Name: gas chromatography-mass spectrometry paired with total vaporization solid-phase microextraction as a forensic tool
Uploaded: 2021-05-25T13:00:00
Description: Watch this Scientific Journal Video about Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool at JoVE.com

This research was supported by the National Institute of Justice (Award No. 2015-DN-BX-K058 &#38; 2018-75-CX-0035). The opinions, findings, and conclusions expressed here are those of the author and do not necessarily reflect those of the funding organizations.

1. General TV-SPME sample preparation and GC-MS analysis
NOTE: If the sample is already dissolved in a matrix, skip to Step 1.2.
<ol>
	<li>Extract or dissolve the solid sample in enough solvent (water, methanol, acetone, etc.) to reach the desired concentration. Liquid samples can be used &#8220;as is&#8221;. 
	NOTE: The amount of solid sample used depends on the desired concentration of the sample. Concentrations below 1 ppm (w/v) are recommended to avoid overloading the GC column. Ana...

<ol>
	<li>Pawliszyn, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+and+Device+for+Solid+Phase+Microextraction+and+Desorption.">Method and Device for Solid Phase Microextraction and Desorption.</a> United States patent. , (2005).</li><li>Pawliszyn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid phase microextraction: theory and practice. , (1997).</li><li>Rainey, C. L., Bors, D. E., Goodpaster, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+optimization+of+a+total+vaporization+technique+coupled+to+solid-phase+microextraction.">Design and optimization of a total vaporization technique coupled to solid-phase microextraction.</a> Analytical Chemistry. 86 (22), 11319-11325 (2014).</li><li>Sinnott, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Engineering Design: Chemical Engineering. 6, (2005).</li><li>Davis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Detection of Illicit Drugs in Various Matrices Via Total Vaporization Solid-Phase Microextraction (TV-SPME). , (2019).</li><li>Ash, J., Hickey, L., Goodpaster, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+identification+of+novel+derivatives+of+primary+amine+and+zwitterionic+drugs.">Formation and identification of novel derivatives of primary amine and zwitterionic drugs.</a> Forensic Chemistry. 10, 37-47 (2018).</li><li>Sauzier, G., Bors, D., Ash, J., Goodpaster, J. V., Lewis, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+recovery+protocols+for+double-base+smokeless+powder+residues+analysed+by+total+vaporisation+(TV)+SPME/GC-MS.">Optimisation of recovery protocols for double-base smokeless powder residues analysed by total vaporisation (TV) SPME/GC-MS.</a> Talanta. 158, 368-374 (2016).</li><li>Bors, D., Goodpaster, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+smokeless+powder+residue+on+PVC+pipe+bomb+fragments+using+total+vaporization+solid+phase+microextraction.">Mapping smokeless powder residue on PVC pipe bomb fragments using total vaporization solid phase microextraction.</a> Forensic science international. 276, 71-76 (2017).</li><li>Bors, D., Goodpaster, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+analysis+of+racing+fuels+using+total+vaporization+and+gas+chromatography+mass+spectrometry+(GC/MS).">Chemical analysis of racing fuels using total vaporization and gas chromatography mass spectrometry (GC/MS).</a> Analytical Methods. 8 (19), 3899-3902 (2016).</li><li>Beiranvand, M., Ghiasvand, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+optimization+of+the+VA-TV-SPME+method+for+ultrasensitive+determination+of+the+PAHs+in+polluted+water.">Design and optimization of the VA-TV-SPME method for ultrasensitive determination of the PAHs in polluted water.</a> Talanta. 212, 120809 (2020).</li></ol>

TV-SPME has some benefits over liquid injection GC in that large sample sizes (e.g., 100 &#181;L) may be used without instrument modifications. TV-SPME also has some of the same benefits as headspace SPME. Headspace SPME does not require any extraction or filtration because any nonvolatile compounds will remain in the headspace vial and will not be adsorbed onto the fiber, yielding a clean sample. This method also helps to eliminate matrix effects due to this being a two-phase system (headspace and fiber) as opposed to a...

