Determination of Myo-Inositol - AOAC International

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4 Schimpf et al.: Journal of aoac international Vol. 95, no. 4, 2012 Table 1. Free myo-inositol validation data— intermediate precision, mg/100 g HPLC method Day Product type 1 2 3 Hypoallergenic infant formula RTF 8.11 8.06 8.17 n 3 3 3 RSD, % 3.64 5.01 3.40 Soy-based infant formula powder 31.1 31.8 30.9 n 3 3 3 RSD, % 2.49 0.327 1.35 Specialty infant formula RTF 19.7 18.9 19.0 n 3 3 3 RSD, % 1.60 0.976 1.73 Milk-based infant formula RTF 4.36 4.43 4.38 n 3 3 3 RSD, % 1.91 4.26 0.784 Very low-fat infant formula powder 125 126 122 n 3 3 3 RSD, % 1.46 3.37 1.19 Free amino acid-based infant formula powder 65.5 65.0 64.9 n 3 3 3 RSD, % 1.56 1.20 2.78 Pediatric nutritional beverage 10.3 10.5 11.1 n 3 3 3 RSD, % 2.02 1.00 4.19 Adult nutritional beverage 91.6 92.3 NA a n 4 4 RSD, % 1.35 1.22 a NA = No data generated. Table 3. Free myo-inositol validation data—accuracy data Product type HPLC method a , mg/100 g Table 2. Free myo-inositol validation data— intermediate precision, mg/100 g Product type n Avg. Hypoallergenic infant formula RTF 9 a Soy-based infant formula powder 9 a Specialty infant formula RTF 9 a Milk-based infant formula RTF 20 b Very low-fat infant formula powder 9 a Free amino acid-based infant formula powder 9 a Pediatric nutritional beverage 9 a Adult nutritional beverage 8 c a Intralaboratory triplicate analyses on 3 days. HPLC method RSD, % 8.13 3.58 31.3 1.89 19.2 2.22 4.39 2.53 125.0 2.72 65.0 1.82 10 4.25 91.9 1.25 b Intralaboratory single, duplicate, or triplicate analyses on 9 days. c Intralaboratory quadruplicate analyses on 2 days. Thoroughly mix or stir products prior to sampling. For liquid products, accurately weigh 0.5 to 5 g (±10%) of product into a 100 mL volumetric flask and record the weight to the nearest 0.0001 g. For powdered products that do not require reconstitution, accurately weigh 0.25 to 1.5 g powder into a 100 mL volumetric flask and record the weight to the nearest 0.0001 g. Add approximately 10 to 15 mL laboratory water to the volumetric and swirl or stir to completely dissolve the powder. For powdered products that are not homogeneous at the subgram level, reconstitute following the product label instructions and accurately weigh 0.5 to 5 g reconstituted product into a 100 mL volumetric flask. Record the weight to the nearest 0.0001 g. Add enough 0.5% hydrochloric acid to each sample to adjust the sample pH to 4.5 ± 0.2 and swirl to mix. Allow the volumetric flasks to set a minimum of 2 min. Dilute to volume with laboratory water and mix well. Filter samples through Whatman 2V filter paper into 125 mL Erlenmeyer flasks. (Note: Although some samples will filter cloudy, the filtrates can still be used.) Filter an aliquot of sample filtrate through a 0.45 µm syringe filter into an autosampler vial. (b) Sample preparation for phosphatidylinositol determinations.—(1) Extraction.—Weigh 4 g (±10%) liquid Microbiological method b , mg/100 g HPLC/micro, % Hypoallergenic infant formula RTF 8.13 7.4 110 Soy-based infant formula powder 31.3 37.0 84.5 Specialty infant formula RTF 19.2 19.0 101 Milk-based infant formula RTF 4.39 3.7 119 Very low-fat infant formula powder 125.0 140.0 89.2 Free amino acid-based infant formula powder 65.0 62.0 105 Pediatric nutritional beverage 10.6 11.0 96.6 Adult nutritional beverage 91.9 83.0 111 a Multiple intralaboratory analyses on multiple days. b Single analyses.
or reconstituted powder product or 1 g (±10%) homogeneous powder into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Add 10 mL methanol and stir for at least 20 min. Add 20 mL chloroform and stir for at least 5 min. If large clumps form when chloroform is added, cap tube and shake well to mix sample. Add 5 mL 6% metaphosphoric acid and 1 mL 1 N NaCl and mix well. Centrifuge. Remove the bottom chloroform layer and evaporate with nitrogen in a 60°C water bath. (2) Sample cleanup.—Condition a 1 g silica SPE cartridge with 6 mL hexane. Dissolve residue in bottom of centrifuge tube in 1 mL chloroform:methanol (2:1). Quantitatively transfer dissolved residue to the conditioned silica SPE cartridge. Rinse SPE cartridge with 3 mL hexane:diethyl ether (80:20) and discard the eluant. Rinse SPE cartridge with 3 mL hexane:diethyl ether (50:50), 4 mL methanol, and 4 mL methanol:chloroform:water (75:15:10) and collect all eluants in a single 50 mL centrifuge tube. Evaporate eluants collected from SPE cartridge with nitrogen in a 60°C water bath. (3) Hydrolysis.