Development of a Rapid Microbore Metabolic ... - ACS Publications

Apr 26, 2016 - A rapid gradient microbore ultraperformance liquid chromatography–mass spectrometry (UPLC–MS) method has been developed to provide ...
1 downloads 7 Views 1MB Size
Subscriber access provided by SUNY DOWNSTATE

Article

Development of a Rapid Microbore Metabolic Profiling (RAMMP) UPLC-MS Approach for High-Throughput Phenotyping Studies Nicola Gray, Kyrillos Adesina-Georgiadis, Elena Chekmeneva, Robert S. Plumb, Ian David Wilson, and Jeremy K. Nicholson Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00038 • Publication Date (Web): 26 Apr 2016 Downloaded from http://pubs.acs.org on May 1, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Development of a Rapid Microbore Metabolic Profiling (RAMMP) UPLCMS Approach for High-Throughput Phenotyping Studies Nicola Gray1, Kyrillos Adesina-Georgiadis1, Elena Chekmeneva1, Robert S. Plumb1, Ian D. Wilson1*, Jeremy K. Nicholson1,2* 1

Division of Computational and Systems Medicine, Department of Surgery and Cancer,

Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, UK 2

MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine,

Department of Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith Hospital, London, W12 0NN, United Kingdom

*Corresponding Authors: [email protected], [email protected]. Tel: +4420 7594 3195

ABSTRACT A rapid gradient microbore UPLC-MS method has been developed to provide a highthroughput analytical platform for the metabolic phenotyping of urine from large sample cohorts. The rapid microbore metabolic profiling (RAMMP) approach was based on scaling a conventional reversed-phase UPLC-MS method for urinary profiling from 2.1 x 100 mm columns to 1 x 50 mm columns, increasing the linear velocity of the solvent, and decreasing the gradient time to provide an analysis time of 2.5 min/sample. Comparison showed that conventional UPLC-MS and rapid gradient approaches provided peak capacities of 150 and 50 respectively, with the conventional method detecting approximately 19,000 features compared to the ca. 6000 found using the rapid gradient method. Similar levels of repeatability were seen for both methods. Despite the reduced peak capacity and the reduction in ions detected, the RAMMP method was able to achieve similar levels of group discrimination as conventional UPLC-MS when applied to rat urine samples obtained from investigative studies on the effects of acute 2-bromophenol and chronic acetaminophen administration. When compared to a direct infusion MS method of similar analysis time the RAMMP method provided superior selectivity. The RAMMP approach provides a robust and sensitive method that is well suited to high-throughput metabonomic analysis of complex

1 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mixtures such as urine combined with a fivefold reduction in analysis time compared with the conventional UPLC-MS method.

Keywords Metabolic phenotyping, high-throughput analysis, metabolic profiling, xenometabolome, metabonomics, metabolomics

INTRODUCTION Increasingly untargeted metabolic phenotyping (metabotyping), of the type performed in metabonomic/metabolomic studies, is being applied to large scale investigations, often comprising several thousands of samples obtained in preclinical metabolism/toxicological1,2, clinical and epidemiological investigations3,4,5. In pursuit of the analysis of these samples ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) has become an indispensable tool for exploratory metabolic phenotyping6,7 as it provides 3-5 fold increase in throughput compared to conventional HPLC-MS. However, even with the combination of sensitivity, reproducibility and throughput provided by UPLC-MS, there remains an unresolved tension between the desire to have both rapid analysis and comprehensive global metabolite coverage. Currently UPLC analysis times of 10-20 min/sample are used to provide a compromise between throughput and the number of features detected8,9. Faster analyses can be achieved by eliminating the chromatographic separation using direct infusion/injection of samples into the MS (DIMS)10,11,12. However, although data acquisition via these approaches is fast, DIMS data processing and interpretation is a time-consuming and complicated step due to the presence of multiple components such as molecular ions, adducts, in-source fragments and multiply charged species, all present in the same spectrum. In addition, DI methods remain prone to ion suppression/enhancement effects and are unable to distinguish isomeric species. Whilst DIMS is undoubtedly useful as a rapid diagnostic technique, LC-MS, particularly UPLC-MS, still provides a more comprehensive metabolic phenotyping tool13,14,15. A halfway house between the current UPLC-MS profiling methods and DIMS is to use short chromatographic analysis times, thereby ameliorating some of the disadvantages of DIMS, whilst accepting the loss of some metabolome coverage that inevitably results from reduced chromatographic resolution. Indeed, an early study on the metabolite profiling of rodent urine that compared short chromatographic separations with “conventional” UPLC-MS-based methods clearly showed the viability of this approach, maintaining class separation even with a reduction in the number of features detected16. 2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Faced with the need for higher sample throughput, we have therefore reinvestigated the use of this type of rapid UPLC-MS, but in addition have combined the approach with microbore LC, which we have recently shown to combine the robustness required for large scale LCMS-based metabotyping with enhanced sensitivity and reduced solvent usage17,18,19. This was undertaken with the aim of developing methodology that provides similar performance to conventional LC that, when applied to screening large batches of samples of rodent urine, also reduces analysis time, cost per sample and environmental impact whilst providing the same results in terms of major biomarkers.

