Development and validation of a GC-FID method for the analysis of short chain fatty acids in rat and human faeces and in fermentation fluids

https://doi.org/10.1016/j.jchromb.2020.121972Get rights and content

Highlights

  • Simple method to quantify short chain fatty acids in biological samples by GC-FID.

  • Number of extractions to be performed was evaluated.

  • Method validation (recovery, sensitivity, repeatability, etc.) gave good results.

  • Method is adequate also when small quantity of sample is available (down to 20 mg)

  • Method was applied to human, mice, rat faeces and fluid fermentation samples.

Abstract

Short-chain fatty acids (SCFAs) are gut microbiota metabolites recognized for their beneficial effects on the host organism. In this study, a simple and rapid sample preparation method combined to SCFAs analysis by direct injection and gas chromatography coupled with flame ionization detection (GC-FID), for the determination and quantification of eight SCFAs (acetic, propionic, i-butyric, butyric, i-valeric, valeric, i-caproic and caproic acids) in rat, mice and human faeces and in fermentation fluids samples, has been developed and validated. The method consists of extraction of the SCFAs by ethyl ether after acidification of the samples. The effect of the number of extractions has been assessed in order to optimize the procedure and to obtain a satisfactory yield for all the analyzed SCFAs. The increase of the extracted analytes quantity was significant passing from 1 to 2 and from 2 to 3 extractions (P < 0.05), while no significant differences were found performing 3, 4 or 5 extractions (P > 0.05). The SCFAs extracted are directly analyzed by GC-FID without derivatization and separated on a polyethylene glycol nitroterephthalic acid modified coated capillary column, with a chromatographic run time of 13 min. The proposed method showed good sensitivity, with limits of quantifications in the range 0.14–0.48 µM for SCFAs from propionic to caproic acids and 2.12 µM for acetic acid; recovery was between 80.8 and 108.8% and intraday and interday repeatability in the range 0.6–5.0% of precision (RSD, %) The optimized method is suitable for the quantitative analysis of SCFAs in real samples of rat, mouse and human faeces and in fermentation fluids, and it can be applied also to very small amount of faecal sample (20 mg).

Introduction

The relation between the health state of an animal organism and the intestinal content of short chain fatty acids (SCFAs) is providing continuously evidences of their beneficial effects. SCFAs are organic acids with an aliphatic chain of up to 6 carbons, produced by the gut microbiota in the large bowel from the fermentation of the unabsorbed components of food. The most abundant SCFAs produced are acetic (C2), propionic (C3) and butyric (C4) acids, representing 90–95% of the total SCFAs in the colon [1].

They derive essentially from carbohydrates, but some amino acids (especially valine, leucine and isoleucine) can be converted into branched-chain SFCAs, such as isovalerate or isobutyrate, that contribute slightly to the total amount of SCFAs [2]. In the last decades they received great attention because they seem to play an important role in the prevention and the treatment of some diseases, such as bowel disorders, colon cancer and metabolic problems [3]. In fact, the amount of SCFAs in the faeces can be used as a biomarker of the health status of an individual, because they depend on the gut microbiota status, the intestinal transition time and mostly on the diet. Both long-term and short-term diets can affect the human gut microflora. Specifically, it was noticed that a diet rich in fiber and low in fat brings to higher amounts of faecal SFCAs [2]. The presence of SCFAs in the intestinal tract has several advantages, for instance they reduce the luminal pH, favoring the nutrients absorption and reducing the formation of some pathogenic microorganisms. It is also known that SCFAs prevent colon cancer and adenoma development. In particular, butyrate is responsible for the apoptosis and the contrast of tumor cell progression, while acetate and propionate show a lower antiproliferative activity against carcinogenic cells [4]. Furthermore, butyrate and propionate control the intestinal inflammation, preventing inflammatory bowel diseases, such as colitis or diarrheas [4]. Acetic acid, in the liver, is converted to acetyl-CoA, a precursor of lipogenesis that also stimulates gluconeogenesis, while propionate inhibits the process and seems to reduce plasma cholesterol concentration [5].

The characterization of the SCFAs is important also in other fields, such as in the evaluation of the quality of food. In fact, volatile fatty acids affect the aromas and thus food quality. Acetic, propionic and butyric acid can be generated by fermentation during the production or the storage of some foods where their concentrations give important indications. For instance, acetic acid in wine is important in the formation of various acetate esters, producing fruity flavors. But, when it is present in a concentration higher than 0.05 g per 100 mL of wine, acetic acid is responsible of the vinegar defect [6]. Another case is the presence of butyric acid in cheese. A high concentration of this volatile organic acid can be responsible for the rancidity defect [7].

