Agilent Analysis of Natural Food Colorants By Electrospray Atmospheric Pressure Chemical Ionization LC/MS Manual
Contents
1. h e 6 e e e e 20 e Natural Food Colorants s Agilent Technologies t Innovating the HP Way Analysis of Natural Food Colorants by Electrospray and Atmospheric Pressure Chemical lonization LC MS Introduction Many kinds of natural colors are used in beverages jellies and candies In many coun tries food regulations have been recently revised to cover natural colorants to the same degree as synthetic ones Accordingly it has become necessary to develop reliable analyti cal methods for various natural colorants in food In this study LC MS methods using elec trospray ionization ESI or atmospheric pres sure chemical ionization APCI were devel oped to identify major pigments in four natural colorants red cabbage paprika Monascus and lac Experimental Paprika and Monascus colorants were dissolved in acetone the other colorants were dissolved in deionized water Eachcolorant was filtered through a 0 2 ym filter A 10 1 portion was injected into the system which consisted of an Agilent 1100 Series binary pump thermostatted column compartment vacuum degasser autosampler and LC MSD The LC MSD used either an ESI or APCI source Complete system control and data handling were done on the Agilent ChemStation for LC MS Operating conditions were optimized for each sample Results and discussion Red cabbage colorant Figure 1 shows the structure of seven major pigments of red cabbage The p2. a UNH 6 c A CH2 6CHs 1 Rubropunctatin 2 Monascin 3 Monascorubrin 4 Ankaflavin 5 Rubropunctamine 6 Monascorubramine Figure 4 The major pigments of Monascus colorant Abundance 550000 450000 350000 250000 150000 50000 2 00 6 00 10 00 14 00 R Time min 1 Rubropunctatin 2 Monascin 3 Monascorubrin 4 Ankaflavin 5 Rubropunctamine 6 Monascorubramine 495 18 00 Figure 5 The total ion chromatogram of Monascus colorant LC conditions Column 250 x 2 1 mm Inertsil 0083 5 um Mobile phase A 1 formic acid B acetonitrile Gradient Start with 50 B At 10 min 90 B Flow rate 0 2 ml min Column temp 40 C Injection vol 10 ul MS Conditions Source ESI lon mode positive Vcap voltage 4000 V Nebulizer 50 psig Drying gas flow 10 l min Drying gas temp 350 C Corona 4 uA Vaporizer temp 350 C Scan range 100 1200 amu Step size 0 1 Peak width 0 15 min Time filter on Fragmentor 100 V Natural Food Colorants 1 Rubropunctatin 357 2 Monascin 359 80 40 160 200 240 280 320 360 m z M H 3 Monascorubrin 385 4 Ankaflavin 387 160 200 240 280 320 360 mz Figure 6 Mass spectra of major pigments in Monascus colorant Three major peaks with base peaks at m z 439 467 and 495 were not identified Figure 6 shows the mass spectra of the identified pigments Protonated molecular ions M H
3. C CHCH CH NH COOH Abundance 150000 110000 90000 70000 50000 30000 Laccaic acid A B Laccaic acid C 2 00 6 00 R Time min 4 00 Trey retry erry retry e 10 00 12 00 Figure 10 The strucuture of major pigments of lac colorant Figure 11 The total ion chromatogram of lac colorant Laccaic acid 90 c Laccaic acid A B 200 280 360 440 538 520 Figure 12 Mass spectra of the major pigments in lac colorant LC conditions Column Mobile phase Flow rate Column Temp Injection vol MS Conditions Source lon mode Vcap voltage Nebulizer Drying gas flow Drying gas temp Scan range Step size Peak width Time filter Fragmentor 250 x 2 1 mm Inertsil 0DS3 5 wm 30 acetonitrile in 5 mM dibutylamine isocratic 0 2 ml min 40 C 10 yl ESI Negative 4000 V 50 psig 10 l min 350 C 100 1200 amu 0 1 0 15 min On 100 V lt Agilent Technologies Innovating the HP Way Natural Food Colorants Conclusion Four commercial natural colorants were analyzed using ESI and APCI LC MS The MS data provided molecular weight information and some structural information for the major pigments Masahiko Takino is an applications chemist at Yokogawa Analytical Systems Inc Windows NT is a U S registered trademark of Microsoft Corporation Windows is a U S registered trade mark of M
4. C Column temp 40 C Injection vol 10 pl MS Conditions Source ESI lon mode positive 200 600 1000 m z Vcap Voltage 4000 V D 919 Nebulizer 50 psig 80 Drying gas flow 10 l min 757 Drying gas temp 350 C Corona 4uA 40 287 Vaporizer temp 350 C Scan range 100 1200 amu Step size 0 1 Peak width 0 15 min G 1125 Time filter on 80 Fragmentor 200 V L gt gt E gt E i D 963 Figure 3 shows the mass spectra of the seven major pigments in red cabbage colorant For these pigments the singly charged molecular F 1155 ion is observed rather than the more typical a M H ion because the cyanidin group already has a positive charge on an oxygen 40 993 In source collision induced dissociation CID can be used to generate fragment ions to pro vide structural confirmation Using CID mass E 1185 spectra of these pigments show common frag 80 ments corresponding to the loss of a glucose as well as cyanidin m z 287 and cyanidin 3 40 1023 glucoside m z 449 ions 400 800 1200 mz Monascus colorant Monascus contains six major pigments their structures are shown in Figure 4 Four pig ments were identified from the mass spectra of major peaks in the TIC See Figure 5 Figure 3 Mass spectra of major pigments in red cabbage colorant Natural Food Colorants Osc CH2 4CH3 WH p oO a CH2 4CH 3 c 7 CH2 6CH3 Omg C H26CH3 CH CH3 a QO O n _ CH2 4CH3 SCH oN
