Measuring complete isotopomer distribution of aspartate using gas chromatography/tandem mass spectrometry

We have developed a simple and accurate method for determining the complete positional isotopomer distribution of aspartate carbon atoms by gas chromatography/tandem mass spectrometry for (13)C-metabolic flux analysis. First, we screened tandem mass spectrometry (MS) spectra of the tert-butyldimethylsilyl (TBDMS) derivative of aspartate for daughter fragments with the necessary carbon atom fragmentations to fully resolve all 16 isotopomers of aspartate. Tandem MS scanning parameters were optimized for each daughter fragment, and the accuracy of tandem MS measurements were evaluated. We selected five accurate fragments that provided a redundant set of 47 labeling measurements to quantify the complete isotopomer distribution of aspartate by least-squares regression. The validity of the approach was demonstrated using six (13)C-labeled aspartate standards and natural aspartate.     

Determination of clenbuterol in bovine liver by combining matrix solid-phase dispersion and molecular imprinted solid-phase extraction followed by liquid chromatography/electrospray ion trap multiple-stage mass spectrometry

Matrix solid-phase dispersion (MSPD) is a new sample pretreatment for solid samples. This technique greatly simplifies sample pretreatment but, nonetheless, the extracts often still require an extra cleanup step that is both laborious and time-consuming. The potential of combining MSPD with molecularly imprinted solid-phase extraction (MISPE) was investigated in this study. Liver samples were ground in a mortar with C18 sorbent and the homogenized mixture packed into an SPE cartridge and placed on top of a MISPE cartridge. Subsequently, clenbuterol was eluted from the MSPD cartridge onto the MISPE cartridge using acetonitrile containing 1% acetic acid. The ability of the molecularly imprinted polymer to selectively adsorb analyte in acetonitrile was exploited for re-extracting clenbuterol directly from this acetonitrile extract via the double cartridge tandem system. The analyte was eluted from the MISPE cartridge using acidified methanol. A clear eluate was obtained, which was subsequently evaporated, redissolved, and analyzed by HPLC electrochemical detection (ECD) or ion trap mass spectrometry (LC/IT-MS). The MISPE cartridge used in this study was imprinted using bromoclenbuterol, a structural analogue of clenbuterol, as the template. These MISPE cartridges showed excellent stability. The complete extraction procedure was rapid, and recoveries exceeded 90% for the target analyte. The method detection limit for the LC/IT-MS procedure was < 0.1 microg/kg. This method, therefore, satisfies the stringent requirements of European Union regulation EEC 2377/90.     

Determination of parabens in pharmaceutical formulations by solid-phase microextraction-ion mobility spectrometry

Solid-phase microextraction (SPME) coupled with ion mobility spectrometry (IMS) was used for the detection and quantitation of 4-hydroxybenzoate preservatives, methylparaben, ethylparaben, propylparaben, and butylparaben, in commercial pharmaceutical products. For the first time, SPME-IMS is described for the simultaneous detection, separation, and quantitation of multiple analytes in complex matrixes. The parabens are extracted from the samples using SPME, and the analytes on the fiber are heated by the IMS desorber unit and vaporized into the drift tube. The four preservatives differing only by a methyl group were separated in less than 18 ms. The analytical procedure was optimized for fiber coating selection, extraction time, sample pH, sample volume, ionic strength, and IMS conditions. Separation characteristics such as resolution, theoretical plates, and drift times of the parabens were also evaluated based on the direct interfacing of SPME to IMS. The conditions were tested using six over-the-counter topical products containing various combinations of preservatives. Analysis of the samples by SPME-IMS using benzyl paraben as an internal standard yields good comparison to an HPLC method, thereby reinforcing the applicability of this technique as a method for routine analysis. Limits of detection were 10 ng/mL for methylparaben and ethylparaben and 5 ng/mL for propylparaben and butylparaben. Good linearity range and reproducibility of less than 8% were obtained.     

