Journal of Chromatography B (v.899, #C)

Simultaneous determination of lercanidipine, benazepril and benazeprilat in plasma by LC–MS/MS and its application to a toxicokinetics study by Keguang Chen; Jing Zhang; Sha Liu; Dujuan Zhang; Yanni Teng; Chunmin Wei; Benjie Wang; Xiaoyan Liu; Guiyan Yuan; Rui Zhang; Wenjing Zhao; Ruichen Guo (1-7).
► Simultaneous determination of LER, BEN and benazeprilat in plasma with single IS. ► The method was fully validated to meet the requirements of FDA and GCP guidelines. ► It can meet the requirement in analyzing large number of samples from clinical trials.We aim to develop a rapid, simple, sensitive and specific LC–MS/MS method for the simultaneous quantification of lercanidipine, benazepril and benazeprilat in plasma. It is performed on the Agilent 6410 LC–MS/MS under the multiple-reaction monitoring (MRM) mode with electrospray ionization. Gliclazide was used as the internal standard (IS). Analytes and IS were extracted from plasma by solid-phase extraction. The reconstituted samples were chromatographed on a Diamond C18 (150 mm × 4.6 mm, 5 μm) column. The mobile phase was composed of 0.1% acetic acid–acetonitrile (50:50, v/v), with gradient flow rates: 0.6 mL/min (0–4.55 min); 4.55–4.65 min, 1 mL/min; 1 mL/min (4.65–9.5 min); 9.5–9.6 min, 0.6 mL/min; 0.6 mL/min (9.6–10 min). Method validation demonstrated that the method was of satisfactory specificity, sensitivity, precision and accuracy in linear ranges of 1–2000 ng/mL for lercanidipine, 1–2000 ng/mL for benazepril and 1–1600 ng/mL for benazeprilat, respectively. The precision (RSD%) was better than 15, and the lower limit of quantitation was identifiable and reproducible at 1 ng/mL for the three analytes. The plasma samples were stable after being stored for more than 60 days and after two freeze–thaw cycles (−20 to −25 °C). It is demonstrated that this method was successfully applied to samples from a toxicokinetics study of a compound of lercanidipine and benazepril in beagle dogs.
Keywords: Lercanidipine; Benazepril; Benazeprilat; Determination; LC–MS/MS;

Interference by metabolites and the corresponding troubleshooting during the LC–MS/MS bioanalysis of G004, a bromine-containing hypoglycemic agent by Linlin Hu; Li Ding; Xiaobing Li; Na Zhou; Shuisheng Zhong; Guanzhong Wu; Huibin Zhang (8-13).
► G004, a novel bromine-containing hypoglycemic agent with antithrombosis effect. ► The detection of an interfering metabolite in the LC–MS/MS bioanalysis of G004. ► MS conditions were optimized to eliminate the interference from the metabolite. ► Metabolite-related matrix effect was evaluated by the standard addition method. ► Pharmacokinetics of G004 in beagle dogs was reported for the first time.The quantitative determination of drugs in bio-samples may be interfered by the drug-related metabolites during the high-throughput LC–MS/MS analysis. When quantifying bromine or chlorine containing compounds, the 81Br/37Cl isotopic forms of their mono-hydroxylated metabolites after in-source dehydration could produce ions which are isobaric with the precursor ions of the parent compounds at the 79Br/35Cl isotopic form. In this report, we described the identification of an interfering hydroxylated metabolite of G004, a novel bromine-containing hypoglycemic agent, during LC–MS/MS analysis of plasma samples. Several different MRM transitions were tested and evaluated to minimize the metabolite influence on the quantification of G004. Furthermore, the standard addition method using incurred samples was used to evaluate the matrix effect caused by the interfering metabolite. The lower limit of quantitation of the established method was 0.2 ng/ml, which was 10 times lower than the existing one. The method was successfully applied to investigate the single-dosing pharmacokinetic profile of G004 in beagle dogs. The above results indicated that when quantifying chlorine or bromine containing compounds, evaluation of the interference from mono-hydroxylation or dehydrogenation metabolites should be undertaken, and if such metabolites existed, their impact on quantification of the parent compounds could be eliminated by the proper selection of the MRM transitions.
Keywords: G004; Liquid chromatography–tandem mass spectrometry; Bromine or chlorine containing compounds; Metabolite interference; Metabolite-related matrix effect; Pharmacokinetics;

