Milk-based Nutritional Products Research Articles

Rapid Analysis of Taurine in Infant Formula and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. A rapid compliance method for the analysis of taurine that is applicable to infant formula and milk-based nutritional products is described. Following protein precipitation with Carrez solutions, taurine in the sample extract is separated by hydrophilic interaction liquid chromatography (HILIC) with detection by triple quadrupole mass spectrometry using multiple reaction monitoring (MRM). Stable isotope-labeled taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. The method was shown to be accurate, with acceptable recovery of 99.6% (range = 91.1-106.5%). Results for National Institute of Standards and Technology (NIST)-certified reference materials showed no statistical bias for NIST 1849a (P = 0.96) and NIST 1869 (P = 0.88) when compared with reference values. No bias was found when results were compared with those of an international reference method, AOAC Official MethodSM997.05 (P = 0.18). Repeatability was estimated to be 3.1% RSDr (range: 2.4-4.0%, HorRat: 0.3), and intermediate precision was estimated to be 4.9% RSDiR (range: 2.2-7.7%). Successful single-laboratory validation demonstrates that this rapid method is suitable for use in high-throughput laboratories as part of routine product compliance release testing of taurine in nutritional products. A method for the analysis of taurine in infant formula and adult nutritionals by hydrophilic interaction liquid chromatography-mass spectrometry (LC-MS) is described. The method is suitable for use in high-throughput laboratories for routine product compliance testing of taurine. A single-laboratory validation study demonstrated the method to be accurate, precise, and fit for purpose.

Comparison of LC-MS/MS and Enzymatic Methods for the Determination of Total Choline and Total Carnitine in Infant Formula and Milk Products.

Choline and l-carnitine are classified as pseudo-vitamins because of their conditionally essential status. As they are involved in multiple physiological metabolic pathways in the human body, they are routinely fortified in infant and adult nutritional formulas. The performance of an LC-MS/MS method for the analysis of choline and carnitine, compared with enzymatic methods in routine use for the analysis of total carnitine and total choline, is described. Powder samples were reconstituted, with release of carnitine and choline facilitated by both acid and alkaline hydrolysis and the extract analyzed by LC-MS/MS. Quantitation was by internal standard technique using deuterium-labeled carnitine and deuterium-labeled choline. Method range, specificity, sensitivity, precision, recovery, accuracy, and ruggedness were assessed for milk powders, infant formulas, and soy- and milk-based nutritional products. Spike recoveries of 94.0-108.4% were obtained for both total carnitine and choline, and no statistical bias (α = 0.05) between measured results and certified values (choline: P = 0.36; free carnitine: P = 0.67) was found for NIST 1849a certified reference material (NIST1849a). Precision, as repeatability relative standard deviation (RSD), was 2.0% RSDr for total carnitine and 1.7% RSDr for total choline. Equivalent results for total choline and total carnitine were obtained by LC-MS/MS and enzymatic methods (n = 30). The described LC-MS/MS method is fit for purpose for routine product compliance release testing environments. This validation study has confirmed that alternative enzymatic assays can be used with confidence in laboratories in which LC-MS/MS platforms are unavailable. An LC-MS/MS method was evaluated and found to be fit-for-purpose for routine product compliance release testing of infant formula. The LC-MS/MS method was compared with enzymatic methods for the analysis of total carnitine and total choline. Alternative enzymatic assays can be used with confidence in laboratories in which LC-MS/MS platforms are unavailable.

Rapid Method for the Determination of Thiamine and Pantothenic Acid in Infant Formula and Milk-Based Nutritional Products by Liquid Chromatography‒Tandem Mass Spectrometry.

Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography‒tandem mass spectrometry (LC-MS/MS), with quantitation accomplished by internal standard technique. The method was shown to be accurate, with acceptable recovery (thiamine, 99.3-101.1%; pantothenic acid, 99.2-108.6%). A certified reference material (NIST 1849a), showed no statistical bias (α = 0.05) for thiamine (P = 0.64); although a statistically significant bias (P < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC-MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (α = 0.05; thiamine, P = 0.399; pantothenic acid, P = 0.058). Acceptable precision was demonstrated with a repeatability of 7.2% repeatability relative standard deviation (RSDr) (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.

