Menthol Analysis in Cough Drops

While cold and flu season are over for now, allergy season is here.  When my allergies are in full force I get quite a tickle in my throat that causes me to cough quite frequently.  To deal with this I like to keep cough drops on hand to help ease the cough. This got me to wondering what was the active ingredient in these cough drops and could I analyze them using the Lucidity GC-FID. 

Looking at the package, the active ingredient is listed as menthol.  Menthol is a compound found in mints such as peppermint and spearmint, but is now easily synthesized in a laboratory. A quick internet search revealed that the GC-FID was the instrument of choice for menthol analysis.

I got to work by first purchasing a pure sample of menthol from a chemical supply company and preparing the samples.  While I waited on the menthol to arrive I did some calculation to see where I should build a standard curve.  Reading the label, it stated that there was 5.8 mg of menthol in each piece, which has an average mass of about 3 grams. With the amount of analyte of interest known, I just needed to get that analyte into solution and figure out how much of a dilution needed to be done.

I knew that I wanted to dilute the cough drop enough that any flavor peaks would be small and not overload the column but make sure the menthol peak would be large enough to see. I ran some preliminary chromatograms and found that there were some visible peaks at a dilution of 3 grams in 50 mL.  Using this knowledge, I found that my ideal concentration would be around 0.3 mg/mL of menthol, so I would build a calibration curve around that concentration.

Once the menthol arrived I started by making a standard curve. I made a 10 mg/mL stock standard, then a 1 mg/mL working standard, from there I made a four point curve that ranged from 0.05 mg/mL to 0.4 mg/mL.  The R squared value was 0.9991, so a decent calibration curve.

Calibration curve of the menthol from 0.05 to 0.04 mg/mL

The method I was following used THF, or tetrahydrofuran, and ethanol to extract the menthol from a gel, so that is where I started. I first ground the cough drops with a mortar and pestle, which left me with a sticky mess of a sample. I ended up grinding about six cough drops to get the mass of one cough drop into a 50 mL volumetric flask.

Top view of ground cough drops in a mortar and pestle

Adding 10 mL of THF to the flask, I shook the flask for 5 minutes to ensure that the menthol was extracted. After shaking, I diluted to the mark with ethanol and gently shook the solution to ensure a uniform mixture. The solution was cloudy and the majority of the cough drop did not dissolve, so the solution was centrifuged at 5000 rpm for 5 minutes, the supernatant was then filtered through a 0.45 μm filter and injected onto the GC using the method below.

While the first sample was running, I decided that maybe I should try a different extraction method, because I don’t use THF to ingest a cough drop.  So, I took a whole cough drop and placed it into a 150 mL beaker and added 20 mL of HPLC grade water, added a stir bar, and let it mix for about 15 minutes. While the cough drop was on the stir plate there was a strong smell of menthol in the area around the beaker.

Cough drop dissolving in water on a stir plate

After the cough drop was fully dissolved I added that to a 50 mL volumetric flask and rinsed the beaker with ethanol twice, adding the rinses to flask and then diluted to the mark with ethanol. This sample prep allowed the cough drop to be completely in solution, but when the ethanol was added there was a precipitate and the solution had to be filtered with a 0.45 μm filter as well.

Three solutions of cough drops using different solvent ratios

Once filtered, the solution was run on the Lucidity GC-FID using the same method as above and the following chromatogram was obtained.

Chromatogram showing the cough drop with the menthol peak highlighted

There are some other peaks that do elute very close to the menthol, I’m not sure what they could be as the cough drop has quite a lot of ingredients that they could be, but a clear peak was visible at the same retention time as the menthol standard and the software picked it out as menthol and calculated a concentration of 0.216 mg/mL.  Taking out the dilution I get a concentration of 3.6 mg/mL.

While this result isn’t what is shown on the label, it was only one small sample point in a large batch of product.  Ideally, I would be able to have access to multiple sample points throughout the entire production run and average the results to get a labeled amount within a small margin of error. The loss of menthol may be from the sublimation process that menthol can undergo at ambient conditions. If I were to do this again, I would try to do the sample preparation in a sealed container, hopefully avoiding any loss of analyte of interest.