White Paper - Rapid Infield Detection of the Hotness of Chillies - Principles of Electrochemical Capsaicin Detection

In this first white paper from the ZP Chilli Group we describe the core technologies that have been licensed from the Richard Compton Group at the University of Oxford - all data presented has been gathered on the ChillIPot Scoville Meter by the ZP Chilli Technology Group.

 

In subsequent whitepapers we will describe the ChilliPot Scoville Meter performance  versus HPLC, and the preparation of samples from chilli peppers, oils, sauces and powders etc.  If you have a particular technical or buisness question then please contact us, and you will find that we are extremely comprehensive in our technical  responses.

In the rest of this page we have a technical white paper on the chilli sensing technology, but immediately below we have some data from a recent trial at company where we did a test 5 x to find out the precision in the customers' hands, the answer in that case it was 5 %.


Paper

The ZP Chilli group are hot for fiery chillies, not only do we appreciate the spice we are serious about the science and characterisation of chillies.

 

In the first of our series of whitepapers on chilli sensing, we introduce the substances responsive for the fiery heat of chillies, Capsaicinoids, and the principle means of testing for the most prevalent capsaicinoid, Capsaicin. In future whitepapers we will look at the characterisation of the ChilliPot and the extraction of capsaicin from food products. 

Capsaicinoids are a group of structurally related lipophilic alkaloids which are the causative agents in the pungency (heat) of peppers. The most of common of the capsaicinoids is capsaicin, which together with dihydrocapsaicin constitutes approximately 90% of the total capsaicinoid content of chilli peppers [1]. Other members of the capsaicinoids group include nordihydrocapsaicin, homodihydrocapsaicin and homocapsaicin, however these are scarcely prevalent and of lower potency [1].  As the capsaicinoid content of peppers is directly related to the pungency of the peppers, Capsaicin quantity is often determined for commercial quality testing of foods and pharmaceutical products.

Figure 1. Capsaicin


The traditional method for determining the pungency of chillies is the Scoville Organoleptic Test which requires a group of five testers to taste a diluted chilli sample and report the pungency flavour level. Depending on the testers experience of the chilli sample and the dilution level, the chilli pepper is assigned a Scoville Heat unit (SHU). Despite the flaws in the Scoville Test, the Scoville scale has become the standard unit of measurement for the pungency of chillies.

Analytical techniques for quantifying capsaicin content include HPLC, gas chromatography, capillary electrophoresis, and colorimetry. These techniques are however laboratory based, resource-heavy, and require considerable investment from the user. Despite this HPLC has become the routine technique due to ASTA requirements of reporting pungency units.  Although HPLC has high selectivity and sensitivity its suffers from a number of disadvantages such as slow sample turnover time, re-analysis delays and the need for complicated expensive equipment. This along with the subjectivity of the Scoville Organoleptic Test has incentivised the need for alternative means of quantifying capsaicin.

We introduce the ZP ChilliPot which is the first commercial rapid, portable electrochemical system for capsaicin detection. The ChilliPot analyses solutions of capsaicin in the appropriate buffer on a microelectrode. The device is combined with a user-interface app which reports the capsaicin concentration in equivalent Scoville heat units. The ChilliPot is designed for on-site testing and does not require laboratory-based equipment for operation. A value of capsaicin-related pungency can be provided in several minutes and measurements can be continued in succession. The ChilliPot can be operated readily and intuitively by anyone be they food technologists, pharmaceuticals or hobbyists.

 

The potential for electrochemical detection of capsaicin was first realised by the elucidation of the oxidation/reduction mechanism of capsaicin and its structural analogue, catechol [2]. Kachoosangi et al. demonstrated that the first electrochemical oxidation of capsaicin is linked with an irreversible hydrolysis step, followed by the production of o-benzoquinone on capsaicin which then undergoes a reversible redox loop [2]

Figure 2.  Mechanism for the electrochemical oxidation/reduction of capsaicin [2].


