Stakeholder Panel On Infant Formula And Adult Nutritionals Research Articles

Challenge to evaluate regulatory compliance for nutrients in infant formulas with current state-of-the-art analytical reference methods

Infant formulas are strictly regulated and rigorously tested for compliance. Recently, new official analytical methods/standards have been established for nutrient analyses in these product categories through the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN), governed by AOAC INTERNATIONAL. Many of these methods have been adopted or are in the process of being adopted as reference methods by Codex Alimentarius. The purpose of this paper is to assess the ability of these cutting-edge analytical methods to deliver acceptable results in the context of established regulatory limits for nutrients in food standards and regulations. For this evaluation, the analytical method variability is considered as one of the three main sources of overall process variability, which also includes variation in raw materials/ingredients and the manufacturing process.The process capability (Cp) is a concept for determining the overall process variability relative to specification limits for a parameter in the final product. Based on this principle an analytical method capability (Cm) was defined and calculated for SPIFAN methods. Global regulatory requirements were evaluated including minimum and maximum limits and tolerances from the declared label values. Compared to these requirements, analytical methods for vitamins A, B12, D and folic acid are of particular concern in relation to the requirements in China, some EU member states, Pakistan, Russia, Singapore, South Africa and Thailand. For a product with a manufacturing target at the midpoint of the regulatory range for these nutrients, the probability of obtaining an analytical result outside the regulatory requirements due to analytical variation alone can be as high as 19%. This does not consider variability caused by the production process and/or raw materials/ingredients.These currently used analytical methods are state-of-the-art and represent the latest developments in technology. However, this work demonstrates that continuous method improvements for the nutrients identified must be pursued. In addition, this work supports a risk management approach that takes into consideration analytical method capability when establishing regulatory limits for nutrients in infant formulas. Ongoing efforts towards harmonization of regulatory requirements across global markets will facilitate evaluation of regulatory compliance in infant formulas.

Single-Laboratory Validation of AOAC Official Method 2011.10 for Vitamin B12 in 'Indian' Infant and Pediatric Formulas and Adult Nutritionals.

Ensuring the quality of infant and pediatric formulas and adult nutritionals is of utmost importance for the health and safety of rapidly urbanizing Indian population. B12 is an important water-soluble vitamin, which is fortified externally in such nutritional formulations. The Bureau of Indian Standards (BIS) has a recommended microbiological assay-based method for determination of vitamin B12 that is not precise and accurate enough to meet the label claim requirements of infant, adult, and/or pediatric nutritionals. The AOAC Official Method 2011.10 was originally developed under the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) for the determination of vitamin B12 in infant and pediatric formulas and adult nutritionals. However, those SPIFAN matrixes did not contain malt and other indigenous cereal and legume flour (with or without cocoa powder), which are commonly found in Indian formulations. Thus, there is a need to replace this method with a more precise and accurate method. This study was undertaken to validate the AOAC Official Method 2011.10 on vitamin B12 in 'Indian' infant and pediatric formulas and adult nutritionals. The single-laboratory validation (SLV) of AOAC Method 2011.10 was carried out as per the AOAC Guidelines in six Indian pediatric and adult nutritional formulas to verify its fitness for purpose. Cobalamin in the sample was converted to cyanocobalamin on treatment with potassium cyanide. The sample was then subjected to clean up through a C18 cartridge. Vitamin B12 in the eluted extract was separated from other components using size-exclusion column chromatography followed by a C18 column. The HPLC analysis was carried out at 550 nm. Diastase treatment and C18 solid-phase extraction cleanup satisfactorily removed the matrix interference. The relative standard deviation of the determined values in 30 samples each from 6 selected Indian products and NIST SRM 1849a was <20%. The average recoveries for the spiked recovery samples ranged from 91.75 to 101.14%. Method 2011.10 met the standard method performance requirements set forth by the AOAC SPIFAN. Therefore, we recommend the Method 2011.10 for adoption as the BIS official method for the analysis of vitamin B12 in 'Indian' infant and pediatric formulas and adult nutritionals. This was the first SLV project that the AOAC India section undertook to extend the scope of the AOAC Method 2011.10 for vitamin B12 analysis by validating it in 'Indian' infant and pediatric formulas and adult nutritionals.

