Identification and quantification techniques of polymorphic forms - A review.

In the pharmaceutical industry, the unexpected appearance of crystalline forms could impact the therapeutic efficacy of an Active Pharmaceutical Ingredient (API). For quality control, a thorough qualitative and quantitative monitoring of pharmaceutical solid forms is essential to ensure the detection and the quantification of crystalline forms, wither different or with the same chemical composition (polymorphs) at a low detection level. The purpose of this paper was to review and highlight the importance of choosing adequate solid-state techniques for detection and quantification APIs that present polymorphism - based on limits of detection (LOD) and quantification (LOQ), pharmacopeias specifications, international guidelines and studies reported in the literature. To this study, the powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), Infrared and Raman spectroscopies and solid-state nuclear magnetic resonance (NMR) were the solid-state techniques analyzed. Additionally, the Argentine, Brazilian, British, European, International, Japanese, Mexican and the United States of America pharmacopeias were reviewed. Based on the analysis performed, the advantages and disadvantages of these techniques, as well as the LOD and LOQ values of APIs were reported. In comparison to these solid-state techniques, reference material used for identification analyses should be previously identified with the corresponding polymorph. Without this previous procedure, the patterns, the spectra, and DSC curves of the reference material can only be used to confirm the mixture of solid forms, not being able to specify which polymorphs are contained in the sample. A major advantage of PXRD is the use of the calculated diffraction patterns obtained from the Crystallographic Information Frameworks (CIFs) files which could be used as a reference pattern without any other information, assistance technique, or physical standards. Regarding the quantification aspect, different pharmacopeias suggest various methods such as the PXRD combining with Rietveld method, which can be used to obtain lower LOD values for minority phases in the mixture of different substances without the need for a calibration curve. Raman spectroscopy can detect polymorphs in small particles and solid-state NMR spectroscopy is a powerful technique for quantification not only crystalline but also crystalline-amorphous mixtures. Finally, this review intends to be a useful tool to control, with efficiency and accuracy, the polymorphism of APIs in pharmaceutical compounds.

Exploring the potential of Mustard (Brassica spp.) seeds through 'Kolhu' traditional method of extraction and novel identification of an anti-cancer dipeptide, Aurantiamide acetate (Asperglaucide) on ultra-performance liquid chromatography-mass spectrometry coupled with quadrupole time-of-flight (UPLC/MS-QToF) analytical platform.

Mustard (Brassica spp.) is one of the world's oldest condiments in the food basket, which holds a significant place in the global culinary landscape due to historical prominence and perceived health benefits. This study explores the extraction of oils from Mustard seeds by employing traditional 'Kolhu' method, modern supercritical fluid, and solvent extraction techniques. This study, for the first-time, identified Aurantiamide acetate, a potent anti-cancer dipeptide in Mustard seeds using ultra-performance liquid chromatography-mass spectrometry coupled with quadrupole time-of-flight (UPLC/MS-QToF) analytical platform. The analytical methodology was meticulously validated encompassing optimal parameters such as limit of detection, limit of quantification, precision, accuracy, linearity and robustness, within the range. Interestingly, 'Kolhu' method of oil extraction exhibited better yield of Aurantiamide acetate, suggesting superior efficiency of traditional methods. This study accentuates the importance of classical extraction methods, used traditionally, and emphasizes that naturally occurring substances indeed could be harnessed for better health.

Using QuEChERS and HPLC Method to Monitor the Background Concentration of Polycyclic Aromatic Hydrocarbons in Commercial Black Tea Leaves and Infusions in Taiwan.

Tea is an integral part of Taiwanese culture and is a popular drink as it contains many beneficial compounds. However, during the processing of tea, polycyclic aromatic hydrocarbons (PAHs) may form. This study investigated the concentrations of PAH4 in different black tea leaves and tea infusions based on the origin of the tea. The samples were extracted using QuEChERS, while the content of PAH4 was analyzed by high performance liquid chromatography coupled to a fluorescence detector (HPLC-FLD). The content of PAH4 in the tea leaves ranged from 2.88 µg/kg to 218.2 µg/kg (dry weight), with the highest concentration being found in teas from Vietnam. The concentration of BaP ranged from ND to 47.92 µg/kg. The release of PAH4 from tea leaves to tea infusions was significantly low, with the highest transfer being 25.8%. In this study, all PAH4 compounds in commercial black tea leaves can be detected by QuEChERS extraction with a simple HPLC method.