Cyclic Voltammetric quantification of capsaicin

Exploiting the electrochemical activity of capsaicin, the capsaicin content in solution can be determined using cyclic voltammetry.  Cyclic Voltammetry is an electroanalytical technique whereby the voltage is increased to a selected maximum voltage and then cycled back down to the starting voltage. During this voltage cycle, the characteristic current response which develops due to oxidation/reduction reaction is recorded on a voltammogram. Data from the voltammogram can be used to calculate concentrations of sample. Here we describe the quantification of capsaicin using cyclic voltammetry, performed using the ZP ChilliPot. 

Figure 3. ChilliPot Capsaicin quantification system


Capsaicin solutions are prepared in optimum pH buffer solution and then diluted to various concentrations. With the electrode inserted into the ChilliPot, 50µl of the sample capsaicin solution is added to the electrode surface and the measurement is initiated using optimised voltammetry conditions. A characteristic voltammogram is generated for the capsaicin. The method is then repeated with varying concentrations of capsaicin. The resulting voltammogram (Figure 4) demonstrates a consistent cathodic and anodic peak for all capsaicin concentration where the peak current increases with increasing concentration of capsaicin.

The voltammogram data is converted to parts per million using a proprietary calibration algorithm. The ppm value is converted to the Scoville heat units using the standard conversion of 1ppm equivalent to 15 SHU [3]. The duration of each test once the sample is on the electrode is 1.5 minutes. The nature of the test is simple, requiring little technical expertise to use the ChilliPot and minimal equipment. The ChilliPot is lightweight and portable, numerous samples can be tested in succession and repeat testing on a batch sample can be performed without the need to reconfigure test equipment or reagents. 

 

Figure 4. Cyclic voltammogram of different capsaicin concentrations. 


Validation

The ChilliPot method was validated according to the ICH Harmonised Tripartite Guidance Q2(R1) by evaluating precision, linearity, LOQ, LOD and range parameters. To better demonstrate the relationship, standard curves of peak current signal against capsaicin concentration were constructed. 

Linearity was evaluated by assaying 10 concentrations of capsaicin in pH-optimised buffer. The testing was repeated over many weeks and the peak current values were collated. Regression analysis was performed on the data; as per classical linearity criteria, acceptable linearity was defined as a correlation coefficient close 1, and y-intercept does not differ significantly from zero. Limit of detection (LOD) and Limit of quantification (LOQ) was determined from the regression analysis of data. The range was defined as the limit of detection up to the highest concentration at which good linearity was observed.

 

Figure 5. Standard curve of peak current at varying Capsaicin concentrations. 


Precision (or repeatability) was determined by repeat measurements of select concentrations of Capsaicin preformed as described above. The relative standard deviation of 8.8% meets the ICH criteria RSD<10% for a validated test. The results were analysed by Cochran C test demonstrating homogeneity of variance at the same concentration using different electrodes. The validated parameters are summarised in Table 1. 

Table 1. Validation Summary 


Summary

To overcome the flaws encountered in the Scoville Organoleptic test and the challenging HPLC test, a rapid and easy method of capsaicin quantification has been described. The device has been validation showing low limit of detection, good linearity and precision. Results can be generated in minutes and expressed either as molar concentrations, ppm, or Scoville heat units. Considering the ease of use, the small footprint and usability, the ChilliPot system surpasses the conventional methods of capsaicin detection. The ChilliPot is ideal for anyone for wants to determine capsaicin-related pungency in a reliable way. 

[1] V. S. Govindarajan and M. N. Sathyanarayana, “Capsicum — production, technology, chemistry, and quality. Part V. Impact on physiology, pharmacology, nutrition, and metabolism; structure, pungency, pain, and desensitization sequences,” Crit. Rev. Food Sci. Nutr., vol. 29, no. 6, pp. 435–474, Jan. 1991.

[2] R. T. Kachoosangi, G. G. Wildgoose, and R. G. Compton, “Using capsaicin modified multiwalled carbon nanotube based electrodes and p-chloranil modified carbon paste electrodes for the determination of amines: Application to benzocaine and lidocaine,” Electroanalysis, vol. 20, no. 23, pp. 2495–2500, 2008.

[3] D. R. Tainter and A. T. Grenis, Spices and seasonings : a food technology handbook. Wiley, 2001.