TechnicalApplication Support Note Note-000 10355 . It is included here as it is known to be It is included here as it is known to be . [3] used in pepper spray products as a flavoring component. can be caused by a number of compounds of natural can be caused by a number of compounds include capsaicinoids, along with many others, origin that, and thymol. piperine, 404-86-4) and related capsaicinoid Capsaicin (CAS 19408-84-5) compounds like dihydrocapsaicin (CAS and are typical occur in plants from the genus Capsicum Due to of hot (chili) and non-pungent (bell) peppers. is a banned capsaicin its stimulating characteristics, 94-62-2) (CAS Piperine substance in equestrian sports. belongs to a group of alkaloids typical of plants from like black pepper (Piper nigrum the Piperaceae family, 89-83-8) is a Thymol (CAS a most popular spice. L.), Thymus naturally occurring monoterpene phenol from Due to strong flavor. known for its distinctive, vulgaris, its antimicrobial attributes it is also used as an antiseptic Trans-cinnamaldehyde ingredient in household products. 104-55-2) occurs naturally in the bark of cinnamon (CAS trees and other species of the genus Cinnamomum and gives the cinnamon powder its typical flavor and for cinnamaldehyde The best known application odor. and an but it is also used as a fungicide is flavoring, antimicrobial Thymol 2 Thermo Fisher Scientific, Singapore Thermo Fisher Scientific, 2 Receptor sensitizing and activation

. Capsaicin [2] . Such receptors can respond to chemical . , Hans-Joachim Huebschmann

1 [1] Piperine

Alpha Analytical Pte. Ltd., Singapore, Alpha Analytical Pte. Ltd., Alex Chen GC-MS/MS Analysis of the Receptor- Receptor- of the Analysis GC-MS/MS Spice Ingredients Active Natural Sensitizing Thymol and Piperine, Capsaicin, 1 -Methyl-t-Cinnamaldehyde stimuli caused by a variety of natural and synthetic stimuli caused by a variety of natural and Upon receptor activation the nerve signal is compounds. a sensation consumers of interpreted as a painful burning, hot dishes recognize Pungent spices are common ingredients for food Pungent spices are common ingredients Spices have been preparations in all cooking traditions. Chinese used as well for a long time in the traditional Beyond that there is a modern use of the medicine (TCM). personal defenseactive ingredients of spices in a variety of due such as pepper spray, and law enforcement products, effects. to their immediate physiological irritation with specific Many of these active ingredients interact of the receptors and modulate the sensing mechanism human body Introduction α Spices, active ingredients, traditional Chinese medicine TCM, traditional Spices, active ingredients, Keywords: fungicides, DTCs, hydrolysis, Thiram, - fungicides, DTCs, hydrolysis, analyte protectants. medical applications, personal defense products, pepper spray, MRM, pepper spray, personal defense products, medical applications, 2

This application note describes the GC-MS/MS analysis acquisition method based on the timed-SRM mode. The of extracts from spices as a highly selective tool for choice of timed-SRM dispenses with the tedious manual the quantitative determination of the representative search and setting of several segment breaks, as required ingredients of natural active spice ingredients capsaicin, by former triple quadrupole systems. Timed-SRM uses a piperine, thymol, and cinnamaldehyde. short window around the compound retention time, the duration of which is user definable. Once the AutoSRM Experimental Conditions process is completed, the generated acquisition method as All measurements have been carried out using the Thermo shown in Table 2 is used immediately for sample analysis. Scientific™ TSQ 8000™ triple quadrupole GC-MS/MS system equipped with the Thermo Scientific™ TRACE™ The molecular structures of the natural active compounds 1310 GC with SSL Instant Connect™ SSL module and under investigation in this application note have very Thermo Scientific™ TriPlus™ RSH autosampler. The polar groups. These polar sites pose a special challenge method details are given in Table 1. to the GC system because of their active nature and long- term instability, especially in real life matrix samples. The TSQ 8000 MS acquisition method has been developed automatically by AutoSRM, a unique MS/MS Table 1. TRACE 1310 GC and TSQ 8000 MS/MS method parameter method development tool included in the TSQ 8000 TRACE 1310 GC software suite. The AutoSRM method development starts from a standard solution vial in the TriPlus RSH Injection mode splitless autosampler and automatically determines retention Splitless Time 1.0 min time, the two most suitable precursor and product ions, GC Column Restek™ RTX™-5Sil MS, and optimizes the collision energy for best sensitivity. 15 m × 0.25 mm × 0.25 μm The program runs automatically and provides the SRM Carrier gas He（99.999 %） Flow 1.2 mL/min, constant flow Temperature program 50 °C, 2 min 20 °C/min to 300 °C, 2 min Transfer line temperature 280 °C Total analysis time 14.6 min

TriPlus RSH Autosampler Injection volume 1 µL

TSQ 8000 MS/MS Ionization mode EI, 70 eV Ion source temperature 250 °C Figure 1. TSQ 8000 with TRACE 1310 GC and TriPlus RSH Scan mode SRM using timed SRM autosampler SRM transition setup automatically build-up by AutoSRM software, transitions see Tab.2

