Journal of Chromatography B (v.932, #C)

Waldenstrom's macroglobulinemia (WM) is considered by the Revised European American Lymphoma (REAL) and World Health Organization (WHO) as a clinical lympho plasmacytic syndrome associated with high monoclonal (IgM) secretion. The hyper viscosity syndrome is associated with several clinical disorders of monoclonal IgM. Patients with clinical symptoms of hyper viscosity should be treated with plasma pheresis, which is limited by its non-selective removal of all plasma components. These limitations have steered efforts to find a more specific removal according to clinical needs and avoiding plasma components replacement. Removal by specific adsorption is the most powerful selective apheresis technique. The active adsorbed ligand is covalently bound to an insoluble matrix through which plasma is passed. Amino acids have been introduced as ligands in clinical apheresis for the removal of auto antibodies associated with autoimmune diseases. The present preliminary study describes the binding of monoclonal IgM antibodies from sera of patients with WM, on histidine immobilized to activated sepharose. The advantages of efficient binding and elution, suggest histidine adsorbents as prospective clinical means suitable for the removal of monoclonal IgM from sera of patients diagnosed with WM. The advantages of efficient adsorption and elution, non toxicity of histidine, good selectivity, good stability, as well as their low cost strongly suggest histidine adsorbents as prospective clinical means suitable for the removal of monoclonal IgM from sera of patients diagnosed with WM.
Keywords: Waldenström's macroglobulinemia; Monoclonal IgM; Histidine; Pseudobioaffinity;

Quantification of vitamin D and 25-hydroxyvitamin D in soft tissues by liquid chromatography–tandem mass spectrometry by Tristan E. Lipkie; Amber Janasch; Bruce R. Cooper; Emily E. Hohman; Connie M. Weaver; Mario G. Ferruzzi (6-11).
Inadequate data on tissue distribution of vitamin D and its metabolites remains a barrier to defining health outcomes of vitamin D intake and 25-hydroxyvitamin D (25(OH)D) status. The purpose of this study was to develop a method for the analysis of vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), 25(OH)D2, and 25(OH)D3 in soft tissues, and determine distribution in select tissues from a dose–response study of vitamin D2 and vitamin D3 in rats. Liver, gastrocnemius muscle, and epididymal fat homogenates were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization following liquid–liquid extraction, solid-phase extraction, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). A dose–response was observed in most tissues for vitamin D and 25(OH)D from both vitamers. Vitamin D concentration was greater in epididymal fat than gastrocnemius muscle and liver, but 25(OH)D concentration was not significantly different between tissues. Soft tissues of rats fed crystalline vitamin D3 had higher concentrations of total vitamin D than those of rats fed yeast-derived vitamin D2, while total 25(OH)D concentrations were similar between vitamin D sources. This method is well suited to more complete studies of vitamin D bioavailability and metabolite tissue distribution.
Keywords: 25-Hydroxyvitamin D analysis; Vitamin D analysis; Ergocalciferol; Cholecalciferol; Tissue distribution; LC–MS/MS;

Measurement of free carnitine and acylcarnitines in plasma by HILIC-ESI-MS/MS without derivatization by Minzhi Peng; Li Liu; Minyan Jiang; Cuili Liang; Xiaoyuan Zhao; Yanna Cai; Huiying Sheng; Zhiying Ou; Hong Luo (12-18).
Measurement of carnitine and acylcarnitines in plasma is important in diagnosis of fatty acid β-oxidation disorders and organic acidemia. The usual method uses flow injection tandem mass spectrometry (FIA-MS/MS), which has limitations. A rapid and more accurate method was developed to be used for high-risk screening and diagnosis. Carnitine and acylcarnitines were separated by hydrophilic interaction liquid chromatography (HILIC) without derivatization and detected with a QTRAP MS/MS System. Total analysis time was 9.0 min. The imprecision of within- and between-run were less than 6% and 17%, respectively. Recoveries were in the range of 85–110% at three concentrations. Some acylcarnitine isomers could be separated, such as dicarboxylic and hydroxyl acylcarnitines. The method could also separate interferent to avoid false positive results. 216 normal samples and 116 patient samples were detected with the validated method, and 49 patients were identified with fatty acid oxidation disorders or organic acidemias.
Keywords: Acylcarnitine isomer; Fatty acid β-oxidation disorders; Organic acidemia; LC–MS/MS; HILIC;

