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

Trimethylamin-detektion i animalsk medicin af 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.

Forfatter Spotlight: En forbedret teknik til trimethylamindetektion i animalsk afledt medicin ved headspace-gaskromatografi-tandem quadrupole massespektrometri  

En forbedret teknik til påvisning af trimethylamin i medicin af animalsk oprindelse ved headspace-gaskromatografi-tandem quadrupolmassespektrometri

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  

Trimethylamindetektion i animalsk afledt medicin ved Headspace GC-MS

Til at begynde med knuse de medicinske materialer Pheretima, Periplaneta americana og Hirudo med en urtekværn. Sigt det medicinske pulver gennem standard lægemiddelsigter af nummer to og nummer fire. Pulveret opsamles mellem de to sigter for at opnå det nødvendige prøvepulver.Tag et gram af pulveret i et 50 ml plastcentrifugerør. Tilsæt 20 ml 5% trichloreddikesyreopløsning og homogeniser ved 1.000 RPM i et minut med en højhastighedsdispersionshomogenisator. Efter homogeniseringen centrifugeres homogenatet ved 1,717 G i fem minutter ved stuetemperatur, og supernatanten filtreres gennem en absorberende bomuldsdyppet glastragt over i en 50 ml målekolbe.Efter gentagelse af ekstraktionsprocessen to gange med 15 og 10 ml 5% trichloreddikesyreopløsning kombineres filtraterne og fyldes op til 50 ml med 5% trichloreddikesyreopløsning. Derefter tilsættes nøjagtigt to milliliter natriumhydroxidopløsning og 0,5 gram fast paraffin i 20 ml headspace hætteglas. Sæt dem derefter i en ovn ved 70 grader Celsius i ca. 30 minutter.Tag hætteglassene ud og lad dem køle af til stuetemperatur, så paraffinen størkner. For at behandle prøvens headspace tilsættes to ml af hver prøveekstraktionsopløsning til de størknede paraffinholdige hætteglas. Tryk på hætten og forsegl hætteglasset.Sæt de forseglede headspace-hætteglas på GCMS til måling. For at opnå standardkurven aspireres en serie på to ml trimethylaminstandardopløsning oven på det størknede paraffinlag. Sæt de forseglede headspace-hætteglas på maskinen til trimethylaminmåling.For at udføre præcisionstesten aspireres to milliliter 0,1 mikrogram pr. milliliter trimethylaminstandardopløsning oven på det størknede paraffinlag. Udfør seks parallelle test i maskinen efter producentens anvisninger. Til genvindingshastighedseksperimentet bages flere partier af prøvepulveret i en ovn, indtil der ikke påvises triethylamin i GCMS som tidligere vist.Tag derefter hvert gram ovnbagt og fint pulver i separate 50 ml plastcentrifugerør. Tilsæt 50 mikroliter trimethylaminstandardopløsning til alle glassene og ekstraher med 5% trichloreddikesyreopløsning som vist tidligere. Retentionstiden for trimethylamin viste sig at være 2,3 minutter med en skarp top og ingen interferens fra andre urenheder.Pheretima-, Periplaneta americana- og Hirudo-prøver, der blev udsat for spikede genopretningstest ved tørring for at reducere triethylamin i urterne, viste gennemsnitlige genvindingshastigheder på henholdsvis 84,49, 94,66 og 85,67%. Triethylamin blev påvist i ni partier urter med koncentrationer fra 13,23 til 271,63 milligram pr. Kg.

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 ...