Journal of AOAC International最新文献

Background: Sucrose is produced in the greatest quantity of all industrially produced organic substances and can be used in almost all processed foods. Glucose is the most abundant monosaccharide and contained in many foodstuffs naturally or added as an ingredient.

Objective: To validate the performance of the Enzytec™ Liquid Combi Sucrose/D-Glucose test kit for the determination of sucrose and D-glucose in juices, chocolate, breakfast cereals, ice cream, sweetened condensed milk, wine, beer, and soft drinks.

Methods: The kit contains all reagents in a ready-to-use format and is suitable for automation. Sucrose is cleaved by β-fructosidase. The resulting D-glucose from sucrose and glucose originally in the sample are phosphorylated and react in a NADH-generating reaction afterwards. β-Fructosidase is not present in the glucose system. The amount NADH produced is equivalent to the amounts of sucrose and D-glucose and is measured at 340 nm within 30 minutes.

Results: The linear measurement range for 100 µL test volume is from 17 mg/L to 2500 mg/L sucrose and 4 mg/L to 2000 mg/L D-glucose. Trueness was checked by using four CRMs and resulted in recoveries from 96% to 105%. Spiking of juices, ice cream, sweetened condensed milk, and shandy resulted in recoveries between 98% and 106% for both systems. Intermediate precision was at 3% or lower for sucrose and glucose.For automation, three applications with different test volumes were validated. Linearity is given from 3.8 mg/L to 9500 mg/L for sucrose and 2.4 mg/L up to 10000 mg/L for glucose.

Conclusions: The method is robust and accurate for manual and automated applications. The method was approved as AOAC Official Method of Analysis℠.

Highlights: The ready-to-use components of the test kit have a shelf of at least 29 months.

Background: Moxidectin is an active pharmaceutical ingredient (API) extensively used in various drug products within the pharmaceutical and animal health sectors. Despite its widespread use, the analytical methods prescribed by the United States Pharmacopeia (USP) and European Pharmacopoeia (EP, Ph. Eur.) exhibit significant limitations. These methods fail to adequately separate key impurities of (23Z)-moxidectin (EP impurity L) and 3,4-epoxy-moxidectin, potentially affecting the quality control, purity assessment, and safety of moxidectin-containing products.

Objective: The objective was to develop and validate an alternative, improved stability-indicating HPLC method for the identification, assay, and quantification of related substances in the moxidectin drug substance, along with the analysis of its degradation pathways.

Methods: High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) were employed to comprehensively examine moxidectin and its two degradation products under specified acidic conditions. The degradation products were isolated and identified using a range of analytical techniques, including HRMS, NMR, and other relevant methods.

Results: The epoxy derivative of moxidectin (RRT = 1.2) was not identified under the studied acidic degradation conditions. HRMS data indicated that the degradant at RRT = 1.2 is an isomer of moxidectin, as it exhibited an identical molecular ion. Detailed NMR studies on moxidectin and its impurity at RRT-1.2 revealed differences in carbon and proton chemical shifts at positions C-22 and C-24, strongly supporting the identification of the structure as an oxime geometric isomer of moxidectin, ie, (23Z)-moxidectin.

Conclusions: The findings revealed specific degradation products formed under acidic conditions, offering valuable insights into the chemical transformations of moxidectin. This information is crucial for assessing the drug's stability profile and ensuring the quality and safety of moxidectin-containing products.

Highlights: The HPLC method developed in this study significantly improves upon existing USP/EP methods with regard to a separation of key impurities of (23Z)-moxidectin and 3,4-epoxy-moxidectin, offering robust performance for routine analysis of bulk moxidectin API batches and stability samples in quality control (QC) laboratories.

Background: Traditional formulations are used extensively throughout the world due to their holistic approach to health and wellness with the fewest possible adverse effects. Itrifal Sana is a traditional unani polyherbal formulation, a unique combination that makes it synergistically potent, capable of providing dual benefits for health and well-being. Even though the formulation is frequently utilised, there is no scientific evidence to support its therapeutic efficiency.

