AOAC Official Method Research Articles

Validation of Enzytec™ Liquid Citric Acid for Enzymatic Determination of Citric Acid in Selected Foods and Beverages: First Action 2024.02.

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. 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. 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. 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). The method is robust and accurate for manual and automated applications. The method was approved as AOAC Official Method of Analysis℠. The ready-to-use components of the test kit have a shelf life of at least 24 months from date of manufacture.

Determination of Aloin A, Aloin B, and Aloe-Emodin in Raw Materials and Finished Products Using HPLC Multi-Laboratory Validation Study, AOAC 2016.09, Final Action Status.

A multi-laboratory validation study was performed on AOAC Official Method of Analysis (OMA) 2016.09, for final action. Eight different laboratories throughout the world participated in the study tested the same set of 6 different laboratory samples of raw materials (commercial) and formulated products (commercial), and all the laboratories successfully generated results within the acceptance criteria. The intention of the study was to evaluate specificity, precision (variation), linearity/range, system suitability, limit of detection (LOD), and limit of quantitation (LOQ) by multiple laboratories to satisfy the requirement of moving the OMA 2016.09 to the final action status. Accuracy and ruggedness were already validated in earlier work of single laboratory validation (1), and it was not necessary to include these validation parameters in the multi-laboratory validation study. Laboratory samples containing Aloe were sent out to participating laboratories. Each lab followed AOAC 2016.09 (First Action status) to analyze contents of aloin A, aloin B, and aloe emodin using high performance liquid chromatography (HPLC) instrument. The results generated by each laboratory were collected and evaluated. The specificity results show that blank and matrix chromatograms do not contain major interfering peaks on the retention time of aloin A, aloin B, and aloe-emodin. The precision (variation) results of duplicated preparations are not more than 0.05 parts per million (ppm) different. The linearity/range results from six standards (10 parts per billion (ppb), 20 ppb, 40 ppb, 80 ppb, 160 ppb, and 500 ppb) have correlation coefficient (R) value of ≥ 0.999. The system suitability results meet the acceptance criteria to show the instrument validity. The limit of detection (LOD) results show that the signal-to-noise (S/N) ratio of 10 ppb standards for all three components are about 3. The limit of quantitation (LOQ) results show that the S/N ratio of 20 ppb standards for all three components are about 10. The validation parameters (specificity, precision (variation), linearity/range, system suitability, LOD, and LOQ) have been successfully analyzed, and it shows that the test method is suitable for its intended use. The test method has been successfully validated by the eight different laboratories around the world (US, UK, Germany, and Canada). Each of the laboratory is managed independently by the site lab management team. This multi-lab study validated the method of Determination of Aloin A, Aloin B, and Aloe-Emodin in Raw Materials and Finished Products Using high performance liquid chromatography (HPLC), and the method fits for its intended purpose.

Inhibition of bleaching of stored red hot pepper through appropriate postharvest technologies and practices

The colour qualities of hot red pepper are the major issue in pepper value chain. Due to poorly coordinated scientific evidence, information on the factors causing colour loss and the inhibition mechanism is not well known. Therefore, this review paper aimed to summarize the inhibition mechanism of stored red hot pepper bleaching through appropriate postharvest technologies and practices. The information in this paper was gathered from a variety of sources, including journal articles, books, book chapters, workshop proceedings, FAO reports, and AOAC official methods of analysis. According to these studies, carotenoids, surface colour, and extractable colour (ASTA value) are the primary colourants that define hot red pepper. The findings demonstrate that low-temperature drying methods, such as open sun drying, are best for preserving the red hot pepper powder’s colour quality, while higher temperatures cause the colour to darken. Blanching, the use of desiccants (CaCl2), and chemical dipping are pretreatments that preserve the best colour quality by hastening the drying time. Similarly, storage of red hot pepper powder at lower temperatures (5oC) resulted in less colour degradation. In other words, materials used for packaging that have a high barrier to light, moisture, and air, such as laminated aluminium, amber or black polyethylene, and high-density polyethylene, maintained a higher level of colour quality. Through their influence on drying and processing times, breeding technologies, varieties, and maturity level also impact colour quality. In conclusion, the colour quality of red hot pepper is highly influenced by environmental, biological, and processing methods. It is, therefore, critical to use appropriate drying and pretreatment techniques, storage time, well-managed storage temperature, appropriate processing methods and packing materials, and improved agronomic practices for the sustainable management of colour fading and adulteration that can occur throughout the value chain.

