Journal of Chromatography B (v.870, #1)

The partitioning behaviour of bovine trypsinogen and alpha-chymotrypsinogen, enzymatic precursors with similar physicochemical properties, was investigated in different polyethyleneglycol/sodium citrate aqueous two-phase systems. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length and temperature was also examined. The increase of pH and the decrease of polyethyleneglycol molecular weight displaced the partitioning equilibrium of both proteins to the top phase. An enthalpy–entropy compensation pattern was observed, indicating the participation of water molecules in the partitioning mechanism. TRPz phase equilibrium showed to be more displaced to the citrate-rich phase than ChTRPz for most of the assayed systems. From a practical view, the aqueous two-phase system formed by polyethylenglycol of molecular weight 1450 and sodium citrate pH 8.20 showed the best capability for separating both proteins. When a mixture formed by equal quantities of both zymogens was partitioned in this system, significant recoveries (about 60%) were obtained. Purity values were improved significantly (84–89%) by either developing a second extractive step or increasing the top–bottom volume ratio.
Keywords: Trypsinogen; Alpha-chymotrypsinogen; Pancreatic proteases; Aqueous two-phase systems;

Validated LC–MS/MS methods for the determination of risperidone and the enantiomers of 9-hydroxyrisperidone in human plasma and urine by Marc De Meulder; Bart M.M. Remmerie; Ronald de Vries; Luc L.A. Sips; Sandra Boom; Edwin W.J. Hooijschuur; Nico C. van de Merbel; Philip M.M.B.L. Timmerman (8-16).
Two liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods are described, one for the quantitative determination of risperidone and the enantiomers of its active metabolite 9-hydroxyrisperidone (paliperidone) in human plasma and the other for the determination of the enantiomers of 9-hydroxyrisperidone in human urine. The plasma method is based on solid-phase extraction of 200 μl of sample on a mixed-mode sorbent, followed by separation on a cellulose-based LC column with a 13.5-min mobile phase gradient of hexane, isopropanol and ethanol. After post-column addition of 10 mM ammonium acetate in ethanol/water, detection takes place by ion-spray tandem mass spectrometry in the positive ion mode. Method validation results show that the method is sufficiently selective towards the enantiomers of 7-hydroxyrisperidone and capable of quantifying the analytes with good precision and accuracy in the concentration range of 0.2–100 ng/ml. An accelerated (run time of 4.3 min) and equally valid method for the enantiomers of 9-hydroxyrisperidone alone in plasma is obtained by increasing the mobile phase flow-rate from 1.0 to 2.0 ml/min and slightly adapting the gradient conditions. The urine method is based on the same solid-phase extraction and chromatographic approach as the accelerated plasma method. Using 100 μl of sample, (+)- and (−)-9-hydroxyrisperidone can be quantified in the concentration range 1–2000 ng/ml. The accelerated method for plasma and the method for urine can be used only when paliperidone is administered instead of risperidone, as there is insufficient separation of the 9-hydroxy enantiomers from the 7-hydroxy enantiomers, the latter ones being present only after risperidone administration.
Keywords: Risperidone; Paliperidone; Chiral separation;

Simultaneous determination of cyanide and volatile alkylnitriles such as acetonitrile, cis- and trans-crotononitrile, allylnitrile and butyronitrile at low ppb concentration on whole blood (rat and mice) by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) with nitrogen phosphorus detection has been achieved for the first time. SPME extraction time and temperature were optimized using a star experimental design. Optimum conditions for cyanide extraction were chosen to analyze unspiked blood samples containing alkylnitriles as that analyte occurs at the lowest concentrations. For all analytes, the developed methodology yielded good quality parameters. In all cases, good reproducibility (relative standard deviation ≤12%), detection limits (<3 ng mL−1) and quantification limits (<4 ng mL−1) were recorded.
Keywords: Headspace; Headspace-SPME; GC-NPD; Volatile alkylnitriles; Cyanide; Crotononitrile; Allylnitrile;