Objective: The present study was designed to detect and identify bioactives responsible for acetylcholinesterase inhibitory activity by TLC-bioautography-MS and its validation using an in silico molecular approach.

Methods: Authentication of the formulation was done using macroscopy and powder microscopy. Quality control was done using UPLC-MS fingerprint analysis. TLC-bioautography-MS was done to detect the bioactives responsible for acetylcholinesterase inhibitory activity and the findings were validated using in-silico approach.

Results: The TLC-MS bioautography revealed the presence of rosmarinic acid, kaempferol, and apigenin as potential bioactive anticholinesterase metabolites. UPLC-MS analysis demonstrated the separation of 48 phytocompounds in the best active fraction of formulation. In silico analysis of identified metabolites showed acetylcholinesterase inhibitory activity of ten identified metabolites, moreover, rosmarinic acid and lobeline showed the best potential activity.

Conclusions: Our findings indicated that Itrifal Sana, which was investigated for the first time, has an enormous potential for managing AD caused by acetylcholinesterase enzyme inhibition. It was derived through successfully attempted TLC-bioautography-MS and in silico approach; however, further research on their full efficacy using in vitro cell line studies, in vivo studies, pharmacokinetics studies, and toxicity studies is still needed.

Highlights: TLC-bioautography-MS and in silico molecular approach offer much more effective, accurate, and reliable results than the conventional methods in the identification and validation of bioactive components from IS, a polyherbal formulation helps to advance the development of natural product-based therapeutics for cholinesterase dysfunctional diseases.

Background: Cronobacter species are opportunistic emerging pathogens associated with diverse foods of plant and animal origin. Considering the diversity of the Cronobacter group of bacteria and their co-existence with closely related Enterobacterales in the aquatic environment, their isolation from fish and shellfish is a challenge.

Objective: This study aimed to investigate the incidence of Cronobacter in finfish, shellfish, and dried fish, and to compare nine combinations of enrichment broth-selective isolation media for efficient isolation of Cronobacter spp.

Methods: Seventy-five seafood samples collected from five different retail markets were subjected to multiple selective-enrichment methods to isolate Cronobacter, which were presumptively identified by biochemical tests followed by confirmation with genus- and species-specific PCRs.

Results: Of 75 seafood samples analyzed, 24 (32%) were positive for Cronobacter spp. The highest incidence was in dried fish (21 samples, 47.72%), followed by 19 (43.18%) fresh finfish and four (9.09%) shellfish samples. Forty-four isolates from these samples were identified as Cronobacter spp. by PCR. Species-specific PCR further categorized these as C. sakazakii (25), C. malonaticus (16) and C. turicensis (1), while two isolates remained unidentified at species level. Enrichment in Cronobacter screening broth or Rappaport Vassiliadis medium, followed by isolation on the chromogenic Cronobacter sakazakii agar was found to be the most effective combination for the isolation of Cronobacter spp. from seafood.

Conclusions: Dried fish is an important reservoir of C. sakazakii owing to its desiccation tolerance and absence of competing microbiota in dried fish. Although C. sakazakii is the only known pathogen among Cronobacter spp., improved and specific methods to identify diverse members of this genus are needed.

Background: Monitoring labs are a fundamental link in the food safety chain, and regulatory demands in a competitive economy call for analytical methods that are simpler, faster, more rugged, and broader in scope. The QuEChERSER mega-method introduced in 2021 meets these monitoring needs, which includes high sample throughput, automated cleanup of extracts, and fast low-pressure gas chromatography (LPGC).

Objective: The goal of this work was to extend the QuEChERSER method to additional matrices and more analytes using LPGC, including comparison of the analytical performances of two different mass spectrometric (MS) analyzers: triple quadrupole tandem MS/MS and orbital ion trap (orbitrap) high-resolution (HR)MS.

Methods: The QuEChERSER mega-method was validated for 245 pesticides and environmental contaminants in barley grains and hemp pellets using automated instrument top sample preparation (ITSP) coupled with LPGC-MS/MS or LPGC-HRMS (orbitrap).

