6. AOACSPIFANMethods-2018Awards

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D RAHER & W HITE : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . nnn , 2017 1

INFANT FORMULA AND ADULT NUTRITIONALS

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis: Single-Laboratory Validation, First Action 2017.03

J ONATHAN D RAHER and N ORMAN W HITE Abbott Nutrition, 3300 Stelzer Rd, Building RP4-3, Columbus, OH 43219

A method for tryptophan (Trp) analysis designed to comply with AOAC Standard Method Performance Requirement 2014.013 is described. Unlike AOAC 988.15, which uses alkaline hydrolysis, this method uses enzymatic hydrolysis to release the Trp from the intact protein. The method achieves an LOQ of 0.18 mg/100 g Trp on a ready-to-feed basis with mean recoveries ranging from 93.8 to 104.9%. Repeatability ranged from 0.2 to 5.0%. Intermediate precision ranged from 1.0 to 6.9%. The analytical range was determined to be 0.18 – 300 mg/100 g, with linearity over eight calibration standard levels giving an average deviation from theoretical levels of 0.3%. No single calibration point had a deviation of >5.0%. Two standard reference materials (SRMs 1849a and 927e) were analyzed, and the average deviation from the certified value was 98.5% for SRM 1849a and 101.2% for SRM 927e. Sample preparation is very similar to existing methods in terms of time and complexity. The use of an internal standard reduces laboratory error and allows for reproducible results. T ryptophan (Trp) is an essential amino acid and plays a critical role in the metabolism of mammals. The human body cannot synthesize Trp and is dependent on dietary sources of Trp. Trp is found in foods that naturally contain protein in dietetic and fortified food products and specific pharmaceutical formulations. Therefore, Trp is found in the content of various food groups, including milk and dairy products, such as infant formula and adult/pediatric nutritional formula. Food-processing and preparation practices include heating processes that may lead to oxidative degradation and cross-linking between protein molecules and decrease the bioavailability of Trp (1 – 3). Accurate Trp determination is Received June 21, 2017. Accepted by SG August 17, 2017. This method was approved by the AOAC Expert Review Panel for Nutrient Methods as First Action. The Expert Review Panel for 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 for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@ aoac.org. Corresponding author ’ s e-mail: jonathan.draher@abbott.com DOI: https://doi.org/10.5740/jaoacint.17-0257

necessary for this important component in the content of food composition databases. Many analytical methods have been developed for specifically determining Trp in foodstuffs. Trp is degraded during the classic amino acid analysis of proteins by acid hydrolysis. Several approaches have been proposed for direct Trp determination after derivatization, such as second-derivative spectroscopy, p- dimethylaminobenzaldehyde, p -phenylenediamine, O -phthaldehyde, and ninhydrin derivatization. Acidic, alkaline, and enzymatic hydrolysis of the protein have been carried out, followed by chromatographic, colorimetric, or fluorometric determination of Trp (4). This paper describes a method for Trp determination in an infant formula and adult/pediatric nutritional formula following enzymatic hydrolysis. This method was developed to meet the specific requirements for amino acid analysis explained in Standard Method Performance Requirement (SMPR ® ) 2014.013. Although the method is not novel, it does compare very well with other techniques used for Trp determination in terms of time of analysis and complexity. Like the majority of methods used to determine amino acids, the hydrolysis time for this method is 16 h. The other aspects of the sample preparation are very simple dilution and filtration steps. The method was used to perform a single-laboratory validation (SLV) on a broad range of infant formula and adult/pediatric nutritional products. AOAC Official Method 2017.03 HPLC Determination of Total Tryptophan in an Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis First Action 2017 [Applicable to the determination of the total tryptophan (Trp) content in infant, pediatric, and adult nutritional products as defined in Standard Method Performance Requirement 2014.013 (1). See Tables 2017.03A – 2017.03C for matrixes for which single-laboratory validation data has been generated, supporting acceptance of the method.]

A. Principle

Trp is released (hydrolyzed) from intact protein using a combination of proteolytic enzymes found in pronase, which was isolated from Streptomyces griseus for this purpose. The pronase enzyme powder contains at least 10 proteolytic enzymes (depending on the source, supplier, etc.) that hydrolyze peptide bonds internally (endoproteases) and externally (exopeptidases)