An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or Nano-ESI mass spectrometry for proteolytic digests

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; Mr = 25,563), a DNA-binding protein that initiates the uracil-excision DNA repair process by catalyzing the release of uracil from the deoxyribose phosphate backbone of DNA. Both on-line and off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liquid chromatography (HPLC)/micro-electrospray MS to assign peptide masses. The off-line approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/collision-induced dissociation (CID) tandem mass spectrometry, to analyze fractions (2-3 microL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at different collision energies with only a marginal increase in sample handling due to the nano-LC step.     

In vivo metabolism study of polygalic acid in rat using HPLC-ESI-MSn

A very simple and direct method has been established for the determination of polygalic acid and its metabolites in rat urine based on HPLC coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC-ESI-MS(n)). The rats were administered a single dose (100 mg/kg) of polygalic acid by oral gavage. The urine samples were collected and purified through a C(18) solid-phase extraction cartridge, and then these pretreated samples were injected into a reversed-phase C(18) column with a gradient elution program, whereas acetonitrile-0.5% aqueous formic acid was used as mobile phase and detected by an on-line MS/MS system. As a result, the parent drug and its four metabolites were identified and characterized in rat urine for the first time by comparing their changes in molecular mass (ΔM), retention times and full-scan MS(n) spectra with those of the parent drug. A possible metabolic pathway of polygalic acid was investigated and proposed. More importantly, the results demonstrated that the newly developed method (HPLC-ESI-MS(n)) was sensitive, simple and suitable for the determination of polygalic acid and its metabolites in biological samples.     

Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometry

Analysis of caffeine and its metabolites is of interest with respect to caffeine exposure, for kinetic and metabolism studies and for opportunistic in vivo estimation of drug metabolizing enzyme activity in humans and animals. For the latter, analysis is usually done by high-performance liquid chromatography (HPLC) with UV detection. However, this method is close to the detection limit for certain of the metabolites and requires very long chromatography, 30-60 min. We have developed a fast method for the quantification of caffeine and its metabolites 1-methylxanthine, 1-methyluric acid, 1,7-dimethyluric acid, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by HPLC tandem mass spectrometry (MS/MS) in urine that requires only its dilution with buffer and centrifugation before injection into the HPLC/MS/MS system. The chromatography lasts 7 min and is followed by 4.5 min for re-equilibration of the HPLC column, giving a total analysis time of 11.5 min. The method provides a great sensitivity improvement with detection limits for all analytes 相似文献   

Simultaneous quantitative cassette analysis of drugs and detection of their metabolites by high performance liquid chromatography/ion trap mass spectrometry

A method using high performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (MS) for simultaneous quantification of multiple drugs and detection of their metabolites is described. The new approach offers a significant increase in analytical throughput and is illustrated with analysis of the in vitro metabolism of 19 alpha-1a receptor antagonists. The compounds were separated into four cassette groups by using a computer program as well as by manual examination. The samples from incubation with dog liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrospray (ESI) ion trap MS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 60 min, respectively. The quantitative results from the cassette analysis procedure agreed well with those obtained from conventional discrete analysis. In addition, the technique allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multi-stage tandem mass spectrometry (MS(n)) on the ion trap instrument allowed confirmation of the detected metabolites.     

Identification and quantification of glutathione adducts of clozapine using ultra-high-performance liquid chromatography with orthogonal acceleration time-of-flight mass spectrometry and inductively coupled plasma mass spectrometry

The application of sulphur-specific detection via ultra-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (UPLC/ICPMS) to detect and quantify the glutathione (GSH)-adducts produced via the in vitro formation of reactive metabolites is demonstrated. The adducts were formed in human liver microsomes supplemented with unlabelled GSH for clozapine. The calculation of adduct concentration was performed via comparison of the peak areas to calibration curves constructed from omeprazole, a sulphur-containing compound over the range of 0.156 to 15.62 μM of sulphur with a detection limit of 1.02 ng of sulphur on-column. Identification of the adducts was performed using conventional UPLC/time-of-flight (TOF)-MS with the calculation of clozapine intrinsic clearance carried out by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). The use of ICPMS in this way appears to offer a novel, rapid and sensitive means of determining the quantity of GSH conjugates with the combined adducts producing 0.9 μM of reactive metabolite out of a total of 3.5 μM of metabolites. The GSH adduct therefore represents 26% of this total produced as a result of the metabolism of drug to reactive species.     

Fast mass spectrometry-based methodologies for pharmaceutical analyses

Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.     

Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis

Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS(2)), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 microL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 microL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [M-nH](n-) ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS(2) mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS(2) analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 microg of total amount of protein).     

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC.     

