Quantitative Analysis of Triptolide in Human Whole Blood by LC–APCI-IT-MS-MS

In this study, the development and validation of an analytical method for triptolide in whole blood using high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization ion trap tandem mass spectrometry (LC–APCI-IT-MS-MS) is reported. This is the first report of the systematic development and validation of an LC–MS-MS method for the quantitation of triptolide in human whole blood using prednisolone as an internal standard (IS). Prior to LC–MS-MS analysis, liquid–liquid extraction with ethyl acetate was used to isolate them from the biological matrix. Validation parameters such as specificity/selectivity, limit of quantitation (LOQ), linearity, precision, accuracy and stability are evaluated for this method. The calibration curve was linear (r ² = 0.9973) in the concentration range of 0.5–100.0 ng mL⁻¹ in human whole blood with a lower limit of quantitation of 0.5 ng mL⁻¹. Intra- and inter-day relative standard deviations (RSDs) were less than 8.6 and 11.7%, respectively. Extraction recoveries of triptolide ranged from 81.5 to 88.1%. This assay can be used to determine trace triptolide in human whole blood.     

Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry

An accurate and selective method for the simultaneous determination of triptolide, tripdiolide and tripterine in human urine using hydrocortisone as an internal standard (IS) by high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization mass spectrometry in negative ion mode has been developed. After triptolide, tripdiolide and tripterine in human urine were extracted with ethyl acetate and cleaned by solid-phase extraction with C(18) cartridges, a satisfactory separation was achieved on an XDB C(18) short column (30 x 2.1 mm i.d., 3 microm) using the mobile phase of acetic acid-ammonium acetate (5 mmol/L, pH = 4.5)-acetonitrile-methanol in gradient elution. Detection was operated by APCI in selected ion monitoring mode. The target ions m/z 359, m/z 375, m/z 449 and m/z 419 were selected for the quantification of triptolide, tripdiolide, tripterine and IS, respectively. The linear range was 1.0-100.0 ng mL(-1), and the limits of quantification in human urine were found to be 0.1-0.5 ng mL(-1) for the three compounds. The precisions (CV%) and accuracies were 6.6-12.9 and 85.1-97.0%, respectively. The developed method could be applied to the determination of triptolide, tripdiolide and tripterine in human urine for diagnosis of the intoxication and for forensic purposes.     

A liquid chromatography–mass spectrometric method for the quantification of azithromycin in human plasma

A liquid chromatographic mass spectrometric assay for the quantification of azithromycin in human plasma was developed. Azithromycin and imipramine (as internal standard, IS) were extracted from 0.5 mL human plasma using extraction with diethyl ether under alkaline conditions. Chromatographic separation of drug and IS was performed using a C₁₈ column at room temperature. A mobile phase consisting of methanol, water, ammonium hydroxide and ammonium acetate was pumped at 0.2 mL/min. The mass spectrometer was operated in positive ion mode and selected ion recording acquisition mode. The ions utilized for quantification of azithromycin and IS were m/z 749.6 (M + H) ⁺ and m/z 591.4 (fragment) for azithromycin, and 281.1 m/z for internal standard; retention times were 6.9 and 3.4 min, respectively. The calibration curves were linear (r² > 0.999) in the concentration ranges of 10–1000 ng/mL. The mean absolute recoveries for 50 and 500 ng/mL azithromycin and 1 µg/ mL IS were >75%. The percentage coefficient of variation and mean error were <11%. Based on validation data, the lower limit of quantification was 10 ng/mL. The present method was successfully applied to determine azithromycin pharmacokinetic parameters in two obese volunteers. The assay had applicability for use in pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.     

LC–APCI–MS–MS for the Determination of Celastrol in Human Whole Blood

A rapid, sensitive high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) assay has been developed and validated for the quantitative determination of celastrol in human whole blood using hydrocortisone as an internal standard (I.S.). The celastrol and I.S. were extracted from human whole blood with ethyl acetate. The separation was performed by reversed-phase HPLC using an isocratic mobile phase consisting of 5 mmol L^?1 aqueous ammonium acetate containing 0.05% acetic acid/methanol (25:75, v/v) on a XDB-C₁₈ column (150 mm × 4.6 mm I.D., 5 μm). Detection was by negative atmospheric pressure chemical ionization (APCI) and ion trap tandem mass spectrometry in multiple reaction monitoring (MRM) mode with a transition of m/z 449.4 → 405.1 for celastrol, and 419.2 → 329.1 for I.S. The calibration curve was linear (r ² = 0.9967) in the concentration range of 1.0–200.0 ng mL^?1 in human whole blood with a limit of quantification of 1.0 ng mL^?1. Intra-day and inter-day relative standard deviations (RSDs) were less than 8.5 and 10.1%. The mean extraction recovery was 89.2% for celastrol and 92.6% for I.S. This assay can be used to determine trace celastrol in human whole blood.     

