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Agilent The Analysis of Triglycerides in Edible Oils by APCI LC/MS

1. 0 C18 1 cis 9 C18 1 trans 9 50000 Tristearin es C18 0 40000 30000 20000 10000 e Oj eee T T T T i T ji T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T ii T 16 00 17 00 18 00 19 00 20 00 21 00 22 00 23 00 Minutes Figure 1 TIC showing separation obtained for four C18 triglyceride standards Note that cis and trans isomers are well resolved LC Conditions the ammonium formate The effect of adding ammo Column Hypersil MOS 200 mm x 2 1mm 5 um nium formate is shown in Figure 2 Additionally the M obile phase A 60 40 water isopropanol 25 mM vaporizer had to be lowered to 300 C to minimize ammonium formate fragmentation and enhance the molecular signal If B 10 10 80 structural information was desired in source colli water ck le eel 25 mM sion induced dissociation CID was employed by ammonium formate i Gradient Start with 30 B raising the fragmentor from 80 volts to 150 volts at 1 5 min 30 B TR at 25 min 60 B Qualitative Spectral Information at 28 min 100 B ca Flow rate 0 25 mL min Under these analytical conditions each triglyceride Column temperature 50 C shows predominantly an M 18 adduct ion and few Injection volume 1 5 uL other major fragments Since triglycerides in plants MS Conditions are made up predominantly of fatty acids with even Source APCI numbers of carbons C12 C14 etc and either 0 1 ian m d Positive or 2 double bonds per acid much can be deter Va Vol
2. 3 min Column temperature 50 C Time filter On Injection volume 15 uL Fragmentor 80 V Lite olive oil Spanish extra virgin olive oil Minutes Figure 5 The TIC of lite and Spanish extra virgin olive oils The data indicate lite oil is more unsaturated or has shorter fatty acids Further examination of ElCs indicates more unsaturation LC Conditions MS Conditions Column Hypersil MOS 200 mm x 2 1mm 5 um Source APC Mobile phase A 60 40 water isopropanol 25 mM lon mode Positive ammonium formate Vcap voltage 4000 V B 10 10 80 Nebulizer 50 psig water isopropanol n butanol 25 mM Drying gas flow 4 L min ammonium formate Drying gas temp 325 C Gradient Start with 30 B Corona 4 pA at 1 5 min 30 B Vaporizer temperature 300 C at 25 min 60 B Scan range 300 1100 m z at 28 min 100 B Step size 0 1m z Flow rate 0 25 mL min Peak width 0 3 min Column temperature 50 C Time filter On Injection volume 1 5 uL Fragmentor 80 V 500 000 400 000 m z 908 8 0 double bonds 7 m z 906 8 1 double bond Io re m z 904 8 2 double bonds m z 902 8 3 double bonds 300 000 M Z 900 8 4 double bonds Al m z 898 8 5 double bonds 200 000 100 000 o T t f i L i i rt Ii ee a 1i a E i 21 22 23 24 25 26 27 Minutes Figure 6 Extracted ion chromatograms EICs for olive oil corresponding to the 3 C18 series Triolein is the major component Con
3. S E E D S E 300 350 400 450 500 550 600 650 700 Figure 2 Effect of ammonium formate on the spectrum of trilaurin from coconut oil LC Conditions acid with two oleic acids is not a likely solution Column Hypersil MOS 200 mm x 2 1 mm 5 um Figure 3 shows a typical TIC trace for coconut oil M obile phase A 60 40 water isopropanol 25 mM with the masses of the base ion annotated over each ammonium formate peak Inserting these masses into the spreadsheet B 10 10 80 produces the results shown in Table 1 Further ae ae al 25 mM confirmation if needed can be obtained using Gradient n KE e in source CID This is shown in Figure 4 0 at 1 5 min 30 B Most oils do not produce as simple a TIC trace as at 25 min 60 B at 28 min 100 B coconut oil Figure 5 shows a trace for two grades of Flaw iste 0 25 mL min olive oil Some differences are visible in the TIC but Coluenn temperatu j 50 C not much useful information is apparent However Injection volume 1 5 uL by looking at extracted ion chromatograms EICs MS Conditi much more can be determined Figure 6 shows the a ee results from extracting the signals for m z 908 8 ne nN 906 8 904 8 902 8 900 8 and 898 8 These corre V volta 9 g 4000 V spond to triglycerides made up from 3 C18 fatty Nebulizer i 50 psig acids with 0 1 2 3 4 and 5 double bonds respec Drying gas flow 4L min tively Not surprisingly the dominant ion is at m z Drying gas temp 325 C 902 8 which correspon
