a mobile phase consisted of A (H2O/methanol = 95/5, v/v) and B (H2O/methanol = 3/97, v/v) – both with 10 mM ammonium acetate and 0.1% acetic acid (pH = 3.2), were used for the separation. A binary flow pumping mode with a total flow rate of 0.400 mL/min was used. A single sample extraction with 1-chlorobutane for both substance groups was performed. Acceptable linearity in the validated calibration ranges of 0.05–1 ng/mL for SC and 1–50 ng/mL for NDD was achieved. The limit of detection was not greater than 0.02/0.40 ng/mL and the limit of quantification not greater than 0.05/0.50 ng/mL for SC/NDD respectively. The presented study revealed that this method is a very effective way for sensitive SC and NDD identification in human serum and has useful application in hospitals, therapy centres and forensic psychiatric centres. S-MRM ensures a method upgrade with a smaller loss of sensitivity, precision and accuracy in comparison to traditional MRM methods. Also addition of new SC and NDD can be performed in the future.
Keywords: Synthetic cannabinoids; Cathinones; Piperazines; Amphetamines; Pyrovalerones; Scheduled MRM;

Preparation and characterization of sodium dodecyl sulfate doped polypyrrole solid phase micro extraction fiber and its application to endocrine disruptor pesticide analysis by Korcan Korba; Levent Pelit; Füsun Okçu Pelit; K. Volkan Özdokur; Hasan Ertaş; Ahmet E. Eroğlu; F. Nil Ertaş (90-96).
A robust in house solid-phase micro extraction (SPME) surface has been developed for the headspace (HS)-SPME determination of endocrine disruptor pesticides, namely, Chlorpyrifos, Penconazole, Procymidone, Bromopropylate and Lambda-Cyhalothrin in wine sample by using sodium dodecylsulfate doped polypyrrole SPME fiber. Pyrrole monomer was electrochemically polymerized on a stainless steel wire in laboratory conditions in virtue of diminishing the cost and enhancing the analyte retention on its surface to exert better selectivity and hence the developed polymerized surface could offer to analyst to exploit it as a fiber in headspace SPME analysis. The parameters, mainly, adsorption temperature and time, desorption temperature, stirring rate and salt amount were optimized to be as 70 °C and 45 min, 200 °C, 600 rpm and 10 g L−1, respectively. Limit of detection was estimated in the range of 0.073–1.659 ng mL−1 for the pesticides studied. The developed method was applied in to red wine sample with acceptable recovery values (92–107%) which were obtained for these selected pesticides.
Keywords: Polypyrrole fiber; Head space solid phase micro extraction; Endocrine disruptor pesticide; Gas chromatography; Red wine;

Determination of antazoline hydrochloride in Beagle dog plasma by HPLC–UV and its application to pharmacokinetics by Xiaotian Li; Yanle Chu; Yu Ke; Linxi Wang; Tong Yu; Lianqi Hao (97-101).
In order to evaluate the pharmacokinetics characteristic of antazoline hydrochloride in Beagle dogs, a sensitive and specific HPLC method was developed and validated using phenacetin as the internal standard (IS). The analyte and the IS were extracted from dog plasma by ethyl acetate under the basic condition. The analyte was separated by a C18 column and detected with a variable wavelength UV-detector. The mobile phase consisted of methanol–5 mmol L−1 tetrabutyl ammonium bromide (45:55, v/v) containing 0.5% glacial acetic acid in a flow rate of 1.0 mL min−1. Standard calibration graph for antazoline was linear over a curve range of 20–1600 ng mL−1 (R  > 0.99) and the lower limit of quantification was 20 ng mL−1 using a plasma sample of 500 μL. The intra- and inter-day precision values were less than 14.3% relative standard deviation (RSD). The intra-day assay accuracy was in the range of 98.1–100.6% and the inter-day assay accuracy in the range of 99.2–101.1%. The extraction recoveries were on the average of 88.4% for antazoline and 76.8% for IS. Plasma samples were stable at least for 1 month at −20 °C. This method was successfully applied to pharmacokinetics study of antazoline after intravenous administration to Beagle dogs.
Keywords: Antazoline hydrochloride; Reversed phase ion pair chromatography; Dog plasma; Pharmacokinetics;

