2012 6 Vol. 30 No. 6 June 2012 Chinese Journal of Chromatography 624 ~ 629 DOI 10. 3724 /SP. J. 1123. 2012. 01015-4 1 1 2* 1. 361005 2. 361005 HPLC Ⅰ Ⅱ ⅢB - SB-C18 / 3 4 81. 3% ~ 100. 4% 0. 8 mg /kg RSD n = 6 2. 6% ~ 3. 8% Ⅰ Ⅱ ⅢB LOD LOQ 0. 009 ~ 0. 054 mg /kg 0. 030 ~ 0. 181 mg /kg HPLC O658 A 1000-8713 2012 06-0624-06 Determination of four Sudan dyes in chili oil by high performance liquid chromatography with on-line photochemical derivatization and fluorescence detection LIU Jun 1 GONG Zhenbin 1 2* 1. State Key Laboratory of Marine Environm ental ScienceXiam en UniversityXiam en 361005China 2. College of Oceanography & Environm ental ScienceXiam en UniversityXiam en 361005China Abstract A method for the measurement of Sudan ⅠSudan ⅡSudan Ⅲ and Sudan B in chili oil using high performance liquid chromatography HPLC w ith on-line photochemical derivatizatio n and fluo rescence detectio n has been develo ped. The Sudan dyes w ere separated on an SB-C18 column in a single run by the mixed mobile phase of acetonitrile-w ater w ith a gradient program. A laboratory-built time /energy programmed photochemical reactor PCR w ith an ultraviolet mercury lamp w as installed betw een a photodiode array detector PDA and a fluorescence detector FLD and it w as applied to co nvert the no n-o r w eakly fluo rescent Sudan dyes into fluo rescence em issio n co m po nents. The pho to chem ical derivatizatio n co nditions and fluorescence detection parameters have been investigated and optimized. The recoveries of the standards spiked in real chili oil samples for all the dyes w ere 81. 3% - 100. 4%. The relative standard deviations RSDsn = 6 of the fluorescence signal intensity at the spiked level of 0. 8 mg /kg w ere 2. 6% - 3. 8%. The limits of detection LODs w ere in the range of 0. 009-0. 054 mg /kg and the limits of quantification LOQs w ere 0. 030-0. 181 mg /kgw hich w ere better than those of the commonly used HPLC coupled w ith PDA. The developed method w hich is simplesensitiveand selective can be applied to the routine analysis of Sudan dyes. Key words high performance liquid chromatography HPLC on-line photochemical derivatization fluorescence detection Sudan dyes chili oil *. Tel 0592 2186222E-mail zbgong @ xmu. edu. cn. Agilent. 2012-01-12
6-4 625 / 27 15 W PTFE 1 6 m 0. 3 mm Zeus CorpUSA PLC 2 3 1. 3 4-6 10. 0 g 60 TLC 7-10 - GC- ml 30 min MS 11-13 HPLC 14-20 100 ml - LC-MS 21-24 HPLC- - 25 26 LC-MS UVD 1. 4 Agilent ZORBAX SB C18 150 mm 3. 0 mm 3. 5 μm MS A -B 0. 4 ml /min 0 ~ 4 min90% A 4 ~ 8 min90% A ~ UVD FLD 100% A 8 ~ 20 min100% A 20. 01 ~ 23 min FLD 90% A 20 μl PDA PCR FLD FLD HPLC-FLD PCR 1 / FLD photochemical reactorpcr Analyte Time / λ Ex / λ Em / LC - min nm nm - Ⅰ Ⅱ ⅢB 1 1. 1 2 Tedia 2. 1 Sudan Ⅰ 97% Sudan Ⅱ 90% Sudan Ⅲ 96% Sudan B ph 97% Sigma-Aldrich 100 mg /L - 0. 001% 4 0. 005% 0. 01% 0. 05% 0. 1% - - 1. 2 - LC-20AB - 1 SPD-M20A PDA RF-20A 0. 45 μm 1 Table 1 Operating parameters for PCR and FLD Models 7725iRheodyneUSA 20 μl 4 2. 2 Ⅰ Ⅱ PCR UV lamp-on time /min Lamp voltage /V Sudan Ⅰ 0. 0-7. 0 289 340 0. 0-20. 0 220 Sudan Ⅱ 7. 0-11. 0 337 375 Sudan III 11. 0-15. 0 314 369 Sudan B 15. 0-20. 0 320 373 λ Ex excitation w avelength λ Em emission w avelength. 4
626 30 1 Ⅰ Ⅱ ⅢB a PDA b FLD Fig. 1 a PDA chromatogram and b FLD chromatograms of the standard solution of Sudan ⅠⅡⅢ and B Sudan Ⅰ and Sudan Ⅱ 4 mg /L Sudan Ⅲ 0. 5 mg /L Sudan B 1 mg /L. PDA detection 477 nm for Sudan Ⅰ492 nm for Sudan Ⅱ502 nm for Sudan Ⅲ509 nm for Sudan B. For the conditions of FLD detectionsee Table 1. Ⅲ B ⅢB 2 ⅠⅡ 2 a b Fig. 2 a Excitation and b emission spectra of Sudan dyes before and after irradiation Conditions PTFE tube length6 m irradiation time65 s lamp voltage220 V. Curves 1. UV lamp ON 2. UV lamp OFF.
