Name: 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
Uploaded: 2023-03-10T13:30:00
Description: Watch this Scientific Journal Video about An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry at JoVE.com

Este trabajo contó con el apoyo de subvenciones de la Fundación Nacional de Ciencias Naturales de China (82173991) y del Programa de Ciencia y Tecnología de Sichuan (2022YFS0442).

Ver Tabla 1 para información sobre los materiales medicinales de Pheretima, Periplaneta americana e Hirudo. Fueron identificados por el profesor Xu Runchun, de la Universidad de Medicina Tradicional China de Chengdu, como los cuerpos secos de Pheretima aspergillum (E. Perrier), Periplaneta americana L . y Whitmania pigra Whitman.
1. Extracción de muestras
<ol><li>Triture Pheretima, Periplan...

Detección de trimetilamina en medicina de origen animal mediante Headspace GC-MS

<ol>
	<li>Fan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+basis+of+stench+of+animal+medicine:+a+review.">Material basis of stench of animal medicine: a review.</a> China Journal of Chinese Materia Medica. 47 (20), 5452-5459 (2022).</li><li>Deng, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+on+formation+and+taste-masking+technology+of+stench+of+animal+medicines.">Progress on formation and taste-masking technology of stench of animal medicines.</a> China Journal of Chinese Materia Medica. 45 (10), 2353-2359 (2020).</li><li>Casaburi, A., Piombino, P., Nychas, G. J., Villani, F., Ercolini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+populations+and+the+volatilome+associated+to+meat+spoilage.">Bacterial populations and the volatilome associated to meat spoilage.</a> Food Microbiology. 45 (Pt A), 83-102 (2015).</li><li>Rouger, A., Tresse, O., Zagorec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+contaminants+of+poultry+meat:+sources,+species,+and+dynamics.">Bacterial contaminants of poultry meat: sources, species, and dynamics.</a> Microorganisms. 5 (3), 50 (2017).</li><li>Bali&#241;o-Zuazo, L., Barranco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+liquid+chromatography-mass+spectrometric+method+for+the+simultaneous+determination+of+trimethylamine,+dimethylamine+and+methylamine+in+fishery+products.">A novel liquid chromatography-mass spectrometric method for the simultaneous determination of trimethylamine, dimethylamine and methylamine in fishery products.</a> Food Chemistry. 196, 1207-1214 (2016).</li><li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-efficient+trimethylamine+gas+sensor+based+on+Au+nanoparticles+sensitized+WO3+nanosheets+for+rapid+assessment+of+seafood+freshness.">Ultra-efficient trimethylamine gas sensor based on Au nanoparticles sensitized WO3 nanosheets for rapid assessment of seafood freshness.</a> Food Chemistry. 392, 133318 (2022).</li><li>Bota, G. M., Harrington, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+trimethylamine+in+meat+food+products+using+ion+mobility+spectrometry.">Direct detection of trimethylamine in meat food products using ion mobility spectrometry.</a> Talanta. 68 (3), 629-635 (2006).</li><li>Neyer, P., Bernasconi, L., Fuchs, J. A., Allenspach, M. D., Steuer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization-free+determination+of+short-chain+volatile+amines+in+human+plasma+and+urine+by+headspace+gas+chromatography-mass+spectrometry.">Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.</a> Journal of Clinical Laboratory Analysis. 34 (2), e23062 (2020).</li><li>Mitsubayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimethylamine+biosensor+with+flavin-containing+monooxygenase+type+3+(FMO3)+for+fish-freshness+analysis.">Trimethylamine biosensor with flavin-containing monooxygenase type 3 (FMO3) for fish-freshness analysis.</a> Sensors &amp; Actuators B: Chemical. 103 (1-2), 463-467 (2004).</li><li>National Health and Family Planning Commission of the People's Republic of China. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GB 5009. 179-2016. , 12 (2016).</li><li>Liu, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+material+basis+and+processing+principle+of+fishy+smell+of+Pheretima+aspergillum+by+electronic+nose+and+HS-GC-MS.">Study on material basis and processing principle of fishy smell of Pheretima aspergillum by electronic nose and HS-GC-MS.</a> Chinese Journal of Experimental Traditional Medical Formulae. 26 (12), 154-161 (2020).</li><li>Zheng, X., Sun, F., Du, L., Huang, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+on+changes+of+volatile+components+in+Gecko+before+and+after+processing+by+HS-SPME-GC-MS.">Comparison on changes of volatile components in Gecko before and after processing by HS-SPME-GC-MS.</a> Chinese Journal of Experimental Traditional Medical Formulae. 28 (15), 145-152 (2022).</li><li>Yoshiharu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Odor olfactory measurement. , (2004).</li><li>Jia, Z. W., Mao, B. P., Miao, S., Mao, X. H., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+sulfur+dioxide+residues+in+sulfur+fumigated+Chinese+herbs+with+headspace+gas+chromatography.">Determination of sulfur dioxide residues in sulfur fumigated Chinese herbs with headspace gas chromatography.</a> Acta Pharmaceutica Sinica. 49 (2), 277-281 (2014).</li></ol>

