同位素稀释气相色谱质谱法测定血清总胆固醇

目的建立一种新的同位素稀释-气相色谱-质谱（ID／GC／MS）测定人血清总胆固醇的方法。方法取一定量的血清样品与[3，4-^13 C2]-胆固醇内标溶液充分混匀，水解并提取溶液中的胆固醇后，用N，O-二-（甲基硅烷）三氟乙酰胺（BSTFA）衍生胆固醇为三甲基硅烷醚；气相色谱．四极杆质谱（GC/MS）选择检测目标离子。检测标准溶液和血清样品的m／z368和m／z370的离子强度并积分，校正标准溶液中天然同位素胆固醇对内标的影响，将校正后的胆固醇和内标的m／z368和m／z370面积比对胆固醇标准溶液浓度做线性回归。用拟合的回归方程定量血清样品胆固醇浓度。结果建立的ID／GC/MS测定胆固醇的方法平均批内变异系数0，04％～0．81％。分析美国国家标准物质与技术研究所（NIST）2个浓度水平的标准血清SRM1951a，相对偏差分别是0．19％和0．90％。结论建立的ID／GC/MS测定胆固醇的方法操作简单、精密，可以用做血清胆固醇的准确定量。     

同位素稀释液相色谱串联质谱法测定血清肌酐

目的 建立一种使用同位素稀释液相色谱串联质谱法(ID-LC/MS/MS)测定血清肌酐的候选参考方法.方法 以[2H3]肌酐为内标,用无水乙醇沉淀血清中的蛋白质,用氯仿纯化上清液,用液相色谱串联质谱分离测定,以包括法定量.结果 血清肌酐测定的批内、批间和总变异系数的平均值分别为0.57%(范围0.52%～0.61%)、0.43%(范围0.11%～0.59%)和0.73%(范围0.62%～0.83%).加样回收试验的回收率范围为99.09%～101.13%.分析两种参考物质SRM909b和SRM 967b,测定结果与认定值的偏差小于0.4%.结论 建立了ID-LC/MS/MS法测定血清肌酐的方法,方法准确、精密、简便,可望作为血清肌酐测定的参考方法.     

同位素稀释液相色谱串联质谱法测定血清总甘油

目的 建立同位素稀释液相色谱串联质谱测定血清总甘油的方法.方法 以[13C3]-甘油作内标,用氢氧化钾异丙醇溶液水解血清甘油酯为游离甘油,将游离甘油转化为苯甲酸酯,用同位素稀释液相色谱串联质谱(LC/MS/MS)分离检测,用标准曲线法定量.结果 甘油/内标峰面积比与甘油浓度(0.565～4.517 mmol/L)线性相关系数大于0.999 9;测定不同浓度血清总甘油批内变异系数(CV)平均为0.52%(范围0.21%～2.62%),总CV平均为1.15%(范围0.62%～2.00%);分析国际和国家标准物质,测定值与认证值的偏倚小于1%(-0.20%～1.06%).结论 建立同位素稀释液相色谱串联质谱测定血清总甘油方法,方法特异、精密、准确,可望用作血清总甘油测定参考方法.     

同位素稀释液相色谱串联质谱法测定血清睾酮

目的建立一种用同位素稀释液相色谱串联质谱法(ID-LC/MS/MS)测定血清睾酮的候选参考方法。方法以[16,17,17-d3]睾酮为内标,用重量法准确地与血清混合,用乙酸乙酯-正己烷混合溶剂提取,以羟丙基-β-环糊精水溶液对提取液作净化处理,用液相色谱串联质谱分析,质谱选择离子监测模式检测睾酮与内标的特定碎片离子,用包括法定量。结果血清睾酮测定的批内、批间和总变异系数(CV)的均值(范围)为0.84%(0.22%~2.00%)、1.01%(0.48%~2.37%)和1.37%(0.53%~3.09%)。参考物质ERM DA-345a和NIST SRM 971测定结果与认定值的平均偏差范围为-2.0%~+1.8%。结论用ID-LC/MS/MS建立了血清睾酮的测定方法,方法准确、精密、简便,有望作为血清睾酮测定的参考方法。     

衍生化气相色谱法测定冠心病患者血清中游离脂肪酸的方法学研究

目的建立同时测定冠心病患者血清中11种游离脂肪酸(FFA)含量的测定方法。方法采用N,O-双(三甲基硅烷基)三氟乙酰胺(BSTFA)对11种FFA进行硅烷化反应,并利用气相色谱质谱联用技术(GC/MS)同时测定冠心病患者血清冻干粉中11种FFA的含量。结果 11种FFA色谱峰完全分离,峰形良好,血清中杂质对测定不产生干扰。在测定浓度范围内线性关系良好,11种FFA的回收率均75%,日内、日间精密度和重复性实验的相对标准偏差(RSD)均10%。结论该方法具有操作简单、灵敏度高、准确性好、线性范围宽等优点,可应用于临床实际血清样品中FFA的分析检测。     

