Journal of AOAC International最新文献

Background: Salmonella Infantis is an emerging multidrug-resistant pathogen worldwide due to the acquisition of a megaplasmid, plasmid of emerging Salmonella Infantis (pESI). Reported initially in poultry, the distribution of pESI-harboring S. Infantis in other food types, including seafood, is unknown.

Objective: This study aimed to develop and optimize a PCR assay for detecting the pESI in Salmonella and non-Salmonella Enterobacterales.

Methods: A duplex PCR targeting the hilA gene and a pESI-associated gene of S. Infantis was designed, and the PCR conditions were optimized. The specificity and sensitivity of the assay were established using 119 Salmonella serovars and 51 non-Salmonella bacterial strains.

Results: All Salmonella isolates yielded hilA PCR product, while only pESI S. Infantis was positive for both hilA and pESI genes. No amplification product was obtained with the DNA of 51 non-Salmonella bacterial strains. The detection limit of the duplex PCR was 104 CFU/mL of pure culture of pESI S. Infantis. The sensitivity of detection in artificially spiked shrimp meat was 1 CFU/g after 6 h of enrichment in lactose broth, followed by 12 h of selective enrichment in the Rappaport-Vassiliadis medium.

Conclusion: The duplex assay will help screen seafood for Salmonella in general and pESI S. Infantis in particular. Given its high sensitivity, the PCR will be a valuable tool for seafood quality assurance. This approach decreases the typical 3-6 day identification time of Salmonella to less than 24 h.

Highlights: S. Infantis carrying the highly transmissible megaplasmid (pESI) is a significant food safety concern. Given its rapid geographical spread and high antimicrobial-resistant traits, it is necessary to have a molecular tool that detects pESI-harboring Salmonella. This study successfully developed a duplex PCR assay that simultaneously detects Salmonella enterica and pESI S. Infantis. This molecular tool will help understand the distribution, sources, and spread of the multidrug-resistance (MDR) plasmid in the food environment.

Background: Currently, the most popular technique in gas chromatography (GC) is "temperature programming," where the temperature increases from the start of the injection. This leads to faster elution of analytes compared to isothermal methods. However, isothermal methods are considered optimal for separating compounds with similar retention times. Another interesting technique that provides higher resolution is dynamic thermal gradient gas chromatography (TGGC), where separations are achieved as a decreasing thermal gradient. This gradually decreases the positive gas velocity. Nevertheless, it was proven that GC techniques with negative velocity gradients do not improve the resolution of compounds with nearly identical retention times.

Objective: Optimizing a new GC approach to combine both the short time from positive temperature ramps programming, and the enhanced separation of the negative ramps of the TGGC, a model under the name of "end column reverse chromatography" (ECRC).

Methods: The process simply consists of two steps: the first is a normal positive ramp from the start of the injection, and the second step is a negative thermal ramp at a time that is around the retention time of the first eluting peak. This will decrease the solute velocity almost solely for the second compound, leading to relatively enhanced separation.

Results: The optimized ECRC method increased the resolution of two isomers (trans- and cis-chlordane) from 1 (slightly overlapping) in the case of temperature programming to 2.78 as shown in this study. This comes at the expense of the width and intensity of the peaks, where the intensity decreased about 17 and 12% for cis- and trans-chlordane, and the peak width increased with 37 and 77% for the same compounds, respectively.

Conclusions: ECRC is a novel model for enhanced separation that comes with some drawbacks.

Highlights: It can be an alternative approach to get a fast GC method with enhanced separation for isomers.

Background: Due to its excellent acidifier, antioxidant, and preservative properties, citric acid is added to or is a constituent in a broad range of foods and beverages. Its measurement should be performed to assure the food quality specifications are met.

Objective: To validate the performance of the Enzytec™ Liquid Citric acid test kit for the determination in food and beverages such as wines, juices, and tomato products.

Methods: The kit contains two ready-to-use components, which makes handling very easy and suitable for automation. Citrate is cleaved into oxaloacetate and acetate by citrate lyase. Oxaloacetate reacts to L-malate by L-malate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH). Pyruvate, spontaneously formed from oxaloacetate, is converted by L-lactate dehydrogenase to L-lactate. The NADH consumed is equivalent to the amount of citric acid converted and is measured at a wavelength of 340 nm within 20 min.

Results: The test is specific to citric acid and shows no relevant interferences. Limit of detection and limit of quantification are 15 and 40 mg/L, respectively when using a test volume of 100 µL. The linear measurement range is from 40 to 1000 mg/L citric acid. Trueness was evaluated using materials from FAPAS, NIST, LGC, and two control wines from the German Wine Analysts. Matrix interference was evaluated by spiking tomato ketchup, tomato paste, orange juice, and carbonated beverages and resulted in recoveries around 100%. Intermediate precision is between 6.2 and 8.5% for matrixes with extraction and below 4% for matrixes measured directly. For automation, three applications with different test volumes and different measurement ranges were validated. Linearity is given from 8 mg/L up to 5 g/L (depending on the test volume).

