Off-line solid-phase microextraction and capillary electrophoresis mass spectrometry to determine acidic pesticides in fruits

A method based on solid-phase microextraction (SPME) and capillary electrophoresis/mass spectrometry (CE/ MS) is described for determining simultaneously five acidic pesticides (o-phenylphenol, ioxynil, haloxyfop, acifluorfen, picloram) in fruits. The CE device is coupled to an electrospray interface by a commercial sheath-flow adapter. Emphasis is placed on fulfillment of the speed and sensitivity requirements. The best separation is achieved using 32 mM ammonium formate/acid formic buffer at pH 3.1, with a working voltage of 25 kV. The MS detection of the five pesticides was performed in negative ionization mode. Full-scan spectra with base peaks corresponding to [M-H]- were obtained except for acifluorfen, which gives [M-H-CO2]- as most abundant ion. Compared with the conventional EC-UV, the limits of detection were lower for acifluorfen, haloxyfop, ioxynil, and picloram, by a factor of 20, 20, 50, and 2, respectively. Extraction involved fruit sample homogenization with an acetone-water solution (5:1), filtration, and acetone evaporation prior to fiber extraction. SPME conditions such as time, pH, ion strength, stationary phase of the fiber, sample matrix, and desorption solvents were examined. The recovery of the analytes ranged from 7 to 94%, and the relative standard deviation was between 3 and, 13%. The method was found to be linear between 0.02 and 500 mg kg(-1) with correlation coefficients ranging from 0.992 to 0.997. The limits of quantification were from 0.02 to 5 mg kg(-1). The optimized method was successfully applied to the analysis of acid pesticides in fruit samples.     

Solid-phase microextraction coupled to gas chromatography/mass spectrometry for determining polycyclic aromatic hydrocarbon-micelle partition coefficients

Solid-phase microextraction (SPME) coupled to gas chromatography with MS detection has been employed to study the partition coefficients of PAHs to ionic and nonionic micelles. The results obtained in this work for seven PAHs, using 85-microm polyacrylate- and 100-microm poly(dimethylsiloxane)-coated fibers and anionic (sodium dodecyl sulfate), cationic (cetyltrimethylammonium bromide), and nonionic (polyoxyethylene-10-lauryl ether) surfactants, indicate that SPME is a viable method for estimating the partition coefficients of PAHs to micelle. The procedure could also be potentially extended to the measurement of partition coefficients between a wide variety of semi- or nonvolatile compounds and micellar media.     

Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, Y162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162YnLYEIAR168 and the corresponding decrease in the 162YLYEIAR168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK75, also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.     

Applications of Hadamard transform to gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N, N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced approximately 10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1023 and 511, respectively. The HT-GC/MS was successfully applied to the determination of MDMA in the urine sample of a suspect.     

固相萃取-高效液相色谱串联质谱测定牛奶中9种性激素残留

采用固相萃取-高效液相色谱质谱联用法(SPE-HPLC/MS),建立牛奶中9种性激素(雌酮、雌三醇、17α-雌二醇、诺龙、甲睾酮、丙酸睾酮、醋酸氯地孕酮、醋酸甲地孕酮、醋酸甲羟孕酮)残留的检测方法。样品经甲醇提取,过固相萃取柱净化,氮气吹干,残留物甲醇溶解后测定。其中雌激素(雌酮、雌三醇、17α-雌二醇)采用负模式;其余性激素为正模式,进行多反应监测(MRM)模式定性定量分析。雌激素检出限(LODs)为1.1~1.2μg/L,定量限(LOQs)为3.63~3.96μg/L;其余性激素检出限(LODs)为0.1~0.5μg/L,定量限(LOQs)为0.33~1.65μg/L;分别在20.0~500.0μg/L及2.5~100.0μg/L范围内线性关系良好(r20.994 4)。在50.0~100.0μg/L的添加水平上,9种性激素的平均回收率在82.7%~98.2%之间,变异系数(CV)为4.2%~11.2%。该法操作简单、灵敏度高,可用于牛奶中9种性激素的测定。     

