Standardized Targeted Metabolomics Using the BIOCRATES MxP Quant 500 Kit on the ACQUITY UPLC I-Class PLUS and Xevo TQ-XS Mass Spectrometer

In this application note, we present the validation of the quantitative MxP Quant 500 kit on a Waters Xevo TQ-XS Mass Spectrometer. The assay offers multiplexed MS/MS analysis of up to 630 metabolites and lipids from 26 analyte classes in only a 10-μL sample volume. In addition, 232 pre-defined metabolism indicators, that are biologically meaningful sums and ratios of metabolites and lipids, can be calculated by the BIOCRATES MetIDQ software tool, MetaboINDICATOR. Combined, these are 852 metabolic features that could be determined in one experiment with the MxP Quant 500 kit.

Benefits

• Ready-to-use BIOCRATES quantitative metabolomics kit solution including reagents and software
  
• Broad coverage of multiple metabolic pathways
  
• Validated across multiple sample types

Metabolic signatures can provide crucial insights into physiological mechanisms, as well as a better understanding of diseases. For comprehensive metabolomics and lipidomics analyses, analytical reliability, inter-laboratory comparability, automation, and standardization are of utmost importance.

Here, we present the validation of the quantitative MxP Quant 500 kit on a Waters Xevo TQ-XS Mass Spectrometer. The assay offers multiplexed MS/MS analysis of up to 630 metabolites and lipids from 26 analyte classes in only a 10-μL sample volume. In addition, 232 pre-defined metabolism indicators, that are biologically meaningful sums and ratios of metabolites and lipids, can be calculated by the BIOCRATES MetIDQ software tool, MetaboINDICATOR. Combined, these are 852 metabolic features that could be determined in one experiment with the MxP Quant 500 kit.

While the assay has been initially validated for human plasma, it also allows standardized analysis in a variety of biological sample matrices (e.g., blood, feces, and tissue) and species, including studies of the gut microbiome. The MxP Quant 500 kit combines LC-MS/MS of 13 compound classes, basically small molecules, bile acids, and free fatty acids, followed by FIA-MS/MS of 12 lipid classes (including acylcarnitines) and hexoses, into a single workflow (Figure 1).

The Xevo TQ-XS Mass Spectrometer offers a broader dynamic range and increased sensitivity compared to its predecessors. Analytical validation was performed on a Waters ACQUITY UPLC I-Class PLUS System coupled to a Xevo TQ-XS Mass Spectrometer for human plasma, following EMA and FDA guidelines.

High throughput analysis with a minimal sample volume (10 μL) is achieved by an easy and rapid sample preparation using a patented 96-well filter plate as described in the MxP Quant 500 kit user manual. Blank and zero samples, seven calibration standards, three levels of quality control samples (human plasma-based QCs), and a variety of plasma samples were subjected to two UPLC-ESI-MS/MS analyses in multiple reaction monitoring (MRM) mode, followed by two FIA-MS/MS runs. A Waters ACQUITY UPLC I-Class PLUS System, equipped with a reversed-phase MxP Quant 500 UHPLC Column, was coupled to a Xevo TQ-XS Mass Spectrometer. Figure 2 shows two representative chromatograms (total ion current (TIC), positive, and negative ion modes) for a human plasma sample.

For quantitation, both LC and FIA data were converted and imported directly into the BIOCRATES software, MetIDQ Oxygen, and quantified. MetIDQ includes an automated simple target normalization procedure based on QC or sample pool for batch-to-batch and kit plate-to-plate correction for sample cohort across several kit plates.

MxP Quant 500 kit

The MxP Quant 500 kit is a ready-to-use kit for standardized, multiplexed MS/MS analysis of up to 630 metabolites and lipids. It includes an automated software guided workflow from sample registration to data analysis. The kit is equipped with hardware (patented kit plate, reagents, additives to mobile phase, column set, and USB stick with predefined read-to-use FIA-MS/MS and UHPLC-MS/MS analysis and quantitation methods validated for Xevo TQ-XS) and software (MetIDQ).

Sample preparation

Sample preparation was performed according to the BIOCRATES MxP Quant 500 kit user manual. In brief, 10 μL of sample (blood plasma, serum, tissue, and fecal homogenate), blank, zero sample, kit calibrator, and kit quality control material were each added directly onto the 96-well plate provided with the kit according to the pipetting plan predefined in MetIDQ. After a drying step of 30 min using nitrogen, a derivatization step for one hour using a 5% PITC solution was performed. After another drying step of one hour, 300 μL of 5 mM methanolic ammonium acetate were added as extraction solvent and, after shaking of the kit plate for 30 min, the contents were filtered into a lower sandwich plate by centrifugation at 200 g for 2 min. The sample extracts were diluted for subsequent FIA-MS/MS and UHPLC-MS/MS analysis as specified in the kit user manual.