Los medicamentos de origen animal provienen de todo el cuerpo, órganos o tejidos, productos fisiológicos o patológicos, excreciones o secreciones y productos procesados de animales. La TMA es una fuente importante de olor a pescado en los medicamentos de origen animal; es una sustancia maloliente típica con un umbral olfativo muy bajo (0,000032 × 10-6 V/V) y un fuerte olor a pescado13. En la actualidad, el método HS-GC-MS comúnmente utilizado no puede detectar TMA en medicamentos...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65291">An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry</a>. The Authors section was updated from:
Hui Ye1 
Xuemei Liu1 
Haozhou Huang2 
Lin Huang1 
Yang Bao1 
Hongyan Ma1 
Junzhi Lin3 
Xiaoming Bao4 
Dingkun Zhang1 
Runchun Xu1 
1State Key Laboratory of Southwestern Chinese Medicine Resources, Pharmacy School, Chengdu University of Traditional Chinese Medicine 
2Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine 
3TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine 
4Shimadzu (China) Co., Ltd
to:
Hui Ye1 
Xuemei Liu1 
JiaBao Liao2 
Haozhou Huang3 
Lin Huang1 
Yang Bao1 
Hongyan Ma1 
Junzhi Lin4 
Xiaoming Bao5 
Dingkun Zhang1 
Runchun Xu1 
1State Key Laboratory of Southwestern Chinese Medicine Resources, Pharmacy School, Chengdu University of Traditional Chinese Medicine 
2China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd 
3Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine 
4TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine 
5Shimadzu (China) Co., Ltd

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65291">An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry</a>. The Authors section was updated.

