Simple Extraction Research Articles

A Simple and Cost-Effective Extraction for Piscine Environmental DNA Metabarcoding Using Guanidine Hydrochloride Method.

Environmental DNA (eDNA) metabarcoding is a valuable tool for assessing aquatic biodiversity, but the high cost and complexity of DNA extraction pose challenges for widespread adoption, especially in developing countries. This study presents a cost-effective eDNA extraction method using a guanidine hydrochloride (GuHCl) buffer, proteinase-K digestion, and isopropanol precipitation to improve the detection of fish communities. Comparison with the Qiagen DNeasy Blood & Tissue Kit using MiFish universal primers showed that the GuHCl protocol detected more fish species in freshwater samples, with comparable performance in relative read abundance metrics. However, the GuHCl method exhibited higher PCR inhibition in brackish samples, likely due to salinity and natural inhibitors. The results suggest that the GuHCl-based method is a viable alternative, offering enhanced sensitivity for low-abundance species in freshwater samples and cost savings. This protocol facilitates large-scale eDNA metabarcoding for ecological studies and conservation management efforts.•The GuHCl protocol identified a greater diversity of fish species in freshwater samples than the Qiagen kit, but detected fewer species in brackish water samples.•Both extraction methods demonstrated robust positive correlations in metrics of relative read abundance.

Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 (MS4) gene in Cryptomeria japonica D. Don: toward an environmentally friendly protocol

Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 (MS4) gene in Cryptomeria japonica D. Don: toward an environmentally friendly protocol

Surface monitoring of L. monocytogenes by real-time fluorescence and colorimetric LAMP.

Listeria monocytogenes is a major foodborne pathogen affecting developing, and developed countries. The analysis of food contact surfaces in food industries is key for better controlling this pathogen. The current study focused on the development, optimization, and evaluation of a rapid and simple method for the detection of L. monocytogenes on stainless steel surfaces, suitable for decentralized setups, taking advantage of Loop-mediated isothermal amplification (LAMP). This was accomplished using a general pre-enrichment broth (TSB), with a simple DNA extraction based on a chelating resin, and final isothermal amplification. Two different detection strategies were tested, real-time fluorescence and naked-eye colorimetric, which were evaluated after 5, 7, and 24h of pre-enrichment. Regardless the detection chemistry selected, after 5-7h of pre-enrichment, 103-104CFU/cm2 were needed to obtain a positive result, while after 24h, it was possible to detect concentrations below 10CFU/cm2. Within each given time, all the performance parameters calculated, relative sensitivity, specificity, and accuracy, reached values higher than 80-90%; likewise, a Cohen's k of concordance with a culture-based approach higher than 0.8. Overall, the most sensitive assay can be performed in roughly 25h. This time-to-result outperforms commercial kits with the added value of specifically detecting L. monocytogenes instead of Listeria spp. KEY POINTS: • Real-time fluorescence and naked-eye colorimetric, were compared for the novel assay. • An LOD50 of 3.4CFU/cm2 and 4.2CFU/cm2 was calculated for the two assays. • Three pre-enrichment times were compared providing 24h better results.

Investigating the Presence of Selenoneine, Ergothioneine, and Selenium-Containing Biomolecules in Fish and Fish-Derived Commercial Products.

Two potent antioxidants, selenoneine and ergothioneine, have been determined simultaneously for the first time in the muscle of highly consumed fish and in fish-derived products. The extraction methods were optimized, and their subsequent analysis was performed by HPLC-ESI-MS/MS. Best extraction efficiency (106 ± 3 and 104 ± 5%) was attained by performing a simple aqueous extraction containing 100 mmol L-1 ammonium acetate or ammonium formate as extractants. Selenoneine and ergothioneine were simultaneously detected in tuna (0.306 ± 0.006 and 3.5 ± 0.2 μg g-1) and swordfish (0.14 ± 0.02 and 1.1 ± 0.4 μg g-1) muscle tissues, while in fish roe and in farmed salmon, only ergothioneine was detected (with contents ranging 0.03-3.3 μg g-1). Moreover, the presence of their respective methylated forms was also considered. Additionally, Se-containing biomolecule profiles from water-soluble proteins were obtained by SEC-ICP-MS analysis. Results revealed similar profiles between fish and fish roe, whereas for ultraprocessed fish products, Se seems to be mostly associated with low-molecular-weight biomolecules. The results included in this work may contribute to a better understanding of the relations between selenoneine and ergothioneine in fishery products as well as their fate and potential modifications of Se-containing biomolecule profiles during fish processing.

