Development of a Method for Measuring Phytanic Acid as a Lifestyle-related Disease Biomarker in Rat Serum Using Ultra-fast Liquid Chromatography–Ultraviolet Spectrophotometry Combined with a Modified 2-Nitrophenylhydrazine Derivatization Method

Abstract

A simple and rapid ultra-fast liquid chromatography–ultraviolet spectrophotometry (UFLC-UV) method combined with modified 2-nitrophenylhydrazine (2-NPH) derivatization was developed for determining phytanic acid (Phy) in rat serum. Serum Phy and heptadecanoic acid (the internal standard) were derivatized by 2-NPH at ambient temperature for 20 min and extracted in n-hexane. After extracting derivatized Phy (D-Phy) and derivatized IS from the reaction mixture, the extracts were separated with a YMC-Pack C8 column (150 × 3.0 mm i.d., S-3 μm) using an isocratic mobile phase comprised of acetonitrile:H₂O (90:10; pH 4.4) at 0.5 mL/min. The detection wavelength was 228 nm. Linearity was observed over 1 – 20 μg/mL (r = 0.9997). The intra- and inter-day reproducibilities of D-Phy measurements were ≤13.0%. To our knowledge, this is the first report of the quantitative and qualitative measurement of serum Phy using 2-NPH derivatization and UFLC-UV. This method can be performed rapidly under mild conditions.