Quantitative analysis of illegal dyes in eggs using LC/MS/MS

Transcription

1 PO-CO9E Quantitative analysis of illegal dyes ASMS 0 TP 33 Rashi Kochhar, Shailendra Rane, Shailesh Damale, Deepti Bhandarkar, Shruti Raju, Ajit Datar, Pratap Rasam and Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-0009, Maharashtra, India.

2 Introduction Generally, egg yolk color is an indication of its nutritional value and freshness. Eggs with yellow-orange hue are the most desired ones. Hence, egg producers enhance the color of their products by feeding the egg laying hens with feed containing various synthetic dyes (shown in Figures and ). This can be proved by confirming the presence of illegal dye residues in eggs []. Figure. Duck eggs Figure. sudan dyes in egg yolk A method for pre-treatment and subsequent LC/MS/MS analysis has been developed for highly sensitive quantitation of these dyes from egg using LCMS-800, a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan. Ultra-fast scanning speed of LCMS-800 enables simultaneous analysis of four sudan dyes and para red dye. All these dyes were quantitated at low ppb levels with high specificity and good repeatability even in presence of egg matrix. Sudan and para red dyes consists of a group of synthetic industrial dyes like sudan I, II, III and IV etc. The International Agency for Research on Cancer evaluated the safety of sudan I, II, III and IV in 987 and considered that they were unclassifiable as to their carcinogenicity to humans. Although there is some evidence that sudan dyes may cause cancer in experimental animals and may cause damage to the genes, there is currently inadequate evidence that they cause cancer in humans. Structure of four sudan dyes and para red under study are as shown in Figure 3. Sudan I Sudan II Sudan III O Sudan IV Para Red Figure 3. Structure of sudan dyes and para red used to contaminate egg

3 Method of analysis Preparation of matrix matched calibration curve In g of egg yolk, 9 ml water and 0 ml acetonitrile was added in 0 ml tarson tubes. Mixed it well for one min. For extraction, QuEChERS Kit [] ( packet E ) (Phenomenex Inc.) was added into it and mixed thoroughly for one min. Centrifuged these tubes at 000 rpm for min at -0 C and collected approximately 8 ml of supernatant. Passed the 3 ml of collected supernatant through a preconditioned C-8 SPE cartridge (Orochem), which finally eluted with 3 ml acetonitrile. This eluent was used to prepare dilutions for matrix matched calibration in range of ppb - 00 ppb. LC/MS/MS analysis Compounds were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) exera coupled with LCMS-800 triple quadrupole system (Shimadzu Corporation, Japan). The details of analytical conditions are given in Table. LCMS-800 triple quadrupole mass spectrometer as shown in Figure, with its ultrafast polarity switching and ultrafast MRM, along with high sensitivity is best suited instrument for food safety analysis. These features ensure analysis of multiple components with different ionizing tendency to be analyzed within same run, without any loss of sensitivity. Figure. exera with LCMS-800 triple quadrupole system by Shimadzu 3

4 Table. LC/MS/MS conditions for analysis of sudan dyes and para red Column : Shim-pack XR-C8 (7 mm L x 3 mm I.D.;. µm) Flow rate : 0. ml/min Oven temperature : 0 ºC Mobile phase : A: 0.% formic acid in water B: 0.% formic acid in acetonitrile Gradient program (B%) : min 0-80 (%); min 80 (%);.0 min (%);.0.0 min 00 (%);.0. min 00-0 (%);. min 0 (%) Injection volume : 0 µl MS interface : Electro Spray Ionization (ESI) itrogen gas flow : ebulizing gas 3 L/min; Drying gas 0 L/min MS temperature : Desolvation line 30 ºC; Heating block 00 ºC MRM Transitions : Sudan I: 9.00>9 Sudan II: 77.00>. Sudan III: 30>77.0 Sudan IV: 380>.0 Para red: 9.0>.0 Results Analysis of sudan and para red dyes was carried out in both aqueous and egg yolk matrix. MRM was optimized using automatic MRM optimization feature of LabSolutions. Analysis was also checked for blank interference. The method optimized had substantially reduced background interferences. Result of simultaneous analysis of five dyes in egg yolk is shown in Figure. Overlay of blank, LOQ and highest concentrations are shown in Figure. Linearity and accuracy were checked for both aqueous and matrix matched calibration. These quantitative results are given in Table. LOQ of ppb was obtained for all the five dyes in both aqueous and egg yolk matrix. 7.0 (x0,000) :SUDA-I 9.00>9(+) CE: - :SUDA-II 77.00>.(+) CE: :SUDA-III 30>77.0(+) CE: -8.0 :SUDA-IV 380>.0(+) CE: -.0 :PARA RED 9.0>.0(+) CE: -8.0(0).0 SUDA-II..0. SUDA-I SUDA-III SUDA-IV. PARA RED min Figure. Overlay of sudan dyes and para red for ppb standard in egg yolk

