可溯源干血片样本类固醇激素标准曲线的建立

目的 建立可溯源的干血片样本19种类固醇激素液相色谱-串联质谱(LC-MS/MS)标准曲线。方法收集乙二胺四乙酸抗凝新鲜全血,用0.9%NaCl溶液清洗血细胞,将清洗后的血细胞和无类固醇激素的血浆按0.55∶0.45比例混合,制备模拟全血。根据拟制备标准曲线浓度添加不同量类固醇激素至模拟全血样本中,制备不同浓度的类固醇激素标准曲线干血片。采用液相色谱-串联质谱(LC-MS/MS)检测各标准曲线干血片类固醇激素含量,分析其线性范围,并对类固醇激素标准曲线干血片进行性能验证。结果 类固醇激素标准曲线干血片中孕烯醇酮、孕酮、可的松、脱氢表雄酮的线性范围为0.25～100 ng/mL,11-脱氧皮质酮、21-脱氧皮质醇、18-羟皮质醇、11-酮睾酮的线性范围为0.05～20ng/mL,皮质酮、11-羟睾酮的线性范围为0.025～10 ng/mL,17-羟孕酮、18-羟皮质酮、11-脱氧皮质醇、雄烯二酮、11-羟雄烯二酮、睾酮的线性范围为0.1～40 ng/mL,皮质醇的线性范围为1.5～600 ng/mL,17-羟孕烯醇酮的线性范围为0.2～80 ng/mL,硫酸脱氢表雄酮的线性范围为25～1...     

液相色谱-串联质谱技术的临床应用进展

液相色谱-串联质谱技术(liquid chromatography-tandem mass spectrometry,LC-MS/MS)与免疫学方法相比具有特异性高、灵敏度高、多组分检测能力等方面的优势,已经成为临床检验领域重要的新技术之一。目前LC-MS/MS主要在激素检测、新生儿筛查、治疗药物监测、维生素D代谢物检测、蛋白质与多肽定量等项目应用比较成熟。虽然LC-MS/MS技术凭借其独特的优势,已经在临床检验领域占得一席之地,但也存在自动化及标准化程度低、仪器操作复杂等不足。自动化、标准化、蛋白质定量、高分辨质谱等将是今后LC-MS/MS在临检应用的主要发展方向。     

LC-MS/MS法和HPLC法检测人血清中利培酮和九羟利培酮血药浓度的对比研究

目的 验证液质联用质谱(LC-MS/MS)法和高效液相色谱(HPLC)法检测人血清中利培酮和九羟利培酮血药浓度结果的可靠性,并分析两种方法检测结果的差异性.方法 对实验室目前在用的LC-MS/MS法和HPLC法检测人血清中利培酮和九羟利培酮血药浓度的方法学进行验证,合格后再分别对该院住院患者的60份血清标本进行相关检测.采用配对t检验、Pearson相关分析、散点图和Bland-Altman偏差图对其检测结果的相关性及差异进行评价.结果 LC-MS/MS法和HPLC法的方法学验证均符合要求,检测结果呈正相关;LC-MS/MS法和HPLC法检测利培酮和九羟利培酮结果比较,差异有统计学意义(P<0.05);LC-MS/MS法检测利培酮与九羟利培酮的结果分别比HPLC法检测结果高1.97和1.38 ng/mL,95％ 置信区间分别为-5.81～9.74、-10.28～11.66.结论 LC-MS/MS法和HPLC法可同时运用于日常检测人血清中利培酮和九羟利培酮血药浓度,检测结果可靠但存在差异,如果是需要长期监测的患者建议一直选用同一种方法检测.     

先天性肾上腺皮质增生症致女性性分化异常8例分析

先天性肾上腺皮质增生症（CAH）是一组常染色体隐陛遗传病,临床少见,是由于某些合成肾上腺皮质类固醇激素所需酶的先天性缺乏,导致类固醇激素分泌异常,且因继发性促肾上腺皮质激素分泌过多而致肾上腺皮质增生。CAH的某些类型可引起性分化异常,如假两性畸形、第二性征缺如、原发性闭经等,是性分化异常的重要病因之一。现报道本院1997年1月至2006年12月收治的CAH患者8例,并以此对CAH中不同类型女性性分化异常的诊断和治疗进行讨论。     