Summary recommendations on "Analytical methods for substances in the Watch List under the Water Framework Directive".

The Watch List (WL) is a monitoring program under the European Water Framework Directive (WFD) to obtain high-quality Union-wide monitoring data on potential water pollutants for which scarce monitoring data or data of insufficient quality are available. The main purpose of the WL data collection is to determine if the substances pose a risk to the aquatic environment at EU level and subsequently to decide whether a threshold, the Environmental Quality Standards (EQS) should be set for them and, potentially to be listed as priority substance in the WFD. The first WL was established in 2015 and contained 10 individual or groups of substances while the 4th WL was launched in 2022. The results of monitoring the substances of the first WL showed that some countries had difficulties to reach an analytical Limit of Quantification (LOQ) below or equal to the Predicted No-Effect Concentrations (PNEC) or EQS. The Joint Research Centre (JRC) of the European Commission (EC) organised a series of workshops to support the EU Member States (MS) and their activities under the WFD. Sharing the knowledge among the Member States on the analytical methods is important to deliver good data quality. The outcome and the discussion engaged with the experts are described in this paper, and in addition a literature review of the most important publications on the analysis of 17-alpha-ethinylestradiol (EE2), amoxicillin, ciprofloxacin, metaflumizone, fipronil, metformin, and guanylurea from the last years is presented.

Analyte and matrix method extension of per- and polyfluoroalkyl substances in food and feed.

The development and expansion of analytical methods for per- and polyfluoroalkyl substances (PFAS) in food are essential for the continued monitoring of the United States (US) food supply and assessments of dietary exposure. In March 2022, the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants in Feed and Food (EURL POPs) released a guidance document covering priority PFAS of interest, including analytical method parameters and limits of quantification (LOQs). As a result, the Food and Drug Administration (FDA) began method extension work to incorporate ten new additional analytes to method C-010.02 including long-chain perfluorosulfonic acids, fluorotelomer sulfonates, and perfluorooctane sulfonamide. Four long-chain carboxylic acids were also validated across all foods, which were previously added to C-010.02 but only validated in seafood. In December 2022, the European Union published Commission Regulation 2022/2388, establishing maximum levels for perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) in certain foodstuffs, primarily fish, molluscs, crustaceans, and eggs. As a result, the FDA method was evaluated for performance in reaching LOQs defined in Commission Regulation (EU) 2022/1431. The FDA method was found to be able to reach all required LOQs for analytes in matrices with established maximum levels. Currently, method detection limits (MDLs), which are used by the FDA as the lower limit for reporting PFAS in surveillance samples, were in the same range as defined indicative levels. With further method modifications, required LOQs could be met in fruits, vegetables, and milk. Reaching the lower targeted LOQs for these food matrices will require moving the method to an instrument that can provide increased signal:noise gains at the lower limits of quantification.

Review of scientific literature on available methods of assessing organochlorine pesticides in the environment.

Organochlorine pesticides (OCPs) are persistent organic pollutants (POPs) widely used in agriculture and industry, causing serious health and ecological consequences upon exposure. This review offers a thorough overview of OCPs analysis emphasizing the necessity of ongoing work to enhance the identification and monitoring of these POPs in environmental and human samples. The benefits and drawbacks of the various OCPs analysis techniques including gas chromatography-mass spectrometry (GC-MS), gas chromatography-electron capture detector (GC-ECD), and liquid chromatography-mass spectrometry (LC-MS) are discussed. Challenges associated with validation and optimization criteria, including accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ), must be met for a method to be regarded as accurate and reliable. Suitable quality control measures, such as method blanks and procedural blanks, are emphasized. The LOD and LOQ are critical quality control measure for efficient quantification of these compounds, and researchers have explored various techniques for their calculation. Matrix interference, solubility, volatility, and partition coefficient influence OCPs occurrences and are discussed in this review. Validation experiments, as stated by European Commission in document SANTE/11813/2017, showed that the acceptance criteria for method validation of OCP analytes include ≤20 % for high precision, and 70-120 % for recovery. This may ultimately be vital for determining the human health risk effects of exposure to OCP and for formulating sensible environmental and public health regulations.

Quantification of Ochratoxin A in 90 spice and herb samples using the ELISA method.