非衍生-气相色谱串级质谱法测定饲料中三聚氰胺

针对衍生化-气相色谱串联质谱(选择离子监测法)的操作繁琐费时、过程不易控制的状况,利用离子阱气相色谱质谱联用仪,建立了非衍生化-气相色谱串级质谱直接分析饲料中三聚氰胺的方法.利用三聚氰胺二级质谱进行定性,以二级质谱的特征离子峰m/z85进行定量,检测效率大大提高,定性更加直接.方法精密度为5.9%,回收率为87%-98%,满足饲料中三聚氰胺的检测要求.     

Determination of nicotine and other minor alkaloids in international cigarettes by solid-phase microextraction and gas chromatography/mass spectrometry

Nicotine, nornicotine, anabasine, and anatabine are the most abundant alkaloids in tobacco. Along with the addictiveness of nicotine, other properties, including their occurrence in tobacco at relatively high concentrations, and as the primary precursors for the highly carcinogenic tobacco-specific nitrosoamines, make these chemicals important from a public health standpoint Therefore, developing a fast and accurate quantitative method to screen large numbers of cigarette samples for these alkaloids was important. This report describes the first use of headspace analysis using solid-phase microextraction combined with gas chromatography/mass spectrometry for the unambiguous detection of tobacco alkaloids. Detection and confirmation of each analyte isestablished by both chromatographic retention times and the ratio of reconstructed ion chromatogram peak areas from characteristic quantitation ion and confirmation ion. Twenty-eight cigarette brands from 14 countries were analyzed. Surprisingly, the minor alkaloids' response factors varied considerably among different styles of cigarettes. Accurate quantification was achieved using a three-point standard addition protocol. The standard addition approach was essential to obtain accurate measurements by minimizing matrix effects that would otherwise have contributed to quantitation bias. Significant differences in the alkaloid profiles were measured in the different cigarette brands. These results strongly suggest that such differences reflect variations associated with blend compositions, tobacco quality, and manufacturing practices.     

Determination of alkylbenzyldimethylammonium chlorides in river water and sewage effluent by solid-phase extraction and gas chromatography/mass spectrometry

This study presents a modified method to analyze alkylbenzyldimethylammonium chlorides (ABDACs) in river water and sewage effluent. The method involves mixed samples with linear alkylbenzenesulfonates (LAS) as a counterion to enhance the extraction of ABDAC residues from an RP-18 solid-phase cartridge by formation of hydrophobic ion-pair complexes. The ABDACs were then eluted with methanol-ethyl acetate (1:1, v/v) and formed to their corresponding alkyldimethylamines by the Hofmann degradation with potassium tert-butoxide. The alkyldimethylamines were then identified and quantitated by gas chromatograph/mass spectrometry (GC/MS). The results indicate that, in the presence of LAS, debenzylation of ABDACs occurs selectively at a temperature higher than 90 degrees C to produce the corresponding nonionic alkyldimethylamines. The method proposed herein provides a high precision and sensitivity for ABDACs, to quantitation at < or =0.1 microg/L in 500 mL of the water samples. The average recovery of ABDAC spiked water samples was 95% with relative standard deviations (RSD, n = 7) of 9%. The RSDs of three replicate environmental sample analyses ranged from 5 to 11%. Direct HPLC method was applied to evaluate the GC/MS method, and compatible results were observed.     

Development of a solid-phase microextraction gas chromatography/tandem mass spectrometry method for polybrominated diphenyl ethers and polybrominated biphenyls in water samples

Solid-phase microextraction has been applied for the first time to the determination of trace concentrations of some brominated flame-retardant compounds (BFRs) in water samples. For the development of the method, six polybrominated diphenyl ethers and two polybrominated biphenyls were considered as target analytes. The factors expected to influence the extraction process are fully discussed. Quantification has been performed by gas chromatography/tandem mass spectrometry using an ion trap mass analyzer. This is also the first time that tandem mass spectrometry is applied with these analytes. Unlike conventional methods for BFR analysis, which involve solvent extraction and several cleanup steps before gas chromatography, the proposed method uses headspace extraction and hard contamination of the chromatographic system is prevented. In addition, tandem mass spectrometry provides selectivity and sensitivity in the detection process. The method performs well achieving good linearity (R(2) > 0.997), precision, and detection limits (S/N = 3) ranging from 7.5 to 190 pg/L. The method has been applied to a variety of water samples.     