液相色谱-串联质谱检测血浆3-甲氧酪胺方法的建立

目的建立一种稳定的检测血浆3-甲氧酪胺(3-MT)的液相色谱-串联质谱(LC-MS/MS)方法。方法对LC-MS/MS的分离条件(色谱柱、柱温、p H值)进行优化,建立检测血浆3-MT的方法,并对该方法的线性范围、回收率、精密度、最低检测下限和稳定性进行评价。结果以同位素氘代作为内标,采用BEH HILIC色谱柱进行分离。流动相为100 mmol/L甲酸铵缓冲液(p H值为3)和纯乙腈,梯度洗脱;柱温为35℃。LC-MS/MS检测3-MT的线性范围为5~1 000 pg/mL,定量检出限为5 pg/mL,天间和批间变异系数(CV)分别为6%和7%,回收率为97.1%~109.3%。由于3-MT在室温中稳定性较差,因此需冰浴送检。结论建立了检测3-MT的LC-MS/MS方法,能够灵敏、准确地检测血浆3-MT水平。     

液相色谱串联质谱法和循环酶法测定血清中同型半胱氨酸的相关性

目的分析液相色谱串联质谱(LC-MS/MS)法与循环酶法测定人血清中同型半胱氨酸(homocysteine,HCY)浓度的相关性。方法收集63例患者的血清样本,用LC-MS/MS法和循环酶法分别测定相同样本的HCY浓度,并评价2种方法的相关性。结果 LC-MS/MS法与循环酶法对HCY浓度测定的结果分别为(19.11±15.69)μmol/L和(16.95±14.41)μmol/L,差异有统计学意义(t=6.25,P0.05)。2种方法的线性回归方程为YLC-MS/MS法=1.074X循环酶法+0.892,相关系数(R)=0.987,相关性较好。结论 LC-MS/MS法与循环酶法测定血清中HCY浓度的相关性较好,LC-MS/MS法适用于临床对HCY的检测。     

液相色谱质谱联用技术在临床检测和代谢研究中的应用进展

色谱-质谱联用技术[液相色谱-质谱联用(LC-MS)、气相色谱-质谱联用(GC-MS)等]因其灵敏度高、具有保留时间和质荷比双重分离的选择性和特异度、检测动态范围宽等优势,已成为临床代谢物指标检测和代谢组学研究中不可或缺的分析工具。其中液相色谱串联质谱(LC-MS/MS)可对目标代谢物进行准确定性定量分析,能够在单次分析中获得数千种代谢物的信息。该文结合文献报道,综述了液质联用技术在临床样品的代谢物检测和代谢组学研究的应用进展,介绍了多种代谢标志物与疾病相关性和相应的检测方法,讨论了现阶段液质联用技术在临床转化应用中面临的问题,并展望未来发展趋势。     

液相色谱-串联质谱法检测血浆醛固酮和血管紧张素1筛查原发性醛固酮增多症的应用评价

目的 评价液相色谱-串联质谱法（LC-MS/MS）检测血浆醛固酮和血管紧张素1用于原发性醛固酮增多症（PA）筛查的应用效果。方法 建立LC-MS/MS法检测血浆醛固酮和血管紧张素1的检测体系,并进行性能验证。收集疑似PA患者共58例,分别取卧位和立位进行采血检测血浆醛固酮和血管紧张素1,比较LC-MS/MS法与免疫分析法（CLIA）在卧位和立位条件下筛查PA的效能,并进行一致性评价。结果 LC-MS/MS法在性能验证中表现出良好的线性、精密度、准确度和定量下限;LC-MS/MS法与CLIA法的结果呈中等程度正相关性;筛查PA时LC-MS/MS法在卧位和立位条件下的曲线下面积均优于CLIA法,特别是在卧位条件下,LC-MS/MS法的AUC达到了0.85,具有相对较高的准确性。结论 LC-MS/MS法在筛查PA方面可能具有更高的诊断准确性和临床预测价值,但在实际推广中仍需考虑其设备和操作技术的局限性。     

