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

Detectie van trimethylamine in diergeneeskunde door 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.

Auteur in de schijnwerpers: een verbeterde techniek voor trimethylaminedetectie in diergeneeskunde door Headspace gaschromatografie-tandem quadrupool massaspectrometrie  

Een verbeterde techniek voor trimethylaminedetectie in diergeneesmiddelen door Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

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  

Trimethylamine detectie in diergeneeskunde door Headspace GC-MS

Om te beginnen, plet de medicinale materialen Pheretima, Periplaneta americana en Hirudo met een kruidenmolen. Zeef het medicinale poeder door standaard medicijnzeven van nummer twee en nummer vier. Verzamel het poeder tussen de twee zeven om het benodigde monsterpoeder te verkrijgen.Neem een gram van het poeder in een plastic centrifugebuis van 50 milliliter. Voeg 20 milliliter 5% trichloorazijnzuuroplossing toe en homogeniseer gedurende één minuut bij 1.000 RPM met een hogesnelheidsdispersiehomogenisator. Na homogenisatie centrifugeert u het homogenaat bij 1.717 g gedurende vijf minuten bij kamertemperatuur en filtreer het supernatant door een absorberende glazen trechter met katoen in een maatkolf van 50 milliliter.Na het extractieproces tweemaal te hebben herhaald met 15 en 10 milliliter 5% trichloorazijnzuuroplossing, combineert u de filtraten en vult u het volume aan tot 50 milliliter met 5% trichloorazijnzuuroplossing. Voeg vervolgens nauwkeurig twee milliliter natriumhydroxideoplossing en 0,5 gram vaste paraffine toe in 20 milliliter headspace-injectieflacons. Zet ze vervolgens ongeveer 30 minuten in een oven op 70 graden Celsius.Haal de injectieflacons eruit en laat ze afkoelen tot kamertemperatuur zodat de paraffine stolt. Om de kopruimte van het monster te verwerken, voegt u twee milliliter van elke monsterextractieoplossing toe aan de gestolde injectieflacons met paraffine. Druk op de dop en sluit de injectieflacon af.Plaats de verzegelde headspace-injectieflacons op het GCMS voor meting. Om de standaardcurve te verkrijgen, zuigt u een reeks van twee milliliter trimethylaminestandaardoplossing op de gestolde paraffinelaag. Plaats de verzegelde headspace-injectieflacons op de machine voor trimethylaminemeting.Om de precisietest uit te voeren, zuigt u twee milliliter van 0,1 microgram per milliliter trimethylamine-standaardoplossing op de gestolde paraffinelaag. Voer zes parallelle tests uit in de machine volgens de instructies van de fabrikant. Bak voor het herstelsnelheidsexperiment verschillende batches van het monsterpoeder in een oven totdat er geen triethylamine wordt gedetecteerd in GCMS, zoals eerder is aangetoond.Neem vervolgens elke gram in de oven gebakken en fijn poeder in afzonderlijke plastic centrifugebuizen van 50 milliliter. Voeg 50 microliter trimethylamine standaardoplossing toe aan alle buizen en extraheer met 5% trichloorazijnzuuroplossing zoals eerder gedemonstreerd. De retentietijd van trimethylamine bleek 2,3 minuten te zijn met een scherpe piek en geen interferentie van andere onzuiverheden.Pheretima, Periplaneta americana en Hirudo monsters onderworpen aan spiked recovery tests door drogen om triethylamine in de kruiden te verminderen toonden gemiddelde herstelpercentages van respectievelijk 84,49, 94,66 en 85,67%. Triethylamine werd gedetecteerd in negen partijen kruiden met concentraties variërend van 13,23 tot 271,63 milligram per kilogram.

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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 ...