Application Note Flavors and Fragrances
Profiling Flavors and Fragrances in Complex Matrices Using Linear Retention Indices Without Sample Preparation
Author Abstract Eleazar Rojas Santiago Agilent Technologies, Inc. This Application Note describes a method for the analysis of flavor and fragrance compounds. The method deploys gas chromatography/mass spectrometry (GC/MS) using backflush and a thermal separation probe at constant flow with an Agilent Intuvo 9000 GC and an Agilent 5977B GC/MSD. Flavor and fragrance compounds in complex matrices such as soap, scented candles, toothpaste, cream liqueur, and fabric softener were analyzed without sample preparation. The samples were introduced into the GC/MSD system using the thermal separation probe. Identification was done using deconvoluted mass spectra and incorporating linear retention index results. In addition, a hyperlink was created to connect the compound name with a fragrance and flavor website to obtain organoleptic and cosmetic information. Introduction supplier), temperature program, and, The innovations in the Intuvo 9000 GC to a lesser extent, on the carrier gas include: The Agilent Thermal Separation Probe velocity. Therefore, it is sometimes • A direct heating system, which is (TSP) is ideal for fast GC/MS analysis difficult to reproduce published RIs in faster, uses half the power, and of a variety of dirty liquid and solid different laboratories. Most companies takes half the bench space of a samples in food testing, forensics, and in the flavor and fragrance industry use conventional air-bath oven environmental applications. Advantages in‑house methods based on historical of the thermal separation probe include: choices of columns and conditions. • Ferrule-free direct connections with a plug-and‑play flowpath eliminate • Little or no sample preparation Agilent MassHunter Unknowns Analysis a major source of complexity and required software uses a deconvolution process leaks. that helps identify compounds even • Greater flexibility with less risk than • The unique disposable Intuvo Guard traditional direct sample probes when they are hidden under coeluting matrix compounds. The deconvolution Chip eliminates the need for column • Low risk of contamination or process is automated, and takes trimming. decrease in performance typically approximately 1 to 2 minutes for a total associated with traditional direct ion chromatogram (TIC). The process Experimental sample probes not only allows analysts to get reliable Analyses were performed on an Intuvo • Control sample delivery by adjusting and reproducible results fast, but also 9000 GC equipped with a multimode split flow ratios, eliminating minimizes false positives and false inlet (MMI) and post column backflush. 5 the possibility of overload or negatives . The MMI was set to 60 °C to introduce contamination of the detector Because many retention data are already the TSP at a low temperature, thus • Temperature programming in the available as RIs, it was also evaluated avoiding loss of light components inlet and GC column for improved whether these data could be transferred before the actual injection occured. identification of multicomponent into absolute retention times that match After introducing the TSP, the MMI samples, which is not possible with with locked retention times. It was temperature was ramped to 280 °C traditional direct sample probes shown that RIs from existing retention at 600 °C/min. Separation was • Two ways to use TSP with GC/MS index libraries can be recalculated as done on an Agilent HP‑5MS, 30 m systems: separate complex samples absolute retention times that match with × 0.25 mm id, 0.25 µm column (β = 250) using a longer analytical column, or experimental data. In this study, two (p/n 19091S-433‑INT). Helium at transfer the neat sample to the MS libraries with RIs were used: NIST 2017, approximately 65 kPa (9.43 psi) at using a short deactivated capillary and the