Journal of Chromatography B (v.879, #3-4)

Direct LC–ES-MS/MS determination of phthalates in physiological saline solutions by C. Pérez Feás; M.C. Barciela-Alonso; P. Bermejo-Barrera (231-235).
A method for determining a group of phthalic esters (PAEs) in physiological saline solutions has been developed. The PAEs studied were dimethyl phthalate, diethyl phthalate, butyl benzyl phthalate and dibutyl phthalate. These groups of phthalates were determined by liquid chromatography–electrospray ionization-tandem mass spectrometry, working in positive ion mode. The compounds were separated by liquid chromatography working in gradient mode with acetonitrile–ultrapure water as a mobile phase. The separation was performed starting with 5% of acetonitrile and increasing to 75% in 5 min, followed by isocratic elution for 8 min. The method was precise (with relative standard deviation (RSD) from 1.0 to 6.8%) and sensitive, with LODs of 0.05, 0.38, 0.05 and 0.82 μg L−1 for DMP, DEP, BBP and DBP, respectively. The proposed analytical method has been applied to determine these compounds in different physiological saline solutions commercialized in plastic bottles.
Keywords: Phthalates; LC–ES-MS/MS; Physiological saline solutions;

Budesonide quantification by HPLC coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometry. Application to a comparative systemic bioavailability of two budesonide formulations in healthy volunteers by Ney Carter do Carmo Borges; Rafael Barrientos Astigarraga; Carlos Eduardo Sverdloff; Bruno Carter Borges; Thaís Rodrigues Paiva; Paulo Rebelo Galvinas; Ronilson Agnaldo Moreno (236-242).
In the present study, a novel, fast, sensitive and robust method to quantify budesonide in human plasma using 3-keto-desogestrel as the internal standard (IS) is described. The analyte and the IS were extracted from human plasma by liquid–liquid extraction (LLE) using ether. Extracted samples were analyzed by high performance liquid chromatography coupled to Atmospheric pressure photoionization tandem mass spectrometry (HPLC–APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 μm analytical column. The temperature of the autosampler was kept at 6 °C and the run time was 4.00 min. A linear calibration curve over the range 7.5–1000 pg ml−1 was obtained and the lowest concentration quantified was 7.5 pg ml−1, demonstrating acceptable accuracy and precision. This analytical method was applied in a relative bioavailability study in order to compare a test budesonide 64 μg/dose nasal spray formulation vs. a reference 64 μg/dose nasal spray formulation (Budecort Aqua) in 48 volunteers of both sexes. The study was conducted in an open randomized two-period crossover design and with a one-week washout period. Plasma samples were obtained over a 14 h interval. Since the 90% CI for both C max, AUClast and AUC0-inf were within the 80–125% interval proposed by the Food and Drug Administration and ANVISA, it was concluded that budesonide 64 μg/dose nasal spray was bioequivalent to Budecort Acqua® 64 μg/dose nasal spray, according to both the rate and extent of absorption.
Keywords: Budesonide; Nasal spray; Allergy; LC–MS/MS; Bioavailability; Pharmacokinetics;

Development of an LC–MS/MS method for the quantitation of 55 compounds prescribed in combined cardiovascular therapy by Oskar Gonzalez; Rosa Maria Alonso; Nerea Ferreirós; Wolfgang Weinmann; Ralf Zimmermann; Sebastian Dresen (243-252).
This paper reports an LC–MS/MS method with positive electrospray ionization for the screening of commonly prescribed cardiovascular drugs in human plasma, including compounds with antihypertensive (57), antidiabetic (12), hypolipemiant (5), anticoagulant (2) and platelet anti-aggregation (2) effects. Sample treatment consisted of a simple protein precipitation with MeOH/0.1 M ZnSO4 (4:1, v/v) solution after the addition of internal standard, followed by evaporation and reconstitution. Analytes separation was performed on a Polar-RP column (150 mm × 2 mm, 4 μm) using a gradient elution of 15 min. The MS system was operated in MRM mode, monitoring one quantitation and one confirmation transition for each analyte. The recovery of the protein precipitation step ranged from 50 to 70% for most of the compounds, while some were considerably affected by matrix effects. Since several analytes fulfilled the linearity, accuracy and precision values required by the ICH guidelines, the method proved to be suitable for their quantitative analysis. The limits of quantitation varied from 0.38 to 9.1 μg/L and the limits of detection from 0.12 to 5.34 μg/L. The method showed to be suitable for the detection of plasma samples of patients under cardiovascular treatment with the studied drugs, and for 55 compounds reliable quantitative results could be obtained.
Keywords: LC–MS/MS; Metabolic syndrome; Matrix effect; Cardiovascular drugs;

