色谱法,液相%串联质谱法%脂肪酸类,不饱和%肾上腺脑白质营养不良
色譜法,液相%串聯質譜法%脂肪痠類,不飽和%腎上腺腦白質營養不良
색보법,액상%천련질보법%지방산류,불포화%신상선뇌백질영양불량
Chromatography,liquid%Tandem mass spectrometry%Fatty acids,unsaturated%Adrenoleukodystrophy
目的 建立一种检测血清中极长链脂肪酸的LC-MS/MS方法.方法 收集2009年4-6月35份疑似ALD患者血清样本,采用LC-MS/MS方法检测血清中极长链脂肪酸含量.分析加样回收率、精密度及准确度,研究在常温放置和反复冻融条件下血清样本中极长链脂肪酸含量的稳定性.同时,用该方法测定101份健康人血清中极长链脂肪酸含量,统计测定值并进行分析.随机抽取35份血清,测定结果与德国柏林医学诊断检验中心(MDI)实验室测定数值进行比对.结果 血清样本中的极长链脂肪酸在梯度洗脱条件下分离良好,杂质干扰程度小.山萮酸(behenate,C22:0)的线性范围为2～64 mg/L,加样回收率为99.92%～102.05%,日内RSD≤6%,日间RSD≤9%;木焦油酸(tetracosanoicacid,C24:0)线性范围为2～64 mg/L,加样回收率为95.12%～100.44%,日内RSD≤6%,日间RSD≤7%;蜡酸(hexacosanoic acid,C26:0)线性范围为0～8 mg/L,加样回收率为92.21%～103.71%,日内RSD≤7%,日间RSD≤8%.山萮酸、木焦油酸和蜡酸的准确度均在85%～115%之间.样本在常温条件下放置12 h、反复冻融10次可以保持稳定.检测101份健康人血清中极长链脂肪酸含量服从正态分布,山萮酸含量为(19.43±4.43)mg/L,木焦油酸含量为(19.10±4.58)mg/L,蜡酸含量为(0.21±0.11)mg/L,计算木焦油酸/山萮酸和蜡酸/山箭酸比值分别为(0.99±0.13)和(0.01±0.01).统计结果显示,成年人血清中蜡酸(0.18±0.10)mg/L和木焦油酸/山萮酸比值(1.01±0.10)和未成年人血清中蜡酸(0.21±0.08)mg/L和木焦油酸/山萮酸比值(0.99±0.14)差异无统计学意义(t分别为1.439、0.806,P均＞0.05);男性健康人血清中木焦油酸/山萮酸比值(1.05±0.10)与女性健康人血清中木焦油酸/山萮酸比值(0.97±0.10)差异有统计学意义(t=3.394,P=0.001).与德国MDI实验室比对结果发现,本研究测定的山萮酸含量(16.93±4.30)mg/L和木焦油酸含量(19.57±6.40)mg/L与德国MDI实验室测定的山萮酸含量(13.85±3.17)mg/L和木焦油酸含量(16.10±5.84)mg/L差异有统计学意义(t分别为8.401和9.914,P均=0.000),而本研究测定的蜡酸含量(0.68±0.48)mg/L、木焦油酸/山萮酸比值(1.20±0.40)和蜡酸/山萮酸比值(0.04±0.04)与德国MDI实验室测定的蜡酸含量(0.65±0.67)mg/L、木焦油酸/山萮酸比值(1.19±0.43)和蜡酸/山萮酸比值(0.05±0.05)差异无统计学意义(t分别为0.372、0.317、0.945,P均＞0.05).结论 应用LC-MS/MS方法检测血清中极长链脂肪酸,具有较好的准确度和灵敏性,特异性强,稳定性好,能为临床诊断提供重要的生化依据.
