Abstract
Background: O-aminohippuric acid (OAH) is considered a low-abundance urinary fluorescent metabolite with the potential to be an innovative lung cancer biomarker. There is a lack of simple methods for measuring urinary OAH metabolite, and the measurement needs to be normalized by urinary creatinine, which is produced at a constant rate. Thus, the newly developed method must be able to determine urinary creatinine and OAH simultaneously in the same run. Objective: This work aimed to develop and validate a simple high-performance liquid chromatography (HPLC) method for measuring urinary OAH and to establish the reference intervals of OAH in healthy individuals. Materials and methods: We synthesized OAH standard in a simple route and optimized simple HPLC method for simultaneous measurement of creatinine and OAH in a single run. Analysis was performed on a RP-C18 column with a gradient elution system of acetonitrile - ammonium acetate buffer (pH 6.5, 100 mM). After implementing optimal conditions, the procedure was compiled according to the International Council for Harmonisation (ICH) validation parameters. The developed method was used for the establishment of reference intervals of a total of 120 random urine samples of healthy individuals. Results: The linear range of the calibration curve for creatinine and OAH were 1-1000 µg/mL and 0.1-100 µg/mL, respectively. The recoveries ranged for both metabolites were between 91.35 % and 109.12%. The relative standard deviations (RSDs) for the intra-day and inter-day results ranged from 0.11-0.66 % to 0.16-1.73 %, respectively. The limits of detection (LOD) and quantification (LOQ) were 0.258 µg/mL and 0.783 µg/mL for creatinine, while OAH was 0.045 µg/mL and 0.137 µg/mL, respectively. The method was successfully applied to establish reference intervals of OAH in healthy individuals and was defined as 0.420-2.287 mmol/mol creatinine. Conclusion: According to various validated parameters, the proposed method was proven to quantify urinary OAH and creatinine in a single run. It can also be analyzed noninvasively without additional sample processing. Reported herein is the first establishment of OAH reference intervals in healthy individuals, which may benefit the utilization of OAH as a noninvasive biomarker for lung cancer detection in the future.
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