Data management

MassLynx v4.2 Software with MetIDQ Software

The adaption of the ready-to-use MxP Quant 500 kit to the Xevo TQ-XS LC-MS/MS system comprised the optimization of instrumental parameters from sample preparation setup to mass spectrometric features.

Overall, analysis times were 7 min per LC-MS/MS run and 4 min per FIA-MS/MS run, resulting in a total analysis run time of 36 hours for 80 samples plus quality control samples, calibration standards, a blank, and zero samples on a 96-well plate. Overall the kit run time from sample preparation to data processing and technical validation is about 42 hours.

Validation: Summary

A comprehensive set of defined human plasma samples was used for the validation of the MxP Quant 500 kit on the Waters ACQUITY UPLC I-Class PLUS System coupled to a Xevo TQ-XS Mass Spectrometer. Appendix 1 provides an overview of the analytical performance (classification) of the LC-MS/MS analytes. To determine the validity of the analytes, intra-day (within batch) and inter-day (batch-to-batch) analyses were evaluated in terms of precision and accuracy, as well as detection sensitivity, selectivity, and matrix effects. Excellent intra- and inter-batch accuracy (between 85–115%) and coefficient of variation as a measure for precision (CV <15%) were obtained for all seven-point calibrated analytes and their calibrants above the limit of detection (LOD) in the course of the validation, therefore classified as “quantitative”.

LC analytes that rely on a one-point internal calibration were accepted at a CV <20% and an accuracy between 80–120%, therefore classified as “quantitative with restriction”. If the accuracy criteria were not fulfilled, they were classified as “relative quantitative”. Two analytes could not be validated because many of their measured values were <LOD. No analytes were classified as “invalid”.

The NIST standard reference material (SRM) 1950 was analyzed and the measured concentration values were compared to the certified values to demonstrate the performance of the MxP Quant 500 kit on the Xevo TQ-XS system. Excellent accuracy between 85–115% was obtained for specified analytes in the reference material, amino acids, creatinine, and hexoses (Figure 3). These findings are important in the context of inter-laboratory comparability.

Furthermore, a broad spectrum of metabolites, mostly lipids, present in biological specimens can be determined by FIA-MS/MS analysis with the MxP Quant 500 kit. These metabolites belong to key analyte classes, such as acylcarnitines (including carnitine), carbohydrates (hexoses), and a number of lipid classes (Figure 1). An overview of the analytical performance classification of acylcarnitines in human plasma is provided in Appendix 2. For FIA-MS/MS, the label quantitative implies precision <15% and accuracy between 80–120%. Since not all FIA-MS/MS metabolites are commercially available as external and internal standards, the accurate determination of lipids and a subset of acylcarnitines is limited. Thus, the validity of these analytes is termed “relative quantitative”. The label <LOD means that analytes could not be validated in human plasma because in most cases the measured concentrations were below the detection limits. In a typical human plasma sample approximately 520 smallmolecules and lipids could be routinely analyzed (>LOD) and quantified with the MxP Quant 500 kit. Excellent precision values were obtained for the vast majority of metabolites, making them highly suited for comparative studies of plasma from different sample cohorts.

In addition, the MxP Quant 500 kit on the Xevo TQ-XS Mass Spectrometer was also evaluated for rat plasma, mouse liver homogenate, and human fecal samples. The latter are particularly relevant for microbiome research. In these experiments >220 analytes were detected in a human fecal pool with CVs <20%, among these were acylcarnitines C3:1, C5:1, and C6:1, which are <LOD in plasma samples. Note that there is a separate application note detailing a recommended sample preparation protocol for the analysis of fecal samples with the MxP Quant 500 kit.

Comparison of Xevo TQ-XS and TQ-S

The analytical performance of the MxP Quant 500 kit on the ACQUITY UPLC I-Class PLUS System coupled to a Xevo TQ-XS Mass Spectrometer was compared to their performance on a Xevo TQ-S Mass Spectrometer.

The advantages of the TQ-XS are:

• An improved instrument sensitivity
  
• Related, shorter necessary dwell times
  
• A broader dynamic range
  

This results in approximately 20 analytes more being detectable using the Xevo TQ-XS. The broader dynamic range improves the linearity especially of higher abundant metabolites. The number of FIA injections could be reduced from three to two, resulting in a total instrument run time of about 33 instead of 40 hours per 96-well plate.

The data shown in this application note supports the high reliability and analytical potency of the MxP Quant 500 kit on the ACQUITY UPLC I-Class PLUS, Xevo TQ-XS system, delivering high level of global standardization in metabolomics with excellent lab-to-lab comparability.