An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography–tandem mass spectrometry

Short-chain fatty acids (SCFAs) are produced by anaerobic gut microbiota in the large bowel. Qualitative and quantitative measurements of SCFAs in the intestinal tract and the fecal samples are important to understand the complex interplay between diet, gut microbiota and host metabolism homeostasis. To develop a new LC-MS/MS method for sensitive and reliable analysis of SCFAs in human fecal samples, 3-nitrophenylhydrazine (3NPH) was employed for pre-analytical derivatization to convert ten C₂–C₆ SCFAs to their 3-nitrophenylhydrazones under a single set of optimized reaction conditions and without the need of reaction quenching. The derivatives showed excellent in-solution chemical stability. They were separated on a reversed-phase C₁₈ column and quantitated by negative-ion electrospray ionization – multiple-reaction monitoring (MRM)/MS. To achieve accurate quantitation, the stable isotope-labeled versions of the derivatives were synthesized in a single reaction vessel from ¹³C₆-3NPH, and were used as internal standard to compensate for the matrix effects in ESI. Method validation showed on-column limits of detection and quantitation over the range from low to high femtomoles for the ten SCFAs, and the intra-day and inter-day precision for determination of nine of the ten SCFAs in human fecal samples was ≤8.8% (n = 6). The quantitation accuracy ranged from 93.1% to 108.4% (CVs ≤ 4.6%, n = 6). This method was used to determine the SCFA concentrations and compositions in six human fecal samples. One of the six samples, which was collected from a clinically diagnosed type 2 diabetes patient showed a significantly high molar ratio of branch-chain SCFAs to straight-chain SCFAs than the others. In summary, this work provides a new LC-MS/MS method for precise and accurate quantitation of SCFAs in human feces.     

Development and Validation of a Reliable UHPLC-MS/MS Method for Simultaneous Quantification of Macrocyclic Lactones in Bovine Plasma

A fast, accurate and reliable ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro^® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S^® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.     

An In Vitro Pilot Fermentation Study on the Impact of Chlorella pyrenoidosa on Gut Microbiome Composition and Metabolites in Healthy and Coeliac Subjects

The response of a coeliac and a healthy gut microbiota to the green algae Chlorella pyrenoidosa was evaluated using an in vitro continuous, pH controlled, gut model system, which simulated the human colon. The effect of C. pyrenoidosa on the microbial structure was determined by 16S rRNA gene sequencing and inferred metagenomics, whereas the metabolic activitywas determined by¹H-nuclear magnetic resonancespectroscopic analysis. The addition of C. pyrenoidosa significantly increased the abundance of the genera Prevotella, Ruminococcus and Faecalibacterium in the healthy donor, while an increase in Faecalibacterium, Bifidobacterium and Megasphaera and a decrease in Enterobacteriaceae were observed in the coeliac donor. C. pyrenoidosa also altered several microbial pathways including those involved in short-chain fatty acid (SCFA) production. At the metabolic level, a significant increase from baseline was seen in butyrate and propionate (p < 0.0001) in the healthy donor, especially in vessels 2 and 3. While acetate was significantly higher in the healthy donor at baseline in vessel 3 (p < 0.001) compared to the coeliac donor, this was markedly decreased after in vitro fermentation with C. pyrenoidosa. This is the first in vitro fermentation study of C. pyrenoidosa and human gut microbiota, however, further in vivo studies are needed to prove its efficacy.     

Isomaltulose Exhibits Prebiotic Activity,and Modulates Gut Microbiota,the Production of Short Chain Fatty Acids,and Secondary Bile Acids in Rats

In vitro experiments have indicated prebiotic activity of isomaltulose, which stimulates the growth of probiotics and the production of short chain fatty acids (SCFAs). However, the absence of in vivo trials undermines these results. This study aims to investigate the effect of isomaltulose on composition and functionality of gut microbiota in rats. Twelve Sprague–Dawley rats were divided into two groups: the IsoMTL group was given free access to water containing 10% isomaltulose (w/w), and the control group was treated with normal water for five weeks. Moreover, 16S rRNA sequencing showed that ingestion of isomaltulose increased the abundances of beneficial microbiota, such as Faecalibacterium and Phascolarctobacterium, and decreased levels of pathogens, including Shuttleworthia. Bacterial functional prediction showed that isomaltulose affected gut microbial functionalities, including secondary bile acid biosynthesis. Targeted metabolomics demonstrated that isomaltulose supplementation enhanced cholic acid concentration, and reduced levels of lithocholic acid, deoxycholic acid, dehydrocholic acid, and hyodeoxycholic acid. Moreover, the concentrations of propionate and butyrate were elevated in the rats administered with isomaltulose. This work suggests that isomaltulose modulates gut microbiota and the production of SCFAs and secondary bile acids in rats, which provides a scientific basis on the use of isomaltulose as a prebiotic.     