Table 2. MRM acquisition method created by AutoSRM

CAS Precursor Product Collision Peak Compound name RT Number Mass Mass Energy Width [min] [m/z] [m/z] [V] [min] Thymol 89-83-8 6.24 135.1 91.1 15 5 Thymol 89-83-8 6.24 150.1 135.1 10 5 α-Methyl-trans-cinnamadehyde 101-39-3 6.51 145.1 91.1 25 5 α -Methyl-trans-cinnamadehyde 101-39-3 6.51 145.1 115.1 20 5 Capsaicin 404-86-4 12.64 137.0 94.0 20 5 Capsaicin 404-86-4 12.64 137.0 122.0 15 5 Dihydrocapsacin 19408-84-5 12.89 137.0 94.0 20 5 Dihydrocapsacin 19408-84-5 12.89 137.0 122.0 15 5 Piperine 94-62-2 14.08 200.8 115.1 20 5 Piperine 94-62-2 14.08 285.0 172.7 10 5 3

Figure 2. TRACE 1310 GC method setup SSL injector

RT: 12.64 Sample Measurements AA: 54365 SN: 59 100 RT: 12.89 The active compounds capsaicin and dihydrocapsacin AA: 44242 SN: 39 elute with only a short retention time difference. A good

e 80

nc separation free from peak tailing is necessary for a reliable da 60 un peak integration for low RSD values at low concentration Ab

ve 40 levels (Figure 3). It was found with different types of GC ti la columns that the quality of the column deactivation, age Re 20 (A) A 15 m column, well deacvated of the column and matrix deposits have a detrimental 0 RT: 14.70 effect on the capsaicin and dihydrocapsacin peak shape RT: 14.44 AA: 80196 AA: 81163 SN: 23 and quantitative reproducibility. Also, piperine was 100 SN: 22 affected while thymol always showed symmetrical peak 80 shapes, apparently being unaffected by the increasingly 60 active column film conditions.

40

20 (B) A 30 m column, with acve sites To preserve inert conditions with the inlet liner and analytical column for high quantitative precision and 0 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 reproducible results with a high number of samples, an Time (min) analyte protectant was co-injected with the extract of active analytes [4, 5, 6]. These compounds are known to be Figure 3. Capsaicin and dihydrocapsacin elution, (A) from a short 15 m well deactivated used in pesticides analysis, also comprising a number of capillary column at 100 ppb, (B) from a 30 m column with active sites at 500 ppb, active and polar compounds. A concentration of 2 ppm resulting in poor peak shape and low S/N value. Both column dimensions are 0.25 µm of sorbitol was added to the extracts in all experiments. film thickness, 0.25 mm ID, and no analyte protectant was added. 4

Results RT: 14.49 RT: 14.75 AA: 27108 100 CapsaicinDAA: 24730 ihydrocapsacin All measurements were carried out using the above described instrumental setup with a co-injection of

50 (A) 14.79 sorbitol as analyte protectant. Symmetrical peak shapes 14.54 14.84 14.94 15.14 14.06 14.14 14.35 15.21 for all compounds of interest, including the critical pair 0 capsaicin and dihydrocapsacin, could be achieved, as 14.0 14.2 14.4 14.6 14.8 15.0 15.2 15.4 Time (min) shown in Figure 4. The individual peaks for selected RT: 14.51 AA: 79906 compounds of the calibration runs, normalized to 100% 100 RT: 14.77 AA: 72753 each, are given in Figures 5-8. The linear quantitative calibrations with a zoom into the low concentration (B) 50 10 ppb range of 10-200 ppb are shown in Figure 9.

0 14.0 14.2 14.4 14.6 14.8 15.0 15.2 15.4 Time (min)

Figure 4. Capsaicin and dihydrocapsacin peak shape, (A) without and (B) with analyte protectant, both runs at 10 ppb concentration, 30 m column of Figure. 3.

RT: 7.92 RT: 8.24 AA: 96993015 AA: 33817857 SN: 82873 SN: 825929

RT: 8.24 AA: 6346374 SN: 310523 RT: 7.86 AA: 1791377 SN: 1561 1000 ppb 1000 ppb

RT: 7.92 RT: 8.24 AA: 8920897 AA: 2972548 SN: 18416 SN: 221745 200 ppb 200 ppb

RT: 7.93 RT: 8.24 AA: 913005 AA: 338618 SN: 1913 SN: 13200 RT: 8.27 100 100 AA: 83321 e SN: 924

nc e 100 ppb 80 80 100 ppb da

60 undanc 60 bun A Ab 40 40 ve ve ti ti la 20 la 20 Re 10 ppb Re 10 ppb 0 0 7.8 7.9 8.0 8.1 8.0 8.1 8.2 8.3 8.4 8.5 Time (min) Time (min)

Figure 5. Thymol calibration peaks 10-1000 ppb. Figure 6. α-Methyl-trans-cinnamaldehyde calibration peaks 10-1000 ppb.