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

Highlights: The ready-to-use components of the test kit have a shelf life of at least 24 months from the date of manufacture.

Background: Astragali Complanati, known in Chinese as Shayuanzi, is a common medicinal material in traditional Chinese medicine, mainly used for tonifying the kidney, supporting yang, consolidating essence, reducing urine, and other diseases.

Objective: The ultra performance liquid chromatography (UPLC) fingerprint of Astragali Complanati Semen (ACS) was established, and the Q-markers of ACS were analyzed by network pharmacology.

Methods: First, a UPLC fingerprint detection method was established for ACS, and the common peaks were identified by UPLC-MS/MS. The "component-target-pathway" network relationships of characteristic components of ACS were constructed by network pharmacology, and the potential quality markers (Q-markers) were predicted.

Results: A total of 24 common peaks were identified from the UPLC fingerprint of ACS, and 12 chromatographic peaks were identified by UPLC-MS/MS. A total of 12 Q-markers candidate components were screened out. Through network pharmacological analysis, it is predicted that myricetin 3-O-β-D-xylopyranosyl-(1-2)-[α-L-rhamnopyranosyl-(1-6)]-β-D-glucopyranoside, myricetin 3-O-β-D-xylopyranosyl(1-2)-β-D-glucopyranoside, myricetin 3-β-D-glucopyranoside, cannabiscitrin, laricitrin-3-O-glucoside, leucoside, complanatoside B, complanatuside, complanatuside 6''-malonate, clycosin, rhamnocitrin 3-O-β-D-apiofuranosyl(1→2)-β-D-glucopyranoside, and 3-O-[5'''-O-feruloyl-beta-D-apiofuranosyl(1'''->2'')-beta-D-glucopyranosyl] rhamnocitrin are the Q-markers of ACS.

Conclusion: The method established in this study was accurate, reliable, simple, and practical and could be used as a reference method for ACS quality detection. Twelve Q-markers selected by network pharmacology could provide support and references for ACS QC.

Background: Detection methods for GMO events are required because of regulatory compliance requirements. Efficient detection and quantification of GMO events saves time and resources. Multiplex digital PCR (dPCR) allows detection and quantification of more than one GMO event at the same time.

Objective: The study used two tetraplex droplet digital PCR (ddPCR) assays for the detection of 19 soybean GMO events.

Methods: Two multiplex dPCR assays were developed and optimized for the detection of 19 soybean GMO events. The first tetraplex ddPCR assay contained four element-specific targets commonly found in GMO plants (P-35S, T-nos, tE9, and Pat). The second event-specific tetraplex ddPCR assay targeted four soybean GMO events that are not detected with the element-specific tetraplex ddPCR (CV127, DP305423, MON87701, and MON87751).

Results: The element-specific tetraplex ddPCR assay detected all the expected 15 soybean GMO events. The element-specific tetraplex ddPCR assay also detected selected soybean GMO events at the 0.01% level. The event-specific tetraplex ddPCR assay was successfully used to quantify the four soybean GMO events at the 0.1, 1, 2, and 5% levels. The event-specific tetraplex ddPCR assay also detected the four soybean GMO events at the 0.01% level.

Conclusions: The two tetraplex ddPCR assays can be used for the detection of 19 soybean GMO events.

Highlights: An element-specific tetraplex ddPCR assay was used to detect 15 soybean GMO events, and an event-specific tetraplex ddPCR assay was used to detect and quantify four soybean GMO events that are not detected by the element-specific ddPCR assay.

Background: The 11+Myco MS-PREP® Immunoaffinity Column (IAC) contains a gel suspension of monoclonal antibodies specific to the toxins of interest. Following sample extraction, the IAC is used for cleanup and concentration of mycotoxins prior to analysis by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS).

Objective: This study evaluated the IAC with LC-MS/MS method for Performance Tested Methods  SM certification for simultaneous determination and confirmation of Aflatoxins B1, B2, G1, G2, and M1; Deoxynivalenol, Fumonisins B1, B2, and B3; Ochratoxin A; T-2; HT-2; and Zearalenone from corn, wheat, cereal-based baby food (with and without dairy ingredients), paprika, chili powder and animal feed.

Methods: A single extraction method using 50% Acetonitrile and water was used for all matrixes. The Method Developer validated all matrixes and an independent laboratory verified method performance on corn and animal feed. Data were analyzed for recovery, repeatability precision, LOD, LOQ, confirmation of identity, and method selectivity.

Results: Recovery (72%-138%) and repeatability (0.46%-24%), with the exception of sporadic data points, were within acceptance criteria. LOQ was estimated as AFB1 0.018-0.32 ꙡg/kg, AFB2 0.037-0.28 ꙡg/kg, AFG1 0.019-0.14 ꙡg/kg, AFG2 0.036-0.28 ꙡg/kg, DON 4.0-75 ꙡg/kg, Fumonisin B1 4.9-37 ꙡg/kg, Fumonisin B2 4.0-32 ꙡg/kg, Fumonisin B3 2.0-16 ꙡg/kg, OTA 0.15-4.4 ꙡg/kg, T-2 0.5-7.5 ꙡg/kg, HT-2 0.70- 7.5 ꙡg/kg), and ZON 1.3-7.2 ꙡg/kg), depending on matrix. Method performance was verified with reference and quality control materials. Selectivity and confirmation of identity were also demonstrated.

