2015 10 Vol.33 No.10 October 2015 Chinese Journal of Chromatography 1040 ~ 1045 DOI 10.3724 / SP.J.1123.2015.06010 - / 1 1* 2 1 1 1 1 1 1. 210001 2. 210001 - / 0. 22 μm Carbohydrate 100 mm 2. 1 mm 2. 6 μm 0. 1% Target-MS / MS m / z 4. 0 30 ev - GF 2 GF 3 GF 4 5 10-6 5 ppm 10 min GF 2 GF 3 100 μg / kg GF 4 55 μg / kg 0. 998 5 10 20 mg / kg 3 GF 2 GF 3 GF 4 75. 8% ~ 107. 3% RSD 1. 6% ~ 8. 3% - / O658 A 1000-8713 2015 10-1040-06 Determination of fructo-oligosaccharides in milk powder by high performance liquid chromatography- quadrupole / electrostatic field orbitrap high resolution mass spectrometry LIU Yun 1 DING Tao 1* XU Suli 2 WU Bin 1 SHEN Chongyu 1 ZHANG Rui 1 WANG Yan 1 FEI Xiaoqing 1 1. Animal Plant and Food Inspection Center Jiangsu Entry-Exit Inspection and Quarantine Bureau Nanjing 210001 China 2. School of Public Health Nanjing Medical University Nanjing 210001 China Abstract A method of high performance liquid chromatography-quadrupole / electrostatic field Orbitrap high resolution mass spectrometry HPLC-Q / Orbitrap MS was developed to deter- mine fructo-oligosaccharides in milk powder. The milk powder samples were dissolved in deion- ized water. Subsequently an aqueous solution of zinc acetate was used to precipitate protein. After centrifugation the final aqueous solution was filtered by a polytetrafluoroethylene PTFE membrane with pore size of 0. 22 μm. The analytes were separated on a Carbohydrate column 100 mm 2. 1 mm 2. 6 μm through gradient elution with the combination of acetoni- trile and 0. 1% formic acid aqueous solution. The target-ms / MS templates were performed at isolation window of m / z 4. 0 and collision energy of 30 ev in positive mode to extract the accu- rate product ion mass of analytes. Under the optimal condition 1-kestose GF 2 nystose *.Tel 025 52345183 E-mail dingt@ jsciq.gov.cn. 2015KJ29 2014BAK19B00 2014BAK19B02. 2015-06-08

10 - / 1041 GF 3 and 1-F-β-fructofuranosyl nystose GF 4 were well separated and the accuracy of extracted mass routinely detected was below 5 10-6 5 ppm. The whole analysis time is only ten minutes. The detection limits for GF 2 and GF 3 were 100 μg / kg and the detection limit for GF 4 was 55 μg / kg. Good linearities were obtained in their respective linear ranges with correla- tion coefficients higher than 0. 998. The average recoveries at three spiked levels 5 10 and 20 mg / kg were in the range of 75. 8% -107. 3% and the relative standard deviations RSDs were in the range of 1. 6% -8. 3%. The proposed method is simple sensitive fast and only in need of precipitation of proteins. The interference of matrix can be eliminated through the selection of product ion. The results were convenient and reliable and thus can be used in the large batch determination of any milk powder. fructo-oligosaccharides FOS - 11 HPLC-RID 12-1 ~ 3 β-1 2 HPLC-ELSD 13 14 DP 2 ~ 9 16 HPLC-RID 1-kestose GF 2 nystose GF 3 1-F-β-fructofuranosyl nystose 15 17 Key words high performance liquid chromatography-quadrupole / electrostatic field orbitrap mass spectrometry HPLC-Q / Orbitrap MS high resolution mass spectrometry HRMS fruc- to-oligosaccharides milk powder GF 4 1 2 0. 5% ~ 2% HPLC- RID HPLC-ELSD HPLC-RID B 3 4-8 HPLC-ELSD 9 GF 2 GF 3 GF 4 SPE 30 min inulo-triose F 2 inulo-tetraose F 3 inulo- - / pentaose F 4 9 AOAC 997. 08 10 1 1.1 GF 2 GF 3 GF 4 Q-Exac- tive HESI-Ⅱ Dionex 3000

1042 33 XW-80A ms m / z 4. 0 Sigma LP403 2 2.1 99. 9% 99. 9% 99. 9% Merck TEDIA Milli-Q - 1.2 A 6% v / v B 10 10. 0 mg 0. 1 mg mmol / L C ph 5 mol / L ph 1. 90±0. 50 2 min 10 ml 1. 0 g / L 5 mol / L ph 4. 70±0. 50 3 4 S1 0. 1 0. 2 S2 0. 5 1. 0 mg / L 4 0. 22 μm 10 - Milk sample Precipitation 1.4-1.4.1 Agilent Carbohydrate 100 mm 2. 1 mm 2. 6 μm 0. 1% A v / v acetic acid aqueous solution B 10 mmol / L zinc ace- B 0. 60 ml / min 10 μl tate solution C adjusting the ph based on the isoelectric 25 point. 0 ~ 2 min 80% B 2 ~ 5 min 80% B ~ 2.2 20% B 5 ~ 8. 5 min 20% B 8. 5 ~ 9 min 20% B ~ 80% B 9 ~ 10 min 80% B 1.4.2 HESI-Ⅱ 320 N 2 50 1 1.3 1 g 50 ml 20 ml 30 s 1 10 mmol / L ml 10 mmol / L 20 min 1 2 000 r / min 5 min Table 1 Comparison of different precipitation methods for milk powder samples Recoveries / % GF 2 GF 3 GF 4 S1 A 78.3 80.9 84.1 B 83.5 85.6 80.2 C 65.9 70.7 73.3 S2 A 75.2 78.3 73.1 B 78.9 82.4 75.8 C 64.2 68.9 60.6 S1 domestic milk sample S2 foreign milk sample. A 6% Carbohydrate 100 mm L / min N 2 8 L / min N 2 2. 1 mm 2. 6 μm Carbohydrate 150 mm 4. 6 5 L / min 3 kv 50 V mm 5 μm Target-MS / MS R = 35 000 30 ev AGC target 2e 5 100

