Determination of unconjugated aromatic acids in urine by capillary electrophoresis with dual electrochemical detection – Potential application in fast diagnosis of phenylketonuria

Abstract

A novel method of CE coupled with dual electrochemical detection has been developed for the determination of pathological metabolites of phenylalanine in urine samples. Factors influencing the separation and detection were examined and optimized. Five aromatic acid metabolites and a major coexisting interfering compound uric acid could be well separated within 23 min at a separation voltage of 16 kV using a 35 mmol/L SDS/60 mmol/L H(3)BO(3)-Na(2)B(4)O(7) running buffer (pH 8.2). Highly linear response was obtained for these five biomarker compounds over three orders of magnitude with detection limits ranging from 6.6 to 0.064 μg/mL (S/N=3). The average recovery and RSD were within the range of 92.6-121.0 and 1.0-12.0%, respectively. The proposed method has been used to detect the unconjugated aromatic acids simultaneously in urine samples with the advantages of obtaining more information about target analytes and avoiding redundant measurements and high assay cost, thus could find potential applications involving assays of biomarker compounds for the purpose of fast diagnose of some metabolic diseases including phenylketonuria.

Study on urinary metabolic profile of phenylketonuria by micellar electrokinetic capillary chromatography with dual electrochemical detection—Potential clinical application in fast diagnosis of phenylketonuria

“…Other four PKU marker compounds were also determined in different kinds of urine samples. As listed in Table 3, the content of PPA in the tested urine samples of PKU patients was in the range of 1.6 × 10 −5 -1.5 × 10 −4 g mL −1 ; the urinary content of other four marker compounds found in PKU patients were generally higher than those in healthy babies [29]. Besides, the assay results showed that the abnormal metabolism of phenylalanine could be maintained at a stable level with a long-time low-phenylalanine diet.…”

“…The MECC-C 4 D/AD system used in this work was laboratorybuilt with C 4 D as an upstream detector and AD as a downstream one [29]. A ±30 kV high-voltage power supply (Shanghai Institute of Nuclear Research, China) provided a voltage between the ends of the capillary.…”

“…The design of AD was based on the end-column approach and has been described previously [29,30]. The carbon disc electrode was made of polymer leads (diameter 0.3 mm, hardness HB, Sakura, Japan).…”

“…posed method [29]. According to literature the urinary content of PPA in healthy newborns is the range of 10 −6 -10 −7 g mL −1 [14].…”

“…A variety of dual detection schemes have been proposed for chromatographic methods such as HPLC [26] and CE [27], while, to the best of our knowledge, CE coupled with C 4 D and AD (CE-C 4 D/AD) has not been employed for the determination of marker compounds of PKU disease [28]. In our previous work, CE with dual electrochemical detection method was applied for the determination of PKU marker compounds in the spiked urine samples of healthy volunteers [29].…”