Journal of AOAC International最新文献

Background: The genus Usnea (Parmeliaceae; lichenized Ascomycetes) is pale grayish-green fruticose lichens which grow as leafless mini-shrubs and comprise about 360 species. Most of the Usnea species are edible and is utilized in preparation of traditional foods as well as in medicines to combat wide range of ailments.

Objective: The goal of this work was to quantify usnic acid in three Usnea spp. [Usnea ghattensis (UG), Usnea orientalis (UO) and Usnea undulata (UU)] using HPTLC-MS and chemical profiling of acetone extracts using UPLC-QTof-MSE resulted in the identification of sixteen compounds based on their MS/MS fragmentation patterns.

Methods: Hyphenated techniques, HPTLC-MS and UPLC-QTof-MSE have been proposed to quantify usnic acid and analysis of metabolites in the crude extracts qualitatively. This method allowed tentative characterization of metabolites from Usnea spp.

Results: The quantification study showed the excellent linearity of the usnic acid at 0.25-1 µg/band with a correlation coefficient r  2>0.99, and LOD, LOQ was found to be 51.7 and 156.6 ng/band, respectively. Further, UPLC-QTof-MSE analysis of crude extract led identification of lichen substances through their exact molecular masses and MS/MS fragmentation studies.

Conclusions: The present study summarizes HPTLC method for quantification of usnic acid in three different Usnea spp. Along with two herbal formulations containing Usnea spp. as the ingredient and developed method was validated as per the ICH guidelines and further UPLC-QTof-MSE analysis provides characterization of the sixteen different secondary metabolites based on their mass fragmentation studies.

Highlights: Rapid HPTLC method for quantification of usnic acid in three different Usnea spp. along with two herbal formulations and metabolite profiling using UPLC-QTof-MSE.

Background: Antibiotic residues in milk are a well-known hazard in the dairy food chain. Detection methods for these residues, such as nonspecific microbiological inhibitor tests or group-specific receptor tests, are relatively inexpensive, easy to use, and widely applied to ensure food safety. In contrast, specific detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-although a critical, complimentary method to confirm the results of nonspecific testing-is relatively costly, time-consuming, and laborious. Furthermore, sample processing before LC-MS/MS analysis requires unique preparation procedures for different groups of antibiotic compounds.

Objective: To simplify and speed up specific antibiotic residue detection, a low-cost, passive, and single-step method to fractionate analytes in raw milk was developed.

Methods: Untreated raw milk was fractionated into its water and fat/protein phases using a Fractionation of Milk for Trace Analysis of Contaminants and Residues for Antibiotics (FraMiTrACR® AB) fractionation unit. The water fraction was then analyzed by LC-MS/MS. The analyte fractionation method was evaluated against a Quick Easy Cheap Effective Rugged and Safe (QuEChERS)-based method for sample preparation.

Results: Our method allows qualitative and quantitative detection of substances from the penicillin, cephalosporin, macrolide, lincosamide, sulfonamide, tetracycline, and fluoroquinolone groups of antibiotics. Detection limits are below the legally prescribed maximum residue levels, allowing reliable, specific, and rapid validation of a positive result in nonspecific microbiological inhibitor tests.

Conclusion: Analyte fractionation by FraMiTrACR AB is a faster alternative to QuEChERS-based sample preparation for the detection of antibiotic substances in milk.

Highlight: This method describes a low-cost, environmentally friendly, passive, and single-step milk analyte fractionation. As an alternative to QuEChERS-based preparation, this fractionation method simplifies and speeds up the process for specific antibiotic residue detection.

Background: Arcae concha and Meretricis concha cyclinae concha are two marine shellfish herbs with similar composition and efficacy, which are usually calcined and used clinically.

Objective: This study investigated variations in the inorganic and organic components of Arcae concha and Meretricis concha cyclinae concha from different production regions, both Arcae concha and Meretricis concha cyclinae concha. The aim was to enhance the understanding of these two types of marine shell traditional Chinese medicine (msTCM) and provide a foundation for their future development and application.

Method: Spectroscopic techniques, including infrared spectroscopy, X-ray spectroscopy, and X-ray fluorescence spectroscopy, were used to analyze the calcium carbonate (CaCO3) crystal and trace elements. Thermogravimetric analysis was used to investigate the decomposition process during heating. The proteins were quantified using the BCA protein assay kit. Principal component analysis (PCA) was used to classify inorganic elements in the two marine shellfish traditional Chinese medicines.

