A P P L I C AT I O N N O T E Gas Chromatography/ Mass Spectrometry Author: Adam Patkin PerkinElmer, Inc. Shelton, CT Analysis of FAMEs Using Cold EI GC/MS for Enhanced Molecular Ion Selectivity Introduction Characterization of fatty acid methyl esters (FAMEs) is used in several important fields ranging from biofuel analysis to fat content in foods and blood. They are generally derivatized from free fatty acids and mono-, di-, and triglycerides. FAMEs may be saturated, mono- or polyunsaturated, linear or branched, and of variable chain lengths. Electron Ionization Gas Chromatography/Mass Spectrometry (EI GC/MS) is often used to characterize FAMEs, but may fail to produce a useful molecular ion (M+•) for short, unsaturated, or branched chains, making compound identification more difficult. Contrastingly, Cold Electron Ionization GC/MS (Cold EI GC/MS) can substantially increase the M+• intensity of these compounds, while retaining the EI fragmentation pattern for spectral library searching without modification to established GC methodologies. Experimental Cold EI GC/MS Ion Source All work was carried out on a PerkinElmer AxION iQT® GC/MS/ MS system. Experimental conditions are shown in Table 1. The enhanced Cold EI M+• abundance allows use of this ion for more selective compound analysis, even for short-chained or polyunsaturated FAME isomers which may have unstable molecular ions with low or no intensity in EI. Results and Discussion A mixture of FAMEs was characterized using EI and Cold EI GC/MS. The Cold EI chromatogram is shown in Figure 2. Figure 1. Cold EI GC/MS Ion Source. • Velocity is increased (kinetic energy in the range of 1-20 eV) • Velocity is directed along the beam (jet separation) • Vibrational Energy is decreased (supercooling “Cold EI”) • GC column flow rate compatibility to 100 mL/min Molecules exit the GC column and are mixed with makeup gas. Supersonic expansion through a nozzle then ‘cools’ the analyte molecules from about +250 ºC to about -255 ºC by adiabatic expansion, forming a molecular beam. Excess carrier gas is skimmed off, and cold analyte molecules enter the ion source for ionization, molecular ion formation and subsequent mass analysis. When used in a novel MS/MS configuration the enhanced molecular ion in Cold EI provides enhanced selectivity and valuable information on FAMEs molecular weight. The compound retention times were identified using the NIST 2014 and Wiley 10th Edition mass spectral libraries. Of the 37 FAMEs in the test mix, all except 3 (coeluting at 12.21 min) were chromatographically resolved or could provide “clean” spectra through background subtraction. Specific attention is given to those compounds which have a relative intensity percentage, RI (%), of <5% of the M+• in NIST, as increasing their M+• intensity would be most beneficial to identification and quantification. These are highlighted red in Figures 2, 5 and Table 2, and include a number of the shorter chain FAMEs such as Methyl hexanoate and Methyl decanoate, and polyunsaturated isomers such as cis-5,8,11,14,17Eicosapentaenoic acid methyl ester and cis-4,7,10,13,16,19Docosahexaenoic acid methyl ester. Also shown are the Chemical Abstracts Service number (CAS No.), the Cold EI RI (%) of the M+•, the enhancement ratio of the Cold EI to