4. The Analysis of Triglycerides in Edible Oils B e s Food Author Doug Mcintyre Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19806 1610 USA Originally published M ay 2000 Abstract Triglycerides are the major components in edible oils This application describes a method for analyzing intact triglyc erides by LC MS and the type of information available Introduction Triglycerides are found in both plant oils and animal fats Their characterization is important for nutri tional reasons where the amount of unsaturation must be known as well as for determining other characteristics of the oil such as product purity suitability for deep frying etc Traditionally they have been analyzed by hydrolyz ing the triglycerides to yield the fatty acids which are then derivatized and analyzed as methyl esters by GC MS 1 This offers only indirect analysis of the original triglycerides Triglycerides can be chro matographed intact by HPLC but their lack of a chromophore unless conjugated makes detection dif ficult 2 In addition in order to identify each indi vidual triglyceride in an oil a larger number of standards than is practical would be required and the analysis time would be quite lengthy LC MS analysis offered the ease of an LC separation plus the specificity of mass detection Due to the _ Application by APCI LC MS non polar nature of the molecules atmospheric pressure chem
5. clusions References LC MS using APCI as an ionization process provides a simple way to analyze oils and fats for triglyc erides Information on the type of fatty acids including the degree of unsaturation can be obtained directly rather than indirectly This infor mation can be used for determining nutritional value and other physical properties of the oil or fat It also has the potential of determining the amount of processing the oil has undergone extra virgin versus pure Little or no sample cleanup is required allowing for a rapid analysis 1 M M Mossoba and D Firestone New Methods for Fat Analysis in Foods Food Testing and Analysis 1996 2 2 24 32 2 R Schuster Multicomponent Analyses of Fats and Oils Using Diode Array Detection Hewlett Packard Application Note 1987 publication number 59546269 For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent 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 specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Printed in the USA May 20 2008 5989 8441 EN Agilent Technologies
6. ds to the ammonium adduct Corona 4 pA ion for triolein 3 C18 1 Oleic acid is the major Vaporizer temperature 300 C fatty acid component in olive oil Using EICs in this Scan range 300 1100 m z way different oils can be compared to see how they Step size 0 1 m z differ in degree of unsaturation Peak width 0 3 min Time filter On Fragmentor 80 V 684 7 700000 2 600000 x 500000 400000 4 300000 S 200000 J 100000 J S Y e E F T T T t T IE T 8 10 12 14 16 18 20 Minutes Figure 3 TIC showing mass of the base peak of the spectrum from peak apex Peaks differ by m z 28 corresponding to difference in the number of CH CH2 groups on sidechains LC Conditions MS Conditions Column Hypersil MOS 200 mm x 2 1 mm 5 um Source APC M obile phase A 60 40 water isopropanol 25 mM lon mode Positive ammonium formate Vcap voltage 4000 V B 10 10 80 Nebulizer 50 psig water isopropanol n butanol 25 mM Drying gas flow 4 L min ammonium formate Drying gas temp 325 C Gradient Start with 30 B Corona 4 pA at 1 5 min 30 B Vaporizer temperature 300 C at 25 min 60 B Scan range 300 1100 m z at 28 min 100 B Step size 0 1m z Flow rate 0 25 mL min Peak width 0 3 min Column temperature 50 C Time filter On Injection volume 1 5 uL Fragmentor 80 V Table 1 Structure Calculations for the M ajor Peaks in Coconut Oil Observedion Calc MW Backbone removed m n Possible sidechai
