This work was supported by grants from the National Natural Science Foundation of China (82173991), and Sichuan Science and Technology Program (2022YFS0442).

See Table 1 for information on the medicinal materials of Pheretima, Periplaneta americana, and Hirudo. They were identified by Prof. Xu Runchun, Chengdu University of Traditional Chinese Medicine, as the dried bodies of Pheretima aspergillum (E.Perrier), Periplaneta americana L., and Whitmania pigra Whitman.
1. Specimen extraction
<ol>
	<li>Crush Pheretima, Periplaneta americana, and Hirudo with an herbal grinder (see Table of Materials), sift the medicinal powder through No. 2 (sieve aperture: 0.8 mm) and No. 4 (sieve aperture: 0.25 mm) standard drug sieves, and collect the powder between the two sieves to obtain the required sample powder. 
	NOTE: The Pheretima is fluffy after crushing, so its powder does not need to be sieved.</li>
	<li>Take 1 g of powder (accurate to 0.001 g) in a 50 mL plastic centrifuge tube, add 20 mL of 5% trichloroacetic acid (TCA) solution (see Table of Materials), and homogenize at 1,000 rmin-1 for 1 min with a high-speed dispersion homogenizer.</li>
	<li>After homogenization, centrifuge at 1,717 x g for 5 min at room temperature, add a little absorbent cotton in the glass funnel, and filter the supernatant into a 50 mL volumetric flask.</li>
	<li>Repeat the above extraction process twice with 15 mL and 10 mL of 5% TCA solution. Combine the filtrate and dilute it to 50 mL with 5% TCA solution.</li>
</ol>
2. Reagent preparation
<ol>
	<li>Prepare 20% sodium hydroxide solution: weigh 20 g of sodium hydroxide and use deionized water to fix the volume in a 100 mL volumetric flask.</li>
	<li>Prepare 5% TCA solution: weigh 25 g of TCA and use deionized water to fix the volume in a 500 mL volumetric flask.</li>
</ol>
3. TMA standard stock solution preparation
<ol>
	<li>Prepare TMA standard stock solution: weigh 0.0162 g of TMA hydrochloride standard sample, dissolve it in 5% TCA solution, and fix the volume to 100 mL, equal to the concentration of 100 &#181;g/mL of TMA standard stock solution. Store it at 4 &#176;C.</li>
	<li>Prepare TMA standard use solution: take a certain volume of TMA standard stock solution and dilute it step by step with 5% TCA solution to concentrations of 0.1 &#181;g/mL, 0.5 &#181;g/mL, 1 &#181;g/mL, 2 &#181;g/mL, 5 &#181;g/mL, and 10 &#181;g/mL TMA standard solution.</li>
</ol>
4. Sample headspace processing
<ol>
	<li>Accurately weigh 2 mL of sodium hydroxide solution and 0.5 g of solid paraffin (melting point: 58-60 &#176;C) in a 20 mL headspace vial (see Table of Materials).</li>
	<li>Place the headspace vial in an oven at 70 &#176;C for about 30 min. The solid paraffin completely melts.</li>
	<li>Take it out, and let it cool down to room temperature so that the paraffin solidifies. The solidified paraffin will seal sodium hydroxide.</li>
	<li>Take 2 mL of each sample extraction solution and put it on top of the paraffin layer, press the cap, and seal.</li>
	<li>Put the sealed headspace vial on the machine (see Table of Materials) for measurement.</li>
</ol>
5. Setting of HS-GC-MS/MS analysis conditions
<ol>
	<li>See Table 2 for headspace conditions and GC-MS conditions.</li>
	<li>See Table 3 for ion information.</li>
</ol>
6. Standard curve drawing
<ol>
	<li>Refer to the sample headspace processing in steps 4.1-4.3 to prepare the headspace vial containing lye and paraffin sealing layer.</li>
	<li>Aspirate 2 mL of 0.1 &#181;g/mL, 0.5 &#181;g/mL, 1 &#181;g/mL, 2 &#181;g/mL, 5 &#181;g/mL, and 10 &#181;g/mL TMA standard solution into a 20 mL headspace vial, seal the cap, and measure on the machine.</li>
</ol>
7. Precision test
<ol>
	<li>Refer to the sample headspace processing in steps 4.1-4.3 to prepare the headspace vial containing lye and the paraffin sealing layer.</li>
	<li>Aspirate 2 mL of 0.1 &#181;g/mL TMA standard solution into a 20 mL headspace vial and seal the cap. Carry out six parallel tests in the machine following the manufacturer&#39;s instructions (see Table of Materials).</li>
</ol>
8. Recovery rate experiment
<ol>
	<li>Take a batch of Pheretima, Periplaneta Americana, and Hirudo (S02, S05, S07; Table 1) as the representative medicines for the recovery rate experiment.</li>
	<li>Take several batches of sample powder (S02, S05, S07) and bake them in an oven at 50 &#176;C for 72 h until no TMA is detected.</li>
	<li>Refer to the sample preparation method in sections 4-6 to detect the content of TMA in baked medicine powder.</li>
	<li>Take 1 g of baked powder (accurate to 0.001 g), put it into a 50 mL plastic centrifuge tube, and add 50 &#181;L of TMA standard solution. 
	NOTE: The concentration of TMA standard solution is 100 &#956;g/mL, 1000 &#956;g/mL, and 10000 &#956;g/mL.</li>
	<li>Add 20 mL of 5% TCA solution and homogenize at 1000 rmin-1 for 1 min.</li>
	<li>After homogenization, centrifuge at 1717 x g for 5 min, add a little absorbent cotton in the glass funnel and filter the supernatant into a 50 mL volumetric flask.</li>
	<li>Repeat the above extraction process twice with 15 mL and 10 mL of 5% TCA solution; combine the filtrate and dilute it to 50 mL with 5% TCA solution.</li>
</ol>
9. Determination of the limits of detection (LOD) and quantification (LOQ)
<ol>
	<li>Determine the LOD by the corresponding concentration when the signal-to-noise ratio (S/N) = 3.</li>
	<li>Determine the LOQ by the corresponding concentration when the S/N = 10.</li>
</ol>
10. Determination of sample TMA content
<ol>
	<li>Take about 1 g of fine powder of Pheretima, Periplaneta americana, and Hirudo, respectively, extract the sample according to the above method, and determine it on the machine.</li>
</ol>

Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS

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J., Villani, F., Ercolini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+populations+and+the+volatilome+associated+to+meat+spoilage.">Bacterial populations and the volatilome associated to meat spoilage.</a> Food Microbiology. 45 (Pt A), 83-102 (2015).</li><li>Rouger, A., Tresse, O., Zagorec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+contaminants+of+poultry+meat:+sources,+species,+and+dynamics.">Bacterial contaminants of poultry meat: sources, species, and dynamics.</a> Microorganisms. 5 (3), 50 (2017).</li><li>Bali&#241;o-Zuazo, L., Barranco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+liquid+chromatography-mass+spectrometric+method+for+the+simultaneous+determination+of+trimethylamine,+dimethylamine+and+methylamine+in+fishery+products.">A novel liquid chromatography-mass spectrometric method for the simultaneous determination of trimethylamine, dimethylamine and methylamine in fishery products.</a> Food Chemistry. 196, 1207-1214 (2016).</li><li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-efficient+trimethylamine+gas+sensor+based+on+Au+nanoparticles+sensitized+WO3+nanosheets+for+rapid+assessment+of+seafood+freshness.">Ultra-efficient trimethylamine gas sensor based on Au nanoparticles sensitized WO3 nanosheets for rapid assessment of seafood freshness.</a> Food Chemistry. 392, 133318 (2022).</li><li>Bota, G. 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D., Steuer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization-free+determination+of+short-chain+volatile+amines+in+human+plasma+and+urine+by+headspace+gas+chromatography-mass+spectrometry.">Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.</a> Journal of Clinical Laboratory Analysis. 34 (2), e23062 (2020).</li><li>Mitsubayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimethylamine+biosensor+with+flavin-containing+monooxygenase+type+3+(FMO3)+for+fish-freshness+analysis.">Trimethylamine biosensor with flavin-containing monooxygenase type 3 (FMO3) for fish-freshness analysis.</a> Sensors &amp; Actuators B: Chemical. 103 (1-2), 463-467 (2004).</li><li>National Health and Family Planning Commission of the People's Republic of China. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GB 5009. 179-2016. , 12 (2016).</li><li>Liu, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+material+basis+and+processing+principle+of+fishy+smell+of+Pheretima+aspergillum+by+electronic+nose+and+HS-GC-MS.">Study on material basis and processing principle of fishy smell of Pheretima aspergillum by electronic nose and HS-GC-MS.</a> Chinese Journal of Experimental Traditional Medical Formulae. 26 (12), 154-161 (2020).</li><li>Zheng, X., Sun, F., Du, L., Huang, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+on+changes+of+volatile+components+in+Gecko+before+and+after+processing+by+HS-SPME-GC-MS.">Comparison on changes of volatile components in Gecko before and after processing by HS-SPME-GC-MS.</a> Chinese Journal of Experimental Traditional Medical Formulae. 28 (15), 145-152 (2022).</li><li>Yoshiharu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Odor olfactory measurement. , (2004).</li><li>Jia, Z. 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The authors have nothing to disclose.

