Characterisation of allergens in cosmetics by GC GC TOF MS with Select-eV variable-energy electron ionisation

Transcription

1 Application Released: January 214 Application Note 522 Characterisation of allergens in cosmetics by GC GC TF MS with Select-eV variable-energy electron ionisation Summary This Application Note shows that BenchTF instruments, coupled with GC GC, can provide a high-performance solution for the detection and quantitation of allergens in complex cosmetics extracts. Revolutionary variable-energy electron ionisation technology was also shown to enhance both the sensitivity and selectivity of analyses by generating mass spectra containing structurally-significant fragment ions with an improved molecular ion signal. This Application Note describes the use of Select-eV revolutionary MS source technology that enhances compound identification by enabling efficient electron ionisation at much lower (softer) energy levels without compromising sensitivity. We also show how Select-eV complements the other fundamental advantages of Markes BenchTF time-of-flight mass spectrometers for the GC GC identification of allergens. Background to BenchTF instruments Markes BenchTF time-of-flight mass spectrometers are designed specifically for gas chromatography. They are particularly appropriate for the GC GC analysis of complex samples, such as cosmetics extracts, because they offer exceptional sensitivity, spectral quality and speed: Sensitivity: Highly efficient direct-extraction technology allows BenchTF instruments to acquire full-range spectra with SIM-like sensitivity, allowing them to reliably detect trace-level analytes in a single run, which would be difficult or impossible on a quadrupole instrument. Introduction In 23, an EU Directive 1 restricting the use of allergenic compounds in fragrances was released. The Directive named a total of 27 allergens, stating that they should be labelled if present at > ppm in wash-off products (such as shower gels), or >1 ppm in leave-on products (such as perfumes). Compliance with this Directive therefore requires that these compounds are identified and quantified accurately. Two-dimensional gas chromatography with time-of-flight mass spectrometry (GC GC TF MS) is well-suited for the analysis of allergens within complex cosmetics extracts. The enhanced separation provided by the coupling of two columns of different stationary phase minimises the requirement for tedious sample preparation steps, which can also introduce error into the analytical process. Despite the superior separation afforded by GC GC, the identification of individual compounds in complex samples remains challenging when multiple compounds in a chemical class have similar spectra, or weak molecular ions. This problem can be addressed by the use of soft ionisation to reduce the degree of ion fragmentation, but this approach has been cumbersome to implement until now. Markes flagship BenchTF-Select instrument features Select-eV 12 ev ion-source technology as standard. Select-eV breaks new ground by allowing ionisation energies to be reduced on a sliding scale from to 1 ev, without impacting sensitivity, simply by changing a parameter in the method. This low-energy (i.e. soft ) electron ionisation reduces analyte fragmentation, which benefits a wide range of GC and GC GC analyses by enhancing selectivity, sensitivity, and aiding structural elucidation all while avoiding the inconvenience of reagent gases, ion source pressurisation, or changes in hardware setup typically associated with other soft ionisation techniques for GC MS. Formerly ALMSC Application Note 22. T: +44 () F: +44 () E: enquiries@markes.com

