Direct determination of free methionine in soy-based infant formula

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.     

Determination of total fat in milk- and soy-based infant formula powder by supercritical fluid extraction

Commercially available simple benchtop systems using CO2 supercritical fluid extraction (SFE) eliminate expensive organic solvent disposal problems and offer potential to meet a demand for rapid, accurate high-volume gravimetric determinations of total fat content of infant formula powders. A Data Quality Objectives (DQOs) approach was used to evaluate the performance characteristics of instrumental SFE extraction for determination of total gravimetric fat in infant formula. The established DQOs included the following: ACCURACY: Correct values were obtained for a suitable reference material, SRM 1846 Infant Formula [National Institute of Standards and Technology (NIST), Gaithersburg, MD]. RUGGEDNESS: Variables were defined as (1) extraction time (35 min optimum); (2) ratio of sample size to diatomaceous earth support material (1 g sample/2 g support); (3) ratio of distilled water to alcohol (50% isopropanol optimum for both milk- and soy-based infant formula samples); (4) extraction flow rate was 3-3.5 mL/min optimum. PRECISION: Relative standard deviations of multiple determinations fell within the Horwitz limits of acceptability of < or = 2.8% at the level of analyte determined (0.34-2.5% obtained). SCOPE OF APPLICABILITY: Includes milk- and soy-based infant formula powders. Research data were obtained by use of a commercially available fat analyzer. Samples of the SRM, 2 commercial milk-based and 3 commercial soy-based infant formula products were distributed to 2 additional collaborating laboratories. Very good agreement was obtained among the submitting and collaborating laboratories for these samples. The use of clearly defined DQOs to establish method performance characteristics, along with the commercially available reference material, provided the mechanism for verification and validation of analytical methodology.     

Determination of pesticides in soy-based infant formula using liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive method using liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed and validated to quantify and confirm 13 pesticides, including aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, formetanate, 3-hydroxycarbofuran, carbendazim, thiabendazole, aldicarb, propoxur, carbofuran, carbaryl, and methiocarb, in soy-based infant formula. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring of 2 fragment ion transitions to provide a high degree of sensitivity and selectivity for both quantitation and confirmation. Different approaches to constructing calibration curves were compared and discussed to address issues of the extraction efficiency or recovery, and matrix effects. Matrix-matched standard calibration curves with the use of isoprocarb as an internal standard were finally used to achieve the best accuracy of the method. Under most circumstances, recoveries of 13 pesticides, spiked at 5.0, 25.0, and 45.0 microg/kg, were close to 100%. The method detection limits (signal-to-noise ratio > or =3:1; microg/kg) of 13 pesticides were 0.2 for thiabendazole and methiocarb, 0.6 for aldicarb, and 0.1 for the others.     

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.     

Interlaboratory verified liquid chromatographic method for analysis of vitamins A and E in soy-based infant formula powder

An interlaboratory verified, liquid chromatographic (LC) method is presented for the analysis of all-rac-alpha-tocopheryl acetate and retinyl palmitate in soy-based infant formula. The extraction procedure uses sample dehydration with magnesium sulfate followed by extraction with isopropanol, hexane-ethyl acetate (85 + 15, v/v). After evaporation and filtration, the sample extract is injected directly onto a normal-phase LC system with fluorescence detection. All-rac-alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing 0.50% (v/v) and 0.125% (v/v) isopropanol, respectively. A zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" is the minimum level of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in 21 Code of Federal Regulations 107.100. The following recoveries and RSD values represent an average (n = 25) of the 5 levels for each analyte: all-rac-alpha-tocopheryl acetate, 100% (RSD = 3.5%); retinyl palmitate, 97.2% (RSD = 2.1%). Two additional laboratories analyzed the fortified ZRM samples. Average recoveries (n = 24) of all-rac-alpha-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-alpha-tocopheryl acetate, 99.0% (RSD = 4.0%), and retinyl palmitate, 96.2% (RSD = 1.4%) at the second laboratory. Average recoveries (n = 24) of all-rac-alpha-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-alpha-tocopheryl acetate, 102% (RSD = 1.4%) and retinyl palmitate, 95.7% (RSD = 2.0%) at the third laboratory. In addition, 6 replicates of the same commercial soy-based infant formula powder were run by the 3 laboratories.     

Determination of choline in infant formula by ion chromatography.

