This work was supported by the intramural research project (SFP #1323-103) at the German Federal Institute for Risk Assessment (BfR).

1. Tattoo ink preparation and sample mounting
<ol>
	<li>Use a 25 mm hollow glass pyrolysis tube as a sample holder and quartz wool for sample preparation.
	<ol>
		<li>Grab the pyrolysis tube with the specialized tweezers for pyrolysis tubes (bake out for decontamination) and insert the necessary amount of quartz wool with pointed tweezers into the tube.</li>
		<li>Insert two steel sticks (bake out for decontamination) at each side of the pyrolysis tube and compress the wool into a 1&#8211;2 mm thick stopper. The stopper must be positioned at the lower third of the pyrolysis tube to achieve adequate heating during the pyrolysis process.</li>
		<li>Ignite a gas burner and bake the pyrolysis tube and filling for 2&#8211;3 s from each side to remove contaminants. 
		NOTE: Use clean gloves and do not touch any item before handling the pyrolysis tube and wool. Use eye protection and remove all flammable liquids and items during the pyrolysis tube bake out. The protocol can be stopped here. Store the prepared pyrolysis tubes in a clean glass dish until further use.</li>
	</ol>
	</li>
	<li>Shake the tattoo ink bottles vigorously for 1 min by hand to ensure homogeneity. Additionally, they can be placed in an ultrasonic water bath for 1 min. Some inks may still show sedimented pigments after conducting both steps and may need prolonged homogenization.</li>
	<li>Obtain a 2 &#181;L microcapillary with a diameter below the inner diameter of the pyrolysis tube. Dip the capillary tip in the ink and aspirate about 1 &#181;L of ink by filling half of the capillary.</li>
	<li>Insert the capillary into the pyrolysis tube and stain the quartz wool stopper with the ink. A clear color staining must be visible without adding too much ink to the sample.</li>
	<li>Obtain a steel transport adapter for the automated injection unit and attach the prepared pyrolysis tube to it using tweezers for pyrolysis tubes.
	<ol>
		<li>Check that the pyrolysis tube is perfectly vertical and does not fall off during shaking.</li>
		<li>Place the transport adapter in the tray at the desired position. 
		NOTE: The ink might contaminate the steel transport adapter to which the pyrolysis tube is attached; therefore, this needs more thorough cleaning afterwards.</li>
	</ol>
	</li>
</ol>
2. Analysis of ink samples by py-GC-MS
<ol>
	<li>Set up the GC-MS-system equipped with a Thermal Desorption Unit (TDU) and a pyrolyzer module on top of the Cold Injection System (CIS) according to the manufacturer&#8217;s instructions. Use an inert electron impact (EI) ion source at 70 eV and helium with a purity of 99.999% as carrier gas (1 mL/min). Set the split ratio of the CIS to 1:50.</li>
	<li>Install an HP-5MS column and a guard column for separation. Set the following analysis parameters in the instruments&#8217; control software: start the oven temperature at 50 &#176;C, hold for 2 min, and ramp at 10 &#176;C/min to 320 &#176;C. Hold the final temperature for additional 5 min. Set the transfer line temperatures to 320 &#176;C.</li>
	<li>Run each sample in a solvent vent mode without pyrolysis to analyze for semi-volatile compounds.
	<ol>
		<li>Use the solvent vent option of the TDU/injector and ramp the TDU temperature after 0.5 min starting at 50 &#176;C to 90 &#176;C at a rate of 100 &#176;C/min. The solvent vent will be shut off after 1.9 min.</li>
		<li>Heat the temperature of the TDU to 320 &#176;C at a rate of 720 &#176;C/min for 3.5 min. The volatile compounds are captured in the CIS at -150 &#176;C.</li>
		<li>Hold the CIS temperature for 2 min and ramp to 320 &#176;C at a 12 &#176;C/min rate. 
		NOTE: Extended time between sample preparation and analysis leads to evaporation of volatile compounds since the sample holder is open. Analyze samples within a few hours after preparation if volatile compounds are being targeted.</li>
	</ol>
	</li>
	<li>Conduct a second run of the same sample in which the pyrolysis unit is used to investigate non-volatile compounds.
	<ol>
		<li>Use the same oven program as for the first desorption run.</li>
		<li>Keep the temperature of the CIS constant at 320 &#176;C and use a split ratio of 1:100.</li>
		<li>Ramp the TDU directly from 50 &#176;C to 320 &#176;C at a 720 &#176;C/min rate and keep constant for 1.6 min.</li>
		<li>Program a 6 s pyrolysis at the desired temperature (here 800 &#176;C) in the final temperature segment of the TDU. 
		NOTE: Make sure to use an adequate amount of sample and split ratio to prevent contamination of the column and MS. Adapt the split ratios for individual instrument set-ups if necessary.</li>
	</ol>
	</li>
	<li>Use a polystyrene standard to check the performance of the instrument. Run at least three polystyrene samples before and after each experiment. Insert 2 &#181;L of a 4 mg/mL polystyrene standard in dichloromethane into the glass wool and perform a pyrolysis at 500 &#176;C for 0.33 min.</li>
	<li>Check if the peak area ratio of the polystyrene monomer and the dimer is between 3 and 4 and the tailing of the dimer is below 2 in the resulting chromatogram (also called a pyrogram). Keep historical data of polystyrene parameters and calibrate the pyrolysis temperature if the peak ratio is out of range. 
	NOTE: Values for polystyrene monomer and dimer ratio as well as tailing should be taken from historical values of well operating systems.</li>
</ol>
3. Data evaluation approaches
NOTE: Data evaluation should be adapted depending on the individual analytical questions, e.g., the search for volatiles, non-volatile compounds, hazardous cleavage products from azo pigments, or similar.
<ol>
	<li>Data evaluation for volatile compounds
	<ol>
		<li>For data evaluation of volatile compounds, start the GC-MS analysis/MS library searching software (see Table of Materials) and open the chromatogram of the desorption run.</li>
