这项工作得到了国家自然科学基金（82173991）和四川省科学技术计划（2022YFS0442）的资助。

有关Pheretima，Periplaneta americana和Hirudo的药材的信息，请参见表1。成都中医药大学徐润春教授将他们鉴定为Pheretima aspergillum（E.Perrier），Periplaneta americana L.和Whitmania pigra Whitman的干燥尸体。
1. 标本提取
<ol><li>用草药研磨机（见材料表）粉碎Pheretima，Periplaneta americana和Hirudo，通过2号（筛孔径：0.8毫米）和4号（筛孔径：0.25毫米）标准药物筛筛选药粉，并在两个筛子之间收集粉末以获得所需的样品粉末。 注意： Pheretima 在粉碎后蓬松，因此其粉末不需要过筛。</li>
	<li>在50 mL塑料离心管中取1 g粉末（精确到0.001 g），加入20 mL 5%三氯乙酸（TCA）溶液（见 材料表），并用高速分散均质器在1,000 rmin-1 下匀浆1分钟。</li>
	<li>匀浆后，在室温下以1,717× g 离心5分钟，在玻璃漏斗中加入少许吸收棉，并将上清液过滤到50mL容量瓶中。</li>
	<li>用 15 mL 和 10 mL 的 5% TCA 溶液重复上述提取过程两次。合并滤液，用5%TCA溶液稀释至50mL。</li>
</ol>2. 试剂制备
<ol><li>准备20%氢氧化钠溶液：称取20g氢氧化钠，用去离子水将体积固定在100 mL容量瓶中。</li>
	<li>准备 5% TCA 溶液：称取 25 g TCA，并使用去离子水将体积固定在 500 mL 容量瓶中。</li>
</ol>3. TMA标准储备液制备
<ol><li>制备TMA标准储备溶液：称取0.0162g盐酸盐TMA标准样品，溶于5%TCA溶液中，将体积固定至100 mL，等于100 μg/mLTMA标准储备液的浓度。将其储存在4°C。</li>
	<li>制备TMA标准用液：取一定体积的TMA标准储备液，用5%TCA溶液逐步稀释至浓度为0.1μg/mL、0.5μg/mL、1μg/mL、2μg/mL、5μg/mL和10μg/mL的TMA标准溶液。</li>
</ol>4. 样品顶空处理
<ol><li>在 20 mL 顶空小瓶中准确称取 2 mL 氢氧化钠溶液和 0.5 g 固体石蜡（熔点：58-60 °C）（参见 材料表）。</li>
	<li>将顶空小瓶置于70°C的烤箱中约30分钟。固体石蜡完全熔化。</li>
	<li>将其取出，让它冷却至室温，使石蜡凝固。固化的石蜡将密封氢氧化钠。</li>
	<li>取 2 mL 每种样品提取溶液，将其放在石蜡层顶部，按下盖子并密封。</li>
	<li>将密封的顶空小瓶放在机器上（参见 材料表）进行测量。</li>
</ol>5. HS-GC-MS/MS 分析条件的设置
<ol><li>有关顶空条件和GC-MS条件，请参见 表2 。</li>
	<li>有关离子信息，请参见 表3 。</li>
</ol>6. 标准曲线图
<ol><li>参照步骤4.1-4.3中的顶空处理样品，制备含有碱液和石蜡密封层的顶空小瓶。</li>
	<li>将 2 mL 的 0.1 μg/mL、0.5 μg/mL、1 μg/mL、2 μg/mL、5 μg/mL 和 10 μg/mL TMA 标准溶液吸入 20 mL 顶空小瓶中，密封盖子，并在机器上测量。</li>
</ol>7. 精密度测试
<ol><li>参照步骤4.1-4.3中的顶空处理样品，制备含有碱液和石蜡密封层的顶空小瓶。</li>
	<li>将 2 mL 的 0.1 μg/mL TMA 标准溶液吸入 20 mL 顶空小瓶中并密封盖子。按照制造商的说明在机器中进行六次平行测试（请参阅 材料表）。</li>
</ol>8. 回收率实验
<ol><li>取一批 Pheretima，Periplaneta Americana 和 Hirudo （S02，S05，S07; 表 1）作为回收率实验的代表药物。</li>
	<li>取几批样品粉末（S02，S05，S07），并在50°C的烤箱中烘烤72小时，直到没有检测到TMA。</li>
	<li>参考第4-6节中的样品制备方法，以检测烘焙药粉中TMA的含量。</li>
	<li>取1 g焙烧粉（精确至0.001 g），放入50 mL塑料离心管中，加入50 μLTMA标准溶液。 注意：TMA标准溶液的浓度为100μg/mL，1000μg/ mL和10000μg/ mL。</li>
	<li>加入 20 mL 的 5% TCA 溶液，并在 1000 rmin-1 下匀浆 1 分钟。</li>
	<li>均质化后，以1717× g 离心5分钟，在玻璃漏斗中加入少许吸收棉，并将上清液过滤到50mL容量瓶中。</li>
	<li>用 15 mL 和 10 mL 的 5% TCA 溶液重复上述提取过程两次;合并滤液，用5%TCA溶液稀释至50mL。</li>
</ol>9. 检测限 （LOD） 和定量限 （LOQ） 的测定
<ol><li>当信噪比 （S/N） = 3 时，通过相应的浓度确定 LOD。</li>
	<li>当信噪比=10时，通过相应的浓度确定LOQ。</li>
</ol>10. 样品TMA含量的测定
<ol><li>分别取Pheretima，Periplaneta americana和Hirudo的细粉约1g，按上述方法提取样品，并在机器上测定。 </li>
</ol>