EI M+• RI (%), and the number of double bonds in the compound. To better understand any pattern of EI M+• relative intensities, the compounds were sorted by the number of double bonds and then molecular weight, as shown in Figure 3. Table 1. Cold EI GC/MS Ion Source. Gas Chromatograph: PerkinElmer Clarus® 680 Mass spectrometer: PerkinElmer AxION® iQT™ MS/MS Injector Type: Injector temperature: Injection: Programmable Split/Splitless 250 ˚C 0.4 µL, split 200:1 GC Transfer Line Temperature: Ion Source Temperature: 250 ˚C 200 ˚C Oven Program: 50 ˚C, ramp to 240 ˚C at 20 ˚C/min Acqusition Range: Acqusition Time: m/z 40-400 0.20 sec Solvent Delay: none Analytical Column: PerkinElmer Elite™ - 5MS 30 m x 0.25 mm ID x 0.25 μm Carrier Gas: 1 mL/min Helium 99.999+% purity Supelco 37 Component FAME Mix Ion Source Type: Ion Source Mode: Background Noise Removal: Cold EI makeup gas: Ion Source Mode: Cold EI makeup gas: Cold EI Cold EI On 50 mL/min Classical EI 2 mL/min Filament: 5 µA Sample: 2 6e7 Intensity 5e7 4e7 3e7 2e7 1e7 0 3 4 5 6 7 8 9 10 11 Retention Time (minutes) 12 13 14 15 16 Figure 2. Cold EI chromatogram of FAMEs mixture. FAMEs with NIST EI molecular ion relative intensities below 5% are highlighted in red. Table 2. FAMEs with NIST EI molecular ion relative intensities below 5% are highlighted in red, and compared to their Cold EI results. FAMEs in retention time order. Compound Name CAS No. Formula MW NIST EI RI % Cold EI RI % Cold EI Enhancement Double Bonds 2.33 Methyl butyrate 623-42-7 C5H10O2 102.1 1.4 48 655 0 4.48 Methyl hexanoate 106-70-7 C7H14O2 130.1 0.5 4 7 0 6.33 Methyl octanoate 111-11-5 C9H18O2 158.1 3.8 16 4 0 7.83 Methyl decanoate 110-42-9 C11H22O2 186.2 1.5 50 44 0 8.50 Methyl undecanoate 1731-86-8 C12H24O2 200.2 2.0 85 57 0 9.14 Methyl laurate 111-82-0 C13H26O2 214.2 6.0 130 37 0 9.74 Methyl tridecanoate 1731-88-0 C14H28O2 228.2 4.0 196 84 0 10.24 Methyl myristoleate 56219-06-8 C15H28O2 240.2 4.0 96 358 1 10.31 Methyl myristate 124-10-7 C15H30O2 242.2 4.0 259 117 0 10.79 Methyl cis-10-pentadecenoate 90176-52-6 C16H30O2 254.4 4.2 102 374 1 10.84 Methyl pentadecanoate 7121-64-1 C16H32O2 256.2 6.0 405 140 0 11.26 Methyl palmitoleate 1120-25-8 C17H32O2 268.2 6.0 89 356 1 11.36 Methyl palmitate 112-39-0 C17H34O2 270.3 10.0 590 115 0 11.76 cis-10-Heptadecenoic acid methyl ester 77745-60-9 C18H34O2 282.3 3.5 77 808 1 11.85 Methyl heptadecanoate 1731-92-6 C18H36O2 284.3 7.9 698 216 0 12.11 Methyl γ-linolenate 16326-32-2 C19H32O2 292.2 23.0 269 22 3 12.18 Methyl linolelaidate 2566-97-4 C19H34O2 294.3 30.0 358 162 2 12.23 cis-9-Oleic acid methyl ester 112-62-9 C19H36O2 296.3 6.0 56 650 1 12.32 Methyl stearate 112-61-8 C19H38O2 298.3 14.0 993 172 0 12.92 cis-5,8,11,14-Eicosatetraenoic acid methyl ester 2566-89-4 C21H34O2 318.3 0.8 41 78 4 12.96 cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester 2734-47-6 C21H32O2 316.2 0.2 4 28 5 13.00 cis-8,11,14-Eicosatrienoic acid methyl ester 21061-10-9 C21H36O2 320.3 3.0 340 234 3 13.07 cis-11,14-Eicosadienoic acid methyl ester 2463-02-7 C21H38O2 322.3 10.0 194 523 2 13.09 Methyl cis-11-eicosenoate 2390-09-2 C21H40O2 324.3 2.8 31 1965 1 13.11 cis-11,14,17-Eicosatrienoic acid methyl ester 55682-88-7 C21H36O2 320.3 8.0 242 51 3 13.20 Methyl arachidate 1120-28-1 C21H42O2 326.3 10.0 880 338 