—Add 40 µL concentrated acetic acid and 2 mL concentrated hydrochloric acid to residue in centrifuge tube from the sample cleanup step. Tightly cap tube. Heat in a 110°C oven for 2 h. Cool. Dilute mixture with approximately 10 mL H 2O and then add 1.25 mL 50% (w/w) sodium hydroxide. Transfer sample to a 50 mL volumetric flask and dilute to volume with water. Filter an aliquot of sample filtrate through a 0.45 µm syringe filter into an autosampler vial. (c) HPLC analysis.—(1) See Tables 2011.18A and B for instrument operating conditions and PAD settings, respectively. (2) Instrument startup.—The HPLC system should be located in an area where temperature fluctuations will be minimal throughout the run. Prepare mobile phases. Helium sparge mobile phases Schimpf et al.: Journal of aoac international Vol. 95, no. 4, 2012 5 and/or pressurize mobile phase reservoirs. If necessary, clean and polish the gold working electrode. Turn on the detector and pump mobile phase over the columns at a flow rate of 0.40 mL/min for at least ½ h to equilibrate the system. Verify that the detector is stable before beginning an analysis. Inject 20 μL of the most concentrated standard at least 5 times and note the peak areas or heights. If the system is equilibrated, the RSD of the peak areas or heights of the last three standard injections should be ≤2.0%. (3) Standard and sample analysis.—Once the system has equilibrated, inject one standard at each concentration. After a set of standards has been injected, a control sample and up to 14 samples can be injected before another set of standards should be injected. (4) System shutdown.—After all samples and standards have been analyzed, inject one vial of water to clean out the autosampler needle and tubing. Store the analytical columns in mobile phase [0.12% (30 mM) sodium hydroxide]. Turn off the electrochemical cell. Flush the pump heads with water to remove sodium hydroxide. F. Calculations Before calculating myo-inositol concentrations in samples, compare the myo-inositol standard peaks with the myo-inositol sample peaks and confirm that there are not any interfering compounds and that the myo-inositol sample peak areas or heights are within the range of the myo-inositol standard peak areas or heights. See Figure 2011.18C for examples of typical chromatograms. The concentration of myo-inositol cannot be calculated if there are interferences or if the separation is poor. The myo-inositol retention time should be 11 to 13 min (depending on the individual analytical column). (a) Concentration of working standards. Table 4. Free myo-inositol and myo-inositol from phosphatidylinositol data, mg/100 g Product type n SRM 1849 9 a Myo-inositol from phosphatidylinositol Free myo-inositol Total (free + phosphatidyl bound) myo-inositol Myo-inositol from phosphatidylinositol, % RSD, % 0.909 41.3 42.2 2.15 5.62 Hypoallergenic infant formula RTF Not detected 8.87 8.87 0 Hypoallergenic infant formula powder Not detected 69.1 69.1 0 Milk-based infant formula RTF 3 b 0.428 3.83 4.26 10.1 1.91 Milk-based infant formula powder 3 b 2.81 31.4 34.2 8.21 1.09 Soy-based infant formula RTF 3 b 0.995 4.05 5.05 19.7 1.46 Soy-based infant formula powder 3 b 11.3 31.9 43.2 26.1 2.05 Specialty infant formula RTF 3 b 0.416 27.5 27.9 1.49 0.60 Specialty infant formula powder 3 b 1.67 mg/100 g 208.0 209.7 0.80 2.69 Lactose-free infant formula RTF 3 b 0.333 3.90 4.23 7.86 0.76 Lactose-free infant formula powder 3 b 0.258 31.7 32.0 0.81 8.12 Specialty infant formula powder 3 b 1.51 mg/100 g 132.0 133.5 1.13 2.13 Rice starch infant formula RTF 3 b 0.318 4.13 4.45 7.15 5.97 Rice starch infant formula powder 3 b 0.233 33.1 33.3 0.70 4.72 Milk-based infant formula RTF 3 b 0.672 4.60 5.27 12.7 8.92 Milk-based infant formula powder 3 b 1.75 37.2 39.0 4.50 4.20 Pediatric nutritional beverage 3 b 0.751 12.4 13.2 5.71 2.08 a Intralaboratory triplicate analyses on 3 days. b Intralaboratory triplicate analyses on 1 day.
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Page 3: Table 2011.18B. PAD settings with g

Determination of Myo-Inositol - AOAC International

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