A rapid method has been developed to analyse CP 47, 497 in human urine. Urine samples were diluted with water:acetonitrile (90:10, v/v) and sample aliquots were analysed by triple quadrupole tandem mass spectrometry with a runtime of 5 min. Multiple reaction monitoring (MRM) as survey scan was performed. The method was validated in urine, according to an in-house validation protocol based on the criteria defined in Commission Decision 2002/657/EC. Three MRM transitions were monitored. The decision limit (CCα) was 0.01 μg mL−1 and for the detection capability a (CCβ) value of 0.02 μg mL−1 was obtained. The measurement uncertainty of the method was 21%. Fortifying human urine samples (n  = 18) in three separate assays, show the accuracy of the method to be between 95 and 96%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (0.1, 0.15 and 0.2 μg mL−1) was less than 10% respectively. The method proved to be simple, robust and time efficient. To the best of our knowledge there are no LC–MS methods for the determination of CP 47, 497 with validation data in urine.
Keywords: Spice; Synthetic cannabinoids; Human urine; Liquid chromatography mass spectrometry; Method validation;

A simple and sensitive method was developed for the determination of cytochrome P450 2E1 (CYP2E1) activity based on the liquid chromatography–mass spectrometry (LC–MS) analysis of 6-hydroxychlorzoxazone generated by 6-hydroxylation of chlorzoxazone under specific catalysis of CYP2E1. In the proposed method, 2-benzoxazolinone was chosen as internal standard and isopropyl ether was used as extraction solvent for sample preparation. The inter-day and intra-day precisions at low, medium and high concentrations of 6-hydroxychlorzoxazone were below 20.0%, and the LOD (S/N = 3) was 0.05 ng/mL. This method was applied to analyze the CYP2E1 activity of rat in different brain regions including frontal cortex (FC), cerebellum (CB), brain stem (BS), hippocampus (HC), striatum (ST), thalamus (TH), and olfactory bulb (OB). The results confirmed that chlorzoxazone was a suitable probe for the determination of CYP2E1 activity in brain regions and samples with low content of CYP2E1.
Keywords: Cytochrome P450 2E1; Chlorzoxazone; 6-hydroxychlorzoxazone; Liquid chromatography–mass spectrometry;

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3′-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography–tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3′-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1 ng/mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.
Keywords: Nicotine; Cotinine; trans-3′-Hydroxycotinine; Cytochrome P450 2A6 (CYP2A6); Tobacco; Secondhand smoke;

