Name: 저자 스포트라이트: 헤드스페이스 가스 크로마토그래피-탠덤 사중극자 질량 분석법을 이용한 동물 유래 의학에서 트리메틸아민 검출을 위한 개선된 기법
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

이 작업은 중국 국립 자연 과학 재단(82173991)과 쓰촨 과학 기술 프로그램(2022YFS0442)의 보조금으로 지원되었습니다.

Pheretima, Periplaneta americana 및 Hirudo의 의약 재료에 대한 정보는 표 1을 참조하십시오. 그들은 청두 중국 전통 의학 대학의 Xu Runchun 교수에 의해 Pheretima aspergillum (E.Perrier), Periplaneta americana L. 및 Whitmania pigra Whitman의 건조 된 시체로 확인되었습니다.
1. 검체 추출
<ol><li>Pheretima, Periplaneta americana 및 Hir...

Headspace GC-MS를 이용한 동물 유래 의약품의 트리메틸아민 검출

<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>

동물 유래 의약품은 전신, 장기 또는 조직, 생리학적 또는 병리학적 산물, 배설물 또는 분비물, 동물의 가공 제품에서 유래합니다. TMA는 동물성 의약품에서 비린내를 유발하는 중요한 원인입니다. 후각 역치가 매우 낮고(0.000032 × 10-6 V/V) 강한 비린내를 풍기는 전형적인 악취 물질이다13. 현재 일반적으로 사용되는 HS-GC-MS 방법은 동물 유래 의약품에서 TMA를 안정적이고 정?...

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

동물 부위 및/또는 그 부산물에서 추출한 제품(여기서는 동물 유래 의약품이라고 함)을 활용하여 인간의 질병을 치료하는 것이 점점 더 주목받고 있습니다. 암, 심혈관 질환, 간경변, 유방염 및 기타 질병을 치료하는 데 중요한 역할을 하며 강력한 효과, 소량, 중요하고 구체적인 임상 효능의 장점이 있습니다. 그러나 동물성 의약품은 일반적으로 비린내가 두드러져 환자의 순응도에 큰 영향을 미치며 특히 어린이 1,2에게 불리합니다. 비린내는 주로 약에 함유된 단백질, 아미노산, 지방 및 기타 물질에서 비롯되며, 지방산 산화, 아미노산 분해 및 기타 비린내를 가진 다양한 물질을 생성하는 기타 방법을 통해 분해됩니다 2,3,4. 그 중 트리메틸아민(TMA)은 썩거나 썩은 동물 유래 식품에 널리 존재하는 비린내를 동반한 휘발성 가스이다5.
지금까지, 가스 크로마토그래피 (GC), 액체 크로마토 그래피 (LC), 이온 크로마토 그래피, 분광 광도법, 액체 크로마토 그래피 - 질량 분광법 (LC-MS) 및 센서 방법은 환경, 식품 및 소변에서 TMA를 검출하기 위해 일반적으로 사용되어 왔다 6,7,8,9. GC 컬럼 및 주입 시스템의 낮은 오염과 높은 감도, 재현성 및 낮은 검출 한계(0.1-1 mg/kg)를 고려할 때, 헤드스페이스 가스 크로마토그래피-질량분석법(HS-GC-MS) 방법은 식품 및 생물학적 분석에 선호되었다8. 현재 중국만이 식품의 TMA에 대한 국가 표준을 제정했으며 HS-GC-MS는 GB5009.179-2016 표준10의 첫 번째 방법입니다. 따라서 동물유래 의약품에서 TMA를 검출하기 위해 상기 HS-GC-MS 방법을 선택하였다. 초기 단계에서 우리 연구 그룹은 식품의 TMA에 대한 HS-GC-MS 검출 표준이 여러 동물 유래 의약품에서 비린내를 감지할 수 있음을 발견했습니다. 연구결과와 11,12 TMA가 동물 유래 의약품에서 비린내를 유발하는 일반적인 핵심 물질임을 증명할 수 있습니다. 그러나 실험 결과의 재현성이 떨어지고 TMA 이탈, 안정성 불량 등의 문제가 있어 방법론으로 검증할 수 없는 문제점이 있는 것으로 나타났다. 이는 잿물이 헤드스페이스 바이알에 주입되고 빠른 산-염기 반응으로 인해 바이알의 압력이 증가하여 TMA가 주입 기공에서 빠져나와 TMA의 안정적이고 정확한 검출을 방해했기 때문일 수 있습니다. 따라서 본 연구에서는 이러한 문제를 해결하기 위해 개선된 헤드스페이스 가스 크로마토그래피-탠덤 사중극자 질량분석법(HS-GC-MS/MS) 검출 방법을 제안했습니다.
이 프로토콜은 우수한 고체-액체 상 변화 물질인 고체 파라핀의 도움으로 전처리에서 산-염기 반응물을 분리하여 샘플 전처리를 개선합니다. 온도 조절로의 온도 상승에 따라 파라핀이 서서히 액화됨에 따라 TMA도 밀봉된 헤드스페이스 바이알에서 천천히 방출되어 격렬하고 빠른 산-염기 반응으로 인한 압력 증가를 방지하고 안정적이고 정확한 TMA 검출을 보장합니다. 또한 GC-MS/MS에서 다중 반응 모니터링(MRM) 모드와 결합된 헤드스페이스 주입은 매트릭스 화학적 간섭을 효과적으로 억제하고 결과의 신뢰성을 보장했습니다. 방법론적 검증의 결과는 개선된 검출 방법의 선형성, 정밀 테스트 및 회수율이 우수한 재현성과 높은 감도로 요구 사항을 충족할 수 있음을 입증했습니다.

