Preanalytical Benchmarks for Solvent-Based GC–MS Profiling of Volatile Organic Compounds in Diabetic Foot Ulcer Swabs

Gas chromatography–mass spectrometry (GC–MS) is widely applied to wound-associated volatile organic compounds (VOCs), yet the preanalytical steps that govern its sensitivity remain poorly characterized, limiting comparison across studies of diabetic foot ulcer (DFU) infection. We benchmark a controlled solvent-based extraction workflow with direct internal-standard addition for GC–MS profiling of clinical DFU swabs. A six-compound multiclass panel (butanoic acid, 3-methyl-; 1-octen-3-ol; nonanal; 5-hepten-2-one, 6-methyl-; dimethyl disulfide; and heptane) was spiked onto sterile cotton swabs with toluene-d8 as an internal standard (IS). At day 0, diethyl ether (DE) gave the highest IS-normalized response for every analyte (F(2,6) = 12.4–131.0), most markedly for hydrogen-bond-active species. A 50% nitrogen blowdown step produced volatility-dependent enhancement the lowest enrichment in every solvent (1.0–1.9-fold), whereas high-boiling analytes exceeded 2-fold in DE. Frozen storage at −20 °C for 15 days preserved the IS-normalized response within ±20% of day 0 for most analyte–solvent combinations; hexane gave the tightest precision (mean %RSD 2.3–8.0%), whereas DE was poor for heptane (25.7%). Applied to 28 infected DFUs, the workflow returned 13 VOCs meeting combined retention index (|ΔRI| ≤35) and spectral-library (match factor ≥75) criteria, spanning fatty acids, fermentation products, host lipid-peroxidation aldehydes, phenylalanine catabolites, and palmitamide. These class-stratified preanalytical benchmarks provide an analytical baseline against which clinical wound-VOC studies can be designed and compared.