Tyrosinase inhibitors play an important role in cosmetic products, food supplements and medicinal industries. In this study, a new facile method based on tyrosinase immobilized magnetic fishing (IMF) coupled with high performance liquid chromatography–diode array detector–tandem mass spectrometry (HPLC–DAD–MS/MS) was developed to screen and identify tyrosinase binders from Glycyrrhiza uralensis root. Experiments were carried out to select optimal immobilization and incubation conditions. The activity of immobilized tyrosinase retained 76.3% after ten consecutive cycles, and remained over 95% when stored at 4 °C for about two months. Eleven tyrosinase binders, including original compounds and transformation products were successfully screened and characterized from G. uralensis root, while dihydrodaidzein and pratensein were reported for the first time. Compared with ultrafiltration-HPLC assay, IMF-HPLC displayed many attractive advantages, including convenient solid–liquid separation ability, good stability, high reusability and activity, which indicated that IMF-HPLC had a broad applicability. The results indicated that IMF-HPLC method is a facile, effective and reproductive way to screen and identify active components from complex mixtures.
Keywords: Tyrosinase; Glycyrrhiza uralensis; HPLC–DAD–MS/MS; Enzyme immobilization; Reverse-ultrafiltration;

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral–chiral and chiral–chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0 × 0.46 cm I.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0 × 0.46 cm I.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis.
Keywords: Multidimensional chromatography; Multivariate analysis; Microsomal fraction; Albendazole metabolites; Polysaccharide chiral phase; Restricted access media (RAM);

LC–MS/MS determination of pramipexole on rat dried blood spots: A pharmacokinetic study by R. Nageswara Rao; B. Sravan; K. Ramakrishna; B. Raju; R. Srinivas (34-39).
A simple and selective liquid chromatography–tandem mass spectrometric method for determination of pramipexole on rat dried blood spots was developed and validated. Chromatographic separation was achieved on a Synergy polar-RP column using 10 mM ammonium acetate and methanol (50:50, v/v) as mobile phase in an isocratic mode of elution at a flow rate of 1.0 mL/min. LC–MS was performed in a positive ion electro spray ionization mode and the MS/MS ion transitions 212.10 → 153.03 for PRX and 198.10 → 153.03 for internal standard (2-amino-4,5,6,7-tetrahydro-6-ethyl-amino-benzthiazole) were monitored. The developed method exhibited a linear dynamic range over 100–5000 pg/mL for PRX on dried blood spots. The overall extraction recovery of PRX from DBS was 96.7%. The intra- and inter-day accuracy and precision were within the pre-defined limits of ≤15% at all concentrations. Influence of hematocrit and spot volume on dried blood spot was also evaluated and found to be well within the acceptable limits. The method was successfully applied to pharmacokinetic studies of PRX in rats.
Keywords: Parkinson's disease; Pramipexole; Dried blood spots; LC–MS/MS; Pharmacokinetics;

A rapid and sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the determination of valproyl-1-O-acyl glucuronide (VPA-G) levels in hepatocyte culture medium. Chromatographic separation was achieved using a Waters Acquity UPLC® BEH C18 column (1.7 μm, 2.1 mm × 50 mm) with gradient elution and a total run time of 4 min. [2H6]-VPA-G was used as internal standard (IS). Quantification was performed in the multiple reaction monitoring (MRM) mode using the total ion current of the MRM transition pairs m/z 319.1 → 142.7 and m/z 319.1 → 175.2 for VPA-G, and m/z 325.1 → 149.3 and m/z 325.1 → 174.9 for the IS under negative electrospray ionization mode. The assay was linear over the VPA-G concentrations of 0.5–500 ng/mL, with a r 2 value of 0.995 ± 0.002 (mean ± SD). The intra- and inter-day accuracy (% deviation) ranged from −10.2% to 11.1%, whereas the intra- and inter-day precision (% RSD) were ≤7.43%. The method was applied successfully to the quantification of VPA-G levels in culture supernatants of sandwich-cultured rat hepatocytes treated with valproic acid (VPA). No significant difference in the levels of VPA-G over a culture period of 6 days was observed in an experiment that investigated the effect of the age of hepatocyte culture on the extent of VPA glucuronidation. The method presented here for the direct quantification of VPA-G is an improvement of existing methods in the literature and offers a shorter run time and greater sensitivity that enables the use of small volumes of sample. To the best of our knowledge, this is the first validated UHPLC–MS/MS method applied to the quantification of VPA-G in cell culture supernatants.
Keywords: Valproic acid; Valproyl 1-O-acyl glucuronide; UHPLC–MS/MS; Rat hepatocytes;