Study on urinary profile of inborn errors of metabolism by 18-crown-6 modified capillary electrophoresis with laser-induced fluorescence detection by Jun-Bo Zhang; Meng-Jie Li; Zhuo Li; Xiao-Jing Yan; Jia-Qun Yuan; Wen-Xia Dong; Yu Zhang; Qing-Cui Chu; Jian-Nong Ye (102-106).
Newborn screening in urine is important for the diagnosis of many inborn errors of metabolism (IEM). Capillary electrophoresis with laser-induced fluorescence detection (CE–LIF) is a major technological advance in screening IEM. It has the advantage of sensitive and simultaneous multiple disease screening with minimal sample requirement. The analytes were derivatized with fluorescein isothiocyanate (FITC) prior to CE–LIF analysis. In urine samples, free amino acids (AAs) were well separated from other coexisting components, exhibiting a linear calibration over the concentration range 0.01–5.0 μmol/L with the limits of detection (LODs) ranging from 0.005 to 0.010 μmol/L. The relative standard deviations (RSDs) were in the range 0.1–1.0% for peak area, and 0.2–1.0% for migration time, respectively. Under optimized conditions, the method presented here has been successfully used for the simultaneous and sensitive analysis of seven AAs in urine samples of newborn babies, and evaluating the effect of therapy as well.
Keywords: Capillary electrophoresis; LIF; IEM; FITC;

A simple and cost-effective pre-treatment procedure was developed for 18 sulfonamides in pork using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra high-performance liquid chromatography–positive electrospray ionisation tandem mass spectrometry (UHPLC–ESI-MS/MS). After extraction with phosphate buffer (pH 6.0), a dSPE procedure was conducted with MWCNTs. The pH value of the extract, extraction time with MWCNTs, type and amount of MWCNTs and type of eluent were optimised to increase the sample throughput and sensitivity. The samples were quantified using sulfamethazine-13C6 as an internal standard. The recoveries of the target compounds from the pork samples were most efficient when 150 mg of MWCNTs with an outer diameter of less than 8 nm and a length of 0.5–2 μm was used. A mixture of acetonitrile/50 mM ammonium acetate (95:5, v/v) was shown to be the most suitable solvent for desorbing the compounds from the MWCNTs. The proposed method was validated according to the European Commission Decision 2002/657/EC, which determines linearity, specificity, decision limit (CCα), detection capability (CCβ), recovery, precision and stability.
Keywords: Sulfonamides; Pork; LC–MS/MS; dSPE; MWCNTs;

Quantification of urinary folate catabolites using liquid chromatography–tandem mass spectrometry by Mareile Niesser; Ulrike Harder; Berthold Koletzko; Wolfgang Peissner (116-124).
Folate catabolites p-aminobenzoylglutamate (pABG) and p-acetamidobenzoylglutamate (apABG) in human urine result from break-down of endogenous folate pools and are potential biomarkers of folate status. There is growing interest in analysis of these non-invasive indicators of folate status, since widespread diseases such as cancer, arteriosclerosis and dementia may be linked to disturbed availability of folates. Determination of pABG and apABG in human urine is challenging due to their low urinary concentrations and due to interferences with other urinary compounds. To address these analytical difficulties, we developed an improved LC–MS/MS method with chemical derivatization for fast, selective and sensitive quantification of pABG and apABG in human urine. Forming butyl esters of urinary folate catabolites pABG and apABG improves ionization efficiency as well as enables selective chromatographic separation on standard C18 reversed-phase column material. In contrast to some previously proposed methods for folate catabolites, the new method allows precise differentiation of apABG from pABG. Partial degradation of apABG during derivatization is exactly accounted for using a second differentially labeled stable isotope internal standard. This method is highly sensitive and covers the full range of physiologically occurring concentrations (from 2 to 1000 nmol/L), with volume requirements of only 80 μL urine. Method performance has been validated according to widely accepted standard recommendations. Use of two stable isotope-labeled internal standards and qualifier ion monitoring for both analytes ensure correct identification and unbiased quantification. With run times of less than 2.5 min per sample and cost-efficient sample preparation, this method allows exact quantitation of urinary folate catabolites pABG and apABG for large-scale non-invasive screening of folate status in clinical and epidemiological trials.
Keywords: Folate catabolite; Urine; Folate status; Derivatization; LC–MS/MS; Qualifier ion monitoring;