This study used a combination of zonal elution and frontal affinity chromatography on immobilized human serum albumin (HSA) high-performance affinity chromatography (HPAC) column to examine the association constants of various compounds that have been studied by equilibrium dialysis or ultra filtration. A standard plot was generated from retention factors of reference compounds using zonal elution chromatography against association constants of reference compounds using frontal affinity chromatography. The linear relationship was established (r 2  = 0.9993) between retention factors and association constants of reference compounds. This standard plot was later used for rapid determination of association constants of various drugs which show low to medium binding affinity to HSA. Association constants of those drugs from this study were compared to that of more generally used methods (i.e., equilibrium dialysis or ultra filtration) from literature and resulted in a relatively high correlation (r 2  = 0.945) value. This combination of zonal elution and frontal affinity chromatography method for determining association constants showed several advantages against traditional methods. Depending on drugs of interest, an association constant of drug to HSA can be measured as fast as 1.5 min. Other notable advantages include an ease of automation and its ability to distinguish association constants of chiral compounds at the same time. The same approach could be used for studying interaction of other drugs and proteins and should further improve overall drug screening process.
Keywords: Frontal chromatography; Zonal elution; Human serum albumin; Association constants; Drug–protein interaction; Affinity chromatography;

A development of LC–MS method combining ultrafiltration and lyophilization for determination of r-RGD-Hirudin in human serum by Shengmin Su; Yunqiu Yu; Wei Mo; Yanling Zhang; Houyan Song; Qinfen Chen; Yi Xie (27-31).
A reliable and validated LC–MS method was established for determination of r-RGD-Hirudin in human serum. Ultrafiltration was used instead of liquid–liquid extraction or solid phase extraction for water solubility drug r-RGD-Hirudin extraction. Freeze drying was used for concentration. The experiment conditions, including pre-processing procedure and LC–MS, have been investigated and optimized. Comparing with reported assays, the current method showed significant improvement in specificity, linearity, precision and sensitivity. This method has been successfully applied in clinical research of r-RGD-Hirudin.
Keywords: r-RGD-Hirudin; Quantitative assay; LC–MS; Ultrafiltration; Freeze drying; Clinical research;

A reversed phase HPLC-MS/MS method has been developed and validated for the quantitative bioanalysis of acetaminophen in dried blood spots (DBS) prepared from small volumes (15 μL) of dog blood. Samples were extracted for analysis with methanol. Detection was by positive ion TurboIonSpray™ ionisation combined with selected reaction monitoring MS. The analytical concentration range was 0.1–50 μg/mL. The intra-day precision and bias values were both less than 15%. Acetaminophen was stable in DBS stored at room temperature for at least 10 days. The methodology was applied in a toxicokinetic (TK) study where the data obtained from DBS samples was physiologically comparable with results from duplicate blood samples (diluted 1:1 (v/v) with water) analysed using identical HPLC-MS/MS conditions. This work demonstrates that quantitative analysis of a drug extracted from DBS can provide high quality TK data while minimising the volume of blood withdrawn from experimental animals, to an order of magnitude lower than is current practice in the pharmaceutical industry. This is the first reported application of DBS analysis to a TK study in support of a safety assessment study. The success of this and similar, related studies has led to the intent to apply DBS technology as the recommended analytical approach for the assessment of pharmacokinetics (PK)/TK for all new oral small molecule drug candidates, which have previously demonstrated a successful bioanalytical validation.
Keywords: Dried blood spots; Acetaminophen; Bioanalytical; Validation; Toxicokinetic; HPLC-MS/MS;

Simple quantification of lansoprazole and rabeprazole concentrations in human serum by liquid chromatography/tandem mass spectrometry by Takanori Hishinuma; Kaori Suzuki; Hiroaki Yamaguchi; Hatsushi Yamagishi; Tomoyuki Koike; Shuichi Ohara; Toru Shimosegawa; Nariyasu Mano; Junichi Goto (38-45).
A rapid, simple and highly sensitive method was developed for the quantitative determination of lansoprazole and rabeprazole concentrations in 20 μL of human serum using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an internal standard (lansoprazole deuterium derivatives), were separated using a mobile phase of acetonitrile/1 mM ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 0.25 ng/mL. A good linear response was observed for each analyte (from 0.25 ng to 2.5 μg/mL). This method was useful for therapeutic drug monitoring and pharmacokinetic studies.
Keywords: Lansoprazole; Rabeprazole; LC/MS/MS; Human; Serum; Determination;