Determination of Soluble Calcium and Phosphorus in Commercial Milled Hydroxyapatite

A method for the determination of soluble calcium and soluble phosphorus in commercial milled hydroxyapatite is described. The Ca and P residing in the supernatant after high-speed centrifugation of a 1.00 % (w/w) suspension in pH 6.8 HEPES buffer are quantified by cresolphthalein and phosphomolybdenum complex colorimetry, respectively. Method performance has been defined by assessments of linearity (r 2 averages for Ca and P, respectively, were 0.998 ± 0.001, n = 15, and 1.000 ± 0.000, n = 15), intermediate precision (soluble Ca rsd = 12 % at 0.500 mg/g to 0.5 % at 8.04 mg/g; soluble P rsd = 18 % at 0.0526 mg/g to 0.5 % at 3.5 mg/g; soluble Ca/P molar ratio rsd = 18 % at 63 to 0.5 % at 1.88; n = 3 days), and accuracy (spike recovery average = 99.7 ± 3.6 % for soluble Ca, and 97.8 ± 0.6 % for soluble P; n = 4). A hydroxyapatite milled to successively smaller particle sizes exhibited increases in soluble Ca, P, and Ca/P as the particle size median decreased from 2.339 to 1.375 μm. Application of the method to seven commercial lots of milled hydroxyapatite found soluble Ca from 0.118 to 1.98 % of theoretical Ca, soluble P from 0.386 to 1.80 % of theoretical P, and soluble Ca/P (molar) from 0.258 to 3.54. The interaction of these lots with sodium caseinate in a simulated retort model was strongly correlated with their soluble Ca concentration (r 2 > 0.9). The method provides a simple and inexpensive means for identifying potentially problematic lots of milled hydroxyapatite, i.e., lots with soluble Ca and P levels sufficiently high to adversely affect the physical stability of retort sterilized milk-based liquid nutritional products.

A New Micellar Mediated Cloud-Point Extraction Procedure for Sensitive and Selective Determination of Trace Amounts of Total Iodine in Milk-Based Nutritional Products by Means of Indirect Spectrophotometry

A new micellar mediated cloud-point extraction procedure (CPE) has been developed for sensitive and selective determination of trace amounts of total iodine by means of indirect spectrophotometry. The method is based on selective charge transfer complex formation of I3, which is produced by the reaction of iodate in presence of 25-fold excess periodate with excess iodide and with acridine orange (AOH+) being a selective fluorescent cationic dye at pH 3.0 phthalate buffer, and then the CPE of charge transfer complex formed from aqueous solution at phthalate buffer using nonionic surfactant, Triton X-114. The calibration graphs were rectilinear in the ranges of 1.5–20 and 20–160 μg L−1 for IO3 −, with r 2 higher than 0.9996 in two linear regions with changing sensitivity. Under optimal conditions obtained, the limits of detection and quantification were 0.4 and 1.5 μg L−1 and the precision (as RSD) for determination of 10, 50, and 100 μg L−1 of iodate was in the range of 2.4–4.7 % (n 5). The method was successfully applied to selective and reliable determination of total iodine in milk-based nutritional products. The accuracy of the method was controlled by analyzing two standard reference materials (SRMs) at suitable matrix to milk samples.

Determination of phosphatidylserine in milk-based nutritional products using online derivatization high-performance liquid chromatography

Phosphatidylserine (PS) has received interest for its ability to improve cognitive abilities and behaviors. A new method for determining PS in milk-based nutritional products has been developed. The method includes a quick and simple sample preparation procedure, followed by high-performance liquid chromatography (HPLC) fluorescence detection (FLD) with an on-line 9-fluorenylmethyloxycarbonyl (FMOC) derivatization. The method allows PS to be determined in raw materials, milk powder and liquid milk products. The day-to-day (n=3 days) average recovery of over spike-in (at 100% PS content level) was 100%, and the method quantification limit is 53mg per kg milk powder.