Total Folate in Infant Formula and Adult Nutritionals by Trienzyme Extraction and LC-MS/MS Quantitation: A Multilaboratory Testing Study, Final Action 2011.06

Background: The need for an updated reference method for folate was identified as a priority by the AOAC's Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) in 2011. An Expert Review Panel (ERP) found AOAC Official MethodSM 2011.06 suitable for the purpose and approved it as a First Action Official Method. Objective: To determine the repeatability and reproducibility of Method 2011.06: Total Folate in Infant Formula and Adult Nutritionals by Trienzyme Extraction and LC-MS/MS Quantitation. Methods: A multilaboratory collaborative study was conducted. Eleven laboratories located in five countries participated and completed analysis of all multilaboratory testing (MLT) samples. The study was divided into two parts. In the first part, the laboratories analyzed two practice samples (blindly coded) using the updated folate Method 2011.06. The laboratories providing results within the expected range qualified for part two, in which they analyzed 11 MLT samples in blind duplicates. Results: The results were compared with the Standard Method Performance Requirements (SMPR 2011.006) established for folate. The precision results met the requirements stated in the SMPR for all of the samples. Repeatability and reproducibility relative standard deviations ranged from 3.5 to 6.6 and from 9.0 to 15.7%, respectively. Horwitz ratio values for all of the samples were well below 2 (0.61-1.06). Conclusions: The ERP determined that the method performance met the SMPR requirements in September 2017 after reviewing the presented MLT data. Highlights: The ERP recommended the method for Final Action status.

Determination of Lutein and β-Carotene in Infant Formula and Adult Nutritionals by Ultra-High-Performance Liquid Chromatography: Single-Laboratory Validation, First Action 2016.13

An ultra-HPLC method for the determination of lutein and β-carotene in infant formula and adult nutritionals was validated using both unfortified and fortified samples provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). All experiments showed separation of all-trans-lutein and β-carotene from their major cis isomers, apocarotenal, α-carotene, lycopene, and zeaxanthin. Samples spiked with all-trans-lutein and β-carotene showed no isomerization during sample preparation. Linearity of the calibration solutions correlated to approximately 0.8-45 μg/100 g (reconstituted basis) for samples prepared for the lowest sample concentrations. With dilutions specified in the method, the range can be extended to approximately 2250 μg/100 g. The LOD for both lutein and β-carotene was 0.08 μg/100 g, and the LOQ for both was 0.27 μg/100 g. For all measurements in the range of 1-100 μg/100 g, repeatability RSD was ≤5.8% for lutein and ≤5.1% for β-carotene. For measurements >100 μg/100 g, repeatability RSD was ≤1.1% for lutein and ≤1.7% for β-carotene. Accuracy was determined by recovery from spiked samples and ranged from 92.3 to 105.5% for lutein and from 100.1 to 107.5% for β-carotene. The data provided show that the method meets the criteria specified in the Standard Method Performance Requirements for carotenoids (SMPR 2014.014).

Analysis of Vitamin D₂ and Vitamin D₃ in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2016.05.

A method for the determination of vitamin D2 and vitamin D3 in fortified milk powders and infant and adult nutritional formulas is described. Samples are saponified at high temperature and lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to derivatize the vitamin D to form a high-molecular-mass, easily ionizable adduct. The vitamin D adduct is then re-extracted into a small volume of acetonitrile and analyzed by RPLC. Detection is by tandem MS, using multiple reaction monitoring. Stable isotope-labeled vitamin D2 and vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies. A single-laboratory validation of the method using AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) kit samples was performed and compared with parameters defined according to the vitamin D Standard Method Performance Requirements (SMPR(®)). Linearity was demonstrated over the range specified in the SMPR, with the LOD being estimated at below that required. Method spike recovery (vitamin D2, 97.0-99.2%; and vitamin D3, 96.0-101.0%) and RSDr (vitamin D3, 1.5-5.2%) were evaluated and compared favorably with limits in the vitamin D SMPR. Acceptable bias for vitamin D3 was demonstrated against both the certified value for National Institute of Standards and Technology 1849a Standard Reference material (P(α = 0.05) = 0.25) and AOAC INTERNATIONAL reference method 2002.05 (P(α = 0.05) = 0.09). The method was demonstrated to meet the requirements of the vitamin D SMPR as defined by SPIFAN, and was recently approved for Official First Action status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods.

