为了满足该标准物质的需求,进行了第二次尿酸血清标准物质的复制.针对复制的尿酸血清标准物质,基于单四极杆质谱的液相色谱-同位素稀释质谱法(LC-IDMS),用乙腈沉淀法去除蛋白质,BEH C18色谱柱和电喷雾离子源负离子模式(ESI-),同位素稀释的单点校准法进行定值、均匀性检验、稳定性检验以及不确定度评定等研究.此定值方法经过CCQM-K109(血清中尿酸分析)国际关键比对进行验证.复制的2种不同浓度水平尿酸(肾病患者和正常人)血清标准物质的定值结果分别为(73.5±1.3)μg/g,(47.5±1.1)μg/g,其均匀性和稳定性评估结果良好.

C-reactive protein (CRP) is one of the most commonly used biomarkers for inflammation. The standardisation of a procedure for the detection of CRP has attracted significant attention globally, and primary reference materials of CRP based on the recombinant expression of E. coli that exist in the form of monomers have been developed. However, a primary reference material of natural CRP is still required to achieve the exact matching of CRP measurements in secondary reference materials (e.g. CRP in frozen human serum). Herein, the development process for a certified reference material of natural CRP is reported, namely GBW09228. The raw material employed in this study was CRP extracted and purified from human body fluid, and exhibits a natural and verified pentameric structure. Through the use of amino acid analysis isotope dilution mass spectrometry (AAA-IDMS) and signature peptide-IDMS, this reference material was certified, and its certification results can be traced to SI units. The developed method was evaluated for its accuracy using the international comparison tests of the National Metrology Institute of Japan (NMIJ) and the Korea Research Institute of Standards and Science (KRISS). Overall, a CRP primary certified reference material (CRM) of well-characterised purity was determined that could be used to calibrate an IDMS-based reference method, that could then be used to assign target values to secondary CRMs. These secondary CRMs could in turn be used to calibrate and verify the accuracy of immunoassays, thereby giving a good foundation for establishing a complete traceability chain for CRP.

Due to the widespread use of synthetic peptide drugs, their quantification and the analysis of impurities have become increasingly important in clinical and medical settings. Moreover, quantifying proteins using synthetic peptides as internal or external standards is a general approach, and the key to this approach is the knowing purities of the peptides. In this paper, synthetic glucagon was quantified using a mass balance method. The impurities in glucagon were analyzed and then accurately quantified separately. Karl Fischer (KF) titration and ion chromatography (IC) were used to determine the water and trifluoroacetic acid (TFA) contents in the samples, respectively. Furthermore, the inorganic ion content in the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS). The sequence of peptide impurities was identified by a Thermo Fisher Orbitrap mass. Samples were determined to be 896.36 ± 0.68 mg/g after subtracting all impurity masses from the sample mass. The result can be traced to SI units.

介绍甲醇中五氯硝基苯溶液标准物质的研制方法.以五氯硝基苯农药纯度标准物质为原料,采用重量–容量法制备溶液标准物质,以液相色谱法(DAD检测器)对所制备的标准物质进行均匀性和稳定性检验,并对该标准物质定值结果的不确定度进行评定.结果表明,该标准物质均匀性良好,在12个月内量值没有显著性变化,具有良好的稳定性,标准值为0.100 mg/mL,扩展不确定度为1.8％(k=2).该标准物质可用于食品和环境样品中五氯硝基苯分析方法的确认与评价.

尿液样品离心后经WATERS C18固相萃取柱净化,收集洗脱液.采用二维超高效液相色谱-串联质谱法测定样品溶液中8-羟基脱氧鸟苷的含量.以Waters BEH C18色谱柱(一维色谱)和Waters BEH HILIC色谱柱(二维色谱)为固定相,以0.1％(体积分数,下同)甲酸溶液与乙腈(体积比8:2)组成的混合液(一维色谱)和0.1％甲酸溶液与乙腈(体积比9:1)组成的混合液(二维色谱)为流动相,串联质谱分析中采用电喷雾离子源正离子模式和多反应监测模式.采用内标法定量.8-羟基脱氧鸟苷的线性范围为0.25～300.0 ng·g-1,检出限(3S/N)为0.10 ng·g-1.以空白样品为基体进行加标回收试验,所得回收率为96.6％～103％,测定值的日内相对标准偏差(n=6)和日间相对标准偏差(n=4)依次为0.87％～3.3％,1.9％～4.9％.