This study addressed the challenge of accurately detecting mycotoxins in herbs and spices, which have gained popularity as alternative medicines but pose health risks due to potential contamination. We used a competitive direct ELISA kit (Art No. 8610), Veratox for Ochratoxin, to quantify Ochratoxin A in the herb and spice samples. The samples were first prepared using solid-liquid extraction with 70% methanol. The resulting filtrate was then subjected to ELISA analysis. The results of the analysis were then further analyzed using principal component analysis (PCA). In this study, PCA was used to classify the concentration levels of Ochratoxin A based on various factors, such as the packaging type, country of origin, shelf life, and sample weight. The limits of detection (LOD) and quantification (LOQ) values indicate the lowest amount of Ochratoxin A that can be detected and quantified, respectively, with high accuracy and precision. The range of the LOD and LOQ values (0.43-0.58 µg/kg and 1.45-1.95 µg/kg, respectively) suggests that the method used was capable of detecting and quantifying Ochratoxin A in the herb and spice samples at different concentrations with a high degree of accuracy and precision. These results suggest that while most of the samples (73.33%) were below the maximum residue limit (MRL) for Ochratoxin A, a significant number of samples (26.67%) had concentrations of Ochratoxin A that were higher than the MRL. This highlights the importance of monitoring Ochratoxin A in herb and spice samples and ensuring the products are safe for consumption.

A coumarin hydrazone appended rotatable phenolic scaffold as fluorescent chemosensor for Ag+ ions and its bio imaging applications.

A coumarin hydrazone-phenol conjugate, COH4 was designed, synthesized and utilized for the cation sensing studies by fluorimetry studies. The synthesized chemosensor was completely characterized by the usual spectroscopic and analytical studies. The COH4 receptor was examined for the detection of metal ions, in which it had a noticeable blue shifted fluorescence enhancement for Ag+ ions. Upon binding towards Ag+ ions, the photoinduced electron transfer (PET) process is inhibited via intramolecular charge transfer (ICT) process assisted by the arrest of the carbon-carbon single bond rotation. The binding stoichiometry of COH4 + Ag+ complexation ratio is noted to be 1:2, which was further confirmed by jobs plot method. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.41 µM and 0.13 µM respectively. Moreover, COH4 was successfully utilized for the practical applications of Ag+ ion detection in bacterial cell lines.

Chemical and in vitro toxicological comparison of emissions from a heated tobacco product and the 1R6F reference cigarette.

It has previously been found that, compared with cigarette smoke, the aerosols generated by heated tobacco products contain fewer and lower harmful and potentially harmful constituents (HPHCs) and elicit lower biological activity in in vitro models and lower smoking-related exposure biomarker levels in clinical studies. It is important to accumulate such scientific evidences for heated tobacco products with a novel heating system, because different heating system may affect the quantitative aspect of the amount of HPHCs and the qualitative aspect of the biological activity of the aerosol generated. Here, the chemical properties of, and toxicological responses to aerosols emitted by DT3.0a, a new heated tobacco product with a novel heating system, and cigarette smoke (CS) were compared, using chemical analyses, in vitro battery (standardized genotoxicity and cytotoxicity) assays, and mechanistic (ToxTracker and two-dimensional cell culture) assays. Regular- and menthol-flavored DT3.0a and standard 1R6F reference cigarettes were tested. Selected HPHC yields were lower in DT3.0a aerosol than 1R6F CS. The genotoxicity-related assays indicated that DT3.0a aerosol was not genotoxic, regardless of metabolic activation. The other biological assays indicated that less cytotoxicity induction and oxidative stress response were elicited by DT3.0a aerosol compared with 1R6F CS. Similar results were found for both regular and menthol DT3.0a. Like previous reports for heated tobacco products with other heating systems, the results of this study indicated that DT3.0a aerosols have chemical and biological properties less likely to be harmful than 1R6F CS.

Method validation of multi-element panel in whole blood by inductively coupled plasma mass spectrometry (ICP-MS).