7. ical ionization APCI was employed Experimental The system included an 1100 Series binary pump vacuum degasser autosampler thermostatted column compartment and LC MSD The LC MSD was used with the APCI source Complete system control and data evaluation were done on the ChemStation for LC MS Earlier work to demon strate feasibility was done on the 1090 HPLC and 5989B MS Engine equipped with the 5987A Electro spray Accessory and the G1075A APCI source A variety of vegetable oils as well as animal fat products such as butter was purchased at local supermarkets All products were dissolved in iso propanol at a concentration of 20 uL of oil per 10 mL of IPA or 20 mg of fat per 10 mL IPA Triglyc eride standards were purchased from Sigma and prepared by dissolving 10 mg of standard in 5 mL of chloroform then further diluted to the desired con centration with IPA Results and Discussion Separation and Ionization In order to have the nonpolar triglycerides elute in a reasonable mobile phase composition a Hypersil MOS C8 column was used Even this column required using isopropanol and n butanol to elute the larger triglycerides The final gradient was able to resolve cis and trans isomers of C18 1 triglyceride standards as shown in Figure 1 a Agilent Technologies Abundance 100000 Trilinolein 90000 C18 2 cis cis 9 12 80000 a 70000 7 Triolein Trielaidin 6000
8. ns 516 50 498 52 280 42 10 0 C8 C8 C10 544 55 526 52 308 42 11 0 C8 C10 C10 or C8 C8 C12 572 55 554 52 336 42 12 0 3 x C10 600 55 582 52 364 44 13 0 C10 C10 C12 628 55 610 52 392 44 14 0 C10 C12 C12 or C10 C10 C14 656 70 638 67 420 59 15 0 3 x C12 684 75 666 72 448 64 16 0 C12 C12 C14 712 75 694 72 416 64 17 0 C12 C14 C14 or C12 C12 C16 740 80 722 71 504 69 18 0 3 x C14 768 75 750 72 532 64 19 0 C14 C14 C16 796 75 778 72 560 64 20 0 C14 C16 C16 or C14 C14 C18 No double bonds oO oooococococoaCcCoeo 684 65 a Fragmentor 80 volts 684 65 Fragmentor 150 volts Loss of C12 fatty acid 439 Loss of C14 fatty acid 411 495 0 n L l of te T rA o Er 300 350 400 450 500 550 Figure 4 The effect of raising the Fragmentor voltage on a mixed triglyceride in coconut oil The fragment masses and ratios are consistent with two C12 fatty acids and one C14 LC Conditions MS Conditions Column Hypersil MOS 200 mm x 2 1 mm 5 um Source APCI M obile phase A 60 40 water isopropanol 25 mM lon mode Positive ammonium formate Veap voltage 4000 V B 10 10 80 Nebulizer 50 psig water isopropanol n butanol 25 mM Drying gas flow 4 L min ammonium formate Drying gas temp 325 C Gradient Start with 30 B Corona 4 uA at 1 5 min 30 B Vaporizer temperature 300 C at 25 min 60 B Scan range 300 1100 m z at 28 min 100 B Step size 0 1 m z Flow rate 0 25 mL min Peak width 0
9. ta 9 z 4000 V mined from this single adduct ion A spreadsheet N Sauer 50 psig was created to determine the possible combinations Drying gas flow 4L min of double bonds and carbon chain length This was Drying gas temp 325 C determined from the basic formula Corona 4 uA Vaporizer temperature 300 C M 218 03 28 03 m 26 02 n Scan range 300 1100 m z Step size 0 1 m z Where M is the mass of the observed adduct ion m Peak width 0 3 min is the number of ethylene CH2 CH2 groups and Time filter On n is the number of ethenyl CH CH groups Since Fragmentor 80 V only integer solutions are allowed the number of It was found that the addition of ammonium formate to the mobile phase allowed the formation of an ammonium adduct M 18 that was more stable than the protonated M 1 species formed without 2 possible answers is typically very short The choice is reduced even further if you assume fatty acids of the same length and unsaturation as the most likely solution followed by only small differences in the fatty acids For example an unsaturated butyric 439 10000 8000 No ammonium formate 6000 4000 J ai 2000 467 J 383 495 639 656 J 699 are ann OEA MEN O OEA AER D a E 300 350 400 450 500 550 600 650 700 10000 ore 8000 J 25 mM ammonium formate 6000 4000 4 2000 439 a 383 411 474 496 695 a a aa aa a o oa a a OS S O S O E S S S O B EOS OOE ONS O S S S O SS O D OS O O S S

Agilent The Analysis of Triglycerides in Edible Oils by APCI LC/MS

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