超高效液相色谱-三重四级杆串联质谱法快速测定水中苯胺和联苯胺

建立了直接进样-超高效液相色谱-三重四级杆串联质谱法(UPLC-MS/MS)快速测定水中苯胺和联苯胺的分析方法。样品经0.45μm聚醚砜(PES)滤膜过滤除去颗粒性杂质后,采用ACQUITY UPLC?BEH C8(1.7μm,2.1 mm×50 mm)为分离柱,甲醇-0.1%甲酸水溶液(v/v)为流动相进行梯度洗脱,电喷雾离子源(ESI)电离,多反应监测模式(MRM)进行定性和定量分析。经条件优化,苯胺和联苯胺可在3 min内分析完毕,检出限分别为0.1μg/L和0.03μg/L,且在0.2～20.0μg/L范围内线性关系良好(r> 0.999)。对3组不同浓度(0.5μg/L,2.0μg/L,5.0μg/L)加标的实际水样平行测定6次,苯胺和联苯胺的相对标准偏差在1.0%～5.4%范围内,回收率为97.6%～113%。该方法简便快捷,灵敏度高,适用于实际水样的分析。     

Multiclass determination of sunscreen chemicals in water samples by liquid chromatography-tandem mass spectrometry

A novel analytical method based on solid-phase extraction (SPE) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for the determination of UV sunscreen agents in the water environment is presented. After a thorough investigation of SPE and LC-MS/MS conditions, it permits the enrichment and determination of nine of these compounds in a single methodology, including three very polar sulfonates (e.g., 2-phenylbenzimidazole-5-sulfonic acid, PBSA) and six other less polar compounds (e.g., benzophenone-3, BP-3; octocrylene, OC,...). Other important matters of concern in the determination of UV filters at trace levels in water, i.e., adsorption on glassware and blank contamination problems, have also been discussed and minimized. This methodology affords detection limits between 7 and 46 ng L-1 and SPE recoveries in the range 63-102% from different real water matrixes, except for butylmethoxydibenzoylmethane (BM-DBM), which was not determinable in wastewater samples due to adsorption problems. The application of the method allowed reporting the levels of benzophenone-4 (BP-4) in environmental water samples for the first time, where it was identified as one of the most important in concentration among the UV filters studied, particularly in wastewater (237-1481 ng L-1).     

Supercritical fluid chromatography and high-performance liquid chromatography/tandem mass spectrometric methods for the determination of cytarabine in mouse plasma

The separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by packed-column supercritical fluid chromatography (pSFC) was achieved on bare silica stationary phase with an isocratic mobile phase composed of CO2/methanol solvent with addition of ammonium acetate. SFC is commonly assumed to be only applicable to nonpolar and relatively low-polarity compounds. In this work, a broader range of compound polarities amenable to pSFC with appropriate mobile-phase modifiers and additives under normal-phase retention mechanism was demonstrated. The pSFC was integrated with an atmospheric pressure chemical ionization source and a tandem mass spectrometer (MS/MS) to enhance the sensitivity, selectivity, and speed of the assay. The influence of mobile-phase components on chromatographic performance and ionization efficiency of the test compounds was investigated for improving the sensitivity and separation for the analyte and the internal standard. The pSFC-MS/MS approach requiring approximately 2.5 min/sample for the determination of ara-C at nanograms per milliliter in mouse plasma was partially validated with respect to stability, linearity, and reproducibility. The mouse plasma levels of ara-C obtained by the pSFC-MS/MS method were found to be consistent with those determined by various reversed-phase, high-performance liquid chromatography methods using a porous graphite carbon column, a mixed-mode column, or a C18 column in conjunction with an ion-pairing agent coupled to a tandem mass spectrometer.     

液相色谱串联质谱同位素稀释法测定污水中卡马西平

卡马西平作为主要的水环境药物污染物,具有极高的检出率.为准确测定污水中卡马西平,该文建立同位素稀释液相色谱串联质谱法.采用卡马西平同位素内标,有效地消除样品前处理过程的干扰,实现卡马西平的准确定量检测.方法的检出限为0.045 ng/mL,方法线性系数r2=0.999(0.5～20 ng/mL).3个污水厂样品测定结果显示,在当前的工艺条件下,卡马西平不能被有效消除,排放水中卡马西平的最高质量浓度可达128.86 ng/L.     