The CCQM Organic Analysis Working Group (OAWG) agreed on a Track A comparison for the measurement of polar organic in biological matrix as part of its 10-year strategic plan. As a model for this comparison, two polar clinical biomarkers: urea and uric acid, in human serum were chosen. This comparison was designed to enable participating National Metrology Institutes (NMIs) or Designated Institutes (DIs) to demonstrate their measurement capabilities in the determination of analytes with molecular mass of 50 to 500 g/mol, having the polarity pKOW > 2 in the range of 10 to 2,000 mg/kg in a biological matrix such as human serum, blood and urine. Two pools of human serum materials with different concentration levels of urea and uric acid were used as the comparison materials. Fifteen NMIs/DIs participated in this Track A comparison. All of them submitted the results for urea and 14 NMIs/DIs submitted the results for uric acid. With the exception of one NMI, all participating institutes employed isotope dilution mass spectrometry (IDMS) for the measurement of both urea and uric acid. Protein precipitation, liquid-liquid extraction and/or clean-up were applied, followed by instrumental analyses using GC-HRMS, GC-MS/MS, GC-MS, LC-HRMS, LC-MS/MS, LC-MS or HPLC-DAD. The Laplacian weighted medians were used as the Key Comparison Reference Values (KCRVs). The assigned KCRVs were the weighted medians of 13 results for urea (both serum pools), 10 results for uric acid (Serum I) and 11 results for uric acid (Serum II). Urea (Serum I) was assigned a KCRV of 1,486.0 mg/kg with a standard uncertainty of 9.0 mg/kg, urea (Serum II) was assigned a KCRV of 334.7 mg/kg with a standard uncertainty of 1.8 mg/kg, uric acid (Serum I) was assigned a KCRV of 136.50 mg/kg with a standard uncertainty of 0.98 mg/kg, and uric acid (Serum II) was assigned a KCRV of 39.39 mg/kg with a standard uncertainty of 0.11 mg/kg. The degree of equivalence (with the KCRV) and its uncertainty were calculated for each result. The majority of the participating institutes in CCQM-K109 demonstrated their capabilities in the measurement of the clinical markers in the biological matrix using IDMS. KEY WORDS FOR SEARCH Clinical biomarkers; urea; uric acid; human serum, IDMS Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described.

Glucose and creatinine are two of the most frequently measured substances in human blood/serum for assessing the health status of individuals. Because of their clinical significance, CCQM-K11 glucose in human serum and CCQM-K12 creatinine in human serum were the fourth and fifth key comparisons (KCs) performed by the Organic Analysis Working Group (OAWG). These KCs were conducted in parallel and were completed in 2001. The initial subsequent KCs for glucose, CCQM-K11.1, and creatinine, CCQM-K12.1, were completed in 2005. Measurements for the next KCs for these two measurands, CCQM-K11.2 and CCQM-K12.2, were completed in 2013. While designed as subsequent KCs, systematic discordances between the participants' and the anchor institution's results in both comparisons lead the OAWG to request reference results from two experienced laboratories that had participated in the 2001 comparisons. Based on the totality of the available information, the OAWG converted both CCQM-K11.2 and CCQM-K12.2 to 'Track C' KCs where the key comparison reference value is estimated by consensus. These comparisons highlighted that carrying out comparisons for complex chemical measurements and expecting to be able to treat them under the approaches used for formal CIPM subsequent comparisons is not an appropriate strategy. The approach used here is a compromise to gain the best value from the comparison; it is not an approach that will be used in the future. Instead, the OAWG will focus on Track A and Track C comparisons that are treated as stand-alone entities. Participation in CCQM-K11.2 demonstrates a laboratory's capabilities to measure a polar (pKow > 2), low molecular mass (100 g/mol to 500 g/mol) metabolite in human serum at relatively high concentrations (0.1 mg/g to 10 mg/g). Participation in CCQM-K12.2 demonstrates capabilities to measure similar classes of metabolites at relatively low concentrations (1 μg/g to 30 μg/g). The capabilities required for the analysis of complex biological matrices include sample preparation (protein precipitation, extraction, derivatization), gas chromatographic (GC) or liquid chromatographic (LC) separation, and quantification using an isotope dilution mass spectrometry (IDMS) approach. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

采用免疫磁珠富集法,通过结合C反应蛋白单克隆抗体磁珠将血清中C反应蛋白提纯浓缩,用蛋白酶切质谱法对提纯物进行测定.同时对C反应蛋白酶切过程进行优化,提高酶切后肽图覆盖率至86％,选取特征肽段为ESDTSYVSLK.通过质谱定量特征肽段的浓度,得到血清中C反应蛋白的浓度为2.06μg/g.本实验建立血清中C反应蛋白的定量方法,血清中C反应蛋白的质谱定量测定奠定了基础.