Here we evaluate the utility of a rapid microbore metabolic profiling (RAMMP) method based on the use of a microscale reversed-phase UPLC separation, coupled to a fast scanning high resolution accurate QToF MS. The samples were obtained from rats administered with either a single, acute, dose of 2-bromophenol or chronic administration of acetaminophen (7 days) from studies that formed part of those undertaken by the Consortium of Metabonomic Toxicology (COMET)20,21. The positive ion mode results of the RAMMP methodology are also compared with the outcomes obtained via both “conventional” UPLC-MS and chipbased nano-electrospray DIMS analysis.

EXPERIMENTAL Chemicals and Reagents. Optima grade LC-MS water was purchased from Fluka (Leicester, UK). Acetonitrile (LC-MS grade), methanol (LC-MS grade), formic acid (LC-MS grade), leucine enkephalin acetate salt hydrate and sodium formate solution were purchased from Sigma Aldrich (Gillingham, UK).

Study Details and Sample Preparation Acute 2-Bromophenol Study.

Urine samples were collected from three groups of male

Sprague-Dawley rats (10 rats/group) administered a single intraperitoneal dose of 2bromophenol at either 100 mg/kg or 200 mg/kg in corn oil. A control group was treated with corn oil alone. Rats were housed in metabolic cages to enable the collection of urine and samples were taken at -16, 0, 8, 24, 48, 72, 96, 120, 144 and 168 hrs after administration. The urine samples were stored at -40 °C prior to analysis. A 120 µL aliquot of urine was mixed with 120 µL water to dilute the salt concentration (before protein removal with acetonitrile (1:3 v/v). These samples were vortex mixed and left at -20 °C overnight before centrifugation for 5 min at 15,000 g at 4 °C. For analysis, 50 3 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

µL was removed and added to 250 µL water in 350 µL 96-well plates. A pooled quality control (QC) sample was prepared by combining 100 µL of each sample and diluting 1:4 with water8,22,23. The 96-well plates were stored at -20 °C until analysis and the plates were centrifuged again for 5 min at 700 g before being placed into the autosampler at 4 °C. Chronic Acetaminophen Study. Urine samples were collected from four groups of male Sprague-Dawley (10 rats/group) rats dosed orally with acetaminophen (APAP) by gastric intubation once daily, for seven days. The rats were treated with 200 mg/kg, 400 mg/kg or 800 mg/kg in 0.2% carboxymethyl -cellulose and a control group was treated with 0.2% carboxymethylcellulose alone. The animals were housed in metabolic cages and urine was collected for metabonomic analysis on at -16, 0, 8, 24, 32, 48, 56, 72, 96, 80, 104, 120, 144, 168, 192, 216, 240, 264, 288, and 312 hrs after administration. Samples were stored at 40 °C prior to analysis. For LC-MS a 20 µL aliquot of each urine sample was mixed with 60 µL methanol for protein removal (methanol was used for this study, rather than acetonitrile, because of occasional instances of phase separation that were observed when the latter was used). The samples were vortex mixed and left at -20 °C overnight before centrifugation for 5 min at 15,000 g at 4 °C. For analysis, 20 µL was removed and added to 180 µL water in 250 µL 384-well plates. A pooled quality control (QC) sample, used for system conditioning and assessment of analytical performance was prepared by combining 5 µL of each sample and diluting 1:9 with water8,22,23. The well plates were centrifuged again for 5 min at 700 g before being placed into the autosampler at 4 °C. For DIMS analysis 20 µL of both protein precipitated rat urine samples and the pooled QC sample were diluted with ultrapure water by a factor of 80. An aliquot of 50 µL of each diluted sample was pipetted, in a randomized order, into the 96 well-plates followed by 100 µL of ultrapure methanol/0.1% formic acid (v/v) to give a sample of 1:2 water-methanol. The sample plates were sealed with foil and then centrifuged at 1500g and 4 °C for 10 min before DIMS analysis. Liquid Chromatography-Mass Spectrometry (LC-MS) and Direct Infusion-Mass Spectrometry (DIMS) Liquid Chromatography. Liquid chromatographic analysis was performed on an Acquity Iclass UPLC system, equipped with a binary solvent manager, sample manager and column heater (Waters Corp., Milford, MA, USA), interfaced with a Synapt G2-S HDMS mass spectrometer (Waters Corp., Wilmslow, UK). The chromatographic separations were performed on a HSS T3 1.8 µm stationary phase of either 2.1 x 100 mm for the conventional 4 ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

