Name: Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS
Uploaded: 2023-03-10T13:30:00
Description: Watch this Scientific Journal Video about Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS at JoVE.com

The video outlines a laboratory procedure for extracting and measuring trimethylamine levels in medicinal materials: Pheretima, Periplaneta americana, and Hirudo. It details grinding, homogenization, centrifugation, and gas chromatography-mass spectrometry (GCMS) techniques used to assess recovery rates and trimethylamine concentration in the samples.

trimethylamine detection in animal-derived medicine by headspace gc-ms

Headspace GC-MS를 이용한 동물 유래 의약품의 트리메틸아민 검출

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.

Watch this Scientific Journal Video about Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS at JoVE.com

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

Headspace GC-MS에 의한 동물 유래 의학의 트리메틸아민 검출

시작하려면 약초 분쇄기로 약재 Pheretima, Periplaneta americana 및 Hirudo를 분쇄하십시오. 2번과 4번의 표준 약물 체를 통해 약용 분말을 체로 치십시오. 필요한 샘플 분말을 얻기 위해 두 체 사이에 분말을 수집합니다.50 밀리리터 플라스틱 원심 분리기 튜브에 1 그램의 분말을 섭취하십시오. 5 % 트리클로로 아세트산 용액 20 밀리리터를 첨가하고 고속 분산 균질화기로 1, 000 RPM에서 1 분 동안 균질화한다. 균질화 후, 실온에서 5분 동안 1, 717 G에서 균질액을 원심분리하고, 상청액을 흡수성 면-담근 유리 깔때기를 통해 50 밀리리터 부피 플라스크 내로 여과한다.15 밀리리터와 10 밀리리터의 5 % 트리클로로 아세트산 용액으로 추출 과정을 2 회 반복 한 후, 여과 액을 합치고 5 % 트리클로로 아세트산 용액으로 50 밀리리터의 부피를 구성한다. 다음으로, 20 밀리리터의 헤드 스페이스 바이알에 2 밀리리터의 수산화 나트륨 용액과 0.5 그램의 고체 파라핀을 정확하게 첨가한다. 그런 다음 섭씨 70도의 오븐에 약 30분 동안 넣습니다.바이알을 꺼내 실온으로 식혀 파라핀이 굳도록 합니다. 샘플 헤드스페이스를 처리하려면 각 샘플 추출 용액 2밀리리터를 바이알을 함유하는 응고된 파라핀에 추가합니다. 캡을 누르고 바이알을 밀봉합니다.측정을 위해 밀봉된 헤드스페이스 바이알을 GCMS에 놓습니다. 표준 곡선을 얻기 위해, 응고된 파라핀 층 상부에 일련의 2밀리리터의 트리메틸아민 표준 용액을 흡인한다. 트리메틸아민 측정을 위해 밀봉된 헤드스페이스 바이알을 기계에 놓습니다.정밀 테스트를 수행하기 위해, 응고된 파라핀 층 위에 밀리리터당 0.1 마이크로그램의 트리메틸아민 표준 용액 2밀리리터를 흡인한다. 제조업체의 지침에 따라 기계에서 6 개의 병렬 테스트를 수행하십시오. 회수율 실험의 경우, 이전에 설명한 대로 GCMS에서 트리에틸아민이 검출되지 않을 때까지 여러 배치의 시료 분말을 오븐에서 굽습니다.다음으로, 오븐에서 구운 미세한 분말 1g을 별도의 50밀리리터 플라스틱 원심분리기 튜브에 넣습니다. 모든 튜브에 50 마이크로 리터의 트리메틸 아민 표준 용액을 추가하고 이전에 시연 된대로 5 % 트리클로로 아세트산 용액으로 추출합니다. 트리메틸 아민의 머무름 시간은 2.3 분으로 피크가 급격하고 다른 불순물의 간섭이 없는 것으로 밝혀졌습니다.Pheretima, Periplaneta americana 및 Hirudo 샘플은 허브의 트리 에틸 아민을 감소시키기 위해 건조하여 스파이크 회수 테스트를 실시한 결과 각각 평균 회수율이 84.49, 94.66 및 85.67 %였습니다. 트리 에틸 아민은 킬로그램 당 13.23에서 271.63 밀리그램 범위의 농도를 가진 9 개의 허브 배치에서 검출되었습니다.

Research

JoVE Journal

Chemistry

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

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 ...