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

Detección de trimetilamina en medicina de origen animal mediante 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 destacado: Una técnica mejorada para la detección de trimetilamina en medicina animal mediante cromatografía de gases en el espacio de cabeza-espectrometría de masas cuadrupolar en tándem  

Una técnica mejorada para la detección de trimetilamina en medicina de origen animal mediante cromatografía de gases en el espacio de cabeza-espectrometría de masas cuadrupolar en tándem

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  

Para empezar, tritura los materiales medicinales Pheretima, Periplaneta americana e Hirudo con un molinillo de hierbas. Tamizar el polvo medicinal a través de tamices de medicamentos estándar del número dos y el número cuatro. Recoja el polvo entre los dos tamices para obtener el polvo de muestra requerido.Tome un gramo del polvo en un tubo de centrífuga de plástico de 50 mililitros. Añadir 20 mililitros de solución de ácido tricloroacético al 5% y homogeneizar a 1.000 RPM durante un minuto con un homogeneizador de dispersión de alta velocidad. Después de la homogeneización, centrifugar el homogeneizado a 1.717 G durante cinco minutos a temperatura ambiente y filtrar el sobrenadante a través de un embudo de vidrio absorbente sumergido en algodón en un matraz aforado de 50 mililitros.Después de repetir el proceso de extracción dos veces con 15 y 10 mililitros de solución de ácido tricloroacético al 5%, combine los filtrados y aumente el volumen a 50 mililitros con una solución de ácido tricloroacético al 5%. A continuación, agregue con precisión dos mililitros de solución de hidróxido de sodio y 0,5 gramos de parafina sólida en viales de 20 mililitros de espacio de cabeza. Luego colócalos en un horno a 70 grados centígrados durante unos 30 minutos.Saca los viales y déjalos enfriar a temperatura ambiente para que la parafina se solidifique. Para procesar el espacio de cabeza de la muestra, agregue dos mililitros de cada solución de extracción de muestra a los viales que contienen parafina solidificada. Presione la tapa y selle el vial.Coloque los viales de espacio de cabeza sellados en el GCMS para su medición. Para obtener la curva estándar, aspire una serie de dos mililitros de solución estándar de trimetilamina sobre la capa de parafina solidificada. Coloque los viales de espacio de cabeza sellados en la máquina para la medición de trimetilamina.Para realizar la prueba de precisión, aspire dos mililitros de 0,1 microgramos por mililitro de solución estándar de trimetilamina sobre la capa de parafina solidificada. Realice seis pruebas paralelas en la máquina siguiendo las instrucciones del fabricante. Para el experimento de la tasa de recuperación, hornee varios lotes del polvo de muestra en un horno hasta que no se detecte trietilamina en GCMS como se demostró anteriormente.A continuación, tome cada gramo de polvo horneado y fino en tubos de centrífuga de plástico de 50 mililitros separados. Añadir 50 microlitros de solución estándar de trimetilamina a todos los tubos y extraer con una solución de ácido tricloroacético al 5% como se ha demostrado anteriormente. Se encontró que el tiempo de retención de la trimetilamina era de 2,3 minutos con un pico agudo y sin interferencia de otras impurezas.Las muestras de Pheretima, Periplaneta americana e Hirudo sometidas a pruebas de recuperación con picos de secado para reducir la trietilamina en las hierbas mostraron tasas de recuperación promedio de 84.49, 94.66 y 85.67% respectivamente. Se detectó trietilamina en nueve lotes de hierbas con concentraciones que oscilaron entre 13,23 y 271,63 miligramos por kilogramo.

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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 ...