analysis or 1 x 50 mm for the RAMMP analysis (Waters Corp., Milford, MA, USA). The chromatographic mobile phase was composed of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). The column temperature was maintained at 40 °C and linear gradient elution was performed at either 0.5 mL/min for the 2.1 x 100 mm column or 0.4 mL/min for the 1 x 50 mm column. For the conventional method the starting composition was 1 % B, held for 1.0 min before increasing to 15 % at 3.0 min, 50 % at 6.0 min, 95 % at 9.0 min for a 1.0 min wash and returning to 1 % B for a 2 min re-equilibration step (total cycle time 12 min). A 2 µL injection of sample was performed using the flow through needle. For the RAMMP method the starting composition was 1 % B, held for 0.14 min before increasing to 15 % at 0.42 min, 50 % at 0.83 min, 95 % at 1.25 min for a 0.25 min wash and returning to 1 % B for a 1 min re-equilibration step (a total cycle time of 2.5 min). A 1 µL (2-bromophenol study) or 0.2 µL (acetaminophen study) injection of sample was performed using the flow through needle. In both cases the purge solvent was 95:5 (v/v) H2O/CH3CN and sample manager wash was CH3OH. For each study, prior to the analysis of the samples themselves 50 consecutive injections (2 µL volume) of the pooled samples were made at the start of the chromatographic run to ‘condition’ the column to ensure that the analytical system was fully equilibrated9,24. The QC sample was injected at the beginning of the analytical run and every 10 injections thereafter to monitor instrument stability. For use of 1 mm i.d. columns, minor system modifications were necessary, as described in detail elsewhere19, to reduce peak dispersion. Briefly, for the microbore separations the standard outlet tubing i.d. (0.004”) was reduced to 0.0025” of minimum length and the ESI stainless steel capillary (125 µm i.d.) was replaced with a narrow bore variant (50 µm i.d.). LC-Mass Spectrometry. Mass spectrometry for metabolic profiling was performed on a Synapt G2-S HDMS accurate mass instrument (Waters Corporation, Wilmslow, UK) operated with electrospray ionization (ESI) operated in positive (ESI+) ion mode. The capillary voltage was 1.0 kV, cone voltage was 25 V, source temperature was set at 120 °C with a cone gas (nitrogen) flow rate of 50 L/h, a desolvation gas temperature of 600 °C and a nebulization gas (nitrogen) flow of 1000 L/h. The Synapt G2-S was operated in resolution (V optics) mode and was set to acquire data over the m/z range 50-1200 with a scan time of 0.1 s for the conventional method or 0.05 s for the RAMMP method. All mass spectral data were collected in centroid mode using the MSe data acquisition25 function to obtain fragmentation data simultaneously. In function one a low collision energy (4 eV) was used and in the second function a high collision energy (ramp 15-45 eV) was used for fragmentation. For mass accuracy, leucine enkephalin (MW = 555.62) was used as a lock mass at a 5 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

concentration of 200 pg/µL (in 50:50 CH3CN/H2O, 0.1 % formic acid) infused at a flow rate of 20 µL/min via a lock spray interface. Lockmass scans were collected every 15 s and averaged over 3 scans to perform mass correction. The instrument was calibrated before analysis with 0.5 mM sodium formate solution. These data were collected using MassLynx V 4.1 software (Waters Corp., Manchester, UK).

