液相色谱-串联质谱方法检测血清中极长链脂肪酸含量

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.

Abstract：

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.

Determination of the concentration of very long chain fatty acids in serum by liquid chromatography-tandem mass spectrometry

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.