Simultaneous determination of short‐chain fatty acids in human feces by HPLC with ultraviolet detection following chemical derivatization and solid‐phase extraction segmental elution

Short‐chain fatty acids are currently the most studied metabolites of gut microbiota, but the analysis of them, simultaneously, is still challenging due to their unique property and wide concentration range. Here, we developed a sensitive and versatile high‐performance liquid chromatography with ultraviolet detection method, using pre‐column derivatization and solid‐phase extraction segmental elution, for the quantification of both major and trace amounts of short‐chain fatty acids in human feces. Short‐chain fatty acids were converted to 3‐nitrophenylhydrazine‐derived analytes, and then solid‐phase extraction segmental elution was used for extraction of major analytes and enrichment of trace analytes. The method validation showed limits of quantitation ?0.04 mM, and coefficient of determination > 0.998 at a wide range of 0.04–8.0 mM. The intra‐ and interday precision of analytes were all within accepted criteria, and the recoveries were 96.12 to 100.75% for targeted analytes in fecal samples. This method was successfully applied in quantification of eight analytes in human feces, which therefore could provide a sensitive and versatile high‐performance liquid chromatography with ultraviolet detection method for precise and accurate quantitation of short‐chain fatty acids in human feces.     

Optimised Method for Short-Chain Fatty Acid Profiling of Bovine Milk and Serum

Short-chain fatty acids (SCFA, C2-C5) in milk and serum are derived from rumen bacterial fermentation and, thus, have the potential to be used as biomarkers for the health status of dairy cows. Currently, there is no comprehensive and validated method that can be used to analyse all SCFAs in both bovine serum and milk. This paper reports an optimised protocol, combining 3-nitrophenylhydrazine (3-NPH) derivatisation and liquid chromatography-mass spectrometry (LC-MS) analysis for quantification of SCFA and β-hydroxybutyric acid (BHBA) in both bovine milk and bovine serum. This method is sensitive (limit of detection (LOD) ≤ 0.1 µmol/L of bovine milk and serum), accurate (recovery 84–115% for most analytes) and reproducible (relative standard deviation (RSD) for repeated analyses below 7% for most measurements) with a short sample preparation step. The application of this method to samples collected from a small cohort of animals allowed us to reveal a large variation in SCFA concentration between serum and milk and across different animals as well as the strong correlation of some SCFAs between milk and serum samples.     

Optimization,Validation and Application of HPLC-PDA Methods for Quantification of Triterpenoids in Vaccinium vitis-idaea L.

Triterpenoids have regained much attention as promising multi-targeting bioactive agents of natural origin in the treatment of numerous disorders. Due to the high potential for phytopharmaceutical development, accurate qualitative and quantitative analysis of triterpenoids for screening and quality control is required. Vaccinium vitis-idaea L. (lingonberry) raw materials have aroused interest as a rich source of triterpenoids. However, currently, no validated, rapid, and easy-to-perform quantification method is available for the routine control of these compounds in lingonberries. This research aimed at developing and validating HPLC-PDA methods for the determination and screening of triterpenoids in extracts of lingonberry leaves, fruits, and flowers. The developed methods were deemed satisfactory by validation, which revealed acceptable analytical specificity, linearity (r² > 0.9999), precision (RSD < 2%), trueness (94.70–105.81%), and sensitivity (LOD: 0.08–0.65 µg/mL). The real sample analysis demonstrated established methods applicability for quantification of 13 triterpenoids in lingonberries and emphasized differences between raw materials. Lingonberry fruits were distinguished by the richness of ursolic acid; lingonberry flowers by similar profile to fruits, but low content of neutral triterpenoids; whereas lingonberry leaves by the particularly high level of α-amyrin. Thus, the proposed methods proved to be reliable and applicable for quantification and routine analysis of triterpenoids in lingonberry samples.     

