LC-MS_MS法测定人尿液中乳清酸的浓度及临床应用.pdf

31药学与临床研究PharmaceuticalandClinicalResearchLC-MS/MS法测定人尿液中乳清酸的浓度及临床应用杨文斌，赵雯雯苏州大学附属第一医院药学部，苏州1215000摘要目的：建立测定人尿液中乳清酸浓度的方法，并应用于临床。方法：尿液样本加水稀释10 0 倍，经0.2 2 m滤膜过滤后，取滤液10 0 L加入甲醇40 0 L沉淀蛋白，以卡马西平（0.50 gmL-)2L为内标，经涡旋1min，130 0 0 r min-1离心5min，取上清液10 0 L采用液相色谱-串联质谱(LC-MS/MS)法测定。色谱柱为WatersAtlantisdCis(4.6mm250mm,5m），流动相为5mmoi.L-1甲酸铵溶液-乙睛（梯度洗脱），流速为0.50 mLmin=,柱温为4,进样量为3L。采用电喷雾离子源以正离子及负离子模式，检测方式为FullMS,FullMS分辨率为7 0 0 0 0,质量扫描范围m/z130360。乳清酸和内标卡马西平的检测离子分别为m/z（-）155.0 0 9 8 2、（+)2 37.10 2 2 4。结果：乳清酸尿液浓度在0.0 16 1.0 gmL-1范围内线性关系良好（P=0.9967），定量下限为0.0 16 gmL-；日内、日间精密度的RSD15%，RE 15%；基质效应为8 8.7 0%9 8.6 2%；提取回收率为9 5.9 3%103.08%；乳清酸尿液样品室温放置2 4h、处理后样品4放置2 4h的稳定性良好（RE10。2.4.3精密度与准确度试验取空白尿液9 0 L,加入10 L“2.2.2项下高、中、低质控溶液（8.0 0、1.25、0.30 gmL-)，按2.3”项下方法处理后，平行测定5次，考察日内精密度；连续测定3d，根据当日标准曲线计算各样品的实测质量浓度，考察日间精密度。将实测质量浓度与理论质量浓度进行比较，计算相对误差，考察准确度。结果显示，各质控样品日内、日间相对标准差（RSD)15%，标准误(RE)15%,符合相关指导原则的基本要求,详见表1。表1精密度与准确度试验结果（x土s）日内精密度（n=5)日间精密度（n=3）浓度(ugmL-)测量值RSDRE测量值RSDRE(gmL-1)(%）(%）(gmL-1)(%)(%）0.300.27 0.014.27-9.490.31 0.039.852.041.251.33 0.064.586.591.32 0.011.105.888.007.70 0.050.60-3.747.58 0.445.78-5.262.4.4基质效应和提取回收率试验取空白尿液90L，加入10 L2.2.2项下高、中、低质控溶液（8.0 0、1.2 5、0.30 gmL-），按“2.3 项下方法处理后，进样分析，得相应色谱峰面积(A）；取空白尿液90L,加人40 0 L甲醇,涡旋1min,13000rmin-离心5min，取全部上清液于另一离心管，并加人10 L“2.2.2项下高、中、低质控溶液（8.0 0、1.2 5、0.30 gmL-),2L内标溶液(0.50 gmL-),涡旋1min,进样分析，得相应色谱峰面积(B)；以水为溶剂，配制上述质量浓度的乳清酸对照品溶液，进样分析，得相应色谱峰面积(C)。各样品平行测定5次，基质效应（%）=B/C100%，提取回收率（%）=A/B100%。结果显示，乳清酸的基质效应为8 8.7 0%98.62%，提取回收率为9 5.9 3%10 3.0 8%(RSDA突变，最终患者被诊断为OTCD。该患者在临床样本送检后1天即报告了血清氨基酸浓度检测结果，送检后3天即报告了尿液乳清酸含量异常。尽管OTCD诊断的金标准是OTC基因突变检测,但基因检测耗时长（本病例从送检至发放报告耗时1月余）。利用本方法对OTCD辅助诊断的过程体现了质谱检测平台的优势与价值本研究使用的QExactive质谱仪具有高灵敏度、高分辨率、高质量准确度和高质谱图采集速度等特点，对物质分子量可精确到万分之一，因此用的是单离子检测扫描采集模式，未使用多反应检测扫描采集子母离子。此外由于OA的极性大，使用普通C18色谱柱分离时，保留时间短，易受基质效应影响。在本实验中我们对比了普通Ci：色谱柱、Hillic色谱柱、硅胶基质反相Cis色谱柱,结果显示OA在硅胶基质反相C18色谱柱中保留时间长，色谱峰形对称，基质效应低，结果重现性好。由于OTCD的发病率很低，本研究中仅检测了1例患者的尿液OA数据，后续需要积累更多的患者数据为临床诊断提供精细化的参考参考文献1 顾学范.临床遗传代谢病 M.北京：人民卫生出版社，2015.2 LICHTER-KONECKI U,CALDOVIC L,MORIZONO H,et al.Ornithine transcarbamylase deficiencyAJ/ADAMMP,ARDINGER HH,PAGON RA,et al.Gene Re-views.Seattle(WA):University of Washington,1993.3 HAUSER ER,FINKELSTEIN JE,VALLE D,et al.Al-lopurinol-induced orotidinuria.A test for mutations atthe ornithine carbamoyltransferase locus in womenJ.NEngl JMed,1990,322:1641-5.4 BANDITT P.Determination of orotic acid in serum byhigh-performance liquid chromatography J.J Chro-matogr B Biomed Appl,1994,660(1):176-9.5 MCCANN MT,THOMPSON MM,GUERON IC,et al.Quantification of orotic acid in dried filter-paper urinesamples by stable isotope dilutionJ.Clin Chem,1995,41(5):739-43.6TAKEDA T,SHIMADA M,NISHI M,et al.Mass-screening of neuroblastoma using urine from infants byhigh-performance liquid chromatographic method:reslts of first(6th month)and second(14th month)screeningJ.J Neurooncol,1997,31(1-2):165-70.7NISHI M,MIYAKE H,TAKEDA T,et