A method for the direct determination of ascorbic acid (AA) in individual rat hepatocyte based on capillary electrophoresis (CE) coupled with electrochemical detection (ECD) using a new kind of homemade carbon fiber micro-disk bundle electrode has been described. Individual rat hepatocytes were injected into a fused-silica capillary with an inner diameter of 25 μm, and lysed by 0.1% sodium dodecylsulfate (SDS) as cell lysis solution. The following conditions were suitable for the determination of AA: running buffer, 1.83 × 10−2  mol/l Na2HPO4–1.70 × 10−3  mol/l NaH2PO4 (pH 7.8); separation voltage, 20.0 kV; detection potential, 0.80 V (vs. saturated calomel electrode (SCE)). The concentration limit of detection (LOD) of the method was1.7 × 10−6  mol/l at a signal-to-noise (S/N) ratio of 3, and the mass LOD was 3.0 fmol. The linear dynamic range was from 5.0 × 10−6 to 5.0 × 10−4  mol/l with a correlation coefficient of 0.9962 for the injection voltage of 5.0 kV and injection time of 10 s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the peak current. This method was successfully applied to AA determination in rat hepatocyte. The recovery was between 91% and 97%, and the amount of AA in single rat hepatocyte ranged from 28 to 63 fmol.
Keywords: Ascorbic acid; Capillary electrophoresis; Electrochemical detection;

Affinity purification of egg yolk immunoglobulins (IgY) with a stable synthetic ligand by Dexian Dong; Haoran Liu; Qishi Xiao; Rongxiu Li (51-54).
Chicken IgY (egg yolk immunoglobulin) is a functional equivalent of mammalian IgG. Traditional methods for IgY purification involve multi-step procedures that result in low recovery of IgY. After a large scale screening of our 700-member synthetic ligand library synthesized by epichlorohydrin and cyanuric chloride methods, a high efficiency ligand of IgY was found. By one-step purification with this ligand, the purity of IgY could reach 92.1%, and the recovery of IgY could reach 78.2%. This synthetic ligand had a higher binding capacity of 74.8 mg IgY/ml and had no negative effects on immunoreactivity. Remarkably, this ligand was also highly stable and could resist 1 M NaOH, thus having great potential for the industrial-scale production of IgY.
Keywords: Affinity purification; Synthetic ligand; Immunoglobulin; IgY; Chicken;

Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G by Georgeen Gaza-Bulseco; Sagar Faldu; Karen Hurkmans; Chris Chumsae; Hongcheng Liu (55-62).
Oxidation of methionine (Met) residues is one of the most common protein degradation pathways. Two Met residues, Met256 and Met432, of a recombinant fully human monoclonal IgG1 antibody have been shown to be susceptible to oxidation. Met256 and Met432 are located in the antibody CH2–CH3 interface and in close proximity to protein A and protein G binding sites. The effect of oxidation of these susceptible Met residues on the binding to protein A and protein G was investigated in the current study. Incubation of the antibody with 5% tert-butyl hydroperoxide (tBHP) resulted in a nearly complete oxidation of Met256 and Met432, while incubation with 1% tBHP resulted in mixed populations of the antibody with different degrees of Met oxidation. Oxidation of Met256 and Met432 resulted in earlier elution of the antibody from protein A and protein G columns when eluted with a gradient of decreasing pH. Analysis by ELISA and surface plasmon resonance (SPR) revealed decreased binding affinity of the oxidized antibody to protein A and protein G. It is therefore concluded that oxidation of the Met256 and Met432 residues of the recombinant monoclonal antibody altered its interaction with protein A and protein G resulting in a decrease in binding affinity.
Keywords: Recombinant monoclonal antibody; Methionine oxidation; Mass spectrometry; Protein A; Protein G;

