Journal of AOAC International最新文献

Background: Cysteine (Cys) is an essential amino acid for the human body. It naturally exists in various foods and serves as a crucial additive in the food industry, so accurate quantification of Cys is vital for food. Several probes for detecting Cys have been developed. However, the majority of fluorescent probes exhibit limitations in food applications.

Objective: This study aimed to develop a dual-channel near-infrared fluorescent probe (NC-NIR) for selective and sensitive quantification of Cys in various food samples.

Methods: The detection performance of the NC-NIR probe was systematically evaluated, including its dual-channel response spectra, reaction mechanism, limit of detection (LOD), selectivity, response time, and feasibility for analyzing actual samples.

Results: The probe (NC-NIR) exhibits high selectivity and low LOD = 34 nM) for the detection of Cys. The NC-NIR probe exhibits dual-channel fluorescence response toward Cys, exhibiting emission wavelengths at 550 nm and 825 nm. Furthermore, NC-NIR exhibits near-infrared characteristics that can overcome background fluorescence in food, and has been successful in detecting Cys content in apples, jujubes, broccoli, pears, onions, and milk slices.

Conclusions: This dual‑channel NIR probe offers a promising near-infrared strategy for in-situ quantification of Cys in food samples.

Highlights: A dual-channel, low background, high selectivity and low LOD probe for the detection of Cys in food samples.

Background: Rapid laboratory detection methods need to be tested against reference methods to confirm their efficiency and suitability.

Objective: In an unpaired study, the IEH Salmonella-Cronobacter spp. and Cronobacter sakazakii-Salmonella Multiplex PCR Assays' performance was compared with the FDA-BAM reference methods Chapter 5 for Salmonella and Chapter 29 for Cronobacter sakazakii detection on stainless-steel (SS) and sealed concrete (SC) surfaces.

Methods: Seeking a shorter enrichment time for the candidate method, the Salmonella-Cronobacter broth enrichments for 18, 20, 22, and 24 h at 35 °C were analyzed with the IEH Multiplex PCR Assays. Two pathogen cocktails were prepared: one including S. Typhimurium ATCC 19585, S. Senftenberg 775W, and S. Enteritidis PT30, and a second one including C. sakazakii MEI 27583, C. sakazakii ATCC 29544, and C. sakazakii ATCC 29004. The Salmonella and C. sakazakii cocktails were independently inoculated onto separate SS and SC following the AOAC guidelines. Additionally, a competing microorganism was added at a concentration 10-100-times higher than that of the pathogens.

Results: The candidate method yielded zero false negatives and zero false positives, with 100% sensitivity and 100% specificity for both SS and SC surfaces, regardless of the inoculation level (high, low, and uninoculated) and the enrichment time (i.e. 18, 20, 22, and 24 h for Salmonella, and 20, 22, and 24 h for C. sakazakii). The candidate method successfully detected both target microorganisms on both surface types with a minimum enrichment of 20 h. Statistical analysis using the probability of detection (POD) confirmed the equivalency between the candidate method and the reference methods at either inoculation level or enrichment time (P > 0.05).

Conclusion: The IEH Salmonella-Cronobacter spp. and Cronobacter sakazakii-Salmonella Multiplex PCR Assays demonstrated performance comparable to the FDA-BAM reference methods for the detection of Salmonella and C. sakazakii on SS and SC, with a significantly shorter turnaround time.

Background: Estazolam is a benzodiazepine drug widely used in clinical practice. Currently, estazolam tablets on the Chinese market are generic drugs. To meet the requirements of national standards and uniformity, the impurity analysis methods were developed for active pharmaceutical ingredient (API) and tablets of estazolam.

Objective: A high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed to achieve enhanced sensitivity and resolution for the quantitative analysis of related substances. This method was applied to determine impurity levels in generic estazolam tablets from 12 Chinese manufacturers and APIs from 4 manufacturers. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the impurity profiles. The impurity content and impurity profiles were used as evaluation indicators to trace the correlation between the differences in impurity profiles and the production process.

Methods: The content of both eight known and unknown impurities was quantitatively determined by the HPLC-UV method. A principal component external standard method with correction factor was used for calculation, and detailed methodological validation was performed according to ICH guidelines. The structures of impurities in Chinese marketed products and the innovator drugs were qualitatively identified by the LC-MS/MS method, and differences in impurity profiles were compared.

Results: This study identified two USP-listed process impurities, two unknown process impurities, and one non-pharmacopeial degradation product. The unknown impurities were successfully separated and preliminarily characterized.

Conclusions: Three API process impurities were key contributors to preparation impurity profile variations. Impurity levels showed close correlation with API synthesis routes and purification processes. A novel degradation product emerged during formulation, generated under light/heat stress, but minimally impacted tablet impurity variations.

