Struys E A, Jansen E E W, Gibson K M, Jakobs C
Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam. The Netherlands.
J Inherit Metab Dis. 2005;28(6):913-20. doi: 10.1007/s10545-005-0111-0.
Succinic semialdehyde (SSA) accumulates in the inborn error of meta- bolism succinic semialdehyde dehydrogenase deficiency owing to impaired enzymatic conversion to succinic acid. We developed a stable-isotope dilution liquid chromato- graphy-tandem mass spectrometry method for the determination of SSA in urine and cerebrospinal fluid samples. Stable-isotope-labelled [13C4]SSA, serving as internal standard, was prepared by reaction of ninhydrin with L-[13C5]glutamic acid. SSA in body fluids was converted to its dinitrophenylhydrazine (DNPH) derivative, without sample purification prior to the derivatization procedure. The DNPH derivative of SSA was injected onto a C18 analytical column and chromatography was performed by isocratic elution. Detection was accomplished by tandem mass spectrometry operating in the negative multiple-reaction monitoring mode. The limit of detection was 10 nmol/L and the calibration curves over the range 0-500 pmol of SSA showed good linearity (r2 > 0.99). The intra-day coefficient of variation (n = 10) for urine was 2.7% and inter-day coefficient of variation (n = 5) for urine was 8.5%. The average recoveries performed on two levels by enriching urine and cerebrospinal fluid samples ranged between 85 and 115%, with coefficients of variation < 8%. The method enabled the first determination of normal values for SSA in urine and pathological values of SSA in urine and cerebrospinal fluid samples derived from patients with succinic semialdehyde dehydrogenase deficiency.
由于向琥珀酸的酶促转化受损，琥珀半醛（SSA）在遗传性代谢病琥珀半醛脱氢酶缺乏症中蓄积。我们开发了一种稳定同位素稀释液相色谱 - 串联质谱法，用于测定尿液和脑脊液样本中的SSA。通过茚三酮与L-[13C5]谷氨酸反应制备稳定同位素标记的[13C4]SSA作为内标。体液中的SSA在衍生化程序之前无需样品纯化即可转化为其2,4 - 二硝基苯腙（DNPH）衍生物。将SSA的DNPH衍生物注入C18分析柱，并通过等度洗脱进行色谱分析。通过在负多重反应监测模式下运行的串联质谱法进行检测。检测限为10 nmol/L，0 - 500 pmol SSA范围内的校准曲线显示出良好的线性（r2>0.99）。尿液的日内变异系数（n = 10）为2.7%，尿液的日间变异系数（n = 5）为8.5%。通过富集尿液和脑脊液样本在两个水平上进行的平均回收率在85%至115%之间，变异系数<8%。该方法首次测定了尿液中SSA的正常值以及琥珀半醛脱氢酶缺乏症患者尿液和脑脊液样本中SSA的病理值。