Analysis of colistin A and B in fishery products by ultra performance liquid chromatography with positive electrospray ionization tandem mass spectrometry by Yingjiang Xu; Xiuhui Tian; Chuanbo Ren; Hui Huang; Xiuzhen Zhang; Xianghong Gong; Huihui Liu; Zhaoqiang Yu; Limin Zhang (14-20).
A rapid and simple method for the determination of colistin A and B in fishery products by reversed phase ultra performance liquid chromatography with positive electrospray ionization tandem spectrometry (UPLC–ESI-MS/MS) method was described. The samples were extracted with 1.0 mol/L of hydrochloric acid in methanol–water and then purified on the PLS solid phase extraction columns. Then the eluate was evaporated to less than 1 mL under a gentle stream of nitrogen at 40 °C and formic acid–acetonitrile–water (0.2/10/90, v/v/v) was added to adjust volume to 1 mL final volume. An aliquot (10 μL) was injected onto the LC column for analysis with the mobile phase of 0.2% formic acid in acetonitrile and 0.2% formic acid in water at 0.20 mL min−1. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 200 ng/mL to 2000 ng/mL for colistin A and B. Mean recoveries were between 72.9% and 82.9%. The LOD was 10.0 μg/kg and LOQ was 40.0 μg/kg. The intra-day assay precision values for QC samples were between 2.17% and 9.00%, and inter-day values were between 2.80% and 6.97%. The method has merits of simplicity, sensitivity and rapidity, and it can be used for the determination of colistin A and B in fishery products.
Keywords: Colistin A; Colistin B; UPLC–ESI-MS/MS; Fishery products;

An LC–MS/MS method for determination of jujuboside A in rat plasma and its application to pharmacokinetic studies by Changhui Liu; Yingyi Li; Yanhua Zhong; Xiaotao Huang; Xia Zheng; Neng Li; Shaoling He; Suiqing Mi; Ningsheng Wang (21-26).
► It firstly developed a LC–MS/MS method for measurement of jujuboside A in rat plasma. ► It firstly reported the pharmacokinetic profile of jujuboside A in rats. ► The lower limit of quantification (LLOQ) of developed LC–MS/MS method was lower than the previous report.A novel, simple, and sensitive method for the determination of jujuboside A in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. Following solid-phase extraction, measurement of jujuboside A was performed by negative ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The limit of detection was 1.25 ng/mL, and the lower limit of quantification was 5 ng/mL in rat plasma. Good linearity was obtained over the range of 6.25–500 ng/mL, and the correlation coefficient was better than 0.998. The intra- and inter-day precisions ranged 4.4–7.5% and 2.9–10.7%, respectively. The accuracy derived from QC samples ranged 3.2–7.8% and 2.2–3.5%, respectively. The recovery ranged from 72.9 to 75.1% and the matrix effect from 96.7 to 105.3%. The analyte was stable under various conditions (at room temperature, during freeze–thaw, in the autosampler and under deep-freeze conditions). The developed method was successfully applied to the pharmacokinetic study in rats.
Keywords: Jujuboside A; LC–MS/MS; Rat plasma; Pharmacokinetics;

Simultaneous quantification of metronidazole, tinidazole, ornidazole and morinidazole in human saliva by Yongqing Wang; Peipei Zhang; Ningling Jiang; Xiaojian Gong; Ling Meng; Dewang Wang; Ning Ou; Haibo Zhang (27-30).
► Simultaneous quantification of metronidazole, tinidazole, ornidazole and morinidazole. ► Ornidazole and morinidazole (500 mg) reach more than 4100 ng/ml after i.v. infusion. ► The method is simple, sensitive and specific, with a short analysis time (6 min). ► It was used to find out who had taken other 5-nitroimidazole derivatives before trial.The aim of this study was to develop a rapid and sensitive method for the simultaneous quantification of metronidazole (MEZ), tinidazole (TNZ), ornidazole (ONZ) and morinidazole (MNZ) in human saliva. A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection at 318 nm was carried out on a C18 column, using a mixture of potassium dihydrogen phosphate buffer, acetonitrile, and methanol (55:15:30, v/v/v) as a mobile phase with a flow rate of 1.0 ml/min. The saliva samples (100 μl) were firstly deproteinized by precipitation with methanol (400 μl), after which they were centrifuged and the supernatants were directly injected into the HPLC system. This method produced linear responses in the concentration ranges of 25.2–5040.0, 23.9–4790.0, 25.4–5080.0, 25.0–5000.0 ng/ml with detection limits of 6.0, 17.6, 10.0 and 11.3 ng/ml for MEZ, TNZ, ONZ and MNZ (S/N = 3), respectively. The methods were validated in terms of intra- and inter-batch precision (within 7.3% and 9.1%, respectively), accuracy, linearity, recovery and stability. The study proved that HPLC is both sensitive and selective for the simultaneous quantification of MEZ, TNZ, ONZ and MNZ in human saliva using a single mobile phase.
Keywords: Metronidazole; Tinidazole; Ornidazole; Morinidazole; Saliva; Periodontitis;

