Name: 著者スポットライト:ヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析による動物由来医療におけるトリメチルアミン検出の改良技術
Uploaded: 2023-03-10T13:30:00
Description: 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

この研究は、中国国家自然科学財団(82173991)と四川科学技術プログラム(2022YFS0442)からの助成金によってサポートされました。

フェレティマ、ペリプラネタアメリカーナ、ヒルドの医薬品については表1を参照してください。それらは、成都中医薬大学のXu Runchun教授によって、フェレティマアスペルギルム(E.ペリエ)、ペリプラネタアメリカーナL.、ホイットマニアピグラホイットマンの乾燥体として特定されました。
1. 検体抽出
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ヘッドスペースGC-MSによる動物由来医薬品のトリメチルアミン検出

<ol>
	<li>Fan, H., 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=Material+basis+of+stench+of+animal+medicine:+a+review.">Material basis of stench of animal medicine: a review.</a> China Journal of Chinese Materia Medica. 47 (20), 5452-5459 (2022).</li><li>Deng, Y. J., 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=Progress+on+formation+and+taste-masking+technology+of+stench+of+animal+medicines.">Progress on formation and taste-masking technology of stench of animal medicines.</a> China Journal of Chinese Materia Medica. 45 (10), 2353-2359 (2020).</li><li>Casaburi, A., Piombino, P., Nychas, G. 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. M., Harrington, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+trimethylamine+in+meat+food+products+using+ion+mobility+spectrometry.">Direct detection of trimethylamine in meat food products using ion mobility spectrometry.</a> Talanta. 68 (3), 629-635 (2006).</li><li>Neyer, P., Bernasconi, L., Fuchs, J. A., Allenspach, M. 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. W., Mao, B. P., Miao, S., Mao, X. H., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+sulfur+dioxide+residues+in+sulfur+fumigated+Chinese+herbs+with+headspace+gas+chromatography.">Determination of sulfur dioxide residues in sulfur fumigated Chinese herbs with headspace gas chromatography.</a> Acta Pharmaceutica Sinica. 49 (2), 277-281 (2014).</li></ol>

動物由来の医薬品は、動物の全身、臓器または組織、生理学的または病理学的産物、排泄物または分泌物、および加工製品から得られます。TMAは、動物由来の医薬品に含まれる魚臭の重要な原因です。これは、嗅覚閾値が非常に低く(0.000032 × 10-6 V / V)、強い魚臭13を持つ典型的な悪臭物質です。現在、一般的に使用されているHS-GC-MS法では、動物由来医薬品中のTMAを?...

