6. AOACSPIFANMethods-2018Awards

12

Jing et al.: J ournal of AOAC I nternational V ol. 98, N o . 5, 2015  1395

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Free and Total Carnitine and Choline in Infant Formulas and Adult Nutritional Products by UPLC/MS/MS: Single-Laboratory Validation, First Action 2014.04 Wei Jing, Joseph J. Thompson 1 , Wesley A. Jacobs, and Louis M. Salvati Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219

the present submission. In September 2012, SMPR 2012.010 was developed and approved for carnitine. Abbott Nutrition submitted AOAC 2012.17 , a carnitine-only LC/MS-based method (1) in response to the Call for Methods, and this was the only carnitine entry for some time. In 2013, the Expert Review Panel (ERP) clarified that, for a total choline method, it was not necessary to prove recovery of the individual inherent choline species such as phosphocholine and sphingomyelin; therefore, the choline method could be made much simpler and combined with carnitine. Carnitine was added as an analyte, similar to Andrieux et al. (2), but now using MS/MS for added specificity. Fu et al. (3) reported a choline single-laboratory validation (SLV) in infant formula using MS/MS but did not look at simultaneous carnitine measurement. Microwave digestion was added to speed up the typical 3 h digestion, similar to Phillips and Sander (4), but again the present work utilizes MS/MS instead of single quadrupole technology. The combined method was substantially different than either AOAC 2012.19 or 2012.17 , and so both methods were withdrawn from the SPIFAN process. The present submission of AOAC 2014.04 has a full set of SLV data with the method in its final form. It is submitted in response to a second Call for Methods for carnitine, but full SLV data are also presented for choline in case the ERP also wants to advance it for this use. AOAC Official Method 2014.04 Simultaneous Determination of Choline/Carnitine in Infant Formulas and Adult Nutritional Products A. Principle Reconstituted test sample is weighed into a microwave reaction vessel. Microwave heating accelerates an acidic hydrolysis process to release bound choline. A subsequent alkaline degradation is performed to release L-carnitine from inherent acylcarnitines. Choline and L-carnitine can be determined quantitatively in nutritional products and raw materials by hydrophilic interaction ultra-performance LC with tandem mass spectrometry (HILIC-UPLC/MS/MS). B. Apparatus and Materials ( a )  Column. —Acquity UPLC ethylene bridged hybrid HILIC LC/MS/MS First Action 2014

Received April 26, 2015. Accepted by SG April 24, 2015. The method was approved by the AOAC Expert Review Panel for Infant Formula and Adult Nutritionals as First Action. The AOAC Expert Review Panel for SPIFAN Nutrient Methods invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. 1 Corresponding author’s e-mail: joseph.thompson@abbott.com DOI: 10.5740/jaoacint.15102 for Methods to find suitable test methods for global dispute resolution purposes for nutritional products. Choline was considered early in the process. Standard Method Performance Requirement (SMPR ® ) 2012.013 was developed and approved in 2012, describing the performance requirements needed for a choline dispute-resolution method. Three methods were chosen for consideration from those submitted. Abbott Nutrition submitted AOAC 2012.19 , a UPLC-MS/MS method similar to A single-laboratory validation (SLV) has been performed for a method that simultaneously determines choline and carnitine in nutritional products by ultra performance LC (UPLC)/MS/MS. All 11 matrixes from the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) were tested. Depending on the sample preparation, either the added (free, with a water dilution and filtering) or total (after microwave digestion at 120 ° C in nitric acid and subsequent neutralization with ammonia) species can be detected. For nonmilk containing products, the total carnitine is almost always equal to the free carnitine. A substantial difference was noted between free and total choline in all products. All Standard Method Performance Requirements for carnitine and choline have been met. This report summarizes the material sent to the AOAC Expert Review Panel for SPIFAN nutrient methods for the review of this method, as well as some additional data from an internal validation. The method was granted AOAC First Action status for carnitine in 2014 (2014.04), but the choline data are also being presented. A comparison of choline results to those from other AOAC methods is given. O ver the last 3 years, as part of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) project, AOAC has issued regular Calls