Simple Extraction Method Research Articles

Gallic Acid Derivatives are the Active Ingredients of the Optimized Phenolic Extracts of Chinese Tallow Tree.

The Chinese tallow tree (Sapium sebiferum (L.) Roxb) is a valuable forest resource, and its leaves have long been utilized in Traditional Chinese Medicine for the treatment of various diseases, primarily due to the antioxidant activity of phenolic compounds. In this study, a simple ultrasonic-assisted phenolic extraction method was established using response surface methodology. The highest total phenolic content was obtained by extracting with 40% ethanol (v/v) in a solid-liquid ratio (g/mL) of 1:48 for 20 min at 30 °C, employing an ultrasonic bath with a power of 270 W and a frequency of 40 kHz. Using the optimized phenolic extract, a non-target metabolomics approach was adopted to evaluate the antioxidant ingredients of the Chinese tallow tree. Most of the identified phenolic compounds were depleted after reacting with free radicals, indicating their significant contribution to the antioxidant activity of Chinese tallow tree leaves. Among them, gallic acid derivatives, geraniin, and ethyl gallate were identified as the predominant antioxidant ingredients. Overall, a simple ultrasonic-assisted phenolic extraction method was optimized, and the antioxidant ingredients of the valuable Chinese tallow tree leaves were identified. These findings significantly promote the development and application of Chinese tallow tree leaves.

Intelligent Intrusion Detection System Against Various Attacks Based on a Hybrid Deep Learning Algorithm.

The Internet of Things (IoT) has emerged as a crucial element in everyday life. The IoT environment is currently facing significant security concerns due to the numerous problems related to its architecture and supporting technology. In order to guarantee the complete security of the IoT, it is important to deal with these challenges. This study centers on employing deep learning methodologies to detect attacks. In general, this research aims to improve the performance of existing deep learning models. To mitigate data imbalances and enhance learning outcomes, the synthetic minority over-sampling technique (SMOTE) is employed. Our approach contributes to a multistage feature extraction process where autoencoders (AEs) are used initially to extract robust features from unstructured data on the model architecture's left side. Following this, long short-term memory (LSTM) networks on the right analyze these features to recognize temporal patterns indicative of abnormal behavior. The extracted and temporally refined features are inputted into convolutional neural networks (CNNs) for final classification. This structured arrangement harnesses the distinct capabilities of each model to process and classify IoT security data effectively. Our framework is specifically designed to address various attacks, including denial of service (DoS) and Mirai attacks, which are particularly harmful to IoT systems. Unlike conventional intrusion detection systems (IDSs) that may employ a singular model or simple feature extraction methods, our multistage approach provides more comprehensive analysis and utilization of data, enhancing detection capabilities and accuracy in identifying complex cyber threats in IoT environments. This research highlights the potential benefits that can be gained by applying deep learning methods to improve the effectiveness of IDSs in IoT security. The results obtained indicate a potential improvement for enhancing security measures and mitigating emerging threats.

Efficient Extraction of Phenols from Coal Tar and Preparation of Phenolic Resin-Based Porous Carbon for Advanced Supercapacitor Application.

Developing simple and efficient extraction methods for phenolic substances from coal tar, which facilitate their direct transformation into high-performance electrode materials, holds considerable practical significance. In this study, amide-zinc chloride deep eutectic solvents are employed for efficient phenol extraction. The optimal phenol extraction process is subsequently investigated, and it is found that the robust hydrogen bonding interactions between solvents and phenols significantly enhance extraction efficiency. Notably, without the need for back-extraction, formaldehyde and tetraethyl orthosilicate are added to obtain phenolic resin, which can subsequently be directly carbonized to fabricate hydrangea-like porous carbon. The carbonization mechanism of the phenolic resin is studied, and the templating and activating roles of tetraethyl orthosilicate and zinc chloride assist in the formation of this unique structure. Furthermore, the flexible supercapacitor assembled using the prepared porous carbon and gel electrolyte achieves a high energy density of 31.0Wh kg-1 and demonstrates broad temperature applicability ranging from -25 to 100°C. This work directly converts the extracted phenolic compounds into phenolic resin and shows potential for fabricating porous carbon materials with diverse structures and enhanced capacitive performance.

Swift and portable detection of soybean mosaic virus SC7 through RNA extraction and loop-mediated isothermal amplification using lateral flow device.

