共找到 20 条结果
Bromoform is the bioactive compound present in Asparagopsis seaweed responsible for methane reduction in ruminants. Although several analytical methods are available, no AOAC Official Method exists. A study was performed to validate a GC-MS/MS-based method for determination of bromoform in fresh and freeze-dried seaweed, canola oil containing bromoform (oil herein), and liquid and dry cattle feed. The method was evaluated according to AOAC guidelines for calibration curve validity, selectivity, extraction protocol robustness, and matrix and extract stability. Repeatability, recovery, and limits of detection and quantification were also determined for each matrix. The calibration curve was linear over the range 0.000124-0.020 µg/mL (R2= 0.999), and residual analysis demonstrated suitability of the linear model. There was no interference from compounds similar to bromoform (dibromomethane, dichloromethane, dibromochloromethane, and dibromoacetic acid). The analyte was identified by comparison of retention time and ion abundance profiles between calibration standards and unknown samples. The LOQs were 0.057 mg/g, 0.423 mg/g, 0.033 mg/g, 0.022 mg/g, and 0.041 mg/g for fresh seaweed, freeze-dried seaweed, oil, liquid feed, and dry feed, respectively. Bromoform recovery among matrixes across multiple fortified concentration levels was 93-104%, and repeatability was 0.75-5.4%. The method was robust to minor changes in extraction conditions. This method was demonstrated to be accurate and precise with or without the inclusion of an internal standard. The GC-MS/MS method for bromoform determination presented in this report was shown to be fit for purpose and has been accepted as a First Action AOAC Official Method  SM. This method is the first bromoform analysis method to successfully complete the AOAC validation process and will serve to standardize the determination of bromoform in feed ingredients and finished supplements. The adoption of this method is timely given the recent interest in methane reduction in cattle using feed fortified with bromoform.
Per- and Polyfluoroalkyl Substances (PFAS) are synthetic compounds widely used due to their water, grease, and dirt repellent properties. They are persistent and resistant to degradation, therefore they can be found in the environment, the food chain, and in human fluids. The European Union (EU) has established limits of quantification (LOQs) for four PFAS in produce, seafood, meat, eggs, fish oil, milk, and ready-to-eat food for infants and young children and set maximum levels (MLs) for the same analytes in eggs, fish, and meat products. The study describes a quantitative LC-MS/MS method for the determination of 57 PFAS in food. The 55 analytes are quantified by isotopic dilution while two of the compounds, namely Capstone A and Capstone B are quantified using 2-level standard addition. Seven of the compounds have only one transition, which need another technique for their confirmation in case of a positive result in a sample. The scope comprises analytes included in the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPRs®) 2023.003, cited in the EU Recommendation 2022/1431, and analytes surveyed by service laboratories in Europe or mentioned in the literature.Representative samples from several food categories (baby food puree, milk-based infant formula (powder), full cream milk powder, fish meat and fish oil, whole egg, soluble coffee, pet food and edible offal) were validated. Targeted performance requirements for LOQ, recovery and precision were as defined per AOAC SMPR 2023.003. The protocol involves an acetonitrile/water extraction. For 55 PFAS analysis in all matrices, a solid phase extraction (SPE) method is applied. Detection is conducted by LC-MS/MS and isotopic dilution is applied for quantification. Confirmation for single-transition analysis is performed by LC-HRMS. For infant formula and baby food, the extract is diverted to two workflows, one for the 55 PFAS and one for Capstone A & Capstone B, where a diluted aliquot is analyzed by LC-MS/MS and quantified by the standard addition procedure. The method was single laboratory validated in the nine food matrices with results generally in agreement with the criteria of the AOAC SMPR 2023.003, in terms of LOQs, recovery and precision. LOQs were generally in the low ng/kg range, and recoveries typically fell within 70-120%, with acceptable repeatability and intermediate precision. The performance of the method was further demonstrated by analyzing 27 additional food matrices. The method described covers a wide range of food matrices and analytes compares favorably with most of the requirements of the SMPR 2023.003, it contains as well a larger scope of analytes compared to the SMPR 2023.003, giving an advantage of searching other potential sources of PFAS contamination. Following independent scientific review, the AOAC Expert Review Panel for PFAS in Food Methods determined that the method met the requirements of AOAC SMPR 2023.003 and approved it for First Action status as AOAC Official Method 2025.10. This work presents a validated LC-MS/MS method for 57 PFAS across diverse food matrices, meeting or exceeding most of the AOAC SMPR 2023.003 performance requirements and demonstrating broad applicability for routine monitoring. The method includes additional PFAS of emerging interest and was approved for AOAC First Action status, designated AOAC Official Method 2025.10.
Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. The fortification of taurine to infant formulas is necessary to provide sufficient nutrient to maintain serum levels equivalent to those of their counterparts fed on their mother's milk. To evaluate method reproducibility of AOAC 2022.03 Official First Action method for compliance with the performance requirements described in AOAC Standard Method Performance Requirements (SMPR®) 2014.013. Following protein precipitation with Carrez solutions, taurine is extracted and analyzed by hydrophilic interaction liquid chromatography-tandem mass spectrometry using multiple reaction monitoring. Stable isotope labelled taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. Thirteen laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2014.013 (≤ 8%) for all seven test samples and all had acceptable HorRatR values ranging from 0.4 to 0.7. The method described is suitable for the quantification of taurine in infant formulas and adult nutritionals. The AOAC Expert Review Panel for the Stakeholder Program for Infant Formula and Adult Nutritionals evaluated the method and accompanying validation data from this multi-laboratory testing study in June 2025 and recommended Official Method 2022.03 for adoption as a Final Action Official Method. A multi-laboratory testing study of a HILIC-MS for the determination of taurine is described.
AOAC INTERNATIONAL SMPRs® 2023.003 outlines method performance requirements for the determination of per- and polyfluoroalkyl substances (PFAS) across a broad range of food and feed matrices. The objective of this study was to develop and validate the EMR LC-MS/MS method in accordance with AOAC INTERNATIONAL SMPR 2023.003 using a single-laboratory approach. The method features QuEChERS extraction followed by a novel enhanced matrix removal (EMR) mixed-mode passthrough cleanup, an online injection program, sensitive LC-MS/MS detection, and accurate, precise quantitation using neat calibration curves with isotopically labelled internal standards. Method performance was evaluated for suitability and selectivity, calibration linearity, limit of quantitation (LOQ), recovery and repeatability, and the method was successfully validated in a single laboratory validation (SLV) across 16 diverse food matrixes, encompassing all 11 required food categories. Acceptable PFAS background cleanliness was demonstrated, supporting accurate and reliable quantitation in all food matrixes.Validated or calculated LOQs for all analytes across all food matrices were equal or below the required LOQs, with an exception rate of < 1%. Recovery and single laboratory repeatability also satisfied the acceptance criteria specified in the AOAC SMPR guideline, again with an exception rate of < 1%. Method selectivity was confirmed through baseline separation of cholic acid interferences from PFOS isomers and through secondary confirmation of PFBA and PFPeA using LC-QTOF analysis. The EMR LC-MS/MS method met all performance criteria, supporting its advancement as a First Action AOAC Official Method of AnalysisTM (OMA) 2025.11 (1). A novel EMR LC-MS/MS method was developed and successfully validated for the determination of 30 PFAS analytes across a wide range of food and feed matrices, fulfilling the requirements of AOAC SMPR 2023.003.