Quantification of monoacylglycerols (MAG) and free fatty acids (FA) is of interest in biological systems, in food, cosmetic and pharmaceutical products. This manuscript describes and validates a reversed phase liquid chromatography–tandem mass spectrometry based approach for simultaneous quantification of these analytes in fats and oils. Purification and concentration of MAG/FA were performed using cation exchange solid phase extraction, which allowed elimination of the abundant triacylglycerols. Following cleanup and concentration, the analytes were separated and detected with the aid of volatile ammonium-formate buffer. MAG were detected in positive ion mode, while FA were detected in negative ion mode. The method was validated by the method of standard additions and using stable isotope labeled internal standards. The results confirm the feasibility of quantifying these two classes of analytes simultaneously without any chemical derivatization. The obtained main quantitative features include: (1) lower limits of quantification 1–30 ppm for MAG analytes, (2) lower limits of quantification 90–300 ppm for FA analytes, (3) averaged inter-batch precision 6%, and (4) averaged bias −0.2% for MAG and 0.5% for FA. Various animal fat and vegetable oil samples were characterized for their MAG/FA profile indicating the usefulness of the method to address quality and authenticity of fats and oils.
Keywords: Monoacylglycerol; Free fatty acid; Oil; Animal fat; Tandem mass spectrometry;

Quantitation of leukotriene B4 in human sputum as a biomarker using UPLC–MS/MS by Wenying Jian; Richard W. Edom; Xiaohua Xue; Mike-Qingtao Huang; Anne Fourie; Naidong Weng (59-65).
Leukotriene B4 (LTB4) is a potent mediator of inflammation and has been recognized as an important target for therapeutic intervention for treatment of diseases such as asthma. In the current work, a highly selective and sensitive UPLC–MS/MS assay was developed for quantitation of LTB4 in human sputum as a biomarker for LTB4 biosynthesis inhibition. A fit-for-purpose strategy for method development, assay qualification, and study support was adopted for this biomarker project. A surrogate matrix (protein buffer) was used for preparation of calibration samples and certain levels of quality control (QC) samples to avoid interference from endogenous analyte, while the low QC was prepared in authentic matrix, human sputum. The analytical methodology utilized a liquid–liquid extraction procedure in 96-well plate format. Chromatographic separation was achieved with a reversed-phase ultra high pressure liquid chromatography (UPLC) column using gradient elution, and the run time was 4.5 min per sample. The lower limit of quantitation (LLOQ) was 0.2 ng/mL, and the calibration curve range was 0.2–20 ng/mL. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (6 h), freeze–thaw stability (3 cycles at −20 °C), and autosampler stability (97 h at ambient temperature) all met acceptance criteria. Frozen long-term stability for 166 days at −20 °C in sputum did not meet acceptance criteria by showing only ≥75% of nominal concentration and the information was taken into consideration for study support. Two important observations in the current work were: (1) LTB4 was unstable in sputum in the presence of liquification reagent dithiothreitol (DTT). Therefore, a non-DTT treatment method for sputum processing was developed and applied to the bioanalytical assay and clinical study support; and (2) chromatographic separation of LTB4 from its three non-enzymatically derived isomers, i.e. 6-trans-LTB4, 12-epi-LTB4, and 6-trans-12-epi-LTB4, was achieved. This assay was successfully applied to a Phase II clinical study for proof-of-concept of a LTA4 hydrolase inhibitor for treatment of asthma.
Keywords: Leukotriene B4; Sputum; Bioanalysis; UPLC–MS/MS; Biomarker; Fit-for-purpose;

A sensitive and selective liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of four flavonoids (schaftoside, isovitexin, luteolin, and apigenin) and one phenolic acid (ferulic acid) in rat plasma using sulfamethoxazole as the internal standard (IS). The separation was performed using a Diamonsil C18 column, which was eluted with methanol (A) and 0.1‰ acetic acid (B). The gradient condition was as follows: 0–5 min, 40–60% A; 5–6 min, 60–95% A; and 6–10 min, maintained at 95% A. The analytes were detected using a hybrid quadrupole linear ion trap mass spectrometer that was equipped with an electrospray ionization source in the negative ion and multiple-reaction monitoring modes. A full validation of the method was performed. The linearity of the analytical response was good, with correlation coefficients greater than 0.9925 for all of the compounds within the concentration range. The lower limits of quantitation (LLOQ) of schaftoside, isovitexin, luteolin, apigenin, and ferulic acid in rat plasma were 1.66, 0.84, 3.69, 1.70, and 3.91 ng/mL, respectively. The intra-day and inter-day precisions of the investigated components exhibited an RSD within 13.20%, and the accuracy (RE%) ranged from −8.47% to 10.90%. The results indicated that the developed method is sufficiently reliable for the pharmacokinetic study of schaftoside, isovitexin, apigenin, luteolin, and ferulic acid in rats following oral administration of the Herba Desmodii Styracifolii extract.
Keywords: LC–MS/MS; Flavonoids; Phenolic acid; Desmodii Styracifolii; Pharmacokinetics;

