Univeristy of Oklahoma Health Sciences Center, Department of Pediatrics, Section of Genetics, 1122 NE 13(th) Street, Suite 1400, O' Donoghue Research Building, Oklahoma City, OK 73117, USA.
Univeristy of Oklahoma Health Sciences Center, Department of Pediatrics, Section of Genetics, 1122 NE 13(th) Street, Suite 1400, O' Donoghue Research Building, Oklahoma City, OK 73117, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Oct 15;1097-1098:101-110. doi: 10.1016/j.jchromb.2018.09.007. Epub 2018 Sep 5.
Glutaric aciduria type 1, a deficiency of glutaryl-CoA dehydrogenase, causes an accumulation of neurotoxic metabolites glutaric acid and 3-hydroxyglutaric acid (3-HGA). Testing of these analytes is routinely done by GC-MS but seldom account for interference from isomers or compounds with similar ion transitions. We developed a liquid chromatography tandem mass spectrometry method that accurately measures 3-HGA in urine and plasma specimens, while utilizing similar reagents and instrumentation used for the routine performance of amino acid and acylcarnitine analysis in determining the diagnosis of several metabolic disorders.
Plasma and urine samples were added aliquots of the deuterated 3-HGA internal standard and acetonitrile. The protein-free supernatant was brought to dryness, and the residue derivatized using 3 M HCL in 1-butanol with heating. The dried derivative was then reconstituted in 50% methanol-water solution and aliquot transferred to an HPLC vial for analysis by LC-MS/MS. Separation was performed using a C8 HPLC column under flow gradient conditions of 0.2% formic acid in water and methanol, respectively. Ionization was by ESI and detection of selected precursor-product ion transitions by multiple reaction monitoring (MRM) in positive mode.
The butyl-ester derivative of 3-HGA eluted at 7.82 min while 2-hydroxyglutaric acid (2-HGA) eluted at 8.21 min. This was equivalent to a separation factor of 1.05 and a resolution of 1.03, respectively. The 3-HGA calibration curve was linear over the range 6.20-319 ng mL (r = 0.9996), and the reportable range determined by the linearity was found to be 1.54-384 ng mL. The calculated limits of detection and quantitation were 0.348 and 1.56 ng mL, respectively. Intra- and Inter-assay %CVs for quality control plasma and urine samples ranged from 2 to 18%, with recoveries of 66-115%. The method correlated to the gold standard GC-MS method for both serum (r ≥ 0.996) and urine analysis (r ≥ 0.949). The concentration of 3-HGA in normal, non-GA1 individuals was ≤25.2 ng mL (in plasma) and ≤ 35.0 μmol mmol of creatinine (in urine).
This LC-MS/MS method accurately quantified plasma and urine 3-HGA concentration after successful resolution from 2-HGA and other compounds with similar ion transitions. This method is suitable for confirmatory testing of 3-HGA, as a follow-up to an abnormal newborn screen test result, with concern for GA type 1.
戊二酸血症 1 型是由于谷氨酰辅酶 A 脱氢酶缺乏引起的，会导致神经毒性代谢物戊二酸和 3-羟基戊二酸（3-HGA）的积累。这些分析物的检测通常通过 GC-MS 进行，但很少考虑到异构体或具有相似离子跃迁的化合物的干扰。我们开发了一种液相色谱串联质谱法，可以准确测量尿液和血浆标本中的 3-HGA，同时利用用于常规进行氨基酸和酰基肉碱分析的试剂和仪器来确定几种代谢紊乱的诊断。
向血浆和尿液样本中加入氘代 3-HGA 内标和乙腈的等分试样。将无蛋白上清液干燥，然后用 1-丁醇中的 3M HCL 加热衍生化。将干燥的衍生物重新溶解在 50%甲醇-水溶液中，并转移等分试样至 HPLC 小瓶中，通过 LC-MS/MS 进行分析。使用 C8 HPLC 柱在分别为 0.2%甲酸和甲醇的流动梯度条件下进行分离。离子化通过电喷雾进行，通过正模式下的多重反应监测（MRM）检测选定的前体-产物离子跃迁。
3-HGA 的丁基酯衍生物在 7.82 分钟洗脱，而 2-羟基戊二酸（2-HGA）在 8.21 分钟洗脱。这相当于分离因子为 1.05，分辨率为 1.03。3-HGA 的校准曲线在 6.20-319ngmL 范围内呈线性（r=0.9996），通过线性确定的报告范围为 1.54-384ngmL。计算得出的检测限和定量限分别为 0.348 和 1.56ngmL。质控血浆和尿液样本的日内和日间%CV 范围为 2-18%，回收率为 66-115%。该方法与血清（r≥0.996）和尿液分析（r≥0.949）的金标准 GC-MS 方法相关。非 GA1 个体的正常血浆 3-HGA 浓度≤25.2ngmL，尿液 3-HGA 浓度≤35.0μmolmmol 肌酐。
本研究通过与 2-HGA 和其他具有相似离子跃迁的化合物成功分离，准确地定量了血浆和尿液中的 3-HGA 浓度。该方法适用于 3-HGA 的确认性检测，作为对异常新生儿筛查结果的后续检测，怀疑为 1 型戊二酸血症。
