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Quantitation of plasma and urine 3-hydroxyglutaric acid, after separation from 2-hydroxyglutaric acid and other compounds of similar ion transition, by liquid chromatography-tandem mass spectrometry for the confirmation of glutaric aciduria type 1 B Analytical technologies in the biomedical and life sciences
- Simon, Garfield A., Wierenga, Andrea
- Journal of chromatography 2018 v.1097-1098 pp. 101-110
- acetonitrile, amino acids, blood serum, butanol, creatinine, detection limit, formic acid, gas chromatography-mass spectrometry, glutaric acid, high performance liquid chromatography, instrumentation, ionization, isomers, metabolites, methanol, monitoring, neonates, neurotoxicity, quality control, tandem mass spectrometry, urinalysis, urine
- 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⁻¹ (r² = 0.9996), and the reportable range determined by the linearity was found to be 1.54–384 ng mL⁻¹. The calculated limits of detection and quantitation were 0.348 and 1.56 ng mL⁻¹, 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 (r² ≥ 0.996) and urine analysis (r² ≥ 0.949). The concentration of 3-HGA in normal, non-GA1 individuals was ≤25.2 ng mL⁻¹ (in plasma) and ≤ 35.0 μmol mmol⁻¹ 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.