Determination of the active metabolite of moguisteine in human plasma and urine by LC–ESI-MS method and its application in pharmacokinetic study by Yanni Teng; Haibo Song; Fanlong Bu; Chunmin Wei; Wenjing Zhao; Rui Zhang; Guiyan Yuan; Xiaoyan Liu; Benjie Wang; Ruichen Guo (31-35).
► It is the first LC–MS method to determine main active metabolite of moguisteine. ► This method is quicker, more accurate and specific than the previous literatures. ► It is the first pharmacokinetic study of moguisteine in healthy Chinese volunteers.In this study, a sensitive and reproducible electro-spray ionization liquid chromatography–mass spectrometry (LC–ESI-MS) method was established to determine the concentration of M1, the main active metabolite of moguisteine in human plasma and urine. The analysis was performed on a Diamonsil® C18(2) column (150 mm × 4.6 mm, 5 μm) with the mobile phase consisting of 0.1% formic acid–acetonitrile (57:43, v/v, pH = 3.0) at a flow rate of 0.8 mL min−1. The pseudo-molecular ions [M+H]+ (m/z 312.2 for M1 and 446.3 for glipizide) were selected as the target ions for quantification in the selected ion monitoring (SIM) mode. Plasma samples were analyzed after being processed by acidification with formic acid and protein precipitation with acetonitrile. Urine samples were appropriately diluted with blank urine for analysis. Calibration curve was ranged from 0.02 to 8 μg mL−1. The extraction recovery in plasma was over 90%. Both the inter- and intra-day precision values were less than 7.5%, and the accuracy was in the range from −6.0% to 6.0%. This is the first reported LC–ESI-MS method for analyzing M1 in human plasma and urine. The method was successfully applied to the pharmacokinetic study after oral administration of single-dose and multiple-dose of moguisteine tablets in healthy Chinese subjects.
Keywords: Moguisteine; LC–ESI-MS; Pharmacokinetics; Plasma; Urine;

► We develop HPLC–DAD–MS method for analysis of carotenoids in human serum. ► 30 carotenoids are separated within 45 min, identified and quantified. ► Sensitivity, recovery and reproducibility of the developed method are good. ► Method applied to samples from subjects fed with carotenoid-rich capsules.An improved high performance liquid chromatography–diode array detection–mass spectrometry method was developed for determination of various carotenoids and their precursors phytoene and phytofluene in human serum. A polymeric C30 column and mobile phase of (A) methanol/acetonitrile/water (84:14:4, v/v/v) and (B) dichloromethane (100%) were employed with the gradient condition of 100% A and 0% B initially, raised to 10% B at 4 min, 18% B at 12 min, 21% B at 17 min, 30% B at 20 min and maintained until 25 min and increased further to 39% B at 28 min, 60% B at 40 min and returned to 100% A and 0% B at 45 min. A total of 30 carotenoids, including 6 all-trans forms, 20 cis-isomers, 2 β-carotene epoxides, phytoene and phytofluene, were resolved within 45 min at a flow-rate of 1 mL/min, column temperature 25 °C and detection wavelengths 450, 348 and 286 nm. Identification of carotenoids was carried out by comparing retention behavior, absorption and mass spectral data with those of reference standards, isomerized standards and reported values. An internal standard parared was found appropriate for quantitation of all the carotenoids. The developed method provided high sensitivity with low detection and quantitation limits (2–14 and 6–43 ng/mL), high recovery (91–99%), and small intra-day and inter-day variations (0.14–6.01% and 0.31–7.28%). Application of the developed method to Taiwan subjects supplemented with carotenoid-rich capsules revealed β-carotene plus its cis isomers as well as epoxide derivatives to be present in largest amount (1069.8–2783.1 ng/mL) in serum, followed by lutein plus its cis isomers (511.6–2009.5 ng/mL), phytofluene plus its cis isomer (515.0–1765.0 ng/mL), lycopene plus its cis isomers (551.1–1455.1 ng/mL), β-cryptoxanthin plus its cis isomers (458.0–965.0 ng/mL), all-trans-zeaxanthin (110.0–177.0 ng/mL), phytoene (41.8–165.0 ng/mL) and all-trans-α-carotene (37.5–95.9 ng/mL).
Keywords: Carotenoid; Phytoene; Phytofluene; Human serum; HPLC–DAD–MS;

► HPLC–ESI-MS method to quantify dextromethorphan, dextrorphan and doxylamine. ► Method was applied in pharmacokinetic study in human plasma. ► Extraction performed with a simple liquid–liquid extraction. ► High sensitivity, specificity and high throughput.In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid–liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0 min at a flow-rate of 1.5 mL min−1 into a Phenomenex Gemini® C18, 5 μm analytical column (150 × 4.6 mm i.d.). The calibration curve was linear over the range from 0.2 to 200 ng mL−1 for dextromethorphan and doxylamine and 0.05 to 10 ng mL−1 for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5 mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study.
Keywords: Quantification; Volunteer; LC–MS/MS;