Vitamin B1 (thiamine), D2 (ergocalciferol), and D3 (cholecalciferol) are essential nutrients in animal diets, critical for health and the maintenance of physiological processes. Deficiencies or excesses can lead to severe health issues, necessitating reliable quantification methods for quality control, label claim compliance, and safety assurance in the pet food industry. The aim of this study was to extend and validate AOAC Official Methods 2015.14 (for vitamin B1) and 2016.05 (for vitamins D2 and D3) to animal feed matrices using LC-MS/MS in a single-laboratory study. Thiamine was extracted via enzymatic digestion with acid phosphatase, while vitamins D2 and D3 were extracted using saponification, followed by liquid-liquid extraction. The methods were validated for cat kibble, dog kibble, and wet dog feed, demonstrating high sensitivity (limits of detection: 0.018 ng/mL for B1, 0.005 ng/mL for D2 and D3; limits of quantification: 0.06 ng/mL for B1, 0.016 ng/mL for D2, 0.018 ng/mL for D3), excellent linearity (R2 = 0.997-0.999), and acceptable recoveries (88-115%). Quantification revealed vitamin B1 concentrations exceeding the Association of American Feed Control Officials minimum recommendations, with the lowest at 8.99 mg/kg on a dry matter basis (DMB) in wet dog food. Vitamin D levels were within recommended ranges, with the lowest in cat kibble at 18.13 µg/kg DMB for vitamin D3, while vitamin D2 contributed minimally to total vitamin D, at 0.27 µg/kg DMB. The AOAC Official Methods 2015.14 and 2016.05 were suitable for the analysis of vitamins B1 and vitamins D2/D3, respectively, in cat kibble, dog kibble, and wet dog food. These results support the utility of the methods for routine analysis of animal feeds to verify label claims, nutritional adequacy, and safety.
The positional distribution of the fatty acids on the triacylglycerol influences uptake of calcium, fat, and energy. The positional distribution of breast milk is similar, but not identical, to that of cow and goat milk, and is different to that of vegetable oil. Often, in infant formula a mixture of milk fat with certain vegetable oils is used, to mimic the positional distribution of breast milk. A method for the analysis of fatty acids at the sn-2 position in infant formula and adult/pediatric nutritionals by enzymatic transesterification is presented. The aim of this study is to demonstrate the suitability and applicability of the method to determine fatty acids at the sn-2 position of triacylglycerol molecules in infant formula and adult/pediatric nutritionals by enzymatic transesterification. The sample is extracted to isolate the fat, which is treated with a regiospecific enzyme to remove the sn-1(3) positions of the triacylglycerol, resulting in 2-monoacylglycerols. These are then separated using solid phase extraction, transesterified to fatty acid methyl esters, and analyzed using gas chromatography with flame ionization detection. The relative standard deviation repeatability (RSDr) and intermediate precision (RSDiR) were in the range of 1.2-5.2% (RSDr) and 2.0-13.3% (RSDiR). The accuracy was assessed by spiking experiments and model triglycerides, and recoveries were on average 95% (spiked addition) and 97% (model triglycerides). The limit of quantification (LOQ) was found at 0.003 g/100g on reconstituted product. The SLV demonstrates that it meets the criteria set in the Standard Method Performance Requirements (SMPRSM) 2022.004. A method to determine the content of fatty acids at the sn-2 position by use of an enzyme with good 1(3) regiospecificity was developed. The method complies to international and governmental regulations. The method was evaluated by the Expert Review Panel on Nutrient Methods, and granted first action status.
The BioSystems Gluten is an automated quantitative method based on immunoturbidimetry for quantification of the 33-mer peptidic immunotoxic fraction of gluten in food and beverages, crucial for ensuring safety for those with gluten-related disorders like celiac disease (CD). An AOAC Performance Tested MethodSM validation study was performed to evaluate the BioSystems Gluten method for the quantification of gluten from wheat, barley, and rye flours across selected food and beverage commodities, including both non-processed and processed products. Validation covered non-processed (rice flour, corn flour, wine, and sausage) and incurred matrixes (cornbread and rice cookies), spiked with wheat, barley, and rye gluten (0-30 mg/kg). Twelve test portions were analyzed for each matrix and contamination level using three different kit lots. The study evaluated linearity, bias, recovery, precision, LOD and LOQ, selectivity, and interferences, evaluated by recovering gluten in a defined gluten-free matrix group, stability studies of the kit components, and method robustness following AOAC Guidelines for Quantitative Gluten Methods. The method met AOAC performance requirements for the quantitative determination of gluten from wheat, barley, and rye across a variety of representative food commodities and spiking levels. The validated working range spans the LOQ (2.5 mg/kg) to 200 mg/kg. The method achieved satisfactory recovery rates within the established acceptance limits, showed no evidence of analytical interferences, and yielded comparable results between the method developer and an independent laboratory, supporting its robustness and reproducibility. The validation study conclusively establishes the test kit as an automated, precise, rapid, and user-friendly method for quantifying gluten concentrations in both food and incurred matrixes derived from wheat, barley, and rye flours. A novel automated method is presented for gluten quantification based on spectrophotometric analysis in a Biosystems Y15 analyzer with ready-to-use and liquid reagents.
