Method for absolute quantification of short chain fatty acids via reverse phase chromatography mass spectrometry

Short chain fatty acids (SCFAs; including acetate, propionate, and butyrate) are an important class of biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules in their native form by liquid chromatography mass spectrometry is challenging due to their relatively poor chromatographic properties. Herein, we introduce SQUAD, an isotope-based strategy for absolute quantification of SCFAs in complex biological samples. SQUAD uses aniline derivatization in conjunction with isotope dilution and analysis by reverse-phase liquid chromatography mass spectrometry. We show that SQUAD enables absolute quantification of biologically relevant SCFAs in complex biological samples with a lower limit of detection of 40 nM and a lower limit of quantification ranging from 160 nM to 310 nM. We observed an intra- and inter-day precision under 3% (relative standard deviation) and errors in intra- and inter-day accuracy under 10%. To demonstrate this quantification strategy, we analyzed SCFAs in the caecal contents of germ free versus conventionally raised specific pathogen free (SPF) mice. We showed that acetate was the most abundant SCFA in both types of mice and was present at 200-fold higher concentration in the SPF mice. We also illustrated the use of our quantification strategy in in vitro microbial cultures from five different species of bacteria grown in Mueller Hinton media. This study illustrates the diverse SCFA production rates across microbial taxa with acetate production serving as one of the key differentiating factors across the species. In summary, we introduce an isotope dilution strategy for absolute quantification of aniline-dativized SCFAs and illustrate the utility of this approach for microbiome research.

biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules by liquid chromatography mass spectrometry (LC-MS) is challenging due to their relatively poor chromatographic properties and low mass. Herein, we introduce SQUAD, a quantitative strategy for analyzing SCFAs using an aniline-based derivatization approach in conjunction with reverse-phase LC-MS/MS analysis. We show that this approach, when coupled to quantification by stable isotope dilution (SID), enables absolute quantification of biologically relevant SCFAs in complex biological samples. We evaluate the limits of detection using this approach, analyze sources of error affecting quantification, and provide practical guidelines for using this strategy. Moreover, we illustrate the utility of this technique via two representative biological applications where SCFA analysis is common: 1) SCFA quantification in the caecal contents of germ free versus conventionally raised specific pathogen free mice and 2) in an analysis of in vitro microbial cultures.

Main text
Short chain fatty acids (SCFAs) are produced by the fermentation of indigestible dietary fiber by the commensal gut microbiota. Acetate (Ac), propionate (Prop), and butyrate (But) make up the majority of SCFAs in the gut, and are present at various ratios throughout the mammalian gastrointestinal tract. [1][2][3] SCFAs are known to directly influence both the epigenome and the immune system, and play an important role in host energy balance. [4][5][6] They have thus become an important topic in microbiome-based research. Accurately quantifying the abundance of SCFAs is therefore an important objective for a wide transect of researchers investigating the interaction between gut microbiota and the host.
The analysis of SCFAs has been accomplished using a number of analytical techniques.
Traditionally, gas chromatography (GC) coupled to a number of detection platforms has served as the primary analytical strategy. [7][8][9][10] Though effective, these GC-based analytical approaches are not transferable to liquid chromatography mass spectrometry (LC-MS), which has become the preferred platform used by most metabolomics-oriented analytical facilities. Unfortunately, SCFA are challenging for LC-MS due to their low molecular weight. Acetate, for example, has a monoisotopic parent mass of 48 in negative ion mode, which is below the scanning range of orbitrap mass spectrometers. Moreover, SCFAs have poor chromatographic properties on most conventional chromatography columns (e.g. C18 and hydrophilic interaction liquid chromatography). These challenges have led several groups to develop alternative approaches for profiling SCFAs and other related molecules. [11][12][13][14] One of the main analytical strategies for making SCFAs compatible with LC-MS has been to chemically derivatize samples to improve the chromatographic separation and MS properties of SCFAs (e.g. parent mass and ionization efficacy). Two successful strategies have included derivatization by 3-nitrophenylhydrazines 12 and aniline. 15 Both methods produce high yields and enable SCFA analyses to be conducted on conventional C18 chromatography. These methods are also convenient because they are compatible with LC solvents and are conducted under relatively mild reaction conditions. Both of the established derivatization methods have shared an isotope-based normalization strategy in which standards are prepared independently from the test samples using an isotopeencoded derivatization reagent. These independently prepared standards are then mixed with the test samples prior to LC-MS analysis. The principle advantage of this isotope-based quantification strategy is that it corrects for MS-related variables 16 , and allows readily-available 12 C commercial molecules to be used as standards. Although this approach provides the greatest possible scope for metabolomics applications 12,15,17 , derivatizing standards independently from the test sample introduces several potentially significant sources of quantitative error. Most importantly, any differences in derivatization efficiency between standards and test samples will directly affect quantification. Moreover, standards added after the sample preparation process cannot correct for errors associated with extraction efficiency and sample handling. Although these sources of error can be controlled by carefully monitoring and optimizing the sample preparation process, these practices are challenging for routine biological applications.
The primary objective of this study was to develop a practical SCFA quantification method that corrects for the greatest scope of errors while minimizing experimental complexity.
Importantly, most biological studies related to SCFAs have focused on just three molecules (acetate, propionate, and butyrate) 18 , all of which are commercially available as 13 C-labeled standards. With this practical consideration in mind, we propose an alternative stable isotope dilution (SID) workflow 19,20 for absolute quantification of SCFAs in biological samples. The primary advantage of our workflow over the established methods is that isotope-labeled standards are added prior to any sample handling. This approach supports robust LC-MS quantification of SCFAs irrespective of differences in derivatization efficiency and sample handling. Herein we validate this method, measure limits of detection, investigate potential sources of experimental error, provide guidelines for conducting biological studies using this approach, and apply the method to two typical biological studies in which SCFA analysis is important.