Background: Analytical methods to measure trace and toxic elements are essential to evaluate exposure and nutritional status. A ten-element panel was developed and validated for clinical testing in whole blood. Retrospective data analysis was conducted on patient samples performed at ARUP Laboratories. Methods: A method was developed and validated to quantify ten elements in whole blood by ICP-MS. Fifty microliters of sample were extracted with 950 µL of diluent containing 1 % ammonium hydroxide, 0.1 % Triton X-100, 1.75 % EDTA along with spiked internal standards. Four calibrators were used for each element and prepared in goat blood to match the patient specimen matrix. Samples were analyzed with an Agilent 7700 ICP-MS with a Cetac MVX 7100 µL Workstation autosampler. Results: The assay was linear for all elements with inter- and intra-assay imprecision less than or equal to 11% CV at the low end of the analytical measurement range (AMR) and less than or equal to 4% CV at the upper end of the AMR for all elements. Accuracy was checked with a minimum of 40 repeat patient samples, proficiency testing samples, and matrix-matched spikes. The linear slopes for the ten elements ranged from 0.94 to 1.03 with intercepts below the AMR and R2 ranging from 0.97 to 1.00. Conclusions: The multi-element panel was developed to analyze ten elements in whole blood to unify the sample preparation and increase batch run efficiency. The improved analytical method utilized matrix-matched calibrators for accurate quantification to meet regulatory requirements. The assay was validated according to guidelines for CLIA-certified clinical laboratories and was suitable for clinical testing to assess nutritional status and toxic exposure.

Development of an automated chemiluminescent immunoassay for cancer antigen 72-4 and the evaluation of its analytical performance.

Objectives: Cancer antigen (CA) 72-4 assay is widely used for monitoring gastric and ovarian cancers. The antigen is a mucin-like, tumor-associated glycoprotein known as TAG-72. It has been identified and characterized using two different monoclonal antibodies, CC49 and B72.3, which recognize its glycochain epitopes, Galß(1-3) sialyl-Tn and sialyl-Tn antigens, respectively. This study describes the quantitative analytical performance of a newly developed CA 72-4 assay, ARCHITECT CA 72-4. Design: and Methods: The ARCHITECT CA 72-4 assay was developed using the ARCHITECT i2000SRs and three ARCHITECT i1000SRs. The assay performance was evaluated based on guidance from CLSI (Clinical and Laboratory Standards Institute) and correlation against Elecsys CA 72-4. Results: In the total precision study, the minimum coefficient of variation (CV) for Control/Panel samples over 4 U/mL was 1.1%. The measuring interval was from 0.95 to 200 U/mL with good linearity; and limits of blank (LoB), detection (LoD), and quantitation (LoQ) were 0.09, 0.18, and 0.95 U/mL, respectively. High dose hook effect; differences among specimen tube types; and interference of common drugs, potential cross-reactants, and endogenous substances were not observed. Significantly, this assay has high biotin tolerance at 4875 mg/mL and correlates well with the Elecys CA 72-4 assay (correlation coefficient: 0.95). Conclusions: ARCHITECT CA 72-4 is a highly sensitive and precise assay for CA 72-4 measurement in human sera and plasma.

Postmortem sampling time effect on toxicity biomarkers in rats exposed to an acute lethal methomyl dose.

Regulations often are imposing long postmortem times before autopsy leading to certain toxicity-unrelated changes in biomarkers, which in turn may affect the reliability of toxicity evaluation during forensic investigations. Since methomyl pesticide shows significant toxicity and is frequently encountered in poisoning cases, the current study evaluated different parameters in methomyl intoxicated rats at three different postmortem intervals (Hour 0, Hour 3 and Hour 6). Eighteen adult Sprague Dawley rats were poisoned with methomyl to simulate actual methomyl poisoning cases. The time of death was assigned as Hour 0. The animals were divided into 3 groups (n = 6) to collect blood and tissue samples at the selected time points. Body weight, relative organ weight, protein concentration, methomyl concentration and acetylcholinesterase activity (AChE) were assessed in blood and different tissues (liver, spleen, kidney, brain, eye, and bone marrow) to evaluate the effect of postmortem sampling time. Outcomes revealed significant decreases in methomyl concentration in blood and bone marrow with advanced sampling time (P < 0.001). Similarly, there were significant reductions in AChE activity in the kidney (P < 0.01), while the enzyme activity significantly increased in brain samples (P < 0.05). Findings illustrated the importance of sampling time in toxicity studies because it could alter experimental results and impact consequent interpretations, as well as it may alter postmortem biomarkers in related forensic cases.

A single centre experience of local perforator flaps in oncoplastic breast surgery; a cross-sectional study.

