血清尿素同位素稀释气相色谱质谱法的建立和研究

目的 建立一种基于同位素稀释/气相色谱/质谱技术(isotope dilution/gas chromatography/mass spectrometry,ID/GC/MS)的血清尿素候选参考方法.方法 以[13C,15N2]尿素为内标,用无水乙醇沉淀、去除血清中的蛋白类物质,依次使用丙二醛-二甲基缩醛和N-甲基-(三甲基硅烷基)-三氟乙酰胺(MSTFA)将尿素衍生成为三甲基硅烷氧基嘧啶,用气相色谱/质谱(GC/MS)分析衍生产物,以包括法定量.结果 血清尿素测定的批内、批间和总变异系数的平均值分别为0.38%(范围0.12%～0.47%)、0.62%(范围0.49%～0.87%)和0.73%(范围0.51%～0.93%),回收率范围为99.37%～100.95%,分析美国国家标准和技术研究院(NIST)2个水平的血清标准物质SRM 909b,测定结果与靶值的偏差小于0.2%.结论 建立了ID/GC/MS技术测定血清尿素的方法,方法准确、精密、简便,可望作为血清尿素测定的参考方法.     

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.     

同位素稀释气相色谱质谱法测定血清孕酮

目的建立一种血清孕酮测定候选参考方法。方法以[3，4-^13 C2]孕酮为内标，用正己烷提取血清，以羟丙基-β-环糊精水溶液对提取液作净化处理，用七氟丁酸酐对孕酮进行衍生化。用气相色谱分离衍生产物，质谱选择离子监测模式检测孕酮与内标的特定碎片离子，用包括法定量。结果血清孕酮测定的总变异系数为0．69％～2．12％，平均分析回收率为98．3％～100．1％。分析血清孕酮标准物质，测定结果与靶值的偏差小于1．5％。结论建立的同位素稀释气相色谱质谱测定血清孕酮的方法，准确、精密，可望作为测定血清孕酮的参考方法。     

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。     

Precision of glucose measurements in control sera by isotope dilution/mass spectrometry: proposed definitive method compared with a reference method

This improved isotope-dilution gas chromatographic/mass spectrometric (GC/MS) method, in which [13C]glucose is the internal standard, meets the requirements of a Definitive Method. In a first study with five reconstituted lyophilized sera, a nested analysis of variance of GC/MS values indicated considerable among-vial variation. The CV for 32 measurements per serum ranged from 0.5 to 0.9%. However, concentration and uncertainty values (mmol/L per gram of serum) assigned to one serum by the NBS Definitive Method (7.56 +/- 0.28) were practically identical to those obtained with the proposed method (7.57 +/- 0.20). In the second study, we used twice more [13C]glucose diluent to assay four serum pools and two lyophilized sera. The CV ranged from 0.26 to 0.5% for the serum pools and from 0.28 to 0.59% for the lyophilized sera. In comparison, results by the hexokinase/glucose-6-phosphate dehydrogenase reference method agreed within acceptable limits with those by the Definitive Method but tended to be slightly higher (up to 3%) for lyophilized serum samples or slightly lower (up to 2.5%) for serum pools.     