Preparation of calibration curves
A standard mixed solution containing all 6 SCFA standards ( 12 C-and corresponding 13 Cstandards) at a final concentration of 1 mM each was prepared in H2O/ACN (50:50, v/v). A 100 L aliquot was derivatized as described above and was then diluted with H2O/MeOH (50:50, v/v) to prepare 15 solutions with a concentration range of 100 M to 20 nM.

Animal procedures
SPF C57BL/6(J) mice were bred and maintained in the specific pathogen-free facility at the University of Calgary Animal Resource Centre. Germ free C57BL/6 mice were bred and maintained in flexible film isolators at the International Microbiome Centre (IMC), University of Calgary. Germ-free status was routinely monitored by culture-dependent and -independent methods and all mice were independently confirmed to be pathogen-free. 10-12 week old mice were used in this study. Animals were humanely euthanized and caecal contents were removed via a longitudinal incision of the caecal wall. Contents were immediately snap-frozen in liquid nitrogen and stored at -80 °C until processed. All animal experiments were approved by the University of Calgary Animal Care Committee (protocols AC16-0235, AC17-0011, and AC17-0090) and were performed in accordance with the guidelines established by the Canadian Council for Animal Care.

Analysis of SCFAs in caecal samples
Caecal samples were removed from liquid nitrogen storage and placed on ice. Ice-cold extraction solvent (H2O/ACN (50:50, v/v) containing 13 C-SCFA internal standards (IS) was added to each sample, as detailed in Table S

Analysis of SCFAs in microbial cultures
All bacterial strains were clinical isolates acquired from Calgary Laboratory Services (CLS, Calgary) pathogen repository. Bacterial samples were grown in 96-well culture plates (Corning, New York, N.Y. USA) containing Mueller Hinton medium. Exponential phase bacterial cultures were used to seed medium to a starting OD600 ~ 0.07. Cultures were incubated in a humidified incubator (Heracell VIOS 250i Tri-Gas Incubator, Thermo Scientific, Waltham, Mass. USA) under a 5% CO2 and 21% O2 atmosphere for 4 hours. Growth was measured at OD600 (Multiskan GO, Thermo Fisher Scientific, Waltham, Mass. USA). After incubation, samples were transferred to a 96-well PCR plate (VWR), and centrifuged for 10 min at 4000 × g at 4 °C to remove cells.
Supernatant was removed, mixed 1:1 with 100% LC-MS grade MeOH, and either frozen at 80 °C for further processing, or centrifuged again for 10 min at 4000 × g at 4 °C to remove any protein precipitate. 20 µL of each supernatant were then dispensed into a 96-well plate. 5 µL of the IS solution in H2O/MeOH (50:50, v/v), with final concentrations of 2 mM for 13 C-acetate, 5 µM for 13 C-propionate, and 25 µM for 13 C-butyrate, was added to each well, followed by 1. Effect of the EDC molar equivalent on observed 12 C: 13

C ratio
Five different molar equivalents (0.1, 0.5, 1, 5 and 10) of EDC were each tested on six different 12 C/ 13 C SCFA standard mixed solutions with a 12 C: 13   Effect of the reaction time on observed 12 C: 13

C ratio
Six different 12 C/ 13 C SCFA standard mixed solutions with a 12 C: 13 C concentration ratio of 0.5, 0.8, 1, 1.25, 2 and 5 were prepared in H2O/ACN (50:50, v/v) (see Table S Effect of the reaction solvent on observed 12 C: 13

C ratio
Four different 12 C/ 13 C SCFA standard mixed solutions with a 12 C: 13  Effect of the reaction volume and assay format on observed 12 C: 13

C ratio
Four different 12 C/ 13 C SCFA standard mixed solutions with a 12 C: 13  Data analyses, on the converted mzXML files, were conducted in MAVEN 21,22 . In short, for each SCFA, the determination of the native SCFA concentration was based on the 12 C: 13 C signal intensity ratio and the respective 13 C-IS concentration.