Choline was determined in infant formula by ion chromatography with suppressed conductivity detection. Samples were digested with 1M hydrochloric acid, filtered, diluted, and injected into the chromatographic system. Choline and the alkali and alkaline earth metals were separated on a high-resolution cation-exchange column and detected by suppressed conductivity. The method was linear between 2 and 200 mg/L (r2 = 0.9999), the concentration range of the diluted samples. This method accurately determined choline in powdered, concentrated, and ready-to-feed infant formulas. Recoveries of choline spikes into powdered infant formula at approximately 1, 0.8, 0.5, and 0.2 times the labeled value ranged from 85 to 114%. This method had good agreement for 8 blind duplicates. The values determined for these samples, which were used in an AOAC collaborative study of an enzymatic method, were consistent with the values determined by the enzymatic method.     

Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay

Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.     

Determination of niacin in infant formula by solid-phase extraction and anion-exchange liquid chromatography.

A peer-verified, solid-phase extraction (SPE)/anion exchange liquid chromatographic method is presented for the determination of niacin in milk-based and soy-based infant formula. Analysis is in 3 steps: test sample digestion, extraction/cleanup, and liquid chromatography (LC). Digestion uses a standard AOAC digestion procedure that involves autoclaving at 121 degrees C for 45 min in (1 + 1) H2SO4 to free endogenous niacin from protein and to convert added niacinamide to niacin. The digest solution is adjusted to pH 6.5 with 7.5M NaOH. Acidification to pH <1.0 with (1 + 1) H2SO4 precipitates the protein. The clarified solution is then filtered, and the filtrate is brought to volume. SPE of niacin is accomplished by passing an aliquot of the digest solution through an aromatic sulfonic acid-SPE (ArSCX-SPE) column. After the column is washed with methanol and water to remove extraneous material, the niacin is eluted with 0.25M sodium acetate/acetic acid buffer at pH 5.6. An anion-exchange polystyrene-divinylbenzene column with 0.1 M sodium acetate/acetic acid buffer at pH 4.0 is used for LC. Niacin is determined by UV detection at 260 nm. A standard curve is prepared by passing known amounts of niacin through the ArSCX-SPE columns used for niacin extraction. The following values for x and relative standard deviation (RSD) were obtained for National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula with a certified value for niacin of 63.3 +/- 7.6 microg/g: Submitting laboratory.-- x = 59.7 +/- 4.0 microg/g; RSD = >6.7%; confidence interval (CI) = +/- 1.4 microg/g; n = 27. Peer laboratory.--x = 56.6 +/- 6.6 microg/g; RSD = >11.7%; CI =+/- 4.1 microg/g; n = 8.     

Determination of folate in infant formula and adult/pediatric nutritional formula by optical biosensor assay: First Action 2011.05

After a review of data from a single-laboratory validation (SLV) study published in the International Dairy Journal 21, 783-789 (2011), a method for folate in infant formula and adult/pediatric nutritional formula was submitted for consideration of adoption by AOAC as an automated assay that is rapid and simple. The method uses an optical biosensor assay to quantitate total folate content in milk and milk-based pediatric and adult nutritional products. The assay uses folate binding protein and a functionalized sensor surface. The SLV showed an instrumental LOD of 0.1 ng/mL (equivalent to 2.5 microg/100 g for a typical infant formula). The method detection limit was 6.5 microg/100 g with a repeatability of 3.48% and an intermediate reproducibility of 4.63% RSD.     

An interlaboratory-verified method for the determination of vitamins A and E in milk- and soy-based infant formula by liquid chromatography with matrix solid-phase dispersion extraction

An interlaboratory-verified, liquid chromatographic (LC) method is presented for determination of all-racemic alpha-tocopheryl acetate and retinyl palmitate in infant formula. The extraction procedure uses matrix solid-phase dispersion. A sample is mixed with C18, and the mixture is packed into a reservoir and eluted with selective solvents to extract the analytes. After evaporation and filtration, the sample extract is injected directly into a normal-phase LC system with fluorescence detection. All-racemic alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing isopropanol at 0.2% (v/v) and 0.125% (v/v), respectively. A nonfortified zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" represents the minimum levels of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in Title 21 of the Code of Federal Regulations, part 107.100. Recoveries of retinyl palmitate ranged from 83.8 to 107%, and those of all-racemic alpha-tocopheryl acetate ranged from 87.7 to 108%. Two additional laboratories analyzed the ZRM samples at 4 spiking levels with 6 replicates. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 92.2 to 104% and from 91.7 to 101%, respectively, in the second laboratory. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 85.3 to 97.0% and from 86.6 to 110%, respectively, in the third laboratory. Relative standard deviations for all 3 laboratories ranged from 0.2 to 7.5% with an average of 2.9%. In addition, each laboratory analyzed a commercial milk- and commercial soy-based infant formula. Excellent agreement in results was obtained between the 3 laboratories for vitamins A and E in all matrixes.     