Results: No significant differences were found among the various production regions. The crystal structure of CaCO3 in the raw products was aragonite, but it transformed into calcite after calcination. The contents of Ca, Na, Sr, and other inorganic elements were highest. The protein content was significantly reduced after calcination. Therefore, these factors cannot accurately reflect the internal quality of TCM, rendering qualitative identification challenging. CaCO3 dissolution in the decoction of Arcae concha and Meretricis concha cyclinae concha increased after calcination, aligning with the clinical application of calcined shell TCM. PCA revealed the inorganic elements in them, indicating that the variation in trace element composition among different drugs leads to differences in their therapeutic focus, which should be considered during usage.

Conclusions: This study clarifies the composition and structure changes of corrugated and clam shell before and after calcining, and it lays the foundation for the comprehensive utilization of marine traditional Chinese medicine.

Highlights: These technical representations reveal the differences between raw materials and processed products, which will provide support for the quality control of other shellfish TCM.

Background: Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk or dosage form.

Objective: The objective of this study was the investigation of oxidation reactions of GDV with five inorganic oxidizing reagents and the employment of the reactions in the development of five green microwell spectrophotometric methods (MW-SPMs) with simple procedure and high throughputs for determination of GDV in its bulk and dosage forms (capsules).

Methods: The reactions were carried out in 96-well plates, and the absorbances of reaction solutions were measured by an absorbance microplate reader. Variables influencing the reactions were carefully investigated and optimized.

Results: Under the refined optimum conditions, Beer's law with excellent correlation coefficients (0.9992-0.9997) was followed in GDV concentrations in a general range of 5-700 µg/mL, and the limits of detection were ≥1.8 µg/mL. All validation parameters of all methods were acceptable. The methods were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision; the recovery percentages were 98.6-101.2 ± 0.58-1.14%. The greenness of MW-SPMs was evaluated by three comprehensive metric tools, which demonstrated the adherence of MW-SPMs to the principles of the green analytical chemistry (GAC) approach.

Conclusions: The proposed MW-SPMs combined the advantages of microwell-based practice and the use of common laboratory reagents for the analysis. The advantages of microwell analysis were the high throughput, readily available for semi-automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of using common laboratory reagents were the availability, consistency, compatibility, safety, and cost-effectiveness.

Highlights: Overall, the proposed MW-SPMs are versatile, valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Background: Busulfan is the most effective medication for treating chronic myelogenous or granulocytic leukemia because it has cytotoxic properties that harm or kill hematopoietic cells. It cannot absorb light in the Ultraviolet range due to its structure. Because of this, it is very challenging to quantify using traditional HPLC coupled with UV/Photodiode Array detectors. So, using sodium diethyldithiocarbamate, a derivatization method was developed to quantify related impurities. A significant unknown impurity was identified in derivatized samples of busulfan and a noticeably high percentage level was discovered during routine drug testing.

Objective: We aimed to isolate, and characterize the unknown impurity observed in the samples and to identify its root cause.

Methods: Preparative HPLC was used to isolate the unidentified, derivatized impurity, and 1H NMR, 13C NMR, and MS were used to decipher its structural components.

Results: The spectral characterization data analysis showed that the unknown impurity was related to busulfan. Additionally, it was noted that the impurity developed as a result of the residual buffer used to prepare the derivatizing reagent.

Conclusion: The isolated impurity was found to be same as comparable to that found in busulfan drug substances, according to the results of the characterization tools. An alternative method of reagent preparation was optimized and deemed satisfactory because the buffer used in reagent preparation is the only factor contributing to the formation of impurities.

Highlights: Using cutting-edge analytical characterization tools, it was possible to explain the structural characteristics of an unknown impurity and discover that it was a novel impurity, which undoubtedly contributes to the comprehension of drug substance reaction properties.

Background: Determining the concentration of nanoparticles (NPs) in marine organisms is important for evaluating their environmental impact and to assess potential food safety risks to human health.

Objective: The current work aimed at developing an in-house method based on single-particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) suitable for surveillance of NPs in mussels.

Methods: A new low-cost and simple protease mixture was utilized for sample digestion, and novel open-source data processing was used, establishing detection limits on a statistical basis using false-positive and false-negative probabilities. The method was validated for 30 and 60 nm gold NPs spiked to mussels as a proxy for seafood.

Results: Recoveries were 76-77% for particle mass concentration and 94-101% for particle number concentration. Intermediate precision was 8-9% for particle mass concentration and 7-8% for particle number concentration. The detection limit for size was 18 nm, for concentration 1.7 ng/g, and 4.2 × 105 particles/g mussel tissue.