SCFAs analysis has been performed by several techniques, such as gas chromatography (GC) [8], [9], high-performance liquid chromatography (HPLC) [10], [11], capillary electrophoresis (CE) [12], [13] or nuclear magnetic resonance (NMR) [14], [15]. Among these, GC remains the most commonly used technique because of the volatility of SCFAs and because of the high sensitivity, good resolution and relatively low cost of the technique and of the sample pre-treatment. The GC can be coupled to a flame ionization detector (FID) [16], [17], [18], moreover, the coupling of the GC with a mass spectrometer (MS) that can further enhance the selectivity and sensitivity of the method. Before the SCFAs analysis, sample pre-treatment usually encompasses extraction [19] and in some cases purification and/or derivatization [20], [21], [22]. Physical pretreatments, such as filtration [23], [24], ultrafiltration [25], [26] or centrifugation [27], [28] can be also exploited to reduce the presence of contaminants from the sample matrix. Other separation techniques are steam distillation [29] and vacuum distillation [30], [31], but they are time consuming and the possibility of losing volatile acids is high. Acidification allows SCFAs to be present mainly in their undissociated form, thus increasing both their hydrophobicity (as compared to the dissociated forms) and their volatility and thus facilitating their extraction by means of an apolar solvent [32], [16] or also by means of a solid–phase microextraction (SPME) device exploiting in this case their volatility [33], [34]. Another possible step in the sample pretreatment is the derivatization, which reduces SCFA polarity and improves their volatility and stability, making them more suitable for the GC analysis and eliminating also the need of using specific stationary phases of the chromatographic column. However, derivatization requires longer time for sample preparation, introduces new deviations in the analysis and requires the use of organic solvents and reagents, which sometimes are toxic and not eco-friendly, such as chloroformates [35], [36]. Alternatively to the extraction with different solvents, a solvent-less extraction technique can be applied, such as SPME. The SPME extraction allows a higher sample purity and a longer life span of the chromatographic systems, but it shows some critical points too, e.g. the fibers are not equally sensitive to all the compounds, but they show an absorption selectivity and they are not always reliable for a quantitative use [37]. Then, the SPME technique generally needs longer time for the extraction as compared to a classical solvent extraction, thus losing its convenience especially when there is a huge number of samples to be analyzed. For example, Olivero et al. [38] proposed a conditioning time of 10 min and an exposure of the fiber of 60 min, Noventa et al. [39] a conditioning time of 10 min and an exposure time of 40 min, while Fiorini et al. [33] employed 15 min of conditioning and 30 min of exposure.

In the present study it was aimed to develop and validate a GC-FID method for the determination of eight SCFAs in biological samples such as faeces and fermentation fluids, applying an easy sample preparation procedure, without any derivatization. In order to optimize the extraction procedure, the assessment of the number of extractions, from 1 to 5, was performed to identify the best conditions for all of the analytes, considering that the broad range of polarity of the analytes causes a different partitioning between water and ethyl ether and thus a different extraction extent for each of them in each extraction step. After that, the method was validated by assessing linearity, limits of quantification and detection (LOQ and LOD, respectively), recovery and interday and intraday repeatibility. The proposed procedure was then applied to rat, mouse and human faecal samples and fermentation fluids, where the content of SCFAs is important to determine the overall status of health of the individuals.

Section snippets

Standards, reagents and solvents

The analytical standards acetic acid (C2, purity ≥ 99%), propionic acid (C3, purity ≥ 99.5%), i-butyric acid (iC4, purity ≥ 99%), n-butyric acid (C4, purity ≥ 99%), i-valeric acid (iC5, purity ≥ 98%), n-valeric acid (C5, purity ≥ 99%), i-caproic acid (iC6 purity ≥ 98%) and n-caproic acid (C6, purity ≥ 98%) were purchased from Sigma–Aldrich (Milan, Italy). Sulfuric acid was purchased from Carlo Erba (Milan-Italy) and ethyl ether from J.T. Baker (Phillipsburg-New Jersey- USA). Water used in this

Method procedure

The sample preparation employed in the method here presented suspends the sample in aqueous sulfuric acid and then performs three subsequent extractions with ethyl ether. SCFAs extracted in the collected organic phase are directly analysed by gas chromatography without any derivatization. The previous acidification is important to move the dissociation equilibrium toward the undissociated forms thus incrementing the concentration of the extracted SCFAs in the organic phase [17], [41]. Several

Conclusions

The proposed method for the accurate identification and quantification of a total of eight SCFAs in faecal samples and fermentation fluids allows to perform the analysis with a relatively short time, of about 25 min, exploiting an easy sample pretreatment and simple and commonly available instrumentation (GC-FID). The extraction method separates SCFAs from the other compounds present in the matrix, avoiding the need of extra sample treatment and can be applied successfully also to small amount

CRediT authorship contribution statement

Serena Scortichini: Visualization, Investigation, Software, Writing - review & editing. Maria Chiara Boarelli: Supervision, Validation. Stefania Silvi: Conceptualization, Methodology, Data curation, Writing - original draft. Dennis Fiorini: Conceptualization, Methodology, Data curation, Writing - original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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