A Modular HPLC System for Routine Analysis of Capsaicin from Hot Sauces

Michael Waite, Andrew Aubin, Tanya Jenkins Waters Corporation, Milford, MA, USA

INTRODUCTION METHODS RESULTS AND DISCUSSION The Scoville Organoleptic Test was the first method used to determine pungency of a pepper1. However, it can be less Capsaicin, and several other related compounds Extraction A total of six samples were analyzed; all were commercial A reliable since it is subject to human perception. In this

such as dihydrocapsaicin, are called grade hot sauces purchased at a local supermarket (two red method, a solution of pepper extract is diluted in sugar water Capsaicin and dihydrocapsaicin (Figure 1) were extracted from capsaicinoids and are produced as secondary pepper based, two habanero pepper based, one jalapeno until the "heat" is no longer detected by a panel of tasters. six commercial hot sauces. A 10 mL aliquot of each hot sauce metabolites by chili peppers. Because of the pepper based and one cayenne pepper based sauce). The degree of dilution gives its measure on the Scoville scale. was transferred into a 50 mL centrifuge tube. Next, 2 mL of burning sensation caused by capsaicin when it Injections of sample extracts were bracketed by replicate For example, bell peppers which contain no capsaicin have a comes in contact with the mucous membranes dichloromethane was added and the tube was vortexed for injections of the capsaicin and dihydrocapsaicin standards Scoville rating of zero, meaning no there was no heat detected of the mouth, it is frequently used in food approximately 1 minute. The tube was then placed in a (Sigma-Aldrich Chemical Co.) The Breeze 2 System has a very even undiluted. However, the hottest chilies, such as a products to give them added spice or centrifuge for 10 minutes at 3000 RPM. Following simple-to-use interface which allows the user to easily set-up habanero pepper, can have a rating of 200,000 or more, "heat" (pungency). Capsaicin can also be found centrifugation, 1 mL of extract was withdrawn from the tube samples for analysis, process and report the final results in a indicating it had to be diluted 200,000-fold before the heat was and evaporated to dryness using nitrogen gas. The material in topical lotions where it acts as a pain reliever Figure 6. First blank injection after the highest concentration preformatted report (Figure 3). Example chromatograms for undetectable.2 According to Tainter and Grenis, 1ppm for minor aches and pains that may be left in the test tube was then reconstituted in 2 mL of standard displayed carryover of 0.02%, below 2707 instrument the two standards and a sample are found in Figure 4. capsaicin (1mg/kg) is equivalent to about 15 Scoville units. specification of 0.05%. associated with backaches, strains, sprains and methanol. Sample extracts were passed through a 0.45μm B This provides for a comparison between the HPLC results and arthritis. The degree of heat found within a syringe tip filter prior to HPLC analysis. the listed Scoville numbers on the product labels (Table 1). The food can be measured on the Scoville scale, CONCLUSIONS however high performance liquid Scoville number as determined by HPLC was within the ranges HPLC Conditions • Confirmation and quantification of capsaicin and chromatography (HPLC) is a more accurate quoted by the manufacturers. By utilizing HPLC,

dihydrocapsaicin from commercial grade hot sauces method of calculating heat pungency resulting manufacturers can more accurately and precisely monitor the HPLC System: Breeze 2 System (Figure 2) was demonstrated using a modular HPLC system. from capsaicin content in a food product. This amount of capsaicin in their products and therefore better poster describes the analysis of capsaicin and 1525 Binary HPLC Pump control the Scoville rating in each batch of product. • Retention time reproducibility was less than 0.10 % dihydrocapsaicin by HPLC utilizing the Breeze 2 2707 Autosampler RSD and the area count repeatability was less than System, a simple-to-use, modular 2489 Dual Wavelength UV/Vis 0.30% for both compounds. chromatographic system. The resulting method • Calibration curves were linear (R2 > 0.999) for both possesses excellent area count reproducibility, Column: 4.6mm X 100mm, Atlantis T3 3μm capsaicin and dihydrocapsaicin. retention time reproducibility, reliable o Column Temp: 40 C C quantification, and low carryover performance, • The habanero based sauces, showed higher Injection Vol: 25 μL demonstrating the excellent performance of this concentrations of capsaicin and dihydrocapsaicin than modular system. Needle Wash: 90/10 Water/Methanol (500μL) the red pepper, cayenne pepper, and jalapeno pepper Mobile Phase A: Water based hot sauces. Figure 3. Acquisition view on the Breeze 2 System Mobile Phase B: Methanol • The Scoville heat units determined by HPLC were

Flow Rate: 1.50 mL/min within the range quoted on the hot sauce labels. A Excellent reproducibility was observed with this methodology • Blanks showed little carryover @ 0.02%, Gradient: Time %A %B for both capsaicin and dihydrocapsaicin. Both compounds had demonstrating the excellent carryover management 0.00 60.0 40.0 a %RSD for area of less than 0.30%, and a %RSD for capabilities of the 2707 autosampler. 8.00 15.0 85.0 retention time of less than 0.10%. It was confirmed that Figure 4. Example chromatograms for a Capsaicin Standard • The simple-to-use modular Breeze 2 HPLC system, B 10.00 1.00 99.0 capsaicin and dihydrocapsaicin were present in all of the hot (A); a Dihydrocapsaicin Standard (B); and a Habanero Based Figure 5. Calibration Curve of Capsaicin in the Review window consisting of the 2707 autosampler, 1525 Binary Hot Sauce (C) Figure 1. Chemical Structure of Capsaicin (A) and Dihdydro- 13.00 1.00 99.0 sauces analyzed. The amounts were calculated from a six of the Breeze 2 Software HPLC pump, 2489 UV/VIS detector is an excellent capsaicin (B) point linear calibration curve and are displayed in Table 1. 15.00 60.0 40.0 choice for this analysis and for other analyses in the

Standard concentrations ranged from 0.028 to 0.84 mg/mL for Capsaicin Dihydrocapsaicin Calculated Scoville Scoville Heat Units QC laboratories of the food and beverage industry. Wavelength: 281 nm both compounds. Excellent linearity was observed for both Amount Amount Heat Units (Manufacturer Label) 2 2 Data Rate: 5 Hz Capsaicin (R = 0.999963) and dihydrocapsaicin (R = Red Pepper Based Sauce 0.228 mg/mL 0.122 mg/mL 3,420 2500-5000 0.999961), (Figure 5). Time Constant: 0.4 sec (Normal) Jalapeno Based Sauce 0.069 mg/mL 0.037 mg/mL 1,035 600-1200 References Habanero Based Sauce 0.479 mg/mL 0.067 mg/mL 7,185 7000-8000 Methanol blanks were injected throughout the sample set to Cayenne Based Sauce 0.046 mg/mL 0.030 mg/mL 690 747 1. The Journal of the American Pharmacists Association 1912; assess carryover. Using a wash solvent of 90/10 water/ Red Pepper Based Sauce 0.266 mg/mL 0.039 mg/mL 3,990 3600 1:453–4. methanol, extraction solvent blanks analyzed after the most 2. Tainter, Donna R.; Anthony T. Grenis (2001). Spices and Habanero Based Sauce 0.323 mg/mL 0.098 mg/mL 4,845 Not stated concentrated standard showed carryover below instrument Seasonings. Wiley-IEEE, p.30. Figure 2. Breeze 2 Modular HPLC System Table 1. Amounts of Capsaicinoids present in the Hot Sauce Samples and the corresponding Scoville Heat Units specification (Figure 6).