Chip-Based Nanoelectrospray DIMS. Chip-based nanoelectrospray infusion analysis was performed using the TriVersa NanoMate system (Advion BioSciences, Ithaca, New York) coupled to a Waters Synapt® G2-S (Waters MS Technologies, UK). The nanoelectrospray was created and maintained by applying 1.4 kV high voltage and 0.8 psi nitrogen flow controlled by ChipSoft software (version 8.3.1). The sample plate temperature was maintained at 4 °C. The data were collected in resolution continuum mode with the scan time of 1 s over the mass range of 40 – 600 m/z in negative (ESI-) and positive (ESI+) ion modes with automatic polarity switch infusing 5 µL of sample. The MSe data independent acquisition function was used with the low collision energy of 4 eV and a high collision energy ramp 15-45 eV in the second function for fragmentation. The sampling cone voltage was set at 40 V, and the source offset at 80 V. Total data acquisition time was of 40 seconds for each ionization mode (first ESI-, and then ESI+) but the overall turnaround time for each sample was 2 minutes in order to let the instrument automatically switch the polarity and enable the voltage to settle before acquiring the data in the second ion mode. The data for the negative and positive mode were acquired in two separate files in the MassLynx™ software. The total time for the analysis of a 96 wellplate was 4.5 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 well-characterized endogenous metabolites present in all urine samples. MS/MS was performed on discriminant peaks in DIMS in resolution mode using a pooled urine sample. The optimal CID energy was selected for each peak between 10-30 eV. For the endogenous and drug-related metabolites the spectra were compared to the metabolite fragmentation patterns available in online databases (HMDB26 and Metlin27) and to the spectra acquired in-house using DIMS for the standards in neat methanol containing 0.1 % formic acid (v/v).

6 ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Data Analysis. The raw data obtained via UPLC-MS in positive ion mode were processed by Progenesis QI data analysis software (Non-Linear Dynamics, Newcastle, UK) for peak picking, alignment and normalization, to produce peak intensities for retention time (RT) and m/z data pairs. In the case of DIMS, basic data visualization and quality control was achieved using MassLynx 4.1 software (Waters Corporation, UK). For DIMS data analysis, the raw data (ESI+) were converted to the mzML format with ProteoWizard software28 followed by processing using in-house scripts as described previously29. In both cases further statistical analysis was performed on the resulting normalized peak intensities using SIMCA P 14.0 (Umetrics, Umea, Sweden).

RESULTS AND DISCUSSION As shown previously16, the use of a relatively short chromatographic run is likely to result in some loss of metabolome coverage compared to the conventional 12 min UPLC-MS-based method routinely used by us for metabolic profiling of urine9. Here we have employed a 1 mm i.d. column format19, previously optimized to be equivalent to our generic 2.1 mm i.d. separation9, and combined it (ensuring appropriate scaling of chromatographic factors) with a reduction in column length to 50 mm, an increased flow rate and a short 2.5 min gradient (with both 2.1 and 1.0 mm-based separations employing a gradient profile of 12 column volumes). This combination of column geometry, flow rate and reduced run time thereby enables significant gains in both sensitivity and reduced solvent consumption compared to the existing “conventional” 2.1 and 1 mm methods to be obtained. In order to evaluate the utility of the RAMMP method, and determine how it compared to the conventional UPLC-MS method, a subset of samples of rat urine from animals dosed with 2-bromophenol were analyzed using both approaches. Subsequent analysis of these data showed that, as would be expected, the use of the RAMMP method resulted in a decrease in the number of ions detected from 18,823 features seen using conventional UPLC-MS to 6,188. However, as shown in Figure 1, when principal component analysis (PCA) was performed, the higher throughput RAMMP analysis (shown in Figure 1B) gave a similar degree of discrimination between the dose groups as provided by the longer conventional method (shown in Figure 1A). So, although a higher number of detected metabolites may be seen as advantageous in metabolic phenotyping, with the chance of detecting potential biomarkers thereby increased, discrimination between groups may still be observed with fewer metabolites as seen when

