The effect of liquid-liquid extraction on metabolite detection and analysis using NMR spectroscopy in human synovial fluid

• Published in: Journal of pharmaceutical and biomedical analysis 2023-03, Vol.226, p.115254-115254, Article 115254
• Main Authors: Jaggard, Matthew K.J., Boulangé, Claire L., Graça, Gonçalo, Akhbari, Pouya, Vaghela, Uddhav, Bhattacharya, Rajarshi, Williams, Horace R.T., Lindon, John C., Gupte, Chinmay M.
• Format: Article
• Language: English
• Subjects: Acetonitrile; Acetonitriles - metabolism; Human synovial fluid (HSF); Humans; Liquid-Liquid Extraction; Magnetic Resonance Spectroscopy - methods; Methanol - chemistry; NMR Spectroscopy; Protein precipitation; Synovial Fluid - chemistry; Synovial Fluid - metabolism
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2023.115254

The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible. •Synovial fluid is of recent interest and presents an analytical challenge due its complex macromolecular composition.•The sensitivity, protein precipitation and analyte recovery of liquid-liquid extraction using NMR detection is reported.•Acid liquid-liquid extractions provided superior protein precipitation at the expense of the analyte recovery.•Methanol washing was an unreliable method inconsistently causing a gelatinous substrate within the samples.•Acetonitrile was preferred, giving superior recovery and detection of metabolites whilst removing macromolecules.