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

Summary

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.

Abstract

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.

Introduction

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' compliance, and are especially unfavorable for children^1,2. 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 odor^2,3,4. 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 urine^6,7,8,9. 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 studies^11,12, 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.

Protocol

See Table 1 for information on the medicinal materials of Pheretima, Periplaneta americana, and Hirudo. They were identified by Prof. Xu Runchun, Chengdu University of Traditional Chinese Medicine, as the dried bodies of Pheretima aspergillum (E.Perrier), Periplaneta americana L., and Whitmania pigra Whitman.

1. Specimen extraction

1. Crush Pheretima, Periplaneta americana, and Hirudo with an herbal grinder (see Table of Materials), sift the medicinal powder through No. 2 (sieve aperture: 0.8 mm) and No. 4 (sieve aperture: 0.25 mm) standard drug sieves, and collect the powder between the two sieves to obtain the required sample powder.
   NOTE: The Pheretima is fluffy after crushing, so its powder does not need to be sieved.
2. Take 1 g of powder (accurate to 0.001 g) in a 50 mL plastic centrifuge tube, add 20 mL of 5% trichloroacetic acid (TCA) solution (see Table of Materials), and homogenize at 1,000 rmin^-1 for 1 min with a high-speed dispersion homogenizer.
3. After homogenization, centrifuge at 1,717 x g for 5 min at room temperature, add a little absorbent cotton in the glass funnel, and filter the supernatant into a 50 mL volumetric flask.
4. Repeat the above extraction process twice with 15 mL and 10 mL of 5% TCA solution. Combine the filtrate and dilute it to 50 mL with 5% TCA solution.

2. Reagent preparation

1. Prepare 20% sodium hydroxide solution: weigh 20 g of sodium hydroxide and use deionized water to fix the volume in a 100 mL volumetric flask.
2. Prepare 5% TCA solution: weigh 25 g of TCA and use deionized water to fix the volume in a 500 mL volumetric flask.

3. TMA standard stock solution preparation

1. Prepare TMA standard stock solution: weigh 0.0162 g of TMA hydrochloride standard sample, dissolve it in 5% TCA solution, and fix the volume to 100 mL, equal to the concentration of 100 µg/mL of TMA standard stock solution. Store it at 4 °C.
2. Prepare TMA standard use solution: take a certain volume of TMA standard stock solution and dilute it step by step with 5% TCA solution to concentrations of 0.1 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL, 5 µg/mL, and 10 µg/mL TMA standard solution.

4. Sample headspace processing

1. Accurately weigh 2 mL of sodium hydroxide solution and 0.5 g of solid paraffin (melting point: 58-60 °C) in a 20 mL headspace vial (see Table of Materials).
2. Place the headspace vial in an oven at 70 °C for about 30 min. The solid paraffin completely melts.
3. Take it out, and let it cool down to room temperature so that the paraffin solidifies. The solidified paraffin will seal sodium hydroxide.
4. Take 2 mL of each sample extraction solution and put it on top of the paraffin layer, press the cap, and seal.
5. Put the sealed headspace vial on the machine (see Table of Materials) for measurement.

5. Setting of HS-GC-MS/MS analysis conditions

1. See Table 2 for headspace conditions and GC-MS conditions.
2. See Table 3 for ion information.

6. Standard curve drawing

1. Refer to the sample headspace processing in steps 4.1-4.3 to prepare the headspace vial containing lye and paraffin sealing layer.
2. Aspirate 2 mL of 0.1 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL, 5 µg/mL, and 10 µg/mL TMA standard solution into a 20 mL headspace vial, seal the cap, and measure on the machine.

7. Precision test

1. Refer to the sample headspace processing in steps 4.1-4.3 to prepare the headspace vial containing lye and the paraffin sealing layer.
2. Aspirate 2 mL of 0.1 µg/mL TMA standard solution into a 20 mL headspace vial and seal the cap. Carry out six parallel tests in the machine following the manufacturer's instructions (see Table of Materials).

8. Recovery rate experiment

1. Take a batch of Pheretima, Periplaneta Americana, and Hirudo (S02, S05, S07; Table 1) as the representative medicines for the recovery rate experiment.
2. Take several batches of sample powder (S02, S05, S07) and bake them in an oven at 50 °C for 72 h until no TMA is detected.
3. Refer to the sample preparation method in sections 4-6 to detect the content of TMA in baked medicine powder.
4. Take 1 g of baked powder (accurate to 0.001 g), put it into a 50 mL plastic centrifuge tube, and add 50 µL of TMA standard solution.
   NOTE: The concentration of TMA standard solution is 100 μg/mL, 1000 μg/mL, and 10000 μg/mL.
5. Add 20 mL of 5% TCA solution and homogenize at 1000 rmin^-1 for 1 min.
6. After homogenization, centrifuge at 1717 x g for 5 min, add a little absorbent cotton in the glass funnel and filter the supernatant into a 50 mL volumetric flask.
7. Repeat the above extraction process twice with 15 mL and 10 mL of 5% TCA solution; combine the filtrate and dilute it to 50 mL with 5% TCA solution.

