Determination of Bovine Lactoferrin in Powdered Infant Formula and Adult Nutritionals by Heparin Affinity Extraction and Reverse-Phase High-Performance Liquid Chromatography/Ultraviolet Detection (HPLC/UV): Single-Laboratory Validation, First Action 2021.10.

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.

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

BACKGROUND: Numerous methods are routinely applied for sugar profile analysis. There is a need for a method that can analyze for the common mono- and disaccharides in human food, pet food, and animal feed. There was no compendia method that had such a large scope of coverage. This requires a method that can overcome the common issues seen with the methods available today, which can have interferences or issues with precision and accuracy when applying them to other matrices. OBJECTIVE: To develop and validate a method that can meet the Standard Method Performance Requirements (SMPR®) outlined by the AOAC INTERNATIONAL Stakeholder Panel on Strategic Food Analytical Methods (SMPR 2018.001). METHODS: The current work describes an optimized high-performance anion exchange with pulsed amperometric detection method that builds on the previously published work from this laboratory for the analysis of nutritionally relevant sugar compounds including galactose, glucose, fructose, sucrose, isomaltulose, lactose, and maltose. This method was optimized to provide coverage across a variety of different matrices, including human food, dietary supplements, pet food, and animal feed. A global multilaboratory validation was conducted to validate the method and compare against the SMPR requirements. RESULTS: A summary of the validation data is presented. The requirements set forth by AOAC SMPR 2018.001 were all met with this method. CONCLUSIONS: The method and data from the global multilaboratory validation were reviewed by the AOAC Expert Review Panel, and determined the method met the SMPR requirements. HIGHLIGHTS: The method was granted AOAC First Action Official MethodsSM status.

Analytical Method for Lactoferrin in Milk-Based Infant Formulas by Signature Peptide Quantification with Ultra-High Performance LC-Tandem Mass Spectrometry.

Background: There is a need for a standardized method for quantification of lactoferrin in infant formulas, and manufacturers have started fortifying lactoferrin to mimic the higher levels found in human milk. A variety of current methods exist, but specificity and accuracy are challenging with the infant formula matrix. The use of signature peptides and MS is becoming more prevalent in the realm of analytical chemistry for quantification of proteins. Objective: The objective of this work was to develop and validate a method through a single-laboratory validation for quantification of lactoferrin in milk-based infant formula and begin to lay the foundation for a standardized method. Methods: The method presented uses signature peptides to quantify lactoferrin in milk-based infant formulas by ultra-high performance LC-tandem mass spectrometry (MS/MS). These peptides are produced through tryptic digestion, and fragments produced from these peptides through MS/MS allow the specific quantification using correlating isotopically labeled peptides. Results: The validation parameters were all met with precision RSDr ranging from 2.1 to 7.1 and intermediate RSDR ranging from 7.0 to 10.4 across different fortified milk-based infant formulas. Accuracy with certified reference material resulted in mean recoveries of 91.7-96.4%. Conclusions: The results from this study demonstrate the method is fit for purpose to support manufacturing specifications and nutritional labeling requirements.

Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs.

This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.