A highly sensitive liquid chromatography electrospray ionization mass spectrometry method for quantification of TMA, TMAO and creatinine in mouse urine

• Published in: MethodsX Vol. 4; no. C; pp. 310 - 319
• Main Authors: Veeravalli, Sunil, Karu, Kersti, Phillips, Ian R., Shephard, Elizabeth A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017; Elsevier
• Subjects: Biochemistry, Genetics and Molecular Biology; Creatinine using LC-ESI-MS; Electrospray ionization; FMO3; Liquid Chromatography; Mass spectrometry; Mouse urine; Quantification of TMA; TMAO and; Trimethylamine; Trimethylamine N-oxide; Creatinine using LC-ESI-MS; Liquid Chromatography; Trimethylamine; Trimethylamine N-oxide; Quantification of TMA; Electrospray ionization; TMAO and; FMO3; Mass spectrometry; Mouse urine; TMAO
• ISSN: 2215-0161; 2215-0161
• DOI: 10.1016/j.mex.2017.09.004

[Display omitted] Our method describes the quantification in mouse urine of trimethylamine (TMA), trimethylamine N-oxide (TMAO) and creatinine. The method combines derivatization of TMA, with ethyl bromoacetate, and LC chromatographic separation on an ACE C18 column. The effluent was continuously electrosprayed into the linear ion trap mass spectrometer (LTQ), which operated in selective ion monitoring (SIM) modes set for targeted analytes and their internal standards (IS). All validation parameters were within acceptable ranges of analytical method validation guidelines. Intra- and inter-day assay precision and accuracy coefficients of variation were <3.1%, and recoveries for TMA and TMAO were 97–104%. The method developed uses a two-step procedure. Firstly, TMA and TMAO are analyzed without a purification step using a 5-min gradient cap-LC- SIMs analysis, then creatinine is analyzed using the same experimental conditions. The method is robust, highly sensitive, reproducible and has the high-throughput capability of detecting TMA, TMAO and creatinine at on-column concentrations as low as 28pg/mL, 115pg/mL and 1ng/mL, respectively. The method is suitable for analysis of TMA, TMAO and creatinine in both male and female mouse urine. The key benefits of the method are: •The small sample volume of urine required, which overcomes the difficulties of collecting sufficient volumes of urine at defined times.•No sample pre-treatment is necessary.•The quantification of TMA, TMAO and creatinine using the same cap-LC-MS method.