9. Determination of the limits of detection (LOD) and quantification (LOQ)

1. Determine the LOD by the corresponding concentration when the signal-to-noise ratio (S/N) = 3.
2. Determine the LOQ by the corresponding concentration when the S/N = 10.

10. Determination of sample TMA content

1. Take about 1 g of fine powder of Pheretima, Periplaneta americana, and Hirudo, respectively, extract the sample according to the above method, and determine it on the machine.

Representative Results

Schematic diagrams of the pre-processing principle and operation of this protocol are shown in Figure 1 and Figure 2, respectively. The peak time of TMA was 2.3 min, with a sharp peak shape and no interference from other impurities (Figure 3). Measuring the linear range of 0.1-10 µg/mL TMA standard solution, with TMA concentration as the abscissa and peak area as the ordinate, a standard curve was drawn. The linear regression equation was obtained as y = 2522482x + 24255, with the correlation coefficient (R²) = 0.9998, showing a good linear relationship. The LOD and LOQ were calculated with S/N = 3 and S/N = 10, respectively. The LOD was 0.03 mg/kg and the LOQ was 0.11 mg/kg. To investigate the precision of this method, the content of TMA (0.1 µg/mL) was determined six times in parallel with a relative standard deviation (RSD) of 5.84%, which proved the good precision of this method. A group of samples from Pheretima, Periplaneta americana, and Hirudo were selected as representative samples for the recovery experiment (S02, S05, S07, respectively); these were subjected to spiked recovery tests by drying to reduce TMA in the herbs, and the average recovery rates were 84.49%, 94.66%, and 85.67%, respectively, with the accuracy meeting the analysis requirements (Table 4). TMA was detected in nine batches of herbs from Pheretima, Periplaneta Americana, and Hirudo, with concentrations ranging from 13.23-271.63 mg/kg (Table 5). This protocol method has good methodological validation results and also detected TMA content in animal-derived drugs with an obvious fishy odor.

Figure 1: Schematic diagram of reaction principle of lye-paraffin-extraction solution. (1) Accurately weigh 2 mL of sodium hydroxide solution in a 20 mL headspace vial. (2) Add 0.5 g of solid paraffin to the headspace vial. (3) Heat to melt the solid paraffin, which is layered with sodium hydroxide solution and floats above the sodium hydroxide solution. (4) After cooling, the paraffin solidifies and seals firmly over the sodium hydroxide solution. (5) Take 2 mL of sample extraction solution and put it on top of the paraffin layer, press the cap, and seal. (6) Put the sealed headspace vial on the machine for measurement. The heating of the thermostatic oven melts the paraffin layer, and the acid-base reactants above and below the paraffin layer react to produce TMA in a sealed environment. The headspace processing of the sample is roughly carried out in sections 1 to 6. Please click here to view a larger version of this figure.

Figure 2: Schematic diagram of sample pretreatment operation. (A) Sealing lye: step 4.1-4.3 in the protocol. (B) Sample extraction: section 1 and step 4.4 in the protocol. (C) TMA detection: step 4.5 and section 5 in the protocol. Please click here to view a larger version of this figure.

Figure 3: Total ion chromatogram of TMA. Spectrogram of 1 µg/mL TMA standard solution. Please click here to view a larger version of this figure.

	Batch	Origin
Pheretima	S01	Leshan City, Sichuan Province
Pheretima	S02	Dianbai City, Guangdong Province
Pheretima	S03	Maoming City, Guangdong Province
Periplaneta americana	S04	Xichang City, Liangshan Yi Autonomous Prefecture, Sichuan Province
Periplaneta americana	S05	Midu County, Dali Prefecture, Yunnan Province
Periplaneta americana	S06	Bozhou City, Anhui Province
Hirudo	S07	Weishan County, Jining City, Shandong Province
Hirudo	S08	Kunshan City, Jiangsu Province
Hirudo	S09	Laiwu District, Jinan City, Shandong Province

Table 1: Animal-derived medicine information.

Headspace condition
Temperature of thermostatic oven	80 °C
Time for sample b thermostatting	30 min
Headspace needle temperature	100 °C
Sample size	1 mL
GC-MS conditions
Chromatographic Column	SH-Volatile Amine,30m×0.32mm×5µm
Column temperature program	40 °C (0.5 min) _20 °C /min _200 °C (5 min)
Injector temperature	200 °C
Carrier gas control mode	constant linear speed
Injection mode	split injection
Split ratio	10:01
Column flow	2 mL/min
Sample size	1 mL
Ionization mode	EI
Ion source temperature	200 °C
GC-MS interface temperature	230 °C
Detector voltage	Tuning voltage +0.6 kV
Acquisition mode information	MRM

Table 2: Headspace condition and GC-MS/MS conditions.