同位素稀释液相色谱串联质谱法测定血清葡萄糖

目的 建立一种使用同位素稀释液相色谱串联质谱法(ID-LC/MS/MS)测定血清葡萄糖的候选参考方法.方法 以[~(13)C_6]葡萄糖为内标,用重量法准确地与血清混合,除去蛋白后在碱性条件下与1-苯基-3-甲基-5-吡唑酮反应,用LC/MS/MS测定葡萄糖和内标衍生产物,以包括法定量.结果 血清葡萄糖测定的批内、批间和总变异系数的平均值分别为0.36%(范围0.28%-0.42%)、0.47%(范围0.20%-0.67%)和0.61%(范围0.42%-0.76%).加样回收试验的回收率范围为99.0%-100.9%.分析参考物质SRM 965a,测定结果与认定值的平均偏差为-0.20%(范围-0.39%-+0.11%).结论 建立了ID-LC/MS/MS法测定血清葡萄糖的方法,方法准确、精密、简便,可望作为血清葡萄糖测定的参考方法.     

血清醛固酮候选参考测量程序的建立及性能评估

目的建立基于同位素稀释液相色谱-串联质谱(ID-LC-MS/MS)的血清醛固酮候选参考方法。方法以甲基叔丁基醚为萃取剂,采用液液萃取方式进行样本前处理。以醛固酮-2H7为内标,采用电喷雾离子源(ESI)负离子模式建立检测血清醛固酮的ID-LC-MS/MS方法,并对方法的准确度、精密度、灵敏度、线性等基本分析性能进行验证。结果 ID-LC-MS/MS测定血清醛固酮的线性范围为0.08～3.55 nmol/L,检测限和定量限(LOQ)分别为0.012 nmol/L和0.045 nmol/L。批内和批间变异系数(CV)分别为5.0%和4.1%。平均加标回收率为98.52%～100.14%。测定国际临床化学和检验医学联合会参考实验室外部质量评价计划(RELA)样本的结果偏移1.6%。结论建立的测定血清醛固酮的ID-LC-MS/MS方法准确、精密,有望作为测定血清醛固酮的参考方法。     

同位素稀释液相色谱串联质谱法测定人血清尿酸

目的建立一种使用同位素稀释液相色谱串联质谱（ID-LC/MS/MS）测定人血清尿酸的候选参考方法。方法用[1,3]1-5N2尿酸作内标,与一定量的血清充分混匀,加入等量乙腈沉淀血清蛋白并提取尿酸,氯仿纯化上清液后用液相色谱串联质谱（LC/MS/MS）分离测定。优化LC/MS/MS的色谱条件,评价该法的精密度、回收率和偏倚,并计算不确定度。结果对3个浓度水平的室间质量评价用质控血清测定3批,批内、批间和总变异系数（CV）的均值（范围）分别为0.54%（0.44%-0.69%）、0.66%（0.45%-0.79%）和0.86%（0.66%-1.02%）。加样回收试验回收率范围为98.38%-102.64%。美国国家标准与技术研究所（NIST）标准物质SRM 909b-Ⅰ、Ⅱ的测定均值与靶值的偏倚分别为0.04%和0.25%。3种质控血清的相对扩展不确定度分别为1.80%、2.19%和2.04%。结论建立的ID-LC/MS/MS测定血清尿酸的方法精密、准确,有望作为血清尿酸测定的参考方法。     