液相色谱串联质谱法检测人血清雌酮、雌二醇

目的建立同时测定人血清中雌酮(E1)及雌二醇(E2)水平的液相色谱串联质谱(LC-MS/MS)法,并初步应用于临床血清样品的雌激素检测。方法血清样品经乙酸乙酯提取,丹磺酰氯衍生化后进行LC-MS/MS分析。用Agilent ZORBAX SBC18色谱柱进行线性梯度洗脱,流动相为乙腈(0.05%甲酸)-水(0.05%甲酸),流速为0.3 m L/min。用多重反应离子监测(MRM)正离子模式对血清中的E1、E2进行质谱检测,并根据"生物样品定量分析方法验证指导原则(中国药典,2015)"对该方法的特异性、线性范围、灵敏度、精密度、提取回收率和稳定性等进行评价。用本法对172例健康成年女性血清标本进行E1、E2检测,用百分位数法计算不同月经周期E1、E2的浓度区间。结果 LC-MS/MS法可同时检测人血清中E1、E2的含量,在0.05~10 nmol/L范围内线性关系良好。该法的定量限为0.05 nmol/L,日内与日间不精密度均小于15%,稳定性良好。初步临床应用显示,用LC-MS/MS法计算所得的E1、E2浓度区间符合女性月经周期生理性变化规律。结论 LC-MS/MS法能有效分离并定量检测人血清中E1、E2的浓度水平,其线性范围广、灵敏度高、精密度好,有望作为临床血清雌激素检测的参考方法。     

串联质谱法测定血清中20种游离氨基酸含量

目的建立串联质谱法(LC-MS/MS)测定血清中20种游离氨基酸含量,为临床血清氨基酸检测提供可靠方法。方法血清样本未经衍生化处理,采用LC-MS/MS法,于电喷雾源(ESI)正离子模式下使用选择反应性离子扫描(SRM)进行定量。结果 20种氨基酸线性相关系数为0.9957～0.9999,日内、日间变异系数分别为0.5%～9.0%和0.6%～12.3%,加样回收率为87.6%～115.5%。样本在常温放置24h,8次冻融循环及-20℃冻存20d等条件下稳定性良好。结论该方法简便、稳定、定量准确,适用于血清氨基酸的临床检测,为氨基酸代谢疾病的临床诊断提供了重要的生化依据。     

Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots. METHODS: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d(8)-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening. RESULTS: Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 microg/L. At a cutoff for 17-OHP of 12.5 microg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified. CONCLUSION: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.     

Determination of 17alpha-hydroxyprogesterone in serum by liquid chromatography-tandem mass spectrometry and immunoassay

17Alpha-hydroxyprogesterone (17OHP) is the most important serum marker for congenital adrenal hyperplasia (CAH). 17OHP is usually measured by immunoassay but its detection by mass spectrometry (MS) is a potentially superior method. An LC-MS (liquid chromatography-mass spectrometry) method was developed which utilizes 0.5 ml serum spiked with 6-alpha-methylprednisolone (6-MP) or deuterated 17OHP (d8-IS) as the internal standard. The samples were extracted with ether/ethylacetate, and the extract was evaporated to dryness and analysed by LC-MS/MS operating in the positive mode after separation on a reversed-phase C18 column. The calibration curves for analysis of serum 17OHP exhibited consistent linearity and reproducibility in the range of 5-250 nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23 nmol/l, respectively. The detection limit was 1 nmol/l (signal-to-noise ratio=3). The mean recovery of 17OHP added to serum ranged from 76 to 89% and that of internal standards from 75 to 82%. The regression equation for the LC-MS/MS (x) and in-house radioimmunoassay (RIA) (y) methods was: y=0.87x+0.26 (r=0.97; n=100) and for a commercial RIA it was: y=1.32x+0.02 (r=0.97; n=26).     

Rapid screening assay of congenital adrenal hyperplasia by measuring 17 alpha-hydroxyprogesterone with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry from dried blood spots

A rapid, simple, and specific method was developed for the diagnosis of congenital adrenal hyperplasia (CAH) from dried blood spots on newborn screening cards based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). The usefulness of 17 alpha-hydroxyprogesterone (17 OH-P) determination on dried filter-paper blood samples from patients with CAH caused by 21-hydroxylase deficiency was evaluated. The LC/MS/MS detection of 17 OH-P was rapid, <4 min. The intra- and interday accuracy and precision of the method were <7%. Our procedure maintained good linearities (R(2) > 0.992) and recovery rate (>83%). We used this new method to directly determine the 17 OH-P levels in dried blood specimens from abnormal children of various ages, with a detection limit of 20 ng/ml (approximately 240 pg), to avoid the time-consuming derivatization steps required by the gas-chromatography/mass spectrometry (GC/MS) method. Four dried filter-paper blood samples of CAH patients (three girls and one boy, 1-14 years old) were all quantified in an LC/MS/MS study and revealed high 17 OH-P levels (>90 ng/ml). After treatment, all of the elevated 17 OH-P levels either decreased or disappeared. Compared with CAH patients, 17 OH-P was nearly undetectable (<20 ng/ml) in the normal infants by LC/MS/MS. This LC/MS/MS assay is not only useful for both diagnosis and monitoring of treatment of CAH in all other age groups, it also can be used as a screening test for CAH infants. In this study, we provided the first data on 17 OH-P in dried blood specimens affected with CAH using HPLC/ESI-MS/MS.     