气相色谱-质谱测定化妆品中的硝基麝香类化合物

建立了化妆品中5种硝基麝香含量的气相色谱-质谱联用检测方法。采用丙酮/正己烷混合液超声提取化妆品中的硝基麝香,提取液经过浓缩,由气相色谱-质谱联用仪检测,以氘代二甲苯麝香内标法定量。该方法对化妆品中5种硝基麝香的加标回收率在85.21%~110.2%之间,相对标准偏差(RSD)不大于7.15%,在50μg/L~5000μg/L范围内呈现良好的线性关系,灵敏度高,最低检测限分别达到20~50μg/kg;选择性好,能有效消除复杂基体干扰。可用于常见化妆品中硝基麝香类化合物含量的检测。     

基质固相分散萃取-气相色谱同时测定茶叶中有机氯及拟除虫菊酯类农药残留

建立了气相色谱-电子捕获检测器(GC-μECD)同时测定茶叶中14种有机氯和8种拟除虫菊酯类农药残留。采用基质固相分散(Matrix Solid Phase Disperse,MSPD)技术对茶叶样品进行前处理,用气相色谱-电子捕获检测器快速定性定量分析。MSPD集提取、过滤、净化于一步完成,避免了样品均化、转溶、乳化、浓缩造成待测农药组分的损失,大大提高了方法的准确性和精密度。添加回收实验结果表明:22种农药的平均回收率为83.5%~112.6%,相对标准偏差为2.5%~10.6%,方法检出限为0.002~0.02mg/kg。该方法灵敏度、准确度和精密度均符合农药残留测定的要求。     

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。     

Analysis of perchlorate in human urine using ion chromatography and electrospray tandem mass spectrometry

Because of health concerns surrounding widespread exposure to perchlorate, we developed a sensitive and selective method for quantifying perchlorate in human urine using ion chromatography coupled with electrospray ionization tandem mass spectrometry. Perchlorate was quantified using a stable isotope-labeled internal standard ((18)O(4)-perchlorate) with excellent assay precision (coefficient of variation <5% for repetitively analyzed quality control material). Analytical accuracy was established by blind analysis of certified proficiency testing materials prepared in synthetic urine matrix; calculated amounts deviated minimally from true amounts, with percent differences ranging from 2% to 5%. Selective chromatography and tandem mass spectrometry reduced the need for sample cleanup, resulting in a rugged and rapid method capable of routinely analyzing 75 samples/day. The lowest reportable level (0.025 ng/mL) was sufficiently sensitive to detect perchlorate in all human urine samples evaluated to date, with a linear response range from 0.025 to 100 ng/mL. This selective, sensitive, and rapid method will help elucidate any potential associations between human exposure to low levels of perchlorate and adverse health effects.     

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.     

Microbial metabolomics with gas chromatography/mass spectrometry

An analytical method was set up suitable for the analysis of microbial metabolomes, consisting of an oximation and silylation derivatization reaction and subsequent analysis by gas chromatography coupled to mass spectrometry. Microbial matrixes contain many compounds that potentially interfere with either the derivatization procedure or analysis, such as high concentrations of salts, complex media or buffer components, or extremely high substrate and product concentrations. The developed method was extensively validated using different microorganisms, i.e., Bacillus subtilis, Propionibacterium freudenreichii, and Escherichia coli. Many metabolite classes could be analyzed with the method: alcohols, aldehydes, amino acids, amines, fatty acids, (phospho-) organic acids, sugars, sugar acids, (acyl-) sugar amines, sugar phosphate, purines, pyrimidines, and aromatic compounds. The derivatization reaction proved to be efficient (>50% transferred to derivatized form) and repeatable (relative standard deviations <10%). Linearity for most metabolites was satisfactory with regression coefficients better than 0.996. Quantification limits were 40-500 pg on-column or 0.1-0.7 mmol/g of microbial cells (dry weight). Generally, intrabatch precision (repeatability) and interbatch precision (reproducibility) for the analysis of metabolites in cell extracts was better than 10 and 15%, respectively. Notwithstanding the nontargeted character of the method and complex microbial matrix, analytical performance for most metabolites fit the requirements for target analysis in bioanalysis. The suitability of the method was demonstrated by analysis of E. coli samples harvested at different growth phases.     