The soybean mosaic disease-caused by the soybean mosaic virus (SMV)-significantly impacts soybean quality and yield. Among its various strains, SMV-SC7 is prevalent in China. Therefore, rapid and accurate diagnosis is deemed critical to mitigate the spread of SMV-SC7. In this study, a simple and rapid magnetic bead-based RNA extraction method was optimized. Furthermore, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay that requires no specialized equipment such as PCR Amplifier was proposed, employing a lateral flow device (LFD) for visual interpretation of SMV-SC7. The RT-LAMP-LFD approach facilitated specificity testing of SMV-SC7. Moreover, the limit of detection (LOD) of this method was as low as 10-5 ng (2.4 copies). The sensitivity of RT-LAMP-LFD was 10-fold higher than that of the colorimetric RT-LAMP method. In 194 field samples tested, the RT-LAMP-LFD detection of the SMV-SC7 had accuracy of 98.45% in comparison to RT-qPCR. In conclusion, the assay exhibited high specificity, sensitivity, and rapidity, enabling economical and portable detection of SMV-SC7 and providing technical support to identify SMV-SC7-infected soybeans.

Development and validation of a quantification method for direct oral anticoagulants from capillary blood using volumetric absorptive microsampling and online SPE-LC-MS.

The number of prescriptions for new direct oral anticoagulants (DOACs) apixaban, edoxaban, rivaroxaban and dabigatran has increased exponentially in recent years, increasingly replacing the old gold standard, vitamin-K-antagonists. Due to their wide therapeutic range, therapeutic drug monitoring (TDM) is not required, although it has been proven that this could significantly reduce side effects. In order to develop a cost-efficient and simple method for the simultaneous detection of the DOACs and phenprocoumon, a new technology for sample preparation from capillary blood in the ambulant sector named VAMS® was integrated and an LC-MS detector with on-line solid phase extraction (SPE) applying a Turboflow HTLC CycloneTM 1.0x50 mm column was used. The mobile phase consisted of methanol with water (3/97 v/v) and 0.1% ammonia solution with a flow rate of 2.5mL/min. For the chromatographic separation, a Phenomenex LTD Kinetex 2.6µm C18 100Å, 100x3.0mm column with a flow rate of 0.3mL/min in gradient mode was utilized. The mobile phase consisted of acetonitrile, water and formic acid (A: 10:90:0.1 v/v and B: 95:05:0.1 v/v). The method was fully validated in the therapeutic range of the substances according to current guidelines. The LLOQ ranged from 3.5µg/L for rivaroxaban to 88µg/L for phenprocoumon and the intra-day and inter-day precision was less than 13% and 12%, while the accuracy was within a range of 85.7-113% and 88.7-106%, respectively. Samples could be stored in the Mitra® devices for at least seven days at room temperature except of dabigatran. Because the Mitras® were used, exactly 10µL of blood could be drawn and no significant haematocrit effect was observed. A reliable, simple and cost-effective extraction and analysis LC-MS method could be developed and validated. This method is therefore applicable in ambulatory care.

Development of a Copro-RPA-CRISPR/Cas12a assay to detect Echinococcus granulosus nucleic acids isolated from canine feces using NaOH-based DNA extraction method.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (E. granulosus s.l.), remains a significant zoonotic parasitic disease affecting both livestock and humans. It arises from the ingestion of food and water contaminated with canine feces containing E. granulosus eggs. The detection of these eggs in canine feces is essential for guiding effective preventative measures against the disease. Therefore, the development of a novel accurate, rapid, and visually interpretable point-of-care test is crucial for controlling CE. We combined recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with a CRISPR-associated protein 12a (Cas12a) system, forming the RPA-CRISPR/Cas12a assay. This assay targeted the E. granulosus mitochondrial nad2 gene and utilized a lateral flow strip for visual readout. To improve field applicability, we integrated a simple and cost-effective NaOH-Based DNA extraction method. Clinical validation included testing DNA extracted from eighteen canine fecal samples, followed by comparison with quantitative PCR (qPCR) and two commercial enzyme-linked immunosorbent assay (ELISA) kits. The RPA-CRISPR/Cas12a assay showed a detection limit of 1 fg/μL DNA, without any cross-reactivity with related tapeworms such as Echinococcus multilocularis, Dipylidium caninum, Taenia hydatigera, Taenia multiceps, and Taenia pisiformis. When applied to 62 clinical fecal samples from dogs, the RPA-CRISPR/Cas12a assay demonstrated 68% sensitivity, while the developed RPA-CRISPR/Cas12a-NaOH assay exhibited 45% sensitivity. In the field performance comparison of the RPA-CRISPR/Cas12a and the RPA-CRISPR/Cas12a-NaOH assay with qPCR and two ELISA kits, the sensitivity, consistency rate, and Youden's index suggested good or fair agreement with the currently employed detection methods. This study describes the development and validation of the RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-NaOH assays for detecting E. granulosus in canine feces. The developed assays surpassed previous detection methods in providing enhanced diagnostic sensitivity and enabling point-of-care testing. Moreover, these assays hold potential for surveilling E. granulosus in low-income countries.