目的 建立一種檢測血清中極長鏈脂肪痠的LC-MS/MS方法.方法 收集2009年4-6月35份疑似ALD患者血清樣本,採用LC-MS/MS方法檢測血清中極長鏈脂肪痠含量.分析加樣迴收率、精密度及準確度,研究在常溫放置和反複凍融條件下血清樣本中極長鏈脂肪痠含量的穩定性.同時,用該方法測定101份健康人血清中極長鏈脂肪痠含量,統計測定值併進行分析.隨機抽取35份血清,測定結果與德國柏林醫學診斷檢驗中心(MDI)實驗室測定數值進行比對.結果 血清樣本中的極長鏈脂肪痠在梯度洗脫條件下分離良好,雜質榦擾程度小.山萮痠(behenate,C22:0)的線性範圍為2～64 mg/L,加樣迴收率為99.92%～102.05%,日內RSD≤6%,日間RSD≤9%;木焦油痠(tetracosanoicacid,C24:0)線性範圍為2～64 mg/L,加樣迴收率為95.12%～100.44%,日內RSD≤6%,日間RSD≤7%;蠟痠(hexacosanoic acid,C26:0)線性範圍為0～8 mg/L,加樣迴收率為92.21%～103.71%,日內RSD≤7%,日間RSD≤8%.山萮痠、木焦油痠和蠟痠的準確度均在85%～115%之間.樣本在常溫條件下放置12 h、反複凍融10次可以保持穩定.檢測101份健康人血清中極長鏈脂肪痠含量服從正態分佈,山萮痠含量為(19.43±4.43)mg/L,木焦油痠含量為(19.10±4.58)mg/L,蠟痠含量為(0.21±0.11)mg/L,計算木焦油痠/山萮痠和蠟痠/山箭痠比值分彆為(0.99±0.13)和(0.01±0.01).統計結果顯示,成年人血清中蠟痠(0.18±0.10)mg/L和木焦油痠/山萮痠比值(1.01±0.10)和未成年人血清中蠟痠(0.21±0.08)mg/L和木焦油痠/山萮痠比值(0.99±0.14)差異無統計學意義(t分彆為1.439、0.806,P均＞0.05);男性健康人血清中木焦油痠/山萮痠比值(1.05±0.10)與女性健康人血清中木焦油痠/山萮痠比值(0.97±0.10)差異有統計學意義(t=3.394,P=0.001).與德國MDI實驗室比對結果髮現,本研究測定的山萮痠含量(16.93±4.30)mg/L和木焦油痠含量(19.57±6.40)mg/L與德國MDI實驗室測定的山萮痠含量(13.85±3.17)mg/L和木焦油痠含量(16.10±5.84)mg/L差異有統計學意義(t分彆為8.401和9.914,P均=0.000),而本研究測定的蠟痠含量(0.68±0.48)mg/L、木焦油痠/山萮痠比值(1.20±0.40)和蠟痠/山萮痠比值(0.04±0.04)與德國MDI實驗室測定的蠟痠含量(0.65±0.67)mg/L、木焦油痠/山萮痠比值(1.19±0.43)和蠟痠/山萮痠比值(0.05±0.05)差異無統計學意義(t分彆為0.372、0.317、0.945,P均＞0.05).結論 應用LC-MS/MS方法檢測血清中極長鏈脂肪痠,具有較好的準確度和靈敏性,特異性彊,穩定性好,能為臨床診斷提供重要的生化依據.
목적 건립일충검측혈청중겁장련지방산적LC-MS/MS방법.방법 수집2009년4-6월35빈의사ALD환자혈청양본,채용LC-MS/MS방법검측혈청중겁장련지방산함량.분석가양회수솔、정밀도급준학도,연구재상온방치화반복동융조건하혈청양본중겁장련지방산함량적은정성.동시,용해방법측정101빈건강인혈청중겁장련지방산함량,통계측정치병진행분석.수궤추취35빈혈청,측정결과여덕국백림의학진단검험중심(MDI)실험실측정수치진행비대.결과 혈청양본중적겁장련지방산재제도세탈조건하분리량호,잡질간우정도소.산유산(behenate,C22:0)적선성범위위2～64 mg/L,가양회수솔위99.92%～102.05%,일내RSD≤6%,일간RSD≤9%;목초유산(tetracosanoicacid,C24:0)선성범위위2～64 mg/L,가양회수솔위95.12%～100.44%,일내RSD≤6%,일간RSD≤7%;사산(hexacosanoic acid,C26:0)선성범위위0～8 mg/L,가양회수솔위92.21%～103.71%,일내RSD≤7%,일간RSD≤8%.산유산、목초유산화사산적준학도균재85%～115%지간.양본재상온조건하방치12 h、반복동융10차가이보지은정.검측101빈건강인혈청중겁장련지방산함량복종정태분포,산유산함량위(19.43±4.43)mg/L,목초유산함량위(19.10±4.58)mg/L,사산함량위(0.21±0.11)mg/L,계산목초유산/산유산화사산/산전산비치분별위(0.99±0.13)화(0.01±0.01).통계결과현시,성년인혈청중사산(0.18±0.10)mg/L화목초유산/산유산비치(1.01±0.10)화미성년인혈청중사산(0.21±0.08)mg/L화목초유산/산유산비치(0.99±0.14)차이무통계학의의(t분별위1.439、0.806,P균＞0.05);남성건강인혈청중목초유산/산유산비치(1.05±0.10)여녀성건강인혈청중목초유산/산유산비치(0.97±0.10)차이유통계학의의(t=3.394,P=0.001).여덕국MDI실험실비대결과발현,본연구측정적산유산함량(16.93±4.30)mg/L화목초유산함량(19.57±6.40)mg/L여덕국MDI실험실측정적산유산함량(13.85±3.17)mg/L화목초유산함량(16.10±5.84)mg/L차이유통계학의의(t분별위8.401화9.914,P균=0.000),이본연구측정적사산함량(0.68±0.48)mg/L、목초유산/산유산비치(1.20±0.40)화사산/산유산비치(0.04±0.04)여덕국MDI실험실측정적사산함량(0.65±0.67)mg/L、목초유산/산유산비치(1.19±0.43)화사산/산유산비치(0.05±0.05)차이무통계학의의(t분별위0.372、0.317、0.945,P균＞0.05).결론 응용LC-MS/MS방법검측혈청중겁장련지방산,구유교호적준학도화령민성,특이성강,은정성호,능위림상진단제공중요적생화의거.