► We compared SPE and QuEChERS methods to determine seven cephalosporins in cow muscle. ► An experimental design was used to optimize the two clean-up methods. ► The validation of the two methods was done according to the Directive 2002/657/EC. ► The separation and detection of the cephalosporins was carried out by LC–MS/MS. ► Comparable results were found for two methods in terms of quality parameters.This work was focused on the comparison of two clean-up methods to be used for the simultaneous determination of seven cephalosporins in cow muscle. In particular, the performance of novel dispersive solid phase extraction (d-SPE) procedures based on QuEChERS methodologies was assessed and compared with conventional SPE. The separation and detection of the analytes using both methods was carried out by LC–MS/MS to reach enough sensitivity to be compatible with the detection of the maximum residue limits (MRL) of cephalosporins as regulated by EU directives. The optimization of the clean-up step relied on experimental design in order to find the most suitable conditions with a reduced number of assays. Besides, multi-objective responses were used to reach an overall compromise in the recovery of all analytes simultaneously. The validation of the two methods was done according to the Directive 2002/657/EC. Linearity, decision limit, detection capability, detection and quantification limits (4–50 μg kg−1), precision (RSD less than 15% except for PIR) and recoveries were determined and adequate results with comparable values using QuEChERS and SPE methodologies. LOQ were better for SPE method (0.1–10 μg kg−1) but both methods show LOQ below MRL values. Precision was slightly better for the QuEChERS method, that also presents better recoveries, higher than 85% except for cephalexin.
Keywords: Cephalosporins; Beef tissues; QuEChERS; SPE; Experimental design; LC–MS/MS; Validation according 2002/657/EC;

► LDS-DLLME presented a high recovery for the extraction of warfarin in human plasma. ► LDS-DLLME–HPLC-UV yielded good sensitivity for warfarin determination. ► Wide linear range of this method can be applied in therapeutic drug monitoring.Extraction and determination of warfarin, a widely used anticoagulant drug, in human plasma were performed using a new generation of dispersive liquid–liquid microextraction (DLLME) and high performance liquid chromatography (HPLC). The extraction procedure is based on extraction solvents lighter than water and performing of extraction in a specially designed extraction cell. Some important parameters, including kind and volume of extraction and disperser solvents, pH of the sample solution, salt concentration in the sample solution and extraction time were investigated and optimized. Under the optimized conditions (150 μL 1-octanol as extraction solvent, 150 μL methanol as disperser solvent, pHsample  = 2.3, extraction time of 2 min, without salt addition), limit of detection (LOD) of 5 ng mL−1 and extraction recovery of 91.0% were obtained. The calibration curve was linear within the range of 15–3000 ng mL−1 with the square of correlation coefficient (R 2) of 0.998. Repeatability and reproducibility of method based on five replicate extraction and determination were 2.8% and 6.5%, respectively. The proposed method was applied successfully for the determination of warfarin in plasma sample from a patient under treatment with this drug, and was demonstrated to be sensitive, efficient, and convenient.
Keywords: Low-density solvent; Dispersive liquid–liquid microextraction; Warfarin; HPLC; Sample preparation; Plasma sample analysis;

Optimization and correlation of HPLC-ELSD and HPLC–MS/MS methods for identification and characterization of sophorolipids by Isabel A. Ribeiro; M. Rosário Bronze; Matilde F. Castro; Maria H.L. Ribeiro (72-80).
► TLC, HPLC-ELSD and HPLC–ESI-MS/MS were optimized and correlated for SLs analysis. ► HPLC–ESI-MS/MS analysis allowed identification of SLs from different sources. ► A [M+Na]+ fragmentation pattern of acidic and lactonic SLs was established. ► For the first time C24:0 monoacetylated and diacetylated SLs were identified.High-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) methods were implemented and optimized to separate and identify sophorolipids (SLs) produced by Rhodotorula bogoriensis and Starmerella bombicola. SLs are carbohydrate-based amphiphilic biosurfactants with increased interest in pharmaceutical and environmental areas. Rhodotorula bogoriensis and Starmerella bombicola are mainly producers of respectively C22, and C16 and C18 SLs. Mass fragmentation patterns of SLs produced by both yeasts were investigated by HPLC–ESI-MS/MS in the positive mode for [M+Na]+. Based on the established fragmentation pattern, SLs produced by both yeasts were identified and characterized. A correlation between HPLC-ELSD and HPLC- ESI-MS/MS methods was established and made possible the identification of SLs by the HPLC-ELSD technique. TLC is a common tool for the analysis of SLs mixtures. In this work, TLC scrapped bands were analysed by HPLC-ELSD and HPLC–MS allowing the correlation between R F values and the identification of sophorolipids by this technique. Identification of monoacetylated and diacetylated C24:0 hydroxy fatty acids sophorolipids produced by Rhodotorula bogoriensis was for the first time accomplished with this study. Although present in lower quantity these longer chain SLs can assume special importance regarding their biological activity and surface active properties.
Keywords: Sophorolipid; TLC; HPLC-ELSD; HPLC–ESI-MS/MS; Rhodotorula bogoriensis; Starmerella bombicola;