Molecular Networks as Strategy for Dereplication of Steroidal Alkaloids of Herbarium Samples of Solanum jabrense Agra and M. Nee, an Endemic and Unexplored Species.

Solanum jabrense is an endemic species from Brazil, distributed in the phytogeographic domains of the Caatinga and Atlantic Forest, in the states of Northeast. Solanum L. species have great economic importance not only because they are used in human food, but also because they present several secondary metabolites, especially glycosylated steroidal alkaloids, giving them medicinal properties. Recently, dry herbarium specimens have been used to identify metabolites of interest preserved even after years of storage, using a simple and fast method of extraction and analysis by liquid chromatography (LC) coupled to mass spectrometry (MS). Dereplication techniques aided by molecular networks (MNs) were used to analyze the chemical composition from samples of S. jabrense herbarium specimens and to identify chemical markers and bioactive molecules with potential medicinal use. From the LC-MS/MS dataset of the crude extracts and a standard (solasodine), an MN was generated that resulted in the dereplication of 19 spirosolane-type alkaminas. Our results suggest that dereplication using fragments of dried Solanum specimens is a quick tool to identify potential conserved metabolites, being useful not only for chemotaxonomy and metabolomic but also for the discovery of new molecules in natural products.

Stable pedestrian trajectory prediction method with dynamic fusion of multiple features in dense crowds

Abstract Predicting pedestrian trajectories is an essential task within the realms of computer vision and artificial intelligence, aiming to forecast the future motion paths of pedestrians based on historical data. Current research primarily focuses on extracting factors that influence pedestrian movement and independently modeling these factors to predict future pedestrian trajectories. However, existing methods overlook the varying degrees of influence that different factors may have on pedestrian movement at different time steps. To address this issue, we, after extracting the influencing factors, employ a model-adaptive approach to model the importance of different factors at each time step and further predict pedestrian trajectories. Specifically, we utilize a simple multi-factor feature extraction method to extract various influencing factors. Subsequently, we employ a Transformer-based dynamic weight fusion mechanism to dynamically adjust the weights of different factors on pedestrian movement at different time steps, thereby achieving pedestrian trajectory prediction. Our method demonstrates significant superiority in dense crowds, with experimental results showing an improvement of 6% and 31% over state-of-the-art approaches.

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.

Establishment and Validation of Fecal Secretory Immunoglobulin A Measurement for Intestinal Mucosal Health Assessment in Wild Lemurs.

The measurement of biomarkers in blood and excreta can enable immune status assessment and provide prognostic information on individual health outcomes. In this respect, the fecal measurement of secretory immunoglobulin A (sIgA), the primary mammalian antibody for mucosal defense, has recently received increased interest in a few anthropoid primates, but a fecal sIgA assay for use in strepsirrhine primates has not yet been reported. Here, we develop and analytically validate a cost-effective in-house sandwich enzyme immunoassay for the extraction and measurement of sIgA in feces of redfronted lemurs (Eulemur rufifrons). We also tested a simple method for sIgA extraction that can be used under remote field conditions and undertook experiments to assess the robustness of sIgA concentrations to variation in processing and storage conditions of fecal extracts. Our analytical validation revealed that the assay recognizes immunoreactive sIgA in redfronted lemur feces, that sIgA can be measured accurately with no potential interference from the fecal matrix, and that assay reagents and performance are highly stable over time. The field-friendly extraction procedure produced sIgA results strongly correlated with those generated by a standard laboratory extraction method. Short-term storage at room temperature resulted in a slight decline in sIgA concentrations, whereas freezing extracts at -20°C kept sIgA levels stable for at least 3 months. Longer-term storage of >5 months, however, led to a significant decline of sIgA concentrations. Multiple freeze-thaw cycles did not affect sIgA levels. This study, therefore, provides the basis for measuring fecal sIgA in lemurs and possibly other strepsirrhines. When samples are processed properly and stored frozen, and when sIgA analysis can be performed within 3 months upon sample collection, fecal sIgA measurements can become a valuable tool for monitoring aspects of immunity and health in both zoo-housed and wild-living lemurs.

