Analytical Science and Technology (분석과학)

The Korean Society of Analytical Sciences (한국분석과학회)
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An analysis of benzoic acid, methyl paraben and butyl paraben in soy sauce using isotope dilution liquid chromatography mass spectrometry

동위원소희석 질량분석법을 이용한 간장 중의 안식향산, 메틸파라벤, 부틸파라벤의 분석

  • Ahn, Seonghee (Center for Analytical Chemistry, Korea Research Institute of Standards and Science)
  • 안성희 (한국표준과학연구원 분석화학표준센터)
  • Received : 2018.09.06
  • Accepted : 2018.10.29
  • Published : 2018.12.25
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Abstract

Benzoic acid, methyl paraben, and butyl paraben are preservatives that have been used in pharmaceutical, cosmetic, and food products. However, as their toxicities for human have been reported, many nations and organizations including Korea have established a regulation limit for thier usage of these preservatives in food products. The present study developed the isotope dilution liquid chromatography mass spectrometry method for accurate determination of three target preseratives in soysauce. In this study, the isotope dilution liquid chromatography mass spectrometry method was developed for accurate determination of three target preservatives in soy sauce. LC separation was optimized considering the pKa of benzoic acid which is lower than those of methyl and butyl parabens. A C18 column was used with 5 mM ammonium acetate and methanol as mobile phases. Mass spectrometry was operated in negative mode and selected reaction monitoring mode (SRM). Soy sauce sample was cleaned-up with C18 SPE cartridge for removing matrix inferences and color material. Optimized conditions and the method were validated with soy sauce reference materials for the analysis of food preservatives from Health Science Authority in Singapore. The measured values of benzoic acid, methyl and butyl paraben agreed well with reference values within their uncertainties.

안식향산, 메틸파라벤, 부틸파라벤은 식품, 의약품뿐 만 아니라 화장품 분야에서도 사용되는 보존료이다. 그러나 이들 보존료들의 여러 가지 독성이 보고되면서, 한국을 비롯하여 여러 나라들에서 식품 중의 이들 보존료의 사용을 규제하고 있다. 본 연구에서는 간장 중의 안식향산, 메틸파라벤, 부틸파라벤의 정확하고 정밀한 분석을 위하여 세가지 분석물질의 동위원소를 내부표준물질로 이용하는 동위원소 희석 액체크로마토그래피 질량분석법 (Isotope Dilution Liquid Chromatography Mass Spectrometry, ID-LC/MS)을 개발하였다. 메틸, 부틸 파라벤보다 pKa가 낮은 안식향산을 고려하여 acetic acid로 pH 4.0으로 조정한 5 mM ammonium acetate를 이동상 용매로 사용하여 C18 컬럼으로 분리하였다. 질량분석 조건으로는 전기분무이온화법으로 음이온을 생성하여 음이온 모드에서 분석하였으며, 방해물질로부터 선택성을 향상하기 위해 선택반응분석법 (Selected Reaction Monitoring)을 이용하여 분석하였다. 간장의 색깔과 간장 중의 여러 가지 방해물질들을 제거하기 위하여 C18 카트리지를 이용하여 정제하였다. 최적화된 조건과 방법을 이용하여 싱가포르 Health Science Authority(HSA)가 제공하는 간장 기준시료를 분석하였다. 본 연구원에서 측정한 결과값은 HSA가 제공하는 기준값과 불확도 내에서 일치하였다.

Keywords

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Fig. 1. Chromatograms of benzoic acid with 5 mM ammonium acetate (a) and 20 mM ammonium acetate (b) solution as an aqueous mobile phase. The mass fraction of benzoic acid was 40 mg/kg.

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Fig. 2. Comparison of chromatograms for benzoic acid (3.4 min), methyl paraben (4.8 min) and butyl paraben (10.3 min) with 5 mM ammonium acetate solution (a) at pH 4.5 and (b) at pH 4.0. The mass fractions of benzoic acid, methyl paraben and butyl paraben were 40 mg/kg, 10 mg/kg and 10 mg/kg, respectively.

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Fig. 3. Total ion chromatograms and benzoic acid chromatograms of soy sauce after (a) dilution, (b) Carb SPE cartridge and (c) C18 SPE cartridge. The soy sauce sample for (a) solution was from HSA. The soy sauce sample for (b) and (c) soy sauce was spiked with standard mixture solution into the soy sauce from the local market. The mass fraction of benzoic acid was 20 mg/kg, and the mass fractions of methyl paraben and butyl paraben were 4 mg/kg.

Table 1. Structures, pKa, solubilities and regulation limits for benzoic acid, methyl paraben, and butyl paraben

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Table 2. Channels and collision energies for selected reaction monitoring of ID-LC/MS/MS

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Table 3. ID-LC/MS/MS measurement results of 5 sub-samplings from soy sauce reference material

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Table 4. Comparison of measured values by KRISS with reference values of benzoic acid, methyl paraben, and butyl paraben in soy sauce sample

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