		<li>Select commercial libraries by clicking on Spectrum and Select Library. Load the library of interest.</li>
		<li>Select integration parameters and perform a library search by clicking on Spectrum and Library Search Report. 
		NOTE: Take special care to compare library spectra of putative identified compounds with the spectra obtained in the analysis of the ink. Sometimes good matches can be achieved despite the presence of additional molecular mass peaks in the unknown spectra. For unequivocal identification, analytical standards have to be analyzed using the set up and instrument parameters to verify retention times and spectra.</li>
	</ol>
	</li>
	<li>Fast screening for non-volatile compounds 
	NOTE: Identification of non-volatile compounds from pyrolysis is based on the presence of multiple specific decomposition products of the parent compound within the same pyrogram. Since pyrograms can contain up to several hundreds of compounds, manual evaluation is hardly feasible. Start with a quick data evaluation approach using the average mass spectra (AMS). This is useful for identification of the most abundant pigment or polymer within the sample. This approach is only suitable for fast screening for ink declaration fraud and for having a starting point for manual pyrogram evaluation.
	<ol>
		<li>For pyrolysis data evaluation, mark the whole chromatogram in the GC-MS evaluation software (see Table of Materials) with the right mouse button pressed down to obtain an AMS.</li>
		<li>Create a library of obtained spectra of known reference substances of all compounds of interest, e.g., pigments and polymers, by clicking on Spectrum and Edit Library (click Create New Library if none is present).</li>
		<li>Select Add New Entry and fill in all information of interest. 
		NOTE: Select the AMS spectra of standards or inks, and click on Spectrum and NIST Search if forwarding to another software capable of MS library searches is desired.</li>
		<li>Generate an AMS of the investigated ink pyrogram and use the library search for comparison to the self-made AMS library15. Exclude masses from column bleed or other column noises. 
		NOTE: The highest match in the library search list will likely be the most abundant pigment or polymer in the inks. To verify this, compare the single characteristic decomposition products of the substance seen in the pyrolysis of the standard substance in the pyrogram of the analyzed ink (see section 3.4 and Supplementary Table 1).</li>
	</ol>
	</li>
	<li>Identification of non-volatile compounds with specialized pyrogram evaluation software 
	<ol>
		<li>Convert the pyrograms of interest to the needed format as stated in the software instructions. Integrate the pyrogram in a way that a maximum of 200 compounds are found. Otherwise, data evaluation time in the pyrogram evaluation sofware increases significantly.</li>
		<li>Build a folder on your computer with all pyrogram entries that should serve as a library, e.g., pigment pyrogram library for pigment identification or pyrograms of an original ink to compare it to putative counterfeit products.</li>
		<li>Load the unknown pyrogram in the tab Library Search by clicking on Browse.</li>
		<li>Load the library folder and select only MS matching and RT matching in the Search options, since the overall abundance will vary compared to pyrogram of reference pigments.</li>
		<li>Click on advanced in the Search options. Select the parameter &#8220;use only peaks with specified MS spectra&#8221; and use a fit threshold of 850 in the pyrogram evaluation software.</li>
		<li>Click on Add to save specified MS spectra (3&#8211;5 MS spectra of the most abundant peaks) from each pyrogram of reference pigments or polymers from the library in the advanced search options (cf. Supplementary Table 1). In this way only pigment related peaks are compared even in a multi-component ink with otherwise interfering high-abundant peaks.</li>
		<li>Press OK to return to the main window.</li>
		<li>Click on Search to start the comparison.</li>
		<li>If needed, go to Chromatogram Match tab, select a compound, and click Send to NIST to forward the spectra to the MS library software and identify the compound. 
		NOTE: Click send to MS options to include the spectra in the advanced Search options (cf. step 3.3.6).</li>
	</ol>
	</li>
	<li>Manual substance identification 
	NOTE: If no specialized pyrogram evaluation software is available use the data evaluation by standard MS library search program and commercial library together with reported fragments (Supplementary Table 1) and our pyrolysis library15. Manual comparison of pyrogram compounds with known decomposition products is also carried out to verify the hits given in the AMS.
	<ol>
		<li>For data evaluation for non-volatile compounds, start the GC-MS analysis/MS library searching software and open the chromatogram of the pyrolysis run.</li>
		<li>Select commercial and (self-made) pyrolysis libraries (e.g., our provided pyrolysis library15) by clicking on Spectrum and Select Library. Load all libraries of interest.</li>
		<li>Integrate the pyrogram in the GC-MS evaluation software and consider all peaks with an area &#8805;0.2% of the total area (may be adapted in a way that a manageable amount of peaks will be integrated).</li>
		<li>Start the library search by clicking on Spectrum and Library Search Report.</li>
		<li>Manually compare all library matches to specific pigment and polymer decomposition products in Supplementary Table 1 or fragments stated in literature16,17,18,19,20,21,22,23,24,25,26. For pigments, 2 to 3 decomposition products are needed to unambiguously identify the pigment used. 
		NOTE: All spectral matches with corresponding libraries must be carefully evaluated. Additional peaks higher than the mass peak often account for similar structures with additional side groups. If possible, reference substances should be analyzed to obtain the individual retention time in the analytical set-up.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