Validation Study on Spectrophotometric Measurement of Ethanol from Beer and Non-Alcoholic Beer Samples Distilled by Micro Steam Distillation Method

The method validation technique for determining the ethanol percentage (v/v) in beer and non-alcoholic beer is presented in this study. The underlying idea is to use the micro water vapor method to distill the ethanol from beer and then use the spectrophotometric method, which uses the oxidation of sodium dichromate to measure the amount of ethanol. The Harmonized Guidelines for Single-Laboratory Validation of Methods of Analysis were used to validate the analytical method that we provided. The following aspects of the method were assessed: precision, recovery, linearity, measuring range, detection and quantification limitations, method detection limit, and measurement uncertainty. The following were the limits of detection (LOD) and quantification values (LOQ), and the method detection limit (MDL): ethanol, 0.04%, 0.05%, and 0.15. For both repeatability and within-laboratory reproducibility, the relative standard deviation values were less than 2.36 and 4.12%, respectively. The spiked samples had recovery rates ranging from 97% to 102%. These findings fulfilled the minimal performance standards set forth in AOAC Official Methods of Analysis Appendix F: Guidelines for Standard Method Performance Requirements. As a result, the process can be used for the regular analysis of ethanol in the beer and non-alcoholic beer under study.

Simultaneous microwave digestion for total arsenic and inorganic arsenic in local shrimp and prawn commodities of Brunei Darussalam for regulatory and safety monitoring

The data gap in food safety regulations have created misinformation leading to the rejection of commodities for trade. The evidence presented is the local regulation of arsenic in sea produce which is based on total arsenic, tAs, instead of toxic inorganic arsenic, iAs. Furthermore, tAs data in animal origin seafood has been widely proven to be dominated by the non-toxic Arsenobetaine, AsB. Therefore, if arsenic regulatory limit was set based only on tAs without reference to iAs data, seafood products might be wrongfully rejected for trade because of non-compliance to tAs limit. We provided analysis of tAs and iAs of 14 local prawn and shrimp commodities from three shrimp/prawn sector namely aquaculture (n = 3), capture (n = 5) and processed (n = 6) using effective extraction, as well as, a fit-for-purpose analytical method for iAs. A HVG-AAS method was developed and validated for iAs with LoQ of 1.6 ppb, analytical range of 0–6 ppb, repeatability RSDr of 0.5–3.1 %, coefficient of determination R2 of 0.9975, and percentage recovery of 90.9 %, while an existing method using ICP-MS was used to verify the tAs. Based on the AOAC Official Method 999.10 2005 with minor adjustments, seafood samples were digested with concentrated nitric acid and hydrogen peroxide under pressure in a closed vessel heated by a microwave digester. An additional step for iAs determination was necessary to ensure compatibility in HVG-AAS analysis. Further subdivision of the aquaculture and capture samples was done by dividing them into 3 fractions, namely head, flesh and peel. Comparison of tAs in all the three fractions indicated that for aquaculture sector, the highest tAs were found in the flesh (2nd highest in % weight) whereas for the capture sector, the highest amount of tAs correlated with the highest % weight of the fraction. On regulatory aspects, speciation analysis on the iAs indicated samples with quantifiable iAs value were in-compliance despite tAs were initially found to be higher than the national limits. Risk assessment of iAs indicated there were no risk for human daily intake based on the BDML0.5 value of 3.0 μg/kg b.w per day for an average 70 kg man. All findings concluded the need for doing arsenic speciation analysis of iAs along with tAs for routine monitoring of prawn/shrimp samples and to revise the local limits from tAs to iAs particularly for seafood commodities.

Paired metagenomic and chemical evaluation of aflatoxin-contaminated dog kibble.