Tomato seeds, a by-product of the tomato-processing industry, are a rich source of bioactive compounds with promising nutritional and functional potential. This study aimed to optimize the supercritical CO2 (SC-CO2) extraction of phenolic compounds from Solanum lycopersicum L. seeds using a Full Factorial Design (FFD) within the framework of Response Surface Methodology (RSM). The effects of extraction pressure (100-500 bar) and temperature (40-80 °C) were evaluated, with oil extraction yield and total phenolic content (TPC) as response variables. A one-factor-at-a-time (OFAT) approach assessed the effect of ethanol (0.25-1.0 mL) as a co-solvent under constant optimal extraction conditions. Derringer's desirability function was applied to identify simultaneous optimal conditions to maximize both responses. The main phenolic compounds were putatively annotated via high-performance liquid chromatography coupled with diode array detector and time-of-flight mass spectrometer (HPLC-DAD-TOF). Extraction pressure and co-solvent volume significantly affected both oil extraction yield and TPC, while temperature primarily influenced yield. Multi-response optimization achieved an overall desirability value of 1.0 at 425 bar and 78 °C with 1 mL ethanol, resulting the highest oil extraction yield and phenolic recovery. SC- CO2 extraction combined with desirability-based RSM is an efficient, green technique for valorizing tomato-processing residues, supporting circular bioeconomy principles through the sustainable recovery of high-value bioactives. Optimized SC-CO2 extraction using RSM revealed that the factors most influencing oil extraction yield and the recovery of phenolic compounds were pressure and the quantity of co-solvents used. HPLC-DAD-TOF analysis putatively annotated the main phenolic compounds, thus emphasizing the sustainable valorization of by-products from tomato processing.
The introduction of human milk oligosaccharides (HMOs) as ingredients in infant formula requires that suitable methods are available for their determination. Three methods were proposed to AOAC INTERNATIONAL for this analysis. Of the three, two methods (AOAC 2022.02 and 2022.07) were essentially based on the same approach. To conduct multi-laboratory testing (MLT) with the consolidated method from AOAC methods 2022.02 and 2022.07 for analysis of Human Milk Oligosaccharides (HMOs) in Infant Formula and Adult Nutritionals. The optimal procedural steps and settings were taken from each method to provide a method that uses ultra-high performance liquid chromatography (UHPLC) with 2-aminobenzamide (2AB) derivatization followed by hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection for quantitative analysis of seven HMOs in infant formula and adult nutritionals. A summary of the method parameters selected from each method along with reasoning is provided, as well as a comparison study of the previous methods. The data demonstrated that results do not differ from those of the previous methods, supporting appropriate trueness. The data also suggests the precision of the new method is likely comparable to the parent methods. The method was reviewed by the AOAC Expert Review Panel on Nutrients and determined that the method provided trueness to the previous methods, and additional data will be collected within the MLT to further support precision. The method was granted AOAC First Action Official MethodsSM status.
Oxytetracycline (OTC) is used as a therapeutic agent against bacterial pathogens in aquaculture, typically administered via medicated feed. Insufficient withdrawal time following drug treatment will result in antibiotic residue deposition in the tissues. Antibiotic residues are determined from freshly harvested fish tissues or processed fish samples for export purposes. But sufficient data is not available on the effect of different processing methods on the fate of antibiotic residues in fish tissues. This study is aimed at understanding the effects of various processing/cooking methods on the retention of OTC residues in the edible fillets of OTC-fed tilapia. Medicated feed was prepared by coating different doses of OTC into commercially available feed pellets and was administered to the fish in all the treatment groups for 10 days, and edible fillet samples were collected for various processing techniques, including boiling, microwave cooking, frying, and drying. The processed samples were analysed for OTC residues using Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). A Limits of detection (LOD) of 1.75 µg/kg and quantification (LOQ) of 5.82 µg/kg was achieved by the developed LC-MS/MS method for the quantification of OTC in fish samples. The results revealed a significant reduction of OTC in fish tissues following various processing methods. The maximum degradation was observed towards microwave processing and frying. This study provides insights into the effectiveness of various cooking/processing methods for the efficient reduction of OTC residues in edible fish fillets. The present study highlights the effects of different processing methods and demonstrates that even when some levels of drug residues are present in fish tissues, they can be effectively degraded using appropriate processing methods.