Pharmacokinetic Characterization of (Poly)phenolic Metabolites in Human Plasma and Urine after Acute and Short-Term Daily Consumption of Mango Pulp

Pharmacokinetic (PK) evaluation of polyphenolic metabolites over 24 h was conducted in human subjects (n = 13, BMI = 22.7 ± 0.4 kg/m²) after acute mango pulp (MP), vitamin C (VC) or MP + VC test beverage intake and after 14 days of MP beverage intake. Plasma and urine samples were collected at different time intervals and analyzed using targeted and non-targeted mass spectrometry. The maximum concentrations (C_max) of gallotannin metabolites were significantly increased (p < 0.05) after acute MP beverage intake compared to VC beverage alone. MP + VC beverage non-significantly enhanced the C_max of gallic acid metabolites compared to MP beverage alone. Pyrogallol (microbial-derived metabolite) derivatives increased (3.6%) after the 14 days of MP beverage intake compared to 24 h acute MP beverage intake (p < 0.05). These results indicate extensive absorption and breakdown of gallotannins to galloyl and other (poly)phenolic metabolites after MP consumption, suggesting modulation and/or acclimation of gut microbiota to daily MP intake.     

Saffron Pre-Treatment Promotes Reduction in Tissue Inflammatory Profiles and Alters Microbiome Composition in Experimental Colitis Mice

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract with an incompletely understood pathogenesis. Long-standing colitis is associated with increased risk of colon cancer. Despite the availability of various anti-inflammatory and immunomodulatory drugs, many patients fail to respond to pharmacologic therapy and some experience drug-induced adverse events. Dietary supplements, particularly saffron (Crocus sativus), have recently gained an appreciable attention in alleviating some symptoms of digestive diseases. In our study, we investigated whether saffron may have a prophylactic effect in a murine colitis model. Saffron pre-treatment improved the gross and histopathological characteristics of the colonic mucosa in murine experimental colitis. Treatment with saffron showed a significant amelioration of colitis when compared to the vehicle-treated mice group. Saffron treatment significantly decreased secretion of serotonin and pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in the colon tissues by suppressing the nuclear translocation of NF-κB. The gut microbiome analysis revealed distinct clusters in the saffron-treated and untreated mice in dextran sulfate sodium (DSS)-induced colitis by visualization of the Bray–Curtis diversity by principal coordinates analysis (PCoA). Furthermore, we observed that, at the operational taxonomic unit (OTU) level, Cyanobacteria were depleted, while short-chain fatty acids (SCFAs), such as isobutyric acid, acetic acid, and propionic acid, were increased in saffron-treated mice. Our data suggest that pre-treatment with saffron inhibits DSS-induced pro-inflammatory cytokine secretion, modulates gut microbiota composition, prevents the depletion of SCFAs, and reduces the susceptibility to colitis.     

Efficient sample clean‐up and online preconcentration for sensitive determination of melamine in milk samples by capillary electrophoresis with contactless conductivity detection

Based on an efficient sample clean‐up and field‐amplified sample injection online preconcentration technique in capillary electrophoresis with contactless conductivity detection, a new analytical method for the sensitive determination of melamine in milk samples was established. In order to remove the complex matrix interference, which resulted in a serious problem during field‐amplified sample injection, liquid–liquid extraction was utilized. As a result, liquid–liquid extraction provides excellent sample clean‐up efficiency when ethyl acetate was used as organic extraction by adjusting the pH of the sample solution to 9.5. Both inorganic salts and biological macromolecules are effectively removed by liquid–liquid extraction. The sample clean‐up procedure, capillary electrophoresis separation parameters and field‐amplified sample injection conditions are discussed in detail. The capillary electrophoresis separation was achieved within 5 min under the following conditions: an uncoated fused‐silica capillary, 12 mM HAc + 10 mM NaAc (pH = 4.6) as running buffer, separation voltage of +13 kV, electrokinetic injection of +12 kV × 10 s. Preliminary validation of the method performance with spiked melamine provided recoveries >90%, with limits of detection and quantification of 0.015 and 0.050 mg/kg, respectively. The relative standard deviations of intra‐ and inter‐day were below 6%. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine contamination in milk products.     