液相色谱-串联质谱法测定蜂产品中吡虫啉及其3种代谢物

建立了蜂蜜和蜂花粉中吡虫啉及其代谢物吡虫啉烯烃、吡虫啉脲、6-氯烟酸的液相色谱-串联质谱(LC-MS/MS)检测方法.在QuEChERS方法基础上,针对目标物化学性质和样品杂质情况,对提取溶液的pH值、提取次数、净化材料等参数进行了优化.最终以5％甲酸-乙腈溶液提取两次,无水MgSO4、NaCl盐析分层,提取液经增强型脂类去除材料(EMR)净化,以LC-MS/MS进行测定,基质外标法进行定量分析.结果表明,蜂蜜和蜂花粉中吡虫啉、吡虫啉烯烃、吡虫啉脲、6-氯烟酸的平均加标回收率为86.0％～111.5％, 相对标准偏差在1.7％～9.6％之间, 检出限分别为0.20, 3.50, 0.40和14.00 μg/kg,定量限分别为0.60, 11.64, 1.20和45.00 μg/kg.本方法分析速度快、灵敏度高、重现性好,适用于蜂蜜和蜂花粉中吡虫啉及其3种代谢物的快速测定.     

Direct determination of the ethanol metabolites ethyl glucuronide and ethyl sulfate in urine by liquid chromatography/electrospray tandem mass spectrometry

A liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) method for the determination in urine samples of two ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), was developed and validated. Pentadeuterated EtG was used as internal standard for both EtG and EtS. In addition to the surviving ions, two MS/MS reactions were monitored for each analyte, with the deprotonated molecule as precursor ion: m/z 221 --> 75, m/z 221 --> 85 (EtG), and m/z 125 --> 97, m/z 125 --> 80 (EtS). Sample pretreatment, though very simple and rapid (1:50 water dilution and centrifugation of 50 muL of urine), was found to contain the occurrence of matrix effects. The method was accurate and precise over the linear dynamic range (0.05-10 mg/L). The analytes were stable in frozen urine for at least 1 month. The assay was applied to several authentic urine samples from social drinkers and to alcoholic beverages.     

超高效液相色谱串联质谱法测定花生、粮油中18种真菌毒素

采用同位素稀释法并结合凝胶色谱净化技术,建立了花生、粮油中18种常见真菌毒素污染的超高效液相色谱串联质谱分析方法.样品中添加同位素内标U-[13C17]-黄曲霉毒素 B1和U-[13C15]-脱氧雪腐镰刀菌烯醇,经乙腈-水溶液(84:16,体积比)均质提取,凝胶渗透色谱净化,Waters ACQUITY UPLCTM ...     

气相色谱-三重四极杆串联质谱法检测环境空气中的多环芳烃

建立了气相色谱-三重四极杆串联质谱检测环境空气中多环芳烃的方法,并利用同位素稀释法对多环芳烃进行了测定。将该方法应用于华南地区某大型石化企业周边环境空气中多环芳烃的检测,并与气相色谱-质谱方法进行了对比。结果表明,该方法的仪器检出限（0.01~0.15 μg/L）和定量限（0.03~1.5 μg/L）均优于气相色谱-质谱法（0.1~0.8 μg/L和0.3~3.5 μg/L）,并有更好的灵敏度与选择性。当利用气相色谱-质谱作为检测手段时,回收率指示物氘代菲和进样内标六甲基苯均受到了杂质的严重干扰,影响了定量结果的准确性,而三重四极杆串联质谱很好地解决了这些问题。实际样品分析时,标准曲线中16种多环芳烃相对响应因子的相对标准偏差为2.60%~15.6%,氘代化合物的回收率为55.2%~82.3%,空白加标样品的回收率为98.9%~111%,平行样品的相对标准偏差为6.50%~18.4%,采样空白含量范围为未检出~44.3 pg/m³,实验室空白含量范围为未检出~36.5 pg/m³。上述研究表明,分析环境空气中的多环芳烃时,气相色谱-三重四极杆串联质谱方法值得推广。     

同位素稀释高效液相色谱-串联质谱法测定水产品中甲苯咪唑及其代谢物的残留量

建立了同时分析水产品中甲苯咪唑及其代谢物羟基甲苯咪唑和氮基甲苯咪唑的同位素稀释高效液相色谱串联质谱法.向样品中添加磷酸二氢钠溶液后用乙酸乙酯提取,提取液用氮气吹干后经2 mL甲醇-0.1%甲酸溶液(体积比1:1)溶解,正己烷去脂.以Hypersil GOLD为色谱分离柱,甲醇-0.1%甲酸溶液为流动相,流速为0.2 m...     

Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.     

液相色谱串联质谱法测定养殖水体中孔雀石绿及其代谢物

研究利用液相色谱-串联质谱(LC-ESI-MS/MS)测定水产品养殖水体中孔雀石绿及其代谢物隐性孔雀石绿的方法.通过一系列实验对样品前处理条件进行了优化研究,养殖水样经乙酸酸化,净化后,采用HPLC-ESI-MS/MS检测分析.在多反应监测模式(MRM)下,外标法定量,定量限均为0.5μg/kg.在0.5～50μg/L...     