Results: Targeted MS/MS detection proved to be more sensitive than orbitrap using full data acquisition, leading to lower limits of quantification (LOQs) with more analytes yielding acceptable recoveries (70-120%) and repeatabilities (RSDs <20%). In barley, 89% of the compounds met validation criteria in MS/MS and 74% in HRMS, which in hemp were 81% and 66%, respectively. Qualitatively, orbitrap HRMS yielded 1% false positives compared to 3-4% in MS/MS, but due to the higher LOQs, the rates of false negatives were 14-17% in orbitrap vs. 6-10% in MS/MS for the different matrices.

Conclusion: The QuEChERSER mega-method including ITSP+LPGC coupled with MS/MS or orbitrap analysis is a robust approach for multiple applications. In the comparison, MS/MS outperformed the orbitrap in terms of sensitivity, but the orbitrap advantages of easier method development, greater selectivity, and possibility for nontargeted/retrospective analysis permit even broader expansion of analytical scope in the future.

Highlights: ITSP+LPGC- MS/MS or HRMS (orbitrap) analysis as part of the QuEChERSER mega-method is a useful and efficient way to monitor for contaminants in foods.

Background: HPTLC is a widely used and accepted technique for identification of botanicals. Current best practices involve subjective comparison of HPTLC-generated images between test samples and certified botanical reference materials based on specific bands.

Objective: This research was designed to evaluate the potential of cutting-edge machine vision-based machine learning techniques to automate identification of botanicals using native HPTLC image data.

Method: HPTLC images from Ginger and its closely related species and common adulterants were used to create large, synthetic datasets using a deep conditional generative adversarial network. This synthetic dataset was used to train and validate a deep convolutional neural network capable of automatically identifying new HPTLC image data. Performance of both neural networks was evaluated over time using appropriate loss functions as an indicator of their progress during learning. Validation of the overall system was measured via the accuracy of the learned model when applied to real HPTLC data.

Results: The machine vision system was able to generate realistic synthetic HPTLC images that were successfully used to train a deep convolutional neural network. The resulting learned model achieved high-accuracy identification from HPTLC images corresponding to Ginger and six other related species.

Conclusions: A proof-of-concept HPTLC image-based machine vision system for the identification of botanicals was proven to be feasible and a fully working prototype was validated for several species related to Ginger.

Highlights: This use of an autonomous machine-vision system for botanical identification removed the subjectivity inherent to human-based evaluation. The learned model also accurately evaluated botanical HPTLC images significantly faster than its human counterpart, which could save both time and resources.

Background: There are several globally recognized methods for preparing laboratory samples. Of these, the QuEChERS and QuPPe methods are commonly used for food laboratory sample preparation. As an alternative, we developed the fractionation method using FraMiTrACR.

Objective: We present a life cycle assessment for the QuEChERS-, QuPPe- and FraMiTrACR methods. Our objective was to collect data to evaluate the carbon footprint of each method. However, as the ecological factors alone do not inform suitability of any given method, we also evaluated economic factors.

Methods: Our life cycle assessments followed ISO 14040/44 to determine the carbon footprint of each method. Also, we have analyzed existing data to support our comparison of all three methods.

Results: The mass of consumables and packaging for our FraMiTrACR method was observed to decrease by 45% and 34% from those required for the QuPPe and QuEChERS methods, respectively. Furthermore, we calculated a 43% reduction in carbon footprint when using FraMiTrACR compared to QuPPe and a 31% reduction compared to QuEChERS. In addition, we determined that our method offers time savings >87% and >71% compared to QuEChERS and QuPPe, respectively. The main economic benefit of FraMiTrACR comes from 84% and 70% labor cost savings compared to QuEChERS and QuPPe, respectively. The laboratory using fractionation method can process 320 samples with FraMiTrACR within 8 hours, an 87% increase in potential compared to QuEChERS and a 71% increase compared to QuPPe.

Conclusions: Fractionation using FraMiTrACR is a more sustainable method for analytical sample preparation, offering the same quality of results and far-reaching economic advantages.