Erratum: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

El tratamiento de las enfermedades humanas mediante la utilización de productos derivados de partes de animales y/o sus subproductos (denominados aquí medicamentos de origen animal) está recibiendo cada vez más atención. Desempeñan un papel importante en el tratamiento del cáncer, las enfermedades cardiovasculares, la cirrosis hepática, la mastitis y otras enfermedades, con las ventajas de un efecto fuerte, una dosis pequeña y una eficacia clínica significativa y específica. Sin embargo, los medicamentos de origen animal generalmente tienen un olor a pescado prominente, lo que afecta en gran medida el cumplimiento de los pacientes y son especialmente desfavorables para los niños 1,2. El olor a pescado proviene principalmente de las proteínas, aminoácidos, grasas y otras sustancias contenidas en el medicamento, que se descomponen a través de la oxidación de ácidos grasos, la degradación de aminoácidos y otras formas de producir una variedad de sustancias con olor a pescado 2,3,4. Entre ellos, la trimetilamina (TMA) es un gas volátil con olor a pescado que existe ampliamente en alimentos de origen animal podridos o podridos5.
Hasta ahora, la cromatografía de gases (GC), la cromatografía líquida (LC), la cromatografía iónica, la espectrofotometría, la cromatografía líquida-espectrometría de masas (LC-MS) y los métodos de sensores se han utilizado comúnmente para detectar TMA en el medio ambiente, los alimentos y la orina 6,7,8,9. En vista de la baja contaminación de la columna de GC y del sistema de inyección, así como de la alta sensibilidad, reproducibilidad y bajo límite de detección (0,1-1 mg/kg), se prefirió el método de cromatografía de gases-espectrometría de masas en el espacio de cabeza (HS-GC-MS) para el análisis alimentario y biológico8. En la actualidad, solo China ha establecido un estándar nacional para TMA en alimentos, y HS-GC-MS es el primer método en el estándar GB5009.179-201610. Por lo tanto, se seleccionó el método HS-GC-MS anterior para detectar TMA en medicina de origen animal. En la etapa inicial, nuestro grupo de investigación descubrió que el estándar de detección HS-GC-MS para TMA en alimentos podría detectar el olor a pescado en varios medicamentos de origen animal. Combinado con los resultados de los estudios11,12, se pudo demostrar que la TMA es la sustancia clave común del olor a pescado en los medicamentos de origen animal. Sin embargo, se encontró que la reproducibilidad de los resultados experimentales era pobre, y existían problemas como el escape de TMA y la poca estabilidad, los cuales no pudieron ser verificados por la metodología. Esto podría deberse al hecho de que la lejía se inyectó en el vial del espacio de cabeza y la rápida reacción ácido-base condujo a un aumento de la presión en el vial, por lo que la TMA escapó del poro de inyección, lo que impidió la detección estable y precisa de la TMA. Por lo tanto, este estudio propuso un método mejorado de detección de cromatografía de gases en el espacio de cabeza-espectrometría de masas cuadrupolar en tándem (HS-GC-MS/MS) para abordar estos problemas.
El protocolo mejora el pretratamiento de la muestra al separar los reactivos ácido-base en el pretratamiento con la ayuda de parafina sólida, un buen material de cambio de fase sólido-líquido. A medida que la parafina se licuaba lentamente con el aumento de temperatura del horno termostático, la TMA también se liberaba lentamente en el vial sellado del espacio de cabeza, evitando el aumento de presión causado por la violenta y rápida reacción ácido-base y asegurando una detección estable y precisa de la TMA. Además, la inyección de espacio de cabeza combinada con los modos de monitoreo de reacciones múltiples (MRM) en GC-MS/MS suprimió eficazmente la interferencia química de la matriz y garantizó la confiabilidad de los resultados. Los resultados de la validación metodológica demostraron que la linealidad, la prueba de precisión y la tasa de recuperación del método de detección mejorado podían cumplir con los requisitos, con buena reproducibilidad y alta sensibilidad.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter Trading (China) Co.</td>
			<td>SSC-2-0213</td>
			<td></td>
		</tr>
		<tr>
			<td>Chinese herbal medicine grinder</td>
			<td>Zhejiang Yongkang Xi&#39;an Hardware and Pharmaceutical Factory</td>
			<td>HX-200K</td>
			<td></td>
		</tr>
		<tr>
			<td>Convection oven</td>
			<td>Sanyo Electric Co., Ltd</td>
			<td>MOV-112F</td>
			<td></td>
		</tr>
		<tr>
			<td>Decapper for 20 mm Aluminum caps</td>
			<td>ANPEL Laboratory Technologies (Shanghai) Inc</td>
			<td>V1750004</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Shimadzu Corporation Japan</td>
			<td>AUW220D</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatography mass spectrometry</td>
			<td>Shimadzu Corporation Japan</td>
			<td>TQ-8050 NX</td>
			<td></td>
		</tr>
		<tr>
			<td>Headspace Vial</td>
			<td>ANPEL Laboratory Technologies (Shanghai) Inc</td>
			<td>25760200</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Shanghai biaomo Factory</td>
			<td>FJ200-SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Preassembled Cap</td>
			<td>ANPEL Laboratory Technologies (Shanghai) Inc</td>
			<td>L4150050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample sieve</td>
			<td>Zhenxing Sieve Factory</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-Volatile Amine</td>
			<td>Chengdu Meimelte Technology Co., Ltd</td>
			<td>227-3626-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Chengdu Chron Chemicals Co., Ltd</td>
			<td>2022101401</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid paraffin wax</td>
			<td>Shanghai Hualing Kangfu apparatus factory</td>
			<td>20221112</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Chengdu Chron Chemicals Co., Ltd</td>
			<td>2022102001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimethylamine hydrochloride</td>
			<td>Chengdu Aifa Biotechnology Co., Ltd</td>
			<td>AF22022108</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure water system</td>
			<td>Sichuan Youpu Ultrapure Technology Co., Ltd</td>
			<td>UPR-11-5T</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved technique for trimethylamine detection in animal-derived medicine by headspace gas chromatography-tandem quadrupole mass spectrometry

Los medicamentos de origen animal tienen características distintivas y efectos curativos significativos, pero la mayoría de ellos tienen un olor obvio a pescado, lo que resulta en un cumplimiento deficiente de los pacientes clínicos. La trimetilamina (TMA) es uno de los componentes clave del olor a pescado en la medicina de origen animal. Es difícil identificar la TMA con precisión utilizando el método de detección existente debido al aumento de la presión en el vial del espacio de cabeza causado por la rápida reacción ácido-base después de la adición de lejía, lo que hace que la TMA escape del vial del espacio de cabeza, deteniendo el progreso de la investigación del olor a pescado de la medicina de origen animal. En este estudio, propusimos un método de detección controlada que introdujo una capa de parafina como capa de aislamiento entre el ácido y la lejía. La tasa de producción de TMA podría controlarse de manera efectiva licuar lentamente la capa de parafina a través del calentamiento termostático del horno. Este método mostró linealidad satisfactoria, experimentos de precisión y recuperaciones con buena reproducibilidad y alta sensibilidad. Prestó apoyo técnico para la desodorización de medicamentos de origen animal.