RT: 16.49 AA: 8088783 SN: 12182

RT: 14.50 RT: 16.49 AA: 23410769 SN: 38532 AA: 1341210 SN: 5366

RT: 14.50 AA: 3808879 RT: 16.49 SN: 10960 AA: 665343 1000 ppb RT: 14.66 SN: 1975 AA: 1029630 SN: 777 1000 ppb RT: 14.50 AA: 1859072 SN: 8500 RT: 16.50 RT: 14.66 AA: 66903 200 ppb AA: 189269 SN: 260 SN: 301 100 200 ppb RT: 14.51 e 80 AA: 203012 SN: 1352 100 RT: 14.66

e AA: 90016

undanc 60 100 ppb nc 80 SN: 213

Ab 100 ppb da un

ve 40 60 ti Ab la 40 ve ti

Re 20 10 ppb la 20 Re 0 0 10 ppb 16.3 16.4 16.5 16.6 14.0 14.2 14.4 14.6 Time (min) Time (min)

Figure 8. Piperine calibration peaks 10-1000 ppb. Figure. 7. Capsaicin calibration peaks 10-1000 ppb. 5

Figure 9. Quantitative calibrations on low concentration side 10-200 ppb.

Table 3. Precision of a spiked spice sample analysis

Day 1 Day 2 Day 3 Compound Name RSD% [area cts] [area cts] [area cts] Thymol 96.513 94.128 91.462 2.70% alpha-Methyl-trans-cinnamadehyde 97.665 93.579 92.918 2.70% Capsaicin 100.669 105.363 99.392 3.10% Dihydrocapsacin 102.752 103.852 101.089 1.40% Piperine 104.307 106.685 103.274 1.70%

Precision Conclusions For a reproducibility study, a series of three The described method using the TSQ 8000 GC-MS/MS measurements on three consecutive days was performed, system provides a very sensitive and precise assay for and the precision of the area results calculated as relative the trace analysis of receptor-sensitizing and active standard deviation (RSD %). The peak area precision for compounds like capsaicin, piperine, and thymol. Excellent thymol, α-methyl-trans-cinnamadehyde, capsaicin, and symmetrical and stable peak shape can be achieved for piperidine was determined for the low level calibration these polar spice components by using sorbitol as analyte points up to 200 ppb. Thymol shows values in all cases protectant. of 3% RSD and below. This compound is less affected by potential active sites in the GC system. The active Analyte protectants can reduce the phenomenon of compounds α-methyl-trans-cinnamaldehyde, capsaicin, poor chromatographic peak shapes and keep the and piperidine also show excellent precision data ranging chromatographic integrity over long sample series with from 0.5% to 8% RSD over the length of the study. This symmetrical peaks and very stable results with excellent excellent area precision of the other active analytes at low precision. Automatic peak integration is facilitated, peak concentration levels is achieved by the use of an analyte areas are increased, and the reproducibility improved protectant in the applied extracts. significantly.

A spiked real life spice sample has been measured All the investigated compounds, in particular capsaicin, on three consecutive days, as well, to calculate the piperine, and thymol, can be detected with high signal- precision of the measurements. The peak area data in to-noise ratio even at the low 10 ppb level. A matrix Table 3 indicate a low level spike below 10 ppb. The sample with measured concentrations well below 10 ppb reproducibility over three days for all compounds tested demonstrated the excellent reproducibility of the TRACE is in the range of 1-3%. 1310 GC system. The precision in all measured levels including the low 10 ppb concentration was below 10% RSD. References 1. Vriens, J., Nilius, B., and Vennekens, R., Herbal Compounds and Toxins Modulating TRP Channels, Curr Neuropharmacol. 6(1) (2008) 79-96. 2. Vriens, J., Appendino, G., Nilius, B., Pharmacology of Vanilloid Transient Receptor Potential Cation Channels, Mol. Pharmacol. 75 (2009) 1262-1279. 3. Ooi, L., Li, Y., et al, Antimicrobial Activities of Cinnamon Oil and Cinnamaldehyde from the Chinese Medicinal Herb Cinnamomum cassia Blume, Am. J. Chin. Med. 34 (2006) 511. 4. Anastassiades, M., Maštovská, K., Lehotay, S.J., Evaluation of analyte protectants to improve gas chromatographic analysis of pesticides, J. Chromatogr. A 1015 (2003) 163-184. 5. Mastovska, K., Lehotay, S.J., Anastassiades, M., Combination of Analyte Protectants To Overcome Matrix Effects in Routine GC Analysis of Pesticide Residues in Food Matrixes, Anal. Chem. 77(24) (2005) 8129-8137. 6. Li, Y., Chen, X., Fan, C., Pang, G., Compensation for matrix effects in the gas chromatography–mass spectrometry analysis of 186 pesticides in tea matrices using analyte protectants, J. Chrom. A 1266 (2012), 131-142.

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