0 13.61 Methyl heneicosanoate 6064-90-0 C22H44O2 340.3 20.0 861 203 0 13.74 cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester 301-01-9 C23H34O2 342.3 0.0 2 na 6 13.94 cis-13,16-Docosadienoic acid methyl ester 61012-47-3 C23H42O2 350.3 12.0 139 301 2 13.95 Methyl erucate 1120-34-9 C23H44O2 352.3 6.0 22 816 1 14.06 Methyl behenate 929-77-1 C23H46O2 354.3 16.0 746 260 0 14.57 Methyl tricosanoate 2433-97-8 C24H48O2 368.4 16.0 999 281 0 15.04 Methyl nervonate 2733-88-2 C25H48O2 380.4 3.4 20 985 1 15.18 Methyl lignocerate 2442-49-1 C25H50O2 382.4 14.0 110 390 0 Ret. Time 3 EI vs. Cold EI Molecular Ion Relative Intensity NIST EI Molecular Ion Relative Intensity 0 Methyl butyrate Methyl hexanoate Relative Intensity % 10 15 20 25 5 1.5 Methyl undecanoate 2.0 1200 Methyl octanoate 16 Methyl undecanoate Methyl laurate Methyl laurate 6.0 4.0 Methyl myristate 4.0 6.0 Methyl palmitate 10 Methyl heptadecanoate 7.9 Methyl stearate Methyl heneicosanoate Methyl behenate 16 Methyl tricosanoate 16 Methyl cis-10-pentadecenoate 4.2 Methyl palmitoleate Methyl lignocerate Methyl myristoleate Methyl cis-10-pentadecenoate 1 Double Bonds 3.5 cis-9-Oleic acid methyl ester 6.0 2.8 Methyl erucate 6.0 Methyl nervonate 3.4 2 Double Bonds 10 cis-13,16-Docosadienoic acid methyl ester 12 Methyl γ-linolenate 3 Double Bonds 3.0 8.0 0.8 861 746 999 110 96 102 Methyl palmitoleate 89 cis-10-Heptadecenoic acid methyl ester 77 cis-9-Oleic acid methyl ester 56 Methyl cis-11-eicosenoate 31 Methyl erucate 22 358 cis-11,14-Eicosadienoic acid methyl ester cis-13,16-Docosadienoic acid methyl ester 194 139 Methyl γ-linolenate cis-8,11,14-Eicosatrienoic acid methyl ester 23 cis-11,14,17-Eicosatrienoic acid methyl ester 993 880 Methyl linolelaidate 30 cis-8,11,14-Eicosatrienoic acid methyl ester 698 Methyl nervonate 20 Methyl linolelaidate cis-11,14-Eicosadienoic acid methyl ester 590 Methyl heptadecanoate Methyl tricosanoate 6.0 cis-10-Heptadecenoic acid methyl ester 405 Methyl palmitate Methyl behenate 14 4.0 259 Methyl pentadecanoate Methyl heneicosanoate 20 Methyl lignocerate 196 Methyl myristate Methyl arachidate 10 Methyl myristoleate 130 Methyl stearate 14 Methyl arachidate 85 Methyl tridecanoate 0 Double Bond Methyl tridecanoate Methyl pentadecanoate 4 Double Bonds 5 Double Bonds 6 Double Bonds cis-11,14,17-Eicosatrienoic acid methyl ester 269 340 242 cis-5,8,11,14-Eicosatetraenoic acid methyl ester 41 cis-5,8,11,14,17-Eicosapentaenoic acid methyl…3.6 cis-4,7,10,13,16,19-Docosahexaenoic acid…2.0 NIST EI Relave Intensity Cold EI Relave Intensity Figure 3. NIST EI molecular ion relative intensity % sorted by number of double bonds and molecular weight. Compounds are color coded by number of double bonds. Figure 4. Much larger Cold EI (hashed pattern) molecular ion relative intensity % compared to NIST EI (solid pattern). Values >100 have the next largest peak defined as 100%. Sorted in this manner and extrapolating with this limited data set, it appears that very short-chained and unsaturated FAMEs with 4 or more double bonds are more likely to produce small molecular ions, while longer chain saturated FAMEs and those with 2 or 3 double bonds tend to produce larger ones. This enhanced M+• in Cold EI provides improved selectivity and less ambiguous molecular weight determination