Introduction

Trimethylamine is ubiquitous in all major kingdoms of life, performing a notable physiological and pathophysiological role (Loo, Chan, Nicholson, & Holmes, 2022). Trimethylamine plasma concentrations directly correlate to several human diseases, such as chronic kidney disease (CKD), cardiovascular diseases, etc. (Yanget al. , 2019). The primary source of trimethylamine and TMAO in humans are various precursors such as carnitine, choline, betaine, etc. (Koeth et al. , 2013). In the human gut, microbes convert dietary precursors into trimethylamine. This trimethylamine gets converted into TMAO by FMOs (Falony, Vieira-Silva, & Raes, 2015) in the liver for excretion. Trimethylaminuria is an accumulation of trimethylamine that causes a fish-like odor among people who lack a functional FMO.
Trimethylamine and TMAO are significant carbon and nitrogen sources for methylotrophs (Colby & Zatman, 1973; Loo et al. , 2022). Methylotrophic bacteria are primarily found in the marine environment and possess FMOs. The FMO, trimethylamine monooxygenase (Tmm ) that converts trimethylamine into TMAO (Liet al. , 2017) is an NADPH-dependent mono-oxygenase (Chen, Patel, Crombie, Scrivens, & Murrell, 2011). This enzyme is assayed by monitoring NADPH concentration. Most of the reported assays monitor NADPH conversion into NADP+­­­. Even techniques like the isothermal calorimetry (Catucci, Sadeghi, & Gilardi, 2019) and detection of NADPH (Dixit & Roche, 1984) have limitations. Although sensitive, these methods can’t be used with crude samples because other enzymes also use NADPH (Spaans, Weusthuis, van der Oost, & Kengen, 2015). Hence it is normal to subtract background signals to obtain enzymatic activity measurements. The stability of NADPH is also sensitive to low pH and high temperatures (Wu, Wu, & Knight, 1986). These limitations make NADPH-dependent assays inaccurate.
Quantifying TMAO and trimethylamine directly in a sample where their concentrations change is quite challenging. Given that TMAO and trimethylamine are simultaneously found in clinical samples (Gątarek & Kałużna-Czaplińska, 2021a), a precise quantification of TMAO requires trimethylamine separation (Awwad, Geisel, & Obeid, 2016a). Existing methodologies take long durations to quantify TMAO precisely as extraction and separation techniques result in sample loss. The amount of TMAO or trimethylamine present in biological samples like urine and blood plasma usually lies in the range of nM - µM (Gątarek & Kałużna-Czaplińska, 2021b). GC-MS detection conditions often convert TMAO to trimethylamine (daCosta, Vrbanac, & Zeisel, 1990). Sample pre-treatment is, therefore, almost always needed to eliminate trimethylamine. Such procedures require column derivatization in which solvent plays a significant role as these reactions do not proceed in an aqueous media (Wang et al. , 2014). Various other methods use NMR (He et al., 2021a), UHPLC (Awwad, Geisel, & Obeid, 2016b; Ocque, Stubbs, & Nolin, 2015), LCMS/MS (Hefni, Bergström, Lennqvist, Fagerström, & Witthöft, 2021), and HPLC (Lang et al., 1998), but these are not cost-effective.