The method to determine uric acid and urea in serum was established by single quadrupole-based isotope dilution ultra performance liquid chromatography-mass spectrometer, which was used for CCQM-K and P international comparison. Acetonitrile was added in the sample for protein precipitation, a C18 reversed-phase column and electrospray ionization (ESI) negative ion mode was used to determine uric acid, and CN normal-phase column and ESI positive ion mode were used to determine urea, and a single point calibration method for quantitative isotope dilution. This method was verified with GBW 09157, GBW 09169 and NIST SRM 909c certified reference materials, and serum samples (Ⅰ,Ⅱ) were measured to obtain international equivalent consistency. The method is simple in operation, rapid and accurate, it is suitable for quantitative analysis of urea in serum uric acid.

Compared with small molecules ,it's difficult to achieve accurate quantifica-tion of protein biomarkers because of their high molecular weight ,complex structure and unstable nature . Due to that , alpha-fetoprotein (AFP ) reference measurement procedure and high grade standard material has not been established yet .For protein macromolecular compounds ,it is usually needed to split the protein into amino acids or peptide segments then use isotope dilution mass spectrometry to achieve accurate quanti-fication .The method using isotope labeled amino acid as internal standard is relatively mature ,but it is only suitable for high purity samples ,otherwise it will introduce large errors due to the contamination of exogenous substances containing quantitative amino acids .The method using isotope labeled signature peptide as internal standard is more accurate and reliable .Therefore ,a method for the determination of AFP by liquid chro-matography-isotope dilution mass spectrometry based on signature peptides was devel-oped .As a potential value assay method for AFP pure certified reference material (CRM) ,three isotope labeled peptides of AFP were selected as internal standard ,which were added into the digested AFP samples .The samples were separated by Phenome-nexKinetex 2.6 μm C18 column ,and measured by tandem mass spectrometer equipped with an electrospray ionization source operated in multiple reaction monitoring (MRM) mode . The optimal digestion condition and efficiency of enzymatic hydrolysis were investigated and the determination of uncertainty was calculated and analyzed .The con-tent of the AFP reference material was measured to be (0.329 ± 0.016) mg/g .The selection of the signature peptides and the optimization of the digestion conditions were key to the experiment . T he signature peptides should have high and reproducible response ,no missed cleavages and suitable sequence length . The optimization of the digestion conditions need repeated experiments based on a large number of literatures .

We developed and evaluated a candidate serum reference material to help improve clinical routine measurement, and to provide traceability of the measurement results. D8-Homocystine, dithiothreitol, and acetonitrile were used as an internal standard, the reducing agent, and the protein precipitating agent, respectively. A triple quadrupole mass spectrometer with an electrospray ionization source was used for monitoring the transitions (m/z 140.0 → 94.0, 136.0 → 90.0) in multiple-reaction-monitoring mode. We used a calibration model relying on bracketing and gravimetric measurements to give SI-traceability and higher accuracy to serum value assignments. The method was evaluated for accuracy using NIST Standard Reference Material SRM1955. The results of the three concentrations (1, 2, and 3) of total homocysteine in human serum samples were determined by an isotope-dilution liquid chromatography-tandem mass spectrometry method; tHcy 1 is 28.8 ± 1.1 μmol/L, tHcy 2 is 17.93 ± 0.57 μmol/L, and tHcy 3 is 14.38 ± 0.46 μmol/L.

本论文在质检公益性行业科研专项项目的资助下 ,建立同位素稀释质谱技术开展国际比对 ,研制血清葡萄糖国家标准物质 ,并应用于参考测量实验室的血糖能力评估 ,验证我国参考测量实验室血糖测量能力 ,为参考测量程序提供可溯源性 ,报道如下.