in complex mixtures. It also provides a more intense and unique ion which can enhance MS/MS selectivity and detection limits. Cold EI was found to enhance the molecular ions in all FAMEs. Large M+• intensity enhancements are noted for mid-length unsaturated linear chains. Nearly all the compounds with EI M+• RI (%) below 5% now are above 5%. This is seen in Figure 4. The most unsaturated compound (cis-4,7,10,13,16,19Docosahexaenoic acid methyl ester) exhibited no M+• at all in EI, but shows a discernable one in Cold EI. The average increase of the M+• RI (%) was a factor of 33. The large observed enhancements make the M+• clearly more unique and thus selective for Cold EI than for EI. Cold EI M+• mass chromatograms can be used for quantification with better selectivity than a lower mass fragment ion which might otherwise have to be used for EI. 4 1000 Methyl decanoate 50 Methyl decanoate cis-4,7,10,13,16,19-Docosahexaenoic acid… 0.0 Relative Intensity % 400 600 800 Methyl hexanoate 4 3.8 cis-5,8,11,14,17-Eicosapentaenoic acid methyl… 0.2 200 Methyl butyrate 48 0.5 Methyl cis-11-eicosenoate 0 35 1.4 Methyl octanoate cis-5,8,11,14-Eicosatetraenoic acid methyl ester 30 Cold EI spectra of some FAMEs with smaller EI M+• relative intensities are shown in Figure 5 with their NIST library search results, and red highlighting of the molecular ion. Some of the names are synonyms. Cold EI cooling is shown to increase the RI (%) of the M+• and reduce fragmentation. This lower internal energy may also change the preferred fragmentation energies and kinetics, leading to the enhancement of mid-mass ions more stable at these lower energies. 74 100 87 100 87 143 102 50 71 43 45 0 40 50 55 49 53 44 42 41 55 57 59 61 60 64 72 69 69 72 75 77 79 75 86 88 90 92 88 96 0 101 87 59 75 43 49 55 60 65 83 69 43 97 50 100 60 70 80 90 Head to Tail MF=620 RMF=627 79 170 177 185 185 200 170 200 169 157 143 100 40 119 133 150 161 119 55 175 161 175 187 87 133 203 80 90 100 110 120 130 140 150 160 Head to Tail MF=577 RMF=584 180 190 Undecanoic acid, methyl ester 348 203 217 233 247 259 318 286 233 247 262 303 3 275 287 318 0 79 80 41 55 67 111 50 91 43 100 100 120 RT12.9269 83 97 111 125 150 67 60 70 50 217 105 100 60 RT8.5085 105 87 41 49 57 67 50 100 50 40 149 149 129 74 Butanoic acid, methyl ester 91 100 41 101 87 74 43 RT2.3314 50 126 115 50 40 0 129 115 109 55 71 100 200 157 101 50 140 160 180 200 Head to Tail MF=760 RMF=771 220 240 260 280 300 320 5,8,11,14-Eicosatetraenoic acid, meth 69 83 305 152 166 180 194 208 222 236 250 264 167 181 195 125 221 236 250 264 380 330 362 2 362 2 380 334 291 306 348 97 55 40 60 80 100 RT15.0414 120 140 160 180 200 220 240 Head to Tail MF=627 RMF=641 260 280 300 320 340 360 Figure 5. Comparison of Cold EI spectra (top, red) to NIST library-searched EI reference spectra (bottom, blue) showing the enhanced Cold EI molecular ion (boxes). Conclusion Cold EI GC/MS enhances FAMEs molecular ion intensity, improving analysis specificity and ease of molecular ion identification for compounds with low EI molecular ion relative intensity. Not for use in diagnostic procedures. * PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2015, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 012360_01PKI 380 15-Tetracosenoic acid, methyl ester, (Z