Development and validation of a liquid chromatography–tandem mass spectrometry method for quantification of decitabine in rat plasma by Haiyan Xu; Shaoqiong lv; Mingxi Qiao; Yao Fu; Xue Jiang; Yi Jin; Chibing Li; Bo Yuan (81-85).
Decitabine is chemically unstable at physiological temperature and pH. In addition, the bioanalysis of decitabine is easily interfered by endogenous 2-deoxycytidine. A simple, sensitive and specific LC–MS/MS method was developed for the analysis of decitabine in rat plasma. No exogenous stabilizers were used to prevent the degradation of decitabine in rat plasma. After deproteinized with acetonitrile at room temperature, rat plasma samples were analyzed on a Hypersil APS-2 NH2 column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Decitabine was completely separated from 2-deoxycytidine using gradient elution of water (solvent A) and acetonitrile (solvent B) at a flow rate of 1 mL/min. To quantify decitabine and daidzin (internal standard), respectively, multiple reaction monitoring (MRM) transitions of m/z 251.1 → 134.7 and m/z 417.3 → 255.3 was performed. The assay was linear over the concentration range of 5.0–2000 ng/mL. The intra- and inter-day precision was within 12.0% in terms of relative standard deviation (RSD%) and the accuracy within 5.9% in terms of relative error. The LC–MS/MS method was fully validated for its sensitivity, selectivity, stability study, matrix effect and recovery. The data indicate that this LC–MS/MS method is a specific and effective method for the pharmacokinetic study of decitabine in rat plasma. Compared with the previously reported analytical methods, this method showed easy and economic sample preparation, good specificity and high sensitivity with less plasma (50 μL).
Keywords: Decitabine; LC–MS/MS; Rat plasma; Pharmacokinetics;

Application of nanoLC–MS/MS to the shotgun proteomic analysis of the nematocyst proteins from jellyfish Stomolophus meleagris by Rongfeng Li; Huahua Yu; Ronge Xing; Song Liu; Yukun Qing; Kecheng Li; Bing Li; Xiangtao Meng; Jinhui Cui; Pengcheng Li (86-95).
► We analyzed the nematocyst proteins of jellyfish Stomolophus meleagris by using the nanoLC–MS/MS with the shotgun proteomic method. ► A total of 181 proteins had been identified with the molecular weight ranging from 5268.06 to 843,487.57 and the pI from 4.49 to 11.39. ► The identified nematocyst proteins were analyzed by bioinformatic method including gene ontology (GO) annotation, pathways and gene network analysis.The nematocyst proteins of jellyfish Stomolophus meleagris, a complicated mixture, contain many important bioactive molecules. In present study, to gain comprehensive insight into the protein component and search some novel bioactive molecules in the nematocyst proteins, shotgun proteomic analysis of the nematocyst proteins was carried out by nano liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) for the first time. Digested peptides of the nematocyst proteins were analyzed by nanoLC–MS/MS and all MS/MS spectra were then automatically searched by the SEQUEST program. A total of 181 proteins had been identified, with the molecular weight ranging from 5268.06 to 843,487.57 and the pI from 4.49 to 11.39. Bioinformatic analysis was also applied to better understand the identified proteins. In the gene ontology (GO) annotation, all the identified proteins were classified into 13, 9 and 7 groups according to biological process, cellular component and molecular function, respectively. Pathways analysis of the identified proteins was conducted with 33 corresponding pathways found. On the basis of pathways analysis, we also constructed the gene network to analyze the relationship of those genes each other, which contained enzyme–enzyme relation, protein–protein interaction and gene expression interaction.
Keywords: Jellyfish; Stomolophus meleagris; Nematocyst proteins; Shotgun; NanoLC–MS/MS; Proteomic analysis;

► Exploring a novel route to a pseudo-biospecific immunoadsorbent – click chemistry. ► Preparing “clickable” reaction modules using l-histidine and original sepharose. ► Click reaction can greatly increase the immobilization efficiency of ligands. ► The 1,2,3-triazole in spacer-arm helps IgG binding without non-specific adsorption. ► The prepared immunoadsorbent exhibits excellent adsorption performance for IgG. l-histidine is a promising alternative to expensive protein ligands for the adsorption of IgG due to its high selectivity, no toxicity and low cost; while click chemistry can improve the reaction selectivity between the ligands and the support matrix under mild reaction conditions. Thus, using l-histidine as a ligand and original sepharose gel as a support, a novel immunoadsorbent possessing pseudo-biospecific affinity for IgG from human plasma, Sep-triazole-His, was designed and prepared according to the principle of Click-reaction between alkyne and azide functional groups; while both sepharose-based control samples Sep-His and Sep-PA were prepared by a conventional method using l-histidine and protein A as a ligand, respectively. The ligand density and IgG adsorption performance of Sep-triazole-His from human plasma were measured and evaluated. The influences of click chemistry on the preparation, structure and performance of sepharose-based immunoadsorbent were also investigated. The results indicate that the ligand density immobilized on Sep-triazole-His is 319.1 μmol/g sepharose gel, almost 4-fold as high as that on Sep-His; the IgG adsorption capacity of Sep-triazole-His from human plasma reaches 16.49 mg/g at pH 7.0, or increases 5.72-fold with respect to Sep-His, and does not decrease noticeably after being repeatedly used for 10 times; and Sep-triazole-His can exhibit high adsorption selectivity for IgG comparable to Sep-PA. The further studies prove that the 1,2,3-triazole ring in the spacer-arm of Sep-triazole-His, can facilitate the binding of IgG without non-specific adsorption.
Keywords: Pseudo-biospecific immunoadsorbent; Click chemistry; Histidine ligand; IgG; Adsorption;