Supercritical fluid extraction and gas chromatography/mass spectrometry for the analysis of tobacco-specific nitrosamines in cigarettes

A method for measuring four tobacco-specific nitrosamines (TSNAs), an important group of compounds in tobacco products, was developed. These compounds were extracted using supercritical fluid extraction (SFE) and purified by a sodium hydroxide wash of the ethyl acetate eluting solvent and solid-phase extraction. Quantitation was performed using gas chromatography/mass spectrometry (GC/MS). Spiking experiments were carried out to determine the recovery, precision, and limits of detection of this method. The detection limits were 0.04 microgram per sample for N'-nitrosonornicotine and N'-nitrosoanatabine and 0.02 microgram for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosoanabasine. This method was used to measure TSNAs in various brands of cigarette tobacco with excellent reproducibility. The variation of TSNA levels among the cigarettes of different packs and types was significantly smaller than that among different brands. Comparable TSNA levels were obtained with SFE and liquid extraction methods. Signal-to-noise levels were similar for GC/MS and GC/thermal energy analysis when low-level tobacco samples were analyzed.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine

We report a hydrophilic interaction liquid chromatography (HILIC) separation with tandem mass spectrometry (MS) detection method for analysis of seven urinary estrogen conjugates. HILIC separation employing a mobile phase with high organic solvent content resulted in enhanced electrospray ionization efficiency and MS sensitivity compared with reversed-phase (RP) LC-MS methods. Solid-phase extraction (SPE) was used to further improve the limit of detection and to eliminate interferences for the analysis of urine samples. No hydrolysis or derivatization was required in the sample pretreatment. This SPE/HILIC-MS/MS method provided limits of quantification (LOQs at S/N = 10) for the seven conjugates ranging from 2 to 1000 pg/mL with only 1 mL of urine sample, representing an improvement of 1 order of magnitude over the RPLC tandem MS methods previously reported. This method provided a linear dynamic range of 3 orders of magnitude, recovery of 92-109%, intraday accuracy of 84-109%, intraday precision of 1-14%, interday accuracy of 80-111%, and interday precision of 1-22%. We have successfully applied this technique to determine the seven estrogen conjugates in urine samples of a pregnant woman and found unique concentration changes of six estrogen conjugates at different stages of pregnancy while the concentration of estriol-3-glucuronide (E3-3G) remained constant. We further studied the profiles of individual estrogen conjugates in breast cancer patients before and after treatment and found patient-dependent effects of aromatase inhibitor treatment on estrogen phase-II metabolism, which have not been reported previously. This study demonstrates the potential clinical application of the HILIC-MS/MS technique for sensitive monitoring of the changes of urinary estrogen conjugates in a clinical setting.     

Liquid chromatography-tandem mass spectrometry application, for the determination of extracellular hepatotoxins in Irish lake and drinking waters

A novel method for the determination of hepatotoxins; microcystins (MCs), and nodularin (Nod) in lake water and domestic chlorinated tap water has been developed using liquid chromatography hyphenated with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). Optimization of the mass spectrometer parameters and mobile-phase composition was performed to maximize the sensitivity and reproducibility of the method. Detection of the hepatotoxins was carried out using multiple reaction monitoring experiments, thus improving the selectivity of the method. A total ion chromatogram and a precursor ion scan on ion m/z 135 was also applied to all samples to detect unknown microcystins or microcystins for which there are no standards available. A comprehensive validation of the LC-ESI-MS/MS method was completed that took into account matrix effects, specificity, linearity, accuracy, and precision. Good linear calibrations were obtained for MC-LR (1-200 microg/L; R2=0.9994) in spiked lake and tap water samples (1-50 microg/L; R2=0.9974). Acceptable interday repeatability was achieved for MC-LR in lake water with RSD values (n=9) ranging from 9.9 (10 microg/L) to 5.1% (100 microg/L). Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked MCs and Nod samples; LOD=0.27 microg/L and LOQ=0.90 microg/L for MC-LR in the "normal linear range" and LOD=0.08 microg/L and LOQ=0.25 microg/L in the "low linear range" in both lake and chlorinated tap water. Similar results were obtained for a suite of microcystins and nodularin. This sensitive and rapid method does not require any sample preconcentration, including the elimination of solid-phase extraction (SPE) for the effective screening of hepatotoxins in water below the 1 microg/L WHO provisional guideline limit for MC-LR. Furthermore, SPE techniques are time-consuming, nonreproducible at trace levels, and offer poor recoveries with chlorinated water. The application of this LC-ESI-MS/MS method for routine screening of hepatotoxins in lake and chlorinated tap water (average Cl2=0.23 mg/L) is achieved and this study represents the first direct method for the screening of hepatotoxins in chlorinated tap water.     