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm × 4.6 mm, 5 μm) at 35 °C under isocratic conditions using 5 mM KH₂PO₄ buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min⁻¹. UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na₂HPO₄ pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 °C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL⁻¹ plasma (r ≥ 0.9927), limits of quantification of 50 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL⁻¹ plasma (r ≥ 0.9900), limits of quantification of 30 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.     

Liquid chromatographic determination of cis-platin as platinum(II) in pharmaceutical preparation, serum and urine samples of cancer patients

Spectrophotometric and high performance liquid chromatographic (HPLC) methods have been developed for the determination of cis-platin and carboplatin based on the pre-column derivatization of platinum(II) with 2-acetylpyridine-4-phenyl-3-thiosemicarbazone. The complex was extracted in chloroform with molar absorptivity of 2.2 × 10⁴ L mol⁻¹ cm⁻¹ at 380 nm. The complex eluted from a Phenomenex C-18 (150 mm × 4.6 mm i.d.) column with methanol:water:acetonitrile:tetrabutyl ammonium bromide (1 mM) (44:30:25:1, v/v/v/v) with a flow rate of 1 ml/min and UV detection at 260 nm. Ruthenium(IV) and selenium(IV) also separated completely. The linear calibration curve was with 0.5-12.5 μg/ml and detection limit of 10 ng/ml platinum(II).The analysis of cis-platin and carboplatin injections by spectrophotometric and HPLC methods indicated relative standard deviation (R.S.D.) of 0.66-2.1%. The method was used for the determinations of cis-platin in serum and urine of cancer patients after chemotherapy and platinum contents were found 148-444 and 50-90 ng/ml with R.S.D. of 0.3-3.0 and 0.6-2.4% for the serum and urine, respectively. The recovery of platinum(II) from serum was 97% with R.S.D. 2.2%.     

Multiresidue analysis of 83 pesticides and 12 dioxin-like polychlorinated biphenyls in wine by gas chromatography–time-of-flight mass spectrometry

A multiresidue method is described for simultaneous estimation of 83 pesticides and 12 dioxin-like polychlorinated biphenyls (PCBs) in red and white wines. The samples (20 mL wine, acidified with 20 mL 1% HCl) were extracted with 10 mL ethyl acetate (+20 g sodium sulphate) and cleaned by dispersive solid-phase extraction (DSPE) with anhydrous calcium chloride and Florisil successively. The final extract (5 mL) was solvent exchanged to 1 mL of cyclohexane:ethyl acetate (9:1), further cleaned by DSPE with 25 mg primary secondary amine sorbent and analyzed by gas chromatography–time-of-flight mass spectrometry (GC–TOF-MS) within 31 min run time. The limits of quantification of most analytes were ≤10–20 μg/L. Acidification of wine prior to extraction prevented hydrolysis of organophosphorous pesticides as well as dicofol, whereas treatment with CaCl₂ minimized the fatty acid co-extractives significantly. Solvent exchange to cyclohexane:ethyl acetate (9:1) further minimized the co-extractives. Recoveries at 5, 10 and 20 ng/mL were >80% for most analytes except cyprodinil, buprofezin and iprodione. The expanded uncertainties at 10 ng/mL were <20% for most analytes. Intra-laboratory precision in terms of Horwitz ratio of all the analytes was below 0.5, suggesting ruggedness of the method. Effectively, the method detection limit for most analytes was as low as up to 1 ng/mL in both red and white wine, except for cyfluthrin and cypermethrin.     