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated for the determination of human plasma homocysteine (Hcy), an important independent risk factor for cardiovascular disease, with a simplified sample pretreatment procedure and a zero blank free of endogenous Hcy for calibrator/QC preparation. Following protein precipitation, chromatographic separation was performed on Hypersil Aquasil C18 column (50 mm × 2.1 mm, 5 μm, Thermo) using mobile phase of aqueous 10% methanol containing 0.02% formic acid at 0.25 mL/min. Hcy and deuterated internal standard were detected in the multiple reaction monitoring mode with precursor to product ion transitions of m/z 136.1/90.0 and 140.1/94.0, respectively. The retention time was 1.2 min, and the total run time was 2 min per injection. A streamlined three-point calibration curve and one-point QC was used. Excellent linearity was observed with correlation coefficient (r) > 0.99. The intra- and inter-batch were ≤3.24% and ≤4.04%, and accuracy was within ±10%. Method comparison between the proposed method (y) and FPIA assay (x) demonstrated a correlation equation of y  = 1.003x  + 0.4318 (r  = 0.9589). The developed method, improved for automation with cost-effective reagents, was proven to be suitable for high-throughput quantitative determination of Hcy in clinical practice by successfully applying it to the cardiovascular disease study.
Keywords: Homocysteine; LC–ESI-MS/MS; Cardiovascular disease; Method comparison;

Determination of d-limonene in adipose tissue by gas chromatography–mass spectrometry by Jessica A. Miller; Iman A. Hakim; Cynthia Thomson; Patricia Thompson; H.-H. Sherry Chow (68-73).
We developed a novel method for analyzing d-limonene levels in adipose tissue. Fat samples were subjected to saponification followed by solvent extraction. d-Limonene in the sample extract was analyzed using gas chromatography–mass spectrometry (GC–MS) with selected ion monitoring. Linear calibration curves were established over the mass range of 79.0–2529 ng d-limonene per 0.1 g of adipose tissue. Satisfactory within-day precision (R.S.D. 6.7–9.6%) and accuracy (%difference of −2.7 to 3.8%) and between-day precision (R.S.D. 6.0–10.7%) and accuracy (%difference of 1.8–2.6%) were achieved. The assay was successfully applied to human fat biopsy samples from a d-limonene feeding trial.
Keywords: d-Limonene; GC–MS; Adipose;

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes by Naoko Goto-Inoue; Takahiro Hayasaka; Yuki Sugiura; Takao Taki; Yu-Teh Li; Mineo Matsumoto; Mitsutoshi Setou (74-83).
Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).
Keywords: Thin-layer chromatography; Matrix-assisted laser desorption/ionization; Glycosphingolipid;

Determination of UDP-glucuronosyltransferase UGT2B7 activity in human liver microsomes by ultra-performance liquid chromatography with MS detection by Hui-Xin Liu; Yu-Qi He; Ying Hu; Yong Liu; Jiang-Wei Zhang; Wei Li; Zheng-Tao Wang; Ling Yang (84-90).
A rapid and specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3′-azido-3′-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm), with phase of acetonitrile–water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5–500 μM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 °C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.
Keywords: Ultra-performance liquid chromatography; UGT2B7; Human liver microsomes;

Development of an automated on-line pepsin digestion–liquid chromatography–tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents by Jeroen Carol-Visser; Marcel van der Schans; Alex Fidder; Albert G. Hulst; Ben L.M. van Baar; Hubertus Irth; Daan Noort (91-97).
Rapid monitoring and retrospective verification are key issues in protection against and non-proliferation of chemical warfare agents (CWA). Such monitoring and verification are adequately accomplished by the analysis of persistent protein adducts of these agents. Liquid chromatography–mass spectrometry (LC–MS) is the tool of choice in the analysis of such protein adducts, but the overall experimental procedure is quite elaborate. Therefore, an automated on-line pepsin digestion–LC–MS configuration has been developed for the rapid determination of CWA protein adducts. The utility of this configuration is demonstrated by the analysis of specific adducts of sarin and sulfur mustard to human butyryl cholinesterase and human serum albumin, respectively.
Keywords: Chemical warfare agents; Protein adduct; Mass spectrometry;