6-4 627 4 Ⅰ FLD PCR Ⅱ Ⅲ B FLD PCR 6. 0 m 1 6. 0 m 2. 3 / PLC 2. 0 4. 0 6. 0 8. 0 m PTFE 130 150 170 180 200 220 V 2. 0 4. 0 6. 0 8. 0 m 3 4 21 42 65 84 s 3 3 2 ~ 6 m Ⅰ 8 m 6. 0 m Ⅰ 6 ~ 8 m 220 V Fig. 3 3 Effects of irradiation time and light intensity on the fluorescence signals of Sudan dyes 0. 001% 0. 005% 0. 01% 0. 05% 0. 1% 4 5 16 ~ 45 5 ⅠⅡ ⅠⅡ ⅢB Ⅲ B 4 16
628 30 4 2 4 Fig. 4 Effect of formic acid content in mobile phase Table 2 Recoveries of Sudan dyes spiked on the fluorescence signals of Sudan dyes in a chili oil sample % The conditions w ere the same as in Fig. 2. Analyte 0. 8 mg /kg 2 mg /kg 4 mg /kg Sudan I 93. 1 89. 8 86. 6 Sudan II 100. 4 99. 4 85. 2 Sudan III 81. 3 93. 4 81. 4 Sudan B 92. 7 86. 9 82. 6 5 Fig. 5 Effect of reaction temperature on the fluorescence signals of Sudan dyes The conditions w ere the same as in Fig. 2. 2. 4 ⅢB RSD 2. 6% 3. 5% 3. 2%3. 8% 4 0. 8 2 4 mg /kg 3 2 3 81. 3% ~ 100. 4% 2. 5 y x mg /L r 2 3 r 2 0. 996 3 1. 3 1. 4 SD n = 10 3 LOD 10 LOQ 3 RSD LOD LOQ 6 PCR PDA 0. 8 mg /kg 1. 3 ⅢB PDA 1. 4 RSD Ⅰ Ⅱ ⅢB 3 Table 3 HPLC-FLD r 2 HPLC-PDA /FLD Linear ranges linear equationscorrelation coefficients r 2 for HPLC-FLD methodand limits of detection LODs limits of quantification LOQs for HPLC-PDA /FLD method Analyte Linear range / Retention time LOD / mg /kg LOQ / mg /kg Linear equation r 2 mg /L ± SD n = 5 /min FLD PDA FLD PDA Sudan Ⅰ 0. 1-10. 0 6. 08 ± 0. 01 y = 227. 98x + 16. 459 0. 9963 0. 052 0. 054 0. 172 0. 180 Sudan Ⅱ 0. 1-10. 0 8. 75 ± 0. 02 y = 183. 58x + 28. 086 0. 9994 0. 054 0. 081 0. 181 0. 270 Sudan Ⅲ 0. 01-2. 0 12. 1 ± 0. 03 y = 10233x - 25. 594 0. 9981 0. 009 0. 092 0. 030 0. 307 Sudan B 0. 02-2. 0 18. 0 ± 0. 02 y = 2342x - 38. 15 0. 9998 0. 022 0. 199 0. 074 0. 664 y peak area x mass concentrationmg /L. 3 - Ⅰ Ⅱ ⅢB
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