5 ame Overlay of matrix blank, ppb, 00 ppb Calibration curve ( ppb 00 ppb) (x00,000) :SUDA-I 9.00>9(+)(0) CE: - AQS_Data_0.lcd :SUDA-I 9.00>9(+)(.00) CE: - AQS_Data_0.lcd :SUDA-I 9.00>9(+) CE: - AQS_Data_.lcd.. Area(x,000,000) r = Sudan I min Conc. (x00,000) 8.0 :SUDA-II 77.00>.(+)(0) CE: -7.0 AQS_Data_0.lcd :SUDA-II 77.00>.(+)(.00) CE: -7.0 AQS_Data_0.lcd :SUDA-II 77.00>.(+) CE: -7.0 AQS_Data_.lcd Area(x,000,000) r = Sudan II min Conc. (x00,000) 3:SUDA-III 30>77.0(+)(0) CE: -8.0 AQS_Data_0.lcd 3:SUDA-III 30>77.0(+)(.00) CE: -8.0 AQS_Data_0.lcd 3:SUDA-III 30>77.0(+) CE: -8.0 AQS_Data_.lcd Area(x,000,000) 7. r =0.99. Sudan III min Conc. (x0,000) :SUDA-IV 380>.0(+)(0) CE: -.0 AQS_Data_0.lcd :SUDA-IV 380>.0(+)(.00) CE: -.0 AQS_Data_0.lcd :SUDA-IV 380>.0(+) CE: -.0 AQS_Data_.lcd.0.0 Area(x00,000).0.0 r = Sudan IV min Conc. (x0,000) :PARA RED 9.0>.0(+)(0) CE: -8.0 AQS_Data_0.lcd :PARA RED 9.0>.0(+)(.00) CE: -8.0 AQS_Data_0.lcd. :PARA RED 9.0>.0(+) CE: -8.0 AQS_Data_.lcd Area(x00,000). r = Para red min Conc. Matrix blank; ppb; 00 ppb Figure. Overlay mass chromatograms and calibration curve for sudan dyes and para red in egg yolk matrix

6 Table. Results of sudan dyes and para red calibration curve ame ominal concentration (ppb) Measured concentration* (ppb) Avg. area* %RSD cal.point area* % accuracy* Sudan I Sudan II Sudan III Sudan IV Para red * Average result for n=3

7 Conclusion Highly sensitive method for simultaneous analysis of illegal dyes in egg yolk like four sudan dyes and para red was developed. LOQ of ppb was obtained for all five dyes by matrix matched calibration standards, with linearity > 0.99 and accuracy between 90-0%. An analytical method was developed to minimize the background interference, hence giving high level of sensitivity. References [] Marta Piatkowska, Piotr Jedziniak, Jan Zmudzki, Bull Vet Inst Pulaway, Volume 8, (0), 7-3. [] Javier Dominguez- Alvarez et al., Journal of Chromatography A, Volume 78, (03), - 7. First Edition: May, 0 For Research Use Only. ot for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 0