Conclusions: The 11+Myco MS-PREP® IAC with LC-MS/MS method demonstrated acceptable performance for simultaneous determination of 12 mycotoxins in 7 matrixes.

Highlight: The data were reviewed by the AOAC Performance Tested Methods  SM Program and approval was granted for certification of the 11+Myco MS-PREP® Method as PTM 112401.

Background: Melatonin supplements are often used to alleviate jetlag and other sleep-depletion related disorders. Recent studies found large inconsistencies between labeled values and actual contents of melatonin within products, which has led to concerns over the quality of melatonin supplements. In order to facilitate the quality control testing of melatonin supplements, an improved and more modern approach to the liquid chromatographic analysis of melatonin is required. In addition, growing public concern over the environmental footprint of analytical laboratories exacerbates the need to modernize legacy analytical procedures with more eco-friendly or greener approaches.

Objective: This study aims to optimize the routine liquid chromatographic analyses that are prescribed in the US Pharmacopeia (USP) melatonin monograph on a High Performance Liquid Chromatography (HPLC) system without fundamentally modifying the methods.

Method: The melatonin assay and the melatonin related compounds (impurities) test were optimized on a C18 column packed with 2.5 µm particles. The column length and the gradient elution parameters were adjusted following the guidelines on Adjustment of Chromatographic Conditions in USP General Chapter <621>. The mobile phase compositions were optimized to meet the system suitability requirements that were specified in the USP melatonin monograph. The flow rate was optimized for better separation efficiency.

Results: The optimized HPLC methods not only met the USP system suitability requirements in relative retention time (RRT), resolution, and relative standard deviation (RSD), but also demonstrated excellent linearity, sensitivity, accuracy, precision (repeatability), and would have a lower environmental impact.

Conclusions: The optimized HPLC methods for assay of melatonin and test of its related compounds achieved significantly increased throughput and a reduced environmental impact, without fundamentally modifying the methods.

Highlights: The optimized HPLC methods are significantly faster and more eco-friendly. These methods can be implemented on HPLC systems without a full re-validation.

Background: White granulated sugar, a significant commodity in bulk trade and widely used raw material, plays a crucial role in the food, energy, and medicine industries. A certified reference material (CRM) of white granulated sugar provides a valuable tool for monitoring and maintaining the safety and quality of white granulated sugar and other related products. The colorimetric value, conductivity ash, sucrose content, and reducing sugar content serve as essential factors that determine the quality of white granulated sugar.

Objective: Develop a CRM of grade one white granulated sugar, conduct a comprehensive study to assess its homogeneity and stability, and determine the designated property values.

Methods: Collaborative certification across 11 laboratories was applied to validate its property values and evaluate corresponding uncertainties.

Results: The designated property values and expanded uncertainties (k = 2) determined to be 75.6 ± 2.8 IU, 0.0100 ± 0.0004 g/100g, 99.71 ± 0.08 g/100g, and 0.0226 ± 0.0024 g/100g for colorimetric value, conductivity ash, sucrose content, and reducing sugar content, respectively.

Conclusion: The reference material (RM) was sufficiently homogeneous between and within bottles and remained stable for up to 20 months.

Highlights: The developed RM of the grade one white granulated sugar could be an effective support for method validation, capability validation, quality control, and metrological traceability in the sugar and food industries.

Background: Rosemary extracts are derived from the leaves of Rosmarinus officinalis and commonly employed as natural food preservative. They serve as natural antioxidants in food, preventing spoilage and extending shelf life.

Objective: This study aimed to develop a modified QuEChERS extraction with liquid chromatography tandem mass spectrometry for the analysis of rosemary extracts in food as sum of its markers Carnosol and Carnosic Acid.

Method: Carnosol and Carnosic Acid in food were extracted by a modified QuEChERS extraction after the addition of analyte protectant during extraction and analyzed by liquid chromatography tandem mass spectrometer via internal standard calibration method. 2,2'-Isopropylidienediphenol and Podocarpic acid were used as internal standards for Carnosol and Carnosic Acid, respectively.

Results: The Limit of Detection of Carnosol and Carnosic Acid were all less than 1 mg/kg, while their corresponding values of Limit of Quantitation ranged from 1.44-3.12 mg/kg in various matrices. Spike recoveries at three fortification levels (10, 50 and 300 mg/kg) were all within 90-110% with %RSD less than 10% in all cases.

Conclusions: A modified QuEChERS extraction with LC-MS/MS detection for the analysis of Rosemary extracts in food was successfully developed, validated and demonstrated to be fast, robust and reliable.

Highlights: The developed modified QuEChERS extraction with LC-MS/MS detection offered a fast and efficiency way to analyse rosemary extracts in various food during routine food surveillance programme.

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.