February LC-MS/MS. Quantitative Analysis of l-ascorbic Acid and Erythorbic Acid in Foods by HPLC, and Confirmation Method by LC-MS/MS

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1 February l-hplc LC-MS/MS * Quantitative Analysis of l-ascorbic Acid and Erythorbic Acid in Foods by HPLC, and Confirmation Method by LC-MS/MS Yuki Sadamasu*, Mari Morikawa, Narue Sakamaki, Kimio Monma and Chigusa Kobayashi Tokyo Metropolitan Institute of Public Health: Hyakunin-cho, Shinjuku-ku, Tokyo , Japan; * Corresponding author Analysis of l-ascorbic acid AsA and erythorbic acid ErA in foods is generally performed by HPLC measurement after extraction with metaphosphoric acid solution. But this method can not always measure the concentrations of AsA and ErA precisely due to the presence of interfering compounds, and the reproducibility of retention time is poor. We considered that quantitative analysis by HPLC and confirmation by LC-MS/MS using an identical extraction solvent might be an effective approach for AsA and ErA analysis. Chelate fiber was added to the sample, followed by extraction with acetic acid solution containing ethylenediaminetetraacetic acid disodium salt, purification with Oasis MCX, and 2-fold dilution with methanol. The resulting solution was used for quantification by HPLC using a ZIC-HILIC column and identification by LC-MS/MS. In recovery tests with 8 kinds of foods, the recovery of AsA was over 91, and that of ErA was over 88. The RSD was 5.1 or less for both analytes. Analysis of 8 kinds of foods by both methods showed that this method gave better RSD values than the conventional method. AsA and ErA in all samples were confirmed by product ion scanning and selected reaction monitoring of LC-MS/MS. Received July 31, 2017 Key words l- l-ascorbic acid erythorbic acid ethylenediaminetetraacetic acid disodium salt chelate fiber hydrophilic interaction liquid chromatography HPLC - LC-MS/MS l- AsA ErA 2 AsA ErA * 1 * Yuki_Sadamasu@member.metro.tokyo.jp * 1 No cnt/3/25190/1/message147.pdf 1 4 AsA ErA HPLC LC-MS LC-MS/MS 5,6 AsA HPLC LC-MS/ MS

2 12 Vol. 59, No AsAErA EDTAEDTA 2Na AsAErA AsAErA 0.05 g 25 μg/ml EDTA 2Na 2 50 ml 1 ml AsA ErA 1,000 μg AsAErA AsA ErA 25 μg/ml EDTA 2Na 2 2 HPLCLC-MS/MS 25 μg/ml EDTA 2Na 2EDTA EDTA 2Na mg 100 ml 1 ml EDTA 2Na 1,000 μg 25 ml20 ml1,000 ml MetaSEP CH-1 1 CH-1 CH Oasis MCX 60 mg, 30 μm Waters EDTA 3 ml 3 EL ICP: Optima 7300DV Perkin Elmer HPLC: 1100 Agilent Technologies LC-MS/MS ACQUITY UPLC/TQD Waters 4 ICP nm 1.0 ml/min 15 L/min 0.2 L/ min 0.7 L/min RF 1,300 W Scheme 1 Analytical method of AsA and ErA 5 HPLC ZIC-HILIC 2.1 mm i.d. 150 mm 3.5 μm 200 Å SeQuant 40 mmol/l - 20 : ml/min UV260 nm 25 2 μl 4 HPLC 6 LC-MS/MS HPLC ESI 2.5 kv 30 kv L/hr 12 ev m/z 175 m/z SRMm/z g CH-1 5 g EDTA 50 ml20 2,330 g 10 5 ml Oasis MCX 6 8 ml/min 3 ml 1 ml 1 ml 0.2 μm HPLC LC-MS/MS Scheme 1 1 AsA ErA 7, LC-MS/MS

3 February 2018 l- 13 Fig 1 Time courses of recovery of AsA and ErA with various extraction solvents Sample sausage, spiked level 0.10 g/kg. 50 μg/ml EDTA containing 2 acetic acid solution, 25 μg/ml EDTA containing 2 acetic acid solution, 10 μg/ml EDTA containing 2 acetic acid solution, 5 μg/ml EDTA containing 2 acetic acid solution, 2 acetic acid. LC- MS/MS 1,2 2 AsA ErA 20 μg/ml HPLC 2 20 μg/ml ErA 2 20 μg/ml 0.5, 1, AsA ErA 7,8 Cu 2 Fe 3 EDTA 2Na μg/mledta 2Na 2 10 g EDTA 2Na 250 ml 30 2,330 g 10 AsA ErA 20 μg/ml Fig. 1 5 AsAErA μg/ml EDTA 2Na 2 EDTA EDTA LC-MS/ MS 25 μg/ml EDTA 2Na 2EDTA 2 EDTA AsA RSD 10 1 EDTA 2Na MetaSEP CH-1 CH-1 AsA Cu 2 8, AsAErA Fe 3 AsA Fe g 50 ml 30 μg/mledta 40 μg/ml 40 ml CH g EDTA 50 ml EDTA 10 ICP CH-12.5, 5, 7.5 g 16.3, 10.0, 7.2 μg/ml CH-1 10 g ErA 0.10 g/kg CH g EDTA 50 ml 30 2,330 g 10 HPLC ErA