The issue of non-specific binding of cabazitaxel by Peter de Bruijn; Inge M. Ghobadi Moghaddam-Helmantel; Walter J. Loos; Ron H.J. Mathijssen; Erik A.C. Wiemer (74-75).
Keywords: Cabazitaxel; Pharmacokinetics; LC–MS/MS; Non-specific binding;

Pharmacokinetic comparisons of different combinations of Shaoyao-Gancao-Decoction in rats: Simultaneous determination of ten active constituents by HPLC–MS/MS by Yang Wang; Changhua Xu; Ping Wang; Xiaoyan Lin; Yan Yang; Donghua Li; Huifen Li; Xianzhong Wu; Hongbin Liu (76-87).
A specific HPLC–MS/MS method was developed and validated for simultaneous determination of ten constituents including albiflorin, oxypaeoniflorin, paeoniflorin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, ononin, glycyrrhizin and glycyrrhetinic acid in rat plasma using genistein as an internal standard (IS). The rat plasma samples were prepared by a one-step direct protein precipitation procedure with methanol. HPLC separation was achieved on a Zorbax XDB-C18 column (2.1 mm × 50 mm i.d., 3.5 μm) with gradient elution (A: 0.1% aqueous formic acid; B: methanol with 0.1% formic acid) at a flow rate of 0.5 mL/min in a run time of 7 min. All analytes and IS were detected by multiple reaction monitoring scanning with electrospray ionization in the negative ion mode. Calibration curves showed good linearity (r  > 0.998) over a wide concentration range for all analytes. The intra- and inter-day precisions were all within 15% and the accuracies were in the range of −6.2% to 10.1%. The validated method was successfully applied to determination and comparative pharmacokinetics investigation of the ten constituents in rat plasma after oral administration of different combinations (Radix Paeoniae Alba:Glycyrrhiza uralensis  = 1:1 or 4:1) of Shaoyao-Gancao-Decoction (SGD) extracts. Pharmacokinetic parameters were evaluated by a compartment model. There were perceptible differences in pharmacokinetic parameters (C max, AUC0−t , CL) of the analytes except for liquiritin between the two groups of SGD.
Keywords: Shaoyao-Gancao-Decoction; Monoterpene glucosides; Flavonoids; Triterpenoid saponins; Pharmacokinetics; HPLC–MS/MS;

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of indapamide enantiomers in rats by Bin Du; Li Pang; Hongyan Li; Sijia Ma; Yang Li; Xin Jia; Zhenzhong Zhang (88-91).
This investigation describes a precise and accurate stereoselective HPLC method for the simultaneous determination of indapamide enantiomers in whole blood to follow its pharmacokinetics in rats up to 24 h after single oral dosing. Enantiomeric resolution was achieved on a cellulose tris (3,5-dichlorophenylcarbamate) column known as Chiralpak IC, with UV detection at 240 nm, and the mobile phase consisted of n-hexane and isopropanol (70:30, v/v). Using the chromatographic conditions described, indapamide enantiomers were well resolved with a resolution factor (Rs) of at least 2.0 and with retention times of 19.2 and 23.3 min, respectively. Linear responses (r  > 0.999) were observed over the range of 0.05–50 μg/mL of indapamide enantiomers, with quantitation limit of 0.05 μg/mL. The mean relative standard deviation (RSD) of within-day precision and accuracy of the drug were <10%. The mean extraction efficiency was greater than 86% for each enantiomer. The assay method shows good specificity to indapamide enantiomers, and it could be successfully applied to its pharmacokinetic studies and to therapeutic drug monitoring.
Keywords: Indapamide; Enantiomer separation; Chiral column chromatography; Pharmacokinetics;