Analysis of N′-nitrosonornicotine and its metabolites in rabbit blood with liquid chromatography/tandem mass spectrometric method by Beibei Zhao; Sheng Wang; Juan Wang; Hailei Lang; Shihao Sun; Jian Mao; Jianxun Zhang (103-108).
► A method to analyze NNN and its metabolites with HPLC–MS/MS was established. ► Hydroxy acid was the major metabolite of NNN in rabbit blood. ► 5′-Hydroxylation of NNN might be the main metabolic pathway of NNN in rabbit blood. ► Metabolic curves and pharmacokinetic parameters were obtained for NNN and its metabolites.N′-nitrosonornicotine (NNN) is a strong carcinogen. The metabolic study of NNN in vivo will help us to further understand it, however, trace detection in complex matrices requires highly sensitive detection methods. After the chromatographic conditions and mass spectrometric conditions had been optimized and confirmed, a method for determining NNN and its metabolites in rabbit blood by high-performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) was established. The results showed that precisions (R.S.Ds) were between 0.5% and 8.62%, the recoveries ranged from 80% to 111%. Linearity was observed for all compounds with detection limits ranging from 0.039 ng mL−1 to 0.217 ng mL−1. Metabolic curves and pharmacokinetic parameters were obtained for NNN and its metabolites. The elimination half-life of NNN was 30 min and the main metabolite of NNN was 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid) and the major metabolic pathway was 5′-hydroxylation and subsequent secondary metabolite formation.
Keywords: N′-nitrosonornicotine (NNN); Metabolites; Pharmacokinetic; HPLC–MS/MS;

► A robust LC/MS metabolic study focused on flavonols and flavanonols is presented. ► For analysis of the metabolites C18, C8, CNP and phenyl-based LC stationary phases was used. ► The study describes retention behaviour and MS data for individual metabolites. ► The metabolites were obtained using an in vitro human hepatocyte model system.Liquid chromatography (LC) on various stationary phases was used for the metabolite profile analysis of quercetin, rutin, isoquercitrin and taxifolin. The metabolites were obtained using an in vitro model system of human and rat hepatocytes in the form of cell suspensions and the primary cultures. For separations of the parent compounds and their metabolites, stationary phases based on C18, C8, cyanopropyl (CNP) or phenyl (PHE) modifications of silica were tested. CNP and PHE stationary phases operating in reversed-phase mode have been shown to be efficient for separation of parent flavonoids and their polar metabolites. Individual metabolites were identified on the basis of an elemental composition determination using electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) on-line connected with an LC system. Detailed analytical parameters such as retention times, selectivity, resolution of chromatographic peaks, MS fragmentation and UV–vis absorption maxima were determined for individual metabolites, namely for phase II biotransformation products. The predominant metabolites were methylated flavonols and flavonol glucuronides. The highest biotransformation rate was found with taxifolin, which was mainly converted to sulfates. The HPLC/ESI-QqTOF MS analyses revealed that quercetin and taxifolin were metabolized more extensively than the studied glycosides, rutin and isoquercitrin.
Keywords: Flavonols; Flavanonol; Taxifolin; Metabolism; Liquid chromatography; Retention behaviour; Mass spectrometry; Hepatocytes;

Design of high productivity antibody capture by protein A chromatography using an integrated experimental and modeling approach by Candy K.S. Ng; Hector Osuna-Sanchez; Eric Valéry; Eva Sørensen; Daniel G. Bracewell (116-126).
► A lumped parameter transport-dispersive model is used. ► An experimental strategy for critical parameter estimation is designed. ► The impact of process variables on productivity is studied via use of model.An integrated experimental and modeling approach for the design of high productivity protein A chromatography is presented to maximize productivity in bioproduct manufacture. The approach consists of four steps: (1) small-scale experimentation, (2) model parameter estimation, (3) productivity optimization and (4) model validation with process verification. The integrated use of process experimentation and modeling enables fewer experiments to be performed, and thus minimizes the time and materials required in order to gain process understanding, which is of key importance during process development. The application of the approach is demonstrated for the capture of antibody by a novel silica-based high performance protein A adsorbent named AbSolute. In the example, a series of pulse injections and breakthrough experiments were performed to develop a lumped parameter model, which was then used to find the best design that optimizes the productivity of a batch protein A chromatographic process for human IgG capture. An optimum productivity of 2.9 kg L−1  day−1 for a column of 5 mm diameter and 8.5 cm length was predicted, and subsequently verified experimentally, completing the whole process design approach in only 75 person-hours (or approximately 2 weeks).
Keywords: Protein A chromatography; Batch chromatography; Antibody capture; Productivity; Integrated process experimentation and modeling;