2 Page 2 Spectral quality: The reference-quality spectra produced by BenchTF instruments are a close match for those in commercial libraries such as NIST or Wiley. This enables quick and confident matching of both targets and unknowns. Speed: The ability to record full-range mass spectral information to extremely high densities (1, transient spectral accumulations per second) enables BenchTF to handle the narrowest peaks encountered in well-optimised GC GC couplings. The high stored-to-disk data rate also enables advanced spectral deconvolution and data-mining algorithms to extract maximum information from weak, matrix-masked signals. Experimental A series of calibration standards containing allergenic compounds were prepared in acetone, ranging from.2 ppm to 1 ppm. Two cosmetics extracts were also prepared; one from a cream and the other from a perfume. GC: Injector: Split/splitless injector Liner: 4. mm i.d. liner, 1 µl injection Carrier gas: He, constant flow at 1. ml/min Mode: Split Inlet temperature: 23 C Septum purge: n, 3 ml/min 2D column set: 1st dimension: SGE SolGelWax, 3 m.25 mm.25 µm 2nd dimension: DB1, 2.6 m.1 mm.1 µm Modulation loop: As for 2nd dimension Column set: Equivalent pneumatic impedance to 35 m.18 mm (calculated from K factor look-up charts for 1st- and 2nddimension columns used) Temperature programme: Main oven: 4 C (1. min), 6 C/min to C, 4 C/min to 25 C (2 min) Secondary oven: Hot jet: No offset 14 C (1. min), 6 C/min to 21 C, 4 C/min to 25 C (hold time matched to total run time) Cold jet: Dewar fill: high, 45%; low, 35% Modulation period: 2.5 s, hot-jet pulse 35 ms Total run time: 64 min TF MS: Instrument: Filament voltage: Ion source: 25 C Transfer line: 28 C BenchTF-Select (Markes International) 1.8 V Mass range: m/z 4 6 Data rate: 5 Hz with 2 spectra per data point Software: Image processing: Results and discussion GC Image (GC Image, LLC) Compound separation and identification A calibration series, consisting of four standard solutions, was analysed using the conditions described. Separation of all 29 target compounds was achieved with the reverse-phase (polar apolar) column set (Figure 1) ISTD Figure 1: Close-up view of the GC GC TF MS contour plot of an allergens standard. Compound numbers are provided in Table 1. T: +44 () F: +44 () E: enquiries@markes.com

3 Page 3 BenchTF instruments are the only GC time-of-flight mass spectrometers capable of delivering classical EI spectra, with no mass discrimination. Spectral comparisons (using the NIST database) were performed for all allergens in the calibration mixture. Example comparisons are shown in Figure 2, with retention times and library match results for all 29 target compounds being summarised in Table 1. 1,2-Dibromobenzene Forward match Reverse match Probability % Quantitation of target compounds Seven target compounds were chosen to demonstrate quantitation by BenchTF-Select citral (two isomers), cinnamyl alcohol, geraniol, limonene and lyral (two isomers). A Br Br No. Compound RT 1 (min) RT 2 (s) Forward match 1 Limonene Linalool Methyl 2-octynoate β-citral α-citral ,2-Dibromobenzene Citronellol Geraniol α-isomethyl ionone Benzyl alcohol Safrole Hydroxycitronellal Methyl eugenol Cinnamaldehyde Lilial Majantol Eugenol Amylcinnamaldehyde p-anisyl alcohol Cinnamyl alcohol Farnesol (isomer 1) Farnesol (isomer 2) Isoeugenol Farnesol (isomer 3) Hexylcinnamaldehyde Coumarin (isomer 1) (isomer 2) Amylcinnamic alcohol Table 1: Summary of results for all target allergens in the 1 ppm calibration standard, identified by matching against the NIST library. Coumarin Forward match 9 Reverse match 93 Probability 51.6% Safrole Forward match 917 Reverse match 918 Probability 28.4% Geraniol Forward match 925 Reverse match 927 Probability 42.3% H Figure 2: Comparison of the deconvolved spectra for four target analytes (top, red) with the NIST 211 library spectra (bottom, blue). T: +44 () F: +44 () E: enquiries@markes.com