Identification of chemical toxins from complex or highly processed foods can present 'needle in the haystack' challenges for chemists. Metagenomic data can be used to guide chemical toxicity evaluations by providing DNA-based description of the wholistic composition (eukaryotic, bacterial, protozoal, viral, and antimicrobial resistance) of foods suspected to harbor toxins, allergens, or pathogens. This type of information can focus chemistry-based diagnostics, improve hazard characterization and risk assessment, and address data gaps. Additionally, there is increasing recognition that simultaneously co-occurring mycotoxins, either from single or multiple species, can impact dietary toxicity exposure. Metagenomic data provides a way to address data gaps related to co-occurrence of multiple fungal species. Paired metagenomic and chemical data were used to evaluate aflatoxin-contaminated kibble with known levels of specific mycotoxins. Kibble was ground to a fine powder for both chemical and molecular analyses. Chemical analyses were performed with Liquid Chromatography Mass Spectrometry (LCMS) and according to the AOAC Official method 2005.08: Aflatoxins in Corn, Raw Peanuts, and Peanut Butter using Liquid Chromatography with Post-Column Photochemical Derivatization. Metagenomes were created from DNA extracted from ground kibble and sequenced on an Illumina NextSeq 2000 with an average sequence depth of 180 million reads per replicate. Metagenomic data demonstrated that the abundance of DNA from putative aflatoxigenic Aspergillus spp. correlated with the levels of aflatoxin quantified by LCMS. Metagenomic data also identified an expansive range of co-occurring fungal taxa which may produce additional mycotoxins. DNA data paired with chemical data provides a novel modality to address current data gaps surrounding dietary mycotoxin exposure, toxigenic fungal taxonomy, and mycotoxins of emerging concern.

FT-IR Characterization and Determination of Mycotoxins (Aflatoxins) (AF’s) from Branded & Unbranded Mixed Spice Samples

Background: The global spice market is essential to food industry and public health due to the widespread use of spices as flavor enhancers, preservatives, and medicinal components. However, the quality of spices can be compromised by the presence of mycotoxins, specifically aflatoxins, which pose significant health risks. Ensuring the quality and safety of spices, both branded and unbranded, is of paramount concern, especially in regions with high humidity and temperature that can facilitate fungal growth and mycotoxin production. Objective: This study aimed to assess the quality of branded and unbranded spice samples from Sindh, Pakistan, by determining their ash content, moisture level, oil composition, and mycotoxin (aflatoxin) content to ensure their suitability for consumption and adherence to food safety standards. Methods: A total of ten spice samples (five branded and five unbranded) were collected from different cities in Sindh province, Pakistan. Ash content was measured by high-temperature oxidation, while moisture content was quantified through oven drying. Oil was extracted using a Soxhlet apparatus, and its quality parameters, including free fatty acid (FFA), peroxide, iodine, and saponification values, were determined using standard AOCS methods. Aflatoxins were quantified by employing an official AOAC method. Additionally, FT-IR spectroscopy was utilized for qualitative analysis of the spice oils. Results: The ash content varied widely, with the highest in unbranded samples at 18.33% and the lowest in branded at 5.67%. Moisture content across all samples was within the safe limit of 5-10%, with the highest at 4.46% and the lowest at 2.22%. The oil content ranged from 5.96% to 14.56%, with unbranded samples showing higher values. FFA values exceeded the standard 0.1% in all samples, indicating a potential quality concern. Peroxide values were higher than the standard 2 meq/mol in most samples, raising concerns about oxidative stability. Aflatoxin levels were within the EU regulatory limits, suggesting acceptable levels for consumption. FT-IR analysis corroborated these findings, revealing no significant differences in the spectral profiles between branded and unbranded samples. Conclusion: The study concluded that while moisture content and aflatoxin levels in the spice samples complied with safety standards, there were notable concerns regarding the ash content, oil quality, and peroxide values. These findings highlight the necessity for regular quality control measures and adherence to food safety regulations to ensure the safety of spice consumption.