Bovine coronavirus (BCoV) is an economically significant pathogen that causes respiratory and enteric infections in cattle and wild ruminants. Although multiple diagnostic methods are available, there is still an urgent need for a sensitive real-time reverse transcription quantitative PCR (RT-qPCR) assay capable of detecting continuously emerging novel strains of BCoV. This study aimed to develop a sensitive and broad-spectrum RT-qPCR assay for the efficient detection of BCoV. Following optimization and comparison of two primer-probe sets targeting the M gene, an RT-qPCR assay was established. Using Decaro's method as a reference, gradient dilutions of transcribed RNA solutions and BCoV-derived cDNA were prepared to test sensitivity, repeatability, and reproducibility. Additionally, four other bovine viruses were used to evaluate specificity. Finally, the diagnostic sensitivity and specificity of both assays were analyzed using 46 field samples. A sensitive and broad-spectrum RT-qPCR assay was developed in this study. Its analytical sensitivity was 10 copies/μL with transcribed RNA as the template, comparable to that of Decaro's method. While the cDNA of an emerging Chinese viral isolate was used as the template, the analytical sensitivity was one order of magnitude higher than that of Decaro's method. Reproducibility testing revealed intra-assay coefficients of variation (CV) ranging from 1.75 to 3.56%, and inter-assay CV values between 3.13 and 4.91%. When evaluating the diagnostic sensitivity and specificity with 46 field samples, our assay exhibited higher diagnostic sensitivity compared to Decaro's method, a difference explained by two mutations within the primer and probe regions of Decaro's method. A highly sensitive and broad-spectrum RT-qPCR assay was successfully developed for the detection of BCoV, especially for the detection of emerging novel strains. The RT-qPCR assay developed in this study exhibited high sensitivity, particularly in detecting BCoV field strains and newly emerging variants circulating in recent years.
Certified reference materials (CRMs) are essential for method validation, quality control, and metrological traceability in analytical chemistry. However, CRMs for plant matrices of regional relevance, such as castor bean (Ricinus communis L.) leaves, remain scarce in tropical and subtropical regions. This study addresses that gap by evaluating the homogeneity of a castor bean leaf RM candidate produced under single-laboratory conditions. To assess between- and within-unit homogeneity of a Ricinus communis L. leaf RM candidate for seven inorganic elements (Al, Ca, Cu, Fe, Mg, P and Zn), following ISO Guide 35:2017. This study does not aim at certification or replacement of full certification procedures as specified in ISO 17034:2016. Castor bean leaf samples were collected in Bahia, Brazil. A total of 400 g of dry material was produced, homogenized, and distributed among 20 units. Elemental mass fractions were determined by microwave-induced plasma optical emission spectrometry (MIP OES) after acid digestion. Data were evaluated by one-way ANOVA (ISO Guide 35:2017) and principal component analysis (PCA) as a complementary exploratory tool. A minimum sample mass of 400 mg was established as adequate for reliable homogeneity evaluation. Ten units were randomly selected for between-bottle assessment, with three subsamples per unit for within-bottle evaluation. The RM candidate was considered homogeneous for all seven elements at the 95% confidence level. PCA provided additional exploratory insights, including identification of atypical subsamples not detectable by univariate ANOVA. Castor bean RM will be of great use for confirming the accuracy of methods, which will facilitate the validation of analytical methods and verification of quality control of samples of vegetable leaves or similar matrix, in addition to allowing metrological traceability.