Conclusion: The performance characteristics of the method were satisfactory compared with numeric Codex criteria. Further, the method was applied to titanium-, chromium- and copper-based particles in mussels.

Highlights: The method demonstrates a new practical and cost-effective sample treatment, and streamlined, transparent, and reproducible data treatment for the routine surveillance of NPs in mussels.

Background: Infant formulas, and pediatric and adult nutritional products, are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards.

Objective: To develop and validate a lactoferrin method capable of meeting the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) 2020.005.

Methods: Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4°C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction (SPE) column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated using an external calibrant.

Results: The LOQ (2 mg/100 g), repeatability (RSD: 2.0-4.8%), recovery (92.1-97.7%), and analytical range (4-193 mg/100 g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin.

Conclusion: The reported single-laboratory validation (SLV) results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas.

Highlights: The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents.

Background: To protect public and animal health against risks provoked by aflatoxins contained therein, maximum limits for aflatoxins are defined. Limit values vary depending on the intended use and regulatory authority, therefore quantitative detection is essential.

Objective: Validation of a one-step competitive lateral flow immunochromatographic assay for quantitative screening of total aflatoxin (B1, B2, G1, and G2) in corn and peanut paste for the high-sensitivity range (0-50 µg/kg).

Methods: Corn or peanut paste test portions are water-based extracted and prepared for testing within 15 min. The AgraStrip® Pro Total Aflatoxin WATEX® test method quantifies the concentration of aflatoxins in the sample. Selectivity, robustness, product consistency, and stability testing were performed in addition to matrix testing.

Results: No cross-reactivity was detected against possible interferants. Corn resulted in a LOD and LOQ of 0.9 and 2.8 µg/kg and overall recoveries between 74 and 108%. Peanut paste resulted internally in a LOD and LOQ of 0.8 and 2.3 µg/kg and recoveries between 86 and 98%. Stability testing showed no influence of the age of the respective lot on the result. Robustness testing demonstrated that varying the amount of water used for extraction, extraction time, and delay between extract dilution and analysis did not significantly affect the result. Due to supply chain issues, a change to the outer cartridge required an increase in the test aliquot size, which had no effect on method performance.

Conclusion: The test kit was validated for the determination of total aflatoxins in corn and peanut paste. Recovery and precision met the requirements laid down in Codex Alimentarius CXG 71-2009 and acceptable robustness, selectivity, and product consistency and stability were demonstrated.

Highlights: The AgraStrip Pro Total Aflatoxin WATEX test kit in the high sensitivity range (0-50 µg/kg) was approved by the AOAC AOAC Research Institute (PTM number 032402).

Background: Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch.

Objective: This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product.

Methods: Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration.

Results: The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP).

Conclusion: In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil.

Highlights: The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

Background: Dietary supplements derived from botanicals are commonly consumed and investigated in biomedical studies for their potential health benefits. Accurate identification and quantification of key chemical constituents from botanical ingredients is necessary for consistent product preparations and reproducible research results. Manufacturers need quantitative reference materials of the chemical constituents of interest to verify the content of ingredients and products. The rigor and reproducibility of biomedical research is enhanced through thorough characterization of the interventions used in mechanistic, clinical, and safety investigations. Quantitative reference materials enable reliable product quality assessments and reproducible research results.

Objective: Solution-based certified reference material (CRM) mixes were developed as calibrants for phytochemicals in ginger and kava. The kava CRM contained yangonin, desmethoxyyangonin, dihydrokavain, DL-kavain, methysticin, dihydromethysticin, flavokawain A, flavokawain B, and flavokawain C. The ginger CRM contained 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol.

Methods: Each phytochemical was sourced as an isolated compound and assigned a purity factor by a mass balance approach accounting for residual impurities. The solution standard mixes were formulated by gravimetric addition of each phytochemical incorporating the purity factor and diluting with acetonitrile to the target concentrations of 500 µg/mL for the gingerols and shogaols, 250 µg/mL for the kavalactones, and 25 µg/mL for the flavokawains.

Results: The concentration accuracy of each component in the solution mixes was analytically verified by ultra high performance liquid chromatography with ultraviolet detection (UHPLC-UV) assay comparison to an independently prepared calibration solution. Each component in the ginger and kava CRMs were within 5 and 7% of the target concentrations, respectively.

Conclusion: Homogeneous kava and ginger phytochemical solution mixes were produced with accurate constituent concentrations and demonstrated good stability over 2 years. These solution mixes were launched as commercially available CRMs.

Highlights: These mixes can be used as accurate concentration stock solutions to prepare calibrators and controls for botanical dietary supplement product testing and standardization.