Determination of vitamin A in infant formula and adult nutritionals by UPLC-UV: First Action 2011.07

Vitamin A, a fat-soluble vitamin, is essential for health and plays an important part in vision, bone growth, reproduction, regulating the immune system, cell function, and skin health. Due to the advances in technology and the expansion of its uses, LC technologies are being studied for effectiveness in detecting and quantifying vitamin A in an effort to help determine the amount of vitamin A in various types of samples. For this reason, an Expert Review Panel agreed on June 29, 2011, at the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," to approve "Determination of Vitamin A in Infant Formula and Adult Nutritionals by UPLC-UV" as AOAC Official Method 2011.07. To move from First to Final Action status, it was recommended that additional information be generated for all types of infant formulas and adult nutritional formula matrixes at varied concentration levels, as indicated in the standard method performance requirements. International units or retinol equivalents typically represent the concentration of vitamin A in food and supplements. However, for the purpose of this method, the concentration represented is presented in microg/100 g.     

Determination of vitamin B12 in infant formula and adult nutritionals by HPLC: First Action 2011.10

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting" held on June 29, 2011, an Expert Review Panel (ERP) reviewed the method "Determination of Vitamin B12 in Infant Formula and Adult Nutritionals by HPLC." Under the new pathway to Official Methods, the ERP adopted the method as Official First Action. The method is applicable to the determination of vitamin B12 in infant formula and adult nutritionals. Data showed an average overall intermediate precision of 6.64% RSD, an estimated quantitation limit of 0.8 microg/kg, and a detection limit of 0.2 microg/kg in prepared samples. The standard range of the method is 2 to 200 microg/L, which corresponds to an analytical range of 0.8 to 500 microg/kg.     

Determination of niacin in infant formula and wheat flour by anion-exchange liquid chromatography with solid-phase extraction cleanup

Niacin content must be included on food labels of infant formula products and bakery products containing enriched flour. Liquid chromatographic (LC) determination of niacin in complex food matrixes is complicated by the presence of endogenous compounds that absorb at the commonly used wave-length of 260 nm. Also, the presence of particulate matter in the standard sulfuric acid extraction procedure results in reduced life of LC columns and precolumns. A simple, rapid, solid-phase extraction (SPE) procedure for separation and cleanup of niacin from a complex food matrix digest has been developed. By using a vacuum manifold with the SPE column system, multiple samples can be processed quickly and efficiently for LC analysis, compared with gravimetric column cleanup. Sulfuric acid sample digest is passed over an aromatic sulfonic acid cation-exchange (ArSCX-SPE) or a sulfonated Florisil SPE column. Niacin is eluted with 0.25M sodium acetate-acetic acid, pH 5.6 buffer in vacuo. LC chromatograms of the resulting eluate are free of interference from other components absorbing at 260 nm at the retention time of niacin. Validation of the method was obtained from agreement of analytical results on available reference materials. For both SPE methods, values for niacin in SRM 1846 Infant Formula (milk-based powder) were within uncertainty ranges of the certified value. Use of several calibration procedures (the LC computer program, a peak area response graphic standard curve, or the method of standard additions) with both SPE procedures resulted in niacin values for 3 RM-Wheat Flours (not certified for niacin) in agreement (90-105%) with their respective values reported in the literature. Several commercial wheat flours showed a broad 260 nm interference, resulting in high niacin values. Niacin recoveries from spiked soy-based liquid infant formulas ranged from 95-107% with the ArSCX-SPE column. Calibration curves of niacin were linear up to 400 micrograms/mL, with a detection limit of 0.2 microgram/mL.     

