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

Detecção de trimetilamina em medicina derivada de animais por Headspace GC-MS

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.

Autor em destaque: Uma técnica aprimorada para detecção de trimetilamina em medicina derivada de animais por cromatografia gasosa headspace-espectrometria de massa quadrupolo em tandem  

Uma Técnica Aprimorada para Detecção de Trimetilamina em Medicina Derivada de Animais por Cromatografia Gasosa Headspace-Espectrometria de Massas em Tandem Quadrupolo

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  

Detecção de Trimetilamina em Medicina Derivada de Animais pelo Headspace GC-MS

Para começar, esmague os materiais medicinais Pheretima, Periplaneta americana e Hirudo com um moedor de ervas. Peneire o pó medicinal através de peneiras de drogas padrão de número dois e número quatro. Recolher o pó entre as duas peneiras para obter o pó de amostra necessário.Pegue um grama do pó em um tubo de centrífuga de plástico de 50 mililitros. Adicionar 20 mililitros de solução de ácido tricloroacético a 5% e homogeneizar a 1.000 RPM durante um minuto com um homogeneizador de dispersão a alta velocidade. Após homogeneização, centrifugar o homogeneizado a 1,717 G durante cinco minutos à temperatura ambiente e filtrar o sobrenadante através de um funil de vidro absorvente mergulhado em algodão num balão volumétrico de 50 mililitros.Depois de repetir o processo de extração duas vezes com 15 e 10 mililitros de solução de ácido tricloroacético a 5%, combinar os filtrados e completar o volume para 50 mililitros com solução de ácido tricloroacético a 5%. Em seguida, adicione com precisão dois mililitros de solução de hidróxido de sódio e 0,5 gramas de parafina sólida em frascos de headspace de 20 mililitros. Em seguida, coloque-os em um forno a 70 graus Celsius por cerca de 30 minutos.Retire os frascos para injetáveis e deixe-os arrefecer até à temperatura ambiente para que a parafina se solidifique. Para processar o headspace da amostra, adicione dois mililitros de cada solução de extracção da amostra aos frascos para injetáveis contendo parafina solidificada. Pressione a tampa e sele o frasco para injetáveis.Coloque os frascos de headspace selados no GCMS para medição. Para obter a curva padrão, aspirar uma série de dois mililitros de solução padrão de trimetilamina sobre a camada de parafina solidificada. Coloque os frascos para injetáveis de headspace selados na máquina para medição de trimetilamina.Para realizar o teste de precisão, aspirar dois mililitros de 0,1 microgramas por mililitro de solução padrão de trimetilamina sobre a camada de parafina solidificada. Realizar seis testes paralelos na máquina seguindo as instruções do fabricante. Para o experimento de taxa de recuperação, asse vários lotes do pó da amostra em um forno até que nenhuma trietilamina seja detectada no GCMS, como demonstrado anteriormente.Em seguida, pegue cada grama de pó cozido no forno e fino em tubos de centrífuga de plástico separados de 50 mililitros. Adicionar 50 microlitros de solução padrão de trimetilamina a todos os tubos e extrair com solução de ácido tricloroacético a 5%, conforme demonstrado anteriormente. O tempo de retenção da trimetilamina foi de 2,3 minutos, com pico acentuado e sem interferência de outras impurezas.As amostras de Pheretima, Periplaneta americana e Hirudo submetidas aos testes de recuperação por secagem para redução de trietilamina nas ervas apresentaram taxas médias de recuperação de 84,49, 94,66 e 85,67%, respectivamente. Trietilamina foi detectada em nove lotes de ervas com concentrações variando de 13,23 a 271,63 miligramas por quilograma.

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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 ...