6. AOACSPIFANMethods-2018Awards

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bound as phosphatidylinositol data. The method was validated for the quantitation of free myo-inositol and myo-inositol from phosphatidylinositol in infant, pediatric, and adult nutritionals. Repeatability was determined from duplicate analyses performed on multiple days. Accuracy was determined from spike recovery experiments (free myo-inositol and myo-inositol from phosphatidylinositol). Instrument LODs and LOQs were determined statistically from injections of low-level standards and by spiking samples with low levels of free myo-inositol. See Tables 2011.18 A – C for results of single- and multilaboratory studies supporting acceptance of the method. Caution : Refer to Material Safety Data Sheets (MSDS) of

Method

A few minor modifications were made to AOAC Official First Action 2011.18 before it was sent to the multilaboratory collaborative study participants. These changes included updating the pulsed amperometric detector (PAD) program and increasing the sodium hydroxide concentration from 750 mM to 1 M. The PAD program listed in AOAC 2011.18 , First Action, was the waveform that was used when the method was originally developed over 15 years ago using equipment that is now obsolete. After First Action status was granted to AOAC 2011.18 and before completion of the SPIFAN SLV and multilaboratory collaborative study, the obsolete PAD was replaced with a newer model, and the waveform listed in AOAC 2011.18 was updated based on the recommendations of the instrument manufacturer. The updated waveform was added to the multilaboratory collaborative study protocol and to AOAC 2011.18 , Final Action, and the previous waveform was removed. It should be noted that whenever changes are made to the PAD, the accuracy of the detector waveform should be verified by analyzing an SRM and performing free myo-inositol spike recovery experiments with an infant or adult formula containing hydrolyzed protein. It should also be noted that although the multilaboratory study protocol specified using a standard (not disposable) gold electrode, some participating laboratories used disposable gold working electrodes. The instruction to not use disposable electrodes was removed from the AOAC 2011.18 Final Action method. After completion of the multilaboratory collaborative study, the modifications noted above were incorporated in the Final Action method along with a few additional modifications based on study results and feedback from study participants and the ERP. For this reason, the method described below is slightly different than the Official Final Action method currently posted on the AOAC website.

chemicals prior to use and follow safe handling procedures and the suggested personal protective equipment. Chloroform is a hazardous chemical and should be handled in a fume hood. Perform the phosphatidylinositol bound myo-inositol extraction and SPE sample cleanup procedure in a fume hood.

A. Apparatus

(a)  Analytical balance. —Minimum weighing capacity of at least 0.0001 g. (b)  Centrifuge. (c)  Desiccator. (d)  N-evap. —With water bath (Organomation Associates, Inc., Berlin, MA) or equivalent.

(e)  Oven. —Capable of maintaining 120°C. (f )  pH meter. —With pH 4 and 7 buffers. (g)  Stir plate. —Multiposition with stir bars. (h)  Vacuum manifold. (i)  Vortex mixer.

(j)  HPLC system. —Corrosion-resistant components, including an autosampler, two isocratic pumps, 6-port switching valve, pulsed amperometry detector with a gold electrode and polyether ether ketone or Teflon 0.007–.01 in. id tubing.Autosampler capable of injecting 20 µL . (k)  Columns. —Dionex CarboPac MA1 (4 × 250 mm) P/N 44066, MA1 (4 × 50 mm) P/N 44067, and PA1 (4 × 50 mm) P/N 43096, or equivalent (www.thermofisher.com/dionex/). B. Materials (a)  Beakers. —Assorted sizes. (b)  Centrifuge tubes. —50 mL with Teflon-coated caps. (c)  Syringe filters. —Nylon, 0.45 and 0.2 μm. (d)  Filter paper. —Whatman 2 V or equivalent (www. whatman.com). (e)  Erlenmeyer flasks. —50 or 125 mL or equivalent. (f )  Volumetric flasks. —Assorted sizes. (i)  SPE cartridges. —Silica, 1 g (J.T. Baker Inc., Phillipsburg, NJ; P/N 7086-07, www.avantormaterials.com) or equivalent. (j)  Syringes. —1 mL disposable and 25 mL gas-tight glass with 4 in. stainless steel needles. (g)  Funnels. —Suitable for use with filter paper. (h)  Pipets. —Volumetric (Class A); assorted sizes.

AOAC Official Method 2011.18 Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant and Pediatric Formula and Adult Nutritionals Liquid Chromatography/Pulsed Amperometry with Column Switching

First Action 2011 Final Action 2014

ISO–AOAC METHOD

The LC method with electrochemical (pulsed amperometry) detection (PAD) allows for the quantitation of myo-inositol in infant, pediatric, and adult nutritional formulas. The concentration of myo-inositol is calculated by comparison with standards of known concentration. Myo-inositol, as defined by AOAC Standard Method Performance Requirement (SMPR ® ) 2011.007 (free and bound as phosphatidylinositol), can be calculated by adding the free myo-inositol and myo-inositol