Determination of hyperforin in mouse brain by high-performance liquid chromatography/tandem mass spectrometry

Hyperforin is one of the essential active ingredients of St. John's wort extract, which is used as an antidepressant for mild to moderately severe depressions. In vitro and in vivo data as well as several clinical studies and meta analyses have confirmed the pharmacological effect of treatment with hyperforin-containing preparations. However, little is known about the brain availability of hyperforin until now. Accordingly, a highly sensitive and selective LC/MS method for this purpose was developed and validated. This method proved suitable for the determination of hyperforin in mouse brain, after oral administration of hyperforin sodium salt and St. John's wort extract. This method involves liquid-liquid extraction of hyperforin with ethyl acetate followed by separation with rapid reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using electrospray ionization. Excellent linearity was obtained for the entire calibration range from 0.25 to 10 ng/mL (corresponding to 2.5-100 ng/g brain tissue concentration, calculated with the factor derived from sample processing) with an average coefficient of correlation of 0.9992. The recovery of hyperforin from mouse brain homogenates was between 71.4 and 75.3% with a relative standard deviation of less than 3%. Validation assays for the lower limit of quantitation yielded an accuracy of 5.8%. Intraday accuracy and precision for the developed method were between 4.6 and 10.6% and 4.3-8.4%, respectively, while the interday parameters varied between 6.7 and 12.2% for accuracy and 2.0-5.0% for precision. After the method validation, hyperforin brain levels in mice, treated with 15 mg/kg hyperforin (either as the sodium salt or as 5% St. John's wort extract), were investigated. The average concentration of hyperforin found for the sodium salt group was 28.8+/-10.1 ng/g of brain (n = 8), which was somewhat higher than the hyperforin concentration of 15.8+/-10.9 ng/g of brain (n = 8), determined in the extract-treated group. This method is robust, selective, and highly sensitive and represents an appropriate tool to further prove the occurrence and distribution of hyperforin in mouse brain.     

Use of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to investigate pesticide residues in fruits

In this paper, the potential of coupling liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF) for the determination of pesticides in a variety of fruit samples (orange peel and flesh, banana skin and flesh, strawberry and pear) has been explored. The quantitative application at residue levels has been proven for two insecticides (buprofezin and hexythiazox), which were satisfactorily determined at three concentration levels, 0.1, 1, and 5 mg/kg, obtaining a suitable linearity range (correlation coefficient>0.99) of more than 2 orders of magnitude. Satisfactory recoveries have been obtained for both compounds at the three levels tested in all sample matrices, with lowest calibration levels (LCL) of 0.075 and 0.01 mg/kg. The excellent potential of QTOF for identification purposes is illustrated by the high number of identification points (IPs) earned, up to 21, at the highest concentration of 5 mg/kg, or between 11 and 21 at the 0.1 and 1 mg/kg levels. The application of LC-QTOF MS to real samples revealed the presence of several positives at concentrations close to the LCL, all of which were confirmed with more than 11 IPs. The potential of QTOF for elucidation of nontarget analytes has also been demonstrated by the finding of one transformation product (TP) of buprofezin in a banana skin sample. This TP was identified by obtaining the full scan product ion spectra at different collision energies with acceptable accurate mass deviation. The work performed in this paper illustrates the suitability and excellent confirmatory potential of LC-QTOF MS for pesticides residues analysis in food samples.     

Determination of boswellic acids in brain and plasma by high-performance liquid chromatography/tandem mass spectrometry

