LC-MS/MS法测定大鼠血浆中的野黄芩苷含量

目的:建立LC-MS/MS的分析方法测定大鼠血浆中的野黄芩苷,研究灯盏生脉胶囊中野黄芩苷在大鼠体内的药动学行为。方法:以噻氯匹定为内标,血浆样品经1%甲酸乙腈沉淀蛋白处理后,用LC-MS/MS法测定血浆中的野黄芩苷浓度。结果:野黄芩苷线性范围为1.31～670.00 ng·mL-1(γ0.999),最低定量浓度为1.31 ng·mL-1,回收率、日内、日间考察均符合生物样品分析要求。实验结果显示,野黄芩苷在大鼠体内出现多峰现象。结论:建立的LC-MS/MS定量分析方法灵敏、准确,可用于大鼠血浆中野黄芩苷的测定及其药代动力学研究。     

Determination of macrocyclic trichothecenes in mouldy indoor materials by LC-MS/MS

Stachybotrys occurring in mouldy indoor environments is associated with the so called “sick building syndrome” in humans or cases of idiopathic pulmonary hemorrhages. Samples of mouldy materials from indoor environments (n=15) were analysed for the occurrence of this fungus and its secondary metabolites by a sensitive LC-MS/MS method. In four samples,Stachybotrys and macrocyclic trichothecenes have been detected. Maximum values for Satratoxin G and H in wallpaper were determined with 9.7 μg/cm² and 12.0 μg/cm², respectively.     

Sensitive and accurate analyses of free 3-nitrotyrosine in exhaled breath condensate by LC-MS/MS

The quantitative determination of 3-nitro-l-tyrosine, a biological marker for inflammatory processes, in exhaled breath condensate (EBC) is described. The clean-up and preconcentration was performed by solid phase extraction (SPE). After liquid chromatography the specific detection was performed by tandem mass spectrometry using electron spray ionisation and selected reaction monitoring (SRM). 13C9-3-nitrotyrosine was used as an internal standard. For reliability, tests for the precision of the method, the losses during preparation, a test for nitrating artifacts and the comparibility of calibrants in EBC and buffer solution were performed. The calibration of the method was linear over a range of 10-500 pg/mL. The within-run coefficients of variation (CV) of the samples were found to be 8.4% at 25 pg/mL and 8.3% at 250 pg/mL. The day-to-day CV was found to be 11.2%. The limit of quantification was 3.9 pg/mL. The losses during preparation were 15%. The discrepancy between the calibration with EBC and buffer solution was below 10%. No artificial production of 3-nitrotyrosine was observed during the procedure. The application of the method on the EBC samples of healthy smokers (N=10) and non-smokers (N=10) showed no difference between the two groups. The concentration of 3-nitrotyrosine ranged between the limit of quantification and 184 pg/mL and was distinctly lower than data detected by an immunoassay procedure. The procedure was proven to be accurate, sensitive and in contrast to GC methods less elaborate and is recommended for the determination of 3-nitrotyrosine in exhaled breath condensate.     

Determination of glabridin in human plasma by solid-phase extraction and LC-MS/MS

Glabridin is a major flavonoid included specifically in licorice (Glycyrrhiza glabra L.), and has various physiological activities including antioxidant and anti-inflammatory effects. We have developed and validated an analytical method for determination of glabridin in human plasma by solid-phase extraction (SPE) and LC-MS/MS. Glabridin was extracted from plasma by SPE using a C8 cartridge and analyzed by LC-MS/MS using mefenamic acid as an internal standard (IS). The analyte were separated by a C18 column on LC, and monitored with a fragment ion of m/z 201 formed from a molecular ion of m/z 323 for glabridin and that of m/z 196 from m/z 240 for IS during negative ion mode with tandem MS detection. The lower limit of quantitation (LLOQ) of glabridin was 0.1 ng/mL in plasma, corresponding to 1.25 pg injected on-column. The calibration curves exhibited excellent linearity (r>0.997) between 0.1 and 50 ng/mL. Precision and accuracy were <17 and <+/-7% at LLOQ, and <11 and <+/-5% at other concentrations. Glabridin was recovered >90%, and was stable when kept at 10 degrees C for 72 h, at -20 degrees C until 12 weeks, and after three freeze-thaw cycles. This is the first report on determination of glabridin in body fluids by the selective, sensitive, and reproducible method.     