► Microdialysis sampling and turbulent-flow chromatography were coupled to LC/MS. ► The system is able to achieve online desalting and concentrating within 1 min. ► Influences of matrix effect and carry-over were eliminated effectively. ► Lifetime of extraction column is much extended. ► The temporal resolution of sampling points is improved by using MD technology.Sample pretreatment is a key step in bioanalytical process because of possible interference and matrix effects in mass spectrometry analysis. In this work, a novel strategy towards high speed and sensitivity was developed combining in vivo microdialysis (MD) sampling, turbulent-flow chromatography (TFC), and liquid chromatography–mass spectrometry (LC–MS). The procedures of cleanup, preconcentration, and separation were completed on-line in one step within 10 min. During the MD optimization procedure, 1% hydroxypropyl-β-cyclodextrin (HP-β-CD) was used to improve the relative recovery of the analyte. Untreated MD samples were directly injected, and a TFC precolumn was flushed for 1 min with aqueous phase of 4 mL/min flow rate to desalt and concentrate biosamples. The retained analytes were then back-flushed by a switching valve onto a fast LC column (4.6 mm × 50 mm, 1.8 μm) for separation. Another diverter valve was employed to prevent the HP-β-CD that interferes with the ESI process from entering the MS. Puqietinone, a lipophilic alkaloid from Fritillaria puqiensis, was used as a case for validation. Results showed that the limit of quantification for puqietinone was 0.10 ng/mL, and good linearity (R 2  = 0.9993) was maintained over the range of 1.02–200.02 ng/mL. Accuracy and precision were satisfactory within the range of the standard curve. This approach was able to effectively eliminate the influences of matrix effect and carry-over as the injection volume increased up to 20 μL. The developed method was successfully applied to pharmacokinetic study of puqietinone after intravenous administration to rat. Results demonstrate the potential of using MD with TFC-LC/MS for in vivo monitoring experiments.
Keywords: Microdialysis; Turbulent-flow chromatography; Mass spectrometry; Desalting; Pharmacokinetic; Puqietinone;

Bile acid profiling in human biological samples: Comparison of extraction procedures and application to normal and cholestatic patients by Lydie Humbert; Marie Anne Maubert; Claude Wolf; Henri Duboc; Myriam Mahé; Dominique Farabos; Philippe Seksik; Jean Maurice Mallet; Germain Trugnan; Joëlle Masliah; Dominique Rainteau (135-145).
► Four extraction protocols were compared to optimize bile acid quantification by HPLC MS/MS. ► The best protocols were the Acetonitrile one for liquid (serum, urine, bile) and the NaOH one for stool samples. ► Allowed to detect 14 different species in serum, 11 in urine and 25 in stool. ► Applied to control and cholestatic patients and useful for quantification in routine analysis.The role of bile acids in cell metabolism, membrane biology and cell signaling is increasingly recognized, thus making necessary a robust and versatile technique to extract, separate and quantify a large concentration range of these numerous molecular species. HPLC–MS/MS analysis provides the highest sensitivity to detect and identify bile acids. However, due to their large chemical diversity, extraction methods are critical and quite difficult to optimize, as shown by a survey of the literature. This paper compares the performances of four bile acid extraction protocols applied to either liquid (serum, urine, bile) or solid (stool) samples. Acetonitrile was found to be the best solvent for deproteinizing liquid samples and NaOH the best one for stool extraction. These optimized extraction procedures allowed us to quantitate as much as 27 distinct bile acids including sulfated species in a unique 30 min HPLC run, including both hydrophilic and hydrophobic species with a high efficiency. Tandem MS provided a non ambiguous identification of each metabolite with a good sensitivity (LOQ below 20 nmol/l except for THDCA and TLCA). After validation, these methods, successfully applied to a group of 39 control patients, detected 14 different species in serum in the range of 30–800 nmol/l, 11 species in urine in the range of 20–200 nmol/l and 25 species in stool in the range of 0.4–2000 nmol/g. The clinical interest of this method has been then validated on cholestatic patients. The proposed protocols seem suitable for profiling bile acids in routine analysis.
Keywords: Bile acids; Extraction; Liquid chromatography–mass spectrometry; Profiling; Routine analysis;

