Determination of Fructans in Infant, Adult, and Pediatric Nutritional Formulas: Single-Laboratory Validation, First Action 2016.06.

A method for fructan analysis designed to comply with AOAC Standard Method Performance Requirements (SMPR®) 2014.002 is described. It is closely related to existing methods for fructan analysis, including AOAC 997.08 and 999.03, as well as a method previously published by Cuany et al. This new method achieves LOQ of 0.03% fructan on a ready-to-feed (RTF) basis with mean recoveries ranging from 93 to 108% in the presence of up to 9% sucrose (even at the 0.03% level of fructan). Repeatability ranged from 1.09 to 3.67%. Intermediate precision ranged from 2.46 to 6.79%. Sample preparation for quantitative analysis is simplified compared to some of the existing methodologies. The method incorporates a qualitative profile analysis to determine fructan size category. This allows assignment of appropriate correction factors without independent knowledge of fructan type.

Determination of Vitamin B12 in Infant, Adult, and Pediatric Formulas by HPLC-UV and Column Switching: Collaborative Study, Final Action 2011.10.

AOAC First Action Method 2011.10, Vitamin B12 in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. This method uses a pH 4.5 sodium acetate buffer and potassium cyanide at 105°C to extract and convert all biologically active forms of vitamin B12 present to cyanocobalamin; octylsilyl (C8) or C18 SPE cartridges to purify and concentrate cyanocobalamin; a combination of size-exclusion and RPLC to isolate cyanocobalamin; and visible absorbance at 550 nm to detect and quantitate cyanocobalamin in infant, pediatric, and adult nutritionals with vitamin B12 concentrations greater than 0.025 µg/100 g ready-to-feed (RTF) liquid. During this collaborative study, nine to 11 laboratories from eight different countries analyzed blind duplicates of 12 infant, pediatric, and adult nutritional formulas. Per the AOAC Expert Review Panel (ERP) on Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Nutrient Methods the method demonstrated acceptable repeatability and reproducibility and met SPIFAN Standard Method Performance Requirements (SMPRs®) for the majority of product matrixes analyzed. Vitamin B12 SPIFAN SMPRs for repeatability were ≤15% RSD at vitamin B12 concentrations of 0.01 µg/100 g RTF liquid and ≤7% RSD at vitamin B12 concentrations of 0.2-5.0 µg/100 g RTF liquid. Vitamin B12 SPIFAN SMPRs for reproducibility were ≤11% RSD in products with vitamin B12 concentrations ranging from 0.3 to 5.0 µg/100 g RTF liquid. During this collaborative study, the RSDr ranged from 2.98 to 9.77%, and the RSDR ranged from 3.54 to 19.5%. During previous single-laboratory validation studies, the method LOQ was estimated to be 0.025 µg/100 g RTF liquid.

Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Liquid Chromatography-Pulsed Amperometric Detection with Column Switching: Collaborative Study, Final Action 2011.18.

AOAC First Action Method 2011.18, Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. With this method free myo-inositol and phosphatidylinositol bound myo-inositol are extracted using two different sample preparation procedures, separated by ion chromatography using a combination of Dionex Carbo Pac PA1 and MA1 columns with column switching, and detected with pulsed amperometry using a gold electrode. Free myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica SPE cartridges. Myo-inositol is then released from the glycerol backbone with concentrated acetic and hydrochloric acids at 120°C. During this collaborative study, nine laboratories from five different countries analyzed blind duplicates of nine infant and pediatric nutritional formulas for both free and phosphatidylinositol bound myo-inositol, and one additional laboratory only completed the free myo-inositol analyses. The method demonstrated acceptable repeatability and reproducibility and met the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs®) for free myo-inositol plus phosphatidylinositol bound myo-inositol for all the matrixes analyzed. SMPRs for repeatability were ≤5% RSD at myo-inositol concentrations of 2-68 mg/100 g ready-to-feed (RTF) liquid. SMPRs for reproducibility were ≤8% RSD in products with myo-inositol concentrations ranging from 2 to 68 mg/100 g RTF liquid. During this collaborative study, repeatability RSDs ranged from 0.51 to 3.22%, and RSDs ranged from 2.66 to 7.55% for free myo-inositol plus phosphatidylinositol bound myo-inositol.

Determination of Vitamin K1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Fluorescence Detection: Single-Laboratory Validation, First Action 2015.09.

This normal-phase HPLC method with postcolumn reduction and fluorescence detection allows for the quantitative determination of trans vitamin K1 in infant, pediatric, and adult nutritionals. Vitamin K1 is extracted from products with iso-octane after precipitation of proteins and release of lipids with methanol. Prepared samples are injected onto a silica HPLC column where cis and trans vitamin K1 are separated with an iso-octane-isopropanol mobile phase. The column eluent is mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and vitamin K1 is reduced to a fluorescent derivative in a zinc reactor column. The resulting hydroquinone is then detected by fluorescence at an excitation wavelength of 245 nm and an emission wavelength of 440 nm. During a single-laboratory validation of this method, repeatability and intermediate precision ranged from 0.6 to 3.5% RSD and 1.1 to 6.0% RSD, respectively. Mean overspike recoveries ranged from 91.9 to 106%. The method demonstrated good linearity over a standard range of approximately 2-90 µg/L trans vitamin K1 with r2 averaging 0.99995 and average calibration errors of <1%. LOQ and LOD in ready-to-feed nutritionals were estimated to be 0.03 and 0.09 µg/100 g, respectively. The method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements® and was approved as a first action method at the 2015 AOAC Mid-Year Meeting.

