Analysis of methylcitrate in dried blood spots by liquid chromatography-tandem mass spectrometry

Abstract

Accumulation of propionylcarnitine (C3) in neonatal dried blood spots (DBS) is indicative of inborn errors of propionate metabolism including propionic acidemia (PA), methylmalonic aciduria (MMA), and cobalamin (Cbl) metabolic defects. Concentrations of C3 in affected newborns overlap with healthy individuals rendering this marker neither specific nor sensitive. While a conservative C3 cutoff together with relevant acylcarnitines ratios improve screening sensitivity, existing mass spectrometric methods in newborn screening laboratories are inadequate at improving testing specificity. Therefore, using the original screening DBS, we sought to measure 2-methylcitric acid (MCA), a pathognomonic hallmark of C3 disorders to decrease the false positive rate and improve the positive predictive value of C3 disorders. MCA was derivatized with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). No separate extraction step was required and derivatization was performed directly using a 3.2-mm disc of DBS as a sample (65°C for 45 min). The reaction mixture was analyzed by liquid chromatography tandem mass spectrometry. MCA was well separated and eluted at 2.3 min with a total run time of 7 min. The median and (range) of MCA of 0.06 μmol/L (0-0.63) were in excellent agreement with the literature. The method was applied retrospectively on DBS samples from established patients with PA, MMA, Cbl C, Cbl F, maternal vitamin B12 deficiency (n = 20) and controls (n = 337). Comparison with results obtained by another method was satisfactory (n = 252). This method will be applied as a second tier test for samples which trigger positive PA or MMA results by the primary newborn screening method.

Fig. 1

ESI-MS spectrum of DAABD-MCA derivative (a) and ESI-MS/MS spectrum of m/z 499 (b). Experimental details are as described in the text

Fig. 2

Extracted mass chromatograms obtained with a DBS from a healthy individual (a) and from an MMA patient (b). Solid lines represent MCA and dotted lines represent the IS. The arrow points at MCA in the healthy individual’s sample

Fig. 3

Stability of MCA in DBS samples stored at different temperatures

Fig. 4

Comparison of MCA concentrations in DBS (n = 252) obtained by the current method versus a reference method after modification (Turgeon et al. 2010). Experimental details are as described in the text