Quantitative analysis of urinary volatile organic compounds by headspace solid-phase microextraction coupled to gas chromatography - mass spectrometry under low sample volume conditions.

In this work, a low-sample-volume analytical method based on headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was developed and evaluated for the targeted quantification of selected volatile organic compounds (VOCs) in urine samples. Optimization of HS-SPME conditions was performed using a Box-Behnken design to evaluate the effects of incubation temperature, incubation time, and extraction time, with temperature and extraction time emerging as the most significant factors. A confirmatory experiment supported shortening the model-derived incubation time from 52 to 15 min without compromising analytical performance. Sample volume was varied from 0.01 to 5.0 mL and, after evaluating extraction efficiency, repeatability, and total analytical response, 0.1 mL was selected as the optimal compromise between analytical response, repeatability, and sample consumption. Under the selected conditions (55 °C incubation temperature, 15 min incubation time and 52 min extraction time), the method enabled reliable extraction of eight chemically diverse VOCs with different volatilities, polarities, and functional groups (including dimethyl disulfide, 4-heptanone, cyclohexanol, cyclohexanone, phenol, p-cresol, nonanoic acid, and 2,4-di‑tert-butylphenol). Cyclohexanol and nonanoic acid were excluded from the final study due to the absence of reproducible EIC peaks under the selected HS-SPME-GC-MS conditions. Method evaluation performed in simulated urine showed good linearity (R2 = 0.987 - 0.999), low limits of detection (0.04 - 22.85 μg L⁻1) and limits of quantification (0.12-76.18 µg L⁻1). Recovery experiments in two real urine samples showed acceptable quantitative performance for most analytes and conditions, although compound- and matrix-dependent deviations were observed. Matrix-related variability was further assessed using multiple headspace solid-phase microextraction (MHS-SPME), as a complementary tool to compare extraction behavior in simulated and real urine. Application to seven healthy urine samples demonstrated the analytical applicability of the method in a real biological matrix. Overall, the proposed method combines trace-level sensitivity, minimal sample consumption, quantitative validation, and applicability to chemically diverse urinary VOCs under sample-limited conditions.