顶空 GC-MS 检测动物源性药物中的三甲胺

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作者没有什么可透露的。

动物源性药物来自动物的全身、器官或组织、生理或病理产物、排泄物或分泌物以及加工产品。TMA是动物源性药物中鱼腥味的重要来源;它是一种典型的恶臭物质，嗅觉阈值非常低（0.000032 × 10-6 V/V）和强烈的鱼腥味13。目前，常用的HS-GC-MS方法无法稳定、准确地检测动物源性药物中的TMA。
该方案在几个方面进行了改进：（1）TMA更具极性和碱性。该方?...

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

利用动物器官和/或其副产品衍生的产品（此处称为动物源性药物）来治疗人类疾病正受到越来越多的关注。在治疗癌症、心血管疾病、肝硬化、乳腺炎等疾病中发挥重要作用，具有疗效强、用量小、临床疗效显著、特异性强等优点。但动物源性药物一般有明显的鱼腥味，影响患者依从性很大，对儿童尤其不利1,2。腥味主要来源于药物中所含的蛋白质、氨基酸、脂肪等物质，经脂肪酸氧化、氨基酸降解等方式分解，产生多种具有鱼腥味的物质2、3、4。其中，三甲胺（TMA）是一种具有鱼腥味的挥发性气体，广泛存在于腐烂或腐烂的动物源性食品中5。
到目前为止，气相色谱（GC）、液相色谱（LC）、离子色谱、分光光度法、液相色谱-质谱联用法（LC-MS）和传感器法已被普遍用于检测环境、食物和尿液中的TMA6,7,8,9。鉴于气相色谱柱和进样系统的低污染，以及高灵敏度、重现性和低检测限（0.1-1 mg/kg），顶空气相色谱-质谱（HS-GC-MS）法是食品和生物分析的首选方法8。目前，只有中国建立了食品中TMA的国家标准，HS-GC-MS是GB5009.179-2016标准10中的第一种方法。因此，选择上述HS-GC-MS方法检测动物源性药物中的TMA。在早期阶段，我们的研究小组发现食品中TMA的HS-GC-MS检测标准可以检测几种动物源性药物中的鱼腥味。结合研究结果11,12，可以证明TMA是动物源性药物中鱼腥味的常见关键物质。但发现实验结果重现性差，存在TMA逃逸、稳定性差等问题，无法通过该方法验证。这可能是由于碱液被注入顶空小瓶中，快速的酸碱反应导致小瓶中的压力增加，因此TMA从注射孔中逸出，从而阻止了TMA的稳定和准确检测。因此，本研究提出了一种改进的顶空气相色谱-串联四极杆质谱（HS-GC-MS/MS）检测方法来解决这一问题。
该方案通过在固体石蜡（一种良好的固液相变材料）的帮助下分离预处理中的酸碱反应物来改进样品预处理。随着石蜡随着恒温炉温度的升高而缓慢液化，TMA也在密封的顶空小瓶中缓慢释放，避免了剧烈而快速的酸碱反应引起的压力增加，保证了TMA检测的稳定准确。此外，GC-MS/MS中的顶空进样结合多种反应监测（MRM）模式有效地抑制了基质化学干扰，保证了结果的可靠性。方法验证结果表明，改进检测方法的线性、精密度和回收率均能满足要求，重现性好，灵敏度高。