al.Therelationship between homovanillic/vanillylmandelic acid ratiosand prognosis in neuroblastomaJJ.Oncol Rep,1998,5(3):631-3.8LA MARCA G,CASETTA B,ZAMMARCHI E.Rapiddetermination of orotic acid in urine by a fast liquidchromatography/tandem mass spectrometric method J.Rapid Commun Mass Spectrom,2003,17(8):788-93.9RASHED MS,JACOB M,AL-AMOUDI M,et al.Rapid determination of orotic acid in urine by liquidchromatography-electrospray tandem mass spectrometryJ.Clin Chem,2003,49(3):499-501.10韩连书，叶军，邱文娟，等串联质谱联合气相色谱-质谱检测遗传性代谢病 J.中华医学杂志，2 0 0 888(30):2122-6.11国家药典委员会.中华人民共和国药典：四部 S.北京：中国医药科技出版社，2 0 15：36 3-8.12杨艳玲，孙芳，钱宁，等尿素循环障碍的临床和实验室筛查研究 ：中华儿科杂志，2 0 0 5，45(5)：331-4.35药学与临床研究Pharmaceuticaland Clinical Research13 韩凤，韩连书，叶军，等鸟氨酸氨甲酰转移酶缺乏症患者临床资料及质谱检测结果分析 .中华医学杂志，2 0 14,9 4(34)：2 6 8 4-6.Determination of the Concentration of OroticAcid in HumanUrineebyLC-MS/MS and Its Clinical ApplicationYANG Wenbin,ZHAO Wenwen*Department of Pharmacy,the First Affiliated Hospital of Soochow University,Suzhou 215000,ChinaABSTRACTObjective:To establish a method to determine concentrations of orotic acid in human urine,and to apply this method in clinic.Methods:Urine sample was diluted 100 times with water and filteredby 0.22 m filter membrane,then 400 L methanol was added to 100 L of the filtrate to precipitate pro-tein,carbmazepine(0.50 gmL-l,2 L)was added as internal standard.After whirled for 1 min and cen-trifuged for 5min at 13 000rminl,the supernatant(100 L)was determined by liquid chromatography-tan-dem mass spectrometry(LC-MS/MS).The chromatographic column was Waters Atlantis dCis(4.6mm 250mm,5 m),the mobile phase was 5mmolL-l ammonium formate solution-acetonitrile(gradient elution),the flow rate was 0.50mLmin,the column temperature was 4,a n d t h e i n j e c t i o n v o l u me w a s 3 L.Thepositive and negative ion modes of the electric spray ion source were used.The detection mode was FullMS,the resolution of Full MS was 70000,and the mass scanning range was m/z 130-360.The detectedions of orotic acid and internal standard carbamazepine were m/z(-)155.009 82 and(+)237.10224,re-spectively.Results:The concentration of orotic acid in urine ranged linearly from 0.016 to 1.0 gmL-I(2=0.9967),and the lower limit of quantification was 0.016 gmL-1;Intra-day and intra-day RSD15%,RE15%.Matrix effect was 88.70%-98.62%.The recovery rate was 95.93%-103.08%.The orotic acid urinesample was kept at room temperature for 24h,and the treated sample was kept at 4 f o r 2 4h w i t h g o o dstability(RE15%).The content of urinary orotic acid in normal people was(3.10 1.23)gmL-l,andthat in ornithine carbamyltransferase deficiency(OTCD)patients was 354.82-2 566.57 gmL-.Conclusion:The research method is simple,specific and sensitive,and can be used for the auxiliary diagnosis of OTCDpatients.KEY WORDSLiquid chromatography-tandem mass spectrometry;Orotic acid;Ornithine transcarbamylasedeficiency