Characterization of Final Action Official MethodSM 2011.19 and First Action Official Method 2015.06 Performance at Analyte Levels Corresponding to CODEX STAN 72 (1981) Minimum Levels.

A limited single-laboratory validation (SLV) was conducted in the authors' laboratory to investigate the performance of AOAC Official MethodsSM 2011.19 Determination of Chromium (Cr), Selenium (Se), and Molybdenum (Mo) in Infant Formula and Adult Nutritional Products by Inductively Coupled Plasma/Mass Spectrometry and 2015.06 Determination of Minerals and Trace Elements in Infant Formula and Adult/Pediatric Nutritional Formula by Inductively Coupled Plasma/Mass Spectrometry at analyte levels below the practical LOQs (PLOQs) already published for these Final Action Official Methods. This work was needed to verify that the actual LOQs were below the minimum requirements for minerals in infant formula as given in CODEX STAN 72 (1981). Linearity studies at low levels were conducted as well as the analysis of blanks over multiple days to establish the LOQs (as opposed to PLOQs) for these nutrients. Several placebo matrixes from the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) program were tested over multiple days at two different sample sizes to quantitate the effect of doubling the sample size given in the original publications. The SLV results indicate that both methods can meet the Codex minimum requirements as-is, without modification of the methods, albeit with a relaxation of the stringent precision criteria originally established for these methods by SPIFAN. Precision can be improved by doubling the sample size, but this step is not necessary to use the method for its intended purpose. A concurrent collaborative study of Method 2015.06 showed that the RSDR obtained across eight laboratories for several infant formula placebos containing mineral concentrations between the PLOQ and LOQ were indeed worse than SPIFAN expectations, but reasonable Horwitz ratios (HorRat) were nonetheless obtained for these analytes.

Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2016.05.

A method for the determination of vitamin D2 and vitamin D3 in fortified milk powders and infant and adult nutritional formulas is described. Samples are saponified at high temperature and lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to derivatize the vitamin D to form a high-molecular-mass, easily ionizable adduct. The vitamin D adduct is then re-extracted into a small volume of acetonitrile and analyzed by RPLC. Detection is by tandem MS, using multiple reaction monitoring. Stable isotope-labeled vitamin D2 and vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies. A single-laboratory validation of the method using AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) kit samples was performed and compared with parameters defined according to the vitamin D Standard Method Performance Requirements (SMPR(®)). Linearity was demonstrated over the range specified in the SMPR, with the LOD being estimated at below that required. Method spike recovery (vitamin D2, 97.0-99.2%; and vitamin D3, 96.0-101.0%) and RSDr (vitamin D3, 1.5-5.2%) were evaluated and compared favorably with limits in the vitamin D SMPR. Acceptable bias for vitamin D3 was demonstrated against both the certified value for National Institute of Standards and Technology 1849a Standard Reference material (P(α = 0.05) = 0.25) and AOAC INTERNATIONAL reference method 2002.05 (P(α = 0.05) = 0.09). The method was demonstrated to meet the requirements of the vitamin D SMPR as defined by SPIFAN, and was recently approved for Official First Action status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods.

Simultaneous Determination of Total Vitamins B1, B2, B3, and B6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS: Single-Laboratory Validation, First Action 2015.14

This method provides simultaneous determination of total vitamins B1, B2, B3, and B6 in infant formula and related nutritionals (adult and infant). The method was given First Action for vitamins B1, B2, and B6, but not B3, during the AOAC Annual Meeting in September 2015. The method uses acid phosphatase to dephosphorylate the phosphorylated vitamin forms. It then measures thiamine (vitamin B1); riboflavin (vitamin B2); nicotinamide and nicotinic acid (vitamin B3); and pyridoxine, pyridoxal, and pyridoxamine (vitamin B6) from digested sample extract by liquid chromatography-tandem mass spectrometry. A single-laboratory validation was performed on 14 matrixes provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) to demonstrate method effectiveness. The method met requirements of the AOAC SPIFAN Standard Method Performance Requirement for each of the three vitamins, including average over-spike recovery of 99.6 ± 3.5%, average repeatability of 1.5 ± 0.8% relative standard deviation, and average intermediate precision of 3.9 ± 1.3% relative standard deviation.

New Methods for the Analysis of Fat-Soluble Vitamins in Infant Formula and Adult Nutritionals.

