RESUMEN
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.
RESUMEN
Blood samples stored as dried blood spots (DBSs) are emerging as a useful sampling and storage vehicle for a wide range of applications. Unfortunately, the surging popularity of DBS samples has not yet been accompanied by an improvement in automated techniques for extraction and analysis. As a first step towards overcoming this challenge, we have developed a prototype microfluidic system for quantification of amino acids in dried blood spots, in which analytes are extracted, mixed with internal standards, derivatized, and reconstituted for analysis by (off-line and in-line) tandem mass spectrometry. The new method is fast, robust, precise, and most importantly, compatible with automation. We propose that the new method can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.
