Quantitative Determination of Short-Chain Fatty Acids in Human Serum by LC-MS

Title: Sensitive and quantitative determination of short-chain fatty acids in human serum using liquid chromatography mass spectrometry

Journal: Analytical and Bioanalytical Chemistry

Published: 2021

Background

Short-chain fatty acids (SCFAs) are the end products of fermentation of dietary fiber by intestinal microbiota. These metabolites are linked to gut health and disease, and their levels are typically measured in feces, but the determination of SCFAs in serum is more challenging due to their low concentrations. SCFAs such as acetic acid, propionic acid, and butyric acid are crucial for health as they act as substrates or signal molecules affecting both gut and systemic health. Although SCFAs can be easily quantified in feces, their lower concentration in serum requires sensitive methods for accurate measurement. The study presents a method for determining eight SCFAs in human serum using liquid chromatography tandem mass spectrometry (LC-QQQ-MS), addressing challenges like isomeric interferences and low recovery.

Materials & Methods

Chemicals and Reagents

LC-MS-grade water and acetonitrile (ACN): Thermo Fisher Scientific (Sydney, Australia).
Butyric acid-D7: Cambridge Isotope Laboratories (Cambridge, MA, USA).
SCFAs and reagents: Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 4-methyl valeric acid, hexanoic acid, 13C2-acetic acid, 2-ethylbutyric acid, MTBE, 3-NPH.HCl, EDC.HCl, and pyridine were purchased from Sigma-Aldrich (Sydney, Australia).

Standards and Internal Standards

Stock Solutions: Prepared in 20% methanol/water at different concentrations.
Calibration Standards: Mixed standards were prepared in the range 0.015–25 μg/mL.
Quality Control Standards: QC standards at three concentration levels.
Internal Standards: 13C2-acetic acid, D7-butyric acid, and 2-ethylbutyric acid, prepared in acetonitrile.

Serum Samples

Blood was collected from healthy volunteers in SST vacutainers, allowed to clot, and centrifuged. The serum was stored at -80 °C.

Sample Preparation for LC-MS

Thawing and Mixing: Serum was thawed, mixed with internal standards in ACN, and placed on ice.
Phase Separation: ACN was separated by centrifugation, and water was added to the supernatant.
Derivatization: SCFAs were derivatized with 3-NPH.HCl and EDC.HCl.
Extraction: SCFAs were extracted with MTBE, dried under nitrogen, and reconstituted in 10% acetonitrile/water.
Blanks and Standards: Calibration standards and blanks were prepared similarly.

Sample Preparation for GC-HRAM-MS

Thawing and Internal Standards: Serum was thawed, mixed with internal standards, and de-proteinated using sulfosalicylic acid.
Extraction: SCFAs were extracted with cold MTBE, vortexed, and centrifuged. The organic layer was back-extracted into water.
Sample Preparation: Samples were adjusted with phosphoric acid and transferred to GC vials.

Instrumentation

LC-HRAM-MS

System: Thermo Scientific Ultimate 3000 LC coupled with Q Exactive Focus Orbitrap MS.
Columns: ACE C18-PFP and Thermo Hypersil aQ.
Mobile Phase: Water (A) and acetonitrile (B).
Flow Rate: 0.2 mL/min and 0.3 mL/min; column temperature: 30 °C or 45 °C.

GC-HRAM-MS

System: Thermo Fisher Scientific Q Exactive GC-MS.
Injection: 1 μL using a split/splitless injector.
Carrier Gas: Helium at 1.0 mL/min.
Column: Thermo Fisher Scientific TG-WAXMS A.
Temperature Ramp: 50 °C to 230 °C, with electron ionisation (EI) at 65 eV.

LC-QQQ-MS

System: Thermo Scientific Ultimate 3000 LC coupled with TSQ Quantiva Triple Quadrupole MS.
Detection: Negative ionisation in MRM mode.

Method Validation

Linearity, LOD, and LOQ: Calibration curves were constructed, and LOD/LOQ were based on signal-to-noise ratios of 3 and 10, respectively.
Precision, Trueness, and Accuracy: Precision was evaluated by intra- and inter-day analysis, while accuracy was tested through spike recovery in serum samples.