Plasma estrone sulfate assay in men: Comparison of radioimmunoassay,mass spectrometry coupled to gas chromatography (GC–MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS)

BackgroundEstrogens are involved in the natural history of the prostate cancer and estrone sulfate, the quantitatively main circulating plasma estrogen in men, has been associated with an aggressive form of this cancer. A convenient and accurate plasma assay of this steroid has become important.MethodsWe simultaneously assayed estrone sulfate in the plasma of one hundred men aged 30–50 years, according to LC-MS/MS, GC–MS after solvolysis of E₁S, radioimmunoassay after a chromatographic purification step, and a direct RIA commercial kit.ResultsEstrone sulfate plasma levels obtained with the first three methods were not significantly different. However, estrone sulfate levels measured by the direct RIA were three-fold higher than those obtained by the first three methods. We showed that the excessively high estrone sulfate levels obtained with the direct RIA kit had two origins: interference by high dehydroepiandrosterone sulfate plasma levels in men, and estrone sulfate inaccurate low concentrations in the standards.ConclusionThe LC-MS/MS method can be considered as an optimum option for clinical laboratory. The GC–MS method requires solvolysis to estrone, but allows simultaneous unconjugated steroid measurement. RIA method, with chromatographic purification, is cumbersome, but less expensive. DSL-5400 kit yielded estrone sulfate plasma levels that were too high.     

High-throughput liquid chromatography-tandem mass spectrometric analysis of sirolimus in whole blood.

Sirolimus appears as a new potent immunosuppressive agent taking advantage of therapeutic drug monitoring to optimize its use in organ transplantation. In the absence of any available commercial immunoassay it was mandatory to develop chromatographic assays. Some methods have already been proposed to quantify sirolimus in whole blood, based either on HPLC-UV, liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). We have developed a new faster and simpler LC-MS/MS method to quantify sirolimus in blood using ascomycin as an internal standard and multiple reaction monitoring (MRM) acquisition mode. This method displays a limit of detection of 0.3 microg/l, and the intra-assay reproducibility ranges from 4.1-7.9%. The pre-analytical preparation steps are quite similar to those required for semi-automated immunoassays. Ascomycin and sirolimus present retention times of 0.89 and 0.93 min, respectively, and the turnaround time for a result (2.5 min) is also similar to that observed using a clinical analyzer. The comparison performed between HPLC-UV and LC-MS/MS displays good correlation (r = 0.949). The LC-MS/MS method described above has been used routinely for more than 2000 patient blood specimens and may present several advantages over existing methods, e.g., specificity with sufficient sensitivity, rapidity, and small blood sampling (10 microl), making it particularly adapted for routine clinical use.     

An automated method on analysis of blood steroids using liquid chromatography tandem mass spectrometry: application to population screening for congenital adrenal hyperplasia in newborns

BackgroundNewborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is warranted.MethodsDried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d₈-17-OHP, d₇-androstenedione, and d₄-cortisol. The final extracts were analyzed for 17-OHP, androstenedione and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode.ResultsMean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9–9.9% to 3.6–10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%.ConclusionsThe three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families.     

Quantification of urinary 8-iso-PGF2alpha using liquid chromatography-tandem mass spectrometry and association with elevated troponin levels