Py-GC-MS is a useful screening method for a broad range of substances in tattoo inks that can also be used for the analysis of other products. Compared to other methods, py-GC-MS can be conducted with only minimal sample preparation. GC-MS devices can be found in most analytical laboratories compared to more specialized methods such as MALDI-ToF-MS and EDX.
The data evaluation of pyrograms may be challenging, since the list of possible ingredients is infinite in theory and library searches tha...

Tattoo inks are complex mixtures consisting of pigments, solvents, binders, surfactants, thickening agents, and, sometimes, preservatives1. The increased popularity of tattooing in the last decades has led to the establishment of legislation addressing tattoo ink safety across Europe. In most instances, color-giving pigments and their impurities are restricted and therefore should be monitored by state laboratory market surveys to control their compliance with law.
Using the approach of online pyrolysis-gas chromatography mass spectrometry (py-GC-MS) described here, multiple ingredients can be identified simultaneously. Since volatile, semi-volatile and non-volatile compounds can be separated and analyzed within the same process, the variety of target compounds is high compared to other methods used for tattoo ink analysis. Liquid chromatography methods are mostly carried out with pigments solubilized in organic solvents2. Raman spectroscopy as well as Fourier-transform infrared (FT-IR) spectroscopy have been described as suitable tools for the identification of pigments and polymers but are limited with multi-ingredient mixtures since no separation technique is used in standard laboratory applications3,4. Laser desorption/ionization time-of-flight mass spectrometry (LDI-ToF-MS) has also been used for pigment and polymer identification5,6. Altogether, most methods lack the analysis of volatile compounds. The lack of suitable commercial spectral libraries is a common disadvantage of all of these methods. The identification of inorganic pigments has often been carried out with either inductively coupled plasma mass spectrometry (ICP-MS)7,8 or energy dispersive X-ray spectroscopy (EDX)4,9. Also, FT-IR and Raman spectroscopy have been used for the analysis of inorganic pigments such as titanium dioxide or iron oxides in other research fields10,11,12,13.
The goal of this study was to establish a method applicable in standard analytical laboratories with moderate financial costs to upgrade existing and common devices. Py-GC-MS as described here is a non-quantitative approach for identification of organic ingredients from mixtures. Upon identification of suspicious substances in a py-GC-MS screening, target substances can be quantified with more specialized approaches. It is especially interesting for the analysis of non-volatile and non-soluble substances like pigments and polymers.
The described method can be adapted for inks and varnishes in other fields of application. The data evaluation methods described are applicable to all pyrolysis investigations. Also, counterfeit products, mostly from Asian markets, display a potential source of risk to the consumer and a financial burden to manufacturers (personal communication at the 3rd ECTP in Regensburg, Germany, 2017). The method described here can be used to compare the characteristics of putative counterfeit inks to an original bottle, similar to published forensic approaches for car varnish identification14.