Determination of six thyroid hormones in the brain and thyroid gland using isotope-dilution liquid chromatography/tandem mass spectrometry

Thyroid hormones (THs) play critical roles in the regulation of growth and development, including brain development, in both humans and animals. Until recently, TH levels were assayed with measurements in serum, using immunoassay (IA)-based methods. IA methods are sensitive but are compromised by the lack of adequate specificity. Furthermore, measurements of TH levels in blood do not necessarily reflect the levels and profiles found in critical organs such as the brain. Measurement of TH levels in the brain is critical for studies that assess the effects of environmental contaminants on TH homeostasis. In this study, we developed a selective and sensitive method for the analysis of six THs, l-thyroxine (T(4)), 3,3',5-triiodo-l-thyronine (T(3)), 3,3',5'-triiodo-l-thyronine (rT(3)), 3,5-diiodo-l-thyronine (3,5-T(2)), 3,3'-diiodo-l-thyronine (3,3'-T(2)), and 3-iodo-l-thyronine (3-T(1)), in the brain and thyroid gland (TG) using isotope ([(13)C]T(4))-dilution liquid chromatography (LC)/tandem mass spectrometry (MS/MS). Proteins in the (rat) brain and TG were digested by pronase, and THs were extracted with a solid-phase extraction method and analyzed by LC/MS/MS. The instrumental calibration range for each TH ranged from 0.5 to 200 ng/mL and showed excellent linearity (r > 0.9995). The instrumental detection limits for THs were in the range of 7.5-13.5 pg, in positive ion mode, and 13.5-16.5 pg, in negative ion mode. The optimized procedural recoveries for THs (except for 3-T(1)), spiked into a pig-brain matrix, were between 97.6% and 109%, with a coefficient of variation (CV) of 1.2-8.2%, for the brain, and between 96.4% and 101%, with a CV of 1.8-8.6%, for the TG. Concentrations of THs in the brain and TG of the five rats were 2.20-3.65 ng/g T(4), 1.56-2.20 ng/g T(3), and below the limit of detection (相似文献   

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.     

Determination of alkylbenzenesulfonates in recent sediments by gas chromatography/mass spectrometry

Alkylbenzenesulfonates together with soap are the most widely used anionic surfactants. Linear alkylbenzenesulfonates (LAS) were introduced in the mid-1960s as substitutes for the poorly biodegradable tetrapropylenebenzenesulfonates (TPS). A method is presented for the selective and quantitative determination of LAS and TPS in recent sediments. Alkylbenzenesulfonates were extracted from sediments using methanol. The methanolic extract was passed through a strong anionic exchange column. The alkylbenzenesulfonates contained in the acidic eluate were then derivatized to their corresponding trifluoroethyl esters and quantitatively determined by gas chromatography/mass spectrometry using positive chemical ionization. Limits of quantitation for 10 g sediment samples varied between 1.5 and 21 μg/kg of dry sediment for single LAS isomers and between 71 and 220 μg/kg for total LAS. Limits of quantitation for the total of TPS were at ～200 μg/kg. Relative standard deviations of replicate analyses typically ranged from 5 to 10%. Recovery rates of LAS in spiked sediment samples ranged from 79 to 113%. The presented method was applied to surface and subsurface sediments also containing long-chain (C(14)-C(16))-LAS and mixtures of LAS and TPS.     

Potential application of gas chromatography/tandem mass spectrometry in the measurement of coeluting isomers

Despite the unprecedented popularity of separation chromatography, the measurement of coeluting isomeric chemicals remains an extremely difficult task. We developed an analytical scheme capable of measuring two coeluting isomers using a single chromatographic column and a gas chromatography/tandem mass spectrometry system. The protocol utilized two product ion fragments generated from a common parent ion associated with the isomers for quantitation. The utility of the analytical scheme was demonstrated with the measurements of several pairs of coeluting polychlorinated biphenyl (PCB) isomers in standard solutions and fish liver samples. Best results were given when a set of stringent constraints for the abundance ratio of the two product ion fragments was satisfied. Analyses of seven fish liver samples collected from nearshore San Diego, CA, indicated that the domain that had been previously reported to comprise PCB 153 and PCB 168 actually contained PCB 153 only. Although only a selected number of PCB congeners were examined, the results presented indicate that the analytical scheme has the potential to be used to determine the concentrations of all chromatographically coeluted isomers.     

