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

Trimetylamin detektion i animalisk härledd medicin med 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.

Författare Spotlight: En förbättrad teknik för trimetylamindetektion i djurbaserad medicin genom Headspace gaskromatografi-tandem kvadrupol masspektrometri  

En förbättrad teknik för trimetylamindetektion i djurbaserad medicin genom gaskromatografi-tandemkvadrupolmasspektrometri

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  

Trimetylamindetektion i djurbaserad medicin med Headspace GC-MS

Börja med att krossa de medicinska materialen Pheretima, Periplaneta americana och Hirudo med en örtkvarn. Sikta det medicinska pulvret genom vanliga läkemedelssiktar av nummer två och nummer fyra. Samla upp pulvret mellan de två siktarna för att erhålla det erforderliga provpulvret.Ta ett gram av pulvret i ett 50 ml plastcentrifugrör. Tillsätt 20 ml 5% triklorättiksyralösning och homogenisera vid 1 000 varv / min i en minut med en höghastighetsdispersionshomogenisator. Efter homogenisering centrifugeras homogenatet vid 1,717 G i fem minuter vid rumstemperatur och filtrerar supernatanten genom en absorberande bomullsdoppad glastratt till en 50 ml mätkolv.Efter att ha upprepat extraktionsprocessen två gånger med 15 och 10 ml 5% triklorättiksyralösning, kombinera filtraten och fyll volymen till 50 ml med 5% triklorättiksyralösning. Tillsätt sedan noggrant två milliliter natriumhydroxidlösning och 0,5 gram fast paraffin i 20 ml huvudutrymme. Ställ dem sedan i en ugn på 70 grader Celsius i cirka 30 minuter.Ta ut injektionsflaskorna och låt dem svalna till rumstemperatur så att paraffinet stelnar. För att bearbeta provets huvudutrymme, tillsätt två ml av varje provextraktionslösning till det stelnade paraffinet som innehåller injektionsflaskorna. Tryck på locket och försegla injektionsflaskan.Sätt de förseglade injektionsflaskorna med huvudutrymme på GCMS för mätning. För att erhålla standardkurvan, aspirera en serie av två milliliter trimetylamin standardlösning ovanpå det stelnade paraffinskiktet. Sätt de förseglade injektionsflaskorna med huvudutrymme på maskinen för trimetylaminmätning.För att utföra precisionstestet, aspirera två milliliter 0,1 mikrogram per milliliter trimetylamin standardlösning ovanpå det stelnade paraffinskiktet. Utför sex parallella tester i maskinen enligt tillverkarens instruktioner. För experimentet med återvinningshastighet, baka flera satser av provpulvret i en ugn tills ingen trietylamin detekteras i GCMS som visats tidigare.Ta sedan varje gram ugnsbakat och fint pulver i separata 50 ml plastcentrifugrör. Tillsätt 50 mikroliter trimetylaminstandardlösning till alla rör och extrahera med 5% triklorättiksyralösning som visats tidigare. Retentionstiden för trimetylamin befanns vara 2,3 minuter med en skarp topp och ingen störning från andra föroreningar.Pheretima, Periplaneta americana och Hirudo prover som utsattes för spikade återhämtningstester genom torkning för att minska trietylamin i örterna visade genomsnittliga återvinningsgrader på 84,49, 94,66 respektive 85,67%. Trietylamin detekterades i nio satser av örter med koncentrationer från 13,23 till 271,63 milligram per kilo.

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 ...