Smartcard: an integrated approach for contaminant monitoring, from field to laboratory.

Effective food safety monitoring requires a multi-step approach from farm to fork, involving different methods, ranging from convenient screening devices to sophisticated laboratory confirmatory testing. However, sample transportation to routine laboratories is time-consuming and expensive. Simplified on-site sampling followed by laboratory analysis offers a potential solution. Dried blood spot (DBS) cards ensure stability and ease of sample transportation and are used in clinical testing. However, the applicability of such an approach could be broader and include the storage of dried extract from more complex (solid) matrices. Therefore, a simplified approach is presented here, using DBS cards for on-site sampling and subsequent laboratory confirmation for food contaminants. To achieve this, an analytical tool (Smartcard) was designed using 3D-printing technology. As a proof of concept, the approach was applied to detect the pesticide fipronil, which is widely used in ornamental flower production to limit pests and on poultry farms. The Smartcard can securely store the sample extracts on a DBS card (dried extract spot (DExS) card), incorporate the lateral flow immunoassay (LFIA) and immediately provide an estimate of contamination levels. After simplified in-syringe extraction of the sample, the LFIA allows direct screening of fipronil (half maximum inhibitory concentration of 6.5µg/l with calibration standards), and the same sample extract can be directly applied to the DExS card for storage and transport to the laboratory, where analyte re-extraction and instrumental analysis is performed using ultra high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detecting fipronil down to 0.8µg/kg.

One-step simultaneous and efficient extraction of multiple active compounds from Lycium barbarum by aqueous three-phase system: Focusing on polysaccharides

In the past few years, the aqueous three-phase system (A3PS) has attracted a lot of attention due to its effectiveness in the efficient separation. Polysaccharides, carotenoids and polyphenols were separated simultaneously from Lycium barbarum by an aqueous three-phase system (A3PS) formed by acetonitrile/betaine/K2HPO4 as a novel extraction technique. The system is characterized by small amount of machine reagents, few consumables and simple extraction steps. The effects of acetonitrile, betaine and K2HPO4 concentrations on the extraction efficiency were investigated by Response Surface Methodology. The optimum composition of A3PS were 26.2 % (w/w) acetonitrile, 10.2 % (w/w) betaine and 27.6 % (w/w) K2HPO4. Under this condition, the extraction efficiencies of carotenoids, polyphenols, and Lycium barbarum polysaccharides (LBP) were 42.12±0.10 %, 88.61±0.95 % and 84.63±0.46 %, respectively. The obtained LBP was characterized by several technologies. The LBP was proved to consist of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a proportion of 4.14:0.80:4.00:4.43:22.20:39.9:12.44:3.22:8.46:0.41. The molecular weight of LBP was 12554 Da. In addition, LBP exhibited excellent antioxidant activity. In general, as a rapid developing method of extraction and separation, A3PS had potential application in the simultaneous separation of multiple substances.

Graphene quantum dots modified electrodes as electrochemical sensing tool towards the detection of codeine in biological fluids and soft drinks.