5. icrosoft Corporation Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and speci fications in this publication are subject to change without notice Copyright 1998 Agilent Technologies Inc All rights reserved Reproduction and adaptation is prohibited Printed in the U S A April 2000 23 5968 2979E
6. igments share the basic cyanidin 3 diglucoside structure with differing R1 and R2 groups Figure 2 shows the total ion chromatogram TIC and extracted ion chromatograms EIC of red cabbage pigments Although every major pig ment can be chromatographically separated using 10 formic acid in the mobile phase the high acid concentration reduces sensitivity Therefore 1 formic acid was used in this study The EICs show the separation of the pigments based on their main ion base peak oo wd Zo OB Ry R2 on OF 2 RO RO OH A H Sinapyl ou OH OH B Sinapyl H C Ferulyl H Cyanidin 3 diglucoside D p Coumaryl H E Sinapyl Sinapyl o To fe F Ferulyl Sinapyl cr i G p Coumaryl Sinapyl HO HO Sinapyl 7 Ferulyl CH O0 e i OCH p Coumaryl Figure 1 The structure of major pigments in red cabbage colorant Natural Food Colorants Abundance 1100000 500000 10 00 14 00 R Time min m z 979 m z 1185 m z 949 m z 1155 m z 919 Gj m z 1125 14 00 20 00 16 00 22 00 R Time min R Time min 8 00 Figure 2 Total and extracted ion chromatograms of red cabbage colorant Natural Food Colorants _ _ A 287 979 LC conditions 80 Column 250 x 2 1 mm Inertsil 449 0DS3 5 wm Mobile phase A 1 formic acid 40 817 B acetonitrile Gradient Start with 5 B At 30 min 50 B Flow rate 0 2 ml min B amp
7. nertsil ODS3 5 wm Mobile phase A acetone B methanol Gradient Start with 10 B At 10 min 90 B Flow rate 0 2 ml min 6 2 Monoeicosanoate 879 Column Temp 40 C Injection vol 10 pl MS Conditions Source APCI lon mode Positive Veap voltage 4000 V 3 Dilaurate Nebulizer 50 psig Drying gas flow 5 I min Drying gas temp 350 C Corona 4uA Vaporizer temp 350 C Scan range 100 1200 amu Step size 0 1 Peak width 0 15 min 4 Lauryl myristate 749 Time filter On 5 Dimyristate Lac colorant Figure 10 shows the structure of the major pigments in lac colorant Laccaic acids A B C are known as the major pigments in lac col orant These compounds have the same basic 6 Myristyl palmitate 805 anthraquinone structure but with different R 80 T77 groups Three major peaks were detected in the TIC See Figure 11 Although laccaic acids A B and C were identified A and B could not be separated Figure 12 shows the mass spectra of two peaks laccaic acid C and a combination of lac caic acids A and B The deprotonated molecu lar ions were observed at m z 495 536 and 538 Fragment ions resulting from the loss of carbon dioxide were observed at m z 451 492 and 494 7 Dipalmitate 805 200 400 600 800 1000 Figure 9 Mass spectra of major pigments in paprika colorant Natural Food Colorants O OH Laccaic acid A CH CH CH NHCOCH Laccaic acid B CH CH CH OH Laccaic acid
8. were observed for the four identified pig ments Paprika color Capsanthin and the mono and di esters of capsanthin with fatty acids are known as the major pigments in paprika colorant See Figure 7 Two monoesters and five diesters of capsanthin were identified in the paprika col orant analyzed in this study See Figure 8 The protonated molecular ions M H were observed for every major pigment See Figure 9 However with the exception of capsanthin monoeicosanoate the intensity of these ions was very low Except for capsanthin monoe icosanoate the pigments show fragment ions resulting from the loss of one or two fatty acid fragments A common fragment ion was observed at m z 567 in the mass spectra of these pigments Natural Food Colorants Capsanthin A R1 R2 Acyl R O Monomyristate Monoeicosanoate Dilaurate Lauryl myristate Dimyristate Myristyl palmitate Dipalmitate NOOR OND Figure 7 The structure of major pigments of paprika colorant Abundance 5 6 170000 150000 3 430000 1 Monomyristate 1 2 4 7 2 Monoeicosanoate 110009 3 Dilaurate 90000 4 Lauryl myristate 70000 5 Dimyristate 50000 6 Myristyl palmitate 7 Dipalmitate 30000 10000 0200 6 00 10 00 14 00 18 00 R Time min Figure 8 The total ion chromatogram of paprika colorant Natural Food Colorants 1 Monomyristate LC conditions 80 Column 250 x 2 1 mm I
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