气-质联用技术测定尿有机酸方法的建立及在遗传代谢病诊断中的应用

目的 利用气-质联用技术测定尿液中有机酸种类及含量的变化,为先天性遗传代谢病,特别是有机酸代谢紊乱提供新的诊断依据.方法 收集临床高度怀疑为有机酸代谢紊乱的195例患儿的尿液标本,根据肌酐含量取相应体积的尿样和内标,加盐酸羟胺对带羟基的有机酸进行圬化反应生成酮体后,用乙酸乙酯和乙醚萃取有机酸,三甲基硅烷衍生;测定采用Agilent GC/MS 6890/5973i气相质谱仪,选择扫描模式检测,测定质荷比(m/z)范围在50～550 m/z的所有有机酸,数据处理采用Agilent GCMSD ChemStationGl701DA软件.用健康对照样本中加人的内标和阳性对照样本中加入的阳性有机酸进行分析方法的线性范围、精密度、准确性、样本回收率和残留分析.结果 该方法可测定尿液中100余种有机酸,以健康对照者尿样中加入的二甲基丙二酸(MMA)和2-苯基丁酸(2-PA)内标为参考,最低检测极限为2.5～2.8μmol/L;批内和批间变异系数均<10%,样本前处理批间变异系数为11.4%;样品回收率为95%～105%,残留分析<1%,所有参数均符合临床检测的要求.用该方法检测的临床患儿尿样中,发现诊断阳性病例12例,包括6例甲基丙二酸血症、1例丙酸血症、3例酪氨酸血症Ⅰ型、1例枫糖尿病和1例Ketosis病(酮症病).结论本研究建立了气-质联用技术分析测定尿液中有机酸的方法,可用于先天性遗传代谢性疾病的筛查诊断.     

同位素稀释液相色谱-串联质谱法检测血清睾酮候选参考方法的建立及应用

目的建立基于液相色谱串联质谱(LC-MS/MS)技术的血清睾酮候选参考方法,并采用该方法对临床常规检测方法(微粒子化学发光法)的正确性进行评价。方法采用睾酮-2,3,4-~(13)C_3作为内标,以等体积比的正己烷-乙酸乙酯溶液对样本进行液液萃取,上清液采用氮气吹干后用流动相复溶,进行LC-MS/MS分析。采用五点包括法计算血清睾酮浓度。参考美国临床实验室标准化协会(CLSI)C62-A文件和EP15-A3文件对建立的同位素稀释液相色谱-串联质谱(ID-LC-MS/MS)方法进行性能评价(方法残留、特异性、线性范围、精密度、准确度),参考CLSI EP9-A3文件对微粒子化学发光法的正确性进行评价。结果建立的ID-LC-MS/MS方法检测血清睾酮的分析时间为5min,特异性好,无残留,线性范围为0.034～22.00ng/mL,批内不精密度≤2.3%,批间不精密度≤2.2%,测定有证参考物质SRM971(Level male)的相对偏移为0.2%,测定2017 RELA-A、2017 RELA-B样本的相对偏移分别为-2.9%、-3.3%,测定2017 RELA-A样本的不确定度为0.11 ng/mL。微粒子化学发光法与ID-LC-MS/MS的相关性较好(r=0.963),但偏差较大[总平均偏差为-24.4%,低浓度样本(≤1 ng/mL)平均偏差为-62.4%,高浓度样本(1 ng/mL)平均偏差为6.3%]。结论成功建立了检测血清睾酮的ID-LC-MS/MS候选参考方法。该方法的精密度、准确度均较好,且操作简单,分析时间短。     

Measurement of urea in human serum by isotope dilution mass spectrometry: a reference procedure.

BACKGROUND: A reference measurement procedure is needed to demonstrate the traceability of results of urea measurements in human serum. We developed a measurement procedure using the principle of isotope dilution gas chromatography/mass spectrometry. METHODS: [(13)C,(15)N(2)]Urea as internal standard was added to a serum sample and equilibrated with endogenous nonlabeled urea. For the preparation of calibrators, the same amount of labeled urea was mixed with known amounts of nonlabeled urea. The serum samples were treated with ethanol to remove proteins by precipitation. The labeled and nonlabeled urea of the samples was converted into a trimethylsilyl derivative of 2-hydroxypyrimidine. The gas chromatography/mass spectrometry system was adjusted to monitor m/z 153 and 168 for the nonlabeled urea derivative and m/z 156 and 171 for the isotopically labeled analogs. The results of the determination were calculated from peak ratios by a hyperbolic calculation function based on the theory of isotope dilution analysis. RESULTS: The procedure was applied to control samples and patient samples and evaluated with respect to its trueness and precision. The standard uncertainty of the results was 0.47-1.72%. CONCLUSIONS:This reference measurement procedure allows values to be assigned to controls and calibrators that are traceable to the primary urea reference material of NIST and, therefore, to the Système International unit "mole" with a low degree of uncertainty. This procedure provides a tool for the highly accurate determination of urea in control materials as well as in patient sera.     