7 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

comparing Figure 1A and B. In the case of both the conventional and RAMMP methods the QCs clustered tightly in the center of the PCA plots indicating good analytical stability for both platforms. Example chromatograms of the same urine sample, taken 24 hr post-dose from a high dose (200 mg/kg) animal, are shown in Figure 2 to illustrate the two types of separation. The chromatographic peaks generated using the RAMMP method had an average width at the base of 1.5 sec, which compares with the base width of 3.6 sec generated by the conventional method. Clearly however, the much shorter run time (2.5 vs 12 min) resulted in reduced peak capacity, going from ca. 150 for the conventional method to 50 in the case of the RAMMP analysis, and this is reflected in the reduced number of features detected, which parallels the reduced peak capacity to a similar extent. As the aim of the RAMMP method was to increase sample throughput, and thereby decrease analysis time for large sample numbers, the robustness and repeatability of the RAMMP assay was investigated by the repeated analysis of a single 96-well plate of rat urine samples from the 2-bromophenol study. This 96-well plate was profiled 5 times in a single analytical run and the pooled quality control (QC) sample, used to assess analytical variation throughout the analysis, analyzed every 10 injections. In addition, as has been discussed elsewhere8,22,24,30,31 column “conditioning”, whereby the LC-MS system is modified by the injection of matrix, was performed to stabilize retention times and signal intensities prior to the start of an analysis. Michopoulos et al.,31 have previously shown for human plasma that increasing the amount of matrix injected on column, and using a more rapid gradient for the conditioning phase, represents an efficient means of reducing the time required to achieve reasonable repeatability. Through the use of a rapid gradient, such as that investigated here, a large number of pooled matrix (QC) injections can be made to improve instrument stability without extending the length of the analysis significantly. Thus, to ensure that the best possible results were obtained, extensive column conditioning was employed before the analysis of the samples was commenced. In addition, in order to maximize system conditioning, following on from the work of Michopoulos et al.,31 the amount of sample injected on column was increased from the 1 µL of sample used for analysis to 2 µL, effectively doubling the number of conditioning injections. In this instance, 50 conditioning injections of the QC sample were performed prior to the commencement of sample analysis, with conditioning completed in ca. 2 hrs. Sample analysis was then performed immediately after the conditioning step. The high degree of similarity of the chromatograms obtained for the last QC sample injected, from each of the 5 replicate analyses of the 96-well plate (shown 8 ACS Paragon Plus Environment

Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

in Supplementary Information Figure S1) provides a good indication of the robustness of the assay for the ca. 500 injections made over the course of the analysis (ca. 20 hrs). The repeatability of the technique is further exemplified in Figure 3 by the PCA plot illustrating the agreement between the replicate analysis (n=5) of selected samples from the control animals in the 2-bromophenol study (this repeatability is also demonstrated in Table S1 for a number of ions selected from both QC samples and selected control animals). The QC samples are also useful in determining the reliability of a particular ion as a potential biomarker by examining the stability of factors such as retention time and response in each of the QC injections throughout the analysis. Assessing the coefficient of variance (CV) of the peak area/height of a particular ion across the QC samples gives an indication of how repeatable it is likely to be if used as a potential biomarker (although, clearly features selected in this way require identification and reanalysis by a validated and specific method which is then applied to study samples to confirm their utility as actual, rather than potential, biomarkers). As we have noted elsewhere, the acceptance criteria for reliability in exploratory metabolic phenotyping are still evolving, but using the QC samples to calculate ion intensity CV throughout a run is now a widely adopted technique, with a CV filter of between 15 and 30 % used to determine acceptable metabolite precision, depending on the rigor of analysis required32. Ion intensity stability is typically greater the more intense the ion (providing it is not saturating the detector) and those features that are subject to ion suppression/enhancement will show greater variation than those that are resolved from interfering matrix components. It might, therefore, be supposed that the conventional method offering, as it does, greater chromatographic resolution would also result in a higher proportion of stable features than the RAMMP method, where greater co-elution could result in greater potential for ion suppression/enhancement. Using the CV filter approach here to examine the data for the same set of samples run on the two LC-based analytical methods we observed that, for the conventional 2.1 mm i.d. separation, 89 % of the total features detected had a CV of