Highlights: The optimized HPLC method demonstrated enhanced sensitivity and separation efficiency. API manufacturers should prioritize purification process optimization for impurities exceeding 0.1% thresholds to ensure drug safety.

Background: 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.

Objective: This study aimed to screen the Q-Markers of NI for treating cardiovascular diseases.

Methods: 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.

Results: 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.

Conclusion: Chlorogenic acid, nodakenin, ferulic acid, phenethyl ferulate, and isoimperatorin are potential Q-Markers of NI for the treatment of cardiovascular diseases.

Highlights: 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.

Background: Cranberry (Vaccinium macrocarpon) contains diverse hydrophilic phytochemicals, but their compositional profiles and cultivar-specific variation have not been comprehensively characterized.

Objective: The objective of this study was to profile hydrophilic metabolites in cranberry cultivars and identify constituents that differentiate among cultivars.

Methods: Water-eluted fractions of Sephadex® LH-20-separated cranberry extracts from six cultivars (Crimson Queen, Demoranville, Haines, Mullica Queen, Stevens, and Welker) were analyzed using ultra-HPLC-high-resolution accurate-mass tandem mass spectrometry (UHPLC-HRAM-MSn). Metabolites were annotated based on accurate mass and MS2 fragmentation data and comparison with literature reports. Sparse partial least-squares discriminant analysis (sPLS-DA) was applied to assess classification performance and identify discriminative features.

Results: Thirty-nine putative hydrophilic metabolites were identified, including hydroxycinnamic acid hexose derivatives, anthocyanins, hydroxycinnamoylquinic acids, and a distinct group of iridoid glycosides. Vaccinoside and several coumaroyl-substituted monotropein derivatives were structurally characterized in cranberry for the first time. sPLS-DA identified iridoid glycosides as key discriminators, with higher abundance observed in the Welker cultivar.

Conclusion: Cranberry cultivars contain previously unreported iridoid glycosides that contribute to chemotaxonomic differentiation. These results expand current knowledge of cranberry hydrophilic phytochemistry and support the relevance of iridoid glycosides as cultivar-informative metabolites.

Highlights: This study characterizes the highly polar, water-eluted fraction of six cranberry cultivars using UHPLC-HRAM-MSn analysis, resulting in the putative identification of 39 compounds, including a prominent series of iridoid glycosides.

Background: 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).

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Highlights: 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.

Background: 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.

Objective: This study aimed to develop a sensitive and broad-spectrum RT-qPCR assay for the efficient detection of BCoV.

Methods: 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.

Results: 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.

Conclusion: 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.

Highlights: 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.

Background: Pantoprazole is a selective proton pump inhibitor used in its sodium form in pharmaceuticals. Determining its impurities is crucial for drug purity and safety.

Objective: This study aims to develop a faster impurity analysis method than the European Pharmacopoeia (EP) method.

Methods: An Agilent Zorbax SB Phenyl (250 × 4.6 mm, 5.0 µm) column was used for HPLC, and a Restek Ultra Biphenyl (100 × 2.1 mm, 3.0 µm) column for Ultra Performance Liquid Chromatography (UPLC). A potassium dihydrogen/dipotassium hydrogen phosphate buffer (pH 7.40)-acetonitrile (65 + 35, v/v) mixture was the mobile phase. Analyses were performed at 290 nm with flow rates of 1.0 mL/min (HPLC) and 0.25 mL/min (UPLC). Injection volumes were 20 µL (HPLC) and 3.5 µL (UPLC).

Results: The analysis times of the impurity determination methods developed are 15 min shorter for the HPLC method and 30 min shorter for the UPLC method compared to the EP method. The developed methods were validated according to International Conference on Harmonisation (ICH) guidelines, demonstrating high linearity (R2 >0.99 over the concentration range of 0.03-2.27 µg/mL), accuracy with mean recoveries ranging from 95.0 to 105.0%, and precision with intra- and inter-day RSD values below 2%. Forced degradation studies under acidic, alkaline, oxidative, thermal, and photolytic conditions confirmed that the methods are stability-indicating, with the ability to separate all major degradation products of pantoprazole sodium.

Conclusion: The method was validated according to ICH guidelines, and forced degradation studies were also performed. This study presents optimized HPLC and UPLC methods that allow faster impurity analysis and detection of an additional non-compendial impurity, pantoprazole-N-oxide.

Highlights: As a result of shorter analysis times, a significant cost reduction has also been proven with numerical data. In addition, a performance-cost comparison of two different analytical technologies was made.

Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: The method is robust and accurate for manual and automated applications. The method was approved as an AOAC Official Method of AnalysisSM.

Highlights: The components of the test kit have a shelf life of at least 24 months.