Determination of patulin in fruit juice and dried fruit samples by in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

A simple and sensitive method for the determination of patulin in fruit juice and dried fruit samples was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). Patulin was separated within 5 min by high-performance liquid chromatography using a Synergi MAX-RP 80A column and water/acetonitrile (80/20, v/v) as the mobile phase. Electrospray ionization conditions in the negative ion mode were optimized for MS detection of patulin. The pseudo-molecular ion [M−H]⁻ was used to detect patulin in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample using a Carboxen 1006 PLOT capillary column as an extraction device. The extracted patulin was readily desorbed from the capillary by passage of the mobile phase, and no carry-over was observed. Using the in-tube SPME LC–MS with SIM method, good linearity of the calibration curve (r = 0.9996) was obtained in the concentration range of 0.5–20 ng/mL using ¹³C₃-patulin as an internal standard, and the detection limit (S/N = 3) of patulin was 23.5 pg/mL. The in-tube SPME method showed >83-fold higher sensitivity than the direct injection method (10 μL injection volume). The within-day and between-day precision (relative standard deviations) were below 0.8% and 5.0% (n = 6), respectively. This method was applied successfully for the analysis of fruit juice and dried fruit samples without interference peaks. The recoveries of patulin spiked into apple juice were >92%, and the relative standard deviations were <4.5%. Patulin was detected at ng/mL levels in various commercial apple juice samples.     

Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25 mL oral fluid and 0.75 mL buffer) was applied to conditioned CEREX^® Polycrom™ THC solid-phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS–electron capture negative chemical ionization (NCI-MS). Linearity was 0.5–50 ng/mL for THC, 11-OH-THC, CBD and 1–50 ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5–500 pg/mL. Intra- and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3–6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex^® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.     

Sandwich-type electrochemiluminescence immunosensor based on N-(aminobutyl)-N-ethylisoluminol labeling and gold nanoparticle amplification

A novel electrochemiluminescence (ECL) sandwich-type immunosensor for human immunoglobulin G (hIgG) on a gold nanoparticle modified electrode was developed by using N-(aminobutyl)-N-ethylisoluminol (ABEI) labeling. The primary antibody, goat-anti-human IgG was first immobilized on a gold nanoparticle modified electrode, then the antigen (human IgG) and the ABEI-labeled second antibody was conjugated successively to form a sandwich-type immunocomplex. ECL was carried out with a double-step potential in carbonate buffer solution (CBS) containing 1.5 mM H₂O₂. The ECL intensity increased linearly with the concentration of hIgG over the range 5.0-100 ng/mL. The limit of detection was 1.68 ng/mL (S/N = 3). The relative standard deviation was 3.79% at 60 ng/mL (n = 9). The present immunosensor is simple and sensitive. It has been successfully applied to the detection of hIgG in human serums.     

Comparative analysis of odorous volatile organic compounds between direct injection and solid-phase microextraction: Development and validation of a gas chromatography–mass spectrometry-based methodology

In this study, the feasibility of GC–MS was evaluated for the quantification of odorous volatile organic compounds (VOCs) in environmental samples. These included methyl ethyl ketone, isobutyl alcohol, methyl isobutyl ketone, and butyl acetate plus benzene, toluene, and xylene (BTX). For this purpose, the gaseous standard for these VOCs were analyzed by GC–MS with the aid of both direct injection (DI) into the GC injector and solid-phase microextraction (SPME). The liquid phase standard prepared independently was tested additionally by the DI method as a reference to gaseous calibration. The detection limit (DL) values, when tested for basic quality assurance in this study, showed large differences between DI (0.002–0.007 ng) and SPME method (1.03–1.81 ng) in terms of absolute mass. The DL values, when expressed in terms of concentration (v/v), showed considerable improvement in SPME (below 0.40 nmol mol⁻¹) relative to the DI method (∼6–15 nmol mol⁻¹). The reliability of the GC–MS method was further validated through an analysis of real environmental samples collected from an industrial area.     

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220 nm was successfully transferred from a conventional reversed phase column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000 ng/mL for KE, 25-1500 ng/mL for NK, and 15-750 ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5 ng/mL, respectively, and for DHNK were 15 and 7.5 ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.     

Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay

A surface plasmon resonance immunoassay has been developed to determine human growth hormone (hGH) directly and without pre-treatment in human serum samples. A binding inhibition immunoassay was employed. Antibody concentration, assay buffer and regeneration solution have been optimized in order to reach the best performance and the lower non-specific binding of the matrix components to the sensor surface. The lowest detection limit was 6 ng/mL, with a working range covering the physiological range. Reproducibility of the assay was excellent with both intra-assay and inter-assay relative standard deviations <5%, while a variation of 2.19% was obtained employing different sensor chips. Reutilization of the sensor surface allows its continuous use over 50 measurements with a signal drop <20%. The SPR immunoassay results were validated using enzyme-linked immunosorbent assay (ELISA) showing an excellent correlation (R² = 0.985). A portable and fully automated system (Sensia SL) was employed in this work. This is the first SPR biosensor assay capable of detecting relevant concentrations of a clinical analyte in serum. This study shows the potentials of this device as a diagnostic tool for the detection of multiple clinical analytes.     

