Determination of homocysteine in dried blood spots using liquid chromatography-tandem mass spectrometry

A high-throughput method to measure homocysteine (Hcy) in dried blood spots (DBS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established. The DBS sample processing includes adding homocystine-D8 as internal standard, dithiothreitol (DTT) as reducing agent for bound Hcy forms, acetonitrile containing 0.1% (v/v) formic acid and 0.05% (v/v) trifluoroacetic acid for Hcy extraction. This procedure was carried out in a 96-well plate format in an automated liquid handling platform to facilitate high-throughput analysis. The processed samples were separated on a Phenomenex CN column and quantitated by LC-MS/MS in multiple reaction monitoring (MRM) mode. The limit of detection was 0.12 mu mol/L (S/N=3) and the limit of quantification was 0.46 mu mol/L (S/N=10). The linear range was 1.16-148.00 mu mol/L (R-2 = 0.994). The average recoveries were (103.0 +/- 4.97)% - (112.0 +/- 2.13)%. The intra-day relative standard deviations (RSDs) were 1.9%-4.6% and the inter-day RSDs were 1.5%-7.1%. DBS sample stability was confirmed by measuring same DBS samples stored for 0, 1, 2, 3, 4, 5, 6 and 14 days at -4, -20, 22 and 37 degrees C, and an overall sample RSD < 15% was found under each temperature. Processed sample stability within 48 hours was also confirmed with an overall sample RSD<5%. The comparison of this method with conventional biochemistry assay was made by measuring 47 blood samples both in an automated biochemistry analyzer (samples in plasma form) and with the LC-MS/MS method (samples in DBS form), which showed excellent correlation (R-2 = 0.982).