Results

Chromatographic Separation of SCFAs

The pooled serum samples were derivatized to form 3-nitrophenylhydrazones and separated on a Thermo Hypersil aQ column. High-resolution Orbitrap mass spectrometry (HRAM-MS) in full scan (FS), selected ion monitoring (SIM), and parallel reaction monitoring (PRM) modes were used for analysis. The quantification of SCFAs was achieved through a one-point calibration with a standard solution and internal standards (13C2-acetic acid, butyric acid-D7, and 2-ethylbutyric acid). The three modes (FS, SIM, and PRM) showed good agreement for most analytes, except for propionic acid, which exhibited a discrepancy in PRM mode due to isobaric interference. In PRM, quantification was determined by fragment ions, enhancing selectivity and resolving co-eluting background ions.

Chromatographic and Mass Spectrometry Results

The Hypersil aQ column successfully resolved SCFAs, including several isobaric or isomeric compounds. However, unresolved interference was detected for acetic acid, visible as a shoulder on the peak. Several column modifications, including an ACE C18-AR column, were tested to resolve the acetic acid interference. This approach successfully isolated acetic acid, allowing for accurate quantification of the interference, which varied across samples.

Gas Chromatography (GC) vs. Liquid Chromatography (LC) Results

SCFAs were also analyzed using GC-HRAM-MS to check for unresolved interferences in the LC method. Although both methods showed good agreement (± 20%) in SCFA concentration, discrepancies were observed for isovaleric acid and isobutyric acid, with GC reporting higher concentrations. This suggested that the interferences in LC-MS were not present in GC-MS and were contributing to the analyte signals.

Transition from HRAM-MS to Tandem MS

The HRAM-MS method was transferred to a lower-resolution triple quadrupole (QQQ) instrument for routine analysis. Excellent agreement (95-106%) was obtained between HRAM-MS (PRM mode) and QQQ (MRM mode) for acetic acid, propionic acid, and isobutyric acid. However, for minor SCFAs such as valeric acid and 4-methyl valeric acid, the agreement was lower (75-131%), particularly for lower concentrations. This highlighted the advantage of HRAM-MS in method development for robust quantification in complex matrices.

Method Validation and Precision

An eight-point calibration curve demonstrated good linearity (r² > 0.999) for all analytes, with limits of detection (LODs) and limits of quantitation (LOQs) well within the range of detected concentrations in the serum samples. The method showed excellent precision, with intra- and inter-day CVs ranging from 0.34% to 12.58%, and trueness within ± 20%. Retention time repeatability was also excellent, with %RSDs as low as 0.02%.

Accuracy and Recovery

The accuracy of the method was assessed through recovery studies, with recoveries for SCFAs in serum ranging from 93.97% to 113.81%, which compares favorably with other methods. This demonstrates the robustness and reliability of the developed LC-MS method for SCFA quantification in serum.

Overlay of chromatograms for fatty acids in a standard mix and serum sample, showing data acquired in PRM mode on a Thermo Hypersil aQ column, with some interference. Overlay of extracted ion chromatograms for various fatty acids in a standard mix and pooled serum sample, acquired in PRM mode on a Thermo Hypersil aQ column.

Overlay of chromatograms for fatty acids in a serum sample, showing acetic acid interference. Overlay of extracted ion chromatograms for fatty acids in a pooled serum sample, separated on an ACE C18-AR column in PRM mode.

Extracted ion chromatograms of MRM transitions (236 > 137 and 236 > 152) monitored for (1) isovaleric acid and (2) valeric acid on an ACE C18-AR column in MRM

Reference

Shafaei, Armaghan, et al. "Sensitive and quantitative determination of short-chain fatty acids in human serum using liquid chromatography mass spectrometry." Analytical and Bioanalytical Chemistry 413 (2021): 6333-6342. https://doi.org/10.1007/s00216-021-03589-w

* For Research Use Only. Not for use in diagnostic procedures.

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