Simultaneous quantitation of glycyrrhetic acid and puerarin in plasma using ultra flow liquid chromatography tandem mass spectrometry and application in a pharmacokinetics study in healthy and alcoholic liver injury rats

A rapid, sensitive, and accurate ultra flow liquid chromatography tandem mass spectrometry (UFLC–MS/MS ) method was developed and validated for simultaneous quantitation of glycyrrhetic acid and puerarin in plasma derived from healthy and alcoholic liver injury rats. Plasma samples from healthy and model rats were deproteinated with methanol using liquiritin as an internal standard. Chromatography separation was performed by a Waters BEH (ethylene-bridged hybrid) C₁₈ column (2.1 × 50 mm; 1.7 μm) using a gradient elution from acetonitrile and water (containing 0.1% formic acid) and at a flow rate of 0.4 mL/min. Quantitation was performed on a Triple Quad 4500 tandem mass spectrometer coupled with an electrospray ionization source in negative multiple reaction monitoring mode. Specificity, carryover, dilution integrity, recovery, linearity, precision and accuracy, matrix effect, and stability were within acceptable limits. The newly established method was successfully applied to a pharmacokinetics study to investigate glycyrrhetic acid and puerarin in healthy and alcoholic liver injury rats.     

Identifying and overcoming bioanalytical challenges associated with chlorine-containing dehydrogenation metabolites

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is a widely utilized analytical tool for quantifying small molecules in complex biological matrices. In certain situations the mass-selection capabilities of the tandem mass spectrometer may be insufficient to discriminate between the analyte of interest and its metabolites, particularly those metabolites that are isobaric with the analyte. One scenario by which isobaric interference may occur is the metabolism of a chlorine- or bromine-containing small molecule to a metabolite with the concomitant loss of 2 Da. This report describes the detection and characterization of two distinct dehydrogenation [M-2] metabolites during LC/MS/MS quantification of a chlorinated small molecule in rat plasma samples derived from a toxicokinetic study. The potential isotope-related impact of these metabolites on quantification of the parent compound was assessed. Several alternate precursor ion and product ion combinations were evaluated and shown to minimize the quantitative impact of the interfering metabolites without having to rely on their stringent chromatographic resolution from the parent compound. These results indicate that when quantifying chlorine- or bromine-containing small molecules from in vivo samples or in vitro metabolic incubations: (1) efforts to detect potential dehydrogenation metabolites should be undertaken and (2) if such metabolites are detected, the judicious choice of alternate multiple-reaction monitoring (MRM) transitions can limit their impact on quantification of the parent molecule without the need for robust chromatographic resolution.     

Simultaneous determination of VD2, VD3, 25(OH) D2, and 25(OH) D3 in human plasma using electrospray LC–MS/MS as well as its application to evaluate VD plasma levels in depressive,schizophrenic patients and healthy individuals

Vitamin D measurements in biological fluids by liquid chromatography–tandem mass spectrometry (LC–MS/MS) have been widely used but remain challenging at very low concentration levels. Rapid, high recovery, sensitive and reliable measurements of vitamin D, as well as its primary metabolites using LC–MS/MS are urgently needed for a routine clinical laboratory. Herein, we reported a novel electrospray LC–MS/MS method for determining vitamin D and its primary metabolites using the supported liquid extraction method to achieve higher recoveries, with optimized pH values to achieve optimal derivatization efficiency for higher sensitivity and selected chromatographic conditions to shorten the separation time. The method has been validated with respect to selectivity, recovery, matrix effects, accuracy and precision, stabilities, carryover and dilution effects. The method has been successfully applied to quantify the VD plasma concentrations of depressive, schizophrenic patients and healthy individuals. The result showed that there were significant differences in plasma VD levels between mental disorder patients with healthy individuals, and the total VD levels in mental disorder patients were much higher than healthy individuals, which might require larger clinical samples for validation.     

Measurement of caffeine and its three primary metabolites in human plasma by HPLC‐ESI‐MS/MS and clinical application

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC‐ESI‐MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects.     

Absolute quantification of Atlantic salmon and rainbow trout vitellogenin by the 'signature peptide' approach using electrospray ionization QqToF tandem mass spectrometry

In this work, we present a simple method for absolute quantification of plasma vitellogenin from both rainbow trout and Atlantic salmon. Plasma samples obtained from control and beta-estradiol induced fish were digested with trypsin. A characteristic 'signature peptide' was selected and analyzed by high performance liquid chromatography (HPLC) coupled to an electrospray quadrupole-time-of-flight tandem mass spectrometer, using a deuterated homolog peptide as an internal standard. The hybrid tandem mass spectrometer was operated in a 'pseudo' selected reaction-monitoring mode in which three diagnostic product ions were monitored for identification and quantification purposes. The reproducibility (coefficient of variation approximately 5%) and sensitivity (limit of quantification (LOQ) of 0.009 mg/ml) achieved by this simple assay allow it to be considered as an alternative to immunological assays.