<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

동물 유래 의약품은 특유의 특성과 상당한 치료 효과를 가지고 있지만 대부분 명백한 비린내이가 있어 임상 환자의 순응도가 낮습니다. 트리메틸아민(TMA)은 동물 유래 의약품의 주요 비린내 성분 중 하나입니다. 잿물 첨가 후 급격한 산-염기 반응으로 인한 헤드스페이스 바이알의 압력 증가로 인해 TMA가 헤드스페이스 바이알에서 빠져나가 동물유래 의약품의 비린내에 대한 연구 진행이 지연되어 기존 검출 방법으로는 TMA를 정확하게 식별하기 어렵습니다. 본 연구에서는 산과 잿물 사이의 격리층으로 파라핀층을 도입한 제어된 검출 방법을 제안하였다. TMA 생산 속도는 자동 온도 조절 용광로 가열을 통해 파라핀 층을 천천히 액화시킴으로써 효과적으로 제어 할 수 있습니다. 이 방법은 우수한 재현성과 높은 감도로 만족스러운 선형성, 정밀 실험 및 회수율을 보여주었습니다. 동물 유래 의약품의 탈취에 대한 기술 지원을 제공했습니다.

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.

저자 스포트라이트: 헤드스페이스 가스 크로마토그래피-탠덤 사중극자 질량 분석법을 이용한 동물 유래 의학에서 트리메틸아민 검출을 위한 개선된 기법  

헤드스페이스 가스 크로마토그래피-탠덤 사중극자 질량분석법에 의한 동물 유래 의학의 트리메틸아민 검출을 위한 개선된 기술

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.

이 프로토콜의 전처리 원리와 작동에 대한 개략도는 각각 그림 1과 그림 2에 나와 있습니다. TMA의 피크 시간은 2.3분이었고, 피크 모양이 뚜렷하고 다른 불순물의 간섭이 없었습니다(그림 3). 0.1-10 μg/mL TMA 표준용액의 선형 범위를 측정하고, TMA 농도를 가로좌표로, 피크 면적을 세로좌표로 하여 표준 곡선을 그렸습니다. 선형 회귀 ?...

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

여기에서는 동물 유래 의약품에서 트리메틸아민(TMA) 측정에 적합한 헤드스페이스 가스 크로마토그래피-탠덤 사중극자 질량분석법(HS-GC-MS/MS) 방법을 설명합니다. 이 프로토콜에는 샘플 전처리, 헤드스페이스 처리, 분석 조건, 방법론적 검증 및 동물 유래 의약품의 TMA 측정이 포함됩니다.

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

우리 연구의 범위는 동물 의학에서 생선 냄새의 변화에 대한 물질적 기초입니다. 이 프로토콜은 간단한 전처리 방법과 동물 의약품에서 트리메틸아민의 정확한 검출을 제공합니다. 이 프로토콜의 HS-GC 질량/질량의 간단한 전처리 방법과 프로그래밍된 매개변수는 더욱 최적화되어야 하며 아직 해야 할 일이 많습니다.당사의 프로토콜은 트리메틸아민 분석을 위한 첫 번째 전처리일 뿐만 아니라 사용되는 첫 번째 분석 모드인 독특하고 간단한 전처리와 결합된 GC 질량/질량의 다방향 모니터링 모드를 채택합니다.

an-improved-technique-for-trimethylamine-detection-animal-derived

Research

JoVE Journal

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.

연구

화학

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.