Gill et al.: Journal of aoaC international Vol. 95, no. 3, 2012 1 Analysis of 5′-Mononucleotides in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography: First Action 2011.20 Brendon d. Gill Fonterra Co-operative Group Ltd, PO Box 7, Waitoa 3380, New Zealand University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand Harvey e. indyk, Maureen C. kuMar , and natHan k. SievwriGHt Fonterra Co-operative Group Ltd, PO Box 7, Waitoa 3380, New Zealand Merilyn Manley-HarriS University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand dawn dowell AOAC INTERNATIONAL, 481 N. Frederick Ave, Suite 500, Gaithersburg, MD 20877-2417 Submitted for publication February 8, 2012. The method was approved by the Expert Review Panel on Infant Formula and Adult Nutritionals as First Action. See “Standards News,” (2011) Inside Laboratory Management, September/October issue. The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) 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. 1 Corresponding author’s e-mail: Brendon.Gill@fonterra.com DOI: 10.5740/jaoacint.CS2011_20 INFANT FORMULA AND ADULT NUTRITIONALS A method for the routine determination of 5′-mononucleotides (uridine 5′-monophosphate, inosine 5′-monophosphate, adenosine 5′-monophosphate, guanosine 5′-monophosphate, and cytidine 5′-monophosphate) in infant formula and adult nutritionals is described. After sample dissolution and addition of internal standard, potential interferences were removed by anion-exchange SPE followed by HPLC-UV analysis. Single-laboratory validation performance parameters include recovery (92–101%) and repeatability (1.0–2.3% RSD). The method was approved for Official First Action status by an AOAC expert review panel. N ucleotides are compounds of critical importance to cellular function, and although not essential dietary nutrients, it has been demonstrated that supplementation of pediatric formulas with nucleotides is of benefit in neonatal nutrition. The described method was developed to provide an accurate, rapid, and robust technique for the routine compliance testing of uridine 5′-monophosphate (UMP), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP), and cytidine 5′-monophosphate (CMP) in infant formula and adult/pediatric nutritional formula, and was recently reported (1). In September 2011, the method was reviewed by an AOAC expert review panel and, based on the published single- laboratory validation (SLV) data as compared with the standard method performance requirements (AOAC SMPR 2011.008; 2) set by the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN), it was approved for Official First Action status as AOAC Official Method SM 2011.20. AOAC Official Method 2011.20 5′-Mononucleotides in Infant Formula and Adult/Pediatric Nutritional Formula Liquid Chromatography First Action 2011 (Applicable to the determination of 5′-mononucleotides in infant formula and adult/pediatric nutritional formula.) Caution: Refer to the material safety data sheets for all chemicals prior to use. Use all appropriate personal protective equipment, and follow good laboratory practices. A. Principle Sample is dissolved in high-salt solution to inhibit protein and fat interactions. The 5′-mononucleotides—uridine 5′-monophosphate (UMP), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP), and cytidine 5′-phosphate (CMP)— are separated from the sample matrix by strong-anion exchange solid-phase extraction (SPE), followed by chromatographic analysis using a C 18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique using thymidine 5′-monophosphate (TMP). B. Apparatus (a) HPLC system.—Equipped with pump, sample injector unit with a 50 μL injection loop, degasser unit, column oven, and photodiode array detector. (b) C 18 column.—Gemini C 18 , 5 μm, 4.6 × 250 mm (Phenomenex, Torrance, CA). (c) Spectrophotometer.—Capable of digital readout to 3 decimal places. (d) pH meter. (e) Polypropylene centrifuge tubes.—50 mL. (f) Disposable syringes.—3 mL.