4 Page 4 Peak area ratio (analyte/istd) Compound Equation R 2 α-citral y =.9x Geraniol y =.85x (isomer 2) y =.42x Limonene y =.4x Cinnamyl alcohol y =.31x β-citral y =.26x (isomer 1) y =.x template file containing qualifier expressions was prepared in GC Image data analysis software to enable automatic identification of these allergens in all samples, allowing fast processing of results. The calibration curves for the seven target analytes (Figure 3) were then used to quantify the allergens present in the cream and perfume samples. Contour plots of the cream and perfume extracts are provided in Figure 4, with identifications and quantitative results annotated. The cosmetics extracts analysed in this work contained fairly low background matrix due to robust sample preparation techniques. However, it can be seen that the GC GC TF MS method has provided the high degree of chromatographic resolution necessary to separate target compounds and potential interferences. For example, a conventional one-dimensional separation would have resulted in the co-elution of citronellol (one of the initial 29 target compounds) with geranyl acetate present in the perfume extract, as shown in Figure Concentration (ppm) Figure 3: Calibration curves for seven example target allergens. A Limonene 3.5 ISTD α-citral 1.7 β-citral 1.8 Geraniol Cinnamyl alcohol 4.4 (isomer 1) (isomer 2) B Limonene 71.5 ISTD α-citral 1.8 β-citral 2. Geraniol (isomer 1) (isomer 2) Figure 4: Quantified allergens in the (A) cream and (B) perfume extracts. Values are given in ppm. T: +44 () F: +44 () E: enquiries@markes.com

5 Page Citronellol Geranyl acetate A B Farnesol C H Citronellol A Forward match 913 H Geranyl acetate Forward match 913 B 41 Ac Figure 5: Top: Separation of citronellol and geranyl acetate two compounds in the perfume extract that would have co-eluted in a one-dimensional GC system. Bottom: The corresponding mass spectra (top, red) compared to those in the NIST library (bottom, blue). C Variable-energy electron ionisation Isomeric compounds commonly have similar mass spectra at, making precise identification of individual isomers near-impossible without the time-consuming analysis of standards to obtain retention time qualification However, Select-eV technology allows the generation of EI spectra at lower energies, so providing an extra dimension of information that allows isomers to be distinguished. This is clearly demonstrated for the farnesol isomers in Figure 6. This capability is especially useful in GC GC TF MS analyses, where the added sensitivity and selectivity delivered by lowenergy electron ionisation complement the highly structured separation space occupied by extremely complex samples. Figure 6: Top: Separation of farnesol isomers A C. Bottom: Comparison of mass spectra obtained at ionisation energies of (top) and (bottom), showing how Select-eV improves discrimination. In the spectra, intensity differences are particularly apparent for ions at m/z, and 136. T: +44 () F: +44 () E: enquiries@markes.com

6 Page 6 Safrole Coumarin S/N 12, S/N S/N 25,568 S/N Figure 7: Comparison of mass spectra obtained at ionisation energies of (top) and (bottom) for safrole and coumarin. The reduced fragmentation increases selectivity and significantly improves signal-to-noise ratios. BenchTF instruments are inherently sensitive, but Select-eV facilitates even lower detection limits in the quantitation of allergens. As an example, Figure 7 compares the and spectra for safrole and coumarin (both identified with confidence by virtue of their library match factor of >9 at ). The stronger molecular ion and reduced fragmentation at mean better selectivity, improved signal-to-noise ratios, and thus lower detection limits for these compounds. Chromatographic background (e.g. column bleed) also remains unionised at lower voltages, reducing noise and improving signal-to-noise ratios even further. Conclusion In this Application Note, we have shown that GC GC TF MS provides the sensitivity, spectral quality and chromatographic resolving power necessary for confident qualitative and quantitative analysis of allergens and other compounds in cosmetic extracts. We have also shown the enhancements that Select-eV can bring generating complementary spectra for enhanced compound identification and further improving system sensitivity and selectivity for this complex analysis. References 1. Directive 23//EC of the European Parliament and of the Council of 27 February 23 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. Trademarks BenchTF and BenchTF-Select are trademarks of Markes International. Select-eV is a registered trademark of Markes International. GC Image is a trademark of GC Image, LLC, USA. Applications were performed under the stated analytical conditions. peration under different conditions, or with incompatible sample matrices, may impact the performance shown. T: +44 () F: +44 () E: enquiries@markes.com AN522_5_18714