Immunocapture and LC–MS/MS for selective quantification and differentiation of the isozymes of the biomarker neuron-specific enolase in serum by Silje Bøen Torsetnes; Sandra Gransbråten Løvbak; Cecile Claus; Hanne Lund; Marianne S. Nordlund; Elisabeth Paus; Trine Grønhaug Halvorsen; Léon Reubsaet (125-132).
NSE, neuron-specific enolase, is an important biomarker for several pathological conditions including small cell lung cancer (SCLC). The current paper presents an LC–MS/MS-based approach for quantification of NSE in serum at both reference levels and elevated levels. The analytical approach utilizes selective sample preparation by immunoextraction of all forms of NSE (αγ, γγ, and γ) followed by tryptic digestion, and separation and detection by LC–SRM-MS. The quantification of NSE is performed through a signature peptide specific for the γ-subunit of NSE (tryptic peptide γ16; ELPLYR). The method is validated and shows satisfactory results (linearity r 2  > 0.999 (range 5–500 ng/mL), intra-day precision <13% RSD, and accuracy >95%), and has a limit of quantification (of 38 pg/mL; S/N = 10) significantly lower than endogenous levels of healthy subjects. In addition, the method simultaneously allows determination of the αγ-heterodimer through a signature peptide specific for the α-subunit (tryptic peptide α12; TIAPALVSK). The method was successfully applied to serum samples from healthy blood donors. In all samples from healthy blood donors both the α- and the γ-subunit was detected (S/N > 200 for both signature peptides), confirming the presence of the αγ-heterodimer in these sample. The level in one of them was determined to be (n  = 5) 7.3 ± 0.45 ng/mL of γ-subunit of NSE.
Keywords: Neuron-specific enolase; Signature peptide; Validation; Serum; Immunocapture;

The rise in amphetamine, ketamine and opiates abuse in Taiwan has created a need for a reliable confirmatory assay. A method that combines superficially porous liquid chromatography tandem mass spectrometry (LC–MS/MS) with solid-phase extraction (SPE) was developed for the simultaneous quantification of amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), ketamine, opiates, and their corresponding metabolites in urine. The total run time of the method was 6.7 min including equilibration time. The method was validated in accordance with the European Commission (EC) Decision 2002/642/EC. The within- and between-day precision was below 13.6% and the accuracy ranged from −17.1% to +9.9% for all analytes. Ion suppression was observed but compensated by using deuterated internal standards. No carryover was detected and the analytes were stable at room temperature for 16 h, and for 72 h at 4 °C, and three-thaw cycles. The method was further validated by comparison with a reference gas chromatography–mass spectrometry (GC–MS) method, using 52 authentic urine samples. The results indicated that for the target analytes studied, the LC–MS/MS analysis was as precise, accurate, and specific as the GC–MS method. In conclusion, the present LC–MS/MS method is robust and reliable, and suitable for use as a confirmation assay in the simultaneous urine drug testing and quantification of amphetamines, ketamines, and opiates.
Keywords: Superficially porous liquid chromatography; Liquid chromatography tandem mass spectrometry; Confirmatory assay; Amphetamine; Opiates; Ketamine;