Agilent flavor and fragrance RTL constant flow was used as the carrier 6 column library . gas. Table 1 summarizes the analytical conditions. GC/MS has been used for many years for the analysis of fragrance and flavor compounds1. GC/MS is probably the Table 1. GC/MS analytical conditions. most powerful technique, and extended Parameter Description mass spectral libraries are available, but Column HP-5MS, 30 m × 0.25 mm id, 0.25 µm (p/n 19091S-433-INT) do not enable complete identification. Injection MMI, split ratio 100:1, 0.2 minutes 40 °C, then 900 °C/min to 300 °C In flavor and fragrance quality control, GC/MS with retention indices (RIs) is Carrier gas Helium (13.4 psi), constant flow still frequently used as a complementary RTL Flow set to 1.46 mL/min to give a retention time of 32.000 minutes for n-hexadecane technique to GC/MS. Several libraries are Oven program 60 to 240 °C at 3 °C/min (60 minutes analysis time) available with RIs for many flavor and Guard Chip Intuvo, multimode inlet 2-4 fragrance compounds . RIs are less Temperature program Track oven dependent on operational parameters MSD XTR 6 mm in scan mode (40 to 400 amu) than absolute retention times, but Detection solvent delay: 0 minutes transfer line: 300 °C they still depend significantly on the column type (stationary phase and
2 Table 2. MassHunter Unknowns Analysis method parameters. An alkanes mix from C6 to C44 was injected using the conditions Parameter Description described in Table 2 to generate the calibration retention time (CRT) file, and RT window size factor 25, 50, 100, 200 MassHunter was used to analyze the file Peak filter SNR threshold 5 and calculate library retention times. Enable Trapezoidal Match factor (RT penalty) Data were processed with MassHunter RT range: 60 seconds Penalty free RT range: 30 seconds Unknowns Analysis software, using a Min match factor 75 deconvolution process and RIs from two Library search type Spectral search libraries. The software calculates the library retention time using the CRT file and the RI from the different libraries, For temperature-programmed GC, These absolute retention times are and calculates the difference between the LRI is calculated by the following not the original retention times used library retention time and real retention equation: for the retention index calculation, but time. By applying the retention time filter calculated values. This means that and the minimum match factor, it was tr(unknown) – tr(n) retention times can be calculated from possible to eliminate misidentification. I = 100 × n + the RIs present in an existing database tr(N) – tr(n) using the locked retention times for Transformation of RIs where: n-alkanes if the column dimensions and the temperature program are the same7. The linear retention index (LRI) requires I = Linear retention index that an n-alkane mixture is analyzed n = Number of carbon atoms in the This study analyzed several samples in the elution range of the analytes of n-alkane eluting immediately before with no sample preparation. Only a interest. Each n-alkane is assigned an the analyte few milligrams were introduced into LRI value based on the number of carbon N = Number of carbon atoms in the the microvial (vial volume 40 µL). The atoms multiplied by 100. For example, n-alkane eluting immediately after selected samples were chosen because the analyte of their organoleptic properties, and octane (n-C8) is assigned an LRI of 800, t = Retention time. because it was not possible to inject nonane (n-C9) is assigned an LRI of 900, r directly into the GC port using a syringe decane (n-C10) is assigned an LRI value From the retention index, the absolute of 1,000, and so on. The LRI of a given retention time was calculated using the due to the complex matrix. analyte was calculated according to retention times of n-alkanes as reference where it elutes relative to the n-alkanes compounds. Results and discussion that eluted immediately before and after Figures 1 and 2 show chromatograms the analyte. with identified compounds and TICs of toothpaste, fabric softener, scented candle, and cream liqueur samples. Figure 3 shows the library search results from the MassHunter Unknowns Analysis software. Also, we can use the information from the Web to find out if the identified compound has a relationship with the flavor and fragrance industry (see Figure 4). If the message Sorry, your search: “Docosyl octyl ether” returned zero results. is displayed, this compound has no relationship with flavor or fragrance ingredients.