Glycemic Index of Sweet Corn and the Characteristics of their Flakes by Adding the Red Bean

Consuming foods with a low glycemic index, such as sweet corn (Zea mays Saccharata), which has a high fiber content, can help lower the risk of diabetes. According to previous research, sweet corn is predicted to have a low glycemic index (GI). The study's goals were to determine the value of the glycemic index of corn and to produce low glycemic index instant flake products made from sweet corn and red beans. The glycemic index value of sweet corn was determined using a glycemic index test technique based on ISO 26642:2010E. The flakes was made with the addition of red beans reported to have the lowest glycemic index among legume, i.e. 26 in order to enhance the crude protein content of the flakes. Flakes were made in two formulas based on the proportion of red beans to sweet corn, namely formula 1 (30:70) and formula 2 (70:30). Proximate analysis was done according to AOAC 2005, total dietary fiber analysis according to AOAC Official Methods 985.29, and total starch analysis according to AOAC 2002.02. Texture analysis of the flakes was carried out using a texture analyzer and color analysis using a chromameter. The sweet corn was found to have a low GI (<55) i.e. 36. Formula 1 flakes contained more fat (db), carbohydrate (db), and total starch (db); while formula 2 flakes had more moisture, protein (db), and resistant starch (db). The resulting flakes have crispy texture (hardness of 553±5.09 gf for formula 1 and 519.05±6.86 gf for formula 2) and have yellow color (both are included in the yellow chromatic area).

Sugar Profile Method by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection in Food, Dietary Supplements, Pet Food, and Animal Feeds: Interlaboratory Validation Study, Final Action 2018.16.

A method for sugar profile analysis granted First Action 2018.16 was subjected to a multi-laboratory study. Perform a multi-laboratory study with this method to determine the performance parameters of repeatability and reproducibility against the AOAC Standard Method Performance Requirements (AOAC SMPR 2018.001) for Final Action status. Eleven laboratories from three different countries participated in the study. Each laboratory was provided practice materials for successful method setup. Each laboratory then proceeded with analysis of blind duplicates of 10 different products covering the scope of the method. Results were reported to the study directors with any modifications and assessed following the procedures of Appendix D of the AOAC Official Methods of AnalysisSM (guidelines for collaborative study procedures). The majority of results from the study met the SMPR requirements. The data is presented along with any outlying observations or modifications. The method was proven to be flexible across different instrumentation and laboratories, and the method was updated to provide further system suitability and guidelines to maintain the performance of the method across the large scope of matrixes. The results from the collaborative study supported the method for Final Action status. The Expert Review Panel reviewed and voted to move the method forward to Final Action and was followed by review from the Official Methods Board and granted approval. The method was granted Final Action Official Methods status.

Influence of heavy metal ions and storage time on hydroxymethylfurfural formation in honey collected from Ilu Woreda, Oromia Regional State, Ethiopia

The effect of heavy metals on the formation of hydroxymethyl furfural (HMF) from degradation of reducing sugars in honey collected from Ilu Woreda, West Shewa Zone of Oromia Regional State, Ethiopia were studied. The concentration level of Pb, Cd, Cr and Ni in honey sample was determined using AOAC Official Method. The concentration of HMF in honey was determined using international honey commission standards. The concentrations of heavy metals in fresh honey were 11 ± 0.001, 454 ± 0.002, 630 ± 0.001 and 1 ± 0.001 µg kg-1 for Pb, Cr, Ni and Cd, respectively. The influence of heavy metals on the formation of HMF in honey samples was studied on 5, 30 and 60 days after spiking at concentration level of 10,000 µg kg-1 of each heavy metal. The concentration of HMF for untreated honey samples was ranged 1380-2200 µg kg-1 for the storage time of 5, 30 and 60 days. The concentration of HMF in honey samples spiked with Pb, Ni, Cr and Cd were found to be in the range of 2100-2150, 1900-2430, 2100-2270 and 2050-2330 µg kg-1, respectively. The results of this study indicate that contamination of honey with heavy metal facilitate the formation of HMF.
 KEY WORDS: Fresh honey, HMF, Heavy metal ions, HPLC-DAD and Honey quality
 Bull. Chem. Soc. Ethiop. 2024, 38(1), 1-10. DOI: https://dx.doi.org/10.4314/bcse.v38i1.1

Variations in the Major Nutrient Composition of Dominant High-Yield Varieties (HYVs) in Parboiled and Polished Rice of Bangladesh.