Recyclable covalent organic frameworks/cellulose aerogels for efficient uranium adsorption

The advancement of efficient, recyclable adsorbents for the economical capture of uranium from seawater is critical for the sustainable progression of nuclear energy. In this work, a unique aerogel composed of covalent organic frameworks (COF-TpTHA)/cellulose nanofibrils (CNF) was synthesized under mild conditions for uranium adsorption. TpTHA/CNF aerogel resolves challenges related to the formability of COF. CNF utilized as the matrix to encapsulate COF-TpTHA in order to improve the dispersion and reinforce the composite materials. The introduction of COF-TpTHA endows CNF aerogel with sufficient active groups for uranium adsorption. X-ray diffraction (XRD) characterization confirmed the successful incorporation of COF while maintaining the type I structure of cellulose. Fourier-transform infrared (FT-IR) spectroscopy further validated the presence of hydrogen bonding interactions between COF and cellulose. The results demonstrated the excellent adsorption efficiency of TpTHA/CNF aerogel towards U(VI), with a maximum adsorption capacity of 177.90 mg g‐1 (experiment) for U(VI). Meanwhile, TpTHA/CNF aerogel exhibited favorable adsorption selectivity and reusability. This cellulose-encapsulated COF approach offers a simple and promising method for uranium extraction from seawater, demonstrating its significant application potential.

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.

Determination of nobiletin and tangeretin in rat plasma by ultra-performance liquid chromatography-tandem mass spectrometry and study of their pharmacokinetics

AbstractAn ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of nobiletin and tangeretin in rat plasma, and the plasma was processed by a simple liquid-liquid extraction method with ethyl acetate. The chromatographic column was UPLC HSS T3 (50 × 2.1 mm, 1.7 μm), the mobile phase was acetonitrile-water (containing 0.1% formic acid). Multiple reaction monitoring mode (MRM) was used for quantitative analysis, nobiletin m/z 403.29 → 373.14 (cone voltage 22v, collision voltage 28v), tangeretin m/z 373.28 → 343.17 (cone voltage 20v, collision voltage 28V), tangeretin m/z 373.28 → 343.17 (cone voltage 20V, collision voltage 28V) and internal standard vitexin m/z 433.14 → 313.03 (cone voltage 32v, collision voltage 26v). The pharmacokinetics of nobiletin and tangeretin were evaluated in rats. The established UPLC-MS/MS method in the range of 2–2,000 ng mL−1 was successfully applied to the pharmacokinetics, and the calculated bioavailability of nobiletin and tangeretin was 63.9 and 46.1%, respectively.

A simple, economical, and high-yield method for polyethylene glycol-based extraction of follicular and serum-derived extracellular vesicles.

The optimization of polyethylene glycol (PEG)-based extracellular vesicles (EVs) extraction from human follicular fluid (FF) and serum was investigated, and their functional analysis was confirmed. The PEG-based EV results were compared to the ExoQuick (ExoQ)-based EV. FF-EVs and serum-EVs were extracted by using different concentrations of PEG (8000). Nanoparticle tracking analysis was used to count the particles, and electron microscopy of EVs was performed for visualization. Exosomes were confirmed by the western blot analysis with exosome-specific markers. RNA and microRNA were extracted from exosomes and quantitative polymerase chain reaction analysis was performed. Fallopian tube epithelial (FTE) cells were used for the EV uptake experiment and an anchorage-independent growth test to confirm that extracted EVs harbor transformation activity. The PEG 8% enriched method produced the highest yield and the lowest carry-over protein. Salt containing PEG 8% produced a higher yield than nonsalted PEG 8%. Overnight enrichment increased four times and 18 times for PEG 8% and ExoQ-based EV extraction from FF. For serum EV, the same overnight enrichment moderately increased yield for both PEG 8% and ExoQ methods. Less carry-over protein resulted in more EV-promoted transformation activity. This study overcomes the time-consuming, expensive, laborious, and complicated machine-dependent EV extraction methods. The study highlights that longer incubation time is needed for EV extraction from FF. PEG 8000-based EV extraction provided a higher yield and less carry-over protein than ExoQ-based EV extraction.

