This work was supported by grants from the National Natural Science Foundation of China (82173991), and Sichuan Science and Technology Program (2022YFS0442).

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Reagent preparation
<ol>
	<li>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.</li>
	<li>Prepare 5% TCA solution: weigh 25 g of TCA and use deionized water to fix the volume in a 500 mL volumetric flask.</li>
</ol>
3. TMA standard stock solution preparation
<ol>
	<li>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 &#181;g/mL of TMA standard stock solution. Store it at 4 &#176;C.</li>
	<li>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 &#181;g/mL, 0.5 &#181;g/mL, 1 &#181;g/mL, 2 &#181;g/mL, 5 &#181;g/mL, and 10 &#181;g/mL TMA standard solution.</li>
</ol>
4. Sample headspace processing
<ol>
	<li>Accurately weigh 2 mL of sodium hydroxide solution and 0.5 g of solid paraffin (melting point: 58-60 &#176;C) in a 20 mL headspace vial (see Table of Materials).</li>
	<li>Place the headspace vial in an oven at 70 &#176;C for about 30 min. The solid paraffin completely melts.</li>
	<li>Take it out, and let it cool down to room temperature so that the paraffin solidifies. The solidified paraffin will seal sodium hydroxide.</li>
	<li>Take 2 mL of each sample extraction solution and put it on top of the paraffin layer, press the cap, and seal.</li>
	<li>Put the sealed headspace vial on the machine (see Table of Materials) for measurement.</li>
</ol>
5. Setting of HS-GC-MS/MS analysis conditions
<ol>
	<li>See Table 2 for headspace conditions and GC-MS conditions.</li>
	<li>See Table 3 for ion information.</li>
</ol>
6. Standard curve drawing
<ol>
	<li>Refer to the sample headspace processing in steps 4.1-4.3 to prepare the headspace vial containing lye and paraffin sealing layer.</li>
	<li>Aspirate 2 mL of 0.1 &#181;g/mL, 0.5 &#181;g/mL, 1 &#181;g/mL, 2 &#181;g/mL, 5 &#181;g/mL, and 10 &#181;g/mL TMA standard solution into a 20 mL headspace vial, seal the cap, and measure on the machine.</li>
</ol>
7. Precision test
<ol>
	<li>Refer to the sample headspace processing in steps 4.1-4.3 to prepare the headspace vial containing lye and the paraffin sealing layer.</li>
	<li>Aspirate 2 mL of 0.1 &#181;g/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&#39;s instructions (see Table of Materials).</li>
</ol>
8. Recovery rate experiment
<ol>
	<li>Take a batch of Pheretima, Periplaneta Americana, and Hirudo (S02, S05, S07; Table 1) as the representative medicines for the recovery rate experiment.</li>
	<li>Take several batches of sample powder (S02, S05, S07) and bake them in an oven at 50 &#176;C for 72 h until no TMA is detected.</li>
	<li>Refer to the sample preparation method in sections 4-6 to detect the content of TMA in baked medicine powder.</li>
	<li>Take 1 g of baked powder (accurate to 0.001 g), put it into a 50 mL plastic centrifuge tube, and add 50 &#181;L of TMA standard solution. 
	NOTE: The concentration of TMA standard solution is 100 &#956;g/mL, 1000 &#956;g/mL, and 10000 &#956;g/mL.</li>
	<li>Add 20 mL of 5% TCA solution and homogenize at 1000 rmin-1 for 1 min.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
9. Determination of the limits of detection (LOD) and quantification (LOQ)
<ol>
	<li>Determine the LOD by the corresponding concentration when the signal-to-noise ratio (S/N) = 3.</li>
	<li>Determine the LOQ by the corresponding concentration when the S/N = 10.</li>
</ol>
10. Determination of sample TMA content
<ol>
	<li>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.</li>
</ol>

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

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	<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. 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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>

The authors have nothing to disclose.

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 &#215; 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.
<p c...

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

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.

<table><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&#039;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

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.

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 &#181;g/mL TMA standard solution, with TMA concentration as the abscissa and peak area as the ordinate, a standard curve was drawn. The linear regression e...

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

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.

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 ...

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

Research

JoVE Journal

Chemistry

1.7K Views.  Chengdu University of Traditional Chinese Medicine. De reikwijdte van ons onderzoek is een materiële basis in de variatie van visgeur in de diergeneeskunde. Dit protocol biedt een eenvoudige voorbehandelingsmethode en nauwkeurige detectie van trimethylamine in diergeneesmiddelen. De eenvoudige voorbehandelingsmethoden en de geprogrammeerde parameters van HS-GC massa/massa van dit protocol, moeten verder worden geoptimaliseerd en we hebben nog veel werk te doen.Ons protocol neemt de multi-direction monitoringmodus van GC-massa / massa o...

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

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

Biochemistry

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Environment

Sampling and Analysis of Animal Scent Signals

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas ...

Behavior

Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including controlled substances, ignitable liquids, and explosives. GC-MS can be coupled with Solid-Phase Microextraction (SPME), in which a fiber with a sorptive coating is placed into the headspace above a sample or immersed in a liquid sample. Analytes are sorbed onto the fiber which is then placed inside the heated GC inlet for desorption. Total Vaporization Solid-Phase Microextraction (TV-SPME) utilizes the same technique as immersion SPME but immerses the fiber into a completely vaporized sample extract. This complete vaporization results in a partition between only the vapor phase and the SPME fiber without interference from a liquid phase or any insoluble materials. Depending upon the boiling point of the solvent used, TV-SPME allows for large sample volumes (e.g., up to hundreds of microliters). On-fiber derivatization may also be performed using TV-SPME. TV-SPME has been used to analyze drugs and their metabolites in hair, urine, and saliva. This simple technique has also been applied to street drugs, lipids, fuel samples, post-blast explosive residues, and pollutants in water. This paper highlights the use of TV-SPME to identify illegal adulterants in very small samples (microliter quantities) of alcoholic beverages. Both gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) were identified at levels that would be found in spiked drinks. Derivatization by a trimethylsilyl agent allowed for conversion of the aqueous matrix and GHB into their TMS derivatives. Overall, TV-SPME is quick, easy, and requires no sample preparation aside from placing the sample into a headspace vial.

Total Vaporization Solid Phase Microextraction (TV-SPME) completely vaporizes a liquid sample whilst analytes are sorbed onto a SPME fiber. This allows for partitioning of the analyte between only the solvent vapor and the SPME fiber coating.

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Bioengineering

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

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

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