A new method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ derivatization and gas chromatography–mass spectrometry (GC–MS) is described for the determination of trace amounts of bisphenol A (BPA) in human urine samples. The detection limit and the quantification limit of BPA in human urine sample are 0.02 and 0.1 ng ml−1 (ppb), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 0.1–50 ng ml−1. The average recoveries of BPA in human urine samples spiked with 1 and 5 ng ml−1 BPA are 101.0 (R.S.D.: 6.7%) and 98.8 (R.S.D.: 1.8%), respectively, with correction using the added surrogate standard, bisphenol A-13C12. This simple, accurate, sensitive and selective analytical method can be applicable to the determination of trace amounts of BPA in human urine samples.
Keywords: Bisphenol A (BPA); Miniaturized; Hollow fiber (HF); Liquid-phase microextraction (LPME); GC–MS; In situ derivatization;

Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide by Bistra Benkova; Valentin Lozanov; Ivaylo P. Ivanov; Antonia Todorova; Ivan Milanov; Vanio Mitev (103-108).
Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5–25 μM for homocysteine and glutathione, and in the range of 0.5–200 μM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 μl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35–4.38% and 0.89–4.13%, respectively.
Keywords: Homocysteine; Cysteine; Glutathione; HPLC; 2-Aminoacridone; Maleimide;

A liquid chromatography–tandem mass spectrometric method (LC–MS/MS) has been developed and validated to determine the concentration of Kendine 91 in mice plasma and tissues. Simvastatin was employed as the internal standard. Separation was performed on a C8 column, with a mobile phase consisting of methanol and aqueous 10 mM formic acid (73:27 v/v). Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via multiple-reaction monitoring (MRM) in positive scanning mode. Quantification was performed using the transitions m/z 444 → 169 and 441 → 325 for Kendine 91 and simvastatin, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic study after intravenous injection of Kendine 91 in mice in a dose of 10 mg/kg.
Keywords: Histone deacetylase inhibitor; Pharmacokinetics; Analytical validation; LC–MS/MS; Kendine 91;

Optimized blood sampling with cytidine deaminase inhibitor for improved analysis of capecitabine metabolites by Thierry Besnard; Nicole Renée; Marie-Christine Etienne-Grimaldi; Eric François; Gérard Milano (117-120).
The 5FU prodrug capecitabine undergoes a 3-step enzymatic conversion, including the conversion of 5′DFRC into 5′DFUR by cytidine deaminase (CDA). The presence of CDA activity in blood led us to analyze the possible ex vivo conversion of 5′DFCR into 5′DFUR in blood samples. We thus examined the impact of the addition of a CDA inhibitor (tetrahydrouridine (THU) 1 μM final) in blood. Blood samples from 3 healthy volunteers were taken on tubes containing or not THU. Blood was spiked with 5′DFCR (20 μM final) (T0) and was maintained at room temperature for 2 h. Plasma concentrations of 5′DFRC and 5′DFUR were analyzed with an optimized HPLC assay. In the absence of THU, 5′DFUR was detectable as early as T0. The percent of 5′DFUR produced relative to 5′DFCR increased over time, up to 7.7 % at 2 h. In contrast, the presence of THU totally prevents the formation of 5′DFUR. The impact of THU for preventing the conversion of 5′DFCR was confirmed by the analysis of blood samples from 2 capecitabine-treated patients. Addition of THU in the sampling-tube before the introduction of blood is thus strongly recommended in order to guarantee accurate conditions for reliable measurement of capecitabine metabolites in plasma, and thus faithful pharmacokinetic data.
Keywords: Capecitabine; 5′DFCR; 5′DFUR; Cytidine deaminase; Tetrahydrouridine; Enzyme conversion; Enzyme inhibitor; HPLC assay;