Animal-derived medicines come from the whole body, organs or tissues, physiological or pathological products, excretions or secretions, and processed products of animals. TMA is an important source of fishy odor in animal-derived medicines; it is a typical malodorous substance with a very low olfactory threshold (0.000032 &#215; 10-6 V/V) and a strong fishy odor13. At present, the commonly used HS-GC-MS method cannot detect TMA in animal-derived medicines stably and accurately.
<p c...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65291">An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry</a>. The Authors section was updated from:
Hui Ye1 
Xuemei Liu1 
Haozhou Huang2 
Lin Huang1 
Yang Bao1 
Hongyan Ma1 
Junzhi Lin3 
Xiaoming Bao4 
Dingkun Zhang1 
Runchun Xu1 
1State Key Laboratory of Southwestern Chinese Medicine Resources, Pharmacy School, Chengdu University of Traditional Chinese Medicine 
2Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine 
3TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine 
4Shimadzu (China) Co., Ltd
to:
Hui Ye1 
Xuemei Liu1 
JiaBao Liao2 
Haozhou Huang3 
Lin Huang1 
Yang Bao1 
Hongyan Ma1 
Junzhi Lin4 
Xiaoming Bao5 
Dingkun Zhang1 
Runchun Xu1 
1State Key Laboratory of Southwestern Chinese Medicine Resources, Pharmacy School, Chengdu University of Traditional Chinese Medicine 
2China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd 
3Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine 
4TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine 
5Shimadzu (China) Co., Ltd

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65291">An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry</a>. The Authors section was updated.