<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

动物源性药物具有鲜明的特点和显著的疗效，但大多有明显的鱼腥味，导致临床患者的依从性较差。三甲胺（TMA）是动物源性药物中关键的鱼腥味成分之一。由于加入碱液后快速的酸碱反应导致顶空小瓶压力增加，导致TMA从顶空小瓶中逸出，阻碍了动物源性药物鱼腥味的研究进展，因此使用现有的检测方法很难准确识别TMA。在这项研究中，我们提出了一种受控检测方法，该方法引入了石蜡层作为酸和碱液之间的隔离层。通过恒温炉加热使石蜡层缓慢液化，可以有效控制TMA的产量。该方法表现出令人满意的线性、精密的实验和回收率，重现性好，灵敏度高。为动物源性药物除臭提供技术支持。

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.

作者聚焦：一种改进的顶空气相色谱-串联四极杆质谱法检测动物源性药物中三甲胺的技术  

一种改进的顶空气相色谱-串联四极杆质谱法检测动物源性药物中三甲胺的技术

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.

该协议的预处理原理和操作原理图分别如图 1 和 图2所示。TMA的峰时间为2.3 min，峰形尖锐，不受其他杂质干扰（图3）。测定0.1-10 μg/mL TMA标准溶液的线性范围，以TMA浓度为横坐标，峰面积为纵坐标，绘制标准曲线。线性回归方程为y = 2522482x + 24255，相关系数（R2）= 0.9998，表现出良好的线性关系。LOD和LOQ的计算分别?...

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

本文描述了一种适用于测定动物源性药物中三甲胺（TMA）的顶空气相色谱-串联四极杆质谱（HS-GC-MS/MS）方法。该方案包括样品预处理、顶空处理、分析条件、方法验证以及动物源性药物中TMA的测定。

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

我们的研究范围是动物医学中鱼味变化的物质基础。该方案提供了一种简单的预处理方法和动物药物中三甲胺的准确检测。该协议的简单预处理方法和HS-GC质量/质量数的编程参数需要进一步优化，我们还有很多工作要做。我们的方案采用气相色谱质量/质量数的多方向监测模式，结合独特、简单的预处理，不仅是三甲胺分析的第一个预处理，也是第一个要使用的分析模式。

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

1.3K 次观看.  Chengdu University of Traditional Chinese Medicine. 我们的研究范围是动物医学中鱼味变化的物质基础。该方案提供了一种简单的预处理方法和动物药物中三甲胺的准确检测。该协议的简单预处理方法和HS-GC质量/质量数的编程参数需要进一步优化，我们还有很多工作要做。我们的方案采用气相色谱质量/质量数的多方向监测模式，结合独特、简单的预处理，不仅是三甲胺分析的第一个预处理，也是第一个要使用的分析模式...