H OH metabolites OH Article A Facile Profiling Method of Short Chain Fatty Acids Using Liquid Chromatography-Mass Spectrometry Ha Eun Song 1, Hyo Yeong Lee 1, Su Jung Kim 1, Sung Hoon Back 2 and Hyun Ju Yoo 1,* 1 Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea 2 School of Biological Sciences, University of Ulsan, Ulsan 44610, Korea * Correspondence: [email protected]; Tel.: +82-02-3010-4029 Received: 27 June 2019; Accepted: 23 August 2019; Published: 28 August 2019 Abstract: Short chain fatty acids (SCFAs) are the main products of dietary fibers that are not digested by the human body, and they have been shown to affect human metabolism and inflammation. The amount of SCFAs in the body is related to many human diseases, and studies have focused on elucidating their roles and target molecules in both metabolic and immune responses. Thus, the quantitation of SCFAs in biological samples becomes crucial in understanding their important roles in the human body. Herein, a facile profiling method of SCFAs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and then applied to biological samples. C2-C6 SCFAs were derivatized while using 4-acetamido-7-mercapto-2,1,3-benzoxadiazole for 5 min. at room temperature prior to LC-MS/MS analysis, and characteristic fragmentation patterns and increased hydrophobicity after chemical derivatization enabled specific discrimination among 12 SCFAs. Derivatization was fast and reliable, and the reaction products were stable for a week at 4 ◦C. The developed method was applied to measure SCFAs in mouse feces, plasma, and human exhaled breath condensates. This fast and simple method can save labor and effort to profile SCFAs from various biological samples. Keywords: short chain fatty acids; 4-acetamido-7-mercapto-2,1,3-benzoxadiazole; chemical derivatization; exhaled breath condensate; feces; plasma 1. Introduction Short chain fatty acids (SCFAs) contain less than six carbons, and they are mainly produced by the fermentation of dietary fibers in the human body [1]. One of the major roles of the gut microbiota is to help catabolize dietary fibers into SCFAs. SCFAs are taken up by the host and are used as energy sources or regulators [2]. The main SCFAs are acetate (C2), propionate (C3), and butyrate (C4), and they constitute 95% of total SCFAs. Straight chain SCFAs are derived from dietary fibers, while the branched chain SCFAs are derived from catabolism of branched chain amino acids [2]. SCFAs are metabolized at various sites in the body, transported from the intestinal lumen into the blood, and found in various tissues. Recent studies demonstrated that the gut microbiota plays an important role in regulating host metabolism and immune responses [3,4]. Thus, measuring the type and amount of SCFAs is important for understanding their roles in complex biological systems. Mass spectrometry (MS) has grown in popularity as an analytical method for the determination of various biomolecules, and it is frequently used in combination with separation techniques, such as GC or HPLC, because biological samples are too complex for direct analysis by MS alone. SCFAs have been derivatized by methyl-, ethyl-, and propyl-chloroformate, as well as trimethylsilylation, and determined by GC-MS [5–8]. Liquid chromatography-mass spectrometry (LC-MS) has been often used in metabolomics studies with minimal sample preparation as compared with GC or GC-MS [9,10]. Metabolites 2019, 9, 173; doi:10.3390/metabo9090173 www.mdpi.com/journal/metabolites Metabolites 2019, 9, 173 2 of 11 However, the quantitation of SCFAs without chemical derivatization requires harsh experimental conditions in LC-MS, such as an aqueous mobile phase containing 1.5 mM hydrochloric acid [11]. In addition, their hydrophilicity results in poor chromatographic separation and insufficient ionization in electrospray ionization (ESI) [11]. Thus, it was difficult to detect SCFAs by LC-MS, because their masses were in the lower mass range in mass spectra, where numerous interfering peaks from solvents and additives were present. To overcome these problems, several chemical derivatization methods have been introduced to quantify SCFAs while using LC-MS. However, these derivatization requires longer reaction time or specific reaction condition. SCFAs were derivatized with 12C- or 13C-labeled aniline and analyzed while using a reversed-phase LC column, where derivatization was performed for 2 h at 4 ◦C, and quenching was necessary to avoid unintended reactions [12]. In other studies, optimal reaction condition for derivatization of SCFAs with 3-nitrophenylhydrazine and O-benzylhydroxylamine was determined in 30 min. at 40 ◦C and 1 h at 25 ◦C, respectively [13,14]. In the present study, we aimed to develop a simple profiling method of SCFAs from various biological samples. SCFAs were derivatized with 4-acetoamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH) for 5 min. at room temperature. Derivatization reaction condition was optimized, and the performance of this method was evaluated with standard solutions and biological samples. 