10 - / 1043 5 10-6 1a Target-SIM 2. 6 μm Target-MS / MS 2.3 30 ev M+H + ESI + 2 GF 2 GF 3 GF 4 GF 2 GF 3 GF 4 M+H + 347. 079 85 R 35 000 1b Fig. 1 1 GF 2 GF 3 GF 4 a b a First-full-scan accurate mass spectra and b second-extracted chromatograms of GF 2 GF 3 and GF 4 in milk samples 2.4 2.4.1 Orbitrap Table 2 1.2 GF 2 GF 3 GF 4 y x 3 S / N = 3 10 S / N = 2 Compound GF 2 GF 3 GF 4 Regression equations correlation coefficients r retention times t R limits of detection LODs n = 5 and limits of quantification LOQs n= 5 of the GF 2 GF 3 and GF 4 compounds Regression equation 10 2 GF 2 GF 3 GF 4 r r t R / min LOD / LOQ / μg / kg μg / kg GF 2 y = -886311+0727.9x 0.9980 7.49 100 308 GF 3 y = 213138+4678.07x 0.9991 7.42 100 308 GF 4 y = 99909.6+2629.8x 0.9992 7.32 55 154 y peak area x mass concentration mg / L. Linear range 0. 1-1. 0 mg / L.

1044 33 2 GF 2 GF 3 GF 4 Fig. 2 Second-full-scan accurate mass spectra of GF 2 GF 3 and GF 4 0. 998 0. 1 ~ 2. 0 mg / L 5 GF 2 GF 3 GF 4 2.4.2 3 S1 75. 8% ~ 107. 3% RSD 1. 6% ~ 8. 3% S2 5 10 20 mg / kg 3 Table 3 3 2 GF 2 GF 3 GF 4 n= 5 Recoveries of GF 2 GF 3 and GF 4 spiked in two different kinds of milk samples n= 5 Milk sample Spiked / mg / 100 g GF 2 Recovery / % RSD / % GF 3 Recovery / % RSD / % GF 4 Recovery / % RSD / % S1 0.5 83.5 3.5 85.6 4.6 80.2 8.3 1.0 91.4 5.1 101.7 7.2 105.8 4.9 2.0 84.7 6.3 94.1 1.6 107.3 5.6 S2 0.5 78.9 3.9 82.4 5.8 75.8 4.1 1.0 79.2 8.0 90.5 7.6 86.4 6.3 2.0 90.2 5.9 93.4 6.2 104.9 3.7 2.5 LC-MS / MS 4 5 5 2 2 1 1. 3

10 - / 1045 Table 4 4 10 n= 5 Determination results and labeled values of fructo-oligosaccharides FOS in ten milk powder samples n= 5 Sample No. GF 2 / mg / 100 g GF 3 / mg / 100 g GF 4 / mg / 100 g Total / mg / 100 g Labeled value / mg / 100 g 1 1.06 1.82 0.05 2.93 3.0 2 1.39 2.11 0.03 3.53 3.0 3 0.37 0.76 0.36 1.49 1.3 4 1.54 0.12 0.07 1.73 1.5 5 0.39 0.13 0.03 0.55 0.5 6 0.87 0.02 0.47 1.36 1.2 7 0.32 0.66 0.03 1.01 1.0 8 0.45 0.23 0.11 0.79 0.7 9 0.63 0.12 0.32 1.07 0.9 10 0.76 0.31 0.18 1.25 1.0 3 7 GB 2760-2007 -8 Robefrorid M B. Am J Clin Nutr 2001 73 / Chromatography 32 12 1380 10 AOAC Official Method 997. 08 Medicine 2009 5 9 20 tography. 1 Zhang W. Journal of Biological. 2000 17 1 7 2 Jiang B Zhang T Wang Z. Journal of Wuxi University for Light Industry. 15 Wang M H Li M Guo J et al. Chinese Journal of Health 2001 20 5 445 Inspection. 3 Chen M X Zhu W J. Biology Teaching. 2008 33 5 61 5 Gnoth M J Kunz C Kinne-Saffran E et al. J Nutr 2000 130 12 3014 6 Gao X J. Liquor Making. 2008 35 4 90 Suppl 406 9 Geng L J Huang J R Feng F et al. Chinese Journal of. 2014 11 Wei F Wu Z Y Ma L Y et al. Asia Pacific Traditional. 12 Ding H L Li C Jin P et al. Chinese Journal of Chroma- 2013 31 8 4 Liu Y F Li D Tang H C et al. Journal of Dairy Science and. 2009 30 18 237 Technology. 17 Bai J Jiang J D Tao D L. Food Science 2012 35 1 40. 2014 35 2 257 804 13 Wang Y Z Yao P J Wei Y A. Food Industry. 2009 2 71 14 Lin S J Xiao H L Guo B K. Chinese Journal of Health Inspection. 2009 19 11 2549 2007 17 6 961 16 Zhang Y Y Nie S P Wan C et al. Food Science