Solid-Phase Microextraction (SPME) is a sampling technique in which a liquid or solid sample is placed into a headspace vial and a SPME fiber, coated with a polymeric material, is then introduced into the sample headspace (or immersed in a liquid sample). The analyte is sorbed onto the fiber and then the fiber is placed inside the GC inlet for desorption1,2. Total Vaporization Solid-Phase Microextraction (TV-SPME) is a similar technique as immersion SPME but completely vaporizes a liquid sample before analytes are adsorbed onto the fiber. This allows for partitioning of the analyte between only the solvent vapor and the coating of the fiber, allowing for more of the analyte to be adsorbed onto the fiber and resulting in good sensitivity3. There are various SPME fibers available and the fiber should be chosen based on the analyte of interest, solvent/matrix, and derivatization agent. See Table 1 for established TV-SPME analytes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Analyte(s)</td>
			<td>Recommended SPME Fiber</td>
			<td>Reference(s)</td>
		</tr>
		<tr>
			<td>Human Hair</td>
			<td>Nicotine, cotinine</td>
			<td>Polydimethylsiloxane/divinylbenzene (PDMS/DVB), polyacrylate (PA)</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Smokeless Powder</td>
			<td>Nitroglycerin, diphenylamine</td>
			<td>Polydimethylsiloxane (PDMS), polyethylene glycol (PEG)</td>
			<td>7, 8</td>
		</tr>
		<tr>
			<td>Racing fuel</td>
			<td>Methanol, nitromethane</td>
			<td>PEG</td>
			<td>9</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Polycyclic aromatic hydrocarbons</td>
			<td>PDMS</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Beverages</td>
			<td>&#611;-Hydroxybutyric acid, &#611;-butyrolactone</td>
			<td>PDMS</td>
			<td>This Work</td>
		</tr>
		<tr>
			<td>Solid Powder</td>
			<td>Methamphetamine, amphetamine</td>
			<td>PDMS/DVB</td>
			<td>Unpublished</td>
		</tr>
	</tbody>
</table>
Table 1. Recommended SPME fibers with established TV-SPME analytes.
To perform TV-SPME, analytes are dissolved in a solvent and an aliquot of this mixture is placed into a headspace vial. Samples do not need to be filtered because only the solvent and volatile analytes will vaporize. Specific volumes of liquid samples must be used to ensure total vaporization of the sample. These volumes are determined by using the Ideal Gas Law to calculate the number of moles of a solvent multiplied by the molar volume of the liquid (Equation 1). 
<img alt="Equation 1" src="/files/ftp_upload/61880/61880equ01.jpg" /> Equation 1
where Vo is the volume of the sample (mL), P is the vapor pressure of the solvent (bar), Vv is the volume of the vial (L), R is the ideal gas constant (0.083145 <img alt="Equation 1" src="/files/ftp_upload/61880/61880equ04.jpg" />), M is the molar mass of the solvent (g/mol), T is temperature (K), and <img alt="Equation 5" src="/files/ftp_upload/61880/61880equ05.jpg" /> is the density of the solvent (g/mL).3
In order to use the correct vapor pressure, the Antoine equation (Equation 2) is used to account for the influence of temperature:4 
<img alt="Equation 2" src="/files/ftp_upload/61880/61880equ02.jpg" /> Equation 2
where T is temperature and A, B, and C are the Antoine constants for the solvent. Equation 2 may be substituted into Equation 1, yielding: 
<img alt="Equation 3" src="/files/ftp_upload/61880/61880equ03v2.jpg" /> Equation 3
Equation 3 gives the volume of the sample (Vo) that can be completely vaporized as a function of the temperature and solvent used.
To perform derivatization with TV-SPME, the SPME fiber is first exposed to a vial containing the derivatization agent for a predetermined amount of time depending on the analyte. The SPME fiber is then exposed to a new vial containing the analyte of interest. This vial is heated inside of a heated agitator. The analyte is then adsorbed onto the fiber with the derivatization agent. The derivatization of the analyte and/or the matrix takes place on the fiber before being inserted into the GC inlet for desorption. Figure 1 shows a depiction of the TV-SPME process with derivatization.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61880/61880fig01.jpg" /> 
Figure 1: Depiction of the TV-SPME process with derivatization.&#160;The SPME fiber first enters the derivatization vial where the derivatization agent (yellow circles) sorb onto the fiber. The fiber is then introduced to the sample (blue circles) and heated. Formation of the derivative (green circles) takes place on the fiber during the extraction time. <a href="https://www.jove.com/files/ftp_upload/61880/61880fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
TV-SPME is beneficial because it allows for the analyte to be derivatized during the extraction process which reduces analysis time. Other methods, such as liquid injection, require that the analyte react with the derivatizing agent in solution prior to being injected into the GC. TV-SPME also requires little to no sample preparation. A matrix containing an analyte may be placed directly into the headspace vial and analyzed. Many compounds of interest are compatible with TV-SPME. Compounds must be soluble in a solvent and sufficiently volatile to allow for vaporization. Additionally, compounds must be thermally stable to be analyzed by GC-MS. TV-SPME has been used to analyze drugs and drug metabolites, racing fuels, polycyclic aromatic hydrocarbons, and explosive materials3,5,6,7,8,9,10.