Method for the determination of vitamin K homologues in human plasma using high-performance liquid chromatography-tandem mass spectrometry

We report here the development of a precise and sensitive method for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human plasma using HPLC-tandem mass-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of stable isotope (18)O-labeled internal standard compounds, which were synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values for PK, MK-4, and MK-7 were <10%. Average spiked recoveries from authentic compounds added to normal human plasma samples for PK, MK-4, and MK-7 were 98-102%. Mean plasma concentrations of PK, MK-4, and MK-7 from healthy subjects (n = 20) were 1.22 +/-0.57, 0.39 +/- 0.46, and 6.37 +/- 7.45 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method should be useful for the evaluation of vitamin K status in postmenopausal women and elderly subjects and provides useful information for the treatment and prevention of osteoporosis with vitamin K.     

高效液相色谱-三重四级杆质谱法测定豆制品中二甲基黄、二乙基黄

建立一种同时测定豆制品中二甲基黄、二乙基黄的高效液相色谱-三重四级杆质谱(HPLC-MS/MS)方法。对提取溶剂及色谱条件进行优化,粉碎后的样品经乙腈提取,提取液以HPLC-MS/MS测定,外标法定量。二甲基黄、二乙基黄在3 min内流出并完全分离,两者在0.05~10μg/L范围内相关系数r2均大于0.999,二甲基黄在0.5~50μg/kg的加标回收率为90.0%~93.4%,RSD为0.5%~5.1%,二乙基黄在0.5~50μg/kg水平的加标回收率在84.6%~93.8%,RSD为1.1%~4.6%,检出限两者均为0.2μg/kg。该方法操作简捷、准确度和精密度高,适用于豆制品中二甲基黄、二乙基黄的检测。     

Liquid chromatography-tandem mass spectrometry determination of nonionic organophosphorus flame retardants and plasticizers in wastewater samples

A comprehensive method for the determination of nonionic organophosphorus flame retardants/plasticizers in wastewater samples by solid-phase extraction (SPE) with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is presented. It allows the determination of 11 organophosphorus compounds, trialkyl and trichloralkyl phosphates, triaryl phosphate, and biphosphates together with triphenylphosphine oxide (TPPO). Limits of quantification after SPE of 100 mL of water are between 3 and 80 ng/L, which are adequate for most aqueous samples. The sensitivity of the LC-ESI-MS approach allows direct injection of aqueous sample without preceding extraction for concentrations in the low microg/L range. The method was finally applied to municipal wastewater samples, showing the occurrence of six phosphoric acid triesters and TPPO in both raw and treated municipal wastewaters.     

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.     

GC-MS结合微量QuEChERS法快速测定土壤中16种多环芳烃

为实现土壤样品中典型持久性污染物的快速、灵敏及批量测定,通过基于改进微量-QuEChERS(μ-QuEChERS)技术结合超声辅助方法,且与气相色谱质谱联用(GC-MS)分析,建立土壤中16种多环芳烃(PAHs)快速测定方法。少量土壤(1.0 g)样品经过2.0 mL乙腈与二氯甲烷(V/V=1∶2)辅助15.0 min超声提取后,利用N-丙基乙二胺(PSA)和C18混合分散剂快速涡旋净化,离心、转换溶剂后进行GC-MS外标法测定。结果表明,在2～800μg/L浓度范围内,16种PAHs呈现良好的线性关系,相关系数均在0.992 0以上,检测限(LOD,S/N=3)低于0.50μg/kg。在8.0,20.0,100.0μg/kg加标浓度下,16种PAHs的加标回收率在70.3%～109.1%(n=3)。该方法快速、简单、准确,需样量少、环境污染小,可为土壤中PAHs的快速筛查提供批量检测方法。     