Background & objectives: Various oncoplastic techniques have emerged over the years to preserve breast cosmesis and symmetry without compromising the principles of tumor excision. One of the newer techniques for breast volume replacement to achieve symmetry and cosmesis is the use of fasciocutaneous pedicled chest wall perforator flaps or local perforator flaps (LPF). The objectives of this study were to document the details of the surgical procedure as well as patient-reported satisfaction and well-being following the procedure using a validated BREAST-Q tool among Pakistani women. Materials & methods: This cross-sectional study was conducted from March 2019 to February 2021 enrolling 25 female patients who underwent breast conservative surgery using LPF for breast tumors at The Aga Khan University Hospital, Karachi. Data related to the procedure was collected on a pre-designed proforma. Cosmetic outcomes and patient satisfaction were evaluated using 2 scales from BREAST-Q BCT domain version 2.0. The questionnaire was self-administered by the patients during their routine follow-up in the clinic. Data was analyzed using the Statistical Package for Social Sciences (SPSS) version 23. Mean (SD)/median (IQR) were computed for quantitative variables and frequency and percentages were calculated for qualitative variables. 2 sample t-test was applied. P-value ≤0.05 was considered significant. Results: 25 patients underwent LPF with a mean age of 47 ± 13.1 years. 8 LICAP, 7 AICAP, and 10 LTAP flaps were performed. Two postoperative complications of wound site erythema were encountered. 23 women were eligible for the BREAST-Q survey. Median (IQR) postoperative satisfaction with breasts and physical well-being chest (equivalent Rasch transformed score) was 100 (41) and 76 [18] respectively. We found high satisfaction with breasts and comparable physical well-being among Pakistani women after LPF surgeries. Conclusion: Local perforator flaps in oncoplastic breast-conserving surgery are a good option showing high satisfaction with breasts and physical well-being in Pakistani women.

Determination of Glyphosate in White and Brown Rice with HPLC-ICP-MS/MS.

Background: In 2017, the European Commission renewed the approval of glyphosate (GLY) but only for five years. GLY remains one of the most controversial and studied molecules. Method: A simplified method was tested for the determination of GLY in white rice (WR) and brown rice (BR), after extraction only with a methanol solution, by liquid chromatography coupled with inductively coupled mass triple quadrupole (HPLC-ICP-MS/MS) with a PRP-X100 anionic column. After performing a test on groundwater, the quantification of GLY in WR and BR was validated in terms of the LOD, LOQ, accuracy, precision, linearity, and the matrix effect. Results: The LOD was 0.0027 mg kg−1 for WR and 0.0136 mg kg−1 for BR. The LOQ was 0.0092 mg kg−1 for WR and 0.0456 mg kg−1 for BR. The mean recoveries were within 76−105% at three fortification levels. The relative standard deviation for the analysis (five replicates for three spike levels) was < 11% for both matrices. A linear response was confirmed in all cases in the entire concentration range (R2WR = 1.000 and R2BR = 0.9818). Conclusion: The proposed method could be considered useful for the determination of GLY in different types of rice and designed and adapted for other cereals. The matrix effect, quantified in BR matrix extraction, could be avoided by using a matrix-matched calibration line.

Quantification and identification analysis of Ziziphus jujuba Mill. peel pigmentation at different developmental stages.

The fruit peel of a color mutant jujube cultivar, 'Sanbianhong' (SBF), was investigated using an ultra-high performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) at five ripening stages (S1, Young fruit stage; S2, swelling stage; S3, white-mature stage; S4, pre-mature stage and S5, mature stage). Lutein, ß-carotene, chlorophyll a, chlorophyll b, and 13 anthocyanins were identified. Chlorophyll a and cyanidin 3-O-galactoside were considered key color metabolites in S1 with the content of 1.083 mg/g of fresh weight (FW) and 4.585 mg/g of FW, respectively. Delphinidin (0.488 mg/g FW) and cyanidin (6.259 mg/g FW) were identified as the key pigments in S3. Delphinidin 3-O-glucoside (0.256 mg/g FW) was identified as the key anthocyanin in maturity S5. Herein, the identification and quantitation of pigment-related metabolites of SBF were studied for the first time, and the results provide a theoretical basis for understanding the pigment changes of jujube fruit during ripening.

Results of the BfR MEAL Study: Highest levels of retinol found in animal livers and of ß-carotene in yellow-orange and green leafy vegetables.

This Total Diet Study (TDS) provides representative data on substance levels in foods, prepared as typically consumed by the population in Germany for future dietary exposure assessment. Vitamin A is essential and must be obtained from the diet, either as preformed vitamin A or as provitamin A carotenoids. Levels of retinol and ß-carotene were analysed in 333 and 271 foods, respectively. Highest mean retinol levels were found in cod liver (25,000 µg∙100 g-1), followed by other animal livers, liver-based products, butter, eel and fortified margarine. In contrast, highest mean ß-carotene levels were found in carrots (4,650 µg∙100 g-1), followed by other yellow-orange fruits and vegetables, green leafy vegetables and fortified fruit nectars. Sampling by production type and seasonality revealed differences in retinol and ß-carotene levels in individual foods. This TDS expands the existing data for ß-carotene and vitamin A extensively by providing representative data on most consumed foods.