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.

En la Figura 1 y en la Figura 2 se muestran diagramas esquemáticos del principio de preprocesamiento y el funcionamiento de este protocolo, respectivamente. El tiempo pico de TMA fue de 2,3 min, con una forma de pico nítida y sin interferencia de otras impurezas (Figura 3). Midiendo el rango lineal de 0,1-10 μg/mL de solución estándar de TMA, con la concentración de TMA como abscisa y el área del pico como ordenada, se dibuj?...

Watch this Scientific Journal Video about An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry at JoVE.com

En este trabajo se describe un método de cromatografía de gases-espectrometría de masas cuadrupolar en tándem (HS-GC-MS/MS) adecuado para la determinación de trimetilamina (TMA) en medicamentos de origen animal. El protocolo incluye el pretratamiento de la muestra, el tratamiento del espacio de cabeza, las condiciones de análisis, la validación metodológica y la determinación de TMA en medicamentos de origen animal.

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

El alcance de nuestra investigación es una base material en la variación del olor del pescado en medicina animal. Este protocolo proporciona un método de pretratamiento sencillo y una detección precisa de trimetilamina en medicamentos para animales. Los métodos simples de pretratamiento y los parámetros programados de HS-GC masa/masa de este protocolo deben optimizarse aún más, y todavía tenemos mucho trabajo por hacer.Nuestro protocolo adopta el modo de monitoreo multidireccional de GC masa/masa, combinado con un pretratamiento único y simple, que no solo es el primer pretratamiento para el análisis de trimetilamina, sino también el primer modo analítico que se utiliza.

an-improved-technique-for-trimethylamine-detection-animal-derived

Research

JoVE Journal

Chemistry

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 poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Investigación

Química

Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS

To begin, crush the medicinal materials Pheretima, Periplaneta americana, and Hirudo with an herbal grinder. Sift the medicinal powder through standard drug sieves of number two and number four. Collect the powder between the two sieves to obtain the required sample powder.Take one gram of the powder in a 50 milliliter plastic centrifuge tube. Add 20 milliliters of 5%trichloroacetic acid solution and homogenize at 1, 000 RPM for one minute with a high speed dispersion homogenizer. After homogenization, centrifuge the homogenate at 1, 717 G for five minutes at room temperature and filter the supernatant through an absorbent cotton-dipped glass funnel into a 50 milliliter volumetric flask.After repeating the extraction process twice with 15 and 10 milliliters of 5%trichloroacetic acid solution, combine the filtrates and make up the volume to 50 milliliters with 5%trichloroacetic acid solution. Next, accurately add two milliliters of sodium hydroxide solution and 0.5 grams of solid paraffin in 20 milliliter headspace vials. Then place them in an oven at 70 degrees Celsius for about 30 minutes.Take the vials out and let them cool down to room temperature so that the paraffin solidifies. To process the sample headspace, add two milliliters of each sample extraction solution to the solidified paraffin containing vials. Press the cap and seal the vial.Put the sealed headspace vials on the GCMS for measurement. To obtain the standard curve, aspirate a series of two milliliters of trimethylamine standard solution on top of the solidified paraffin layer. Put the sealed headspace vials on the machine for trimethylamine measurement.To perform the precision test, aspirate two milliliters of 0.1 micrograms per milliliter trimethylamine standard solution on top of the solidified paraffin layer. Carry out six parallel tests in the machine following the manufacturer's instructions. For the recovery rate experiment, bake several batches of the sample powder in an oven until no triethylamine is detected in GCMS as demonstrated previously.Next, take each one gram of oven baked and fineable powder into separate 50 milliliter plastic centrifuge tubes. Add 50 microliters of trimethylamine standard solution to all the tubes and extract with 5%trichloroacetic acid solution as demonstrated previously. The retention time of trimethylamine was found to be 2.3 minutes with a sharp peak and no interference from other impurities.Pheretima, Periplaneta americana, and Hirudo samples subjected to spiked recovery tests by drying to reduce triethylamine in the herbs showed average recovery rates of 84.49, 94.66, and 85.67%respectively. Triethylamine was detected in nine batches of herbs with concentrations ranging from 13.23 to 271.63 milligrams per kilogram.