Development of a Straightforward and Cheap Ethyl Acetate Based Method for the Simultaneous Determination of Pesticides and Veterinary Drugs Residues in Bovine Liver and Muscle

The optimization and validation study of a qualitative and quantitative multiclass, ethyl acetate (EtOAc) multi-residue method to straightforward monitor 48 compounds in liver (6 veterinary drugs and 42 pesticides) and 54 in muscle (5 veterinary drugs and 49 pesticides) followed by high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) and gas chromatography mass-spectrometry determination (GC–MS) is presented. Several clean-up sorbents were evaluated looking for the best strategy for the removal of the matrix co-extractives. A combination of aluminium oxide, (Al₂O₃), C-18 and magnesium sulphate (MgSO₄) yielded the best analytical results in terms of precision and accuracy. The method was validated at three fortification levels: 10, 100 and 250 µg kg⁻¹. The percentages of recovery were between 70 and 114 % for bovine muscle and 70–118 % for liver. Repeatability and intermediate precision percentages were below 20 % for both matrices. Most of the compounds under study presented good linearity and quantification limits below their corresponding European Union (EU) and Codex Alimentarius maximum residue levels (MRLs). Twenty-two randomly taken real samples were analyzed with the validated methodology, trying to prove its effectiveness and suitability for routine analysis. The validated methodology represents a fast and cheap alternative for the simultaneous analysis of pesticides and veterinary drugs which can be easily extend to other compounds and matrices.

     

Monitoring of Polychlorinated Biphenyls in Surface Water Using Liquid Extraction,GC/MS,and GC/ECD

Abstract

A rapid and reliable analytical method, at trace level concentration was developed and validated for monitoring polychlorinated biphenyls (PCBs) in Jordanian surface water. The method combines the advantage of liquid extraction together with gas chromatography‐mass spectrometry (GC/MS) and gas chromatography‐electron capture detector (GC/ECD). The performance of the method was evaluated by analyzing certified reference material (CRM) of the analytes and applied on real water samples collected from different sites in Jordan. A mixture of 60∶40 dichloromethan‐petroleum ether was chosen as a convenient binary solvent for liquid–liquid extraction. The GC conditions for GC/MS were optimized using He as a carrier gas, temperature programming, and chlorpropham as an internal standard (IS).

The conditions for GC/ECD were performed using N₂ gas and a temperature program from 160 to 280°C with different increasing rates. The method of GC/MS in the selective ion mode (SIM) gave linear relationships for all PCBs tested between 0.60–6.0 µg/l with R ²=0.9934 (n=7×18). Recoveries from spiked water samples ranged between 87.6 and 91.4%. The mean accuracy and precision obtained were 4.9% and 2.16%, respectively. The mean of detection limit was 0.14±0.04 µg/l. In GC/ECD, linear relationships for all PCBs examined over the range of 0.3–2.4 µg/l was verified as characterized by a linear regression equation and correlation coefficient, R ²=0.9915 (n=12). The average precision and accuracy were 4.86% and 5.21%, respectively. Analyses results clarified that none of the examined Jordanian water samples contained any of the searched for PCBs within the detection limit achieved.     

Determination of Microcystins in Cyanobacteria by Supercritical Fluid Extraction and Liquid Chromatography‐Tandem Mass Spectrometry

Abstract

A method for the detection of microcystins (microcystin LR, RR, and YR) in cyanobacteria by supercritical fluid extraction (SFE) and liquid chromatography‐mass spectrometry (LC/MS) has been developed. Supercritical fluids for the analytical extraction of nonvolatile, higher molecular weight compound, and microcystins from cyanobacteria were investigated. The microcystins included in this study are sparsely soluble in neat supercritical fluid CO₂. However, the microcystins was successfully extracted with a ternary mixture (90% CO₂, 9.5% methanol, 0.5% water) at 40°C and 250 atm. The polar carbon dioxide‐aqueous methanol fluid system gave high extraction efficiency for the extraction of the polar microcystins from cyanobacteria. The microcystins were determined by liquid chromatography‐tandem mass spectrometry (LC/MS/MS).     