2. Materials and Methods 2.1. Materials Acetic acid (C2), propionic acid (C3), butyric acid(C4), isobutyric acid (C4; 2-methylpropionic acid), 2-methylbutyric acid (C5), isovaleric acid (C5; 3-methylbutyric acid), valeric acid (C5; pentanoic acid), 2,2-dimethylpropionic acid (C5), caproic acid (C6;hexanoic acid), 2,2-dimethylbutyric acid(C6), 2-ethylbutyric acid (C6), and 2-methylvaleric acid (C6; 2-methylpentanoic acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isotope-labeled internal standards, including acetic acid-d3 (C2-2,2,2-d3), propionic acid-d6 (C3-d6), butyric acid-d7 (C4-d7), valeric acid-d4 (C5-2,2,3,3-d4), and caproic acid-d5 (C6-5,5,6,6,6-d5), were purchased from Sigma-Aldrich (St. Louis, MO). All of the stock solutions were prepared in water and stored at 20 C. − ◦ 4-Acetoamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Triphenylphosphine (TPP), 2,2’-dipyridyl disulfide (DPDS), and other reagents, including mobile phase solvents, were from Sigma-Aldrich or J. T. Bakers (Center Valley, PA, USA). 2.2. Sample Preparation 380 µL water and 20 µL internal standard solution (10 µM each of five internal standards in water) were added to 20–25 mg mouse feces, and the feces sample was homogenized while using a tissueLyzer (Qiagen Inc., Valentia, CA, USA) at 30 Hz for 1.5 min. The sample was centrifuged at 18,900 g for 10 min. at 4 C, and the supernatant was collected. Afterwards, the solution was × ◦ transferred to membrane filter (Nanosep 3K centrifugal device, Omega membrane, Pall Corporation, New York, USA) and it was centrifuged at 19,000 g for 20 min. to remove any floating particulates in × the supernatant. For plasma, 380 µL water and 20 µL internal standard solution were added to 200 µL mouse plasma, and mixed well. For exhaled breath condensate, 20 µL internal standard solution was added to 1 mL human exhaled breath condensate (EBC), and mixed well. The mixtures were centrifuged at 19,500 g for 10 min. at 4 C, and the supernatants were then collected. × ◦ For derivatization of SCFAs, 20 µL each of 20 mM AABD-SH, 20 mM TPP, and 20 mM DPDS in dichloromethane were added to the supernatant of a biological sample in a glass tube, and derivatization was performed at room temperature for 5 min while vortexing. The reaction solution was dried under vacuum, and then reconstituted with 20 µL methanol prior to LC-MS/MS analysis. The calibration curves were generated with standard solutions (100 nM to 1 mM). Metabolites 2019, 9, 173 3 of 11 2.3. Biological Samples Mice were housed at 21–23 ◦C with 12 h light/12 h dark cycles at the SPF Animal Facility at the University of Ulsan, with free access to water and rodent chow. All of the animal care and procedures were conducted according to the protocols and guidelines that the University of Ulsan Animal Care and Use Committee approved. Mice feces were collected from 23-week-old male C57BL/6 mice fed a high-fat chow (60% kcal% fat, Research diets, Inc., New Brunswick, NJ, USA) or low-fat chow (10% kcal% fat, Research diets, Inc.) for 12 weeks. Mice feces were frozen in liquid nitrogen and stored at 80 C until − ◦ processing. Mice plasma were prepared after anesthesia of male C57BL/6 mice at eight weeks of age. Human EBCs were obtained while using RtubeTM (Respiraotry Research Inc., Austin, TX), following manufacturer’s instruction. The study protocol was approved by the Institutional Review Board (IRB) of Asan Medical Center (2018-0789). 2.4. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) An LC-MS/MS system that was equipped with a 1290 HPLC instrument (Agilent Technologies, Glostrup, Denmark), a QTRAP 5500 (ABSciex, Framingham, MA, USA), and a reversed-phase column (Pursuit 5 C18 150 2.0 mm; Agilent Technologies, Santa Clara, USA) was employed. MS was × conducted in positive ion mode with a turbo ion-spray voltage of 5500 V, while using 20 psi curtain gas, 50 psi nebulizer gas, and 50 psi drying gas at a temperature of 400 ◦C. The sample injection volume was 3 µL. LC separation was performed while using mobile phase A (0.1 % formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile), at a flow rate of 500 µL/min and a temperature of 40 ◦C. The separation gradient was as follows: 30% B at 0 min., 30 to 50% B in 30 min., 50 to 30% B in 0.1 min., and 30% B in 4.9 min. A collision energy of 15 V was used for multiple reaction monitoring (MRM), and LC-MS/MS data were analyzed by Analyst 1.5.2 software (AB Sciex).