Ultra-performance liquid chromatographic-electrospray mass spectrometric determination (UPLC-ESI-MS) of O-demethylated metabolite of paeonol in vitro: Assay development, human liver microsome activities and species differences

A simple and sensitive method for determination of the O-demethylation activity of rat, dog, minipig, and human liver micrsomes toward paeonol using ultra-performance liquid chromatography with mass detection (UPLC-MS) has been developed. The method uses chemically synthesized O-demethylated metabolite of paeonol (2,4-dihydroxyacetophenone, DHA) as a standard for method validation. Validation was done 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 30:70). Selective ion reaction (SIR) monitor was specific for paeonol, DHA and I.S. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for DHA was linear from 0.5-100 μM with r² = 0.9999. The newly developed method has good precision and accuracy. The method was successfully used to determine the kinetics of DHA activities toward paeonol in liver microsomes from different species. Dog liver microsomes (DLMs) were the most active in paeonol O-demethylation (709.7 pmol/min/mg protein) followed by rat liver microsomes (RLMs) (579.6 pmol/min/mg protein), HLMs (569.3 pmol/min/mg protein), and then minipig liver microsomes (PLMs) (417.3 pmol/min/mg protein). The developed method was appropriated for rapid screening paeonol O-demethylation activity in liver microsomes from different species.     

Salmonella typhi determination using voltammetric amplification of nanoparticles: A highly sensitive strategy for metalloimmunoassay based on a copper-enhanced gold label

A highly sensitive electrochemical amplification immunoassay for Salmonella typhi (S. typhi) determination has been developed for the first time by using a copper-enhanced gold nanoparticle label coupled with anodic stripping voltammetry. Monoclonal antibodies for S. typhi were first immobilized on polystyrene microwells and then captured by S. typhi bacteria. After an immunoreaction occurred, a polyclonal, antibody-colloidal gold conjugate was added to bind to the S. typhi bacteria. Next, a copper-enhancer solution containing ascorbic acid and copper (II) sulfate was added into the polystyrene microwells. The ascorbic acid was employed to reduce the copper (II) ions to copper (0), which was subsequently deposited onto the gold nanoparticle tags. After the copper was dissolved in nitric acid, the released copper ions were detected by anodic stripping voltammetry. The amount of deposited copper was related to the amount of gold nanoparticle tag present, which was controlled by the amount S. typhi attached to the polyclonal antibody-colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration over concentration range of 1.30 × 10² cfu/mL to 2.6 × 10³ cfu/mL in a logarithmic plot, with a detection limit as low as 98.9 cfu/mL. The influences of the relevant experimental variables, such as the concentration of copper and the reaction time of S. typhi with antibody, were investigated. We also successfully applied this method to determine the presence of S. typhi in human serum. Our results are a step towards developing more sensitive and reliable nanoparticle immunoassays.     

Determination of dye precursors in hair coloring products by liquid chromatography with electrochemical detection

The simultaneous determination of seven aminophenols, resorcinol and p-phenylenediamine in hair coloring products was performed by liquid chromatography (HPLC) with amperometric detection (ED). The aminophenols were separated on a ODS C18 reversed-phase column by isocratic elution with a mobile phase based on 0.1 M acetate buffer pH 4.5-methanol (90:10%, v/v) at a flow rate 0.8 mL min⁻¹. The limit of detection (S/N = 3) for the aminophenols was in the 15-40 pg (injected mass) range at an applied potential of 0.950 V versus Ag/AgCl. Peak heights for the aminophenols and the two others compounds were found to be linearly related to the amount injected, from 0.3 to 300 ng (r > 0.994-0.999).The relative standard deviation (R.S.D., n = 10) for 1 ng injected was comprised in the range from 2.5 to 6.2%, depending on the aminophenol tested. The present method minimizes troublesome and time-consuming pretreatment procedures and it was applied to the determination of aminophenols, resorcinol and phenylenediamine in hair coloring formulations.     