Simple extraction method of magnetic hysteresis curve from measurement data

Simple extraction method of magnetic hysteresis curve from measurement data

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.

Development of a Simple Extraction Method for Proteoglycans

Development of a Simple Extraction Method for Proteoglycans

Development of a Simple Extraction Method for Proteoglycans

Development of a Simple Extraction Method for Proteoglycans

Application of MALDI-TOF mass spectrometry for identification of Nocardia species

BackgroundNocardiosis, despite its rarity and underreporting, is significant due to its severe impact, characterized by high morbidity and mortality rates. The development of a precise, reliable, rapid, and straightforward technique for identifying the pathogenic agent in clinical specimens is crucial to reduce fatality rates and facilitate timely antimicrobial treatment. In this study, we aimed to identify Nocardia spp. in clinical isolates, using MALDI-TOF MS as the primary method, with molecular methods as the gold standard. Clinical Nocardia isolates were identified using 16S rRNA/hsp65/gyrB/secA1/rpoB gene sequencing. Identification performance of the Bruker MALDI Biotyper 3.1 (V09.0.0.0_8468) and MBT Compass 4.1 (V11.0.0.0_10833) for Nocardia identification was evaluated.ResultsSeventy-six Nocardia isolates were classified into 12 species through gene sequencing. The MALDI Biotyper 3.1 (V09.0.0.0_8468) achieved 100% genus-level accuracy and 84.2% species accuracy (64/76). The MBT Compass 4.1 with the BDAL Database (V11.0.0.0_10833) improved species identification to 98.7% (75/76). The updated database enhanced species level identification with scores > 1.7, increasing from 77.6% (59/76) to 94.7% (72/76), a significant improvement (P = 0.001). The new and simplified extraction increased the proportion of strains identified to the species level with scores > 1.7 from 62.0% (18/29) to 86.2% (25/29) (P = 0.016). An in-house library construction ensured accurate species identification for all isolates.ConclusionsThe Bruker mass spectrometer can accurately identify Nocardia species, albeit with some variations observed between different database versions. The MALDI Biotyper 3.1 (V09.0.0.0_8468) has limitations in identifying Nocardia brasiliensis, with some strains only identifiable to the genus level. MBT Compass 4.1 (V11.0.0.0_10833) effectively addresses this shortfall, improving species identification accuracy to 98.7%, and offering quick and reliable identification of Nocardia. Both database versions incorrectly identified the clinically less common Nocardia sputorum as Nocardia araoensis. For laboratories that have not upgraded their databases and are unable to achieve satisfactory identification results for Nocardia, employing the new and simplified extraction method can provide a degree of improvement in identification outcomes.

Fast and Simple Extraction for LCMSMS Analysis of Oxytetracycline Residue in Pork

Pork is very popular as an animal-base protein source of peoples in the Lao PDR. The pig growth promotion, treatment and prevention of diseases frequently relies on the use of antibiotics. In terms of food safety, the analysis of antibiotic residue in pork would be therefore important. Oxytetracycline (OTC) is one of antibiotics used in the pig husbandry. In this study, a fast and simple extraction and analysis method for determining OTC residue in pork was developed. Briefly, 1.0 g of homogenized pork sample in a 50mL-centrifµge tube was extracted with 10 mL of CH3OH: H2O mixture (9:1 ratio) assisted with vortex for 10 min, treated with QuEChERS salt and subjected to centrifugation at 5000 rpm. The methanolic extract (1.5 mL) was then cleaned up with 2mL-dSPE tube (containing MgSO4, PSA, and C18), filtered through syringe filter 0.45 micron and analyzed with ESI-LCMS/MS optimized and operated in MRM positive mode. Calibration was prepared with OTC standard solutions (5-100 ng/g). The analytical method was validated with triplicated recovery tests at a spiked level of 50 ng/g. The results unveiled that the method gave very good recovery of 88.7±3.24%. The limits of detection (LOD) and quantification (LOQ) were achieved with 0.36 ± 0.02 and 1.19 ±0.04 ng/g. In consequence, the developed method would be suitable for OTC determination in pork samples.