An electroanalytical method based on disposable screen-printed carbon electrodes modified with non-toxic carbonaceous nanodots is proposed as a reliable and effective device for codeine determination in biological fluids and soft drinks. Graphene quantum dots (GQDs), carbon quantum dots (CQDs) and carbon nanodots (CNDs) were evaluated as electrode modifiers for the determinationof the drug. The electroactive areas of the modified electrodes were assessed by cyclic voltammetry using potassium ferricyanide. Results demonstrated that GQDs provided the best analytical response for codeine, displaying an intense and well-defined anodic wave approximately 0.9V vs reference electrode. The method exhibits an acceptable linear dynamic range, low limits of detection and quantification (0.21 and 0.73µM, respectively), and satisfactory precision (below 3.9% expressed as relative standard deviation (RSD)) in saliva. Only the analysis of biofluids requires a simple extraction protocol. The feasibility and applicability of this novel approach were assessed by determining codeine in different matrices, with recoveries ranging from 69 to 112%. This cost-effective, simple, easily miniaturised and portable method was applied not only to biofluids but also for the direct detection of codeine in soft drinks combined with a codeine-enriched syrup, a medication that is being used to adulterate beverages, particularly at specific events (drinking and nightclub parties). There is no need forany sample treatment, demonstrating its versatility in analysing beverages for potential adulteration as well.

Revisiting representation learning of color information: Color medical image segmentation incorporating quaternion

Currently, color medical image segmentation methods commonly extract color and texture features mixed together by default, however, the distribution of color information and texture information is different: color information is represented differently in different color channels of a color image, while the distribution of texture information remains the same. Such a simple and brute-force feature extraction pattern will inevitably result in a partial bias in the model's semantics understanding. In this paper, we decouple the representation learning for color-texture information, and propose a novel network for color medical image segmentation, named CTNet. Specifically, CTNet introduces the Quaternion CNN (QCNN) module to capture the correlation among different color channels of color medical images to generate color features, and uses a designed local-global texture feature integrator (LoG) to mine the textural features from local to global. Moreover, a multi-stage features interaction strategy is proposed to minimize the semantic understanding gap of color and texture features in CTNet, so that they can be subsequently fused to generate a unified and robust feature representation. Comparative experiments on four different color medical image segmentation benchmark datasets show that CTNet strikes an optimal trade-off between segmentation accuracy and computational overhead when compared to current state-of-the-art methods. We also conduct extensive ablation experiments to verify the effectiveness of the proposed components. Our code will be available at https://github.com/Notmezhan/CTNet.

Effect of Simple Oral Dental Extraction on Systemic Serum Amyloid A Concentrations in Horses.

The translocation of gingival commensals resulting in measurable systemic inflammation has been described in humans and non-equine veterinary species with dental disorders, particularly periodontal disease. Routine odontoplasty does not result in increased serum amyloid A (SAA) concentration in horses, but a measurable increase in SAA concentration in horses undergoing dental extractions could suggest that local inflammation resulting from more severe dental disease has potential for wider, systemic consequences that warrants further study. To determine whether SAA increases in horses undergoing simple, oral extraction of non-fractured cheek teeth with and without periodontal disease. Prospective cohort study. SAA was measured using a stall-side test in horses undergoing simple oral extraction of cheek teeth with intact clinical crowns at baseline (T = 0), 24 h (T = 24) and 48 h (T = 48) post-extraction. Eight horses and 4 ponies aged between 4 and 23 years underwent cheek tooth extraction. A statistically significant difference in SAA concentration was noted between groups with and without periodontal disease at both 24 h (p = 0.004) and 48 h (p = 0.043). At 24h, the median SAA concentration was 135mg/L (range: 0-260mg/L; IQR: 77.5-174mg/L) in horses with periodontal disease and 27.5mg/L (range: 0-47mg/L; IQR: 4.8-43.5mg/L) in horses without periodontal disease. At 48h, median SAA concentration was 264mg/L (range: 236-440mg/L; IQR: 245.5-300.5mg/L) in horses with periodontal disease and 0mg/L (range = 0-41mg/L; IQR: 0-21.8mg/L) in horses without periodontal disease. Small sample group. Horses undergoing extraction of fractured cheek teeth were not included. Extraction of non-fractured cheek teeth does not result in a remarkable increase in SAA, except in horses with periodontal disease. These results suggest that periodontal disease in horses is associated with a local inflammatory response, which in turn drives the development of systemic inflammation, resulting in detectable increases in inflammatory markers when diseased periodontal tissues are disturbed.

