RESUMEN
BACKGROUND: Human milk oligosaccharides (HMOs) are important components of breast milk and may be responsible for some of the benefits of breastfeeding, including resistance to infections and the development of a healthy gut microbiota. Selected HMOs are now available for addition to infant formula, and suitable methods to control the dosing rate are needed. OBJECTIVE: To develop and validate a suitable method for the analysis of HMOs in infant formula. METHOD: A method was developed for the determination of seven human milk oligosaccharides (2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose (6'SL), 2',3-difucosyllactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT)) in infant formula and adult nutritionals. The oligosaccharides are labeled at their reducing end with 2-aminobenzamide, separated by liquid chromatography and detected using a fluorescence detector. Maltodextrins are enzymatically hydrolyzed before analysis to prevent potential interference; likewise, an optional ß-galactosidase treatment can be used to remove ß-galactooligosaccharides. Fructooligosaccharides or polydextrose do not generally interfere with the analysis. RESULTS: The method has been validated in a single laboratory on infant formula and adult nutritionals. The seven HMOs were spiked into eight matrixes at three or four spike levels, giving a total of 176 data points. Recoveries were in the range of 90.9-109% in all cases except at the lowest spike level in one matrix (elemental formula), where the LNT recovery was 113%, the LNnT recovery was 111%, and the 6'SL recovery was 121%. Relative repeatabilities (RSD(r)) were in the range of 0.1-4.2%. The performance is generally within the requirements outlined in the Standard Method Performance Requirements (SMPR®) published by AOAC INTERNATIONAL. CONCLUSIONS: The method developed is suitable for the determination of seven HMOs in infant formula and demonstrated good performance during single-laboratory validation. HIGHLIGHTS: A method has been developed that is suitable for the determination of seven HMOs in infant formula.
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Microbioma Gastrointestinal , Leche Humana , Adulto , Femenino , Lactante , Humanos , Fórmulas Infantiles , Oligosacáridos , Cromatografía LiquidaRESUMEN
BACKGROUND: ß-Galactooligosaccharides (GOS) are typically used in infant formula and adult nutritionals as a source of nondigestible oligosaccharides, which may bring beneficial effects through modulation of the gut microbiota. However, suitable methods for the determination of GOS in products with a high background of lactose do not exist. OBJECTIVE: The aim of this work was to develop a method suitable for the determination of GOS in infant formula and adult nutritionals and demonstrate suitability through single laboratory validation. METHODS: Reducing oligosaccharides are labeled with 2-aminobenzamide (2AB), separated by hydrophilic interaction LC, and determined assuming that all oligosaccharides give an equimolar response in the detector. The same sample is analyzed a second time after treatment with ß-galactosidase to remove GOS. The difference in the determined oligosaccharides between the two measurements will be the GOS content of the sample. The method was validated in a single laboratory on infant formula and adult nutritionals. RESULTS: Recoveries were in the range 91.5-102%, relative standards of deviation (RSDr) were in the range 0.7-5.99%, and one sample had an RSDr of 8.30%. Except for the one sample with an RSDr of 8.30%, the performance is within the requirements outlined in the Standard Method Performance Requirements, which specifies recoveries in the range 90-110% and RSDr of below 6%. CONCLUSIONS: The method is suitable for the determination of GOS in infant formula and adult nutritionals. HIGHLIGHTS: A method has been developed which is suitable for the determination of GOS in products with a high background concentration of lactose (infant fromula and adult nutritionals). The method does not require access to the GOS ingredient used for the production of the finished product. It is also possible to separately quantify the amount of GOS containing three or more monomeric units in order to support dietary fibre analysis.
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Fórmulas Infantiles , Laboratorios , Adulto , Alimentos Formulados , Humanos , Lactante , Fórmulas Infantiles/análisis , Lactosa , Oligosacáridos , Estándares de ReferenciaRESUMEN
BACKGROUND: Fructans are added to infant formula and adult nutritionals for their prebiotic effect. A method (AOAC 2016.14) was developed for their analysis which has already demonstrated excellent performance during single laboratory validation. OBJECTIVE: To determine repeatability and reproducibility of the method through a collaborative study. METHODS: Fourteen laboratories from 11 different countries enrolled for the study. Participants analyzed a practice sample, then 8 formula or adult nutritionals in blind duplicate. Results and any method modifications were reported to the study director. RESULTS: Twelve laboratories provided results on time for reporting. Precision results for five samples met the requirements of the Standard Method Performance Requirements (SMPR 2014.002), with RSDr ranging from 3.60 to 4.25% and RSDR ranging from 5.90 to 11.7%. The practice sample also met the requirements of SMPR 2014.002, with RSDr and RSDR of 2.53% and 6.70% respectively. Precision results for three test samples did not fully meet the SMPR, with RSDr ranging from 2.27 to 7.65% and RSDR ranging from 12.8 to 15.1%. After review, the AOAC Stakeholder Panel for Infant Formula and Adult Nutritional Expert Review Panel (SPIFAN ERP) concluded that the data presented mostly met the SMPR and hence recommended that the method to be advanced for adoption as an AOAC Final Action method. CONCLUSIONS: The method described in AOAC 2016.14 is suitable for the determination of fructans in infant formula and adult nutritionals.