Rice (Oryza sativa L.) is the principal staple food, a fundamental component of food security, a significant source of energy and major nutrients, and a key player in the overall nutritional status in Bangladesh. Parboiling is a common rice-processing treatment in Bangladesh. Recently, polishing has also become a common practice among millers seeking to attract consumers. Polishing may influence the nutrient composition of rice. The present study aimed to investigate the impact of parboiling and polishing on the nutritional content of the five High Yield Varieties (HYVs) of rice (BR11, BRRI dhan28, BRRI dhan29, BRRI dhan49, and BRRI dhan84) and their percent contributions to the Recommended Dietary Allowances (RDA) of vitamins and minerals. All of the rice samples were analyzed for proximate parameters, vitamins (B1, B2, B3, B6, and folate), and minerals (Ca, Mg, Fe, Zn, Na, K, P). Moisture, ash, fat, and total dietary fiber (TDF) were determined gravimetrically, according to the AOAC Official Methods; protein was measured by the Kjeldahl method; B-group vitamins were measured using Ultra Pressure Liquid Chromatography; and mineral content was determined by ICP-OES. The energy, protein, fat, and total dietary fiber (TDF) content of the samples ranged between 342-357 kcal/100 g, 6.79-10.74 g/100 g, 0.31-1.69 g/100 g, and 2.59-3.92 g/100 g respectively. Thiamin, riboflavin, niacin, pyridoxin, and folate content ranged from 0.11-0.25 mg/100 g, 0.01-0.05 mg/100 g, 2.82-6.42 mg/100 g, 0.12-0.30 g/100 g, and 5.40-23.95 g/100 g respectively. In a comparison of parboiling and polishing, macronutrients and vitamin retention were higher in parboiled unpolished rice than in polished unparboiled rice. The minerals (mg/100 g) Ca, Mg, Fe, Zn, Na, K, and P were in the ranges 32.82-44.72, 30.69-58.34, 0.51-0.70,1.83-2.79, 5.00-5.36, 106.49-112.73, and 162.23-298.03. Minerals of BRRI dhan84 were unaffected by polishing and parboiling. BRRI dhan84 contributed a higher percentage of RDA of all B vitamins and minerals. Therefore, to reduce nutrient loss in rice, industries and consumers should be encouraged to avoid polishing or limit polishing to 10% DOM and to consume unpolished rice, either parboiled or unparboiled.

Valorization of Prunus Seed Oils: Fatty Acids Composition and Oxidative Stability.

Prunus fruit seeds are one of the main types of agri-food waste generated worldwide during the processing of fruits to produce jams, juices and preserves. To valorize this by-product, the aim of this work was the nutritional analysis of peach, apricot, plum and cherry seeds using the official AOAC methods, together with the extraction and characterization of the lipid profile of seed oils using GC-FID, as well as the measurement of the antioxidant activity and oxidative stability using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method. Chemometric tools were required for data evaluation and the obtained results indicated that the main component of seeds were oils (30-38%, w). All seed oils were rich in oleic (C18:1n9c) and linoleic (C18:2n6c) acids and presented heart-healthy lipid indexes. Oil antioxidant activity was estimated in the range IC50 = 20-35 mg·mL-1, and high oxidative stability was observed for all evaluated oils during 1-22 storage days, with the plum seed oil being the most antioxidant and stable over time. Oxidative stability was also positively correlated with oleic acid content and negatively correlated with linoleic acid content. Therefore, this research showed that the four Prunus seed oils present interesting healthy characteristics for their use and potential application in the cosmetic and nutraceutical industries.

Quantification of Total Sulfite in Shrimps by BIOFISH 300 SUL Method: First Action 2021.09.

