Simultaneous quantification of trimethylamine oxide and its precursors from gut microbial metabolic pathway

Using the QTRAP 4500MD system

Cui Jingwen¹, Zhang Yuanyuan¹ and Li Guoqing^1
1SCIEX, China

Introduction

Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, is found in higher concentrations in plasma after ingestion of L-carnitine and phosphatidylcholine. Ingestion of choline, L-carnitine, betaine and foods such as red meat cause significant increase in trimethyl amine (TMA) due to the enzymatic activity of gut microbiota. TMA is absorbed into the bloodstream and metabolized by flavin-containing mono oxygenase (FMO) enzymes to trimethylamine oxide (TMAO) in the liver. TMAO has been implicated in the pathology of multiple diseases, including heart failure.¹

Therefore, establishing a rapid and accurate quantitative method for routine monitoring of metabolites involved in TMAO production is needed to support gut microbiome research and screening of hundreds samples from a large research cohorts.

Here, an LC-MS/MS method for the simultaneous quantification of acetyl-carnitine, carnitine, choline, TMAO and betaine in plasma has been developed. All target compounds were extracted using a simple sample extraction procedure then diluted before analysis using LC-MS/MS.²

Key features of QTRAP 4500MD system for gut microbiome studies

• Robust LC-MS/MS system for routine quantification studies with the Turbo V ion source and Curtain Gas interface
• Simplified plasma sample preparation, using a one-step extraction and dilution
• Fast, reversed-phase chromatography provides good separation of the target analytes
• High accuracy and precision values for all analytes monitored, meeting bioanalytical requirements.

Methods

Sample preparation: A 20 µL plasma sample was extracted with 100 µL of acetonitrile and further spiked with 100 µL of a 5 ng/mL deuterated internal standard mixture. The sample mixture was centrifuged at 14000 rpm for 5 min, then 5 µL of the supernatant was injected on the column for analysis. The intra-day and inter-day variability over 5 days of the assay was evaluated.

Chromatography: A Jasper HPLC system (SCIEX) with a Phenomenex Kinetex C18, 2.6 μm, 2.1x100 mm column (00D-4462-AN) was used for sample separation using gradient conditions (Table 1) and a flow rate of 0.6 mL/min. A 5 µL injection volume was used and the column temperature was maintained at 40°C throughout the analysis. The total run time was 5.5 min.

Mass spectrometry: A QTRAP 4500MD system with a Turbo V ion source was used in positive ionization mode. All source and compound parameters were optimized and are outlined in the SCIEX How method. The MS method included optimized MRM transitions for multiple analytes.

Data processing: The method development and data acquisition was carried out using Analyst MD software. Data processing was performed using MultiQuant MD software.

Rapid analysis of TMAO and related species

First, phosphate buffered saline (PBS) was used to dilute the standards to make a standard calibration curve and obtain a first rough assessment of sensitivity. The concentration range evaluated and the linearity of the calibration curves is outlined in Table 2 along with the observed LLOQ concentrations for TMAO, betaine, L-carnitine, acetyl-carnitine, and choline. All analytes showed good gaussian peak shape (Figure 2).

Triplicate injections of TMAO, betaine, L-carnitine, acetyl-carnitine and choline were each analyzed at different concentrations and across 5 days, as outlined in Table 3. The intra-day reproducibility was analyzed across triplicate injections from a single day. The accuracy for each metabolite ranged from 89.87% to 112.03% and the %RSD ranged from 1.81% to 8.87%. The inter-day reproducibility was measured by comparing results from triplicate injections across days. The inter-day accuracy ranged from 92.93% to 107.15% and the %RSD ranged from 1.77% to 8.01%. The LLOQs observed for each analyte are marked in bold in Table 2. Standard bioanalytical requirements were followed.

Next, this rapid LC-MS method was tested in plasma. A simple extraction protocol was used, which provided good detection and good signal/noise of TMAO and its precursors analytes in plasma (Figure 3).

Conclusions

Here, a method for the rapid quantification of TMAO, betaine, L-carnitine, acetyl-carnitine and choline in plasma has been developed on the QTRAP 4500MD system. This method has the advantages of high specificity, linearity and high accuracy. As shown, this method is suitable to support rapid monitoring of TMAO metabolites:

• Rapid 5.5-minute run time provided good peak separation for detection in a shorter run time than several recently published methods for faster screening.³
• Consistent quantification results were obtained for all standards tested, with correlation coefficients ranging from 0.991 to 0.999
• Inter- and intra-day reproducibility and accuracy meet standard bioanalytical requirements of %CV less than ≤ 15% in for all analytes analyzed in plasma
• Method was evaluated in plasma matrix using a simple extraction protocol and good signal/noise was observed.

References

1. The gut, the heart, and TMAO. Cleveland clinic website.
   
2. Velasquez MT, et al. (2016) Trimethylamine N-oxide: The good, the bad and the unknown. Toxins 8, 326.
3. Katrina et al., (2021) A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors.  J. Pharma. Anal. 11, 523-528.