Highlights: In comparison, FraMiTrACR uses up to 45% less consumables and packaging by weight and a reduction in kg CO2eq of up to 43%. In addition, the fractionation method offers up to 85% time savings and up to an 84% reduction in labor cost per sample.

Background: Increasing restrictions for chemicals of concern in plastic packaging materials have created an urgent need to accurately detect and quantify these chemicals. Total fluorine measurements have been utilized to screen for highly scrutinized per and poly-fluorinated substances (PFAS) in food packaging materials. Inorganic contributions to the total fluorine signal can result in false positive signals exceeding regulatory limits.

Objective: The purpose of this study is to develop a method for determining the contribution of talc inorganic filler to the total fluorine signal.

Method: The influence of talc on total fluorine measurements of plastics was evaluated by compounding talc with virgin polypropylene (PP) then measuring the total fluorine concentration using oxidative pyrohydrolytic combustion ion chromatography. This study provides a framework to predict the contribution of talc in plastic samples to the total fluorine signal.

Results: A near infrared spectroscopy method was developed by employing the full width half height (FWHH) of the interstitial fluorine characteristic band of talc. The FWHH signal of the processed puck specimens was determined to be linearly increase with the measured total fluorine difference as a function of talc concentration (R2 = 0.9619).

Conclusions: This study developed a method to predict contribution of talc fillers to the total fluorine signal of plastic samples. This method is critical for accurately determining the regulatory compliance of talc filled plastic samples for PFAS using total fluorine.

Highlights: Total fluorine is a common regulatory compliance technique as an indicator of PFAS. Talc is a common plastic filler that contains fluorine as a contaminant. The fluorine in talc contributes to the total fluorine signal, which can falsely elevate the total fluorine signal, potentially resulting in the lack of regulatory compliance. The developed method serves as a framework of how to identify the fluorine contribution of inorganic fillers in plastics.

Background: The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method, Performance Tested Method  SM (PTM) 122302, is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica  1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately.

Objective: The objective of the study was to validate the MDA2-SE/ST method for detection of an additional monophasic variant of ST, S. enterica  1,4,[5],12:-:1,2.

Methods: A single strain of S. enterica  1,4,[5],12:-:1,2 was tested as part of a seven-strain inclusivity/exclusivity test panel. Strains were tested after growth in two enrichment media used in the method, buffered peptone water (BPW) and buffered peptone water-ISO formulation (BPW-ISO), and at two incubation temperatures, 35 ± 2 °C and 41.5 ± 1 °C.

Results: S. enterica  1,4,[5],12:-:1,2 produced positive results in the ST assay and negative results in the SE assay in both enrichment media and at both incubation temperatures. Results for the other six test strains were as expected under all conditions.

Conclusions: The MDA2-SE/ST method can detect the monophasic variant S. enterica  1,4,[5],12:-:1,2 as well as S. enterica ser. Typhimurium and the monophasic variant S. enterica  1,4,[5],12:i:-. The MDA2-SE/ST method maintains inclusiveness for S. enterica ser. Typhimurium despite flagellar antigen variation.

Highlights: The data were reviewed by the AOAC PTM Program and approval was granted for modification of the MDA2-SE/ST method, PTM 122302.

Background: The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a rapid, nucleic acid amplification-based test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica  1,4 , [5] , 12: i:-, in select poultry samples. Results for SE and ST are generated separately.

Objective: The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in chicken carcass rinse and raw ground chicken (325 g) for Performance Tested Methods  SM (PTM) certification.

Methods: The study consisted of inclusivity/exclusivity testing and independent laboratory testing of chicken carcass rinse and raw ground chicken using inoculated matrixes. Data were analyzed using a paired probability of detection model to determine if differences in the number of positive results obtained with the MDA2-SE/ST and reference methods were significant.

Results: In inclusivity testing, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1  Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, results for the MDA2-SE/ST and reference methods were in complete agreement for both matrixes.

Conclusions: Results of the validation study showed that the MDA2-SE/ST method is an accurate, specific method for detection of SE and ST in select poultry matrixes.

Highlights: The data were reviewed by the AOAC PTM Program and approval was granted for certification of the Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium method as PTM 122302.