Long-term performance of a gas chromatography/tandem mass spectrometry assay for tebufelone in plasma

An automated gas chromatography/tandem mass spectrometry (GC/MS/MS) based method for the rapid determination of tebufelone (TE) in animal and human plasma has been routinely applied in our laboratory to more than 3000 samples over a 2-year period. The selectivity of MS/MS conducted on a triple quadrupole instrument, combined with the use of a stable-isotope-labeled internal standard, results in excellent analytical figures of merit, as well as minimal sample preparation, rapid analysis, and high assay reliability. The work described here goes beyond initial method development and validation studies by evaluating the long-term performance of quantitative GC/MS/MS. Electron ionization produces M.+ ions for TE and the [13C, 18O]TE internal standard, which are selected in Q1 and undergo collisionally activated dissociation in Q2. Quantitation is based on monitoring daughter ions at m/z 248 and 251, respectively, in Q3. A linear range of 1-3000 ng of TE/sample (20 pg to 60 ng injected) provides access to an effective concentration range of 0.5-30,000 ppb TE in plasma (0.1-2-g samples). The assay shows no bias and less than 10% relative standard deviation over this range. In the automated mode, less than 7 min elapse from injection to report printout and more than 70 plasma samples are routinely prepared and analyzed in a day. Such performance is consistently maintained throughout long-term application.     

Determination of colchicine residues in sheep serum and milk using high-performance liquid chromatography combined with electrospray ionization ion trap tandem mass spectrometry

Colchicine is a naturally occurring alkaloid used in human and veterinary medicine. It shows genotoxicity in in vitro and in vivo systems even at low concentrations. Therefore, no ADI has been established, and colchicine has been included in Annex IV of Council Regulation (EEC) No. 2377/90. No abuse of this drug in intensive livestock farming has yet been reported. However, there may be a natural route of entry for this compound into the food chain when Colchicum autumnale is consumed by animals kept outdoors. To address this concern, we developed and validated a highly sensitive and selective quantitative LC-ESI-MS-MS method for the detection of colchicine in sheep serum and milk. For sample pretreatment, all samples were liquid-liquid extracted with phosphate buffer (pH 8.0) and dichloromethane. LC separation was carried out on an RP C18 column employing a 0.5% formic acid/acetonitrile gradient system. The recoveries in both matrixes at a concentration range from 0.0005 to 1 mg/L were >80% with RSDs of <10%. The applicability of the method was demonstrated in serum and the corresponding milk samples from Albanian sheep. After exposure to C. autumnale, the animals showed clinical signs of intoxication. Significant concentrations (up to 1 mg/L) of the drug were found in serum as well as in milk samples.     

Differential metabolomics using stable isotope labeling and two-dimensional gas chromatography with time-of-flight mass spectrometry

This work describes an approach to differential metabolomics that involves stable isotope labeling for relative quantification as part of sample analysis by two-dimensional gas chromatography/mass spectrometry (GCxGC/MS). The polar metabolome in control and experimental samples was extracted and differentially derivatized using isotopically light and heavy (D6) forms of the silylation reagent N-methyl-N-tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). MTBSTFA derivatives are of much greater hydrolytic stability than the more common trimethylsilyl derivatives, thus diminishing the possibility of isotopomer scrambling during GC analysis. Subsequent to derivatization with MTBSTFA, differentially labeled samples were mixed and analyzed by GCxGC/MS. Metabolites were identified, and the isotope ratio of isotopomers was quantified. The method was tested using three classes of metabolites; amino acids, fatty acids, and organic acids. The relative concentration of isotopically labeled metabolites was determined by isotope ratio analysis. The accuracy and precision, respectively, in quantification of standard mixtures was 9.5 and 4.77% for the 16 amino acids, 9.7 and 2.83% for the mixture of 19 fatty acids, and 14 and 4.53% for the 20 organic acids. Suitability of the method for the examination of complex samples was demonstrated in analyses of the spiked blood serum samples. This differential isotope coding method proved to be an effective means to compare the concentration of metabolites between two samples simultaneously.