3 54. Lauryl alcohol 1,2. Hydrogen isocyanate 28. Methoxyacetic acid, octadecyl ester 55. Pentadecane 3. 3,5-Methanocyclopentapyrazole, 3,3a,4,5,6,6a-hexahydro-3a,4,4-trimethyl- 29. 1-Heneicosyl formate 56. 1-Decanol, 2-hexyl- 4. Phosphonic acid, (p-hydroxyphenyl)- 30. 1-Hexacosene 57. 1-Hexadecanol 5. Eucalyptol 31. Carbonic acid, eicosyl prop-1-en-2-yl ester 58. n-Hexadecane 6. gamma-Terpinene 32. n-Eicosane 59. Dodecyl heptyl ether 7. Linalol 33. Disulfide, di-tert-dodecyl 60. Isobutyl hexadecyl ether 8. Menthone 34. 3,5,5-Trimethylhexyl ethylphosphonofluoridate 61. 10-Heneicosene (c,t) 9. Cyclopentene, 1-isopropyl-4,5-dimethyl- 35. Docosyl octyl ether 62. Oxalic acid, allyl tridecyl ester 10. 1-Decene 36. 2-Ethylthiolane, S,S-dioxide 63. Dodecyl nonyl ether 11. 1-Decanol 37. -[2,3-dihydro-4-hydroxy-2-(2-hydroxyisopropyl)benzofuran-7-yl]chromone 64. Trichloroacetic acid, pentadecyl ester 12. Anethole 38. n-Tetracosane 65. 1-Tricosene 13. Eugenol 39. Butyl triacontyl ether 66. Nonyl tetradecyl ether 14. 7-Tetradecene, (Z)- 40. Borane, diethyl(decyloxy)- 67. Behenic alcohol 15. 1-Undecanol, acetate 41. Aminomethanesulfonic acid 68. Carbonic acid, eicosyl vinyl ester 16. Hydrogen isocyanate 42. Ethyl-2methylbutyrate 69. Silane, trichlorodocosyl- 17. 43. Benzaldehyde Tetradecane, 1-chloro- 70. n-Tetracosanol-1 18. 44. 3,7-Dimethyl-1-octanol Lauryl alcohol 71. n-Eicosane 19. 45. Octane, 4-chloro- Pentadecane 72. Oxalic acid, cyclobutyl octadecyl ester 46. 1-Decanol 20. n-Pentadecanol 73. Docosyl pentyl ether 47. Cyclopropane, octyl- 21. 1-Nonadecene 74. Isobutyl tetracosyl ether 48. 1-Dodecene 22. Lauryl acetate 75. Eicosyl octyl ether 49. gamma-Nonalactone 23. 1-Octadecanol 76. Octacosyl trifluoroacetate 50. 1-Tetradecene 24. Sulfurous acid, butyl undecyl ester 77. Hexacosyl pentyl ether 25. 1-Docosene 51. Decyl acetate 52. 1H-Benzimidazole-2-carboxaldehyde 26. Carbonic acid, hexadecyl prop-1-en-2-yl ester 53. 2',4'-Dihydroxypropiophenone 27. n-Octadecane
×107 A 1.7 1.6 Toothpaste 1.5 1.4 1.3 9 1.2 1.1 12 1.0 18 0.9 Counts 0.8 16 0.7 23 0.6 0.5 13 0.4 14 29 33 35 0.3 8 10 17 21 0.2 1 5 38 39 0.1 2 3 4 15 19 20 22 24 25 26 34 36 37 6 7 11 0 40 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38272840 42 3044313246 48 50 52 54 56 58 Acquisition time (min)
6 54 ×10 B Fabric softener 6.0 5.5 5.0 63 4.5 4.0 61
Counts 3.5 73 3.0 2.5 49 69 77 2.0 64 57 66 1.5 65 70 74 76 1.0 52 71 75 47 51 67 68 44 0.5 46 50 55 56 58 59 62 72 41 42 43 45 48 53 60 0 2 468101214161820 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Acquisition time (min)
Figure 1. Toothpaste and fabric softener chromatograms showing identified compounds and TIC.
4 1. Nitrous oxide 29. Heneicosane 57. Vanillin, isopropyl ether 2. Benzoyl isothiocyanate 30. 1-Phenyl-2-(4-methylphenyl)-diazene 1-oxide 58. Cinnamil acetate 3. 2,3-Heptadien-5-yne, 2,4-dimethyl- 31. Benzyl benzoate 59. 1-Tetradecanol 4. Cyclohexane, 1-butenylidene- 32. n-Octadecane 60. Ethanol, 2-(dodecyloxy)- 5. p-Cresol 33. 2-Methylbenoic acid, 2-formyl-4,6-dichlorophenyl ester 61. n-Pentadecanol 6. Methyl-1-silabenzocyclobutene 34. Pentacosane 62. 1-Hexadecanol 7. Methyl-1-silabenzocyclobutene 35. n-Eicosane 63. Pentadecanoic acid 8. Citronellal 36. Sulfurous acid, octadecyl pentyl ester 64. Octadecane, 1-isocyanato- 9. Menthone 37. Sulfurous acid, 2-ethylhexyl hexadecyl ester 65. Hexadecane, 1,16-dichloro- 10. L-Menthol 38. Borane, diethyl(decyloxy)- 66. 