Determination of Free and Total Carnitine and Choline in Infant Formulas and Adult Nutritional Products by UPLC/MS/MS: Single-Laboratory Validation, First Action 2014.04.

A single-laboratory validation (SLV) has been performed for a method that simultaneously determines choline and carnitine in nutritional products by ultra performance LC (UPLC)/MS/MS. All 11 matrixes from the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) were tested. Depending on the sample preparation, either the added (free, with a water dilution and filtering) or total (after microwave digestion at 120°C in nitric acid and subsequent neutralization with ammonia) species can be detected. For nonmilk containing products, the total carnitine is almost always equal to the free carnitine. A substantial difference was noted between free and total choline in all products. All Standard Method Performance Requirements for carnitine and choline have been met. This report summarizes the material sent to the AOAC Expert Review Panel for SPIFAN nutrient methods for the review of this method, as well as some additional data from an internal validation. The method was granted AOAC First Action status for carnitine in 2014 (2014.04), but the choline data are also being presented. A comparison of choline results to those from other AOAC methods is given.

Evaluation Protocol for Review of Method Validation Data by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Expert Review Panel.

Methods under consideration as part of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals process are to be evaluated against a set of Standard Method Performance RequirementsSM (SMPRs) via peer review by an expert review panel (ERP). A validation protocol and a checklist have been developed to assist the ERP to evaluate experimental data and to compare multiple candidate methods for each nutrient. Method performance against validation parameters mandated in the SMPRs as well as additional criteria are to be scored, with the method selected by the ERP proceeding to multilaboratory study prior to Final Action approval. These methods are intended to be used by the infant formula industry for the purposes of dispute resolution.

Free and total carnitine in infant and adult/pediatric nutritional formulas by liquid chromatography/tandem mass spectrometry: first action 2012.17.

Data from a previously published study in J. AOAC Int. 91,130-142 (2008) was presented to the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) during the AOAC Annual Meeting held in Las Vegas, NV, from September 30 to October 3, 2012. The panel reviewed the data submitted and concluded that the method satisfied the standard method performance requirements established by SPIFAN. Based on this information, the panel approved the method as AOAC First Action. The determination of free carnitine involves dilution and filtration of the sample, followed by determination of L-carnitine by LC/MS/MS. The determination of total carnitine includes a saponification procedure prior to dilution and filtration.

Reactivity of a mononuclear iridium(I) species bearing a terminal phosphido fragment embedded in a triphosphorus ligand.

The first example of an iridium(I) species bearing a terminal phosphido (PR(2)(-)) ligand is reported. This stable compound shows well-behaved reactivity toward various electrophiles, owing to its exposed phosphorus lone pair, allowing reversible protonation, selective alkylation, isolation of a phosphidoborane of iridium, and generation of a phosphido-bridged iridium(I)-gold(I) dinuclear species.

Method for the direct determination of available carbohydrates in low-carbohydrate products using high-performance anion exchange chromatography.

An improved method for direct determination of available carbohydrates in low-level products has been developed and validated for a low-carbohydrate soy infant formula. The method involves modification of an existing direct determination method to improve specificity, accuracy, detection levels, and run times through a more extensive enzymatic digestion to capture all available (or potentially available) carbohydrates. The digestion hydrolyzes all common sugars, starch, and starch derivatives down to their monosaccharide components, glucose, fructose, and galactose, which are then quantitated by high-performance anion-exchange chromatography with photodiode array detection. Method validation consisted of specificity testing and 10 days of analyzing various spike levels of mixed sugars, maltodextrin, and corn starch. The overall RSD was 4.0% across all sample types, which contained within-day and day-to-day components of 3.6 and 3.4%, respectively. Overall average recovery was 99.4% (n = 10). Average recovery for individual spiked samples ranged from 94.1 to 106% (n = 10). It is expected that the method could be applied to a variety of low-carbohydrate foods and beverages.

Single-laboratory validation of a liquid chromatographic/tandem mass spectrometric method for the determination of free and total carnitine in infant formula and raw ingredients.

A reversed-phase liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of free carnitine (FC) and total carnitine (TC) in infant formula and raw ingredients. Preparation of samples for FC determination required only nonvolumetric dilution in water, followed by filtration. Determination of TC in whey-based samples required a common saponification procedure, which hydrolyzed the acylcarnitines to FC. L-Carnitine standards were prepared in water at 3 concentrations (7.5, 30, and 60 ng/mL). L-Carnitine-d3 was incorporated in the sample and standard preparations as a stable isotope internal standard (ISTD), which helped to normalize signal suppression due to fouling of the ionization source, or matrix effects. A short C8 column (50 x 2.1 mm id) was used for liquid chromatography, with mobile phases consisting of 0.1% heptafluorobutyric acid in water and methanol. L-Carnitine and the ISTD eluted at about 2 min, and the injection-to-injection cycle time was about 11 min. Multiple-reaction monitoring was used for quantitation by monitoring the ion transitions m/z 162 > 103 and 162 > 85 for L-carnitine, and m/z 165 > 103 and 165 > 85 for the ISTD. The values for overall method precision for a whey-based infant formula, a protein hydrolyzate infant formula, and a water-soluble vitamin/amino acid premix were relative standard deviations of 1.32, 1.86, and 3.33%, respectively. The overall mean recoveries were between 97.3 and 102.8%. Additionally, the method results showed good correlation with those obtained for a radioenzymatic assay done in-house.