糖类是生命活动主要的能量来源，是生命体的基本原料和主要结构成分。糖分析涉及食品标签、饮料、营养等方面；据JCTLM统计，血糖在日常临床检测中排第二位。血糖检测是糖尿病管理中的重要组成部分；血糖检测关键影响因素包括仪器厂家、样本基质、样品处理以及环境等；量值溯源是检测结果准确的重要保证。欧盟对体外诊断器具要求厂家校准物要向更高等级标准物质或参考方法溯源。

The development of high concentration of serum 17β-estradiol reference material was introduced. The serum standard samples were isolated from collected blood by certain conditions before packing,the checking of uniformity,stability test and certified value were systematically studied by ID–LC–MS/MS. The uncertainty of determination was evaluated. The results showed that the certified value of the reference material of high concentration of serum 17β-estradiol reference material was 3.06 ng/g with an relative expanded uncertainty of 0.08 ng/g (k=2). The uniformity and stability of the reference material of serum 17β-estradiol could meet the technical requirements of the standard reference material. IFCC RELA sample and European Commission CRM were used for confirmation of dermination method, and the standard deviation of analysis result was in the range of uncertainty. The 17β-estradiol reference material value was proven by the Metrology Institute of Japan, and the detection result was (3.03±0.1) ng/g. The quantity value of the reference material is traceable and accuracy.

医学诊断及其治疗都依赖于临床检测指标的准确可靠。医学实验室的结果应可溯源至较高级别的参考物质和／或参考测量程序［1‐2］。参考实验室是指运行参考测量程序，提供测量结果不确定度符合特定要求的测量结果的实验室［1］。按国际检验医学溯源性联合委员会（JCTLM ）要求，参考实验室需符合以下3个条件［3‐4］：（1）采用JCTLM 公布的参考测量程序；（2）通过ISO17025与ISO15195认可；（3）定期参加参考实验室室间比对计划。

The advances in the research for the quantitative method of alpha fetoprotein ( AFP ) home and abroadly in recent years are reviewed , including immunosensor and isotope dilution mass spectrometry , which are applied on the AFP quantitation and standardization .The absence of absolute quantitative method of AFP is discussed , and the significance of certified reference material , traceability and establishing a standard quantitative method of AFP is described .

建立了C-反应蛋白的液相色谱-同位素稀释串联质谱检测方法.作为C-反应蛋白纯品标准物质的潜在定值方法,选取同位素标记亮氨酸和缬氨酸为内标,重量法与待测样品准确混合.利用Zorbax SB-Aq色谱柱分离,电喷雾三重四极杆串联质谱多反应监测模式(MRM)测定.对样品的测定不确定度进行计算与分析,测得结果为(41.5±0.9) mmol/kg.利用该方法参加了基于中、日、韩三国计量院合作框架下的国际比对,并取得了比对结果的等效一致.

Objective To realize the recombination clone ,heterologic high‐efficiency expression and protein purification of human‐source alpha‐fetoprotein(AFP)and to obtain the high purity of AFP with clinical bioactivity by the pPICZaA pichia expression systems .Methods The human recombinant expression plasmid pPICZαA‐AFP was generated by PCR from pReceiver‐AFP ,which AFP sequence was in accordance with GenBank .The AFP protein was expressed by methanol induction in pichia GS115 ,and purified by ammonium sulfate precipitation .The expression and purification of AFP was evaluated by SDS‐PAGE ,western blotting and HPLC ,and the value of AFP was quantified by chemiluminescence immunoassay in clinical hospital .Results Recombinant human AFP expressed in the pichia GS115 was mainly secreted into the extracellular medium supernatant ,and also contained the immunogenicity .87% of AFP was in 55% ammonium sulfate precipitate after ammonium sulfate fractionation ,which purity was 98 .844% and value was 3 .473 ng/mL .Recombinant human AFP showed the same size of the protein molecular weight as the clini‐cal hepatocellular carcinoma(HCC)serum samples ,indicating that the glycosylation level of recombinant AFP was comparable with clinical samples .Conclusion The high purified recombinant human‐resouce AFP with biological ac‐tivity could be used to prepare AFP reference materials ,traceable calibrator ,controls and related diagnostic reagents by Pichia expression system ,which could be used for clinical tumor diagnosis and therapeutic detection .