Metal impurities in pharmaceutical products can originate from various sources, including manufacturing machinery, catalysts used during drug synthesis, and even the container-closure systems. The objective of the present study is to accurately quantify trace levels of 24 metal impurities in the active pharmaceutical ingredients of the anticancer drugs vismodegib and idelalisib using an inductively coupled plasma mass spectrometer. The diluent used in the analysis consists of a mixture of 3% nitric acid and 1% hydrochloric acid, while argon serves as the carrier gas. Helium is utilized as the collision gas at a flow rate of 4.3 mL/min. The plasma gas flow rate is maintained at 16 L/min, and the spray chamber temperature is controlled at 2.0 °C to ensure optimal stability during analysis. The dwell time for each metal impurity is set to 0.3 s, and the instrument is precisely tuned in helium mode to achieve accurate and interference-free detection of trace metal impurities. No interference was observed from the calibration blanks, confirming the method's selectivity and accuracy. The sample recoveries were satisfactory, falling within the USP acceptance range of 70-150%. The analytical procedure was performed in accordance with USP <233> and ICH Q3D(R2) guidelines, and all results were found to be within the established acceptance criteria, thereby validating the reliability and compliance of the method. To date, no research studies have been reported on the determination of 24 elemental metal impurities in the drug substances vismodegib and idelalisib. Therefore, the present research aims to develop a unified and reliable analytical method for the quantification of these metal impurities in the active pharmaceutical ingredients of both drugs, ensuring compliance with regulatory standards and enhancing the overall quality and safety of the pharmaceutical products.
A generalized method for addressing outliers in Proficiency Testing (PT) schemes is presented, along with examples illustrating their impact on the analysis used for payment in the grain industry. Enhance the accuracy and robustness of the assigned values in the newly proposed automated testing protocol. Additionally, provide added value to participants in PT schemes by enhancing the significance of outlier detection, enabling further investigation of the underlying causes. The Grubbs outlier detection method has been expanded to include 35 distinct tests for identifying single, double, and up to multiple-10 outliers. Monte Carlo simulations have been used to determine the critical values for each test, which are suitable for datasets containing up to 1000 observations. This research specifically focuses on the one-sided significance level of 1.25%, but six other levels are included. The application of this method is particularly relevant for analyzing PT scheme data within the grain industry. The study discusses the challenges posed by extreme outlier data that may have identical values due to rounding practices. Additionally, another example from the world's largest grain network in France shows a multiple-5 outlier in a large data set of over 700 observations. This extended Grubbs test provides a more accurate determination of mean values and facilitates the identification of outlying observations, which may reveal significant insights when confirmed to be statistically distinct. The importance of precise testing using the new protocol is underscored by the observation that even a minimal shift of 0.1% in protein content in barley could lead to financial implications amounting to millions of US dollars. Notably, a minor shift of 0.07% was observed when applying the quadruple outlier test that excluded the four highest observations from the PT scheme dataset.
D-gluconic acid is found in honey, ripe fruits, and wine. In the food industry, D-gluconic acid is used as a complexing agent and acidifier in desserts, beverages, fruit, and vegetable products. D-glucono-δ-lactone is derived from gluconic acid, which hydrolyzes slowly with the release of protons. It can be used wherever slow acidification is desired. To validate the Enzytec™ Liquid D-Gluconic acid test kit for the determination of the sum of D-gluconic and D-glucono-δ-lactone in food and beverages such as fruit juices, wine, fermented soft drinks, kombucha, and heated sausage meat. The kit contains two ready-to-use components, which makes handling easy and suitable for automation. Gluconic acid reacts with ATP in the presence of gluconate kinase to form D-gluconate-6-phosphate and ADP followed by an indicator reaction that produces NADH, which is measured at 340 nm. The test is specific to D-gluconic acid and shows no side activities or interferences from relevant acids at or below 6.55 g/L. SO2 does not interfere at or below 0.5 g/L. Linearity for a test volume of 100 µL is given from 6 to 1500 mg/L. For 1000 µL, the LOQ is 0.8 mg/L. Intermediate precision is between 2.73 and 7.2% for different matrixes. Mean recovery for an incurred meat sausage is 84% at 799 mg/kg and 96% for the higher level (7509 mg/kg). Spiking of the other matrixes resulted in recoveries between 96 and 103%. For automation, three applications with different test volumes were validated. Linearity is given from 2 up to 9375 mg/L. The method is robust and accurate for manual and automated applications. The method was approved as an AOAC Official Method of AnalysisSM. The components of the test kit have a shelf life of at least 24 months.