Determination of niacin in infant formula by solid-phase extraction/liquid chromatography: peer-verified method performance-interlaboratory validation

This paper reports the results of the interlaboratory peer validation study of AOAC Peer-Verified Method (PVM) 1:2,000 for the determination of niacin in infant formula by solid-phase extraction/liquid chromatography. We have used a Data Quality Objectives (DQO) approach to address not only method variability and robustness but also accuracy of data through the use of an appropriate reference material in conjunction with the interlaboratory validation study. Our DQO included the following: (1) statistical agreement of analytical results and quantitative recovery between 2 collaborating laboratories; (2) the repeatability relative standard deviation (RSDr) values and the HORRAT (Horwitz ratio) obtained (1.07), which satisfied the criteria of the Horwitz "limits of acceptability" at the analyte level present; (3) validation of lack of interference; and (4) accuracy agreement within assigned values for a certified reference material. National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula, with a certified value of 63.3 +/- 7.6 microg/g for niacin content, was used as a test material for collaborative study and accuracy assessment. Niacin values obtained by the originating laboratory were 59.7 +/- 4.0 microg/g (95% confidence interval [CI] = 1.4 microg/g with a relative standard deviation [RSD] of 6.7%) and by the peer laboratory were 56.6 +/- 6.6 microg/g (95% CI = 4.1 microg/g, with an RSD of 11.7%). Statistical evaluation using the means equivalence test showed that nicotinic acid values obtained by the peer laboratory were equivalent to those values obtained by the originating laboratory. Linear calibration curves and quantitative recovery were obtained. Integration of the PVM process with a readily available certified reference material gives the user confidence in the accuracy of the data generated by the method through traceability to the reference material used.     

Determination of vitamin B12 in fortified bovine milk-based infant formula powder, fortified soya-based infant formula powder, vitamin premix, and dietary supplements by surface plasmon resonance: collaborative study

A collaborative study was conducted on an inhibition-based protein-binding assay using the Biacore Q biosensor instrument and the Biacore Qflex Kit Vitamin B12 PI. The samples studied included infant formula, cereals, premixes, vitamin tablets, dietary supplements, and baby food. The collaborative study, which involved 11 laboratories, demonstrated that the assay showed an RSDr of 1.59-27.8 and HorRat values for reproducibility of 0.34-1.89 in samples with levels ranging from ppm to ppb. The assay studied is a label-free protein binding-based assay that uses the principle of surface plasmon resonance (SPR) to measure the interaction between vitamin B12 and a specific binding protein. A Biacore Q biosensor uses this principle to detect binding directly at the surface of a sensor chip with a hydrophilic gold-dextran surface. The instrument passes a mixture of prepared sample extract and binding protein solution across a covalently immobilized vitamin B12 chip surface, and the response is given as free-binding protein as the mixture binds to the immobilized surface. This technique uses the specificity and robustness of the protein-ligand interaction to allow minimal sample preparation and a wide range of matrixes to be analyzed rapidly. The reagents and accessories needed to perform this assay are provided as the ready-to-use format "Qflex Kit Vitamin B12 PI." The method is intended for routine use in the quantitative determination of vitamin B12 (as cyanocobalamin) in a wide range of food products, dietary vitamin supplements, and multivitamin premixes.     

Determination of biotin by high-performance liquid chromatography in infant formula, medical nutritional products, and vitamin premixes

A solid-phase extraction sample preparation procedure was developed for use with a high-performance liquid chromatography (HPLC) method for biotin analysis. The HPLC method used a reversed-phase C18 column; chromatography run time was 8.5 min. After eluting from the column, biotin went through postcolumn reaction to form a conjugate with streptavidin-fluorescein isothiocyanate, which was then detected by a fluorescence detector. This method was tested with infant formula, medical nutritional products, and vitamin premix samples.     

Rapid analysis of melamine in infant formula by sweeping-micellar electrokinetic chromatography