Sulfite is the oldest and most widely used additive in our food supply with antioxidant and preservative properties. Due to its allergenic-like reactions and other adverse health effects, its use is regulated by international regulatory bodies. Therefore, food industries as well as regulatory laboratories must ensure that the maximum concentration of sulfite permitted is not exceeded. The AOAC INTERNATIONAL-approved official method for the quantification of sulfites is the Optimized Monier-Williams Method (AOAC Official Method 990.28), which consists of a time-consuming titration. The present study aims to demonstrate the reliability of the BIOFISH 300 SUL, a simple, fast, and accurate method, as an alternative, for the quantification of total sulfites in shrimp, with a lower LOQ than that of the OMA 990.28, set at 10 mg/Kg. The BIOFISH 300 SUL method is a highly specific biosensor based on its proprietary enzyme-based electrode, for the rapid quantification of total sulfite. The test kit consists of an electrochemical reader (biosensor BIOFISH 300) and disposable electrodes (Biotest) that are capable of providing an electrical signal proportional to the amount of sulfite in the sample analyzed. The method mainly consists of the extraction of sulfite from the solid matrix in an aqueous solution, and its subsequent quantification by the device in less than 3 min. Comparative studies between BIOFISH 300 SUL and OMA 990.28 were conducted for naturally contaminated and spiked samples of raw and boiled shrimp with sulfite levels covering the 7-150 mg/kg range in order to determine linearity, recovery, repeatability, intermediate reproducibility, and accuracy. The BIOFISH 300 SUL method demonstrated high accuracy and precision for the whole range of quantification (7-150 mg/kg). Its ease of use and fast response make it the ideal technology to be implemented by the industry. BIOFISH 300 SUL was adopted as a First Action Official MethodSM by the AOAC Expert Review Panel for Sulfites in Seafood Methods in February 2021 after rigorous review.

Fluoride Determination in Milk, Soy, and Water-Based Products Using Ion-Selective Electrode and Direct Measurement Technique: Single-Laboratory Validation, First Action 2022.05.

The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals issued a call for methods for the determination of fluoride in infant formula. Standard Method Performance Requirement (SMPR®) 2014.016 was approved. A single-laboratory validation (SLV) of a fluoride ion-selective electrode (F-ISE) method was completed. Five powder samples were reconstituted, and hydrochloric acid was added to dissolve and release any bound fluoride. Samples included infant and adult nutritional products made from milk, whey, or soy, containing intact, partially hydrolyzed, or hydrolyzed proteins. Sodium citrate buffer was added to complex any interfering ions and adjust pH and ionic strength. Samples were analyzed in duplicate over 6 days using F-ISE. Results were calculated based on one of two least-squares (log fit) standard curves (0.02 to 0.1 µg/100 g or 0.1 to 2.0 µg/100 g). The LOQ for the method was determined to be 3.3 µg/100 g, which was far below the minimum analytical value of 30 µg/100 g required in the SMPR. The fluoride concentrations ranged from approximately 3 to 14 µg/100 g reconstituted powder and the intermediate precision, RSDr, ranged from 0.7 to 12.4%. Although all of these results are out of scope for the SMPR due to their low fluoride, it should be noted that the RSDr for formulas with fluoride concentrations ranging from approximately 9 to 14 µg/100 g ranged from 0.7 to 4.3%, and the RSDr for the two formulas with fluoride concentrations at or below the theoretical detection limit of the method were only approximately10 and 12.4%. Recoveries ranged from 94 to 98% for samples spiked at three levels with NIST-traceable standard solutions, meeting the SMPR. The F-ISE method meets the SMPR and was adopted as a First Action Official MethodSM. The fluoride ion-selective electrode method presented here is affordable and easy to run. The addition of acid to the samples dissolves any minerals which may bind the fluoride.

Definition of system suitability and proficiency testing acceptance criteria for the standardization of quality control methodologies of botanical bioactives: A case study for the development of AOAC Official Method of Analysis 2020.05 flavanols and procyanidins

Flavanols and procyanidins are bioactives found in many foods including cocoa. The characterization of cocoa flavanols and procyanidins (CF) has historically been challenged by the lack of commercially available standards and weak chromatographic separation performances. The recent release of a reference material and the final authorization of an AOAC Official Method of Analysis (AOAC 2020.05) technically enables the standardization of CF testing. However, the practical implementation of CF testing for routine testing remains challenging for new laboratories, highlighting the need to define guidelines and acceptance criteria to verify normal method performance and user proficiency. This study leveraged the data generated from the recent multi-laboratory validation to define normal method performances. While the challenges associated with HILIC separation can be alleviated through a thorough system equilibration, monitoring system performances remains critical to routine operation and a laboratory’s ability to generate reliable data. Guidelines for routine analysis were developed for system precision and bracketing standard recovery, as well as acceptance criteria for the analysis of the reference material. These guidelines not only complete a body of work that provide accessible, reliable, and robust CF analysis solution to research and quality laboratories, but also provide an example to facilitate the establishment and implementation of future international testing standards for botanical bioactives.