HPLC-DAD和LC-MS/MS法对各种芦荟样品中蒽醌类成分的分析研究

采用HPLC-DAD和LC-MS／MS对各种芦荟样品中蒽醌类成分进行鉴定,在此基础上建立了同时测定各种芦荟样品中芦荟苷与芦荟大黄素含量的方法。采用Nucleodur-silica色谱柱（250mm＊4．6mm,5μm）,流动相A为甲醇-醋酸（500：1．70）,B为水．醋酸（500：1．70）,梯度洗脱,流速1．0mL／min,DAD扫描波长范围为190～370nm,紫外检测波长为254和356nm。质谱离子源为ESI,采用全扫描一级质谱和选择离子全扫描二级质谱两种方式同时测定。结果表明：各种芦荟样品中主要的蒽醌类成分为芦荟苷A、B与芦荟大黄素,未检测到大黄素、大黄酸、大黄素甲醚、大黄酚;芦荟干粉中所含的芦荟苷含量最高。该法准确、可靠、重现性好,可行性高。     

LC-MS/MS法测定人血浆中伊伐布雷定浓度及药动学

目的：建立人血浆中伊伐布雷定的液相色谱-质谱-质谱联用测定方法,研究健康人体药代动力学。方法：以地西泮为内标物,采用液相色谱-质谱-质谱联用法,电喷雾电离源选择性正离子峰检测。测30名健康志愿者单剂量口服盐酸伊伐布雷定片的体内血药浓度,获得药动学参数。结果：伊伐布雷定在0.101-101 ng·mL-1浓度范围内呈良好的线性关系（r=0.998）,最低检测浓度为0.101 ng·mL-1。高、中、低浓度的方法提取回收率分别为93.2%、86.6%、87.5%,日内、日间精密度RSD均小于15%。结论：LC-MS/MS方法灵敏度高,专属性强,准确,简便,适用于盐酸伊伐布雷定片的人体药代动力学研究。     

Determination of risperidone and enantiomers of 9-hydroxyrisperidone in plasma by LC-MS/MS

A robust and validated liquid-liquid extraction LC-MS/MS method was developed for population pharmacokinetic analysis and therapeutic drug monitoring of risperidone and the enantiomers of its major active metabolite (+)-and (-)9-hydroxyrisperidone in pediatric patients. The method was rapid, sensitive and used a low sample amount (200 microL), which is very desirable for the pediatric population. The assay was validated from 0.2 to 50 ng/mL in plasma for all analytes. LLOQ for all analytes was 0.2 ng/mL. The extracts were analyzed by normal phase LC-MS/MS. The sample run time was 8 min. Intra- and interday precision for all analytes was < or =6%; method accuracy was between 89 and 99%. Additional experiments were performed to analyze matrix effects and identify a proper internal standard for each analyte. The validated method was used to study risperidone and its enantiomer metabolites in plasma as part of a population pharmacokinetic study in pediatric patients with pervasive developmental disorder (PDD).     

LC-MS/MS 法测定人血浆中伊伐布雷定浓度及药动学

目的:建立人血浆中伊伐布雷定的液相色谱-质谱-质谱联用测定方法,研究健康人体药代动力学.方法:以地西泮为内标物,采用液相色谱-质谱-质谱联用法,电喷雾电离源选择性正离子峰检测.测30名健康志愿者单剂量口服盐酸伊伐布雷定片的体内血药浓度,获得药动学参数.结果:伊伐布雷定在0.101-101 ng·mL-1浓度范围内呈良好的线性关系(r=0.998),最低检测浓度为0.101 ng·mL-1.高、中、低浓度的方法提取回收率分别为93.2%、86.6%、87.5%,日内、日间精密度RSD均小于15%.结论:LC-MS/MS方法灵敏度高,专属性强,准确,简便,适用于盐酸伊伐布雷定片的人体药代动力学研究.     

Determination of MK-0767 enantiomers in human plasma by normal phase LC-MS/MS

A sensitive and selective analytical method for the enantioselective determination of MK-0767, a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, in human plasma has been developed and validated. The chromatography is based on normal-phase chiral separation on a Kromasil, 5 microm, CHI-DMB 250 mm x 4.6 mm column. The detection involves the direct introduction of the normal phase eluent into MS/MS without the addition of a post-column reagent. Atmospheric pressure chemical ionization (APcI) mode was selected as the ion source in this method. With proper sample handling and processing procedures, ex vivo interconversion of the enantiomers was kept to minimum during sample collection, preparation and short term storage of frozen human plasma samples. The method was successfully utilized to determine the concentrations of MK-0767 enantiomers in human plasma to support pharmacokinetic investigation in man.     

