Inositol is an important nutrient present naturally in human milk and cow’s milk but in a lesser quantity. It exists predominantly in the free myo-inositol form. myo-Inositol is found in large amounts in plant materials such as soy beans but is in the bound form, predominantly as polyphosphate and in a lesser extent as phosphatidylinositol.
The levels of inositol in infant formula are regulated. CODEX1 defines lower and upper levels of inositol at 1 and 9.5 mg/100 kJ. In the USA there is no prescribed limits2 for inositol in dairy-based IF. The minimum amount of inositol in non-dairy based IF according to the Code of Federal Regulations (CFR) is 4 mg/100 kcal (~1 mg/100 kJ) and the upper limit is not regulated. The levels of inositol in IF in Australia and New Zealand are regulated by the FSANZ 2.9.1 and in China by the GB 10765-2010 Food Standards, Table 1.
There are nine isomers of inositol C6H12O6 with the myo-inositol form being the most abundant and physiologically relevant. The structures of the inositol isomers are presented in the Fig. 1. There is limited data on inositol levels in dairy products. The most substantial survey was done in Japan3. The content of total myo-inositol in human milk was reported as 32.7±15.2 in colostrum, 17.8±1.9 in transitional milk, and 14.9±3.1 mg/100 mL in mature milk. In cow’s milk, it was 10.6±1.0, 7.0±1.1, and 4.1±1.0 mg/100 mL, respectively. Levels of lipid bound myo-inositol in human and cow’s milk were 0.22±0.09 and 0.36±0.10 mg/100 mL, respectively. A small amount of scyllo-inositol was found in both human and cow's milk, while D-chiro-inositol was not found in either. The percent of lipid bound to total myo-inositol in cow’s milk reported was 6.3±1.3%.
There are several testing methods for inositol - GC/FID4,5 LC6,7,8,9 anion exchange chromatography10 and microbiological.11,12 The performance requirements set by the Association of Analytical Communities (AOAC International) with respect to inositol methods describe the determination of the free myo-inositol and phosphatidylinositol, but exclude methyl ethers, glycosides, phosphorylated forms and phytate in all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). In this report we present results of a LCMSMS method for the determination of free and bound myo-inositol in Australia- and EU-originated commercial IF samples.
All reagents were of analytical grade unless otherwise specified. Solvents and reagents for UPLC mobile phases were of LCMS grade. Water for UPLC mobile phases was with TOC <2 ppb and resistivity > 16MΩ·cm, double purified on Integra, Siemens of Barsbüttel, Germany from institute water produced by Millipore, Billerica, MA, USA. Acetonitrile and methanol were from Fisher Scientific and the hydrochloric acid was from Merck KGaA of Darmstadt, Germany. The reference material of myo-inositol cat. No. I5125-50G was from Sigma-Aldrich of St. Louis, MO, USA. myo-Inositol-D6 was from TRC Toronto, Canada.
The Acquity H-class UPLC instrument coupled with Xevo TQ MS mass spectrometer were from Waters of Milford, MA, USA. The chromatographic column used was Acquity UPLC BEH Amide, 2.1 mm × 100 mm × 1.7 μm also from Waters.
Dairy-based IF dry powder products were purchased from different retail outlets of the metropolitan Melbourne during June-August 2014. Before analysis samples were stored according to the label requirements. Free and bound inositols were extracted according to AOAC official methods 2012.1213 and bound inositols were hydrolyzed to the free form according to 2011.18.14 Both AOAC protocols were followed precisely in order to have consistency with data of other laboratories. Extracts were analyzed by UPLC-MS/MS. myo-inositol-D6 (100 μL, 5.0 μg/mL) was added to 200 μL of the filtered sample which was further diluted with 1.7 mL of acetonitrile to facilitate HILIC separation. The injection volume was 10 μL. The analytical system was calibrated from 0.25 to 5 ppm using six standards in solvent. Table 2 shows the UPLC gradient used.
Quality assurance and quality control was performed by analysis of reagent blanks, duplicate samples and SRM 1849a, NIST USA.
The extraction of free and bound inositol was based on official AOAC procedures, and the LCMS determination was developed and validated in-house based on publications of J.-H. Ahn15 et al. and K.-Y. Leung16et al. The run time of the chromatographic separation is 8 min, Fig. 2.
Reference material, SRM1849a with stated myo-inositol level of 405.2 ±7.6 mg/kg was repetitively analyzed to determine accuracy and precision of the assay, the results are listed in Table 3.
Table 4 shows the result of the survey. The results showed a mean value of 43.5 mg/100 g for Australian IF, excluding one outlier (IF21). Thermo17 recently published brief results and method. The microbiological and liquid chromatography methods of total inositol determination have been compared during analysis of dietetic milk powders. Several authors reported systematically higher concentrations of total inositol determined by the LC/PAD technique (420 to 1340 mg/kg) than by the microbiological procedure (270 to 1120 mg/kg). The difference between values given by two methods is significant considering mild extraction conditions applied for free inositol determination of both methods. To the contrary, Indyk and Woollard reported18 similar amounts of free inositol in skimmed milk powders, whole-milk powders and milk infant formulas determined by HPLC and microbiological procedures. Soy beans are known to contain up to 0.9 % of pinitol, a methylated form of D-chiro-inositol that may be separated19,20 from D-chiro- and myo-inositol by gas chromatography. The high fraction of free to total inositol in samples of soya bean meal determined by the chromatographic procedure was not confirmed in the microbiological assay. These findings suggest a stereo selectivity of the latter. Pinitol and D-chiro-inositol, the predominant inositols of soya bean husk, most probably coeluted from the analytical column with myo-inositol while microbiological assay determined only the myo-inositol. The use of the better chromatographic resolution of UPLC and selectivity of mass spectrometry is critical to reducing these errors.
Following the protocols of NATA’s Technical Note 33,21 the MU for myo-inositol results was determined to be 25 mg/kg (7.7%) at the levels present in infant formula. The inositol of lipid fraction was found to be about 10% of the total myo-inositol content.
A UPLC-MS/MS method was developed for the analysis of free myo-inositol which was single-laboratory validated and used for the survey of dairy-based IF samples originated in Australia and EU. The mean±SD of free myo-inositol of 27 IF samples originated in Australia was 43.5±3.6 mg/100 g (excluding one outlier) in dry powders.