MSACL 2022 Abstract

INTRODUCTION:
Inherited disorders of propionate, cobalamin, and methionine metabolism are included in many newborn screening (NBS) programs. First-tier NBS for these disorders using propionyl carnitine (C3) and methionine alone lack sufficient specificity for diagnostic purposes. Our clinical laboratory recently published a UPLC-MS/MS approach to measurement of 2-methylcitric acid (MCA), methylmalonic acid (MMA), and homocysteine (HCY) in dried blood spot (DBS) cards for second-tier NBS.1 This approach has been in active clinical use for NBS purposes since December of 2019 in British Columbia, Canada. The injection-to-injection sample analysis time is currently 7 minutes for MCA and 8 minutes 26 seconds for MMA & HCY, accounting for approximately 30 seconds of required instrument default delay time between injections. To facilitate faster diagnostic screening and make more instrument time available for other assays in the laboratory, significantly quicker chromatographic analyses were developed on a shorter UPLC column.

METHODS:
Two DBS card punches (3mm in diameter, 6.2 µL of blood) were used for standards, QCs, and patient samples. Derivatization using 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE) for MCA and butanol-HCl for MMA & HCY was conducted as previously reported.1 UPLC column length was reduced from 100 mm to 30 mm and modifications were made to the binary solvent program to enable fast chromatographic separations and ensure column re-equilibration.

RESULTS:
Injection-to-injection analysis time for MCA was reduced to 1 minute 59 seconds and for analysis of MMA & HCY to 2 minutes 58 seconds. Chromatographic parameters were adjusted such that separation from interferences and overall assay performance was consistent with the current in use validated longer method. Of note, MMA remained separated from the endogenous isobar succinic acid. Consistent elution of lipid species known to cause matrix effects were monitored across the column gradient and re-equilibration time program. Analysis of MCA and MMA & HCY can be performed on the same UPLC column, batched in series, with a combined analysis time of 4 minutes 57 seconds. The longer method having a combined analysis time of 15 minutes and 26 seconds).

CONCLUSION:
Per sample UPLC-MS/MS analysis time was lowered by 72% for MCA and 65% for MMA & HCY respectively using a 30 mm column (combined analysis time reduction of 67.9%). Approaches for both reducing other NBS second-tier assay injection-to-injection times and integrating instrument parameters are being explored towards a goal of random-axis batch analysis.

REFERENCE:
1. Dubland JA, Rakić B, Vallance H, Sinclair G. Analysis of 2-methylcitric acid, methylmalonic acid, and total homocysteine in dried blood spots by LC-MS/MS for application in the newborn screening laboratory: A dual derivatization approach. J Mass Spectrom Adv Clin Lab. 2021 Mar 17;20:1-10. doi: 10.1016/j.jmsacl.2021.03.001. PMID: 34820666; PMCID: PMC8601015.