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. 2023 Dec 28:38:101045.
doi: 10.1016/j.ymgmr.2023.101045. eCollection 2024 Mar.

Accurate determination of Biotinidase activity in serum by HPLC and its utilization as second tier test for the confirmation of initial positive newborn screening results

Abdul Rafiq Khan  1   2 , Souad Al-Enazi  1 , Areej Al-Gahtani  1 , Saleh Al-Zahrani  1 , Syed Muhammad Saad  2 , Khalid Mohammed Khan  3 , Ali Alothaim  1
Affiliations

Accurate determination of Biotinidase activity in serum by HPLC and its utilization as second tier test for the confirmation of initial positive newborn screening results

Abdul Rafiq Khan et al. Mol Genet Metab Rep. .

Abstract

Diagnosis of Biotinidase deficiency (BTD) is extremely important to avoid several neurodevelopmental problems in early childhood. Colorimetric and fluorometric methods lack specificity and selectivity due to several interferences resulting in a high number of false positive results. We developed an HPLC method for BTD activity in serum with fluorescent detection. In colorimetric assays, biotinidase attacks the amide linkage of the artificial substrate biotinidyl-4-aminobenzoic acid (B-PABA) and releases p-aminobenzoic acid (PABA), which is converted to a purple dye by diazotization reaction. The newly developed method injects the reaction mixture directly into the HPLC column and quantifies using a six-point calibration curve without coupling and diazotization reaction. The method is linear over the 5-1000 μmol/L range. The detection and quantitation limits were 2.5 μmol/L and 5.0 μmol/L, respectively. When compared with colorimetric assay, the correlation coefficient (R2) was 0.9963. The within-assay and between-assay precision was <10.0% for four levels of quality control samples. No significant variation in BTD activity was detected due to hemolysis, icteric, and lipemic samples. The newly developed method eliminates the potential interference due to the presence of aromatic amines and significantly reduces the false positive results observed with the colorimetric method. It is simple, specific, sensitive, faster in sample preparation, and requires a small sample volume. The newly developed HPLC method was used in our laboratory as a secondary tier test for initial positive BTD samples from newborn screening programs. To our knowledge, no similar HPLC method has been reported to date.

Keywords: 4-Aminobenzoic acid; HPLC-FL method; Quantification; Serum Biotinidase; Validation.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1
Chromatograms of Blank, Standard and Serum Sample.
Fig. 2
Fig. 2
Effect of interferences on BTD activity
Fig. 3
Fig. 3
Comparison of HPLC method with colorimetric method
Fig. 4
Fig. 4
Effect of incubation time, serum, and substrate on PABA Formation.
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