Journal of AOAC International最新文献

Background: Immunoglobulin G (IgG) levels in milk are critical indicators of dairy quality. Conventional measurement methods require labeling and complex sample pretreatment, limiting their practical use for real-time monitoring.

Objective: This study aimed to develop a label-free optical biosensor based on ordered porous layer interferometry (OPLI) for the rapid quantification of IgG in milk at different processing stages.

Methods: Test portions were directly applied to the flow tank, where protein A from Staphylococcus aureus was immobilized as a functional ligand via chemical conjugation. Conditions for ligand immobilization, flow rate, contact time, and sensor regeneration were optimized to minimize non-specific binding and enhance accuracy.

Results: The OPLI biosensor demonstrated a linear quantification range of 0.44 μg/mL to 5 mg/mL, with a limit of quantification of 0.44 μg/mL. This dynamic range effectively covers the IgG content from raw milk to finished milk. The method showed high reproducibility, with an instrumental response relative standard deviation (RSD) of 0.05% and an average RSD of 12.1% across batches. The biosensor was successfully applied throughout raw milk and final milk production, facilitating IgG calibration for consumer milk.

Conclusion: The OPLI-based biosensor offers a sensitive, real-time and wide-range analytical method for IgG analysis, enabling effective quality control in dairy production.

Highlights: The study developed a label-free biosensor for IgG detection in milk, with a linear quantification range of 0.44 μg/mL to 5 mg/mL and high reproducibility. The biosensor is applicable across all stages of dairy production for IgG quality assessment.

Background: Proline is one of the most abundant free amino acids in honey and is widely used as a quality indicator. However, its enantiomeric composition has not been systematically investigated in relation to long-term storage.

Objective: This study developed a sensitive and selective method for the determination of D-proline (D-Pro) and L-proline (L-Pro) in honey using 9-fluorenylmethyl chloroformate (FMOC) derivatization followed by liquid chromatography with fluorescence detection (LC/FL), and evaluated storage-associated changes in enantiomeric composition.

Methods: Fresh honeys and honeys stored at ambient temperature for approximately 20 years were analyzed. After solid-phase extraction cleanup and selective masking of primary amino acids, proline enantiomers were derivatized with FMOC and quantified by LC/FL.

Results: The method provided acceptable recoveries (90-98%) with a method detection limit of 0.03 μg/g, representing markedly improved sensitivity compared with a previous liquid chromatography with ultraviolet detection (LC/UV) method. Both enantiomers were detected in all honeys. Long-term storage was associated with a marked decrease in L-Pro and a moderate but statistically significant increase in D-Pro (p < 0.05). Notably, the D-Pro/L-Pro ratio clearly differentiated fresh and long-term storage honeys.

Conclusion: These findings indicate that long-term storage is associated with significant changes in proline enantiomer composition, likely related to non-enzymatic processes such as Maillard-type reactions. The present study introduces stereochemically resolved proline analysis as a complementary perspective for honey quality evaluation.

Highlights: A sensitive FMOC-LC/FL method enabled enantiomer-resolved analysis of proline in honey, revealing storage-associated shifts in enantiomeric composition and demonstrating the potential of the D-Pro/L-Pro ratio as an additional quality indicator.

Background: Latilactobacillus sakei (Lb. sakei) could be isolated from various fermented foods, that could adapted under low-temperature conditions.

Objective: This study investigated the potential probiotic properties of Lb. sakei J15, a strain isolated from kimchi that exhibits high β-galactosidase activity at low temperatures.

Methods: The growth characteristics of Lb. sakei J15 under different temperatures, salt concentrations, and carbon sources were investigated.

Results: These results showed that Lb. sakei J15 exhibited the highest β-galactosidase activity (789.31 mU/mg) at a low temperature (15 °C), along with high survival rates under acidic (pH 3.0) and bile salt (0.3%) conditions, reaching 91.5% and 66.0%, respectively. Additionally, Lb. sakei J15 demonstrated strong tolerance in a simulated gastrointestinal digestion system, with survival rates of 63.0% after 3 h of gastric digestion and 47.0% after 8 h of intestinal digestion. The strain also exhibited notable antioxidant activity, with DPPH and ABTS radical scavenging rates of 43.0% and 71.9%, respectively, as well as a high auto-aggregation ability (96.1%). Furthermore, Lb. sakei J15 effectively utilized fructooligosaccharides (FOS) and galactooligosaccharides (GOS) as carbon sources. It also showed high lysozyme tolerance (93.7% survival rate) and displayed no hemolytic activity.