Improved GC method for the determination of the active principles of Catha edulis by Lucia Dell’Acqua; Gabriella Roda; Sebastiano Arnoldi; Chiara Rusconi; Lorenzo Turati; Veniero Gambaro (142-148).
The GC method previously reported by our research group for the analysis of the active principles of Catha edulis, i.e. cathine, cathinone and phenylpropanolamine, was considerably improved. N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) as derivatizing agent was employed, thus allowing an accurate determination of the analytes and a suitable internal standard for quantitative analyses (nicotinamide) was introduced. Moreover the chromatographic conditions were carefully studied to improve the separation of the alkaloids and sensitivity. To this end different chromatographic capillary columns and temperature gradients were investigated. The optimized GC method was validated and resulted adequate for the application in forensic analysis. Finally on behalf of the Tribunal, C. edulis vegetable material seized by the police in northern Italy was analyzed, the quantity of cathine ranging from 0.095 to 0.29%, the quantity of PPA from 0.010 to 0.21% and the quantity of cathinone from 0.025 to 0.374% of the weight of the vegetable material.
Keywords: Catha edulis; Cathinone; Cathine; Phenylpropanolamine; GC/MS; GC/FID;

A method for the quantification of ethyl glucuronide (EtG) and ethyl sulphate (EtS) in human urine is developed and fully validated according to international guidelines. Protein precipitation is used as sample preparation. During the development of the method on an UPLC–ESI-MS/MS system using a CSH C18 column, special attention was paid to reduce matrix effects to improve assay sensitivity and to improve detection of the second transition for EtS for specificity purposes. The method was linear from 0.1 to 10 μg/mL for both analytes. Ion suppression less than 24% (RSD < 15%) was observed for EtG and no significant matrix effect was measured for EtS. The recovery was around 80% (RSD < 14%) for both compounds. This method provides good precision (RSD r and RSD t  < 10%) and bias (<15%) for internal and external quality control samples. The reproducibility of the method was demonstrated by the successful participation to proficiency tests (z-score < 0.86). This method was finally used to analyze urine samples obtained from twenty-seven volunteers whose alcohol consumption during the 5 days before sampling was monitored. Concentrations between 0.5 and 101.9 μg/mL (mean 10.9, median 1.4) for EtG and between 0.1 and 37.9 μg/mL (mean 3.6, median 0.3) for EtS were detected in urine samples of volunteers who declared having consumed alcohol the day before the sampling. EtG and EtS concentrations in urine were highly correlated (r  = 0.996, p  < 0.001). A moderate correlation between the number of drinks the day before sampling and the concentration of EtG (r  = 0.448, p  < 0.02) or EtS (r  = 0.406, p  < 0.04) was observed. Using a cut-off value at 0.1 μg/mL for EtG and EtS, this method is able to detect social alcohol consumption approximately 24 h after the intake, without showing any false positive result.
Keywords: Ethyl glucuronide; Ethyl sulphate; Validation; UPLC–ESI-MS/MS; Alcohol marker; Prospective study;

Structure-based histidine substitution for optimizing pH-sensitive Staphylococcus protein A by Hideki Watanabe; Hiroyuki Matsumaru; Ayako Ooishi; Shinya Honda (155-160).
Optimizing antibody purification is crucial to overcoming a bottleneck in the costly manufacturing process for antibody therapy. To address this issue, we designed a pH-sensitive Staphylococcus aureus protein A variant that retained its innate stability and affinity toward antibody. On the basis of structural information and mutation analysis data, we identified candidate positions for accumulative histidine substitutions to cause electrostatic repulsion under acidic conditions. The histidine substitutions effectively decreased the dissociation rate under acidic conditions by three orders of magnitude. Avoiding deleterious effects of the substitutions, we successfully engineered a protein A variant that exhibited high pH sensitivity and maintained affinity, thermal stability, and alkaline tolerance. The variant was capable of serving as an affinity ligand that made affinity chromatography under milder acidic conditions possible; the elution peak shifted from pH 4.2 to 5.6. Only two substitutions were needed to achieve this pH sensitivity. This structure-based approach is applicable to other protein-based ligands.
Keywords: Antibody; Affinity purification; Protein A; Protein engineering; pH-sensitive affinity ligand;