Estimation of the Number of Scans Required per Hard-to-Clean Location and Establishing the Limit of Quantification of a Partial Least Squares Calibration Model When the FTIR Is Used for Pharmaceutical Cleaning Verification.

This study aims to identify two critical components required for pharmaceutical cleaning verification when an FTIR is used: (a) the number of scans required per hard-to-clean location, and (b) the limit of quantification (LOQ) of the FTIR instrument when measuring the surface contamination. The current practice in pharmaceutical manufacturing does not require multiple samples as it is standard practice to collect a single swab sample from a 25 × 25 cm area from a difficult-to-reach area of the manufacturing equipment. However, since the FTIR will only scan a tiny portion of the surface compared to the swab, a sufficient number of samples (data points) are required to provide enough confidence to ensure that the measurement results are close to the true value with a maximum degree of certainty. Similarly, calculating the LOQ for a linear regression could be straightforward. However, complexity arises when the experimental data are complex; in this case, the complexity arises due to the nature of the measurement and the lack of the defined peak in the pre-processed spectra. Therefore, this study uses the practical approach of calculating the sample size and the LOQ.

Comparison of ADVIA Centaur ultra-sensitive and high-sensitive assays for troponin I in serum.

Cardiac troponin I (cTnI) is a standard biomarker for the diagnosis of acute myocardial infarction (AMI). While older, ultra-sensitive cTnI (us-cTnI) assays use the 99th percentile as the reference threshold, newer high-sensitive cTnI (hs-cTnI) assays use the limit of detection or functional sensitivity instead. However, little has been done to systematically compare these two methods. The present study also served as a validation of hs-cTnI in our laboratory. Here, we compared the results obtained from the blood serum obtained from 8810 patients using the us-cTnI and the hs-cTnI assays run in tandem on the ADVIA Centaur XP analyser. We found that in 2279 samples the concentration of cTnI measured with the ultra-sensitive method was below the detection limit, while with the high-sensitive method, only 540 were below the detection limit. We also compared results from these assays with the ultimate diagnosis of a subset of individuals. The analysis of the results below cut-off with the ultra-sensitive method showed that this method would not detect 96 cases related to heart disorder. Overall, the main finding of our research is that hs-cTnI is the preferable option and is able to be deployed effectively in the laboratory setting.

Development and evaluation of real-time recombinase polymerase amplification assay for rapid and sensitive detection of Vibro mimicus in human plasma samples.

AIM: We aimed at developing a fast and accurate method to detect Vibrio mimicus using real-time recombinase polymerase amplification assay. METHODS AND RESULTS: Specific primers and probe were designed to target V. mimicus haemolysin (vmh) gene. Target DNA was successfully amplified at 41°C within 20 min. The method exhibited a high level of specificity and the sensitivity was 2.1 × 102 copies/25 µl or 8.4 copies/µl, which is in line with real-time polymerase chain reaction (PCR). The calibration curve plotted by the second-order polynomial regression showed better than the linear curve, as the correlation coefficient was raised to 0.9907, which suggested that the second-order polynomial regressions might be considered to apply to the quantification of real-time recombinase polymerase amplification (RPA). The limit of detection (LOD) was predicted to be 77 copies/25 µl or 3 copies/µl by a probit model. The limit of quantification (LOQ) was calculated to be 28 copies /25 µl or 1 copies/µl by a receiver operating characteristic (ROC) curve, which firstly make LOQ could be available to real-time RPA. For the performance of the real-time RPA in plasma samples, the detection sensitivity of real-time RPA was as good as the real-time PCR. For pretreatment of plasma samples, the boiling method was better than using kits, as it further shortened the time of the real-time RPA in detecting V. mimicus. CONCLUSIONS: The real-time RPA assay developed in our study shows multiple advantages over currently available DNA diagnostic method, including a quicker time-to-result for a single sample, requiring minimal infrastructure and technical support and being tolerant to inhibitors in plasma samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time RPA assay developed here is a potentially valuable tool for point-of-care (POC) diagnosis of V. mimicus infection in endemic field, especially in the resources-limited settings, as combined with portable devices.