Conclusions: In conclusion, Lb. sakei J15 showed potential as a starter culture for low-temperature dairy fermentation.

Highlights: Lb. sakei J15 had potential probiotc effects.

Background: Goatpox is a severe viral disease in goats, and vaccination with live attenuated strains remains the most effective strategy for control. Conventional vaccine production relies on primary or Vero cells, but these substrates are limited by unstable supply, high cost, and suboptimal virus yields.

Objective: This study aimed to establish a scalable suspension culture system using immortalized lamb testis (iLT) cells to support efficient replication of the goatpox virus (GTPV) AV41 strain for vaccine production.

Methods: Adherent iLT cells were adapted to suspension by stepwise passaging under reduced serum. Eight commercial suspension media were screened, and four were selected for growth evaluation in 125-mL shaker flasks (20-mL working volume; 1 × 106 cells/mL), with stability assessed up to the 40th passage. For virus production, suspension cultures at 2-3 × 106 cells/mL were inoculated with GTPV-AV41 at 5-10% (v/v), samples were collected at 72-144 h post-inoculation, and viral replication was quantified by real-time quantitative PCR (RT-qPCR) using a cycle threshold (Ct) -50% tissue culture infectious dose (TCID50) calibration curve, with infection confirmed by indirect immunofluorescence.

Results: Suspension-adapted iLT cells maintained robust growth for over 40 continuous passages, reaching densities exceeding 1.2 × 107 cells/mL with >95% viability.Following GTPV-AV41 inoculation, the suspension culture system supported efficient viral replication, yielding a maximum viral titer of 108.00TCID50 equivalents/mL in cell lysates at 72 h post-inoculation. Efficient viral entry and replication were further confirmed by indirect immunofluorescence assays.

Conclusion: Suspension-cultured iLT cells represent a promising platform for scalable goatpox vaccine production, combining high growth performance with efficient viral replication.

Highlights: This study presents a scalable suspension-culture platform by adapting iLT cells under serum-reduced conditions, enabling stable high-density growth and efficient replication of goatpox virus for cost-effective vaccine manufacturing.

Background: Colistin is a polypeptide antibiotic serving as a crucial "last-resort" agent against multidrug-resistant Gram-negative bacteria.

Objective: This study aimed to develop a reliable analytical method for the determination of colistin A and B residues in food matrices using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Method: Test portions were extracted with methanol-water (1:1) under hydrochloric acid-acidified conditions, followed by a secondary extraction with 0.1 mol/L hydrochloric acid. The extracts were cleaned using a weak cation-exchange cartridge, and chromatographic separation was performed on a biphenyl column with gradient elution, followed by LC-MS/MS.

Result: The developed method produced sharp and symmetrical peaks for colistin A and B, with no significant matrix effects. Recoveries ranged from 81.0% to 105.9% across all analytes and matrices, and both repeatability and reproducibility met the target criteria specified in the Japanese guidelines for method validation. The limits of quantification were 20 ng/g in bovine and swine muscles for colistin A and 10 ng/g for others.

Conclusion: An LC-MS/MS method was established for quantitative determination of colistin A and B residues in food matrices. The validation results met all performance criteria of the Japanese guidelines, confirming that the method provides reliable and precise measurements suitable for food residue monitoring.

Highlights: Reliable quantification of colistin residues was achieved using three distinct retention modes, enabling reliable monitoring of residues in foods.

Background: AOAC 2017.16 and AOAC 2022.01 are CODEX Type I methods for total dietary fiber (TDF) that integrate enzymatic-gravimetric steps with HPLC quantification of low molecular weight soluble dietary fiber (SDFS). As implementation expands, chromatography-related limitations and matrix-specific interferences can affect SDFS determination.

Objective: To evaluate performance of AOAC-recommended and alternative HPLC columns for DP2/DP3 boundary assignment across a broad carbohydrate set, and to investigate maltitol-associated interference leading to apparent SDFS overestimation in starch-containing matrices.