Eco-Friendly UPLC–MS/MS Method for Determination of a Fostamatinib Metabolite,Tamatinib, in Plasma: Pharmacokinetic Application in Rats

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid–liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an Acquity^TM CSH C₁₈ (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1–1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.     

HPLC Determination and Pharmacokinetic Study of Valdecoxib in Human Plasma

A sensitive, simple, and accurate high-performance liquid chromatographic method has been developed for determination of valdecoxib and the internal standard rofecoxib in human plasma. Protein was precipitated from plasma samples by addition of perchloric acid (HClO₄); the drug was then extracted with diethyl ether. Separation was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm i.d., 5 m particles) with ammonium acetate buffer-acetonitrile, 60:40 (v/v), containing 0.1% TEA, pH 6.5, as mobile phase. Detection and quantification were performed by UV-visible detection at 239 nm. Detection and quantification limits were 3 and 5 ng mL^–1, respectively. The linear concentration range for valdecoxib was 5–400 ng mL^–1. The validated RP HPLC method was used for determination of the pharmacokinetic data for the drug in humans.     

Bacteria, colonic fermentation, and gastrointestinal health

The colonic microbiota plays an important role in human digestive physiology and makes a significant contribution to homeostasis in the large bowel. The microbiome probably comprises thousands of different bacterial species. The principal metabolic activities of colonic microorganisms are associated with carbohydrate and protein digestion. Nutrients of dietary and host origin support the growth of intestinal organisms. Short-chain fatty acids (SCFAs), predominantly acetate, propionate, and butyrate, are the principal metabolites generated during the catabolism of carbohydrates and proteins. In contrast, protein digestion yields a greater diversity of end products, including SCFAs, amines, phenols, indoles, thiols, CO2, H2, and H2S, many of which have toxic properties. The majority of SCFAs are absorbed from the gut and metabolized in various body tissues, making a relatively small but significant contribution to the body's daily energy requirements. Carbohydrate fermentation is, for the most part, a beneficial process in the large gut, because the growth of saccharolytic bacteria stimulates their requirements for toxic products associated with putrefaction, for incorporation into cellular proteins, thereby protecting the host. However, as digestive materials move along the gut, carbohydrates become depleted, which may be linked to the increased prevalence of colonic disease in the distal bowel.     

Fecal microbial transplantation and a high fiber diet attenuates emphysema development by suppressing inflammation and apoptosis

Recent work has suggested a microbial dysbiosis association between the lung and gut in respiratory diseases. Here, we demonstrated that gut microbiome modulation attenuated emphysema development. To modulate the gut microbiome, fecal microbiota transplantation (FMT) and diet modification were adopted in mice exposed to smoking and poly I:C for the emphysema model. We analyzed the severity of emphysema by the mean linear intercept (MLI) and apoptosis by the fluorescent TUNEL assay. Microbiome analysis was also performed in feces and fecal extracellular vesicles (EVs). The MLI was significantly increased with smoking exposure. FMT or a high-fiber diet (HFD) attenuated the increase. Weight loss, combined with smoking exposure, was not noted in mice with FMT. HFD significantly decreased macrophages and lymphocytes in bronchoalveolar lavage fluid. Furthermore, IL-6 and IFN-γ were decreased in the bronchoalveolar lavage fluid and serum. The TUNEL score was significantly lower in mice with FMT or HFD, suggesting decreased cell apoptosis. In the microbiome analysis, Bacteroidaceae and Lachnospiraceae, which are alleged to metabolize fiber into short-chain fatty acids (SCFAs), increased at the family level with FMT and HFD. FMT and HFD attenuated emphysema development via local and systemic inhibition of inflammation and changes in gut microbiota composition, which could provide a new paradigm in COPD treatment.Subject terms: Respiratory tract diseases, Translational research     

Method Validation and Evaluation of Safrole Persistence in Cowpea Beans Using Headspace Solid-Phase Microextraction and Gas Chromatography