Development of a simple and safe route to C-3 arylated 1H-pyrazoles

A simple transition-metal-free and highly chemoselective strategy for the preparation of C-3 arylated 1H-pyrazoles with enaminones and p-toluenesulfonyl hydrazide (TsNHNH2) has been developed. The current environmental benign protocols showed that, TsNHNH2 could be served as the safe and easy-to-handle hydrazide for the preparation of C-3 arylated 1H-pyrazoles instead of the explosive and genotoxic hydrazine hydrate under mild conditions, with broad substrate scope and excellent functional group tolerance. More importantly, a gram-scale synthesis of a synthetic equivalent of key intermediate to immuno-oncology agonist BMS-986299 was demonstrated successfully through a simple crystallization or extraction process for the scalable production.

Emerging Micromolecule-based Aqueous Two-phase Systems and Their Application in Biofuels

Aqueous two-phase system (ATPS) is a system composed of two incompatible solutions, which has excellent performance for extracting proteins, antibiotics, and other bioactive molecules, so it is a very simple, efficient, and green extraction and separation method. In this paper, we introduced three different emerging micromolecule-based ATPSs and introduced their characteristics, applications, advantages, and disadvantages respectively. This is of guiding significance for the synthesis of new ATPSs. One of the important prospects of this ATPS in industrial application is the separation and purification of fermentation-based biofuel. Adding a salt, sugar, or ionic liquid to a biofuel solution can remove most of the water, enabling efficient recovery and dehydration of biofuel. The two-step process can replace the traditional distillation and molecular sieve adsorption processes to obtain high-purity bioalcohols, thus introducing a new purification process for biofuel. An in-depth analysis of the separation performance of the emerging micromolecule-based ATPSs as well as an objective indication of their application provides valuable insights for further optimization and application of this kind of ATPSs in separation science.

Bio-Guided Extraction of a Phenolic-Rich Extract from Industrial Peanut Skin with Antioxidant and Hypotensive Potential.

Peanut composition includes phenolic compounds, especially in the skins, which are often not consumed. High blood pressure affects more than one billion people worldwide and is considered a high-risk factor for cardiovascular diseases. Several studies have correlated antihypertensive activity with the total phenolic content present in the plants. This study evaluated the hydroethanolic extraction of phenolic compounds from the industrial residue of peanut skin and evaluated the antioxidant and antihypertensive capacity of these extracts using in vitro models. A rotational central composite design (DCCR) was proposed to study the influence of the variables: (1) the ethanol concentration on the hydroalcoholic extractor solution, and (2) the proportion of solid sample (waste) per liquid in the extraction (mass/volume) in a simple solid-a liquid extraction process. The optimal extraction conditions within this model were 50% ethanol in water, and the proportion of sample to extraction solution (m/v) equaled to 0.2. The extract obtained had significant antioxidant capacity, both in chemical (ORAC) and in cellular models, with potential for free radical scavenging. Significant levels of ACE inhibition were also found, indicating antihypertensive activity.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

Streamlined boiling lysis DNA extraction for Gram-positive aquaculture pathogen Streptococcus agalactiae.