<table><tbody><tr><td>99.999% Helium carrier gas</td><td>Air Liquide, D&amp;uuml;sseldorf, Germany</td><td>-</td><td></td></tr><tr><td>5975C inert XL MSD with Triple-Axis Detectors</td><td>Agilent Technologies, Waldbronn, Germany</td><td>-</td><td></td></tr><tr><td>7890A gas chromatograph</td><td>Agilent Technologies, Waldbronn, Germany</td><td>-</td><td></td></tr><tr><td>AMDIS software (Version 2.7)</td><td>The National Institute of Standards and Technology, Gaithersburg, MD, USA</td><td>-</td><td>can be used for GC/MS peak integration, e.g. for transfer to pyrogram evaluation software</td></tr><tr><td>Cold Injection System (CIS)</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>-</td><td></td></tr><tr><td>electron impact (EI) source</td><td>Agilent Technologies, Waldbronn, Germany</td><td>-</td><td></td></tr><tr><td>Enhanced ChemStation (E02.02.1431)</td><td>Agilent Technologies, Waldbronn, Germany</td><td>-</td><td>used to generate Average Mass Spektra (AMS), can be used for peak integration and standard GC/MS library search</td></tr><tr><td>J&amp;amp;W HP-5MS GC Column, 30 m, 0.25 mm, 0.25 &amp;micro;m, 5975T Column Toroid Assembly</td><td>Agilent Technologies, Waldbronn, Germany</td><td>29091S-433LTM</td><td></td></tr><tr><td>MassHunter Software</td><td>Agilent Technologies, Waldbronn, Germany</td><td>-</td><td>no Version specified, can be used for GC/MS peak integration and standard GC/MS library search</td></tr><tr><td>Microcapillary tube Drummond Microcaps, volume 2 &amp;micro;L</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>P1549-1PAK</td><td></td></tr><tr><td>MS ChromSearch (Version 4.0.0.11)</td><td>Axel Semrau GmbH &amp;amp; Co. KG, Sprockh&amp;ouml;vel, Germany</td><td>-</td><td>specialized pyrogram evaluation software</td></tr><tr><td>NIST MS Search Program (MS Search version 2.0g)</td><td>The National Institute of Standards and Technology, Gaithersburg, MD, USA</td><td>-</td><td>used for MS and AMS library generation and corresponding substance search with selfmade and commercial libraries</td></tr><tr><td>NIST/EPA/NIH Mass Spectral Library (EI) mainlib &amp;amp; replib (Data version: NIST v11)</td><td>The National Institute of Standards and Technology, Gaithersburg, MD, USA</td><td>-</td><td>used commercial mass spectral library</td></tr><tr><td>Polystyrene (average Mw ~192,000)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>430102-1KG</td><td></td></tr><tr><td>Pyrolysis tubes, tube type - quartz glass - lenght 25 mm; 100 Units</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>018131-100-00</td><td></td></tr><tr><td>Pyrolyzer Module for TDU</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>-</td><td></td></tr><tr><td>Quartz wool</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>009970-076-00</td><td></td></tr><tr><td>Steel sticks</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>-</td><td></td></tr><tr><td>Thermal Desorption Unit (TDU 2)</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>-</td><td></td></tr><tr><td>Transport adapter</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>018276-010-00</td><td></td></tr><tr><td>Tweezers for Pyrolysis tubes</td><td>Gerstel, M&amp;uuml;hlheim, Germany</td><td>009970-074-00</td><td></td></tr><tr><td>Zebron Z-Guard Hi-Temp Guard Column, GC Cap. Column 10 m x 0.25 mm, Ea</td><td>Phenomenex Ltd. Deutschland, Aschaffenburg, Germany</td><td>7CG-G000-00-GH0</td><td></td></tr></tbody></table>