The RapidChek® Listeria monocytogenes NextDay™ Plus Test System was designed to detect Listeria monocytogenes on stainless steel and plastic environmental surfaces and in selected foods. It uses a proprietary enrichment followed by a lateral flow immunoassay to qualitatively detect L. monocytogenes. The aim of this study was to validate the Romer Labs RapidChek® Listeria monocytogenes NextDay™ Plus Test System against the USDA-FSIS-MLG and FDA-BAM cultural reference methods for the detection of L. monocytogenes in select foods including hot dogs, frozen breaded chicken, cured ham, ice cream and cooked shrimp, and on environmental surfaces including stainless steel and plastic (polyurethane, food grade) in an unpaired study design. The RapidChek® method uses a proprietary enrichment media system, a 44 to 48 h enrichment at 30 ± 1 °C and detects L. monocytogenes on an immunochromatographic lateral flow device within 10 minutes. Different L. monocytogenes strains were used to spike each of the matrices. Samples were confirmed based on the reference method confirmations and an alternate confirmation method. There were 82 RapidChek® presumptive positives matrices of which 81 were confirmed by the alternate confirmation method. The respective cultural reference methods produced 74 confirmed positives. All non-spiked samples were negative for Listeria monocytogenes by both methods. Probability of Detection (POD) analysis was performed and found no statistically significant differences between methods. The RapidChek® Listeria monocytogenes NextDay™ Plus Test System demonstrated performance comparable to the respective cultural reference methods while providing rapid results, supporting its use for monitoring L. monocytogenes in food and food processing environments. The method provides the end user with a rapid and reliable tool for monitoring and control of L. monocytogenes in RTE foods and environmental surfaces in accordance with their ongoing food safety needs.
Simultaneous optimization of chromatographic resolution and analysis time constitutes a significant analytical challenge in multicomponent pharmaceutical analysis, as resolution-driven optimization strategies may improve peak separation without providing explicit control over retention time, often resulting in unnecessarily prolonged runtimes. To address this limitation, an optimization strategy based on a Box-Behnken experimental design was implemented in conjunction with the Improved Chromatographic Response Function (ICRF), which integrates separation quality and analysis time within a single mathematical objective function. This strategy describes the development of a rapid, chemometrically optimized UPLC-PDA method for the simultaneous determination of dorzolamide hydrochloride (DH) and timolol maleate (TI) in a commercial ophthalmic preparation. A Box-Behnken experimental design and optimization approach was employed in combination with an Improved Chromatographic Response Function (ICRF), which integrates resolution, peak overlap, peak width, and runtime into a single composite objective function. This strategy enabled short runtime (or short retention time of analytes in a chromatogram) while preserving adequate peak separation. Under the optimized conditions, complete chromatographic separation was achieved within 3 min using a BEH C18 column and a mobile phase consisting of acetonitrile and 4 × 10-4 M CCl3COOH (60:40, v/v) at a flow rate of 0.32 mL/min with detection at 275 nm. The method demonstrated excellent linearity over the range of 5.0-40.0 µg/mL (r > 0.999), with limits of detection of 0.51 µg/mL for DH and 0.61 µg/mL for TI. Mean recoveries were 99.9% and 99.5% for DH and TI, respectively, with satisfactory precision and robustness. The proposed ICRF-assisted optimization approach provided high-resolution separation within minimal runtime and was successfully applied to the routine quality-control analysis of a commercial ophthalmic formulation. The study demonstrates the effectiveness of composite response-based chemometric optimization in enhancing analytical efficiency in pharmaceutical drug analysis.