LC-MS/MS定量测定人体血浆中阿托伐他汀浓度

目的:建立定量测定人体血浆中阿托伐他汀浓度的HPLC-MS/MS的方法.方法:以吲哚美辛为内标,采用Shim-packVP-ODS柱(150× 2.0 mm I.D.,5μm,日本Shimadzu Technologies Inc.公司)为固定相;乙腈-0.5％甲酸溶液(90:10,v/v)为流动相,流速为0.3 ml/min;通过电喷雾离子源(ESI),以正离子多反应监测模式进行检测.阿托伐他汀与内标用于检测的离子对分别为m/z 559.4 m/z 250.3和m/z 358.3 rn/z 139.2.结果:阿托伐他汀在0.10～20.00 ng/ml范围内与峰面积比值线性范围良好(r=0.9962),定量下限为0.10 ng/ml,日内日间精密度的RSD均小于12％,平均回收率大于71％.结论:所建方法准确度高,方法灵敏,专属性强且操作简便,可适用于阿托伐他汀的血药浓度测定和临床药代动力学研究.     

LC-MS/MS定量测定人体血浆中氨氯地平浓度

目的:建立定量测定人体血浆中氨氯地平浓度的HPLC-MS/MS的方法.方法:以克林霉素为内标,采用Shim-pack VP-ODS柱(150× 2.0 mm I.D.,5μm,日本Shimadzu Technologies Inc.公司)为固定相;乙腈-10 mmol/L乙酸铵溶液(90∶10,v/v)为流动相,流速为0.4 mL/min;通过电喷雾离子源(ESI),以正离子多反应监测模式进行检测.氨氯地平与内标用于检测的离子对分别为m/z409.3 m/z 238.2和rn/z 425.2 m/z 126.3.结果:氨氯地平在0.10～20.00 ng/mL范围内与峰面积比值线性范围良好(r=0.9968),定量下限为0.10 ng/mL,日内日间精密度的RSD均小于7％,平均回收率大于86％.结论:所建方法准确度较高,灵敏度好,专属性强且操作简便,可适用于氨氯地平的血药浓度测定和临床药代动力学研究.     

LC-MS/MS法检测人头发中利培酮及其代谢物9-羟利培酮含量的实验研究

目的:建立高效液相色谱-三重四级杆质谱联用(LC-MS/MS)法检测人头发中利培酮(RIP)及其代谢物9-羟利培酮(9-OH-RIP)含量的方法。方法:采用同位素内标氘4-利培酮(RIP-d4)及氘4-9-羟利培酮(9-OH-RIP-d4),流动相A为10 mmol/L醋酸胺溶液(甲酸调p H值为4.0),流动相B为乙腈,A/B=70/30,流速为0.3 m L/min,等度洗脱4.00 min。色谱柱为安捷伦Zorbax SB C18(2.1×50 mm,1.8μm),柱温30℃。准确称取20 mg丙酮清洗过晾干剪碎成粉末的头发样本,加1N氢氧化钠(Na OH)超声2 h,等量酸中和后,加200μL1N氢氧化钠溶液及5.0 m L的甲基叔丁基醚(MTBE)提取涡旋1分钟,3000×g离心5 min后取上清液在40度水浴下氮气吹干后用100μL流动相复溶,进样2μL经LC-MS/MS检测。MRM监测离子对:RIP:m/z 411.2→191.0,9-OH-RIP:m/z 427.2→207.1,RIP-d4:m/z 415.2→195.2,9-OH-RIP-d4:m/z 431.2→211.1。结果:利培酮及9-羟利培酮线性范围分别为0.5-25 ng/mg,0.0025-0.15 ng/mg,提取回收率均70.0%,方法回收率均在85.0%-115.0%之间,线性r均0.999,精密度和重现性RSD均15%。结论:本研究建立了采用LC-MS/MS法检测人头发中利培酮及9-羟利培酮含量的方法,该法快速、简单、准确、重现性好。     

Characterization of pertussis toxin by LC-MS/MS

Liquid chromatography-mass spectrometry (LC-MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000 Da. At this mass assignment accuracy level, the sequences of the PT subunits investigated in this study are easily determined based on molecular weight alone. The subunits 1, 2, and 5 of PT were observed to undergo oxidation under normal storage conditions as ammonium sulfate suspension at 2 to 8 degrees C. These oxidized subunits can be separated completely or partially by reverse-phase high-performance liquid chromatography (HPLC) from their native counterparts. For the determination of oxidation sites, the oxidized subunits and their nonoxidized counterparts were fraction collected, trypsin digested, and mapped by LC-MS. The oxidized peptides and their nonoxidized counterparts were further studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm their identities. The methionines at position 212 of subunit 1, at position 89 of subunit 2, and at position 40 of subunit 5 were found to be the primary sites of oxidation.     

Detection of a Serum Siderophore by LC-MS/MS as a Potential Biomarker of Invasive Aspergillosis

Invasive aspergillosis (IA) is a life-threatening systemic mycosis caused primarily by Aspergillus fumigatus. Early diagnosis of IA is based, in part, on an immunoassay for circulating fungal cell wall carbohydrate, galactomannan (GM). However, a wide range of sensitivity and specificity rates have been reported for the GM test across various patient populations. To obtain iron in vivo, A. fumigatus secretes the siderophore, N,N'',N"-triacetylfusarinine C (TAFC) and we hypothesize that TAFC may represent a possible biomarker for early detection of IA. We developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for TAFC analysis from serum, and measured TAFC in serum samples collected from patients at risk for IA. The method showed lower and upper limits of quantitation (LOQ) of 5 ng/ml and 750 ng/ml, respectively, and complete TAFC recovery from spiked serum. As proof of concept, we evaluated 76 serum samples from 58 patients with suspected IA that were investigated for the presence of GM. Fourteen serum samples obtained from 11 patients diagnosed with probable or proven IA were also analyzed for the presence of TAFC. Control sera (n = 16) were analyzed to establish a TAFC cut-off value (≥6 ng/ml). Of the 36 GM-positive samples (≥0.5 GM index) from suspected IA patients, TAFC was considered positive in 25 (69%). TAFC was also found in 28 additional GM-negative samples. TAFC was detected in 4 of the 14 samples (28%) from patients with proven/probable aspergillosis. Log-transformed TAFC and GM values from patients with proven/probable IA, healthy individuals and SLE patients showed a significant correlation with a Pearson r value of 0.77. In summary, we have developed a method for the detection of TAFC in serum that revealed this fungal product in the sera of patients at risk for invasive aspergillosis. A prospective study is warranted to determine whether this method provides improved early detection of IA.     

Quality control in LC-MS/MS

In the last 15 years, MS-based protein characterization has expanded at a rapid rate. This success is built upon constantly improving instrumentation and a variety of ingenious methods applied to numerous biological questions. However, the reproducibility of mass spectrometric results is considered by many as insufficient. In part, inadequate quality control might be responsible for the lack of reproducibility. Quality control is rarely discussed in scientific publications. Here, we briefly present measures undertaken in our laboratory to foster a general discussion of the subject.     

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC-MS/MS

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol-water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI(+)). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127-->110 for AICA, and 172-->128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.     

High Sensitivity and Wide Linearity LC-MS/MS Method for Oxylipin Quantification in Multiple Biological Samples

Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05–0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a 1,000-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.     

Protein identification by syringe pump-driven reversed-phase LC-MS/MS

In typical mass spectrometry-based protein identification using peptide fragmentation fingerprinting, front-end separation plays a critical role in successful peptide sequencing. This separation step demands a great deal of time and usually is the rate-limiting step for the whole process. Here we provide an alternative separation method, based on a simple nanoflow delivery system, that is able to shorten the separation time considerably. This system consists of a 25-mul syringe connected to a manually packed reversed-phase mini-capillary column that can be directly coupled to an electrospray ionization tandem mass spectrometer. A syringe pump is then used to deliver the peptide mixtures at a nanoscale flow rate. We examined the efficiency and efficacy of this method by analyzing the tryptic peptides of bovine serum albumin and of 10 Escherichia coli proteins separated by two-dimensional gel electrophoresis (2DE). The results showed that identification of each protein could be achieved successfully within 25 min by using the disposable mini-capillary column. Moreover, all 2DE-separated E. coli proteins were identified at high confidence levels. Together, our data suggest that this method is a suitable option for mass spectrometry-based protein identification.     

Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS

An LC/MS/MS assay for the determination of tenofovir (TNF) was developed and validated for use with the EDTA anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was used to produce a protein-free extract. Chromatographic separation was achieved with a Polar-RP Synergi, 2.0 mm x 150 mm, reversed-phase analytical column. The mobile phase was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable limit of 10 ng/ml when 250 microl aliquots were analyzed. The usefulness of this LC/MS/MS method to routinely monitor plasma concentrations of TNF was demonstrated along with its ability to assist in the performance of pharmacokinetic studies.