Quantitative Analysis of Porphyrins in Dried Blood Spots Using Liquid Chromatography-tandem Mass Spectrometry with Pre-column Derivatization
Bulletin of the Korean Chemical Society (Print)
Porphyrins are intermediates of the heme biosynthetic pathway in mammalian tissues. The porphyrin molecules consist of cyclic tetrapyrrole. Porphyrin metabolism may be disordered or impaired among some Autism Spectrum Disorder (ASD) children; therefore porphyrin levels might have potential clinical utility as a biomarker for ASD identification and/or classification. 1 ASD, which may develop in infancy or during the first three years, is a neurodevelopmental disorder characterized by impaired
... ized by impaired social relatedness, communication, repetitive behaviors, abnormal movement patterns, and sensory dysfunction. 2-4 In accordance with the most recent studies, the prevalence of ASD in the United States is 1.47% on average. 5 The incidence of ASD in the Ilsan area of South Korea is reportedly 2.64%. 6 Thus, early detection, which leads earlier treatment of the ASD population is important. Genetic factors of ASD have been proposed; 7 however, genetic studies carried out in a specific genetic background have not yielded an early diagnostic test yet. A number of techniques have been developed for determining urinary porphyrins, including spectrofluorometry, 8 high performance liquid chromatography, 9,10 thinlayer chromatography, 11 ion-pair high performance liquid chromatography, 12 capillary electrophoresis with fluorescence, 13 matrix-assisted laser desorption ionization/time-offlight mass spectrometry (MALDI-TOF), 14 and fast atom bombardment mass spectrometry (FAB-MS). 15 Recently, liquid chromatography-mass spectrometry (LC-MS), 16,17 and tandem mass spectrometry (LC-MS/MS) 18-21 have been used to determine porphyrins in biological matrices (blood, urine and feces) because of their high sensitivity and specificity. However, there have been no publications regarding the measurement of porphyrin metabolites in whole blood because the metabolites are endogenous and found in very low concentrations. Considering both the necessity of early treatment and the availability of dried blood spot (DBS) newborn screening tests in clinics, we began to develop a method validation of porphyrin metabolites using LC-MS/MS. A few drops of whole blood were collected on filter paper from a finger prick. We conducted the esterification process of porphyrin to improve the sensitivity of the porphyrin metabolites using LC-MS/MS. Finally, we measured infinitesimal porphyrin metabolite, as the potential biomarker of ASD, in DBS samples with esterification. Here, we report the development and validation of the use of LC-MS/MS to quantify porphyrin metabolites in DBS.