同位素稀释液相色谱串联质谱法测定血清可替宁

目的 建立一种同位素稀释液相色谱串联质谱(ID-LC/MS/MS)测定血清可替宁的方法,以评价不同吸烟状况健康人的吸烟暴露水平分布状况.方法 以[D3]-可替宁作内标,乙腈沉淀蛋白、离心后吸取上清液、氮气挥干、流动相重组,液相分离后进人串联质谱分析;然后,采用多离子反应监测模式,以标准品制作标准曲线,结合同位素内标法定量建立同位素稀释液相色谱串联质谱法.用以对0 68、48.42、94.34、250.95、287.04 μg/L 5个水平血清样本进行5次重复分析,每次每种血清重复分析3份,以考察方法的精密度;同时测定与评价血清样本添加不同浓度标准品的加样回收率和样本在常温、4℃、-80℃保存的稳定性,以及2010年10月至12月期间94名健康志愿者在不同吸烟状况下的血清可替宁分布情况.并用Mann-Whitney U检验分析60名非吸烟、14名戒烟与20名吸烟健康志愿者血清可替宁含量的差异.结果 用ID-LC/MS/MS检测血清可替宁在本试验条件下分离良好,无内源性物质的干扰,具有较好的特异性;血清可替宁、内标峰面积比与可替宁浓度的线性相关系数≥0.9993;测定5个水平血清样本的总变异系数(CV)分别为4.71％、1.40％、1.98％、1.10％和1.03％;批内CV分别为2.19％、0.78％、0.75％、0.65％和0.67％,ID-LC/MS/MS的检测限(LOD)和定量限(LOQ)分别为0.013和0.050 μg/L,具有较高的灵敏度;3次试验的加样回收率范围为99.22％ ～ 102.67％;样本在常温条件下放置2d、4 ℃放置7d以及-80℃冻存保存3个月测定结果的准确度为99.28％～ 100.87％,批间CV均＜5％.检测94名健康人血清可替宁水平呈偏态和尖态分布(偏度2.71,峰度6.65),其中,20名吸烟者的可替宁浓度为116.40 (63.17 ～241.12) μg/L,14名已成烟者为0.67 (0.15～0.95) μg/L,60名非吸烟者为0.22 (0.15 ～0.42) μg/L;已戒烟者(Z=-2.12,P＜0.05)和吸烟者(Z=-6.67,P＜0.001)血清可替宁浓度分别显著高于非吸烟者.结论 建立的ID-LC/MS/MS测定血清可替宁方法操作简便、特异、灵敏,可望在吸烟暴露水平评价及暴露与疾病发生危险的研究中提供有效技术平台.     

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。     

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。     

同位素稀释液相色谱串联质谱法测定血清总胆固醇

目的建立同位素稀释液相色谱串联质谱（LC／MS／MS）测定血清总胆固醇的方法。方法血清样品中加入[3，4-^13 C2]-胆固醇作内标，用氢氧化钠水解血清胆固醇酯为胆固醇，用三氧化铬—硫酸氧化胆固醇为胆甾-4-烯-3，6-二酮，用LC／MS／MS分析胆固醇氧化产物，采用大气压化学电离源，用多反应监测（MRM）模式和选择离子监测（SIR）模式检测，用修正标准曲线法对血清样品进行定量。结果两种检测模式的峰面积比与胆固醇浓度的线性相关系数均大于0．9999。MRM模式测定血清总胆固醇的平均批内变异系数（CV）为0．95％（范围0．92％～0．99％），平均总CV为0．86％（0．82％～0．89％）；SIR模式平均批内CV为0．64％（0．54％～0．77％），平均总CV为0．69％（0．62％～0．81％）。测定国际或国家标准物质结果与靶值的平均偏差在MRM模式下为0．23％（0．14％～1．00％），SIR模式为0．24％（0．07％～1．27％）。结论成功建立了LC／MS／MS测定血清总胆固醇的方法，方法特异、精密、简便，可望用作血清胆固醇测定的参考方法。     

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.     

Determination of steroid hormones in a human-serum reference material by isotope dilution--mass spectrometry: a candidate definitive method for cortisol

We report a method, based on isotope dilution--mass spectrometry, for determining cortisol in a pooled specimen of human serum. Isotopically labeled cortisol is added to 5.0 mL of serum so that the molar concentrations of labeled cortisol and unlabeled cortisol are approximately equal. The specimen and two calibration standards are extracted with dichloromethane, and the extracted cortisol is converted to the methoxime-trimethylsilyl ether derivative. Samples and standards are analyzed by gas chromatography--mass spectrometry by monitoring the peak areas for m/z 605 and 608. The cortisol concentration is calculated by linear interpolation between the two bracketing standards. Variances of data collected during six weeks showed that the overall coefficient of variation (CV) was 0.69% (n = 32); the within-vial CV, 0.63%; the among-vial CV, 0.22%; and the among-day CV, 0.15% (means = 3.973 nmol/vial). Method specificity was demonstrated by liquid chromatographic as well as C8 mini-column cleanup of samples before derivation, by alternative ion monitoring at m/z 636 and 639, and by negative-ion chemical ionization at m/z 459 and 462. Derivatives of all observed degradation products of cortisol under basic, neutral, and acidic conditions did not interfere.     

