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Optimization and Application of Direct Infusion Nanoelectrospray HRMS Method for Large-Scale Urinary Metabolic Phenotyping in Molecular Epidemiology

Large-scale metabolic profiling requires the development of novel economical high-throughput analytical methods to facilitate characterization of systemic metabolic variation in population phenotypes. We report a fit-for-purpose direct infusion nanoelectrospray high-resolution mass spectrometry (DI-nESI-HRMS) method with time-of-flight detection for rapid targeted parallel analysis of over 40 urinary metabolites. The newly developed 2 min infusion method requires <10 μL of urine sample and generates high-resolution MS profiles in both positive and negative polarities, enabling further data mining and relative quantification of hundreds of metabolites. Here we present optimization of the DI-nESI-HRMS method in a detailed step-by-step guide and provide a workflow with rigorous quality assessment for large-scale studies. We demonstrate for the first time the application of the method for urinary metabolic profiling in human epidemiological investigations. Implementation of the presented DI-nESI-HRMS method enabled cost-efficient analysis of >10 000 24 h urine samples from the INTERMAP study in 12 weeks and >2200 spot urine samples from the ARIC study in <3 weeks with the required sensitivity and accuracy. We illustrate the application of the technique by characterizing the differences in metabolic phenotypes of the USA and Japanese population from the INTERMAP study.

Materials and methods

Materials and preparation of standard solutions
All chemicals and solvents were of the highest purity grade. Labelled and non-labelled standards used for quantification (See Table S1 for details) were purchased from Sigma- prepared weekly. Generally, the use of 13 C and 15 N isotope-labelled standards is preferential to deuterated standards in targeted analysis because the latter can interfere with the signal of targeted compound. 1 The stock solutions of the labelled and non-labelled standards were stored at 4 °C in tightly closed containers.
For the preparation of analyte-free matrix in the INTERMAP study, sodium borate solution in ultrapure HPLC-grade water at concentration of 10 g/L (as in the standard procedure for urine sample collection and storage in the INTERMAP project) was diluted 50 times to obtain similar borate concentration as expected in diluted urine samples. For the ARIC study, water with 0.01% of formic acid was used as an analyte-free matrix.

Chip based nanoelectrospray MS system
The nanoelectrospray was created and maintained by applying 1. Total data acquisition time was of 40 s for the INTERMAP study and 30 s for the ARIC study for each ionisation mode (first negative, and then positive ion mode) but the overall turnaround time for each sample was of ca. 2 minutes to let the instrument automatically S-3 switch the polarity and the voltage settle before acquiring the data in the next polarity. The data for the negative and positive ionisation mode were acquired in two separate files in the MassLynx™ software. The total time for the analysis of a 96 well-plate was less than 4 hours.
Sodium formate solution was used to calibrate the mass spectrometer on a daily basis. The lock-mass function was turned off but data in both modes were recalibrated post-acquisition by in-house software using reference signals of endogenous metabolites present in all urine samples.
MS/MS was performed for targeted peaks in DIMS in Resolution mode in non-spiked pooled urine sample and pooled urine sample spiked with the standards. The optimal collision energy (CE) was selected for each peak between 10-60 eV. For the endogenous metabolites the spectra were compared to the spectra acquired for the standards in neat solvent and to the metabolite fragmentation patterns available in online databases (HMDB 2 and Metlin 3 ).
For the analysis, the TM was diluted 10 times with ultrapure water. An aliquot of 50 μL was placed in a well and 100 μL of MeOH containing 0.015% of formic acid (final concentration of formic acid in a well was of 0.01%) were added to maintain 1:2 water-methanol proportion required for a stable nESI signal. The intra-day replicate analysis (n=10) of the TM was done in three separate days (total n=30), and the CV% of signal intensities was assessed intra-and inter-day without and with normalisation (to the sum of the intensities of targeted peaks). The TM was also analysed in two separate days (n=8) to estimate the effect of instrument maintenance (mass spectrometer venting after the nitrogen gas supply shutdown event with subsequent detector setup and chip-alignment procedure in the ChipSoft software) on the signal stability.

Parallel assaystandard addition vs. external calibration curves
In this work, the MS quantification was performed by the back-calculation of the ratio of each selected metabolite to its internal standard and the slope of the calibration curve  (Table S- 2). A similar assessment of matrix effect was undertaken for the ARIC pooled SR samples and analyte-free matrix consisting of water with 0.01% formic acid (Table S-3).

Analysis of Test Mixture
The TM was analysed over three days to assess intra-(n=10) and inter-day (n=30) variability of signal intensities. and subsequent venting of mass spectrometer followed by detector set-up and chip-alignment procedure for the nESI source. Figure S-9(B) shows the normalised intensity values for the n=8 measurements done before and after the instrument maintenance. The CV% values for all the analytes measured in positive and negative ion mode did not exceed 20 which is an acceptable metabolite precision providing the normalisation was done for targeted analytes.

S-26
Figure S-6. Precision (as CV %) of slope values and concentration of the QC samples for some metabolites measured in Japan-F pooled urine samples from three validation series prepared and measured to assess the assay stability (freshly prepared, three freeze-thaw cycles, long-term storage at -80°C). S-28 S-29  S-32