Bioinsecticides are regarded as important alternatives for controlling agricultural pests. However, few studies have determined the persistence of these compounds in stored grains. This study aimed at optimizing and validating a fast and effective method for extraction and quantification of residues of safrole (the main component of Piper hispidinervum essential oil) in cowpea beans. It also sought to assess the persistence of this substance in the grains treated by contact and fumigation. The proposed method used headspace solid-phase microextraction (HS-SPME) and gas chromatography with a flame ionization detector (GC/FID). Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was appraised via the parameters selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The LOD and LOQ of safrole were 0.0057 and 0.019 μg kg⁻¹, respectively and the determination coefficient (R²) was >0.99. The relative recovery ranged from 99.26 to 104.85, with a coefficient of variation <15%. The validated method was applied to assess the persistence of safrole residue in grains, where concentrations ranged from 1.095 to 0.052 µg kg⁻¹ (contact) and from 2.16 to 0.12 µg kg ⁻¹ (fumigation). The levels measured up from the fifth day represented less than 1% of the initial concentration, proving that safrole have low persistence in cowpea beans, thus being safe for bioinsecticide use. Thus, this work is relevant not only for the extraction method developed, but also for the possible use of a natural insecticide in pest management in stored grains.     

A Rapid and Simple UHPLC-MS/MS Method for Quantification of Plasma Globotriaosylsphingosine (lyso-Gb3)

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25–100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as <10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects (p < 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group (p = 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, p-value < 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.     

High-Throughput Analysis of Amino Acids for Protein Quantification in Plant and Animal-Derived Samples Using High Resolution Mass Spectrometry

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of <1% for the retention time and <11% for the peak area of the BSA and quality control (QC) lentil samples. The reproducibility of the total protein levels in the hydrolysis batches 1–4 was <12% for the BSA and the lentil samples. The level of detection on column was below 0.1 µM for most amino acids (mean 0.017 µM).     

Adapting the Accelerated Solvent Extraction Method for Resin and Rubber Determination in Guayule Using the BÜCHI Speed Extractor

Guayule (Parthenium argentatum Gray) is a promising alternative source to Hevea brasiliensis for the production of natural rubber, which can reach levels of 8–9% under industrialized farming conditions. The most common method for determining rubber concentration is by accelerated solvent extraction (ASE), a technique developed by the Dionex Corporation and almost exclusively performed with the Dionex ASE-200 or 350 systems. Herein, it is sought to apply and adapt the most common methods used in the literature for the Dionex system to another extraction platform, the BÜCHI Speed Extractor E-914. Results showed that using a sand sandwich method to confine the sample in the center and exploiting a larger cell volume (80 mL) for extraction prevents the occurrence of overpressure and problems with clogging. Under optimized conditions, the coefficient of variation was <15% for both resin quantification for samples containing 5.0–15.8% of resin and for rubber quantification for samples with 1.7–10.3% rubber content. The extraction time for resin (2 cycles of 5 min each) was smaller than for rubber (2 cycles of 20 min each). It would be interesting to carry out interlaboratory comparisons to standardize the method at an international level.     

A fast,fully validated GC-MS method using a simplified pretreatment for the quantification of short and branched chain fatty acids in human stool

The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism, and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acid) in stool. Therefore, a GC-MS method meeting this criterion was developed. A bias sampling study showed no statistical difference (p > 0.05) in analyte means when comparing 100 mg subsamples of homogenized to non-homogenized samples (n = 6, p values 0.153–0.910). Stool samples were homogenized, diluted with 80:20 water:methanol (v/v), and adjusted to a pH of 1.5–2.5. Samples were vortexed, centrifuged, and directly injected into the GC-MS using pulsed splitless injection offering twofold-to-threefold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses, or retention time shifts after 1,476 stool injections, while other columns failed before 361 injections. Intra- and inter-day accuracy for stool supernatant samples ranged from −10.21% to 8.88% and −13.25% to 9.91%, while intra- and inter-day precision ranged from 0.21% to 1.21% and 0.89% to 2.84% coefficient of variation (CV), respectively. This method demonstrates excellent linearity (0.9999–1.0000) and low limits of quantification (1.50–8.01 μM). Stool samples proved stable stored at −20°C up to 28 days, and recoveries ranged from 85.04% to 106.59%. Matrix effects in stool are non-significant determined by comparing standard and stool supernatant calibration curve slopes (p > 0.05).