10-Heneicosene (c,t) 11. L-Menthol 39. Hentriacontane 67. Carbonic acid, pentadecyl prop-1-en-2-yl ester 12. alpha-Terpineol 40. Dotriacontane 68. Dodecyl nonyl ether 13. Cyclopentane, 1-methyl-1-(2-methyl-2-propenyl)- 41. Tetracosane, 11-decyl- 69. Dodecyl nonyl ether 14. d-Piperitone 42. Pentatriacontane 70. 1-Eicosanol 15. Benzofuran, 2-methyl- 43. Arsenous acid, tris(trimethylsilyl) ester 71. Ethanol, 2-(octadecyloxy)- 16. Menthyl acetate; d,L-methyl-2-(methylethyl)cyclohexyl acetate 44. Cyanogen bromide 72. Methoxyacetic acid, octadecyl ester 17. 2,4-Octadiene, 7,7-dimethyl- 45. 1,2-Propadiene-1,3-dione 73. Octadecane, 1-isocyanato- 18. Tridecane 46. 2,3,5-Trimethylpyrazine 74. 1-Propanamine, 3-dibenzo[b,e]thiepin-11(6H)-ylidene-N,N-dimethyl-, S-oxide 19. Eugenol 47. 2-Propynenitrile, 3-fluoro- 75. Hexadecyl nonyl ether 20. n-Tetradecane 48. 1-Hexene, 3,5-dimethyl- 76. 1-Hexacosene 21. beta-Caryophyllene 49. Nonanal 77. Docosyl pentyl ether 22. 1H-Benzimidazole-2-carboxyaldehyde 50. 1-Pyrroline, 3-ethyl- 78. 1-Propanamine, 3-dibenzo[b,e]thiepin-11(6H)-ylidene-N,N-dimethyl-, S-oxide 23. 3,4-Dihydroxypropiophenone 51. 3,7-Dimethyl-1-octanol 79. Carbonic acid, decyl hexadecyl ester 24. 1-Hydroxy-7-hydroxymethylindane, cyclic sulfite ester 52. 1-Decene 80. Docosyl octyl ether 25. Propanoic acid, 2-methyl-3-[4-t-butyl]phenyl- 53. 1-Decanol 81. 5H-Tetrazol-5-amine 26. Isoamyl salicylate 54. 2-Propenal, 2-methyl-3-phenyl- 82. 1-Propanamine, 3-dibenzo[b,e]thiepin-11(6H)-ylidene-N,N-dimethyl-, S-oxide 27. n-Hexadecane 55. gamma-Nonalactone 83. Chlorotrifluoromethane 28. Fosfosal 56. Decyl acetate 84. Borane, diethyl(decyloxy)-
×107 1.3 A Aromatic candle 1.2 39 31 1.1 36 1.0 11 0.9 0.8 35 0.7 10 42
Counts 15 0.6 9 16 0.5 19 41 0.4
0.3 7 34 18 21 23 28 0.2 3 6 12 14 17 32 4 5 25 26 27 29 40 0.1 1 2 8 13 20 22 24 30 33 37 38 0 2 468101214161820 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Acquisition time (min) 58 ×107 B 67 72 1.2 Cream liqueur 65 1.1 1.0 74 0.9 0.8 73 0.7 Counts 0.6 52 76 0.5 47 64 77 0.4 68
0.3 62 54 75 78 50 56 0.2 49 63 70 79 55 5960 69 81 44 45 48 51 57 61 66 0.1 83 42 43 46 71 80 82 0 53 46810 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Acquisition time (min)
Figure 2. Scented candle and cream liqueur chromatograms showing identified compounds and TIC.
5 Figure 3. MassHunter Unknowns Analysis software enables the evaluation of an alternative using the match factor and retention time. This information can be sorted by the match factor or by the retention time difference between the component RT and the library RT.
Figure 4. Information about eugenol and stearyl alcohol obtained from the Web, and the hyperlink was used to created a MassHunter Unknown Analysis.
6 Figure 5. Analysis of the toothpaste sample. Some components were identified by the Agilent library, others were identified by NIST17. In both cases, the system calculates the retention time using the retention index from the libraries. Also, there are two hyperlinks: the hyperlink for the CAS number goes to the NIST webpage to get chemical information, and the hyperlink for the compound name goes to the Good Scents company webpage to get information about organoleptic and cosmetic properties, suppliers, safety data sheet, and so on.
Figure 6. In this table, the RT match penalty was disabled, resulting in lauryl alcohol being detected 10 times, all of them with a match factor over 84. But only one has the lower delta RT and the highest match factor. This feature made compound identification easier. When this feature was enabled, only one compound appeared in the search results.
7 Figure 7. MassHunter Unknowns Analysis software uses a deconvolution algorithm to separate two coeluting compounds. The undeconvoluted spectrum (A), compound deconvoluted spectrum (B), and library spectrum (C) are displayed in the same window to make data review easier.