LC–MS/MS Method for Detection of a Highly Selective KCa3.1 Blocker,TRAM-34, in Rat Plasma

1-[(2-Chlorophenyl)diphenylmethyl]pyrazole (TRAM-34) is a highly selective K_Ca3.1 channel blocker. TRAM-34 was commonly used to study the role of K_Ca3.1 in the pathogenesis of disease in vivo, but there was no validated analytical method. Here, we describe the first validated LC–MS/MS analytical method for TRAM-34. Solid-phase extraction (SPE) was performed to extract TRAM-34 from the rat plasma. Chromatographic separation was achieved on the phenyl column. A triple quadrupole mass spectrometer was operated in positive-mode electrospray ionization. There were two multiple-reaction monitoring (MRM) transitions for TRAM-34: m/z 277.2 → 165.1 (for quantification) and m/z 277.2 → 241.2 (for qualification). Bifonazole was used as an internal standard. The lower limit of quantification (LLOQ) achieved was 1 ng mL^?1 and the run time was 7.5 min. The linear range was from 1 to 1,000 ng mL^?1. The pharmacokinetics profile was acquired for rats following an intraperitoneal injection of TRAM-34, with the following pharmacokinetics parameters found: C _max 17.03 ± 1.34 ng mL^?1; T _max 8.67 ± 3.06 h; and T _1/2 13.45 ± 2.72 h. In addition, a suspected metabolite of TRAM-34 was found using this LC–MS/MS method. Given the results of the detailed validation process and its application to TRAM-34 pharmacokinetics, it is clear that a fast, selective, precise, and reproducible TRAM-34 LC–MS/MS analytical method was successfully established.     

QuEChERS sample preparation followed by ultra-performance liquid chromatography – tandem mass spectrometry for rapid screening of dioctyl sulfosuccinate sodium salt in avian egg tissue

A quantification method was developed for the determination of dioctyl sulfosuccinate sodium salt (DOSS) in avian egg samples based on a QuEChERS extraction technique followed by UPLC-MS/MS analysis. DOSS is an anionic surfactant that is part of the Corexit^® 9500 dispersive mixture that prevents the formation of oil slicks on water bodies. It was extensively used when the Deepwater Horizon rig exploded and a large amount of crude oil was released into the Gulf of Mexico. QuEChERS provided a simple, effective and time saving sample preparation method prior to analysis without reducing analytical sensitivity and became an excellent substitute to lengthy traditional extraction methods. Weak anionic exchange cleanup significantly reduced matrix effects and improved analyte sensitivity. Ultra-performance liquid chromatography provided an effective separation method, while MS/MS provided the necessary selectivity and increased sensitivity. Our method achieved baseline separation of DOSS, surrogate (sodium octyl sulfate – d17) and the internal standard (sodium dioctyl sulfate – d25), with limits of detection (LOD) and limits of quantitation (LOQ) for DOSS being 260 and 500 pg/mL, respectively. Quality control recoveries were 70.5 ± 7.3% (mean ± standard deviation, n = 3) for the laboratory control sample and 72.4 ± 4.9% (n = 3) for the matrix spike. The extraction efficiency was monitored by adding surrogate compound to every sample with recoveries of 104.6 ± 14.1 for SDS-d1 and 81.8 ± 6.8 for SOS-d17. Currently, limited peer reviewed scientific data are reported on the effects of oil dispersants on the environment. Our analytical method for the determination of DOSS in avian egg matrix can be used to provide reliable data on the fate and effects of DOSS in biological systems.     