Methods: Seventy-four carbohydrate standards (including oligosaccharides, polyols, and sugar alcohols) were analyzed in duplicate across five HPLC column configurations under AOAC 2017.16/2022.01 and AOAC 2009.01/2011.25 conditions. Column performance was scored by correct peak-area assignment relative to the maltose demarcation.Maltitol interference was assessed using food products formulated with/without maltitol and mixtures of maltitol (and other polyols) with wheat starch or wheat flour using scaled AOAC 2017.16 and AOAC 2001.03 procedures.

Results: Across 74 analytes, SugarPak (Waters) provided the highest overall assignment accuracy, with a two-column series configuration scoring highest.Maltitol alone could be excluded by adjusting the DP2/DP3 demarcation; however, maltitol combined with glucose-based polymers produced additional peak area in the SDFS region, yielding up to ∼8% apparent SDFS overestimation in wheat starch mixtures.

Conclusions: Chromatographic selection materially affects DP2/DP3 assignment for SDFS quantification.Maltitol can cause matrix-dependent artifactual SDFS in starch-containing test materials, suggesting formation of low-molecular-weight species during enzymatic processing.Guidance on column equivalency would strengthen AOAC dietary fiber methodology and further work is warranted to further investigate maltitol interaction with glucose-based polymers during TDF analysis.

Background: 5-Methyltetrahydrofolate (5-MTHF), a reduced form of folate (vitamin B9) marketed as a potentially more bioavailable alternative to folic acid, is a key ingredient in dietary supplement (DS), particularly prenatal multivitamin/minerals. Its synthesis may produce racemic mixtures of bioactive L-5-MTHF and inactive D-5-MTHF, raising concerns about labeling accuracy, integrity, and health impacts. DS manufacturers consider D-5-MTHF an impurity and do not use racemic mixtures of 5-MTHF in their products.

Objective: This study aimed to develop and validate an HPLC-MS-based method to separate and quantify the D and L-5-MTHF diastereomers in various DS matrixes and dosage forms.

Methods: A single-laboratory validated method was developed for determination of 5-MTHF isomers in different dosage forms of DS using targeted selected ion monitoring (targeted SIM) with a ChiralPak HSA column. L-cysteine was selected as an extraction stabilizer, and the mobile phase was composed of 100 mM ammonium acetate and 1% (v/v) acetic acid in acetonitrile (ACN) at a 97:3 ratio.

Results: The HPLC/MS method was able to separate D- and L-5-MTHF diastereomers by isocratic elution within 20 min. It showed great linearity with the concentration range of 1 to 160 µg/mL of 5-MTHF with R2 >0.99. The method was validated for the limit of detection (LOD), limit of quantitation (LOQ), linear range, specificity, and recovery according to the guidelines described in Q2(R1) Validation of Analytical Procedures: Text and Methodology Guidance for Industry. The method was then applied for the analysis of 101 samples, and two samples were found to exceed the USP limit of ≤1% D-5-MTHF relative to L-5-MTHF (1.39 and 50.28%).

Conclusions: This performance of HPLC-MS method established effectively determines D- and L-5-MTHF ratios in complex DS, serving as a reliable tool to detect adulteration with racemic mixtures in prenatal and other 5-MTHF-labeled containing DS. It is significantly faster than previously reported methods and possesses great selectivity in separation of the diastereomers in different forms of DS.

Highlights: Successfully validated an HPLC-MS method for quantifying D- and L-5-MTHF diastereomers in diverse DS dosage forms, aiding enantiomeric purity assessment.

Background: Peste des petits ruminants (PPR) is a highly contagious and economically devastating disease affecting sheep and goats. The performance and cost of serological diagnostic kits remain a major challenge for PPR eradication.

Objective: The nucleocapsid (N) protein of PPR virus and its monoclonal antibody (mAb) were engineered for development of a blocking ELISA.

Methods: The N protein was truncated and expressed by the eukaryotic expression system. The mAb 65A8 was recovered, followed by sequencing and cloning of its variable regions into expression vector. The recombinant mAb was expressed in 293F cells. Key characteristics of the recombinant mAb were evaluated. A blocking ELISA was developed and validated. Comparison between the blocking ELISA and a commercial blocking ELISA kit was performed.

