Plasma and urinary inositol isomer profiles measured by UHPLC-MS/MS reveal differences in scyllo-inositol levels between non-pregnant and pregnant women.

Previous studies support that myo- and D-chiro-inositol isomers are promising bioactives for the treatment of women with polycystic ovary syndrome and for lowering the risk of gestational diabetes mellitus in pregnant women, whereas scyllo-inositol may have some benefits for neurological disorders (e.g., Alzheimer's disease). Though potentially useful to better understand inositol isomer metabolism and study their role in health and disease, routine analysis of inositol isomers in plasma and urine with a single analytical method is not yet feasible due to the lack of a suitable analytical assay. To address this, we developed and validated a robust ultra-high-performance-liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of inositol isomers in plasma and urine. This method resolves seven inositol isomers with accurate quantification of total chiro- (D and L enantiomers), myo-, and scyllo-inositols and is semi-quantitative for neo-inositol. For urine and plasma myo-inositol, the method repeatability and intermediate reproducibility were below 6% and 8%, respectively. Then, for both chiro- and scyllo-inositols, repeatability and intermediate reproducibility were below 10% and 14%, respectively. A pilot study was carried out to quantify and compare the pattern of inositol isomers in urine and plasma of non-pregnant and pregnant women and showed for the first time that urinary myo- and scyllo-inositol concentrations were significantly higher for women in the third trimester of pregnancy compared with non-pregnant women. These findings warrant further research to understand the biological significance of the observed differences in inositol profiles and suggest a potential role of scyllo-inositol.Graphical abstract Plasma and urinary inositol isomer profiles measured by UHPLC-MS/MS reveal differences in scyllo-inositol levels between non-pregnant and pregnant women.

Determination of Seven Human Milk Oligosaccharides (HMOs) in Infant Formula and Adult Nutritionals: First Action 2022.07.

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.

Determination of ß-Galactooligosaccharides (GOS) in Infant Formula and Adult Nutritionals: Single-Laboratory Validation, First Action 2021.01.

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.

On-line cleanup for 2-aminobenzamide-labeled oligosaccharides.

Analysis of 2-aminobenzamide-labeled oligosaccharides requires removal of excess labeling reagents before chromatography. Manual cleanup is time-consuming and not optimal for routine analysis, so an on-line solid-phase extraction was developed. Labeled oligosaccharides are trapped on an amide phase in a small guard column, the excess reagents are washed away, and then the sample is transferred to the analytical column for analysis. The on-line protocol shortened the sample preparation time and has been applied for the analysis of oligosaccharides and N-glycans released from glycoproteins.

Development and single-laboratory validation of a method for the determination of total fructans in infant formula.

A number of methods have previously been developed for the determination of fructans in food products, resulting in the frequently used AOAC Official Methods 997.08 and 999.03. Method 997.08 has a lower LOQ, but the accuracy may suffer if significant quantities of sucrose are present. Method 999.03 is less affected by sucrose, but the LOQ is not as low as that of the other method. In this work we have combined these two methods, using the sample preparation steps of AOAC Method 999.03 to remove sucrose and other free sugars, and the detection procedure of AOAC Method 997.08 to achieve a low LOQ. The resulting new method achieved an LOQ of 0.13 g/100 g with powdered products (using 1 g sample weight) and recoveries in the range of 83-103% in the presence of sucrose at 12 g/100 g. Expanded measurement uncertainties were calculated and ranged from +/-17.4% at oligofructose and fructooligosaccharide (FOS) concentrations of around 0.2 g/100 g to -/+21.0% at FOS concentrations of 0.6 g/100 g.

Quantitative determination of non-lactose milk oligosaccharides.