In the present study, an analytical method using capillary electrophoresis with on-line preconcentration technique was developed for rapid determination of melamine in infant formula. Both stacking and sweeping preconcentration techniques had been investigated for the comparison of their effectiveness in melamine analysis. The limit of detection of melamine standard was 0.5 ng/mL for the field amplified sample stacking (FASS) technique and 9.2 ng/mL for the sweeping technique. Although the FASS technique provided better concentration efficacy than the sweeping technique, the matrix effect was more profound with the former. Matrix effect was evaluated by comparing the enhancement factor (EF) of melamine standard and post-extraction spiked infant formula solution. The EF was changed from 429.86 ± 9.81 to the level less than 133.31 with significant peak distortion in the FASS system, and it was remained unchanged in the sweeping system. Sweeping-micellar electrokinetic chromatography (sweeping-MEKC) was demonstrated to be most suitable for real sample analysis. Under optimum sweeping-MEKC conditions, melamine content in infant formulas could be determined within 6 min. The developed solid phase extraction (SPE) procedures coupled with the sweeping-MEKC method was subjected to method validation. Run-to-run repeatability (n = 3) and day-to-day reproducibility (n = 3) of peak area were within 3.6% and 4.8% RSD, respectively. The accuracy was tested by spiking 0.5 and 2 μg/mL of melamine standard in the melamine contaminated milk powder provided by the European Commission, and the recoveries were 93.4 ± 0.5% and 98.7 ± 0.4%, respectively. Results of this study show a great potential for the sweeping-MEKC method as a tool for the fast screening of melamine in infant formulas.     

Measurements of natural radioactivity in infant formula and radiological risk assessment

A set of measurements have been conducted to determine the levels activity of gamma-emitters present in infant formula. The study of different samples shows the presence of the following radionuclides: ²²⁶Ra, ²³²Th and ⁴⁰K with respectively average activity of 0.52 ± ?0.087, 0.51 ± 0.062, 371.98? ± 3.68 Bq/kg. The mean annual doses lead to 0.62 and 0.24 mSv/year whereas the total risk is about 7.91 × 10^?6 and 5.32 × 10^?6 respectively ≤ 1 year and 1–2 years old infants. The obtained results show that no significant radiation dose and no risk to the infants due to consumption of these milks.     

Determination of total iodine in infant formula and nutritional products by inductively coupled plasma/mass spectrometry: single-laboratory validation

A single-laboratory validation by inductively coupled plasma-mass spectrometry was developed for the determination total iodine (m/z 127) in infant formula and adult nutritional products. All samples were digested in nitric acid using a closed vessel microwave oven system; Te (m/z 130) was used as an internal standard. To prevent loss of iodine, ammonium hydroxide solution was added to the samples immediately after digestion. The method quantitation limit for total iodine was 0.3 ng/mL, but a practical LOQ was used at 1.0 ng/mL, a concentration at which there was a negligible bias due to nonlinearity. The total iodine concentrations (112-1900 ng/g) in 14 out of 15 nutritional products were within specification limits. Within-day and day-to-day (6 independent days) precision values were < 10% RSD. The observed precision for the overall mean (18 independent days) of a control sample was approximately 4% RSD. In two National Institute of Standards and Technology standard reference materials, total iodine results were within certified limits. Sample spike recoveries for all 15 nutritional products were 92-105%. The data show that a conventional microwave oven digestion procedure can be used to prepare samples for iodine determination. Therefore, this technique is very compatible with other methods being proposed as modern official methods for the analysis of minerals in nutritional products.     

Determination of vitamin A (retinol) in infant formula and adult nutritionals by liquid chromatography: First Action 2011.15

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," held on June 29, 2011, an Expert Review Panel (ERP) reviewed the method for the "Determination of Vitamins A (Retinol) and E (alpha-Tocopherol) in Foods by Liquid Chromatography: Collaborative Study," published by Jonathan W. DeVries and Karlene R. Silvera in J. AOAC Int. in 2002. After evaluation of the original validation data, an ERP agreed in June 2011 that the method meets standard method performance requirements (SMPRs) for vitamin A, as articulated by the Stakeholder Panel on Infant Formula and Adult Nutritionals. The ERP granted the method First Action status, applicable to determining vitamin A in ready-to-eat infant and adult nutritional formula. In an effort to achieve Final Action status, it was recommended that additional information be generated for different types of infant and adult nutritional formula matrixes at varied concentration levels as indicated in the vitamin A (retinol) SMPR. Existing AOAC LC methods are suited for specific vitamin A analytical applications. The original method differs from existing methods in that it can be used to assay samples in all nine sectors of the food matrix. One sector of the food matrix was powdered infant formula and gave support for the First Action approval for vitamin A in infant and adult nutritional formula. In this method, standards and test samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated by an LC method, using UV detection at 313 or 328 nm for retinol. Vitamin concentration is calculated by comparison of the peak heights or peak areas of retinol in test samples with those of standards.