Results: The truncated N protein exhibited a higher yield compared to the full-length N protein. Recombinant mAb 65A8 demonstrated the same blocking efficacy as that of ascites-derived mAb. The yield of recombinant mAb reached 5.3 mg/100 mL. The affinity constant of recombinant mAb 65A8 was comparable to that of the ascites-derived 65A8 (3.48 × 109 L/mol versus 6.76 × 109 L/mol). Based on the truncated N and recombinant mAb, a blocking ELISA was established. The detection limit of blocking ELISA was 1:128 and 1:32 based on strong positive serum and weak positive serum, respectively. Inter- and intra-assay coefficients of variation were <10%, indicating robust reproducibility. The blocking ELISA kit showed high coincidence with the commercial ELISA kit recommended by the World Organization for Animal Health reference laboratory.

Conclusions: This study successfully developed a reliable and cost-effective blocking ELISA by protein engineering of the antigen and mAb.

Highlights: The mAb of PPR Virus was recombinantly expressed in 239F cells, which was employed in the development of a reliable blocking ELISA. This work provided novel ideas and methods for the development of the blocking ELISA kits.

Background: Increased demand for organic alternatives to soil amendment necessitates improved nitrogen (N) determination, including ammonium (NH4+), which is a readily available nitrogen source in manure. Current methods of NH4+ quantification include Kjeldahl distillation, ion selective electrode (ISE), and colorimetry. The Kjeldahl method has remained the most accurate and reproducible for the analysis of liquid manure agricultural samples, as the latter methods are limited by cation and turbidity interferences. The Kjeldahl method is tedious, waste-generating, presents safety concerns, and only detects exchangeable NH4+ nitrogen; therefore, safer, more efficient, and informative alternatives are needed.

Objective: To create a rapid, accurate, and safer technique that does not generate chemical waste, while providing crucial quantification.

Methods: Three AOAC Methods (973.49 Kjeldahl -N, 992.15 Dumas total nitrogen ([TN]), and 978.02 Kjeldahl total nitrogen [TKN]) were utilized to demonstrate the validity of an alternate technique developed in our laboratory for the detection of -N by Dumas combustion. Known N standards were tested to confirm equivalent detection across methods in this comparative study.

Results: Liquid manure samples were analyzed for TN and NH4+ content using the standard methods and the Dumas-based technique, and quantifications were compared. Values were significantly similar between methods, verifying the comparability and effectiveness of the Kjeldahl and alternative Dumas-based techniques.

Conclusions: The technique of NH4+ determination by Dumas combustion provides equivalent quantifications to AOAC Methods. This technique is faster and as accurate as the standard Kjeldahl method, without chemical waste or significant safety hazards. The technique is applicable to high-throughput requirements and can provide detection of total nitrogen, NH4+, carbon, and sulfur, as an advantage over the Kjeldahl method.

Highlights: The technique of NH4+ quantification described herein is safer, as accurate, and more efficient than the standard Kjeldahl method, allowing high throughput. Additionally, combustion analyzers can be fitted to detect sulfur and carbon, allowing simultaneous macronutrient detection.

Background: Yellow camellias, recognized as health-promoting teas, are underexplored in terms of their flavonoid profiles. This study systematically investigates the flavonoid composition of four yellow camellia species: C. euphlebia, C. ptilosperma, C. nitidissima, and C. tunghinensis.

Objective: The primary aim of this study was to systematically identify and quantify the flavonoid constituents in the leaves of four representative yellow camellia species (C. euphlebia, C. ptilosperma, C. nitidissima, and C. tunghinensis) using UHPLC-based mass spectrometry techniques.

Methods: Flavonoid constituents were identified using UHPLC-Q-Exactive-MS. Quantitative analysis of 13 major flavonoids was conducted with a validated UHPLC-QqQ-MS/MS method.

Results: A total of 189 flavonoid compounds were identified across all species. Quantitative analysis revealed significant interspecies variation in flavonoid content. C. nitidissima exhibited the highest flavonoid content (145.73 mg/g), followed by C. euphlebia (105.19 mg/g), C. ptilosperma (54.51 mg/g), and C. tunghinensis (51.68 mg/g).

Conclusions: This study establishes comprehensive flavonoid profiles for four yellow camellia species, highlighting the species-specific differences in flavonoid levels and emphasizing their rich and diverse phytochemical compositions.

Highlights: This study systematically investigates the flavonoid composition of four yellow camellia species: C. euphlebia, C. ptilosperma, C. nitidissima, and C. tunghinensis.