Analysis of Taurine in Infant Formulas and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: First Action 2022.03.

Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. To evaluate the analytical performance of a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for compliance with AOAC Standard Method Performance Requirements (SMPR®) for taurine analysis described in SMPR 2014.013. Following protein precipitation with Carrez solutions, taurine is extracted and separated by HILIC with detection by triple quadrupole MS using multiple reaction monitoring (MRM). Stable isotope labeled (SIL) taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. The method was shown to meet the requirements specified in the SMPR with a linear range of 0.27-2700 mg/hg RTF (ready-to-feed), a limit of detection of 0.14 mg/hg RTF, acceptable recovery of 97.2-100.1%, and acceptable repeatability of 1.6-6.4% relative standard deviation. Additionally, the method was found to have no statistically significant bias compared with reference values for National Institute of Standards and Technology (NIST) 1849a certified reference material (CRM) (P-value = 0.95) and 1869 CRM (P-value = 0.31), and with results from AOAC 997.05 (P-value = 0.10). A recent review of the method and validation data by the Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) found that this method met all the criteria for analysis of taurine specified in SMPR 2014.013 and voted to adopt this method as First Action AOAC Official MethodSM2022.03. A method for the analysis of taurine in infant formulas and adult nutritionals by HILIC-MS/MS is described. A single-laboratory validation (SLV) study demonstrated the applicability of the method to meet requirements of SMPR 2014.013. In December 2022, the SPIFAN ERP voted to adopt this method as First Action AOAC Official Method 2022.03.

Mining nutri-dense accessions from rice landraces of Assam, India

The Indian subcontinent is the primary center of origin of rice where huge diversity is found in the Indian rice gene pool, including landraces. North Eastern States of India are home to thousands of rice landraces which are highly diverse and good sources of nutritional traits, but most of them remain nutritionally uncharacterized. Hence, nutritional profiling of 395 Assam landraces was done for total starch, amylose content (AC), total dietary fiber (TDF), total protein content (TPC), oil, phenol, and total phytic acid (TPA) using official AOAC and standard methods, where the mean content for the estimated traits were found to be 75.2 g/100g, 22.2 g/100g, 4.67 g/100g, 9.8 g/100g, 5.26%, 0.40 GAE g/100g, and 0.34 g/100g for respectively. The glycaemic index (GI) was estimated in 24 selected accessions, out of which 17 accessions were found to have low GI (<55). Among different traits, significant correlations were found that can facilitate the direct and indirect selection such as estimated glycemic index (EGI) and amylose content (−0.803). Multivariate analyses, including principal component analysis (PCA) and hierarchical clustering analysis (HCA), revealed the similarities/differences in the nutritional attributes. Four principal components (PC) i.e., PC1, PC2, PC3, and PC4 were identified through principal component analysis (PCA) which, contributed 81.6% of the variance, where maximum loadings were from protein, oil, starch, and phytic acid. Sixteen clusters were identified through hierarchical clustering analysis (HCA) from which the trait-specific and biochemically most distant accessions could be identified for use in cultivar development in breeding programs.

