中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2010年
12期
1176-1180
,共5页
甲基丙二酸%色谱法,液相%串联质谱法%代谢缺陷,先天性
甲基丙二痠%色譜法,液相%串聯質譜法%代謝缺陷,先天性
갑기병이산%색보법,액상%천련질보법%대사결함,선천성
Methylmalonic acid%Chromatography,liquid%Tandem mass spectrometry%Metabolism,inborn errors
目的 建立一种LC-MS/MS方法 测定血清MMA,用于MMA血症的诊断以及治疗效果的监测.方法 收集2009年4-12月205份健康体检者和146份患者血清,用液-液萃取方法 萃取样本,用自制饱和盐酸-正丁醇进行衍生,采用氮气吹干,色谱柱为Discovery C18(50 mm×2.1 mm,5 μm),流动相为甲醇和水(含0.1%甲酸,V/V),梯度洗脱,液相分离后进入串联质谱进行分析测定,选择性反应监测模式,以标准品制作标准曲线,同位素内标法定量,以血清样本测定2、25、80μg/L3个浓度的加样回收率,以质控样本测定准确度、精密度和稳定性.检测205份健康人血清,用于临床验证.数字表法随机抽取13份血清,测定结果 与德国MDI实验室进行比对,用配对t检验分析测定数据.结果 方法 线性范围2～100μg/L,标准曲线相关系数R2＞0.995.MMA衍生物保留时间为10.5 min,在此实验条件下丁二酸与MMA不互相干扰,批内RSD≤6.4%,批间RSD≤5.0%,回收率为96.42%～103.33%,检出限为1 μg/L,分析准确度94.2%～108.2%.样本常温放置至少6 h稳定、-20℃下至少可以存放70 d稳定、冻融10次稳定,衍生物在4 ℃下至少存放5 d稳定.溶血样本组与未溶血样本组测定值中位数(四分位数)分别为102.53(13.84～302.33)μg/L和39.52(11.94～203.08)μg/L,差异有统计学意义(T=8,P＜0.05).本实验室与德国MDI实验室测定结果 中位数(四分位数)分别为32.82(24.50～100.42)μg/L和32.20(26.65～93.30)μg/L,差异无统计学意义(T=7,P＞0.05).健康成年人(18～58岁)158名,血清MMA测定值(18.46±10.49)μg/L,健康未成年人(1～17岁)47名,血清MMA测定值(22.38±11.45)μg/L.结论 成功建立了LC-MS/MS方法 准确测定血清MMA浓度,样本前处理简单,方法 灵敏度高,特异性强,可重复性好,可以用于MMA血症的筛查、诊断和治疗效果的监测.
目的 建立一種LC-MS/MS方法 測定血清MMA,用于MMA血癥的診斷以及治療效果的鑑測.方法 收集2009年4-12月205份健康體檢者和146份患者血清,用液-液萃取方法 萃取樣本,用自製飽和鹽痠-正丁醇進行衍生,採用氮氣吹榦,色譜柱為Discovery C18(50 mm×2.1 mm,5 μm),流動相為甲醇和水(含0.1%甲痠,V/V),梯度洗脫,液相分離後進入串聯質譜進行分析測定,選擇性反應鑑測模式,以標準品製作標準麯線,同位素內標法定量,以血清樣本測定2、25、80μg/L3箇濃度的加樣迴收率,以質控樣本測定準確度、精密度和穩定性.檢測205份健康人血清,用于臨床驗證.數字錶法隨機抽取13份血清,測定結果 與德國MDI實驗室進行比對,用配對t檢驗分析測定數據.結果 方法 線性範圍2～100μg/L,標準麯線相關繫數R2＞0.995.MMA衍生物保留時間為10.5 min,在此實驗條件下丁二痠與MMA不互相榦擾,批內RSD≤6.4%,批間RSD≤5.0%,迴收率為96.42%～103.33%,檢齣限為1 μg/L,分析準確度94.2%～108.2%.樣本常溫放置至少6 h穩定、-20℃下至少可以存放70 d穩定、凍融10次穩定,衍生物在4 ℃下至少存放5 d穩定.溶血樣本組與未溶血樣本組測定值中位數(四分位數)分彆為102.53(13.84～302.33)μg/L和39.52(11.94～203.08)μg/L,差異有統計學意義(T=8,P＜0.05).本實驗室與德國MDI實驗室測定結果 中位數(四分位數)分彆為32.82(24.50～100.42)μg/L和32.20(26.65～93.30)μg/L,差異無統計學意義(T=7,P＞0.05).健康成年人(18～58歲)158名,血清MMA測定值(18.46±10.49)μg/L,健康未成年人(1～17歲)47名,血清MMA測定值(22.38±11.45)μg/L.結論 成功建立瞭LC-MS/MS方法 準確測定血清MMA濃度,樣本前處理簡單,方法 靈敏度高,特異性彊,可重複性好,可以用于MMA血癥的篩查、診斷和治療效果的鑑測.