Quantitative measurement of cysteinyl leukotrienes and leukotriene B4 in human sputum using ultra high pressure liquid chromatography–tandem mass spectrometry by Gloria Paola Chappell; Xiaoyao Xiao; Arnaldo Pica-Mendez; Tracey Varnell; Stuart Green; Wesley K. Tanaka; Omar Laterza (277-284).
The role of leukotrienes (LTs) in airway inflammatory diseases, such as asthma, has been extensively reported. The measurement of LTs in sputum supernatants, which is commonly done via enzyme immunoassays (EIAs), may prove to be useful for assessing airway inflammation. Despite the many advantages of EIA, these methods suffer from a lack of selectivity. Therefore, a selective and reliable method for the analysis of LTs in human sputum is needed. In this study we developed and validated a sensitive and specific method using ultra high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS), to measure simultaneously cysteinyl leukotrienes (CysLTs) and leukotriene B4 (LTB4) in human sputum. Sputum supernatants obtained by ultracentrifugation were stabilized by protease inhibitors, spiked with stable isotopic internal standards, and subjected to solid phase extraction (SPE) and UHPLC separation. Multiple reaction monitoring (MRM) transitions were optimized and measured on a mass spectrometer. The limit of detection (LOD) for LTE4 and LTB4 was 9.8 and 19.5 pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE4 and LTB4 was 19.5 and 39.0 pg/mL, respectively. The dynamic range of the LTE4 assay was from 9.8 to 5000 pg/mL, whereas for the LTB4 assay was from 19.5 to 10,000 pg/mL. The intra- and inter-day % coefficient of variation (%CV) was <6.5% and <10%, for both LTE4 and LTB4, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB4. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB4 was detectable in all samples tested and it ranged from 79 to 7220 pg/mL. LTE4 was detectable in most of the sputum samples (12.3–891 pg/mL), whereas LTC4 and LTD4 were below limit of detection for majority of sputum samples. The in vitro conversion of LTC4 and LTD4 into LTE4 was observed. The measurement of LTB4 was sensitive to low pH and high temperature. The use of UHPLC–MS/MS method will allow a more accurate and reliable quantitation of LTs in human sputum, which in turn, may lead to a better understanding of the role of LTs in airway disease pathways and the application in associated clinical treatments.
Keywords: Human sputum; Cysteinyl leukotrienes; Leukotriene B4; Asthma; Chronic obstructive pulmonary diseases; LC–MS/MS;

A sensitive and selective LC–MS/MS based bioanalytical method was developed and validated for the quantification of 3-deazaneplanocin A (DZNep), a novel epigenetic anti-tumor drug candidate, in Sprague–Dawley (SD) rat biosamples (plasma, urine, feces and tissue samples). The method comprises a phenylboronic acid (PBA)-containing solid phase extraction procedure, serving for binding and clean-up of DZNep in rat biosamples spiked with tubercidin (as internal standard). The analytes were separated on an Agilent hydrophilic interaction chromatography (HILIC) column. LC–MS/MS in positive ion mode was used to perform multiple reaction monitoring at m/z of 263/135 and 267/135 for DZNep and tubercidin, respectively. The limit of quantification (LOQ) of DZNep in rat biosamples was 20 ng/mL. The data of intra-day and inter-day accuracy were within 15% of nominal concentration while the precision (relative standard deviation) less than 10% for all biosamples. The extraction recoveries for DZNep and tubercidin were consistent and reproducible (around 80%) and the matrix effects were negligible (around 10% suppression) in all biosamples. This method was demonstrated to be applicable for pharmacokinetic studies of DZNep in SD rats.
Keywords: 3-Deazaneplanocin A; LC–MS/MS; Rat biosamples; Pharmacokinetics; Tissue distribution; Excretion studies;

A simple and novel method of single drop liquid–liquid–liquid microextraction (SD-LLLME) coupled with capillary electrophoresis (CE) for the determination of six fluoroquinolones (FQs) was developed. The method was eventually applied to extraction and preconcentration of FQs in human urine samples. Good linear relationships were obtained for all analytes in a range of 40–1000 μg L−1 with the correlation coefficients from 0.9913 to 0.9995. The limit of detections (LODs) varied from 7.4 to 31.5 μg L−1 at a signal-to-noise (S/N) of 3. The recoveries at two spiking levels were 81.8–104.9% with relative standard deviations <8.3%.
Keywords: Single drop liquid–liquid–liquid microextraction; Capillary electrophoresis; Fluoroquinolones; Urine samples;

Determination of hippuric acid in human urine by ion chromatography with conductivity detection by Fuyong Zhao; Zonghua Wang; Hui Wang; Mingyu Ding (296-298).
A simple, rapid, precise and eco-friendly ion chromatography (IC) method for the determination of hippuric acid (HA) in human urine was proposed in this paper. The separation was carried out an anion exchange column with 2.0 mmol L−1 Na2CO3  + 2.0 mmol L−1 NaHCO3 as mobile phase at the flow-rate 0.7 mL min−1. A suppressed conductivity detector was used and the detection limit was 1.0 μg L−1(S/N = 3) for hippuric acid. The analysis time for one run was 30 min under the optimized IC condition. The recovery of hippuric acid was 93.2–98.0% while the relative standard deviation (RSD) was 1.4–2.3% by seven measurements.
Keywords: Ion chromatography; Hippuric acid; Urine;