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

動物部位由来の製品やその副産物(ここでは動物由来医薬品と呼びます)を利用したヒト疾患の治療がますます注目されています。それらは、癌、心血管疾患、肝硬変、乳房炎、およびその他の疾患の治療において重要な役割を果たし、強力な効果、少量の投与量、および重要かつ特異的な臨床効果の利点を備えています。しかし、動物由来の医薬品は一般に生臭さが顕著であり、これは患者のコンプライアンスに大きく影響し、特に子供にとって不利です1,2。生臭は、主に医薬品に含まれるタンパク質、アミノ酸、脂肪などの物質に由来し、脂肪酸の酸化やアミノ酸の分解などによって分解され、魚臭のあるさまざまな物質が生成されます2,3,4。その中でもトリメチルアミン(TMA)は、腐った動物由来食品や腐った動物由来食品に広く存在する生臭みのある揮発性ガスです5。
これまで、ガスクロマトグラフィー(GC)、液体クロマトグラフィー(LC)、イオンクロマトグラフィー、分光光度法、液体クロマトグラフィー-質量分析(LC-MS)、およびセンサー法は、環境、食品、および尿中のTMAを検出するために一般的に使用されてきました6,7,8,9。GCカラムと注入システムの低汚染性、高感度、再現性、および低い検出限界(0.1-1 mg/kg)を考慮すると、食品および生物学的分析にはヘッドスペースガスクロマトグラフィー-質量分析(HS-GC-MS)法が好まれました8。現在、中国だけが食品中のTMAの国家標準を確立しており、HS-GC-MSはGB5009.179-2016標準10の最初の方法です。そこで、動物由来医薬品中のTMAを検出するために、上記のHS-GC-MS法を選択した。初期の段階で、私たちの研究グループは、食品中のTMAのHS-GC-MS検出基準が、いくつかの動物由来の医薬品の生臭さを検出できることを発見しました。研究の結果11,12と組み合わせると、TMAが動物由来の医薬品における魚臭の一般的な重要な物質であることが証明される可能性があります。しかし、実験結果の再現性が悪く、TMAエスケープや安定性の悪さなどの問題があり、方法論では検証できないことがわかりました。これは、灰汁がヘッドスペースバイアルに注入され、急速な酸塩基反応によりバイアル内の圧力が上昇したため、TMAが注入孔から逃げ出し、TMAの安定した正確な検出が妨げられたことが原因である可能性があります。そこで本研究では、これらの問題に対処するために、改良されたヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析(HS-GC-MS/MS)検出法を提案しました。
プロトコルは、優れた固液相変化材料である固体パラフィンの助けを借りて、前処理中の酸塩基反応物を分離することにより、サンプルの前処理を改善します。パラフィンが恒温炉の温度上昇に伴ってゆっくりと液化するにつれて、TMAも密封されたヘッドスペースバイアル内でゆっくりと放出され、激しく急速な酸塩基反応によって引き起こされる圧力上昇を回避し、安定した正確なTMA検出を保証しました。さらに、GC-MS/MSのヘッドスペース注入と多重反応モニタリング(MRM)モードの組み合わせにより、マトリックスの化学的干渉を効果的に抑制し、結果の信頼性を確保しました。方法論的検証の結果、改善された検出方法の直線性、精度テスト、および回収率が、良好な再現性と高感度で要件を満たすことができることが証明されました。

<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

動物由来医薬品は、独特の特徴と顕著な治療効果を有するが、それらのほとんどは明らかな生臭さを有し、その結果、臨床患者のコンプライアンスが悪くなる。トリメチルアミン(TMA)は、動物由来の薬における重要な魚臭成分の1つです。既存の検出方法では、灰汁添加後の急速な酸塩基反応によるヘッドスペースバイアル内の圧力上昇により、TMAがヘッドスペースバイアルから逃げ出し、動物由来医薬品の生臭さの研究が停滞するため、TMAを正確に同定することは困難である。本研究では,酸と灰汁の分離層としてパラフィン層を導入した制御検出法を提案した.TMAの生成速度は、恒温炉加熱によってパラフィン層をゆっくりと液化することによって効果的に制御することができた。この方法は、良好な直線性、精密な実験、および良好な再現性と高感度の回収率を示しました。動物由来医薬品の消臭に関する技術支援を行いました。

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.

著者スポットライト:ヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析による動物由来医療におけるトリメチルアミン検出の改良技術  

ヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析法による動物由来医薬品におけるトリメチルアミン検出の改良技術

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.

このプロトコルの前処理原理と動作の概略図をそれぞれ図1と図2に示します。TMAのピーク時間は2.3分で、ピーク形状がシャープで、他の不純物からの干渉はありませんでした(図3)。TMA濃度を横軸、ピーク面積を縦軸として、0.1-10 μg/mL TMA標準液の直線範囲を測定し、標準曲線を描画した。線形回帰式はy = 2522482x + 24255として得?...

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

ここでは、動物由来の医薬品中のトリメチルアミン(TMA)の測定に適したヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析(HS-GC-MS/MS)法について説明します。プロトコルには、サンプルの前処理、ヘッドスペース処理、分析条件、方法論的検証、および動物由来医薬品中のTMAの測定が含まれます。

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

私たちの研究の範囲は、動物医学における魚の匂いの変化における物質的な基礎です。このプロトコルは、動物用医薬品中のトリメチルアミンの簡単な前処理方法と正確な検出を提供します。このプロトコルの簡単な前処理方法とHS-GC質量/質量のプログラムされたパラメータは、さらに最適化する必要があり、まだやるべきことがたくさんあります。当社のプロトコルは、GC質量/質量の多方向モニタリングモードを採用し、トリメチルアミン分析の最初の前処理であるだけでなく、使用される最初の分析モードでもある独自の簡単な前処理と組み合わせています。

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Chemistry

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.

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.

化学

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.