Comparison of alternative neutral detergent fiber methods to the AOAC definitive method

Neutral detergent fiber (NDF) is the most commonly reported metric for fiber in dairy cattle nutrition. An empirical method, NDF is defined by the procedure used to measure it. The current definitive method for NDF treated with amylase (aNDF) is AOAC Official Method 2002.04 performed on dried samples ground through the 1-mm screen of a cutting mill with refluxing and then filtration through Gooch crucibles without (AOAC-; reference method) or with (AOAC+) a glass fiber filter filtration aid. Other methods in use include grinding materials through the 1-mm screen of an abrasion mill, using filtration through a Buchner funnel with a glass fiber filter (Buch), and use of the ANKOM system (ANKOM Technology, Macedon, NY) that simultaneously extracts and filters samples through filter bags with larger (F57) or smaller (F58) particle size retentions. Our objective was to compare the AOAC and alternative methods using samples ground through the 1-mm screens of cutting or abrasion mills. Materials analyzed were 2 alfalfa silages, 2 corn silages, dry ground and high-moisture corn grains, mixed grass hay, ryegrass silage, soybean hulls, calf starter, and sugar beet pulp. Samples were run in duplicate in replicate analytical runs performed on different days by experienced technicians. Compared with cutting mill-ground samples, the aNDF% of dry matter results from abrasion mill-ground samples were or tended to be lower for 8 of 11 samples. Method affected aNDF% results for all materials, with method × grind interactions for 6 of 11 samples. For ash-free aNDF% assessed with cutting mill-ground materials, a priori selected contrasts showed that the number of materials for which methods differed or tended to differ from the AOAC methods were 4 (Buch), 8 (F57), and 3 (F58); and 3 for AOAC- versus AOAC+. However, statistically different does not necessarily mean substantially different. For a given feed and grind, a positive value for the absolute difference between the AOAC- mean and an alternative method mean minus 2 times the standard deviation of AOAC- suggests that values for the alternative method fall outside of the range of results likely to be observed for the reference method. The number of observed positive values for materials processed with cutting and abrasion mills, respectively, were 0 and 2 (AOAC+); 2 and 2 (Buch); 8 and 10 (F57); 4 and 7 (F58); and 0 and 4 (AOAC-). With the materials tested, methods in order of agreement with the reference method were Buch, F58, and F57, which often gave lower values. The AOAC+ gave results similar to AOAC-, substantiating it as an allowed modification of AOAC-. Best agreement between the reference method and variant NDF methods was achieved with the 1-mm screen cutting mill grind. The 1-mm abrasion mill grind produced more aNDF% results that were lower than the reference method but with fewer differences when filter particle retention size was smaller. The use of filters that retain finer particles could be explored to improve comparability of variant NDF methods and grinds. Further evaluation with an expanded set of materials is warranted.

Evaluation of the effect of household-like handling on the concentration of ethion pesticide in parsley using GC-MS/MS for environmental monitoring

The main purpose of this study was to estimate the influence of different household treatments in the reduction/removal of ethion pesticide from the leaves of parsley in daily basis before human consumption. The suggested analytical method used for detection and quantitation of ethion pesticide in parsley matrix was based on AOAC Official Method 2007.01 using GC-MS/MS instrument. The limit of quantitation (LOQ) for ethion was presented as the minimum fortification level that fulfills convenient recoveries within 70–120% with precision (RSD ≤ 20%). Pesticide residues were extracted from samples of parsley leaves as a representative commodity for high water content group within leafy vegetables category. Parsley samples were gathered from Al-Rass city markets, Al-Qassim region, Saudi Arabia, and analyzed using the suggested method. Ethion pesticide was detected in parsley leaves with levels exceeding the European maximum residual limit (MRL). Some household-type processing and handling procedures were investigated in order to check their effectiveness in reducing the levels of ethion pesticide contaminant in parsley leaves before consumption. Our findings show that normal washing with tap water for 30-45 s is the best practice to reduce 29.6% the concentration of ethion pesticide in parsley samples.

Measurement of Dietary Fiber: Which AOAC Official Method of AnalysisSM to Use.

A broad range of AOAC Official Methods of AnalysisSM (OMA) have been developed and approved for the measurement of dietary fiber (DF) and DF components since the adoption of the Prosky method (OMA 985.29). OMA 985.29 and other OMA were developed to support the Trowell definition of DF. However, these methods do not measure DF as defined by the "new," physiologically relevant, Codex Alimentarius definition. Methodology to support the Codex definition has been developed and updated in recent years. In this article, the relevance of each OMA in supporting the Codex definition of DF is described and suggestions are presented on the most appropriate method, together with proposals for changes in title and application statements for the "historic" OMA methods.