Compound name	CAS	Retention time (min)	Quantitative ion (m/z)	CE	Reference ion (m/z)	CE
Trimethylamine	75-50-3	2.308	58>42	20	58>30	7

Table 3: TMA compound information.

Sample	Sample concentration (mg/kg)	Spiked concentration (mg/kg)	Measured concentration (mg/kg)	Average recovery rate (%)	RSD (%)
S02	128.99	500.00	548.50	84.49	2.12%
S05	49.08	500.00	520.93	94.66	0.96%
S07	101.36	500.00	527.07	85.67	1.87%

Table 4: Results of recovery rate experiment for TMA in animal-derived medicines.

Sample	Sample concentration (mg/kg)
S01	88.11
S02	137.34
S03	18.63
S04	19.10
S05	40.50
S06	13.23
S07	271.63
S08	69.73
S09	67.70

Table 5: Results of determination for TMA concentration in animal-derived medicines.

Discussion

Animal-derived medicines come from the whole body, organs or tissues, physiological or pathological products, excretions or secretions, and processed products of animals. TMA is an important source of fishy odor in animal-derived medicines; it is a typical malodorous substance with a very low olfactory threshold (0.000032 × 10^-6 V/V) and a strong fishy odor¹³. At present, the commonly used HS-GC-MS method cannot detect TMA in animal-derived medicines stably and accurately.

This protocol is improved in several aspects: (1) TMA is more polar and alkaline. In this protocol, a special column for volatile amine gas chromatography is selected to detect TMA, which ensures the accuracy and sensitivity of TMA detection. (2) In the sample preparation process of the HC-GC-MS method in GB5009.179-2016, a high-concentration sodium hydroxide solution is injected into the sealed headspace vial¹⁰. At this time, the occurrence of the acid-base reaction leads to an increase in pressure in the headspace vial, which may cause the escape of TMA, resulting in inaccurate detection of TMA. This protocol referred to the detection method of sulfur dioxide residue in traditional Chinese medicine¹⁴. In the sample pretreatment, solid paraffin is used as a medium to isolate acid-base reactants. After the headspace vial is sealed, the paraffin melts slowly under the heating of the thermostatic furnace, avoids the severe acid-base reaction, and provides a good airtight environment for TMA reaction, ensuring the stability and accuracy of TMA detection. (3) This protocol uses the MRM mode in GC-MS/MS for acquisition and optimizes the detection parameters (column temperature program, etc.) to ensure analytical efficiency and accuracy.

The following points must be paid attention to in the operation of this protocol: (1) an appropriate amount of solid paraffin wax must be selected. A smaller paraffin dosage will lead to an unsealed paraffin layer and the immediate reaction of acid-base reactants, resulting in the generation and escape of TMA before sealing. A higher paraffin dosage may hinder the release, enrichment, and detection of TMA. (2) The gland must be tight and the seal intact. In addition, there are some limitations of the protocol. TMA in animal-derived medicines is endogenous and cannot be removed cleanly by drying; a low concentration of TMA hydrochloride standard solution was used in the recovery experiment, but the effect was unsatisfactory. Therefore, only the same concentration of TMA hydrochloride standard solution was selected for the recovery experiment in this protocol.

In conclusion, this protocol provided a sample pretreatment method and accurate detection of TMA in animal-derived medicines. The establishment of this method filled the gap in the detection method of TMA in animal-derived medicines and provided technical support for the research of fishy odor substances in animal-derived medicines, which is of great significance for promoting the research, development, and application of animal-derived medicines.

Materials

Name	Company	Catalog Number	Comments
Centrifuge	Beckman Coulter Trading (China) Co.	SSC-2-0213
Chinese herbal medicine grinder	Zhejiang Yongkang Xi'an Hardware and Pharmaceutical Factory	HX-200K
Convection oven	Sanyo Electric Co., Ltd	MOV-112F
Decapper for 20 mm Aluminum caps	ANPEL Laboratory Technologies (Shanghai) Inc	V1750004
Electronic balance	Shimadzu Corporation Japan	AUW220D
Gas chromatography mass spectrometry	Shimadzu Corporation Japan	TQ-8050 NX
Headspace Vial	ANPEL Laboratory Technologies (Shanghai) Inc	25760200
Homogenizer	Shanghai biaomo Factory	FJ200-SH
Preassembled Cap	ANPEL Laboratory Technologies (Shanghai) Inc	L4150050
Sample sieve	Zhenxing Sieve Factory	/
SH-Volatile Amine	Chengdu Meimelte Technology Co., Ltd	227-3626-01
Sodium hydroxide	Chengdu Chron Chemicals Co., Ltd	2022101401
Solid paraffin wax	Shanghai Hualing Kangfu apparatus factory	20221112
Trichloroacetic acid	Chengdu Chron Chemicals Co., Ltd	2022102001
Trimethylamine hydrochloride	Chengdu Aifa Biotechnology Co., Ltd	AF22022108
Ultra-pure water system	Sichuan Youpu Ultrapure Technology Co., Ltd	UPR-11-5T