I think you need to be clear about the problem. Is it that allicin is unstable unless it's kept on ice, or is there some technical problem with the absorbance measurement?

If allicin is unstable, then it would seem to be hopeless to use it in a formulation that has to be used at 37oC. The absorbance measurement would be a lesser problem.

If you think there is a technical problem with the absorbance measurement, can you give more details about how it is done?

Allicin rapidly degrades when kept at temperatures more than 40 deg C , so using UV with Peltier maintaining the temperature at 37 deg C can be used to study invitro release studies

Adam B Shapiro Thank you for your reply, Dr. Shapiro. The half life of allicin at 37 ᴼC was previously reported to be about a day (Fujisawa et al. 2008 as attached), which means that it should be safe to proceed with the in vitro drug release assay. Hence, the problem could be technical as you suspected. I prepared 5 dilutions (0.0125 ug/mL, 0.025 ug/mL, 0.050 ug/mL, 0.075 ug/mL and 0.1 ug/mL at a wavelength of 207.60 nm as determined from the spectrum scan) of my allicin in 1x PBS, pH 7.4. A volume of 3 mL of allicin diluted in PBS is added to a quartz cuvette for each dilution and read in triplicate. Only PBS is used as the blank throughout. The allicin dilution is kept on ice the entire time as 4 ᴼC is the recommended temperature for storage. As previously mentioned, I've noticed that the absorbance slowly increases, if it is kept in the machine slightly longer but I'm not quite sure why. The UV Vis is utilised by other students in my lab but they have no complaints so I don't suppose its not due to that. I would like to get your thoughts on this.

Kandluri Geethika Thank you for your suggestion. Unfortunately, I don't have that type of machine at my university but I should be able to conduct the in vitro assay that way by keeping a close eye on the temperature.

It is not surprising that the absorbance of the allicin solutions at 207.6 nm increases with time. This short wavelength of light is very destructive and probably causes significant chemical changes to the molecule over time. If you can use a longer wavelength, you should get a more stable signal. This may come at the cost of lower detection sensitivity, but you can compensate for that by increasing the concentrations of the solutions.

Can I know How are you studying drug release is it in a dissolution Apparatus ??

Adam B Shapiro I used 207.6 nm because that was the lambda max that appeared in the spectrum scan. Would it be advisable to use a wavelength previously reported in other papers even if there is no lambda max? Additionally, would you recommend measuring the absorbance at more than one wavelength for the in vitro release assay once this has been sorted out? Thank you again for your time, Dr. Shapiro.

Kandluri Geethika I will be using the dialysis membrane method with a dialysis bag of MWCO 2450 Da as I don't have access to a dissolution apparatus.

If you have a spectrophotometer capable of scanning through the spectrum, you can measure the whole absorbance spectrum rather than a single wavelength. This will help to verify that the absorbance being measured is actually due to allicin.

An absorbance measurement is not very specific, because many things absorb in the UV, especially the far UV. It would be better to use a method that can identify allicin specifically and quantify it, such as an LC or LC-MS method. These methods require expensive, specialized equipment.

On the other hand, you could probably also use thin-layer chromatography (TLC) as a fast, low-tech, semi-quantitative method if you have a way of staining for the presence of allicin. Older literature on allicin probably has methods for this technique.

Adam B Shapiro Is there a fixed concentration that I should begin with for the spectrum scan? I previously prepared a concentration of allicin and adjusted it until the reading was below 2.0 before I measured the whole absorbance spectrum. That is how I obtained the lambda max of 207.6 nm.

Unfortunately, I don't have access to LCMS and the previous literature I found on TLC for allicin is to verify its presence not to quantify but I shall keep searching in case. I appreciate your feedback, Dr. Shapiro.

You should use a concentration of allicin that keeps the highest absorbance within the linear range of the spectrophotometer. This depends on the instrument. Try a few different concentrations to find the peak(s) and then check whether the absorbance is directly proportional to the concentration. That will allow you to calculate the extinction coefficient at any wavelength you choose, presumably at a peak, such as 240 nm.

I saw a reference that gave the 1-cm extinction coefficient of 1% allicin in water (10 g/L) at 240 nm to be 145.4, so a 0.01% solution (100 mg/L) should give an absorbance of 1.45 at 240 nm.

if you use direct heating to maintain the temperature the temperature fluctuations can be seen , so if you are using dialysis bag method then you can place the beaker with dialysis bag in water bath , and need to see that the temperature is maintained at 37 deg c within the invitro testing beaker ,

you can collect the samples at 37 deg c

1) you can analyze the samples at room temperature immediately

2) if the release is too less then you can add a known amount of allicin to the aliquots, and measure the allicin content , amount released would be the difference between the added amount and obtained amount.