<table>
	<tbody>
		<tr>
			<td>10 &#181;L Syringe</td>
			<td>Gerstel</td>
			<td>100111-014-00</td>
			<td></td>
		</tr>
		<tr>
			<td>BSTFA + 1% TMCS (10 x 1 GM)</td>
			<td>Regis Technologies Inc.</td>
			<td>50442882</td>
			<td></td>
		</tr>
		<tr>
			<td>eVol XR Sample Dispensing System Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>66002-024</td>
			<td></td>
		</tr>
		<tr>
			<td><img alt="Equation 6" src="/files/ftp_upload/61880/61880equ06.jpg" />-Butyrolactone (GBL)</td>
			<td>Sigma-Aldrich</td>
			<td>B103608-26G</td>
			<td></td>
		</tr>
		<tr>
			<td><img alt="Equation 7" src="/files/ftp_upload/61880/61880equ06.jpg" />-Hydroxy Butyric Acid (GHB)</td>
			<td>Cayman Chemicals</td>
			<td>9002506</td>
			<td></td>
		</tr>
		<tr>
			<td>Headspace Screw-Thread Vials, 18 mm</td>
			<td>Restek</td>
			<td>23083</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Screw-Thread Caps, 18 mm</td>
			<td>Restek</td>
			<td>23091</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima water for HPLC</td>
			<td>Fisher Chemical</td>
			<td>W71</td>
			<td></td>
		</tr>
		<tr>
			<td>SPME Fiber Assembly Polydimethylsiloxane (PDMS)</td>
			<td>Supelco</td>
			<td>57341-U</td>
			<td></td>
		</tr>
		<tr>
			<td>SPME Fiber Assembly Polydimethylsiloxane/Divinylbenzene (PDMS/DVB)</td>
			<td>Supelco</td>
			<td>57293-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Topaz 2.0 mm ID Straight Inlet Liner</td>
			<td>Restek</td>
			<td>23313</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A GBL volume study was performed to demonstrate the sensitivity of TV-SPME compared to headspace and immersion SPME. A 100ppmv sample of GBL in water was prepared and placed into 20 mL headspace vials with volumes of 1, 3, 10, 30, 100, 300, 1000, 3000, and 10,000 &#181;L. The phase ratio of the samples allowed for TV-SPME (1-3 &#181;L), Headspace SPME (10 &#8211; 3,000 &#181;L) and Immersion SPME (10,000 &#181;L). All samples were analyzed in triplicate and the average peak area was plotted against the sample ...

Watch this Scientific Journal Video about Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool at JoVE.com

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

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 ...

The TV-SPME protocol is significant because it can analyze a wide range of samples, including drugs, racing fuels, explosive materials, and polycyclic aromatic hydrocarbons. No filtration is needed because only volatile analytes will vaporize. So dirty samples or suspensions can be analyzed.Very simple matrices can be used, including organic or aqueous solvents. The main advantage of the TV-SPME technique is its high sensitivity. TV-SPME demonstrates better sensitivity than standard liquid injection, headspace SPME, and immersion SPME.TV-SPME also has the benefit of utilizing large sample volumes without any changes to the GC instrumentation. TV-SPME is a very simple technique to perform. The main difficulty comes from ensuring the proper volume has been delivered.The use of small manual or electronic syringes can help, as well as taking your time while sampling. It can also be difficult to ensure the fiber maintains its coating, so fibers must be monitored closely for degradation. Visual demonstration is crucial because both manual and robotic manipulations are required to be successful.To begin, extract or dissolve the solid sample in enough solvent to reach the desired concentration. After ensuring that the sample is fully dissolved, calculate the volume needed to fully vaporize it at the chosen temperature. Transfer the sample volume into a headspace vial, and secure the cap.If derivatizing the sample, prepare the proper derivatization agent by placing approximately one milliliter of the agent into a headspace vial. Set the proper incubation and extraction temperature, ensuring total vaporization, sufficient sample extraction, and complete derivatization. Select GC-MS parameters based on the class of the compounds of interest.Ensure that the proper inlet liner is in the GC inlet and that the SPME fiber has been properly conditioned and is in good working order before beginning the analysis. Prepare a sample of gamma-hydroxybutyrate or gamma-butyrolactone in water, with a concentration of less than one part per million. Transfer one microliter of the sample to a 20-milliliter headspace vial, and cap the vial immediately.Place one milliliter of BSTFA with 1%trimethylchlorosilane into a separate 20-milliliter headspace vial, and cap it. Create a GC-MS method by setting the initial oven temperature to 60 degrees Celsius for one minute, the oven program to 15 degrees per minute, the final oven temperature to 280 degrees for one minute, the flow rate to 2.5 milliliters per minute, and the inlet and transfer line temperatures to 250 and 280 degrees, respectively. Ensure a narrow SPME inlet liner has been placed inside the GC inlet and that the PDMS/DVB SPME fiber has been properly conditioned and is in good working order.Then run the GC-MS on the sample. A GBL volume study was performed to demonstrate the sensitivity of TV-SPME compared to headspace and immersion SPME. Overall, sample volumes that allowed for TV-SPME demonstrated more sensitivity than headspace or immersion SPME for GBL in water.A comparison of the chromatograms for each method is shown here. Samples of wine spiked with an effective dose of GHB and GBL were analyzed. These samples also show the interconversion of GBL and GHB.When TV-SPME is performed properly, a sharp abundant peak is observed. TV-SPME has high sensitivity. Therefore, proper concentrations should be used as to not overload the column.Peak asymmetry occurs when high concentrations are present. In these cases, diluting the sample or using a split injection can improve peak shape. TV-SPME could be paired with liquid chromatography, which would dramatically expand the range of detectable analytes.

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