Peritumoral edema, one of the major causes for neurological disorders in brain tumor patients, is mainly treated with steroids, which unfortunately have significant side effects and interfere with the efficacy of chemotherapy. Boswellic acids, the main active ingredients of Boswellia serrata, are antiinflammatory agents, inhibiting 5-lipoxygenase, the key enzyme of leukotriene biosynthesis and one of the pathophysiological mechanisms of peritumoral edema. Based on positive results in clinical trials and animal studies, B. serrata resin dry extract was designated an orphan drug by the European Commission for the treatment of peritumoral edema resulting from brain tumors. Thus boswellic acids may be alternative drugs to corticosteroids. However, the question of the availability of boswellic acids in brain has not been addressed until now. Accordingly, a highly sensitive LC/MS method has been developed for the simultaneous determination of KBA and AKBA, the most potent boswellic acids, in plasma and brain. This method involves matrix-assisted liquid-liquid extraction on Extrelut NT followed by separation on reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using atmospheric pressure chemical ionization. Excellent linearity was obtained for the entire calibration range from 5 to 1500 ng/mL KBA and AKBA in plasma and 5 to 1000 ng/mL KBA and AKBA in brain. Validation assays of the lower limit of quantification as well as for the intra- and interday precision and accuracy met the international acceptance criteria for bioanalytical method validation. Moreover, the interchangeability of calibration curves generated in pork and rat brain homogenates could be demonstrated. Using the developed analytical method, KBA and AKBA could be detected for the first time in brain up to a concentration of 99 and 95 ng/g of brain, respectively, 3 h after the single oral administration of 240 mg/kg of dry B. serrata resin extract to Wistar rats. The developed method represents an appropriate tool to further study the time-dependent distribution of KBA and AKBA in plasma and brain as well as the absolute brain concentration after multiple doses and contributes thus to the optimization of the dosage regimen and to a better understanding of the therapeutic effects of B. serrata.     

液相色谱串联质谱测定鳗鱼中硝基呋喃的含量

硝基呋喃类药物及其代谢产物可使实验动物发生癌变、基因突变,同时具有生殖毒性,可能对人体产生危害。因此,硝基呋喃含量的检测在食品安全中是至关重要的,本文采用液相色谱串联质谱技术检测四种硝基呋喃类代谢物。硝基呋喃类代谢物在酸性条件下经过2-硝基苯甲醛衍生化,用乙酸乙酯提取纯化,电喷雾离子化,液相色谱-串联质谱检测。结果表明,四种硝基呋喃类药物的色谱峰面积与浓度在0.1～10.0 ng/mL范围内均呈现很好的线性关系,该方法具有操作简便、灵敏、准确等优点。     

液相色谱串联质谱法测定烟草中的游离氨基酸

建立液相色谱串联质谱法测定烟草中20种游离氨基酸的方法。烟草样品经0.1%的盐酸溶液超声萃取并离心后,直接进样测定。色谱柱采用XTerra MS C18(50mm×2.1mm×2.5μm),0.1%甲酸溶液和乙腈为流动相。结果表明:20种氨基酸的检出限为0.001～0.011μg/mL,标准曲线的拟合度均大于0.999,回收率在86.4%～105.9%之间。该方法操作简单,灵敏度高,适用于烟草中游离氨基酸的检测。     

Development and evaluation of a candidate reference method for the determination of total cortisol in human serum using isotope dilution liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

Cortisol is an important diagnostic marker for the production of steroid hormones, and accurate measurements of serum cortisol are necessary for proper diagnosis of adrenal function. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d(3), was added to serum, followed by equilibration and solid-phase and ethyl acetate extractions to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) and liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analyses. (M + H)(+) ions at m/z 363 and 366 for cortisol and its labeled internal standard were monitored for LC/MS. The transitions of (M + H)(+) --> [(M + H)(+) - 2H(2)O] at m/z 363 --> 327 and 366 --> 330 were monitored for LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for cortisol [Certified Reference Materials 192 and 193] with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added cortisol. The results of this method for total cortisol agreed with the certified values within 1.1%. The recovery of the added cortisol ranged from 99.8% to 101.0%. This method was applied to the determination of cortisol in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.3%-1.5% and between-set CVs of 0.04%-0.4% for both LC/MS and LC/MS/MS analyses. The correlation coefficients of all linear regression lines ranged from 0.998 to 1.000. The detection limits (at a signal-to-noise ratio of approximately 3-5) were 10 and 15 pg for LC/MS and LC/MS/MS, respectively. This method, which demonstrates good accuracy and precision, and is free from interferences from structural analogues, qualifies as a candidate reference method and can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