The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) defined fat-soluble vitamins (FSVs) to include vitamins A, D, E, and K. The levels of FSVs have been closely scrutinized because of a number of factors, including nutrition value especially in foods used to provide sole-source nutrition and the potential for health risks associated with content both below and above recommended levels. Stricter scrutiny and emphasis on nutrient-level compliance with regulatory requirements placed an increased demand on analytical methods that were fit-for-purpose, provided accurate and precise results. Over time, compendial methods have been developed and published by organizations such as AOAC INTERNATIONAL, the European Committee for Standardization, the International Dairy Federation, and the International Organization for Standardization, among others. In general, these methods have shown adequate precision for regulatory compliance based on example food matrixes for which they were designed. However, method evaluation for infant formulas and adult nutritional products was limited to very few matrixes within these categories. As such, method applicability gaps were noted and correlated with more complicated or diverse product matrixes. AOAC undertook the task of modernizing test methods for the determination of nutrients in infant formulas and in adult nutritional products formulated specifically for both healthy adults and those requiring specific nutritional intake. Composition of products in this category continued to evolve, which in turn presented increasing and new challenges to analytical methodology. A summary of a new generation of candidate compendial methods for determination of FSVs in these matrixes, identified by SPIFAN, is presented here.

Determination of Chloride in Infant Formula and Adult/Pediatric Nutritional Formula by Potentiometric Titration: Single-Laboratory Validation, First Action 2015.07.

A potentiometric method for determination of chloride was validated against AOAC Standard Method Performance Requirement (SMPR(®)) 2014.015. Ten AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) matrixes, including National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1849a, were tested in duplicate on 6 independent days. The repeatability (RSDr) ranged from 0.43 to 1.34%, and the intermediate reproducibility (RSDiR) ranged from 0.80 to 3.04%. All results for NIST SRM 1849a were within the range of the certified concentration (701 ± 17 mg/100 g). Recovery was demonstrated with two overspike levels, 50 and 100%, in the 10 SPIFAN matrixes. Samples were tested in duplicate on 3 different days, and all results were within the SMPR requirement of 95 to 105%. The LOQs of the method for powdered products and ready-to-feed or reconstituted products were 20 mg/100 g and 2.2 mg/100 mL, respectively. A wide analytical range from the LOQ to 99.5% chlorine content can be reached with an appropriate dilution factor, but in practice, the upper analytical value observed in routine matrix testing was approximately 1080 mg/100 g in skim milk powder. This is a rapid, simple, and reliable chlorine-testing method applicable to infant formula, adult nutritionals, and ingredients used in these dairy-based products, such as skim milk powder, desalted whey powder, whey protein powder, and whole milk powder.

Determination of Vitamin E and Vitamin A in Infant Formula and Adult Nutritionals by Normal-Phase High-Performance Liquid Chromatography: Collaborative Study, Final Action 2012.10.

The main objective of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) project is to establish international consensus methods for infant formula and adult nutritionals, which will benefit intermarket supply and dispute resolution. A collaborative study was conducted on AOAC First Action Method 2012.10 Simultaneous Determination of 13-cis and All-trans Vitamin A Palmitate (Retinyl Palmitate), Vitamin A Acetate (Retinyl Acetate), and Total Vitamin E (α-Tocopherol and D-α-tocopherol acetate) in Infant Formula and Adult Nutritionals by Normal-Phase HPLC. Fifteen laboratories from 11 countries participated in an interlaboratory study to determine 13-cis and all-trans vitamin A palmitate (retinyl palmitate), vitamin A acetate (retinyl acetate), and total vitamin E (α-tocopherol and D-α-tocopherol acetate) in infant formula and adult nutritionals by normal-phase HPLC and all laboratories returned valid data. Eighteen test portions of nine blind duplicates of a variety of infant formula and adult nutritional products were used in the study. The matrixes included milk-based and soy-based hydrolyzed protein as well as a low fat product. Each of the samples was prepared fresh and analyzed in singlicate. As the number of samples exceeded the recommended number to be prepared in a single day, analysis took place over 2 days running 12 samples on day one and 10 samples on day two. The reference standard stock was prepared once and the six-point curve diluted freshly on each day. Results obtained from all 15 laboratories are reported. The RSDR for total vitamin A (palmitate or acetate) ranged from 6.51 to 22.61% and HorRat values ranged from 0.33 to 1.25. The RSDR for total vitamin E (as tocopherol equivalents) ranged from 3.84 to 10.78% and HorRat values ranged from 0.27 to 1.04. Except for an adult low fat matrix which generated reproducibility RSD >40% for some isomers, most SPIFAN matrixes gave results within the acceptance criteria of <16% RSD as stated in the respective Standard Method Performance Requirements.

Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS): Single-Laboratory Validation, First Action 2015.10

Analytical methods for the analysis of both L-carnitine and choline are needed for reliable and accurate determination in infant formula and adult/pediatric nutritional formula. These compounds are different in how they are utilized by the human body, but are structurally similar. L-carnitine and choline are quaternary ammonium compounds, enabling both to be retained under acidic conditions with strong cation exchange (SCX) chromatography. This method analyzes both compounds simultaneously as either the free forms or as a total amount that includes bound sources such as phosphatidylcholine or acetylcarnitine. The free analysis consists of water extraction and analysis by LC/MS/MS, while the total analysis consists of extraction by acid assisted microwave hydrolysis and analysis by LC/MS/MS. Calibration standards used for calculations are extracted with all samples in the batch. A single laboratory validation (SLV) was performed following the guidelines of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) utilizing the kit of materials provided. The results achieved meet the requirements of SMPR 2012.010 and 2012.013 for L-carnitine and total choline, respectively.

Determination of Sodium Fluoroacetate in Dairy Powders by Liquid Chromatography–Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2015.02

A single-laboratory validation (SLV) study was conducted for the determination of sodium fluoroacetate in dairy powders by LC-tandem MS (LC-MS/MS). Linearity of response was confirmed by analysis of samples fortified over the concentration range 0.10-100 μg/kg. The LOD was estimated to be 0.028 μg/kg (0.028 ppb) from the SD of the measured concentrations of infant formula samples fortified at 0.10 μg/kg. The corresponding LOQ calculates at 0.085 μg/kg (0.085 ppb), which ensures excellent reliability of quantification at the limit of reporting of 1.0 μg/kg (1 ppb). Repeatability and intermediate precision were estimated from the SD of the recovery of samples fortified at 0.075, 0.10, 0.20, 0.50, 1.0, and 10.0 μg/kg. The previously mentioned method performance values were established using a representative stage 2 (6-12 months) bovine infant formula, and the robustness of the method was tested by the analysis of 107 unique dairy powders and formulations fortified at 1.0 μg/kg. The data collected in this study satisfy the requirements of SLV studies established by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN), and the method was awarded First Action Official Method(SM) status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods (Contaminants) on March 17, 2015.

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 Labeled Fatty Acids Content in Milk Products, Infant Formula, and Adult/Pediatric Nutritional Formula by Capillary Gas Chromatography: Single-Laboratory Validation, First Action 2012.13.

The method described is intended for the quantification of all fatty acids, including commercially important groups of fatty acids used for labeling reasons [i. e., trans fatty acids, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids (PUFA), omega-3, omega-6, and omega-9] and/or individual fatty acids (i. e., linoleic acid, α-linolenic acid, arachidonic acid, ecosapentaenoic acid, and docosahexaenoic acid) in milk products, infant formula, and adult/pediatric nutritional formula. These products often contain milk fat and/or vegetable oils and are supplemented or not supplemented with oils rich in long-chain PUFA. The determination is performed by direct transesterification of ready-to-feed (RTF) liquid concentrate or powder products without prior fat extraction. Single-laboratory validation (SLV) data were submitted to the AOAC Expert Review Panel (ERP) on Nutrient Methods for review at the AOAC Annual Meeting held on September 30 to October 3, 2012, in Las Vegas, NV. The ERP determined that the data reviewed met the Standard Method Performance Requirements (SMPR® 2012.011) set by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) and was approved as an AOAC Official First Action method. The analytical range for SPIFAN samples was between 0.001 and 7.94 g/100 g reconstituted product or RTF liquid. LOQ was estimated as 0.001 g/100 g, while repeatability and intermediate precision were both less than 1.8% RSD above 0.05 g/100 g and <3.5% RSD at 0.005 g/100 g. Recovery values based on spiking experiments at two different levels of linoleic and linolenic acids ranged from 100.0 to 102.9% for three different SPIFAN products. All the parameters evaluated during the SLV were well within the values defined in SMPR 2012.011.