Highly Sensitive Determination of Butyl Xanthate in Surface and Drinking Water by Headspace Gas Chromatography with Electron Capture Detector

A highly sensitive and convenient method for the determination of butyl xanthate in surface water and drinking water was developed by headspace gas chromatography with electron capture detector (HS–GC–ECD). The analytical method was based on the decomposition of butyl xanthate under an acidic condition, generating carbon disulfide, which could be sensitively detected by gas chromatography with electron capture detector. The signal of CS₂ from the decomposition of potassium butyl xanthate was directly proportional to the concentration of potassium butyl xanthate over the range 0.7–100 ng/mL. The detection limit at a signal-to-noise ratio of three (S/N = 3) for potassium butyl xanthate was 0.3 ng/mL (~1.6 × 10⁻⁹ mol/L), which was more than two orders of magnitude lower than the popular UV methods and close to one order of magnitude lower than the similar headspace gas chromatography–mass spectroscopy method. The relative standard deviation (R.S.D.) within a day and in 3 days for potassium butyl xanthate at both 5 and 50 ng/mL was less than 4.7 %, suggesting good analytical performance of the present method. Good recoveries from 93.3 to 104.7 % were obtained from spiked surface and drinking water samples, indicating that the proposed HS–GC–ECD method was applicable for the quantification of butyl xanthate in surface and drinking water. Compared with other reported methods, the present method is highly sensitive, without sample preparation, and easily extended to the analysis of other xanthates.

     

Fast Determination of Fumonisin B1 and B2 in Corn Using a Modified QuEChERS Method and LC–MS–MS

A fast analytical method for the simultaneous determination of fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in corn using a novel QuEChERS method and LC–MS–MS was developed and validated. Samples were extracted with methanol–water (3:1 v/v) by means of ultrasonic extraction. The extract was purified with a novel modified QuEChERS method. Firstly, FB₁ and FB₂ in the extract were retained with primary secondary amine (PSA). Then, FB₁ and FB₂ were released from PSA with 1.0 % formic acid in methanol. The final eluate was diluted with water, and analyzed by LC–MS–MS on a Waters Acquity BEH C₁₈ column with 0.1 % formic acid in water/methanol as mobile phase with gradient elution. Mean recoveries of 83.5–102.4 % with CVs of 3.6–10.5 % were obtained at fortification levels of 2, 50 and 1,000 μg kg⁻¹. The limit of quantification was 2.0 μg kg⁻¹.

     

Practical Method for Accurate Determination of Alkylphenol Ethoxylates in Household Detergents by Aluminum Iodide Cleavage Pretreatment Followed by GC–MS

A practical procedure was developed for detecting nonylphenol ethoxylates (NPEOs) and octylphenol ethoxylates (OPEOs) in household detergents by gas chromatography–mass spectrometry (GC–MS) with the introduction of a cleavage technique using aluminum iodide (AlI₃) to convert NPEOs and OPEOs to their parent nonylphenol and octylphenol. The reliability of the cleavage process was evaluated using Igepal-210, Triton X-15, Tergitol NP-9, and Triton X-100 as substrates, demonstrating satisfactory efficiency. The effect of the sample matrix on the cleavage process was investigated, and the optimized dose of AlI₃ was estimated. The major advantage of this method is the use of a common analytical tool (GC–MS) for accurate monitoring of APEOs after elimination of the ethoxylate chain, with detection limits of 12.5 mg/kg and 5.0 mg/kg for NPEO₉ and OPEO₉, respectively. Analytical results revealed that NPEOs and OPEOs were found in 12.6 % of 182 household detergents in the concentration range of 18–800 mg/kg.     

The Application and Validation of HybridSPE-Precipitation Cartridge Technology for the Rapid Clean-up of Serum Matrices (from Phospholipids) for the Clinical Analysis of Serotonin,Dopamine and Melatonin

Phospholipids have been shown to cause matrix effects particularly in liquid chromatography–mass spectrometry (LC–MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC–MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep–wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021–3.268 μmol L⁻¹; R ² = 0.9983–0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2–23.5 nmol L⁻¹ and the LOQs of 15.4–70.5 nmol L⁻¹ for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples.

