Development and validation of a green, solvent-free HPLC-UV method for the determination of uric acid in human urine using a 24 full factorial design of experiments
Microchemical Journal, cilt.229, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 229
- Basım Tarihi: 2026
- Doi Numarası: 10.1016/j.microc.2026.119521
- Dergi Adı: Microchemical Journal
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Chemical Abstracts Core, Chimica, Index Islamicus, Academic Search Ultimate (EBSCO), Engineering Source (EBSCO)
- Anahtar Kelimeler: AGREE, Design of experiments, Green analytical chemistry, HPLC-UV, Method validation, Uric acid
- Ankara Üniversitesi Adresli: Evet
Özet
A green, solvent-free HPLC-UV method for the determination of uric acid (UA) in human urine was developed using a 24 full factorial design of experiments (DoE), validated per ICH Q2(R2) and FDA guidance, and applied to six volunteer urine samples. Four chromatographic variables were simultaneously optimised: mobile phase pH, flow rate, phosphate buffer concentration, and sample dilution factor. Centre point experiments revealed peak shouldering at pH 4.0–5.5, attributed mechanistically to co-elution of the neutral and monoanionic forms of UA near its pKₐ₁. SPSS GLM analysis identified pH as the dominant factor (F = 18,928, p < 0.001). Optimised conditions (10 mM phosphate buffer, pH 7.25; 0.60 mL/min; 40× dilution; 290 nm) yielded tR = 3.63 min, intra-day precision 0.58% RSD, inter-day precision 1.38% RSD, and mean spike recovery 95.0 ± 1.4%. Calibration was linear over 0.5–50 mg/L (y = 23,278× + 2592; R2 = 0.9999), with LOD = 0.534 mg/L (3.18 μmol/L) and LOQ = 1.619 mg/L (9.63 μmol/L). The AGREE greenness score was 0.67, reflecting the exclusive use of aqueous mobile phase, short analysis time (∼4 min), and single-step sample preparation. UA concentrations in volunteer urine ranged from 1.09 to 6.19 mmol/L, demonstrating clinical applicability across the full reference range.