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Fructanos , Fórmulas Infantiles , Adulto , Aniones , Niño , Cromatografía , Alimentos Formulados/análisis , Humanos , Lactante , Fórmulas Infantiles/análisis , Reproducibilidad de los ResultadosRESUMEN
Until recently, only two AOAC Official MethodsSM have been available for the analysis of fructans: Method 997.08 and Method 999.03. Both are based on the analysis of the fructan component monosaccharides (glucose and fructose) after hydrolysis. The two methods have some limitations due to the strategies used for removing background interferences (such as from sucrose, α-glucooligosaccharides, and free sugars). The method described in this paper has been developed to overcome those limitations. The method is largely based on Method 999.03 and uses combined enzymatic and SPE steps to remove the interfering components without impacting the final analytical result. The method has been validated in two laboratories on infant formula and adult nutritionals. Recoveries were in the range of 86-119%, with most being in the range of 91-104%. RSDr values were in the range of 0.7-2.6%, with one exception when the fructan concentration was close to the LOQ, resulting in an RSDr of 8.9%. The performance is generally within the requirements outlined in the AOAC Standard Method Performance Requirements (SMPR® 2014.002), which specifies recoveries in the range of 90-110% and RSDr values below 6%.
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Alimentos Formulados/análisis , Fructanos/análisis , Fórmulas Infantiles/análisis , Hidrólisis , LaboratoriosRESUMEN
In 2007, Martin et al. developed a method for the analysis of sialic acids by HPLC following 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatisation (Martín et al., Anal Bioanal Chem 387:2943-2949, 2007). Within the article, the authors noted that lactose interfered with the analysis, giving erroneously high results when lactose-containing products were analysed. Such an observation is important when analysing milk-based products, yet was contradictory to the observations of Nakamura et al. (Chem Pharm Bull 35(2):687-692, 1987) who demonstrated that DMB was specific for α-keto acids and did not react with simple sugars such as glucose or lactose. In order to clarify the situation, this phenomenon was investigated and it was confirmed that lactose does not interfere with the analysis. However, it was found that most commercial preparations of lactose do contain small amounts of sialic acids, either as the free monosaccharide or bound to lactose in the form of 3'- and 6'-sialyllactose.
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Lactosa/química , Fenilendiaminas/química , Ácidos Siálicos/análisis , Cromatografía Líquida de Alta Presión , HidrólisisRESUMEN
Sialic acids are becoming recognized as important components of milk-based products for infants and young children. As such, many companies now label the sialic acid content of their products. To control the labeling, suitable methods are required for this analysis. The objective of this work was to set up a rapid and sensitive method for the determination of the two most commonly occurring sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), using high-performance liquid chromatography (HPLC). The sialic acids were released from their parent oligosaccharides, glycoproteins, or glycolipids by mild acid hydrolysis using formic acid. They were then derivatized using 1,2-diamino-4,5-methylenedioxybenzene (DMB) and subsequently separated on a Zorbax SB-Aq Rapid Resolution column in less than 2 min. The method developed was validated on various milk-based products and ingredients containing sialic acid at levels from 0.3 to 900 mg/100 g. Spiking experiments indicate that the sialic acid recoveries ranged from 87% to 108%. The expanded measurement uncertainty was typically below 15% for Neu5Gc and typically below 10% for Neu5Ac or the sum of the sialic acids, with a few exceptions. The proposed method is fast, specific, and easy to set up for compliance analysis in a routine laboratory.
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Cromatografía Líquida de Alta Presión/métodos , Leche/química , Ácido N-Acetilneuramínico/análisis , Animales , Bovinos , Humanos , Leche Humana/química , Ácidos Neuramínicos/análisis , Fenilendiaminas/químicaRESUMEN
Recently there has been some debate regarding the presence and associated health risk of low molecular weight carrageenan in foodstuffs. Unfortunately measurement of the low molecular weight tail (LMT) of food-grade carrageenans (defined here as the carrageenan having relative molecular mass (Mr) below 50,000) is not trivial, largely due to its low abundance. So far methods employing light scattering have been unsuccessful in producing reproducible results, probably due to the poor detector response at low masses. In this work a method based on high performance size exclusion chromatography coupled to a refractive index detector (HPSEC-RI) has been used for the measurement of the LMT in food-grade carrageenan ingredients and in a carrageenan-containing finished product (a jelly). Over the course of half a year, 19 measurements were made on a reference carrageenan; the results demonstrated that the method had excellent reproducibility. Applied to a number of different carrageenan ingredients, it was found that, in general, the LMT represents less than 8% of the total carrageenan in ingredients, and under the correct conditions increases little during food processing. The data also indicated that pH appears to be a critical factor during food processing and pH levels below 4.0 should be avoided.