Accurate genetic analysis is essential for the detection of pathogens as it necessitates suitable DNA extraction methods tailored to specific microorganisms such as Gram-positive bacteria. This study examined several commercial and simplified DNA extraction methods for their suitability in isothermal downstream applications. Extracted DNA was assessed using spectrophotometry, electrophoresis, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) while its stability was inspected after five months of storage. The findings revealed variations in DNA yield, purity and integrity among the extraction methods. While extraction kits demonstrated high yield and purity, the in-house extraction techniques showed incoherent correlation between yield and purity, yet showed promise for a streamlined extraction process. The DNA acquired from all methods yielded positive amplification in PCR and LAMP. DNA extracted by kits exhibits prolonged stability than those obtained via boiling lysis. Both methods offer distinct advantages, with commercial kits providing longer stability and high-quality DNA while boiling lysis stands out for its simplicity, with shorter handling and processing periods. This study emphasizes selecting ideal extraction methods for Streptococcus agalactiae, in the prospect of aquaculture settings. In particular, successful LAMP reaction suggests that boiled extracts are feasible enough for detection, and suited for point-of-care (POC) testing where prompt detection of aquatic pathogens is often critical. Ultimately, the choice of method should be contemplated on a case-by-case basis such as the study goals, intended settings, and type of samples.

Simple extraction method of magnetic hysteresis curve from measurement data

Simple extraction method of magnetic hysteresis curve from measurement data

Chemical Upcycling of Expired Pharmaceuticals as a Source of Value-Added Chemicals for Organic Synthesis and Medicinal Chemistry.

Pharmaceutical and veterinary products are a class of contaminants of emerging concern, and their presence in the environment is due to continuous and incorrect disposal. Environmental scientists have been accumulating data on their adverse effects on animal populations since toxicological effects on wildlife were first published. Therefore, recycling strategies are needed. Valuable active ingredients can be extracted from expired pharmaceuticals and recycled according to various strategies. In an effort to reveal the potential of the chemical upcycling of expired pharmaceuticals, the active ingredients gabapentin and pregabalin were extracted and used as starting materials to prepare a small collection of promising substrates endowed with functionalities and structural three-dimensionality. Gabapentin 1 was transformed into aminoalcohol 3, spiroamine 4, and the bioactive azaspirolactam 5. The lactam analog 6 was synthesized from pregabalin 2. Due to the biological profile of 5 and the structural similarity of the N-alkylated derivatives 5l and 6b with the drug piracetam, a collection of potentially bioactive structural analogs 5a-l and 6a-b were also prepared. Simple extraction, synthesis, and purification procedures were used as a means of chemical and economic revaluation, resulting in moderate to good yields at a low cost.

Development of a UHPLC-MS/MS Method for the Determination of Moxidectin in Rat Plasma and Its Application in Pharmacokinetics.

The aim of the present study was to establish a simple and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method and apply it for the determination of pharmacokinetics of moxidectin-loaded microspheres (MOX-MS) in rats. Plasma samples were processed using a simplified liquid-liquid extraction method and were separated using an Agilent Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of a 10 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for 5 min. Avermectin B1a was used as an internal standard (IS). The sample was injected at a volume of 10 μL with a column temperature of 35 °C and detected in a positive ion mode. A good linear response across the concentration range of 1.00-200 ng/mL (r2 > 0.99) and a lower limit of quantification (LLOQ) of 1.00 ng/mL were achieved. The extraction recovery of moxidectin exceeded 94.1%, the matrix effect was between 91.2% and 96.2%, the accuracy ranged from 100.1 to 103.6%, and the relative standard deviation (RSD) did not exceed 15% for the intra- and inter-day accuracy and precision. The pharmacokinetic results showed that MOX-MS significantly decreased Cmax, prolonged T1/2, and improved bioavailability. The developed method significantly reduced the assay volume, shortened detection time, simplified sample processing methods and saved assay costs, which may contribute to the development of the new antiparasitic drug.

Paralytic Shellfish Toxin Extraction from Bivalve Meat for Analysis Using Potentiometric Chemical Sensors.

A simple and reliable methodology for the detection of paralytic shellfish toxins (PSTs) in bivalve tissues using potentiometric chemical sensors was developed. Five methods of PST extraction from mussel and oyster tissues were evaluated, including the AOAC-recommended method, which served as the reference. The main objective was to minimize the matrix effect of the extracts on the sensors' responses and ensure efficient toxin recovery. Extraction procedures using acetic acid with heating and water yielded the highest responses from the potentiometric chemical sensors to PSTs. The highest recovery of PSTs from bivalve tissues was achieved with extraction using acetic acid and heating. Further extract purification, which is indispensable for liquid chromatography with fluorometric detection (LC-FLD) analysis, was found to be unnecessary for analysis with chemical sensors. While water extraction can also be used as a rapid and simple PST extraction method, the lower recoveries should be considered when interpreting the results. Further research is needed to identify the compounds remaining in the extracts that cause a decrease in sensor responses and to develop procedures for their elimination.

