Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame.

Takashi Ohtsuki,Kyoko Sato,Satoru Kubo,Akira Otabe,Chiye Tatebe,Mitsuya Yoshida,Yoko Oobayashi,Mika Yoshida,Ryoichiro Nakamura,Shoko Suzuki,Hiroshi Akiyama

doi:10.1371/journal.pone.0152174

Takashi Ohtsuki, Kyoko Sato + Show 9 more

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https://doi.org/10.1371/journal.pone.0152174

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α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.

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IntroductionN-L-α-Aspartyl-L-phenylalanine-1-methyl ester (aspartame, L, L-APM; Fig 1A) is an artificial sweetener that is approximately 200 times sweeter than sucrose [1]
N-L-α-Aspartyl-L-phenylalanine-1-methyl ester is an artificial sweetener that is approximately 200 times sweeter than sucrose [1]
L, L-APM is relatively stable in the dry state at low temperatures, under certain conditions of pH and temperature in aqueous solution it is well known to decompose into its constituent amino acids, 2-(5-benzyl-3,6-dioxopiperazin-2-yl) acetic acid (DKP), and the isomeric dipeptide-like L-asparaginyl-L-phenylalanine [5,6,7]. α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM; Fig 1B), α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM; Fig 1C), and α-D-aspartyl-D-phenylalanine methyl ester (D, D-APM; Fig 1D), the three

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Introduction

N-L-α-Aspartyl-L-phenylalanine-1-methyl ester (aspartame, L, L-APM; Fig 1A) is an artificial sweetener that is approximately 200 times sweeter than sucrose [1]. L, L-APM is used in more than 125 countries as a food additive in various processed foods such as soft drinks, chewing gum, and tabletop sweetener, as well as in pharmaceuticals [2,3,4]. L, L-APM is relatively stable in the dry state at low temperatures, under certain conditions of pH and temperature in aqueous solution it is well known to decompose into its constituent amino acids, 2-(5-benzyl-3,6-dioxopiperazin-2-yl) acetic acid (DKP), and the isomeric dipeptide-like L-asparaginyl-L-phenylalanine [5,6,7]. Α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM; Fig 1B), α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM; Fig 1C), and α-D-aspartyl-D-phenylalanine methyl ester (D, D-APM; Fig 1D), the three. L, L-APM is relatively stable in the dry state at low temperatures, under certain conditions of pH and temperature in aqueous solution it is well known to decompose into its constituent amino acids, 2-(5-benzyl-3,6-dioxopiperazin-2-yl) acetic acid (DKP), and the isomeric dipeptide-like L-asparaginyl-L-phenylalanine [5,6,7]. α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM; Fig 1B), α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM; Fig 1C), and α-D-aspartyl-D-phenylalanine methyl ester (D, D-APM; Fig 1D), the three

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