高效液相色谱测定血清甘油三酯的研究——一种参考方法的建议

目的　建立特异、精密的血清总甘油和游离甘油测定方法 ,以便甘油三酯测定标准化。方法　以 1,2 ,4 丁三醇作内标 ,用柱前衍生高效液相色谱技术分析血清皂化前后的甘油含量 (两者分别代表血清游离甘油和总甘油浓度 )。结果　测定总甘油相对不精密度小于 2 % ,游离甘油小于 4% ;总甘油平均回收率 10 0 0 % ,游离甘油 99 7% ,与同位素稀释 /质谱法的相对偏差不大于± 2 %。结论方法特异、精密、简单 ,可望用作血清甘油三酯测定参考方法。     

Gas-chromatographic determination of cholesterol in serum: candidate reference method

We present a candidate Reference Method for determination of total cholesterol in serum. The method is based on high-resolution capillary gas chromatography, and we took special precautions with respect to weighing, calibration, and chromatographic peak integration. Analytical recovery of added cholesterol was essentially complete (99.99%, SD 0.48%) and reproducibility was excellent (total CV 0.35-0.50%). When cholesterol was determined in a reference serum certified by means of an established candidate Definitive Method, no significant bias could be detected.     

Determination of serum triglycerides by capillary on-column gas chromatography

We report an accurate method, based on capillary on-column gas chromatography, for determining triglycerides in human serum. After serum extraction and chemical hydrolysis, glycerol is directly measured by gas chromatography (GC). With our extraction method no free glycerol is extracted from serum. The accuracy of this method was compared with that of a method based on the original procedure of Carlson (J. Atheroscler. Res. 3, 334-336 (1963)) and which is standardized by the Centers for Disease Control (CDC, Atlanta, U.S.A.). Orthogonal regression analysis of the GC method (y) and the CDC reference method (X) resulted in y = 0.996 X + 0.000 with a correlation coefficient of 0.999. The variances of analytical data, collected over a two month period showed that, for the GC method, the within-day coefficient of variation (CV) was less than 1.43%; the between-day CV less than 1.35%. The data for the CDC method were less than 3.36% (within-day) and less than 6.38% (between-days). The CDC method is linear up to 3.7 mmol/l and the GC method to 22 mmol/l.     

Pilot study for the standardization of insulin immunoassays with isotope dilution liquid chromatography/tandem mass spectrometry

BACKGROUND: An international working group convened by the American Diabetes Association (ADA) called for a reference measurement procedure for use in a trueness-based standardization project of insulin immunoassays. In view of this demand, we conducted a pilot study to investigate the feasibility of such a standardization project with our isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure. METHODS: We evaluated the precision, accuracy, and limit of quantification (LoQ) of the ID-LC/tandem MS procedure by use of insulin-free serum supplemented with insulin to give 3 pools with concentrations of 0.0796, 0.769, and 5.56 microg/L. We conducted a pilot method comparison study with 4 immunoassays and 80 samples from fasting and glucose-stimulated patients. RESULTS: The within-run and total imprecision (CV) ranged from 3.2% to 6.3% and from 4.9% to 12.1% (listing sequence from the high to the low pool). The recovery from supplemented insulin-free sera ranged from 101.8% to 104.1%, and the LoQ was 0.07 microg/L (12 pmol/L). Weighted Deming regression and correlation analysis of the method-comparison data showed considerable between-assay variation for the immunoassays but, with the exception of one assay, excellent correlation with ID-LC/tandem MS. Recalibration of the immunoassay results considerably reduced the between-assay variation. Moreover, after recalibration, 3 of the 4 assays fulfilled the total error specification of 32% proposed by the ADA Workgroup. CONCLUSIONS: Recalibration of insulin assays by regression equations established from method comparison with ID-LC/tandem MS can result in successful standardization and fulfillment of the total error criterion proposed by the ADA Workgroup.     

Emergency toxicology testing (detection, confirmation, and quantification) of basic drugs in serum by liquid chromatography with photodiode array detection.

We investigated the applicability of liquid chromatography with photodiode array detection as a serum drug screening technique in an emergency toxicology setting. Basic compounds are extracted from alkalinized serum with hexane and chromatographed on a cyanopropyl reversed-phase column. The photodiode array detector records the ultraviolet spectrum of each eluting peak for identification and quantification. More than 30 drugs/metabolites including antidepressants, antihistamines, phenothiazines, and analgesics are detected at their therapeutic concentrations or less. Quantitative run-to-run precision (CV) for antidepressant drugs at low therapeutic concentrations is less than 6%. We evaluated the potential for interference of greater than 140 compounds of clinical interest. In a split-sample study, 151 of 154 positive drug findings by the method were confirmed by alternative techniques (gas chromatography/mass spectrometry and liquid chromatography with either normal-phase or reversed-phase columns). In the three unconfirmed findings, this method detected drugs at concentrations below the limit of identification of the comparison method. Also, 275 samples judged negative by this procedure were negative by a different liquid-chromatographic method.