Erratum: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Treating human diseases by utilizing products derived from animal parts and/or their by-products (referred to here as animal-derived medicines) is receiving increased attention. They play an important role in treating cancer, cardiovascular disease, liver cirrhosis, mastitis, and other diseases, with the advantages of a strong effect, small dosage, and significant and specific clinical efficacy. However, animal-derived medicines generally have a prominent fishy odor, which greatly affects patients&#39; compliance, and are especially unfavorable for children1,2. The fishy odor mainly comes from the proteins, amino acids, fats, and other substances contained in the medicine, which are decomposed through fatty acid oxidation, amino acid degradation, and other ways to produce a variety of substances with a fishy odor2,3,4. Among them, trimethylamine (TMA) is a volatile gas with a fishy odor that widely exists in rotting or rotten animal-derived foods5.
Until now, gas chromatography (GC), liquid chromatography (LC), ion chromatography, spectrophotometry, liquid chromatography-mass spectrometry (LC-MS), and sensor methods have commonly been used to detect TMA in the environment, food, and urine6,7,8,9. In view of the low contamination of the GC column and injection system, as well as the high sensitivity, reproducibility, and low detection limit (0.1-1 mg/kg), the headspace gas chromatography-mass spectrometry (HS-GC-MS) method was preferred for food and biological analysis8. At present, only China has established a national standard for TMA in food, and HS-GC-MS is the first method in the GB5009.179-2016 standard10. Therefore, the above HS-GC-MS method was selected to detect TMA in animal-derived medicine. In the early stage, our research group found that the HS-GC-MS detection standard for TMA in food could detect the fishy odor in several animal-derived medicines. Combined with the results of the studies11,12, it could be proved that TMA is the common key substance of fishy odor in animal-derived medicines. However, it was found that the reproducibility of the experimental results was poor, and there were problems such as TMA escape and poor stability, which could not be verified by the methodology. This could be due to the fact that the lye was injected into the headspace vial and the rapid acid-base reaction led to increased pressure in the vial, thus TMA escaped from the injection pore, preventing the stable and accurate detection of TMA. Therefore, this study proposed an improved headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) detection method to address these problems.
The protocol improves the sample pretreatment by separating the acid-base reactants in the pretreatment with the help of solid paraffin, a good solid-liquid phase change material. As the paraffin slowly liquefied with the temperature rise of the thermostatic furnace, TMA was also slowly released in the sealed headspace vial, avoiding the pressure increase caused by the violent and rapid acid-base reaction and ensuring stable and accurate TMA detection. Further, the headspace injection combined with multiple reaction monitoring (MRM) modes) in GC-MS/MS effectively suppressed matrix chemical interference and ensured the reliability of the results. The results of the methodological validation proved that the linearity, precision test, and recovery rate of the improved detection method could meet the requirements, with good reproducibility and high sensitivity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter Trading (China) Co.</td>
			<td>SSC-2-0213</td>
			<td></td>
		</tr>
		<tr>
			<td>Chinese herbal medicine grinder</td>
			<td>Zhejiang Yongkang Xi&#39;an Hardware and Pharmaceutical Factory</td>
			<td>HX-200K</td>
			<td></td>
		</tr>
		<tr>
			<td>Convection oven</td>
			<td>Sanyo Electric Co., Ltd</td>
			<td>MOV-112F</td>
			<td></td>
		</tr>
		<tr>
			<td>Decapper for 20 mm Aluminum caps</td>
			<td>ANPEL Laboratory Technologies (Shanghai) Inc</td>
			<td>V1750004</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Shimadzu Corporation Japan</td>
			<td>AUW220D</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatography mass spectrometry</td>
			<td>Shimadzu Corporation Japan</td>
			<td>TQ-8050 NX</td>
			<td></td>
		</tr>
		<tr>
			<td>Headspace Vial</td>
			<td>ANPEL Laboratory Technologies (Shanghai) Inc</td>
			<td>25760200</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Shanghai biaomo Factory</td>
			<td>FJ200-SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Preassembled Cap</td>
			<td>ANPEL Laboratory Technologies (Shanghai) Inc</td>
			<td>L4150050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample sieve</td>
			<td>Zhenxing Sieve Factory</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-Volatile Amine</td>
			<td>Chengdu Meimelte Technology Co., Ltd</td>
			<td>227-3626-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Chengdu Chron Chemicals Co., Ltd</td>
			<td>2022101401</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid paraffin wax</td>
			<td>Shanghai Hualing Kangfu apparatus factory</td>
			<td>20221112</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Chengdu Chron Chemicals Co., Ltd</td>
			<td>2022102001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimethylamine hydrochloride</td>
			<td>Chengdu Aifa Biotechnology Co., Ltd</td>
			<td>AF22022108</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure water system</td>
			<td>Sichuan Youpu Ultrapure Technology Co., Ltd</td>
			<td>UPR-11-5T</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved technique for trimethylamine detection in animal-derived medicine by headspace gas chromatography-tandem quadrupole mass spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Author Spotlight: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry  

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

The scope of our research is a material basis in the variation of fish odor in animal medicine. This protocol provides a simple pretreatment method and accurate detection of trimethylamine in animal medicines. The simple pretreatment methods and the programmed parameters of HS-GC mass/mass of this protocol, need to be further optimized, and we still have a lot of work to do.Our protocol adopts the multi-direction monitoring mode of GC mass/mass, combined with a unique, simple pretreatment, which is not only the first pretreatment for trimethylamine analysis, but also the first analytical mode to be used.

Schematic diagrams of the pre-processing principle and operation of this protocol are shown in Figure 1 and Figure 2, respectively. The peak time of TMA was 2.3 min, with a sharp peak shape and no interference from other impurities (Figure 3). Measuring the linear range of 0.1-10 &#181;g/mL TMA standard solution, with TMA concentration as the abscissa and peak area as the ordinate, a standard curve was drawn. The linear regression e...

Watch this Scientific Journal Video about An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry at JoVE.com

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

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1.3K Views.  Chengdu University of Traditional Chinese Medicine. The scope of our research is a material basis in the variation of fish odor in animal medicine. This protocol provides a simple pretreatment method and accurate detection of trimethylamine in animal medicines. The simple pretreatment methods and the programmed parameters of HS-GC mass/mass of this protocol, need to be further optimized, and we still have a lot of work to do.Our protocol adopts the multi-direction monitoring mode of GC mass/mass, combined with a unique, simple pretreatm...

Video: Author Spotlight: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry  

Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS  

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.