液相色谱-串联质谱法检测芹菜中氟虫腈及其代谢物残留

建立了芹菜中氟虫腈及其代谢物(氟甲腈、氟虫腈硫醚和氟虫腈砜)残留的液相色谱/串联质谱检测方法。样品以乙腈提取,采用电喷雾离子源负离子检测模式(ESI-)和多重反应监测(MRM)模式测定,基质匹配标准曲线定量。结果表明:芹菜基质中,氟虫腈、氟甲腈、氟虫腈硫醚和氟虫腈砜在0.01~0.1mg/L范围内线性关系均较好(r0.995),方法检出限和定量限分别为0.45~1.20μg/kg和1.5~4.0μg/kg;5、50和100μg/kg三个添加水平下,方法回收率在72.3%~110.2%,相对标准偏差为5.0%~9.8%。该法简单、准确、快速、灵敏,符合法规残留限量监测要求。     

Assay of trace D-amino acids in neural tissue samples by capillary liquid chromatography/tandem mass spectrometry

A sensitive chiral capillary HPLC-MS/MS method well suited for the determination of amino acid enantiomers in biological samples was developed. The method involved precolumn derivatization of the sample with 7-fluoro-4-nitrobenzoxadiazole (NBD-F). After derivatization, NBD-amino acids were stacked on a C18 reversed-phase extraction microcolumn, thus enriching and cleaning up the analytes. Various chiral stationary phases (CSPs) including cyclodextrin-bonded silica, Pirkle-type, vancomycin, and teicoplanin-bonded silica particles were evaluated for resolving NBD-F tagged amino acid enantiomers with mobile phases compatible with MS detection. It was found that only teicoplanin aglycon CSP provided sufficient resolution of NBD-Asp and NBD-Ser enantiomers to quantify trace levels of D-Asp and D-Ser in tissue samples. MS/MS detection of NBD-amino acid derivatives was very sensitive and selective. The high selectivity allowed the use of a stable isotope-labeled analyte analogue (i.e., L-aspartic acid-2,3,3-d3) as internal standard for the quantitation to improve assay reproducibility and reliability. Neural tissue samples dissected from rat brain and the central nervous system (CNS) of Aplysia californica, a widely used neuronal model, were analyzed to determine the chirality of glutamic acid (Glu), aspartic acid (Asp), and serine (Ser). The former two are major excitatory amino acids in the brain, and the last one has been recently identified as a neuromodulator. Both D-Ser and D-Asp were detected in rat brain. While the D-Asp level decreased rapidly through the developmental stages of the rat, the D-Ser level increased steadily from 82.3 microg/g of wet tissue in 3-day prenatal rats to 241.3 microg/g of wet tissue in 90-day-old rats. Interestingly, no D-Ser was detected in the CNS of Aplysia, a "primitive" invertebrate. However, the D-Asp level in this animal was found to be high. In a particular connective nerve sample, D-Asp was at 323.2 microg/g of wet tissue and constituted 60.2% of total Asp. D-Glu was not detected either in rat brain or in Aplysia's CNS.     

Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.     

Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.     

液相色谱–串联质谱法同时测定烟草中的肌醇和奎尼酸

建立同时测定烟草中肌醇和奎尼酸的液相色谱–串联质谱(LC-MS/MS)方法。采用0.1 mol/L的乙酸铵水溶液对烟末进行超声提取后,经0.22μm水相滤膜过滤后直接进行LC-MS/MS分析。结果表明:肌醇在0.11～25 mg/g和奎尼酸在0.18～25 mg/g范围内,线性相关系数均≥0.999 0。肌醇的回收率在95.9%～98.6%之间,相对标准偏差(RSD)小于1.9%(n=6);奎尼酸的回收率在95.2%～97.4%之间,相对标准偏差(RSD)小于2.1%(n=6)。肌醇的方法检出限(LOD)为0.035 mg/g,定量限(LOQ)为0.11 mg/g;奎尼酸的方法检出限(LOD)为0.055 mg/g,定量限(LOQ)为0.18 mg/g。该方法准确、灵敏,适合用于烟草中肌醇和奎尼酸的测定。