Summary:
Phenylketonouria (PKU) and medium-chain acyl-CoA dehydrogenase deficiency
(MCADD) are autosomal recessive disorders, usually included in newborn
screening of most European countries due to their high prevalence. Diagnosis of
PKU is based on determination of phenylalanine and phenylalanine to tyrosine
ratio, whereas of MCADD on determination of medium-chain acyl-carnitines
(hexanoyl-, octanoyl-, decanoyl-carnitine) and recently to their ratios with
acetyl-carnitine, in dried blood spots.
Here, we present an HPLC-MS/MS method for the simultaneous quantitative
determination of all mentioned analytes in newborn blood spots. Neither
derivatization of analytes nor addition of isotopically labeled internal
standards in the sample is required since quantitation is based on a series of
analytes calibrators in blood spots. The detection parameters of MS/MS were
optimized, while appropriate solvents and gradient were selected to achieve
separation of the analytes by HPLC prior to MS/MS analysis. The separation of
the analytes permitted quantification to be based on the precursor ion, thus
increasing detection sensitivity compared to MS/MS methods, including internal
standards, and based on the product ion signal, to overcome false positive
results, obtained due to the presence of isobaric compounds or compounds that
show the same m/z with the analytes determined.
The method developed is characterized by high analytical sensitivity, accuracy
and repeatability. Limits of detection of 1.3 and 4.7 μmol/L for Phe and Tyr,
and 0.40, 0.006, 0.008, 0.008 μmol/L for acetyl-, hexanoyl-, octanoyl-, and
decanoyl-carnitine, respectively, were achieved. The calibration curves were
linear (R2>0,99) in the range of 24 to 800 μmol/L for Phe and Tyr, 3 to 100
μmol/L for C2, and 0.1 to 10 μmol/L for C6, C8, and C10.
Accuracy was assessed by recovery tests and found to range between 93 and 105%,
whereas intra- and inter-day imprecision was less than 10% and 15%,
respectively, for all analytes. The method was highly selective since no
interferences from isobaric compounds or other matrix effects were observed.
Finally, the method was evaluated by analyzing 450 newborn blood spot samples
and by comparing the results with those determined with a commercially
available semi-quantitative MS kit that includes deuterated compounds as
internal standards. The results obtained by the two methods were consistent
concerning the samples with values lying within the physiological range as well
as the few outliers.
Taking into account the excellent analytical performance of the method and the
short assay time (5 min per sample) that permits analysis of more than 250
samples per 24 h, the method developed should find wide application for PKU and
MCADD newborn screening.
Keywords:
Phenylketonouria, Medium-chain acyl-CoA dehydrogenase deficiency, Dried blood spots, HPLC-MS/MS