► JH III was determined in bee hemolymph by LC–MS/MS for the first time. ► The proposed sample treatment was fast and simple. ► A mixture of methanol and phenylthiourea was chosen to dilute bee hemolymph. ► Matrix (bee hemolymph) did not have any influence onto the analyte signal. ► The highest concentration of JH III was found in bees infected with Nosema ceranae. It has been described a fast, simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to measure juvenile hormone III (JH III), which was used to study of the effects of Nosema spp. infection on JH III levels in bee hemolymph. Honey bee hemolymph was extracted by centrifugation and mixed with a solution of phenylthiourea in methanol. This mixture was then centrifuged and the supernatant removed and evaporated to dryness. The residue was reconstituted in methanol containing the internal standard (methoprene) and injected onto an LC–MS/MS (ion-trap) system coupled to electrospray ionization (ESI) in positive mode. Chromatography was performed on a Synergi Hydro-RP column (4 μm, 30 mm × 4.60 mm i.d.) using a mobile phase of 20 mM ammonium formate and methanol in binary gradient elution mode. The method was fully validated and it was found to be selective, linear from 15 to 14,562 pg/μL, precise and accurate, with %RSD values below 5%. The limits of detection and quantification were: LOD, 6 pg/μL; LOQ, 15 pg/μL. Finally, the proposed LC–MS/MS method was used to analyze JH III levels in the hemolymph of worker honey bees (Apis mellifera iberiensis) experimentally infected with different Nosema spp. (Nosema apis, Spanish and Dutch Nosema ceranae strains). The highest concentrations of JH III were detected in hemolymph from bees infected with Spanish N. ceranae.
Keywords: Juvenile hormone III; Honey bee hemolymph; LC–MS/MS; Nosema ceranae; Nosema apis;

► Hair analysis is a tool to evaluate drug exposure in several application fields. ► We present a simple UHPLC–MS/MS method to detect 13 common drugs of abuse in hair. ► The method proved excellent analytical performances and drastic reduction of time. ► High sample throughput is essential for laboratories. ► The increased global productivity makes the workplace testing of hair affordable.A simple procedure for the quantitative determination in hair samples of 13 common drugs of abuse or metabolites (morphine, 6-acetylmorphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, benzoylecgonine, cocaine, buprenorphine, methadone and Δ9-tetrahydrocannabinol) has been developed and fully validated. The analytes were extracted from the matrix by a simple overnight incubation with methanol at 55 °C. An aliquot of the extract was directly injected into an ultra-high performance liquid chromatography system equipped with Waters Acquity UHPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase eluted with a linear gradient (water/formic acid 5 mM:acetonitrile; v:v) from 98:2 to 0:100 in 4.5 min, followed by isocratic elution at 100% B for 1.0 min. The flow rate was 0.6 mL/min and the total run time was 8.0 min including re-equilibration at the initial conditions. The compounds were revealed by a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method proved linear in the interval from the limit of quantification to 5.0 ng/mg (1.0 ng/mg for Δ9-tetrahydrocannabinol) with correlation coefficient values ranging from 0.9970 to 0.9997. Quantitation limits were below the cut-off values recommended by the Society of Hair Testing and ranged from 0.02 to 0.08 ng/mg. Application of the present UHPLC–MS/MS procedure and instrumentation to hair analysis allows high sample-throughput, together with excellent sensitivity and selectivity, in workplace drug-screening controls and forensic investigations. These qualities, combined with minimal sample workup, make the cost of this screening affordable for most private and public administrations.
Keywords: Hair; Ultra-high performance liquid chromatography; Multidrugs; Validation;

► This article describes the development of a more sensitive HPLC method for determination of PZQ in bovine muscle for the first time. ► The purpose of this work was to develop a detection method for PZQ residues in bovine muscle. ► This method was validated and used to detect praziquantel residues in a plan survey. ► The work presented here used HPLC with UV detector for the determination of praziquantel following a solid-phase (SPE) extraction. ► The method is specific and sensitive, with a quantification limit of 0.02 mg/kg at and a detection limit of 0.01 mg/kg.A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of praziquantel in bovine muscle. The sample was extracted with ethyl acetate, cleaned up by alumin B cartridge. Analyses were run at a flow-rate of 0.8 ml/min with the detector operating at a detection wavelength of 220 nm. The method is specific and sensitive, with a quantification limit of 0.02 mg/kg and a detection limit of 0.01 mg/kg. The standard calibration curve of drugs solution was linear in the range of 0.02–2.0 μg/ml (r 2  = 0.9999). At the fortified levels of 0.02, 0.05, 0.2 mg/kg, the mean recoveries of praziquantel ranged from 75% to 85%, while the intra-day and inter-day coefficient of variation (CV) of the assay were all less than 9%.
Keywords: Praziquantel; HPLC; Determination; Bovine muscle;

► The sample preparation step was simplified using protein precipitation. ► The run time was shortened with a more selective LC–MS/MS procedure. ► Isotopically labeled internal standard was used to improve precision and accuracy. ► A rapid method for determination of celecoxib in human plasma has been developed. ► The method was successfully applied to a pharmacokinetic study.A liquid chromatography–electrospray tandem mass spectrometry method was developed and validated to quantitate celecoxib in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography on a C18 column (55 mm × 2 mm, 3 μm), the mobile phase consisted of methanol – 10 mM ammonium acetate (75:25, v/v). Quantification was performed by mass spectrometry in the multiple reaction monitoring mode with negative electrospray ionization at m/z 380 → 316 and 384 → 320 for celecoxib and the internal standard celecoxib-D4, respectively. The lower limit of quantitation was 7.0 ng/ml using 0.1 ml of plasma and linearity was demonstrated up to 1800 ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 6% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.
Keywords: Celecoxib; Plasma; LC–MS/MS; Protein precipitation; Pharmacokinetics;