Objective To establish a method for very long chain fatty acids( VLCFA )with liquid chromatography-tandem mass spectrometry( LC-MS/MS ). Methods One hundred and one healthy cases and 35 suspected ALD patients collected from April to June in 2009 were enrolled into this study. Quantitative analyzed the concentrations of VLCFA in serum was performed using liquid chromatography-tandem mass spectrometry. The precision, accuracy and recovery were analyzed, and the stability of VLCFA concentration of sample under room temperature and repeated freeze-thawing were also investigated. Serum levels of VLCFA in 101 normal cases were determined and analyzed statistically. The results for the 35 randomly chosen serum samples were compared with those from MDI in Germany. Results Serum VLCFA were separated well under these gradient condition with small interference. The linear range of C22:0 was from 2 mg/L to 64 mg/L, the recovery was 99. 92% -102. 05%, and the relative standard deviation ( RSD ) of intra-day and inter-day was less than 6% and 9% respectively. For C24:0 they were 2-64 mg/L. 95. 12%-100. 44%. ≤6%, ≤7%,respectively. For C26:0, they were 0-8 mg/L, 92.21%-103.71%, ≤7%, ≤8%, respectively. The accuracy of C22: 0,C24:0 and C26:0 were among 85% to 115%. The samples could be stable within 12 h at room temperature and repeated 10 times freeze-thawing. The values of VLCFA in 101 normal cases followed a normal distribution and the measured values were C22:0 =( 19. 43 ±4.43 ) mg/L,C24:0 =( 19. 10 ±4. 58 )mg/L, C26:0 = ( 0. 21 ± 0. 11 ) mg/L, the ratio of C24: 0/C22:0 and C26:0/C22: 0 were ( 0. 99 ± 0. 13 )and ( 0. 01 ±0. 01 ) respectively. The statistical analysis showed the concentration of C26:0 in adults ( 0. 18±0. 10 ) mg/L and children ( 0. 21 ± 0. 08 ) mg/L, C24: 0/C22:0 in adults ( 1.01 ± 0. 10 ) and children ( 0. 99 ±0. 14 ) has no significant( t values were 1. 439,0. 806, respectively, all P ＞ 0. 05 ); the ratio of C24:0/C22:0 in male (1.05 ± 0. 10 ) and female (0.97 ± 0. 10 ) has significant difference ( t =3. 394,P =0. 001 ). Compared the values determined by MDI laboratory, the results of C22: 0( 16. 93 ±4. 30 ) mg/L,C24: 0( 19. 57 ± 6. 40 ) mg/L by this method and C22:0 ( 13.85 ± 3. 17 ) mg/L, C24:0( 16. 10 ±5.84 ) mg/L by MDI have significant differences( t = 8. 401 ,P =0. 000;t =9. 914,P =0. 000 ),but C26:0( 0.68 ±0.48 ) mg/L, C24:0/C22:0( 1.20 ±0.40 ), C26: 0/C22:0 ( 0.04 ±0.04 )by this method and C26: 0( 0. 65 ± 0. 67 ) mg/L, C24:0/C22: 0( 1.19 ± 0. 43 ), C26:0/C22: 0 ( 0. 05 ± 0. 05 )by MDI have no differences( t values were 0. 372,0. 317,0. 945 ,respectively ,all P ＞0. 05 ). Conclusions The quantitative analysis method for serum very long chain fatty acid using LC-MS/MS is accurate, sensitive,specific and stable. It could provide important biochemistry information for diagnosis in clinic.