High-performance liquid chromatography/tandem mass spectrometry method for the simultaneous determination of cytarabine and its valyl prodrug valcytarabine in rat plasma by Yongbing Sun; Jin Sun; Bing Wen; Shiliang Shi; Youjun Xu; Ying Chen; Yongjun Wang; Changqi Pan; Chunyu Zhang; Tianhong Zhang; Zhonggui He (121-125).
A sensitive, specific and rapid HPLC–MS/MS method has been developed and validated for the simultaneous determination of cytarabine and valcytarabine (valyl prodrug of cytarabine) in rat plasma in the present study. The analytes were separated on a C18 column (50 mm × 2.1 mm, 1.7 μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. Cation exchange solid-phase extraction cartridge was employed to extract the analytes from rat plasma, with high recovery of cytarabine (>85%). The method was linear over the concentration ranges of 10–20,000 ng/mL for cytarabine and 25–1000 ng/mL for valcytarabine. The lower limit of quantitation (LLOQ) of cytarabine and valcytarabine was 10 and 25 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, the method was successfully applied to support the prodrug pharmacokinetic study after valcytarabine and cytarabine were orally administrated to the Sprague–Dawley rat, respectively.
Keywords: High-performance liquid chromatography/tandem mass spectrometry; Cytarabine; Valcytarabine; Solid-phase extraction; Rat plasma;

A sensitive and specific method using high-performance liquid chromatography (LC)–electrospray tandem mass spectrometry (ESI–MS/MS) for the determination of indapamide in human serum was developed and validated. Indapamide and an internal standard (4-diethylaminobenzoic acid) were isolated from serum samples by solid-phase extraction (SPE) with Oasis®HLB 96-well plates and determined by LC–MS/MS in multiple reaction monitoring (MRM) mode. The calibration curve of serum indapamide was linear in the range of 0.2–20 ng/ml with a correlation coefficient of 0.9999. The repeatability, intermediate precisions, and accuracies at 0.2, 5, and 20 ng/ml in serum were less than 15%. The absolute recoveries of indapamide and the internal standard were 79.4–81.5% and 87.5%, respectively, and the low limit of quantitation of serum indapamide was 0.2 ng/mL. The analytical method was applied to a bioequivalence study of KYD-041 (1 mg as film-coated tablets, test formulations) and Natrix®Tab.1 (1 mg as sugar-coated tablets, reference formulation). The 90% confidence interval of the ratios (test formulation/reference formulation) for log(C max) and log(AUCt) were in the range log(0.80)–log(1.25), which supports the conclusion that KYD-041 is bioequivalent to Natrix®Tab.1 with respect to the rate and extent of indapamide absorption.
Keywords: Indapamide; Liquid chromatography–mass spectrometry; Solid-phase extraction;

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 °C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.
Keywords: Capillary electrophoresis; Intracellular; CHO cells; Nucleotides; Nucleotide sugars;

A rapid, sensitive and specific high performance liquid chromatography–electrospray ionization tandem quadrupole mass spectrometry (HPLC–MS/MS) method was developed and validated for the determination of 3-n-butylphthalide in rat plasma. Following protein precipitation with acetonitrile, 3-n-butylphthalide and glipizide (internal standard, I.S.) were separated using a gradient elution program on a C18 column and detected by mass spectrometry in positive ion mode with the multiple reaction monitoring (MRM) mode using the respective precursor to product ion combinations of m/z 191/145 for 3-n-butylphthalide and m/z 446/321 for glipizide, respectively. The total chromatographic running time was 2.5 min. The method was linear over the concentration range of 11.14–3480.00 ng/mL, using as little as 100 μL plasma. The lower limit of quantification (LLOQ) was 5.57 ng/mL. Finally, the method was successfully used to support a preclinical pharmacokinetic study of 3-n-butylphthalide in rats following intravenous administration.
Keywords: HPLC–MS/MS; 3-n-Butylphthalide; Plasma concentration;

Determination of Fumonisin B1 in animal tissues with immunoaffinity purification by Didier Tardieu; Aliénor Auby; Caroline Bluteau; Jean Denis Bailly; Philippe Guerre (140-144).
Immunoaffinity extraction combined with high-performance liquid chromatography (HPLC) with fluorescence detection was developed to determine Fumonisin B1 (FB1) in duck tissues. The method was linear over a concentration range of 0.013–0.250 μg of FB1/g of liver, kidney and muscle. The limit of quantification was 0.013 μg FB1/g in tissue. The mean percentage of extraction was 75% for liver and kidney and 53% for muscle. This method can be used in duck for the detection of FB1 contamination after exposure, the liver being the most contaminated tissue.
Keywords: Fumonisin B1 (FB1); High-performance liquid chromatography (HPLC); Immunoaffinity; Duck; Residue; Contamination; Mycotoxins; Food quality;