Summary and conclusions

Development of an analysis method to determine peroxides in or released from tooth-whitening/dental-bleaching products.

Development of a catalase bioassay for the determination of peroxide

This project developed a catalase bioassay for the determination of the content of peroxide in tooth-whitening products. The catalase bioassay converts peroxide into oxygen, which is then measured with a probe.

The catalase bioassay was tested on pure peroxides (hydrogen peroxide, urea peroxide, calcium peroxide, sodium perborate and sodium percarbonate), all of which are active substances in tooth-whitening products, as well as on a number of different tooth-whitening products on the Danish market.

The newly developed catalase bioassay was moreover compared to chemical analysis methods. It was concluded that the catalase bioassay has some advantages over the chemical methods.

The project was conducted by the National Environmental Research Institute (NERI), University of Aarhus, Department of Environmental Chemistry and Microbiology.

Need for an improved method to determine active substances in tooth-whitening products

In Denmark today it is possible to buy tooth-whitening agents over the counter. This means there is an increased need to be able to monitor this product type. Almost all tooth-whitening agents contain peroxide as an active substance; either as free hydrogen peroxide or bound to other substances. Some products contain several different peroxide compounds and this is a problem in traditional analytical methods which, typically, can only analyse one substance at a time. Furthermore, there are problems associated with analysing peroxide bound to other substances, as many factors may influence the release of peroxide.

The purpose of this project was to develop and validate a new method of analysis to determine the overall content of peroxides in tooth-whitening agents - irrespective of whether the peroxide is free, bound or appears together with other peroxide compounds.

Establishment of a protocol for the determination of peroxides using an enzymatic bioassay

The newly develop catalase bioassay involves enzymatic conversion of hydrogen peroxide to water and oxygen in an analysis chamber, while continuously measuring changes in oxygen concentrations. In an aqueous solution, hydrogen peroxide will split into oxygen and water spontaneously, but the process is very slow. Adding catalase increases the rate of reaction significantly.

The project involved the design and development of an oxygen measurement and regulator system, which basically consists of a closed chamber and a galvanic oxygen probe for the continuous measurement of oxygen released.

Method development was carried out on six different pure chemicals in order to identify the range of and the optimal conditions for the method. Based on this, a protocol was established, which was subsequently used in the analysis of seven different tooth-whitening products.

Main conclusions

The catalase bioassay can be used as an effective and accurate method to determine the overall content of peroxide in tooth-whitening products.
The lower detection limit for peroxides measured with the catalyse bioassay is 0.0006% urea peroxide, corresponding to 0.0002% hydrogen peroxide.
The catalase bioassay is useful for the measurement of hydrogen peroxide, urea peroxide, calcium peroxide, sodium perborate and sodium percarbonate.
Data from the catalase bioassay correspond well to traditional redox titrations.
In contrast to traditional redox titrations, the catalase bioassay is not affected by any reducing or oxidising chemicals in the products.
Determination of peroxide in tooth-whitening agents through use of the catalase bioassay is quick and easy.
The catalase bioassay is robust, simple, quick and has good reproducibility and accuracy (coefficient of variation < 2%).
The catalase bioassay is suitable for use on tooth-whitening products with hydrogen peroxide as the active substance, no matter whether the peroxide is present as free hydrogen peroxide or bound in releasers.
Products containing several different active substances may be tested.
It is probable that the catalyse bioassay developed may also be suitable for use on other product types containing hydrogen peroxide in free or bound state.

Project results - protocol to determine free or bound hydrogen peroxide in tooth-whitening agents

Based on a number of trial runs using the catalase bioassay, a protocol was described. The protocol is outlined in the following:

In cooperation with Loligo-systems (Tjele), a sealed measuring chamber (approx. 100 ml) was constructed fitted with an oxygen probe, and with inlet ports for the injection of sample material. The chamber is filled with a room-temperature phosphate buffer (pH=7) outgassed with nitrogen, and is sealed with an acrylic lid. Catalase (5,000 units dissolved in 40 µl 50 mM phosphate buffer) and a sample quantity that, as a maximum, can result in the creation of oxygen corresponding to 80% of the buffer’s oxygen saturation are injected through the inlet ports. Changes in oxygen concentrations are measured continuously and when the signal is stable a reading is made and subsequently converted to the peroxide content in the sample. Under the conditions described, reading can be performed after 5 minutes. The assay is run at a constant temperature, set at 22 °C in the protocol, and during stirring in the measuring chamber.