Determination of Casein Allergens in Extensively Hydrolyzed Casein Infant Formula by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: The use of hypoallergenic infant formulas and the need for reliable tests to determine the presence of residual antigens have increased in parallel. OBJECTIVE: An LC-MS method for quantitation of casein was validated using incurred samples and a matrix-matched external standard curve. METHOD: Powdered infant formula samples were extracted in a buffer of sodium deoxycholate and ammonium bicarbonate at 60°C and filtered through 7 kDa desalting columns. Samples were digested overnight with trypsin and precipitated with acid prior to analysis of marker peptides by tandem mass spectrometry. RESULTS: Based on three marker peptides, the linear range for casein was 1.8-42 µg/g of powdered infant formula with an LOQ of 1.8 µg/g. The determination coefficients (R2) for each curve were ≥0.99 for casein peptides. Method repeatability was ≤22% RSD and intermediate precision was ≤23% RSD; recovery of casein from incurred material (2-20 µg/g) ranged from 78% to 118%. CONCLUSIONS: An LC-MS/MS method was developed and validated for confirmation of casein allergens in hypoallergenic infant formula. HIGHLIGHTS: A method was developed to accurately and reliably quantify casein allergens in extensively hydrolyzed casein infant formula by LC-MS without the need for custom peptide standards.

Determination of Lutein, ß-Carotene, and Lycopene in Infant Formula and Adult Nutritionals by Ultra-High Performance Liquid Chromatography: Collaborative Study, Final Action 2016.13 for ß-Carotene and Lycopene Only.

BACKGROUND: Lutein, ß-carotene, and lycopene are among the most common carotenoids present in human milk and are frequently added to infant formula and adult nutritionals. OBJECTIVE: A collaborative study was conducted to assess the interlaboratory performance of AOAC Official MethodSM2016.13 for the determination of lutein, ß-carotene, and lycopene in infant formula and adult nutritionals. METHODS: Thirteen laboratories agreed to participate in the study and 10 laboratories from seven different countries reported results. The study samples included blind duplicates of 6 matrices fortified with lutein, 7 matrices fortified with ß-carotene, and 1 fortified with lycopene. NIST SRM 1869 was included in the sample set as a reference material. RESULTS: After the removal of outliers and invalid data, the repeatability (RSDr) data was ≤10.0% for all-trans-lutein, ≤12.0% for total lutein, ≤4.2% for all-trans-ß-carotene, ≤6.0% for total ß-carotene, and 1.6% for total lycopene. Reproducibility (RSDR) was ≤14.8% for all-trans-lutein, ≤19.9% for total lutein, ≤15.3% for all-trans-ß-carotene, ≤13.7% for total ß-carotene, and 7.4% for total lycopene. CONCLUSIONS: The repeatability and reproducibility values met the criteria in the Standard Method Performance Requirements (SMPRs) for ß-carotene and lycopene and it was recommended that the method be approved as a Final Action for these analytes. Since the method did not meet the SMPR for lutein, it was recommended that it remain a First Action method for this analyte. HIGHLIGHTS: AOAC Official MethodSM2016.13 was validated through a collaborative study to be accurate and reproducible for the determination of ß-carotene and lycopene in infant formula and adult nutritionals.

Determination of Lutein and ß-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 ß-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 ß-carotene from their major cis isomers, apocarotenal, α-carotene, lycopene, and zeaxanthin. Samples spiked with all-trans-lutein and ß-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 ß-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 ß-carotene. For measurements >100 µg/100 g, repeatability RSD was ≤1.1% for lutein and ≤1.7% for ß-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 ß-carotene. The data provided show that the method meets the criteria specified in the Standard Method Performance Requirements for carotenoids (SMPR 2014.014).

Flavones: Food Sources, Bioavailability, Metabolism, and Bioactivity.

Flavones are a class of flavonoids that are a subject of increasing interest because of their biological activities in vitro and in vivo. This article reviews the major sources of flavones and their concentrations in food and beverages, which vary widely between studies. It also covers the roles of flavones in plants, the influence of growing conditions on their concentrations, and their stability during food processing. The absorption and metabolism of flavones are also reviewed, in particular the intestinal absorption of both O- and C-glycosides. Pharmacokinetic studies in both animals and humans are described, comparing differences between species and the effects of glycosylation on bioavailability. Biological activity in animal models and human dietary intervention studies is also reviewed. A better understanding of flavone sources and bioavailability is needed to understand mechanisms of action and nutritional intervention.

Effects of food formulation and thermal processing on flavones in celery and chamomile.

Flavones isolated from celery varied in their stability and susceptibility to deglycosylation during thermal processing at pH 3, 5, or 7. Apigenin 7-O-apiosylglucoside was converted to apigenin 7-O-glucoside when heated at pH 3 and 100°C. Apigenin 7-O-glucoside showed little conversion or degradation at any pH after 5h at 100°C. Apigenin, luteolin, and chrysoeriol were most stable at pH 3 but progressively degraded at pH 5 or 7. Chamomile and celery were used to test the effects of glycosidase-rich foods and thermal processing on the stability of flavone glycosides. Apigenin 7-O-glucoside in chamomile extract was readily converted to apigenin aglycone after combination with almond, flax seed, or chickpea flour. Apigenin 7-O-apiosylglucoside in celery leaves was resistant to conversion by ß-glucosidase-rich ingredients, but was converted to apigenin 7-O-glucoside at pH 2.7 when processed at 100°C for 90min and could then be further deglycosylated when mixed with almond or flax seed. Thus, combinations of acid hydrolysis and glycosidase enzymes in almond and flax seed were most effective for developing a flavone-rich, high aglycone food ingredient from celery.

Endogenous enzymes, heat, and pH affect flavone profiles in parsley (Petroselinum crispum var. neapolitanum) and celery (Apium graveolens) during juice processing.

Flavones are abundant in parsley and celery and possess unique anti-inflammatory properties in vitro and in animal models. However, their bioavailability and bioactivity depend in part on the conjugation of sugars and other functional groups to the flavone core. The effects of juice extraction, acidification, thermal processing, and endogenous enzymes on flavone glycoside profile and concentration in both parsley and celery were investigated. Parsley yielded 72% juice with 64% of the total flavones extracted, whereas celery yielded 79% juice with 56% of flavones extracted. Fresh parsley juice averaged 281 mg flavones/100 g and fresh celery juice, 28.5 mg/100 g. Flavones in steamed parsley and celery were predominantly malonyl apiosylglucoside conjugates, whereas those in fresh samples were primarily apiosylglucoside conjugates; this was apparently the result of endogenous malonyl esterases. Acidification and thermal processing of celery converted flavone apiosylglucosides to flavone glucosides, which may affect the intestinal absorption and metabolism of these compounds.