Optimisation and application of the metabolite extraction method of vitreous humour samples.

Vitreous humour (VH) is a unique, avascular ocular structure whose biochemical composition may reflect retinal pathophysiology. Although VH has been studied using metabolomics, it remains less explored compared to other biological matrices, and existing sample preparation protocols show considerable variation. In fact, most available reports are application-oriented studies that describe the sample preparation conditions used, without providing a rationale for why these particular parameters were chosen, consideration of alternative options, or offering a critical evaluation or systematic comparison of methodologies. Therefore, in this study, we systematically compared and optimised procedures for VH preparation prior to untargeted LC-MS analysis. Key steps, homogenisation, extraction, and preconcentration, were evaluated based on feature coverage, signal intensity, reproducibility, and practical aspects such as throughput and cost. The optimal protocol involved sample disruption using liquid nitrogen combined with ultrasound, protein precipitation and metabolite extraction with acetone, followed by evaporation and resuspension in methanol/water (2:1). This protocol was subsequently applied to VH samples from patients with epiretinal membrane (ERM, n = 14) and macular hole (MH, n = 13), conditions with different clinical manifestation but often combined into a single control group in ophthalmic studies. Univariate and multivariate analyses revealed no statistically robust metabolic differences between ERM and MH samples in this cohort. Simulation-based power analysis indicated that, under the present sample size and multiple-testing correction, the study was primarily powered to detect only large effects; therefore, subtle differences cannot be excluded. These findings support the use of ERM and MH as a pragmatic combined comparator group in LC-MS-based vitreous metabolomics, while warranting validation in larger cohorts. The optimised preparation method provides a practical framework for future metabolomic investigations of VH in ophthalmic research.