4 14 CH g n 5 RSD CH-1 RSD CH-1 CH-1 ErA 5 g 3 1,2 2. CH-1 CH-1 CH-1 5 g μg/ml AsA 10 g CH-1 5 gedta 50 ml ,330 g 10 HPLC AsA AsA CH-1 AsA ErA AsA ErA HPLC AsA ErA AsA ErA Sep-Pak C mg Oasis HLB 150 mg Oasis MAX Oasis MCX 150 mg 110 g EDTA 50 ml 2,330 g μg/ml AsA ErA2.5 ml EDTA 2.5 ml 5 mlhplc Vol. 59, No. 1 Sep-Pak C 18 Oasis MCX AsA ErA Sep-Pak C 18 Oasis MCX 60 mg EDTA 3 ml EDTA 200 μg/ml AsA ErA 7 ml Oasis MCX 60 mg 1 ml HPLC2 7 ml AsA ErA 95 5 ml 3 ml1 ml 5 HPLC 1 4 AsA ErA LC-MS/MS 12 LC-MS/MS HPLC 12 HILIC 3 5HILIC SeQuant ZIC-HILIC 3.5 μm5 μm Cosmosil HILIC Waters Atlantis HILIC Silica ACQUITY UPLC BEH Amide AsA ErA ZIC- HILIC 2.1 mm i.d. 150 mm3.5 μm 200 Å HILIC 2-25 μg/mlhplclc-ms/ms HPLC LC-MS/MS HPLC2

5 February 2018 l- 15 Table 1 Recoveries of AsA and ErA from 8 foods Sample AsA Recovery* ErA RSD* Recovery* RSD* Fig 2 Comparison of retention time, height and peak area on HPLC at various concentrations of CH 3 COONH 4 Sausage Ham Fish sausage Carbonated beverage Blueberry jam Peachs canned in syrup Wine Asparagus canned *Sample were spiked with 0.10 g/kg of each compound, n mmol/l 28 : 72LC-MS/MS AsA ErA 20 : mmol/l 40 mmol/l AsAErA Fig mmol/l 20 : 80 6 HPLC1 500 μg/ml r S/N 10 2 μg/ml 7 AsA ErA 8 AsAErA 0.10 g/kg AsAErA AsA AsA ErA n 5AsA Table 1 Fig. 3 8 AsA 91 ErA 88 RSD AsAErA 8 AsAErA AsA ErA 1 RSD Table 2 F Fig 3 HPLC chromatograms of AsA and ErA spiked in fish sausage at the level of 0.10 g/kg A 10 μg/ml standard solution B Sample solution of fish sausage spiked with AsA and ErA C Sample solution of unspiked fish sausage AsA ErA n 5 p 0.05 RSD FRSD AsA 6t 5 p HPLC PDA μg/g 9 LC-MS/MS AsA ErA LC-MS/MSHPLC ESI AsA ErA M H m/z 175 m/z

6 16 Vol. 59, No. 1 Table 2 Comparison of analytical values of AsA and ErA in foods by conventional method and considered method Indication of food Sample Conventional method Analytical value g/kg RSD Considered method Analytical value g/kg RSD AsA Sausage a Ham Baby food fruits and vegetable paste Fruit juice beverage * Apricot jam Peachs canned in syrup Beer * Mushroom in brine 0.57** ErA Sausage a * n 5 a : Sausage in which AsA and ErA were also used. * p 0.05 in F-test ** p 0.05 in t-test Fig 4 SRM chromatogram m/z A and product ion spectra B SRM m/z SRM Fig. 4 S/N 3 2 μg/ml SRM 1 μg/ml 8 MS/MS SRM AsAErA 8 LC- AsAErA HPLC LC-MS/MS CH-1 EDTAOasis MCX HPLC LC-MS/MSZIC- HILIC40 mmol/l 20 : 80 HPLC LC-MS/MS SRM 8 AsA 91 ErA 88 RSD 5.1 AsA ErA 8 RSD LC-MS/MS SRM AsA ErA 1 4 LC-MS/MS p ISBN p ISBN The Pharmaceutical Society of Japan ed Bitamin. 3 Bitamin C. Eiseishikenho Chukai 2015 Methods of Analysis Health Science 2015, Tokyo, Kanehara Shuppan, 2015, p ISBN Ibe, A., Saito, K., Nakazato, M., Kikuchi, Y., Fujinuma, K., Nishima, T. Simultaneous detection and determination of erythorbic acid and l-ascorbic acid by thin layer chromatography and high performance liquid chromatography and survey of their contents in commercial foods. Tokyotoritsu Eisei Kenkyusyo Kenkyu Nenpo Annual Report of the Tokyo Metropolitan Research Laboratory of Public Health, 36, Somchai, B., Sasiwimon, L., Yaneenart, S., Supaluk, P., Virapong, P. Analysis of ascorbic acid and isoascorbic

7 February 2018 l- 17 acid in orange and guava fruit juices distributed in Thailand by LC-MS/MS. Food Anal. Methods, 9, Kakitani, A., Inoue, T., Matsumoto, K., Watanabe, J., Nagatomi, Y., Mochizuki, N. Simultaneous determination of water-soluble vitamins in beverages and dietary supplements by LC-MS/MS. Food Additives and Contaminants : Part A, 31, Kamiya, S., Nakabayashi, T. Studies on the decomposition of ascorbic acid III Decomposition of ascorbic acid by metal ion. Vitamin, 13, l- 1 l Hirata, A. Studies on the poly phosphates as the additives for foods Part 1 On the effect of phosphates as a preservative for vitamin C. Kaseigaku Zasshi, 19, The Pharmaceutical Society of Japan ed Sankaboushizai. 2 Echirenjiaminyonsakusan oyobi Sonoenrui. Eiseishikenho Chukai 2015 Methods of Analysis Health Science 2015, Tokyo, Kanehara Shuppan, 2015, p. 349 ISBN ISBN Tsuruda, S., Akaki, K. Simultaneous determination of ascorbic and erythorbic acid by hydrophilic interaction liquid chromatography HILIC. Fukuokashi Hokenkankyokenkyusyo Ho, 36,