Determination of Minerals and Trace Elements in Infant Formula and Adult/Pediatric Nutritional Formula by Inductively Coupled Plasma/Mass Spectrometry A Performance Evaluation: Single-Laboratory Validation, First Action 2015.06.

A method for determination of 12 minerals and trace elements (Na, Mg, P, K, Ca, Cr, Mn, Fe, Cu, Zn, Se, and Mo) in infant formula and adult/ pediatric nutritional formula was developed and evaluated in a single-laboratory validation. Some additional reproducibility data were obtained from a small interlaboratory study. The method involves microwave digestion of the sample followed by inductively coupled plasma/MS and uses Ge and Te as internal standards. The method is an extension of Official Method(SM) 2011.19 and was compared to AOAC Standard Method Performance Requirements (SMPRs®) 2011.009 and 2014.004 developed by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Repeatability precision for the 12 elements in 11 SPIFAN matrixes and National Institute of Standards and Technology Standard Reference Material (SRM) 1849a was <5%, meeting the SMPR criterion for repeatability. Intermediate reproducibility (8 days, two analysts, two instruments) in the 11 SPIFAN matrixes was <5% for nine (Na, Mg, P, K, Mn, Fe, Cu, Zn, Se) of the 12 elements in all 11 matrixes. The mean reproducibility across 6-7 laboratories and seven SPIFAN matrixes ranged from 2.5% for Cu to 7.1% for P. Recovery from spiked matrixes varied from 90.1 to 109%, and accuracy of determination using SRM 1849a ranged from 96.2 to 107.7%, meeting the requirement of 90-110% recovery/accuracy.

Determination of Chromium, Selenium, and Molybdenum in Infant Formula and Adult Nutritional Products by Inductively Coupled Plasma/Mass Spectrometry: Collaborative Study, Final Action 2011.19.

AOAC First Action Method 2011.19: Chromium, Selenium, and Molybdenum in Infant Formula and Adult Nutritional Products, was collaboratively studied. This method uses microwave digestion of samples with nitric acid, hydrogen peroxide, and internal standard followed by simultaneous detection of the elements by an inductively coupled plasma (ICP)/MS instrument equipped with a collision/reaction cell. During this collaborative study, nine laboratories from four different countries, using seven different models of ICP/MS instruments, analyzed blind duplicates of seven infant, pediatric, and adult nutritional formulas. One laboratory's set of data was rejected in its entirety. 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 almost all of the matrixes analyzed. The Cr, Mo, and Se SPIFAN requirement for repeatability was ≤5% RSD. The SMPR called for a reproducibility of ≤15% RSD for products with ultratrace element concentrations above the targeted LOQ of 20 μg/kg Cr/Mo and 10 μg/kg Se (as ready-to-feed). During this collaborative study, RSDr ranged from 1.0 to 7.0% and RSDR ranged from 2.5 to 13.4% across all three ultratrace elements.

Determination of Chloride in Infant Formula and Adult/Pediatric Nutritional Formula by Automated Potentiometry: Single-Laboratory Validation, First Action 2015.08.

A direct potentiometric method involving titration against a standard volumetric silver nitrate solution using a silver electrode to detect the end point was evaluated for the determination of chloride in infant formula and adult/pediatric nutritional formula. It was assessed for compliance against AOAC Standard Method Performance Requirements (SMPR®) established by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). A single-laboratory validation (SLV) study was conducted as a first step in the process to validate the method. In this SLV, 17 SPIFAN matrixes representing a range of infant formula and adult nutritional products were evaluated for their chloride content. The analytical range was found to be between 1.4 and 1060 mg/100 g reconstituted product or ready-to-feed (RTF) liquid. The LOQ was estimated as 1.4 mg/100 g. Method repeatability was between 0.03 and 1.60% in the range of 20 to 167 mg/100 g RTF, and intermediate precision was between 0.09 and 2.77% in the same range. Recovery values based on spiking experiments at two different levels of chloride ranged from 99.0 to 103% for 15 different SPIFAN products. Evaluation of trueness was performed on National Institute of Standards and Technology Standard Reference Material 1849a (Infant/Adult Nutritional Formula) and showed 97.2% of the theoretical value, with no bias at the 95% confidence level. Based on the results of the SLV, the method met the SMPR and was approved as a First Action method by the AOAC Expert Review Panel on Infant Formula and Adult Nutritionals on March 17, 2015.

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.