To address medication safety concerns from depleted Notopterygium incisum Ting ex H. T. Chang (NI) resources and inconsistent quality, this study employed the quality markers (Q-Marker) concept of traditional Chinese medicine to screen potential Q-Markers via multistep compositional transfer analysis and network pharmacology. This study aimed to screen the Q-Markers of NI for treating cardiovascular diseases. An approach integrating mass transfer analysis, network pharmacology, and HPLC fingerprinting was employed. First, HPLC fingerprints of 13 batches of NI were established. A representative batch was selected to prepare fresh medicinal materials, processed decoction pieces, and standard decoctions. Additionally, blood-absorbed components were collected, and fingerprints for all sample types were established. The transfer rates of seven index components (chlorogenic acid, nodakenin, ferulic acid, psoralen, bergapten, phenethyl ferulate, and isoimperatorin) were determined via similarity evaluation and least-squares discriminant analysis. Network pharmacology and molecular docking were further used to analyze the associations between NI's bioactive components and therapeutic targets for cardiovascular diseases. This study employed an integrated approach of HPLC fingerprinting, mass transfer analysis, network pharmacology, and molecular docking. After establishing HPLC fingerprints for 13 NI batches, a representative batch was processed from fresh herbs, decoction pieces, standard decoctions, and blood-absorbed components for analysis, after which a determination was made for the transfer rates of seven index components and their associations with cardiovascular disease targets. Chlorogenic acid, nodakenin, ferulic acid, phenethyl ferulate, and isoimperatorin are potential Q-Markers of NI for the treatment of cardiovascular diseases. In this study, mass transfer analysis, network pharmacology, and HPLC fingerprinting were integrated, combined with orthogonal partial least-squares discriminant analysis (OPLS-DA) and molecular docking. Potential Q-Markers of NI for the treatment of cardiovascular diseases were identified.
Aronia melanocarpa fruit is a valuable component of a healthy diet due to its rich antioxidant activity and high content of bioactive compounds. Recently, many food products and food supplements have emerged due to the increasing interest in aronia. In this study, a liquid chromatography method for the simultaneous quantification of the anthocyanins cyanidin-3-galactoside, cyanidin-3-arabinoside, and cyanidin-3-glucoside in aronia was developed. In this HPLC system, the simultaneous analysis of the three active substances was performed on a Phenomenex, Gemini C18, (150 x 4.6 mm, 5 µm) column at 30 °C. As a mobile phase, 1% glacial acetic acid: ACN: phosphoric acid (510:485:5) (h: h: h) and 0.5% phosphoric acid solutions were used, and a gradient system was applied at a flow rate of 0.8 mL/min. The developed method was validated according to ICH Q2 (R1) guidelines. Cyanidin-3-galactoside and cyanidin-3-arabinoside were found linearly in the concentration ranges of 2.5-12.5 µg/mL, and cyanidin-3-glucoside was found in the range of 0.25-4.0 µg/mL (r > 0.99). The developed and validated method was successfully applied to the analysis of samples containing aronia. Moreover, the proposed chromatographic method was assessed in terms of greenness and whiteness using the Analytical Greenness software and the White Analytical Chemistry model. A reliable HPLC method was developed for simultaneous quantification of three major anthocyanins in Aronia melanocarpa. The validated method was successfully applied to real aronia-containing samples.
Despite the high nutritional value of jujubes, their product diversification remains limited. Developing a new functional fermented beverage is a promising strategy for enhancing their economic value and expanding their market appeal. To enhance the value-added processing of jujubes, this study developed a novel co-fermentation process for producing kombucha using both jujubes and tea. Furthermore, the individual and synergistic impacts of these substrates on the microbial, sensory and physicochemical properties of the final beverage were systematically evaluated. Three kombucha types were prepared: traditional (C), pure jujube (Z), and tea-jujube blend (CZ). They were comprehensively characterized by evaluating their physicochemical profiles, microbial compositions, and sensory attributes. Group CZ displayed significantly higher contents of total flavonoids and reducing sugars after fermentation. The addition of jujubes also restructured the microbial community, introducing Saccharomyces and significantly increasing the relative abundance of Lactobacillus. Group CZ achieved the highest sensory acceptance scores. The ethanol content of group CZ conformed to the commercial standard for non-alcoholic beverages. Co-fermentation with jujubes and tea significantly enhances both the functional properties and the palatability of kombucha, presenting a promising strategy for creating high-value fermented products. The synergistic fermentation of tea and jujubes significantly enhances the functional properties and nutritional value of the product; jujubes optimize the microbial community structure during kombucha fermentation.