A liquid chromatography/positive ion tandem mass spectrometry method for the determination of cilazapril and cilazaprilat in human plasma

A simple, rapid, and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for simultaneous determination of cilazapril levels and its active metabolite, cilazaprilat, in human plasma using enalapril as internal standard. The acquisition was performed in the multiple reaction monitoring mode; monitoring the transitions: m/z 418.4 > 211.1 for cilazapril and m/z 390.3 > 211.1 for cilazaprilat. The method involves a simple single-step liquid-liquid extraction with ethyl acetate. The analyte was chromatographed on an YMC C₈ reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (10:90, v/v; pH 3.2 with formic acid). Numerous compounds did not interfere with specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted cilazapril, cilazaprilat, and enalapril within 2 min. This method was validated over 0.1-500 ng ml⁻¹ of cilazapril and 0.5-50 ng ml⁻¹ of cilazaprilat. Cilazapril and cilazaprilat were stable in standard solution and in plasma samples under typical storage and processing conditions. The assay was successfully applied to a pharmacokinetic study of cilazapril given as a single oral dose (5 mg) to healthy volunteers.     

Automated determination of folate catabolites in human biofluids (urine,breast milk and serum) by on-line SPE–HILIC–MS/MS

A rapid, precise and fully-automated method for analysis of folate (vitamin B9) and its catabolites (viz. p-aminobenzoylglutamate and its acetamide derivative) in biofluids is here presented. The method is based on on-line hyphenation of solid-phase extraction (SPE) by a Prospekt 2 system with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The method was analytically characterized by estimation of repeatability (RSD, n = 5, between 0.5 and 4.1%), accuracy (between 96 and 105%), and sensitivity (limits of quantificantion between 0.3 and 8.3 ng/mL (1.1 and 18.8 pmol/mL) or 0.03 and 0.83 ng (0.11 and 1.88 pmol)). The proposed method is suited for routine analysis of folate catabolites as biomarkers to monitor deficiency of vitamin B9.     

Multi-residue analytical method for human pharmaceuticals and synthetic hormones in river water and sewage effluents by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Pollutants such as human pharmaceuticals and synthetic hormones that are not covered by environmental legislation have increasingly become important emerging aquatic contaminants. This paper reports the development of a sensitive and selective multi-residue method for simultaneous determination and quantification of 23 pharmaceuticals and synthetic hormones from different therapeutic classes in water samples. Target pharmaceuticals include anti-diabetic, antihypertensive, hypolipidemic agents, β2-adrenergic receptor agonist, antihistamine, analgesic and sex hormones. The developed method is based on solid phase extraction (SPE) followed by instrumental analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) with 30 min total run time. River water samples (150 mL) and (sewage treatment plant) STP effluents (100 mL) adjusted to pH 2, were loaded into MCX (3 cm³, 60 mg) cartridge and eluted with four different reagents for maximum recovery. Quantification was achieved by using eight isotopically labeled internal standards (I.S.) that effectively correct for losses during sample preparation and matrix effects during LC–ESI-MS/MS analysis. Good recoveries higher than 70% were obtained for most of target analytes in all matrices. Method detection limit (MDL) ranged from 0.2 to 281 ng/L. The developed method was applied to determine the levels of target analytes in various samples, including river water and STP effluents. Among the tested emerging pollutants, chlorothiazide was found at the highest level, with concentrations reaching up to 865 ng/L in STP effluent, and 182 ng/L in river water.     

Quantification of sex pheromone from Anocentor nitens by gas chromatography-mass spectrometry-selected ion monitoring

Two highly sensitive and selective methods based on gas chromatography coupled to mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode have been developed for the quantification of 2,6-dichlorophenol (2,6-DCP), a sex pheromone of the tick females of Anocentor nitens. Standard addition method and calibration curve techniques using 5-bromine-4-hydroxy-3-methoxybenzaldehyde (5-BrV) as internal standard (IS) afforded detection limit of 0.1 ng ml⁻¹. The calibration curve was linear over the concentration range from 0.5 to 500 ng ml⁻¹ for 2,6-DCP. Results show that the concentration range of sex pheromone in the extracts samples was 1.08-10.35 ng ml⁻¹. The methods developed provided reliable procedures to determine amounts of 2,6-DCP present in ticks.