Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process

• Published in: Clinica chimica acta 2011-05, Vol.412 (11-12), p.873-879
• Main Authors: Dhillon, Kuldeep S., Bhandal, Ajit S., Aznar, Constantino P., Lorey, Fred W., Neogi, Partha
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Amino Acids - chemistry; Amino Acids - isolation & purification; Carnitine - analogs & derivatives; Carnitine - analysis; Carnitine - chemistry; Carnitine - isolation & purification; Chemical Fractionation - methods; Cost-Benefit Analysis; Dried blood spot; Genetic disease screening; Heptanoates - analysis; Heptanoates - chemistry; Heptanoates - isolation & purification; Humans; Hydrazones - chemistry; Infant, Newborn; Newborn screening; Succinylacetone; Tandem mass spectrometry; Tandem Mass Spectrometry - methods; Time Factors; Tyrosinemia type I; Tyrosinemias - diagnosis
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2010.12.028

Succinylacetone (SUAC), a specific marker for tyrosinemia type I (Tyr I) cannot be detected by the routine LC-MS/MS screening of amino acids (AA) and acylcarnitines (AC) in newborns. The current derivatized methods require double extraction of newborn dried blood spots (DBS); one for AA and AC and the second for SUAC from the blood spot left after the first extraction. We have developed a method in which AA, AC and SUAC are extracted in a single extraction resulting in significant reduction in labor and assay time. The 3.2mm DBS were extracted by incubating at 45°C for 45min with 100μl of acetonitrile (ACN)-water-formic acid mixture containing hydrazine and stable-isotope labeled internal standards of AA, AC and SUAC. The extract was derivatized with n-butanolic-HCl and analyzed by LC-MS/MS. The average inter-assay CVs for, AA, AC and SUAC were 10.1, 10.8 and 7.1% respectively. The extraction of analytes with ACN-water mixture showed no significant difference in their recovery compared to commonly used solvent MeOH. The concentration of hydrazine had considerable impact on SUAC extraction. We developed a new MS/MS derivatized method to detect AA/AC/ SUAC in a single extraction process for screening Tyr I along with disorders of AA and AC.