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 optical biosensor assay for the determination of folate in milk and nutritional dairy products

An optical biosensor assay utilising folate-binding protein was developed for quantitation of the folate content in milk and milk-based paediatric and adult nutritional products. Samples extracted in reducing buffer were enzymatically deconjugated prior to biosensor analysis. The protein-binding assay was configured under inhibition conditions, utilising a sensor surface functionalised with folic acid, and was subjected to single laboratory validation. Cross-reactivity of folate-binding protein towards the dominant folate vitamers was demonstrated. The method was compared with both established microbiological and high performance liquid chromatographic methods for a range of products. As a rapid, simple and automated technique, the method proved to be a precise and accurate alternative to conventional techniques for application in routine compliance monitoring of milk-based nutritional products.

Comparison of Ion-Selective Electrode and Inductively Coupled Plasma-Mass Spectrometry to Determine Iodine in Milk-Based Nutritional Products

The performances of 2 official methods for iodine analysis based on inductively coupled plasma-mass spectrometry (ICP-MS) and the ion-selective electrode (ISE) method were compared for milk-based products. The aim of the study was to determine the performance characteristics of both methods to check the labeled concentration of iodine. Good precision was found for both methods with highest relative standard deviation of repeatability (RSD(r)) at 2.3 and 2.7% for ISE and ICP-MS, respectively. Intermediate reproducibility (RSD(iR)), single laboratory within 6 different days, was also good with the highest values at 7.3 and 8% by ISE and ICP-MS, respectively. Measurement uncertainty was estimated based on the RSD(iR) data, and it was concluded that both methods were capable of determining iodine concentrations within an uncertainty below +/- 20%. The accuracy of the methods was determined by analyzing certified reference materials, in-house proficiency test samples, and commercial products. Both methods returned similar results when applied on freshly opened samples. In samples that had been opened and kept exposed to air during storage, ISE returned lower iodine concentrations than ICP-MS. In commercial samples, the linear regression between both methods was ISE = 0.95 x ICP-MS -0.060 for freshly opened samples and ISE = 0.85 x ICP-MS + 0.069 for samples exposed to air. The tendency of ISE to return lower results than ICP-MS is explained by the fact that ISE is sensitive to iodide but does not measure iodine that may be bound organically to the matrix. This seems to be more pronounced in samples that were stored longer. Because in most countries iodine is labeled as total iodine, acceptance of an international standard based on the ICP-MS technique which takes all forms of iodine into account, is recommended. This would help to avoid any potential dispute on the accuracy of labeled iodine concentrations in finished products.

Determination Of Tryptophan Content In Infant Formulas And Medical Nutritionals

Abstract A method was developed for determination of total tryptophan content in soy- and milk-based nutritional products. The method uses enzymatic (pronase) digestion of the protein to release tryptophan, which is separated and quantitated by isocratic reversed-phase liquid chromatography with fluorescence detection. Enzymatic digestion is completed for products containing these types of proteins in less than 6 h and is accomplished under chemically mild conditions (pH 8.5,50°C), which do not significantly degrade tryptophan. Chromatographic separation is complete in about 8 min, including an internal standard. The precision of the method is 1-2% relative standard deviation. Accuracy is demonstrated by agreement with theoretical values for standard proteins (amino acid sequence known) and by quantitative recoveries of overspikes, which use either free tryptophan or a standard protein as the spiking material. The method allows determinations on samples containing a wide range of tryptophan values. Appropriate sample size selection and verification of digestion time requirements should allow the method to be applied to different protein types as well. The method allows 24 h turnaround of tryptophan analyses, and quantitative recoveries represent a significant improvement on existing techniques applied to infant formulas and other nutritional products.

Determination of volatile lipid oxidation products by dynamic headspace-capillary gas chromatographic analysis with application to milk-based nutritional products

A dynamic headspace-capillary gas chromatographic analysis technique with cryofocusing was investigated for the quantitation of volatile lipid oxidation products. Alkanals, t-2-alkenals, t,t-2,4-alkadienals, and 1-pentanol were purged, trapped onto Tenax TA, and then back-flushed into a gas chromatograph by thermal desorption. Back-flushed compounds were reconcentrated at -150 o C and then injected into a capillary column by rapid heating, resulting in a satisfactory separation with no band broadening. Volatile compounds were transferred quantitatively during thermal desorption