Bioanalytical LC–MS/MS of protein-based biopharmaceuticals by Irene van den Broek; Wilfried M.A. Niessen; William D. van Dongen (161-179).
Biotechnology increasingly delivers highly promising protein-based biopharmaceutical candidates to the drug development funnel. For successful biopharmaceutical drug development, reliable bioanalytical methods enabling quantification of drugs in biological fluids (plasma, urine, tissue, etc.) are required to generate toxicokinetic (TK), pharmacokinetic (PK), and bioavailability data. A clear observable trend is that liquid chromatography coupled to (tandem) mass spectrometry (LC–MS(/MS)) is more and more replacing ligand binding assays (LBA) for the bioanalytical determination of protein-based biopharmaceuticals in biological matrices, mainly due to improved selectivity and linear dynamic ranges. Practically all MS-based quantification methods for protein-based biopharmaceuticals traditionally rely on (targeted) proteomic techniques and include “seven critical factors”: (1) internal standardization, (2) protein purification, (3) enzymatic digestion, (4) selection of signature peptide(s), (5) peptide purification, (6) liquid chromatographic separation and (7) mass spectrometric detection. For this purpose, the variety of applied strategies for all “seven critical factors” in current literature on MS-based protein quantification have been critically reviewed and evaluated. Special attention is paid to the quantification of therapeutic monoclonal antibodies (mAbs) in serum and plasma since this is a very promising and rapidly expanding group of biopharmaceuticals. Additionally, the review aims to predict the impact of strategies moving away from traditional protein cleavage isotope dilution mass spectrometry (PC-IDMS) toward approaches that are more dedicated to bioanalysis.
Keywords: Bioanalysis; LC–MS; Monoclonal antibodies; Therapeutic protein; Absolute quantification; Sample preparation;

Since the human genomic project, the analysis of biomolecules using biotechnology and biopharmaceuticals has become increasingly important for the investigation of molecular mechanisms of biological processes and disease, as well as for the discovery of new biomarkers and drug targets. Among a number of analytical tools and technologies, capillary electrophoresis (CE) is emerging as a useful analytical tool for analyzing various biomolecules, as well as for performing genome sequencing in biological studies because of the simple sample preparation involved in the method and the efficient resolution of compounds of various sizes. In particular, CE with laser-induced fluorescence (CE-LIF) detection is a promising and relatively rapidly developing tool with the potential to provide a highly sensitive and specific analysis of biological molecules. This paper gives a short overview of the methodological and instrumental developments and applications of microchip-based CE and CE-LIF for the analysis of DNA, RNA, proteins, and peptides in the past 3 years
Keywords: Capillary electrophoresis; Laser-induced fluorescence; Biomolecules;

The present study aimed to develop a novel method for simultaneous assay of cortisol, cortisone and dehydroepiandrosterone sulfate (DHEAS) in human hair. The method was based on liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–MS/MS) in negative mode. Analytes were extracted from 20-mg hair incubated in 1 ml of methanol for 5 days. 100 μl non-SPE supernatant of the incubation solution was utilized in LC–MS/MS analysis. The liquid chromatography separation was performed on a reversed-phase C18 column with a mobile phase of 80:20 (v:v) methanol and deionized water containing 0.1% formic acid. The use of ESI in negative mode and the use of a small volume of the incubation solution significantly improved assay sensitivity. The linear range was 5–250 pg/mg for cortisol and cortisone, and 5–500 pg/mg for DHEAS. The limit of detection was 2 pg/mg for the three analytes in hair. The coefficients of variation for intra-day and inter-day assay were less than 10%. The method was applied to determine the three analytes mentioned above of hair samples from 103 participants. The results indicated that there was no significant effect of age and education level on the hair cortisol, cortisone and DHEAS levels. The simple treatment procedure developed in the present study may facilitate simultaneous measurement of more steroids in hair.
Keywords: Hair matrix; Cortisol; Cortisone; Dehydroepiandrosterone sulfate; Electrospray ionization; Negative mode;