Bu Shen Huo Xue formula (BSHX) is a traditional Chinese medicine prescription used for clinical treatment of chronic kidney diseases. A rapid and selective Ultra fast liquid chromatography with tandem mass spectrometry (UFLC–MS/MS) method was developed for simultaneous determination of four bioactive components of BSHX including formononetin, cryptotanshinone, tanshinone IIA, and emodin in control and unilateral ureteral obstruction (UUO) model rat plasma for the first time. Atorvastatin was used as the internal standard (IS). Plasma samples were extracted by liquid–liquid extraction with ethyl acetate. The chromatographic separation was carried out on a Shim-pack XR-ODS III column with a gradient mobile phase consisting of acetonitrile and 0.1% formic acid. The detection was performed on a triple-quad tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source with positive ionization mode for formononetin, cryptotanshinone, tanshinone IIA, and negative mode for emodin. The method was linear for four analytes over the range of investigated concentration with all coefficients of determination (R 2) greater than 0.9938. The lower limits of quantification (LLOQ) for formononetin, cryptotanshinone, tanshinone IIA, and emodin were defined as 0.3, 0.5, 1.5, and 0.3 ng/mL, respectively. The rapid and sensitive method was fully validated and successfully applied to the pharmacokinetic study of formononetin, cryptotanshinone, tanshinone IIA and emodin in rats following oral administration of Bu Shen Huo Xue formula.
Keywords: UFLC–MS/MS; Pharmacokinetics; Plasma;

Validation of an electrospray ionisation LC–MS/MS method for quantitative analysis of telaprevir and its R-diastereomer by Sujan Dilly Penchala; John Tjia; Omar El Sherif; David J. Back; Saye H. Khoo; Laura J. Else (100-110).
A sensitive high-performance reverse phase liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid–liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H]+ ions, m/z 680.59 → 322.42 for telaprevir and VRT-127394, and 691.15 → 110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5–5000 ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD < 6.5%) and accuracy (94–108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers.
Keywords: Hepatitis C; Telaprevir; VRT-127394; LC–MS/MS; Method validation; Ex vivo interconversion;

LC–NMR, NMR, and LC–MS identification and LC–DAD quantification of flavonoids and ellagic acid derivatives in Drosera peltata by Christina Braunberger; Martin Zehl; Jürgen Conrad; Sonja Fischer; Hamid-Reza Adhami; Uwe Beifuss; Liselotte Krenn (111-116).
The herb of Drosera peltata, commonly named the shield sundew, is used as an antitussive in phytotherapy, although the plants’ composition has not been determined in detail so far. Hence, in this study, we present a validated, sensitive, reliable, and cheap narrow-bore LC–DAD method for the simultaneous quantification of flavonoids and ellagic acid derivatives in this herbal drug. In addition, the structures of 13 compounds have been elucidated by LC–MS, LC–NMR, and offline NMR experiments after isolation: herbacetin-3-O-glucoside (1), gossypitrin (2), ellagic acid (3), quercetin-7-O-glucoside (4), isoquercitrin (5), kaempferol-3-O-(6″-O-galloyl)-glucoside (6), herbacetin-7-O-glucoside (7), astragalin (8), gossypetin (9), herbacetin (10), quercetin (11), 3,3′-di-O-methyl ellagic acid (12), and kaempferol (13). Compounds 1, 2, 4, 5, 6, 7, and 10 have been identified in D. peltata for the first time, and compounds 1, 4, 6, 7, and 10 have not been detected in any Drosera species before.
Keywords: Drosera peltata; LC–DAD; LC–MS; LC–NMR; Flavonoids; Ellagic acid derivatives;

In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2′-deoxy-2′,2′-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2′, 2′-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2′,2′-difluoro-deoxyuridine) by LC–MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100 ng/mL, gemcitabine from 0.25 to 100 ng/mL and dFdU from 1 to 1000 ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from −7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24 h at room temperature, 660 days in frozen storage, and at least 4 freeze–thaw cycles, at both −20 and −70 °C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC–MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis.
Keywords: Prodrug; Gemcitabine; Column switching; LC–MS/MS;

LC–MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites by Pauline Le Faouder; Vincent Baillif; Ian Spreadbury; Jean-Paul Motta; Perrine Rousset; Gerald Chêne; Charlotte Guigné; François Tercé; Stephen Vanner; Nathalie Vergnolle; Justine Bertrand-Michel; Marc Dubourdeau; Nicolas Cenac (123-133).
Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography–tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5 min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6–155 pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators.
Keywords: Citrobacter rodentium; Foam macrophage; LC–MS/MS; Peritonitis; Eicosanoid; Docosanoid;