Validation of the performance of a point of care molecular test for leprosy: From a simplified DNA extraction protocol to a portable qPCR.

The study aimed to optimize qPCR reactions using oligonucleotides from the first Brazilian molecular diagnostic kit for leprosy on a portable platform (Q3-Plus). In addition, we sought to develop a simplified protocol for DNA extraction that met point-of-care criteria. During optimization on the Q3-Plus, optical parameters, thresholds, and cutoffs for the 16S rRNA and RLEP targets of M. leprae were established using synthetic DNA, purified DNA from M. leprae, and pre-characterized clinical samples. For the simplified extraction protocol, different lysis solutions were evaluated using chaotropic agents, and purification was carried out by transferring the lysed material to FTA cards. The complete protocol (simplified extraction + qPCR on the portable platform) was then evaluated with pre-characterized clinical skin biopsy samples and compared with standard equipment (QuantStudio-5). LOD95% for the optimized reactions was 113.31 genome-equivalents/μL for 16S rRNA and 17.70 genome-equivalents/μL for RLEP. Among the lysis solutions, the best-performing was composed of urea (2 M), which provided good dissolution of the skin fragment and a lower Ct value, indicating higher concentrations of DNA. The complete technological solution showed a sensitivity of 52% in reactions. Our results highlight the need for additional optimization to deal with paucibacillary samples, but also demonstrate the feasibility of the portable platform for the qPCR detection of M. leprae DNA in low infrastructure settings.

Protocols for bulk off-line fluid inclusion extraction for the analysis of δ13C-CH4 and δ13C-CO2 using a cavity ring-down spectroscopy (CRDS) analyser

Fluid inclusions are a window into deep geological fluids, providing unique access to their nature and composition. The isotopic composition of CO2 and CH4 hosted in fluid inclusions is a powerful proxy to assess the origin and transformation of deep geological fluids, giving insights into carbon sources, fluxes, and degassing in a wide variety of geodynamic settings. Over the last 5 decades, techniques have been developed to extract fluid inclusions from their host minerals and measure their bulk composition. These techniques are often challenged by analytical artifacts including high blank levels of CO2 and CH4, fluid re-speciation, gas adsorption, and diffusion. Since these processes may alter the pristine composition of gases liberated from fluid inclusions, rigorous protocols are needed in order to evaluate the isotopic integrity of the extracted volatile species. In this study, we introduce new protocols for bulk off-line fluid inclusion extraction for the analysis of δ13C-CH4 and δ13C-CO2 using a Cavity Ring-Down Spectroscopy (CRDS) analyser (Picarro G2201-i). Two mechanical fluid extraction techniques are compared: ball milling in ZrO2 jars and sample crushing in a stainless steel sealed tube under a hydraulic press. Blanks and isotopically labelled tests with the ball milling technique suggest that rotation speed, grinding stock filling degree and filling type alter the CH4 and CO2 concentrations and isotopic compositions measured by the CRDS analyser. In contrast, the crushing technique does not generate measurable quantities of blank CH4 and CO2. The protocols presented in this study allow to extract, detect, and analyse δ13C of CH4 and CO2 for concentrations above 10 and 1,000 ppm respectively. Interlaboratory experiments allowed to replicate previously measured δ13C-CH4 values in natural fluid inclusions within 1‰ with both extraction techniques. This study highlights the potential of combining simple bulk off-line fluid inclusion extraction techniques with a CRDS analyser for δ13C analysis of CO2 and CH4 without gas separation being required.