Method for quantifying SCFAs by LC-MS/MS
A defined mixture of 12 C-and 13 C-labeled acetate, propionate, and butyrate standards (1 and 0.5 mM final concentration for each standard) was subjected to derivatization with aniline, as depicted in the reaction scheme ( Figure 1A) and submitted to LC-MS/MS analysis. As expected, the 13 C-labeled and unlabeled derivatized SCFA standards co-eluted ( Figure 1B), were easily distinguishable by m/z ( Figure 1C), and had peak intensities proportional to the relative concentrations of the 12 C: 13 C isotope ratios. To determine the linear range of detection for both 12 C-and 13 C-labeled aniline-derivatized SCFA standards using our LC-MS/MS method, calibration curves were generated using an equimolar standard solution. Linear relationships were apparent for 12 C-and 13 C-labeled acetate, propionate, and butyrate derivatized standards from 20 nM to 100 µM (Figure S-1; R 2 > 0.995 for all six standards). The slopes of the regression lines for all isotopologues were similar (4.24E+06 / 4.38E+06 for 12 C/ 13 C-Ac, 7.12E+06 / 8.16E+06 for 12 C/ 13 C-Prop and 1.01E+07 / 1.06E+07 for 12 C/ 13 C-But, Figure S-1). The limit of detection (LOD) for Ac, Prop and But were 156, 80 and 40 nM, respectively.

Sources of error affecting quantification
Derivatization-based quantification strategies are frequently subject to quantitative errors resulting from incomplete reactions, differences in reaction efficiencies between samples, and other chemistry-related variables. We used a stable isotope-based dilution strategy-referred to herein as SQUAD (SCFA Quantification Using Aniline Derivatization)-to correct for these sources of error. To assess the efficacy of SQUAD, we prepared 11 mixed solutions with 12 C-and   Although SQUAD can correct for most sources of experimental error, one important variable affecting these experiments is the ratio between 12 C-and 13 C-labeled analytes. To assess the impact these isotope ratios have on SCFA quantification, we prepared mixtures of standards with concentration ratios ranging from 0.001 to 1000 and quantified observed versus expected concentrations for each compound to calculate error. We found that isotope ratios between 0.5 and 20 were necessary to maintain absolute error below 25% (Figure 3). 12 C: 13 C mixing ratios below 0.5 overestimate isotope ratios whereas 12 C: 13 C mixing ratios above 20 underestimate the actual ratio ( Figure 3, insets). These findings indicate that a two-stage workflow may be necessary for robust quantification of SCFAs in biological samples: the first stage used to establish rough concentration ranges for each analyte and the second with mixing ratios between 0.5 and 20 for robust quantification. Error bars indicate standard deviation, n = 3 technical replicates.

Quantification of microbial SCFA production in mouse caecum and in vitro cultures
One of the primary biological drivers for SCFA analyses is in the microbiome research, where microbial-derived SCFAs can have a significant impact on host biology. To test the biological applicability of SQUAD, levels of SCFAs in the caecal contents of germ free (GF) and specific pathogen free (SPF) mice were analyzed. Since gut microbes are the primary producers of SCFAs in mammals, we anticipated that GF mice would show minimal SCFA content relative to their SPF counterparts 23 . To test this, caecal contents were collected and SCFAs were extracted with a solvent spiked with 13 C-standards. Caecal extracts were then derivatized, analyzed by LC-MS/MS, and native SCFA levels were quantified. As predicted, the caecal extract of GF mice showed low levels of SCFAs. Acetate concentration was found to be 100 µM in the extract whereas propionate and butyrate had concentrations approaching the limit of detection. In contrast, the caecal contents of SPF mice contained all three SCFAs at over 200 fold greater levels than observed in GF mice ( Figure 4A). In SPF mice, the relative abundance of these SCFAs followed a 4:1:1 ratio for acetate, propionate and butyrate, respectively. Both the higher abundance of SCFAs in SPF mice and the relative ratios of SCFAs we report here match the patterns reported elsewhere. 24,25   Figure 4B).
These data highlight the flexible nature of SQUAD and the ability to adapt this method to diverse biological applications. To facilitate the use and adaption of this method to other applications, we have provided practical guideline for SQUAD analysis in the supplemental materials.

Conclusion
Herein, we introduce SQUAD, an isotope-based strategy for absolute quantification of SCFAs in complex biological samples. The primary advantage of SQUAD is that it is not sensitive to incomplete derivatization and other chemistry-related variables that affect existing SCFA quantification methods. We demonstrate that SCFAs can be quantified with less than 25% error provided that 12 C: 13 C mixing ratios are maintained between 0.5 and 20. Moreover, we demonstrate that the SQUAD approach is readily amenable to a diverse range of biological applications.
Although the SQUAD approach we present here is primarily focused on SCFA quantification, our underlying aniline derivatization approach is applicable to a wide range of biomolecules containing carboxylic acids. 17           Dilute sample(s) as needed with 50% H2O/MeOH (v/v) before LC-MSMS analysis.

Biological considerations:
SCFAs levels can span many orders of magnitude between biological groups (e.g SPF versus GF mice). Pilot projects should include each condition to calibrate isotope spiking ratios. download file view on ChemRxiv Supporting information for publication.pdf (0.98 MiB)