同位素稀释-超高效液相色谱串联质谱法测定人体血清/全血中维生素B1

目的建立快速、灵敏测定人体血清/全血中维生素B_1的稳定同位素稀释-超高效液相色谱串联质谱(ID-UPLC-MS/MS)法。方法血清/全血样本经蛋白质沉淀处理,上清液用双蒸水稀释,采用Thermo Hypersil GOLD a Q色谱柱分离,以10 mmol/L甲酸铵水溶液~乙腈为流动相,梯度洗脱。采用电喷雾离子源电离,正离子模式下,采用多反应监测(MRM)扫描方式检测,维生素B_1和同位素内标的离子通道分别为m/z 265.2→122.0和269.2→122.0。结果维生素B_1在0.446~89.2 ng/m L范围内线性关系良好,r=0.999 6;低、中、高质控样本的日内、日间精密度的相对标准偏差(RSD)均5.34%;平均加样回收率在96.9%~102.6%,RSD均6.61%。结论建立的方法简单快速、灵敏度好、准确度高,可用于人体血清/全血中维生素B_1的测定。     

Isotope dilution/mass spectrometry of serum cholesterol with [3,4-13C]cholesterol: proposed definitive method

We describe a new gas-chromatographic/mass-spectrometric (GC/MS) isotope-dilution method for determination of serum cholesterol. The method has been fully optimized and documented to provide the high accuracy and precision expected for a Definitive Method. In the presence of [3,4-13C]cholesterol, cholesteryl esters in serum are hydrolyzed under optimum conditions and the entire cholesterol pool is extracted and derivatized to silyl ethers. The cholesterol derivatives are resolved from other sterols by gas-liquid chromatography on a fused silica column, and selected ions characteristic of cholesterol and the [3,4-13C]cholesterol are monitored with a GC/MS quandrupole system. We estimated the cholesterol content of samples by bracketing each sample with standards of comparable cholesterol concentration that also contained the [3,4-13C]cholesterol. The procedure was highly reproducible (CV less than 0.5%), better accuracy and precision being obtained with [3,4-13C]cholesterol than with heptadeuterated cholesterol. Mean values per gram of dry serum for one serum pool assayed by this method and that of the National Bureau of Standards differed by 0.5%. We conclude that the method satisfies the criteria for a Definitive Method.     

Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry

We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of homovanillic acid (HVA), a biochemical marker for catecholamine and neurotransmitter metabolism. Urine specimens are spiked with 5 microg of a stable-isotope labeled internal standard, 13C(6)18O-HVA, and prepared by automated solid phase extraction. Residues were dissolved in acetonitrile: 0.05% aqueous acetic acid and analyzed by MS/MS in the selected reaction monitoring mode (HVA: m/z 181 to m/z 137; 13C(6)18O-HVA: m/z 189 to m/z 145) after separation using a Discovery RP Amide C16 column. Consecutive calibrations (n=7) between 0.52 and 16.7 mg/l exhibited consistent linearity and reproducibility. At a urine concentration of 0.51 mg/l, the signal-to-noise ratio for HVA was 21:1. Inter- and intra-assay CVs ranged from 0.3% to 11.4%, at mean concentrations ranging 1.8 to 22.7 mg/l. Recovery of HVA added to urine ranged between 94.7% and 105% (1.25 mg/l added), 92.0% and 102% (5.0 mg/l), and 96.0% and 104% (10 mg/l). LC-MS/MS is well suited to replace an HPLC method for routine HVA determination, by providing positive identification, faster turn around time, virtually no repeat analyses and a 44% reduction of personnel necessary to perform the testing.     

Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry.

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d(3)-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02+/-1.84 micromol/l (mean+/-S.D.) in adults, 3.91+/-0.76 micromol/l in children age 5-10 years and 11.57, 15.16, 14.36 micromol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7+/-15.25 micromol/l in adults, 58.96+/-22.30 micromol/l in children and 5.37, 8.15, 403.5 micromol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83+/-1.43 and 5.04+/-1.84 micromol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.