OBJECTIVES: Increased lipid peroxidation (i.e. "oxidative stress") has been identified as a central mechanism in the development of atherosclerosis and inflammatory vascular damage. Measurement of 8-iso-PGF(2alpha) has demonstrated to be a reliable indicator of in vivo oxidative stress levels. The purpose of this study was to develop a rapid, sensitive, and specific LC-MS/MS method for detection of urinary 8-iso-PGF(2alpha), establish reference intervals, and correlate isoprostane levels with cardiac troponin I. DESIGN AND METHODS: Urinary 8-iso-PGF(2alpha) was detected after direct injection onto a C18 silica column and monitored in the MRM mode using m/z transitions of 353.2>193.25 (8-iso-PGF(2alpha)) and 357.2>197.25 (8-iso-PGF(2alpha)-d(4)). The LC-MS/MS method was also compared to an ELISA kit. Reference interval studies were evaluated against a separate population of patients presenting with chest pain that had positive cTnI values. RESULTS: Elution of 8-iso-PGF(2alpha) was achieved after 7 min, with a total run time of 10 min. Inter-assay CVs were 13.8-20.0% and intra-assay CVs were 10.9-17.0%. Linearity ranged from 100 pg/mL to 100 ng/mL. Deming regression of ELISA and LC-MS/MS methods for 8-iso-PGF(2alpha) levels yielded poor correlation, with a slope of 0.0265, y-intercept of 0.255 ng/mL, and R(2) value of 0.0434. Urine 8-iso-PGF(2alpha) concentrations in samples obtained from healthy individuals (n=34) ranged from 57 to 390 ng/g creatinine with a mean of 221 ng/g creatinine. 8-iso-PGF(2alpha) levels were statistically significant in troponin-positive (n=35) versus troponin-negative (n=36) patients (p<0.0049). CONCLUSIONS: This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 8-iso-PGF(2alpha) in urine. 8-iso-PGF(2alpha) has potential to be a great prognostic risk indicator in individuals with a high probability for future coronary events.     

Pitfalls in measuring the endocannabinoid 2-arachidonoyl glycerol in biological samples.

BACKGROUND: The endocannabinoid 2-arachidonoyl glycerol (2-AG) undergoes spontaneous isomerization to biologically inactive 1-AG. This effect has not been adequately addressed in previous studies that reported 2-AG concentrations in biological samples. METHODS: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used for 1-AG and 2-AG analyses. RESULTS: Identical collision-induced disintegration spectra were found for 1-AG and 2-AG. For specific detection of both compounds, which share a common mass transition, baseline chromatographic separation is mandatory, even when applying MS/MS technology with its generally high detection specificity. When using standard chromatographic conditions with the very short run times typically used in LC-MS/MS methods, co-elution of 2-AG with 1-AG, which is present in human serum, causes false 2-AG results. CONCLUSIONS: Our data highlight that the analytical specificity of MS/MS can be limited by interference from isobaric isomers with identical disintegration patterns. The specificity of this technology must be carefully evaluated for each individual application.     

Progress in automation of LC-MS in laboratory medicine

Background

LC-MS/MS is an almost universal technology for the quantification of small molecules in human sample materials. The widespread use of this technology in laboratory medicine is so far limited mainly by the extensive occupation of highly trained personnel which is required for method implementation and application. Furthermore, robustness of function and results is still a critical issue of routine quantitative applications of LC-MS/MS.

Content

This article reviews approaches to the automation of essential processes of LC-MS/MS applications in clinical laboratories. Furthermore, perspectives of further steps towards highly robust and fully automated LC-MS/MS methods and instrument configurations are discussed.

Conclusions

There is a variety of efficient approaches to automation of LC-MS/MS methods in use which mainly address sample preparation. Such configurations allow a substantial increase of sample throughput and convenience when compared to standard protocols. However, these applications still have to be implemented for individual methods in heterogeneous instrument configurations and still require highly trained experts. Based on existing technologies, however, the development of fully automated LC-MS/MS front-end modules or MS/MS-based analyzers which offer a degree of user-friendliness and robustness similar to current standard clinical chemistry analyzers seems feasible today. Only such systems will make the entire analytical potential of LC-MS/MS amenable to clinical medicine also outside from tertiary care centres.     

The current role of liquid chromatography-tandem mass spectrometry in therapeutic drug monitoring of immunosuppressant and antiretroviral drugs

Therapeutic drug monitoring of critical dose immunosuppressant drugs is established clinical practice and there are similar good reasons to monitor antiretrovirals. The aim of this article is to review the recent literature (last five years), with particular reference to the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS offers many potential advantages. The superior selectivity of LC-MS/MS over immunoassays for immunosuppressant drugs has been widely reported. Simultaneous measurement of a number of drugs can be performed. It is currently routine practice for the four major immunosuppressants (cyclosporin, tacrolimus, sirolimus and everolimus) to be simultaneously measured in whole blood. While up to 17 antiretroviral drugs have been simultaneously measured in plasma. The exquisite sensitivity of LC-MS/MS also provides the opportunity to measure these drugs in alternative matrices, such as dried blood spots, saliva, peripheral blood mononuclear cells and tissue. However, the clinical utility of measuring these classes of drugs in alternative matrices is still to be determined.