     

Pesticides Residues Rapid Extraction from Panax Ginseng Using a Modified QuEChERS Method for GC–MS

A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) rapid detection method followed by gas chromatography–tandem mass spectrometry (GC–MS) has been developed for the simultaneous determination of 42 pesticides in Panax ginseng. This method can be different from the other QuEChERS methods in the sense that it uses acetone and n-hexane solution rather than acetonitrile to extract and partition pesticides. This acetone, water and n-hexane solution QuEChERS method consists essentially of two steps: extraction/partitioning and purification. In step 1, P. ginseng was mixed with acetone, water and n-hexane solution, and then partitioned by vortex. In step 2, the top layer (n-hexane) was transferred into a centrifuge tube containing primary secondary amine, activated carbon and C₁₈ for purification. After the centrifuge supernatant was injected into GC–MS. The QuEChERS method was applied in P. ginseng detection and we confirmed that this method can easily extract various types of pesticides from P. ginseng. The rates of recovery for pesticides studied were satisfactory, ranging from 75.3 to 119.4 % for most of the pesticides with a relative standard deviation of less than 13 %. The LOQs ranged between 0.5 and 1.2 µg kg⁻¹. The modified QuEChERS method and GC–MS could enable complex pretreatment in P. ginseng analysis quickly and easily.

     

Comparison between GC–MS and GC–ICPMS using isotope dilution for the simultaneous monitoring of inorganic and methyl mercury,butyl and phenyl tin compounds in biological tissues

The aim of this work is to compare simultaneous isotope dilution analysis of organotin and organomercury compounds by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–inductively coupled plasma mass spectrometry (GC–ICP/MS) on certified bivalve samples. These samples were extracted by microwave with tetramethylammonium hydroxide (TMAH). Derivatization with both NaBEt₄ and NaBPr₄ was evaluated, and analytical performances were compared. Two CRM materials, BCR-710 and CRM-477, were analyzed by both techniques to verify accuracy. A mixed spike containing ²⁰¹Hg-enriched methylmercury (MeHg), ¹⁹⁹Hg-enriched inorganic mercury (iHg), ¹¹⁹Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) as well as homemade ¹¹⁶Sn-enriched monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) was used for the isotope dilution analysis of samples. The two techniques studied were compared in terms of classic analytical parameters: linearity, precision or repeatability (i.e., percent relative standard deviation, RSD%), limit of detection (LOD), and limit of quantification (LOQ), showing excellent linearity, precision below 12 % for all analytes, and LOQs of 0.06–1.45 pg for GC–MS and 0.02–0.27 pg for GC–ICP/MS.

Ochratoxin A in Wine: Its Determination and Photostability

Abstract

Due to the growing public concern regarding food safety, reliable, nondemanding and robust analytical methods are needed for quantitative determination of toxic compounds in complex matrices. Sample preparation is frequently a crucial step in determination of ochratoxin A (OTA) in wine, and a simplified and automated procedure is described, using solid‐phase extraction coupled on‐line to high pressure liquid chromatography (HPLC) with fluorimetric detection (λ_ex=333 nm, λ_em=460 nm). While the limit of quantitation is frequently better compared to off‐line procedures (30 ng/L), the decisive advantages of the new procedure are the absence of all sample manipulation during preconcentration and subsequent analysis, and consequentially no risk of analyte loss or sample contamination. Furthermore, using the standard addition method, matrix interferences can be avoided and the determination of extraction efficiency is unnecessary. These improvements have important consequences for the overall uncertainty of the analytical procedure. The developed method was applied for determination of OTA in 12 selected Slovenian wines. The typical relative standard deviation (RSD) was 10%. In none of the samples, did the OTA amount exceeded 2 µg/kg, the limit regulated by the EC.

The photo‐stability of the mycotoxin in solutions was examined. During irradiation of OTA solutions, its content was quickly reduced, while three fluorescent degradation products were detected. The degradation proceeds faster in water and 12% ethanolic solutions than in organic solvents or wine. Identification of the fluorescent degradation products was attempted using LC‐MS/MS with electrospray ionization.     

