LC-MS/MS quantification of 3-O-methyldopa in DBS for the diagnosis of AADC Deficiency using a derivatization method to improve sensitivity.

INTRODUCTION:Aromatic L-amino-acid decarboxylase (AADC) deficiency is a rare autosomal recessive disorder of biogenic amine metabolism. In AADC deficiency L-dopa accumulates and it is methylated to produce 3-O-methyldopa (3-OMD). The final diagnosis must be confirmed by analysis of the deficient AADC enzyme activity, neurotransmitters in CSF or molecular genetic test of the DDC gene. 3-OMD can be detected in dried blood spots (DBS). Quantification of 3-OMD in DBS using LC–MSMS has been described since 2014 (Pin-Wen Chen et al.) with a simple preparation of samples. AIM: To present the validation of a modified method that includes a derivatization step to improve the sensitivity in the detection.MATERIALS AND METHODS: 3-OMD concentration was analyzed in DBS of normal patients (n=50), AADC deficiency patients confirmed with positive molecular tests (n=2) and patients in treatment with L-DOPA without AADC deficiency with negative molecular tests (n=3). Sample Preparation Strategy: 1) extraction the sample with Methanol and internal standar (3-OMDd3) shaking for 30 minute 2) dry under N2 flow 3)derivatization with hydrochloric acid 3N in 1-Butanol at 65°C 20 minutes 4) dry under N2 flow and reconstitute with MEOH:H2O (90:10). The UPLC–MS/MS analyses were performed on a Waters Acquity H-Class UPLC and a Waters Quattro Premier XE Micromass triple quadrupole mass spectrometer.The mass spectrometer was operated in the positive ion mode with multiple reaction monitoring (MRM). The instrument was optimized to monitor the m/z 268.1 → 166.2 and m/z 271.1 → 169.2 transitions for derivatized 3-OMD and 3-OMD-d3 analytes, respectively. RESULTS:Calibration curve were linear over the range of 150–20000nmol/L. A typical standard curve was y = 0.00573792*x + 0.966722 with a regression coefficient (R2) of 0.997.LoQ 150nmol/L and LoD 50nmol/L.Three levels were performed for precision, accuracy and stability parameters. Patient samples revealed increased 3-OMD levels(3248-4272 nmol/L) related to normal controls (mean 556,7nmol/L; ranged 198-1089nmol/L). L-DOPA patient`s showed highly increased 3-OMD levels(mean 9839nmol/L; ranged 8491-11897nmol/L). CONCLUSIONS: During the validation of a method it is probable that new strategies will have to be implemented. Here we use the same workflow as in newborn babies in DBS for acylcarnitines and aminoacids in derivatization methods, in order to improve sensitivity and eliminate matrix effects. It is relevant to have an easy and fast tool to detect AADC deficiency.Thus patients can be treated accordingly and profit from the latest gene therapies, that have advanced so much in recent years.