液相色谱-串联质谱法测定蜂蜜中四环素残留量的不确定度评定

采用液相色谱-串联质谱法依照GB/T18932.23-2003对蜂蜜进行四环素残留量测定。测定结果的不确定度来源主要有标准溶液配制、标准曲线校准、样品称量、样品定容、样品的回收率及结果的重复性等。通过测定过程中各种不确定度分量的计算和分析,测得四环素扩展不确定度为U(CX)=6.70mg/kg,k=2。     

Determination of perchlorate anion in foods by ion chromatography-tandem mass spectrometry

A rapid, sensitive, and specific method was developed for determining perchlorate anion in lettuce, cantaloupe, bottled water, and milk. A test portion of chopped crop homogenate was extracted with diluted nitric acid and filtered. Milk proteins were precipitated with acetonitrile, and the supernatant, after centrifugation, was cleaned up on a graphitized carbon solid-phase extraction column. Water samples were analyzed directly. All test solutions were syringe filtered and mixed with an 18O4-labeled perchlorate internal standard before ion chromatography-tandem mass spectrometry. A strong anion exchange column eluted with 100 mM ammonium acetate in 50:50 (v/v) acetonitrile/water was interfaced via electrospray ionization to a triple stage quadrupole mass spectrometer operated in the negative ion mode. The labeled internal standard corrected for any sample matrix effects on measured signals. Four parent-to-product ion transitions, for loss of oxygen, were monitored for native and 18O4-labeled perchlorate anion, respectively: 35Cl-perchlorate, m/z 99 --> 83 and 107 --> 89; 37Cl-perchlorate, m/z 101 --> 85 and 109 --> 91. The limit of quantitation was 1.0 microg/kg in lettuce, 2.0 microg/kg in cantaloupe, 0.50 microg/L in bottled water, and 3.0 microg/L in milk. Native perchlorate was recovered from fortified test portions in the range 93-107% for lettuce, 107-114% for cantaloupe, 100-115% for bottled water, and 99-101% for milk.     

Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.     

Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry

Tamoxifen (Tam), the antiestrogen used to treat estrogen receptor-positive breast cancer is a pro-drug that is converted to its major active metabolites, endoxifen and 4-hydroxy-tamoxifen (4-OH-Tam) by various biotransformation enzymes of which cytochrome P450-2D6 (CYP2D6) is key. The usual Tam dose is 20 mg daily; however, the plasma active metabolite concentrations vary due to common genetic variants encoding the biotransformation enzymes and environmental factors (e.g., concomitant drugs) that inhibit these enzymes. Effective treatment depends on adequate Tam conversion to its active isomers. To monitor metabolite plasma levels, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantitate Tam, N-desmethyl-tamoxifen (ND-Tam), and tamoxifen-N-oxide (Tam-N-oxide), and the E, Z, and Z' isomers of endoxifen and 4-OH-Tam. Known standards were used to identify each metabolite/isomer. Quantitation of these metabolites in plasma was linear from 0.6 to 2000 nM. Intra- and inter-assay reproducibilities were 0.2-8.4% and 0.6-6.3%, respectively. Accuracy determined by spike experiments with known standards was 86-103%. Endoxifen, 4-OH-Tam, and their isomers were stable in fresh frozen plasma for ≥6 months. This method provides the first sensitive, specific, accurate, and reproducible quantitation of Tam and its metabolite isomers for monitoring Tam-treated breast cancer patients.     

Qualitative determination of synthetic analogues of insulin in human plasma by immunoaffinity purification and liquid chromatography-tandem mass spectrometry for doping control purposes

Synthetic insulins such as Humalog Lispro, Novolog Aspart, or Lantus Glargine, are commonly employed for the treatment of insulin-dependent diabetes mellitus owing to convenient handling and fast or prolonged bioavailability. However, the misuse of insulin in sports has been reported often, and the international doping control system requires a reliable and robust assay to determine the presence or absence of related drugs prohibited by the World Anti-Doping Agency. Qualitative evidence of administered substances, which is preferably obtained by mass spectrometry, is of utmost importance. Plasma specimens of 2 mL were fortified with three synthetic insulin analogues and purified by immunoaffinity chromatography, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This top-down sequencing-based assay allows the assignment of individual fragment ions, in particular, of those comprising modifications that are originating from C-termini of B-chains. Recoveries of synthetic insulins from plasma aliquots ranged from 91 to 98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes.