液相色谱串联质谱法检测法布雷病样本的质量控制研究

法布雷病(FD)是一种罕见的X连锁隐性遗传的溶酶体贮积病.用于检测FD的常用生物学标志物之一是Lyso-GB3,它是一种脱乙酰基的神经酰胺三己糖苷,通常通过高灵敏和特异性的液相色谱-串联质谱法(LC-MS/MS)进行分析.由于LC-MS/MS技术的复杂性,中国大多数医院仍无法自行建立LC-MS/MS对FD进行高效可靠的诊断.患者的样本需要在当地医院采集,然后送到中心实验室进行LC-MS/MS分析.样本经过漫长的运输和储存过程,可能会影响样本质量,从而影响诊断的准确性.我们首先建立并验证了一种新的LC-MS/MS方法以保证FD样本质量,研究了分析前变量对Lyso-GB3检测的影响.这些分析前变量包括血浆的储存温度和时间、血浆的冻融循环、血浆的溶血程度、全血的储存温度和时间,以及离心温度.最后,我们分析了86个FD样本和100个正常人样本,并对Lyso-GB3浓度和与FD疾病分型的关系进行了相关性研究.结果显示,当血浆在20℃下储存时,Lyso-GB3浓度在4 d后开始下降.此外,溶血会显著影响FD样品的质量,重度溶血导致Lyso-GB3浓度下降了57.8%,其他分析前变量对FD样品质量影响不大.本方法在使用分析前标准化操作流程下,筛查FD的灵敏度和特异度为100%,确保了检测的正确性;当Lyso-GB3浓度的cut-off值为55.15μg/L,鉴别经典型、非经典型患者的灵敏度和特异度为71%和100%,ROC曲线下面积为0.83.我们的研究结果有助于医学实验室操作程序的标准化,以保证FD样本的质量,并为临床判定中国法布雷病的分型提供依据.     

Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS

Recent technological advances have made available reverse phase chromatographic media with a 1.7 microm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC-MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10-fold and improvements in method speed were as large as 5-fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC-MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.     

LC-MS/MS quantitation of plasma progesterone in cattle

Quantitation of progesterone (P₄) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P₄ in plasma of cattle with no sample derivatization. The quantitation of plasma P₄ released from three nonbiodegradable, commercial, intravaginal P₄-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P₄ kinetics (P > 0.05) whereas results of P₄ quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P₄ limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P₄ quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies.     

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.     

Standardized LC-MS/MS based steroid hormone profile-analysis

In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R(2)>0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8% and 8.2-18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5% and 109.3-128.2% for 11-deoxycorticosterone, inter-day 91.4-117.2% and 102.3-137.1% for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.     

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。     

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.     

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.     

高效液相色谱-串联质谱分析微量格尔德霉素类似物

安莎类抗生素例如利福霉素和安丝菌素,通常由一组化学结构相似的组分组成。格尔德霉素为苯安莎类抗生素,已经发现4个组分。本研究采用高效液相色谱-串联质谱方法对格尔德霉素(GDM)制品中的微量组分进行了分析,发现5个新的和1个已知的GDM类似物。依据质谱数据、结合GDM生物合成机制,对6个GDM类似物的化学结构进行了推测:分子式为C29H42N2O10的新化合物3个,分别为GDM安莎链上C2-C3、C4-C5和C8-C9之间的C-C双键变为单键并同时单羟基化的GDM衍生物;分子式为C28H38N2O8的新化合物2个,其中1个为17(或12,或4)-去甲氧基格尔德霉素,另1个为4,5-双氢-10,11-脱水-17-去甲基-17-羟基格尔德霉素;分子式为C29H42N2O9的已知化合物1个,为4,5-双氢格尔德霉素。这些GDM类似物的发现有助于加深对GDM生物合成的认识,并对通过基因阻断、组合生物合成技术获得GDM衍生物的研究有启示作用。     

LC-MS/MS 检测癫痫患者神经递质类氨基酸

目的:建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价.方法:选用AAA-C18柱色谱柱,以乙腈水(含有0.01%七氟丁酸、0.1%甲酸)为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测.疾病组与健康组的统计采用t检验和主成份分析.结果:疾病组和健康组氨基酸测定结果显示:Trp、GABA两组间没有显著性差异(P＞0.05),Arg、Glv、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异(P＜0.05),通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸.结论:试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价.     

Analysis of anabolic steroids in human hair using LC-MS/MS

New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the first time. Hair samples from 180 participants (108 males, 72 females, 62% athletes) were screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring (SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range 1-400 pg/mg and 5-400 pg/mg, respectively. The methods were validated for LLOD, interday precision, intraday precision, specificity, extraction recovery and accuracy. The assays were capable of detecting 0.5 pg stanozolol and 3.0 pg nandrolone per mg of hair, when approximately 20 mg of hair were processed. Analysis using LC-MS/MS confirmed 11 athletes’ positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and 1 for nandrolone (14.0 pg/mg) thus avoiding false results from ELISA screening. The results obtained demonstrate the application of these hair analysis methods to detect both steroids at low concentrations, hence reducing the amount of hair required significantly. The new methods complement urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis benefits from non-invasiveness, negligible risk of infection and facile sample storage and collection, whilst reducing risks of tampering and cross-contamination. Owing to the wide detection window, this approach may also offer an alternative approach for out-of-competition testing.     

LC-MS/MS检测癫痫患者神经递质类氨基酸

目的：建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价。方法：选用AAA-C18柱色谱柱,以乙腈水（含有0.01%七氟丁酸、0.1%甲酸）为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测（MRM）扫描方式进行检测。疾病组与健康组的统计采用t检验和主成份分析。结果：疾病组和健康组氨基酸测定结果显示：Trp、GABA两组间没有显著性差异（P〉0.05）,Arg、Gly、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异（P〈0.05）,通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸。结论：试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价。     

Global proteome discovery using an online three-dimensional LC-MS/MS

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.     

Quantitative protein determination for CYP induction via LC-MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.     

Characterization of the human tear metabolome by LC-MS/MS

The tear film overlying the epithelial cells of the eye's surface is vital to visual function, and its composition is reflective of ocular surface health. The ultrasmall volume of tears poses challenges in its analysis, contributing to the limited number of reports on the tear metabolome. In addition, using a standard clinical method of tear collection posed some confounding factors in metabonomic analysis. We sought to establish an analytical platform for the global characterization of human tear metabolites. Following information dependent acquisition (IDA) directed liquid chromatography-tandem mass spectrometry (LC-MS/MS), isotope pattern matched peak mining was performed using Extracted Ion Chromatogram (XIC) manager within the PeakView software. Sixty metabolites representing diverse compound classes were identified in human tears, most of which have not been previously reported. Selected metabolites were verified using pure standards. Unsupervised chemometric analysis showed good separation between tear samples and blanks (PC1 = 87%, R(2) = 0.91, Q(2) = 0.87). The results demonstrated the potential of our platform for untargeted metabonomic studies of eye diseases.     

应用鸟枪法LC-MS/MS鉴定烟粉虱唾液蛋白

烟粉虱是一种刺吸式口器小型昆虫,每年对我国农业生产造成巨大危害。烟粉虱在取食植物时分泌多种唾液蛋白帮助其取食,同时唾液中的效应因子在调控植物防御反应过程中发挥重要作用。由于烟粉虱个体微小,唾液收集十分困难,有关其唾液蛋白的研究难以开展。本研究收集了3万头烟粉虱唾液,利用高精度Q-Exactive质谱,采用shotgun的方法首次鉴定了烟粉虱唾液中蛋白组分。应用双层膜收集装置收集到的烟粉虱唾液经浓缩等处理后其蛋白浓度约为1.698 g/L。唾液蛋白样品经胶内酶解、LC-MS/MS检测、序列分析,最终鉴定出42个烟粉虱唾液蛋白。功能分析表明烟粉虱唾液蛋白组分与蚜虫唾液蛋白组分具有一定的相似性,但多数蛋白为功能未知蛋白。通过质谱鉴定烟粉虱唾液蛋白组分为深入研究烟粉虱唾液蛋白的功能奠定了基础,有利于明确烟粉虱危害寄主植物的分子机制,为开发害虫防治新方法提供新思路。     

Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.     

Development and validation of Lenalidomide in human plasma by LC-MS/MS

A highly sensitive and ultra-fast high performance liquid chromatography- tandem mass spectrometry (LC–MS/MS) assay is developed and validated for the quantification of Lenalidomide in human plasma. Lenalidomide is extracted from human plasma by Liquid- Liquid Extraction by Ethyl Acetate and analyzed using a reversed phase isocratic elution on a XTerra RP18, (4.6 × 50 mM, 5 µm) column. A 0.1% Formic acid: Methanol (10:90% v/v), is used as mobile phase and detection was performed by Triple quadrupole mass spectrometry LC-MS/MS using electrospray ionization in positive mode. Fluconazole is used as the internal standard. The lower limit of quantification is 9.999 ng/mL for Lenalidomide. The calibration curves are consistently accurate and precise over the concentration range of 9.999 to 1010.011 ng/mL in plasma for Lenalidomide. This novel LC–MS/MS method competes with all the regulatory requirements and shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic and bioequivalence studies in humans.     

Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50 pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5 pg/mL while using only 0.175 mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6?×?50 mm, 1.8 μm, 600 bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5 mL per minute. The calibration curve was found to be linear over a range of 5–10,000 pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20 pg/mL), medium (1000 pg/mL), and high (5000 pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ±?15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5 h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5 months at -?70°C), and post-preparative storage (for 48 h).     

Quantification of cidofovir in human serum by LC-MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1-->234.9 and the m/z 288.1-->133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within +/-12.1% for quality controls. The novel LC-MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.