Flow-assisted automated solid-phase microextraction for the determination of chloroethers in aqueous matrices

In this study a method of flow-assisted automated solid-phase microextraction (FA-SPME) was developed for the determination of organic pollutants in aqueous samples. A CTC Combi-PAL autosampler coupled with gas chromatography–mass spectrometry (GC–MS) was used to automate the entire extraction process. In this method, the SPME fibre was exposed to 100 mL of sample in a direct immersion mode for 10 min. After exposure, the fibre was desorbed at the injection port of GC–MS. To demonstrate the applicability of FA-SPME, chloroethers were selected as model compounds. Good linear correlation was found over a concentration range of 0.5–100 µg/L. The detection limits of the method were determined between 0.02 and 0.05 µg/L with the coefficients of determination (R²) from 0.9980 to 0.9996. The relative standard deviations (RSDs) of the FA-SPME for three sequential FA-SPME analyses were determined to be in the range between 1.2% and 6.2% (n = 3). The applicability of the method was assessed by means of recovery studies and satisfactory values for all compounds were obtained. This optimised method was used in the analysis of water and human urine samples to show the matrix effect on FA-SPME. This FA-SPME/GC–MS is substantially faster and suitable for the routine continuous flow-mode environmental monitoring applications.     

Simultaneous Nonchiral Determination of Artemisinin and Arteannuin B in Artemisia annua Using Circular Dichroism Detection

A new method was performed using on-line coupling of nonchiral reversed-phase liquid chromatography (RP-LC) to circular dichroism (CD) spectroscopy for simultaneous determination of artemisinin and arteannuin B in crude plant extracts of Artemisia annua. Analysis was carried out on an LC–CD system equipped with an Agilent TC-C₁₈ column (4.6 mm I.D. × 200 mm L, 5 μm) using gradient of acetonitrile. The method was validated to be practicable and reliable at alterable wavelength in the ranges of 220–420 nm as desired. LOD and LOQ of artemisinin and arteannuin B were 0.08, 0.26 and 0.31, 1.02 μg mL⁻¹, respectively. It was more sensitive than conventional LC–UV and comparatively cheaper than LC–MS in analysis of TCM.

     

Rapid and Sensitive UPLC-MS–MS for the Determination of Trimetazidine in Human Plasma

A specific and sensitive UPLC-MS–MS was developed for the determination of trimetazidine in human plasma. The sample preparation was based on a single-step liquid–liquid extraction with acetic ether. The chromatographic separation was on a C₁₈ analytical column (50 mm × 2.1 mm, 1.7 μm) with acetonitrile and 10 mM ammonium acetate (30:70, v/v) as the mobile phase, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) applied for detection. The method was linear over the concentration ranges of 0.25–100.00 ng mL⁻¹ for trimetazidine, and the lower limit of quantification (LLOQ) was 0.25 ng mL⁻¹. The intra- and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, this method has been successfully applied to analyze plasma samples from a bioequivalence study with 18 volunteers.

     

Automated Mini-Column Solid-Phase Extraction Cleanup for High-Throughput Analysis of Chemical Contaminants in Foods by Low-Pressure Gas Chromatography—Tandem Mass Spectrometry

This study demonstrated the application of an automated high-throughput mini-cartridge solid-phase extraction (mini-SPE) cleanup for the rapid low-pressure gas chromatography—tandem mass spectrometry (LPGC-MS/MS) analysis of pesticides and environmental contaminants in QuEChERS extracts of foods. Cleanup efficiencies and breakthrough volumes using different mini-SPE sorbents were compared using avocado, salmon, pork loin, and kale as representative matrices. Optimum extract load volume was 300 µL for the 45 mg mini-cartridges containing 20/12/12/1 (w/w/w/w) anh. MgSO₄/PSA (primary secondary amine)/C₁₈/CarbonX sorbents used in the final method. In method validation to demonstrate high-throughput capabilities and performance results, 230 spiked extracts of 10 different foods (apple, kiwi, carrot, kale, orange, black olive, wheat grain, dried basil, pork, and salmon) underwent automated mini-SPE cleanup and analysis over the course of 5 days. In all, 325 analyses for 54 pesticides and 43 environmental contaminants (3 analyzed together) were conducted using the 10 min LPGC-MS/MS method without changing the liner or retuning the instrument. Merely, 1 mg equivalent sample injected achieved <5 ng g⁻¹ limits of quantification. With the use of internal standards, method validation results showed that 91 of the 94 analytes including pairs achieved satisfactory results (70–120 % recovery and RSD ≤ 25 %) in the 10 tested food matrices (n = 160). Matrix effects were typically less than ±20 %, mainly due to the use of analyte protectants, and minimal human review of software data processing was needed due to summation function integration of analyte peaks. This study demonstrated that the automated mini-SPE + LPGC-MS/MS method yielded accurate results in rugged, high-throughput operations with minimal labor and data review.