A method for the determination of non-lactose oligosaccharides (NLO) in milk using liquid chromatography has been developed. Oligosaccharides were labelled with a fluorescent tag, 2-aminobenzamide (2AB), and were identified by comparison of their retention times to those of oligosaccharide standards, their mass (as measured by mass spectrometry) and their fragmentation patterns in the mass spectrometer. The concentrations of the NLO in milk have been determined using 2 different approaches: (1) by preparing a calibration curve using genuine standards of each oligosaccharide. (2) by preparing a calibration curve using maltotriose as a universal standard for all NLO, and assuming all 2AB labelled oligosaccharides give an equimolar response in the detector. The accuracy of the method was assessed by spike-recovery experiments. Using genuine NLO standards for calibration, recoveries were in the range 96-114%. Using maltotriose as a universal calibrant, recoveries were in the range 86-120%. Method precision was assessed by determining the relative standard deviation of the results under repeatability (RSD(r)) and intermediate reproducibility (RSD(iR)) conditions. In most cases RSD(r) and RSD(iR) were below 5% irrespective of calibration method, but increased when NLO levels were close to LoQ.

Temporal Change of the Content of 10 Oligosaccharides in the Milk of Chinese Urban Mothers.

Breastfed infants tend to be less prone to infections and may have improved cognitive benefits compared to formula-fed infants. Human milk oligosaccharides (HMO) are the third most abundant component of human milk, but are absent from formulae. They may be partially responsible for the benefits of breastfeeding. In this cross-sectional observational study, the HMO composition of milk from Chinese mothers was studied to determine the impact of stage of lactation, mode of delivery and geographical location. The content of 10 HMO was measured by HPLC in 446 milk samples from mothers living in three different cities in China. Around 21% of the samples contained levels of 2'-fucosyllactose (2'-FL) below the limit of quantification, which is similar to the frequency of fucosyltransferase-2 non-secretors in other populations, but 2'-FL was detected in all samples. Levels of most of the HMO studied decreased during the course of lactation, but the level of 3-fucosyllactose increased. Levels of 2'-FL and 3-fucosyllactose seem to be strongly correlated, suggesting some sort of mechanism for co-regulation. Levels of 6'-sialyllactose were higher than those of 3'-sialyllactose at early stages of lactation, but beyond 2-4 months, 3'-sialyllactose was predominant. Neither mode of delivery nor geographical location had any impact on HMO composition.

Determination of ß -Galactooligosaccharides by Liquid Chromatography.

Beta-galactooligosaccharides (GOS) are oligosaccharides normally produced industrially by transgalactosylation of lactose. They are also present naturally in the milk of many animals including humans and cows. GOS are thought to be good for health, being potential prebiotic fibres, and are increasingly added to food products. In order to control the GOS content of products, the AOAC official method 2001.02 was developed. However, the method has some shortcomings and in particular is unsuited to the analysis of products containing high levels of lactose such as infant formula. To overcome this problem, we developed a new method for application to infant formula and tested it on various GOS ingredients as well as infant formulae. When applied to GOS ingredients the results of the new method compare well with those of the official AOAC method, typically giving results in the range 90-110% of those of the official method and having an expanded measurement uncertainty of less than 15%. For three products, the results were outside this range (recoveries of 80-120% and expended measurement uncertainties up to 20%). When applied to the analysis of infant formula, recoveries were in the range of 92-102% and the expanded measurement uncertainties were between 4.2 and 11%.

Milk oligosaccharides over time of lactation from different dog breeds.

The partnership of humans and dogs goes back to over 10'000 years, yet relatively little is known about a dog's first extra-uterine nutrition particularly when it comes to milk oligosaccharides. We set out to identify and quantify milk oligosaccharides over the course of lactation from different dog breeds (Labrador retriever, Schnauzer and 3 Alaskan husky crossbreeds). To this end, 2 different chromatographic methods with fluorescence and mass spectrometry detection were developed and one was validated for quantification. Besides lactose and lactose-sulphate, we identified 2 different trisaccharides composed of 3 hexose units, 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 2'fucosyllactose (2'FL), and a tetrasaccharide composed of 2 hexoses, an N-acetylhexosamine and a deoxyhexose. 3'SL was present at the highest levels in milk of all dog breeds starting at around 7.5 g/L and dropping to about 1.5 g/L in the first 10 days of lactation. 6'SL was about 10 times less abundant and 2'FL and the tetrasaccharide had rather varying levels in the milk of the different breeds with the tetrasaccharide only detectable in the Alaskan husky crossbreeds. The longitudinal and quantitative data of milk oligosaccharides from different dog breeds are an important basis to further our understanding on their specific biological roles and also on the specific nutritional requirements of lactating puppies.