To begin, crush the medicinal materials Pheretima, Periplaneta americana, and Hirudo with an herbal grinder. Sift the medicinal powder through standard drug sieves of number two and number four. Collect the powder between the two sieves to obtain the required sample powder.
Take one gram of the powder in a 50 milliliter plastic centrifuge tube. Add 20 milliliters of 5%trichloroacetic acid solution and homogenize at 1, 000 RPM for one minute with a high speed dispersion homogenizer. After homogenization, centrifuge the homogenate at 1, 717 G for five minutes at room temperature and filter the supernatant through an absorbent cotton-dipped glass funnel into a 50 milliliter volumetric flask.
After repeating the extraction process twice with 15 and 10 milliliters of 5%trichloroacetic acid solution, combine the filtrates and make up the volume to 50 milliliters with 5%trichloroacetic acid solution. Next, accurately add two milliliters of sodium hydroxide solution and 0.5 grams of solid paraffin in 20 milliliter headspace vials. Then place them in an oven at 70 degrees Celsius for about 30 minutes.
Take the vials out and let them cool down to room temperature so that the paraffin solidifies. To process the sample headspace, add two milliliters of each sample extraction solution to the solidified paraffin containing vials. Press the cap and seal the vial.
Put the sealed headspace vials on the GCMS for measurement. To obtain the standard curve, aspirate a series of two milliliters of trimethylamine standard solution on top of the solidified paraffin layer. Put the sealed headspace vials on the machine for trimethylamine measurement.
To perform the precision test, aspirate two milliliters of 0.1 micrograms per milliliter trimethylamine standard solution on top of the solidified paraffin layer. Carry out six parallel tests in the machine following the manufacturer's instructions. For the recovery rate experiment, bake several batches of the sample powder in an oven until no triethylamine is detected in GCMS as demonstrated previously.
Next, take each one gram of oven baked and fineable powder into separate 50 milliliter plastic centrifuge tubes. Add 50 microliters of trimethylamine standard solution to all the tubes and extract with 5%trichloroacetic acid solution as demonstrated previously. The retention time of trimethylamine was found to be 2.3 minutes with a sharp peak and no interference from other impurities.
Pheretima, Periplaneta americana, and Hirudo samples subjected to spiked recovery tests by drying to reduce triethylamine in the herbs showed average recovery rates of 84.49, 94.66, and 85.67%respectively. Triethylamine was detected in nine batches of herbs with concentrations ranging from 13.23 to 271.63 milligrams per kilogram.