     

A simple,rapid and sensitive liquid chromatography–tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K₂EDTA plasma using levonorgestrel d₆ as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid–liquid extraction. Chromatographic separation was achieved on a Zorbax XDB‐Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile–5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003–200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra‐ and inter‐day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples. Copyright © 2014 John Wiley & Sons, Ltd.     

A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma

A rapid and stereospecific method using high performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) for the separation and determination of PSI‐7851 diastereomers in human K₂EDTA plasma has been developed. The analytical method involves direct protein precipitation with acetonitrile, followed by separation of the diastereomers on a Luna C₁₈ column, positive mode electrospray ionization and selected reaction monitoring mode mass spectrometry detection. The mobile phase composition and pH were investigated for the resolution of the two diastereomers of PSI‐7851. The optimized method showed good resolution (R_s = 4.8) within short analysis time (approximately 8 min). The assay range was 5–2500 ng/mL for both diastereomers using a 1/x² weighted linear regression analysis for standard curve fitting. Replicate sample analysis indicated that intra‐ and inter‐day accuracy and precision were within ±15.0%. The recovery of diastereomers from human plasma was greater than 85% and no significant matrix effect was observed. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolic Fingerprinting of acs7 Mutant and Wild-Type Arabidopsis thaliana Under Salt Stress by Ultra Performance Liquid Chromatography Coupled with Quadrupole/Time of Flight Mass Spectrometry

The metabolic fingerprints of the acs7 mutant and wild-type (WT) of the model plant Arabidopsis thaliana with and without salt stress were compared by ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF MS). Separations were performed on C₁₈ column (2.1 mm × 100 mm, 1.7 µm). A linear gradient elution of acetonitrile and 0.1% formic acid solution was used at a flow rate of 0.4 mL/min. Principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were combined for the data treatment. A clear discrimination was obtained by both PCA and PLS-DA. The acs7 salt-treated group was closer to the control group samples than the WT salt-treated group samples. Several potential stress-induced ions were revealed as markers of salt stress. The markers 12-oxophytodienoic acid (OPDA), arabidopside A, sinapoyl malate, linolenic acid, and abscisic acid were identified by the accurate mass (from TOF MS). Linolenic acid and OPDA are the biosynthetic precursors of jasmonic acid (JA) by the octadecanoid pathway. The JA content determination results indicated that salt stress increased the JA levels in the leaves of the WT plant, but there was no significant increase in the JA content of acs7 after salt treatment. These data suggested the responses to salt stress of the acs7 mutant and WT A. thaliana were different in the octadecanoid pathway.     

LC-MS–MS Determination of Pregabalin in Human Plasma

A rapid, sensitive and specific method to quantify pregabalin in human plasma using metaxalone as the internal standard is described. Sample preparation involved simple protein precipitation by using acetronitrile as solvent. The extract was analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS–MS). Chromatography was performed isocratically on Thermo Hypurity C₁₈ 5 μm analytical column, (50 mm × 4.6 mm i.d.). The assay of pegabalin was linear calibration curve over the range 10.000–10000.000 ng mL^?1. The lower limit of quantification was 10.000 ng mL^?1 in plasma. The method was successfully applied to the bioequivalence study of pregabalin capsules (150.0 mg) administered as a single oral dose.