8 Conclusions References 6. David, F. et al. Analysis of Essential Oil Compounds Using A method was developed for the analysis 1. Tabacchi, R.; Garnero, J. Capillary Retention Time Locked Methods of flavors and fragrances in complex Gas Chromatography in Essential Oil and Retention Time Databases, matrices without sample preparation. A Analysis; Sandra, P., Bicchi, C., Eds.; Agilent Technologies Application small quantity of sample was deposited Huthig: Heidelberg, 1987; 1–11. Note, publication number into the microvial, and inserted in 2. Jennings, W.; Shibamoto, T. 5988‑6530EN, 2002. the inlet port. The method may be Qualitative Analysis of Flavor and 7. Sandy, C.; Butler, I. Incorporating used for quality control analysis. The Fragrance Volatiles by Glass Capillary Retention Index Results in method is retention-time-locked using Gas Chromatography; Academic Deconvoluted GC/MS Library n-hexadecane as the locking standard. Press: New York, 1980. Search Data, Agilent Technologies A retention index database, containing 3. Shibamoto, T. Capillary Gas Data Sheet, publication number approximately 400 compounds, was 5991‑8221EN, 2017. modified from an existing method. Chromatography in Essential Oil This database can be used to identify Analysis; Sandra, P., Bicchi, C., Eds.; 8. Giarrocco, V.; Quimby, B.; constituents based on their absolute Huthig: Heidelberg, 1987; 259–274. Klee, M. Retention Time Locking: retention time under the locked 4. Adams, R. P. Identification of Concepts and Applications, conditions. The locked method also Essential Oil Components by Agilent Technologies Application guarantees retention time stability as a Gas Chromatography - Mass Note, publication number function of time, between columns, and Spectroscopy; Allured Publishing 5966‑2469E, 1997. between instruments. Corporation: IL, USA, 1995. Finally, it is shown that RIs for flavor 5. Ping, X.; Menge, C.‑K.; compounds measured under specific Zslewski, M. Building operational conditions can be transferred Agilent GC/MSD Deconvolution into locked retention times using the Reporting Libraries for Any locked retention times of n-alkanes. Aplication, Agilent Technologies Thus, existing RI databases can be Technical Overview, publication translated into locked retention time number 5989-2249EN, 2005. databases.
Appendix 1. Acquire data files for the n-alkane standard mixture, reference standard mixtures, and test sample. Figure 8 shows the workflow for creating deconvoluted GC/MS libraries with 2. Use MH Unknows Analysis to deconvolute and library search the mass spectra of LRI values, and processing sample components in the n-alkane standard mixture against the NIST14 EI GC/MS library. data files in MassHunter Unknowns
Analysis software. The full procedure 3. Add the deconvoluted n-alkane mass spectra to a user .xml library, and assign nominal can be found in the Agilent Technologies LRI values to each entry . Data Sheet Incorporating Retention Index Results in Deconvoluted GC/MS 4. Create an MH Quant method for the n-alkane standard mixture, and create a .RTC file. Library Search Data, publication number 5991‑8221EN. 5. Use MH Unknows Analysis to deconvolute and library search the standard mixture data files against the NIST14 EI GC/MS library using the .RTC file to calculate LRI values; then, create a user .xml library.
6. Process the sample data file in Unknowns Analysis using the user-created LRI library and NIST14.
Figure 8. Workflow for creating deconvoluted GC/MS libraries with LRI values.
9 To provide qualitative information on 1. Open the QuantAnalysis.exe.config 4. Save the file. the components of complex flavor file in the path: 5. Start MassHunter Unknowns and fragrance mixtures, user libraries ProgramFiles\Agilent\MassHunter\ Analysis software. Now, you have can be searched in combination with Workstation\Quant\bin\ a new hyperlink in the compound commercially available GC/MS libraries and edit the link to the desired URL. name field that connects with (such as NIST17). Deploying retention This file is read-only by default, so the Web to get organoleptic and time locking or linear RIs can improve you need to disable this feature. cosmetic information, data sheet, confidence in analyte identification 2. Open the file, and look for the line: and so on (Figures 9 and 10). by reducing false positives. Mass
Figure 9. Hyperlink connecting a compound name to a web page, in MassHunter Unknowns Analysis software.
10 Figure 10. Example of compound information from an associated webpage (http://www.thegoodscentscompany.com).
11 www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019 Printed in the USA, September 4, 2019 5994-0546EN