목적 건립일충LC-MS/MS방법 측정혈청MMA,용우MMA혈증적진단이급치료효과적감측.방법 수집2009년4-12월205빈건강체검자화146빈환자혈청,용액-액췌취방법 췌취양본,용자제포화염산-정정순진행연생,채용담기취간,색보주위Discovery C18(50 mm×2.1 mm,5 μm),류동상위갑순화수(함0.1%갑산,V/V),제도세탈,액상분리후진입천련질보진행분석측정,선택성반응감측모식,이표준품제작표준곡선,동위소내표법정량,이혈청양본측정2、25、80μg/L3개농도적가양회수솔,이질공양본측정준학도、정밀도화은정성.검측205빈건강인혈청,용우림상험증.수자표법수궤추취13빈혈청,측정결과 여덕국MDI실험실진행비대,용배대t검험분석측정수거.결과 방법 선성범위2～100μg/L,표준곡선상관계수R2＞0.995.MMA연생물보류시간위10.5 min,재차실험조건하정이산여MMA불호상간우,비내RSD≤6.4%,비간RSD≤5.0%,회수솔위96.42%～103.33%,검출한위1 μg/L,분석준학도94.2%～108.2%.양본상온방치지소6 h은정、-20℃하지소가이존방70 d은정、동융10차은정,연생물재4 ℃하지소존방5 d은정.용혈양본조여미용혈양본조측정치중위수(사분위수)분별위102.53(13.84～302.33)μg/L화39.52(11.94～203.08)μg/L,차이유통계학의의(T=8,P＜0.05).본실험실여덕국MDI실험실측정결과 중위수(사분위수)분별위32.82(24.50～100.42)μg/L화32.20(26.65～93.30)μg/L,차이무통계학의의(T=7,P＞0.05).건강성년인(18～58세)158명,혈청MMA측정치(18.46±10.49)μg/L,건강미성년인(1～17세)47명,혈청MMA측정치(22.38±11.45)μg/L.결론 성공건립료LC-MS/MS방법 준학측정혈청MMA농도,양본전처리간단,방법 령민도고,특이성강,가중복성호,가이용우MMA혈증적사사、진단화치료효과적감측.
Objective To establish a LC-MS/MS method for the determination of MMA in serum,and provide a assay for the diagnosis and screening of methylmalonic academia in the clinic. Methods MMA was extracted from 205 serum samples from healthy controls and 146 serum samples from patients with liquidliquid extraction method with MTBE as the extraction solvent. The supernatant was transferred to a tube and dried with nitrogen gas. Then the residual was derived with HCI-BuOH mixed agent to give a product, which was analyzed directly by LC-MS-MS system with a gradient elution, selective reaction monitor, a Discovery C18 column (50 mm × 2. 1 mm ,5 μm) as the isolation column and a mobile phase consisting of methanol and water (0. 1% formic acid, V/V), respectively. The concentration of MMA was detected with the isotope internal standard method. The stand curve was employed with a series of calibrators. The recovery was estimated with the 3 serum samples with the concentrations of 2, 25, 80 μg/L respectively. The accuracy,precision and stability were also tested with quality control samples. Moreover, the range of concentrations in healthy people were detected to investigate the influence of hemolysis on the detection results. Thirteen samples were randomly tested according to the digital chart. The testing results were compared with the results provided by Medical Diagnositic institution (MDI) in Germany. The paired t-test was applied to statistical analysis. Results The linear range of this method was 2-100 μg/L, and the correlation coefficient (R2 ) was more than 0. 995. The retention time of MMA derivative was 10. 5 min. Succinic acid and MMA were not found to interfere with each other. The within-run RSD was less than 6. 4%, and the between-run RSD was less than 5.0%. The recovery rates were from 96. 42% to 103. 33%. The limit of quantification was 1 μg/L.The accuracies of the method were form 94. 2% to 108. 2%. The samples were stable for 6 h at room temperature and stable for 70 d even keep at - 20 ℃. The samples were stable after 10 freeze-thaw cycles. The derivatives of MMA were found to be stable at least for 5 d at 4 ℃. The medians of the hemolysis group and the normal group were 102.53 (13.84-302.33) μg/L and 39.52 (11.94-203.08) μg/L,respectively. There was significant difference between the 2 groups ( T = 8, P ＜ 0. 05 ). The medians of comparison test in our laboratory and the MDI were 32. 82(24. 50-100. 42) μg/L and 32. 20(26. 65-93. 30)μg/L There was no significant difference between the 2 groups( T=7 ,P ＞0. 05 ). The mean value (-x± s)of 158 healthy adults( 18-58 years old) and 47 healthy teenages( 1-17 years old)were ( 18.46 ± 10.49 )μg/L and (22. 38 ± 11.45) μg/L, respectively. Conclusions A LC-MS/MS method for analysis of MMA in serum is established successfully. The quantitative method is simple and accurate with good sensitivity,specificity and repeatability. The method can be applied for diagnosis, screening and monitoring of methylmalonic acidemia.