Evaluating effects of penicillin treatment on the metabolome of rats by Jinchun Sun; Laura K. Schnackenberg; Sangeeta Khare; Xi Yang; James Greenhaw; William Salminen; Donna L. Mendrick; Richard D. Beger (134-143).
Penicillin (PEN) V, a well-known antibiotic widely used in the treatment of Gram-positive bacterial infections, was evaluated in this study. LC/MS- and NMR-based metabolic profiling were employed to examine the effects of PEN on the host's metabolic phenotype. Male Sprague Dawley rats were randomly divided into groups that were orally administered either 0.5% methylcellulose vehicle, 100 or 2400 mg PEN/kg body weight once daily for up to 14 consecutive days. Urine, plasma and tissue were collected from groups sacrificed at 6 h, 24 h or 14 d. The body fluids were subjected to clinical chemistry and metabolomics analysis; the tissue samples were processed for histopathology. The only notable clinical chemistry observation was that gamma glutamyltransferase (GGT) significantly decreased at 24 h for both dose groups, and significantly decreased at 14 d for the high-dose groups. Partial least squares discriminant analysis scores plots of the metabolomics data from urine and plasma samples showed dose- and time-dependent grouping patterns. Time- and dose-dependent decreases in urinary metabolites including indole-containing metabolites (such as 3-methyldioxyindole sulfate generated from bacterial metabolism of tryptophan), organic acids containing phenyl groups (such as hippuric acid, phenyllactic acid and 3-hydroxyanthranilic acid), and metabolites conjugated with sulfate or glucuronide (such as cresol sulfate and aminophenol sulfate) indicated that the gut microflora population was suppressed. Decreases in many host-gut microbiota urinary co-metabolites (indole- and phenyl-containing metabolites, amino acids, vitamins, nucleotides and bile acids) suggested gut microbiota play important roles in the regulation of host metabolism, including dietary nutrient absorption and reprocessing the absorbed nutrients. Decreases in urinary conjugated metabolites (sulfate, glucuronide and glycine conjugates) implied that gut microbiota might have an impact on chemical detoxification mechanisms. In all, these results clearly show that metabolic profiling is a useful tool to better understand the effects of the antibiotic penicillin has on the gut microbiota and the host.
Keywords: Penicillin; Metabolome; Gut microbiota; Host-microbial interaction; LC/MS; NMR;

In this study, a simple, sensitive and reliable HPLC–UV method applying rapid sample preparation technique for the determination of captopril in human plasma was developed and validated. The method is based on pre-column derivatization of captopril and 2-propene-1-thiol (internal standard) with a new reagent 2-naphthyl propiolate. Sample clean-up, derivatization and extraction were carried out in two steps, totally less than 30 min. The extracts were chromatographed on a C18 column (5 μm, 150 mm × 4.6 mm i.d.). The mobile phase consisted of methanol (75%, v/v) and phosphate buffer (25%, pH = 8, 0.01 M). UV detection was performed at 290 nm. To obtain the best reaction yield, the factors that could influence the derivatization process, including the concentration of derivatization reagent, pH of sample solution and temperature were investigated in detail and optimized using Box–Behnken response surface methodology. Under optimized conditions the average extraction recovery of captopril and internal standard were >86%. The achieved lower limit of quantification (LLOQ) was 3 ng/mL; the assay exhibited a linear dynamic range of 3–2000 ng/mL with correlation coefficient (r 2) of ≥0.99. The precision was satisfactory in the whole calibration range with RSD of 5.9–12.4% (accuracy: from 97.5% to 93.6%) and of 6.4–12.8% (accuracy: from 97.3% to 95.2%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze–thaw, short- and long-term stability testing. Lastly, the developed method was successfully applied to the bioequivalence study of captopril administrated as a single oral dose (50 mg) to 12 healthy male volunteers.
Keywords: Captopril; 2-Naphthyl propiolate; Optimization; Pre-column derivatization; Plasma; HPLC;

Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA by Tatsuhiro Ota; Makoto Yasuda; Ryosuke Iijima; Satoru Yui; Tomoko Fukuuchi; Noriko Yamaoka; Ken-ichi Mawatari; Kiyoko Kaneko; Kazuya Nakagomi (152-157).
N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450 nm and 560 nm, respectively. Both intra- and inter-day (n  = 6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576 fmol to 2.0 nmol and 556 fmol to 2.0 nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.
Keywords: N-acetylneuraminic acid; 4-(Acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid; DBD-ED; HILIC; HPLC;