Putative Detection of 4-Acetamidobutanoic Acid in an HPV-16-Positive Pooled Cervical Cytology Sample: An Exploratory LC–HRMS Study Polar Metabolites in HPV-Genotyped Cervical Cytology Fluid

Abstract

Objective: Residual cervical cytology material allows the biochemical environment of human papillomavirus (HPV) infection to be studied without additional sampling. We surveyed polar metabolites in this material, stratified by HPV genotype, to generate candidates for targeted testing.

Methods: Sixty residual cytology specimens were genotyped by multiplex real-time PCR and combined into four pools of fifteen: HPV-negative, HPV-16, HPV-18 and other high-risk. Cells were pelleted, washed twice in buffered saline, and extracted by a biphasic chloroform–methanol–water procedure. The aqueous phase was analysed by hydrophilic interaction chromatography with high-resolution tandem mass spectrometry, all four pools and two blanks in one sequence. The design yields four observations without replication, so no inferential statistics were computed. Three constraints qualify every result: no pooled quality control sample was injected, injections were not randomised, and the eluent was unbuffered, limiting retention of acidic analytes.

Result: Annotation returned 291 features. After removing solvent additives, leachables, pharmaceuticals and non-specific matches, 77 were endogenous metabolites, of which 23 eluted in the void volume and were excluded. Choline, glutamate and a C6H13NO2 feature appeared in all four pools. A feature putatively annotated as 4-acetamidobutanoic acid (N-acetyl-GABA) was observed only in the HPV-16 pool, at 1.34 × 10⁹ arbitrary units, with an MS/MS library match of 79.4 and a mass error of 0.15 ppm, it is resolved by 6.08 min from the only other C6H11NO3 feature present, supporting a putative annotation at Metabolomics Standards Initiative level 2. Pathway over-representation analysis returned no set surviving correction. Detection in one pool is not evidence of absence elsewhere, limits of detection being undetermined.

Conclusion: One putative annotation warrants follow-up: 4-acetamidobutanoic acid in the HPV-16 pool. Nothing reported is a validated biomarker. The report defines what this material yields under an untargeted workflow and what a confirmatory study must meet. Because each group was represented by a single non-randomized injection without pooled QC or technical replication, the study cannot distinguish biological differences from analytical variation.