a two-step pyrolysis-gas chromatography method with mass spectrometric detection for identification of tattoo ink ingredients and counterfeit products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps.

The method includes a two-step chromatographic approach for each sample (Figure 1). In the first run, the sample is dried inside the injector system at 90 &#176;C before volatile compounds are transferred onto the column. Since the drying process is incomplete in most cases, residual solvents and volatile compounds are transferred and analyzed. In the second run, the previously dried sample is subsequently pyrolyzed to facilitate the analysis of non-volatile ...

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A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

This method for two-step pyrolysis online coupled to gas chromatography with mass spectrometric detection and data evaluation protocol can be used for multi-component analysis of tattoo inks and discrimination of counterfeit products.

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

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Research

JoVE Journal

Chemistry

11.0K Views.  German Federal Institute for Risk Assessment (BfR). Tattoo inkten en andere inkten zijn complexe mengsels van ingrediënten. Onze methode biedt een snelle en eenvoudige manier om meerdere componenten tegelijkertijd te analyseren. Pyrolyse en vervolgens gekoppeld aan gaschromatografie met massaspectrometrische detectie maakt de analyse van niet-vluchtige en vluchtige verbindingen in twee opeenvolgende runs mogelijk.Naast een zuivere analyse van inktingrediënten, kan deze methode ook worden gebruikt om originele en nagemaakte producten t...

Video: A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in tea samples have remained unclear. In this work, an adsorbent method combined with UPLC-MS/MS was developed to determine 15 PAs in the weed Ageratum conyzoides L., A. conyzoides rhizospheric soil, fresh tea leaves, and dried tea samples. The average recoveries ranged from 78%-111%, with relative standard deviations of 0.33%-14.8%. Fifteen pairs of A. conyzoides and A. conyzoides rhizospheric soil&#160;samples and 60 fresh tea leaf samples were collected from the Jinzhai tea garden in Anhui Province, China, and analyzed for the 15 PAs. Not all 15 PAs were detected in fresh tea leaves, except for intermedine-N-oxide (ImNO) and senecionine (Sn). The content of ImNO (34.7 &#181;g/kg) was greater than that of Sn (9.69 &#181;g/kg). In addition, both ImNO and Sn were concentrated in the young leaves of the tea plant, while their content was lower in the old leaves. The results indicated that the PAs in tea were transferred through the path of PA-producing weeds-soil-fresh tea leaves in tea gardens.

The present protocol describes the contamination of pyrrolizidine alkaloids (PAs) in tea samples from PA-producing weeds in tea gardens.

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in ...

Environment

Chromatographic Fingerprinting by Template Matching for Data Collected by Comprehensive Two-Dimensional Gas Chromatography

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass spectrometry. The rich information encrypted in the data may be highly valuable but difficult to access efficiently. Data density and complexity can lead to long elaboration times and require laborious, analyst-dependent procedures. Effective yet accessible data processing tools, therefore, are key to enabling the spread and acceptance of this advanced multidimensional technique in laboratories for daily use. The data analysis protocol presented in this work uses chromatographic fingerprinting and template matching to achieve the goal of highly automated deconstruction of complex two-dimensional chromatograms into individual chemical features for advanced recognition of informative patterns within individual chromatograms and across sets of chromatograms. The protocol delivers high consistency and reliability with little intervention. At the same time, analyst supervision is possible in a variety of settings and constraint functions that can be customized to provide flexibility and capacity to adapt to different needs and goals. Template matching is shown here to be a powerful approach to explore extra-virgin olive oil volatilome. Cross-alignment of peaks is performed not only for known targets, but also for untargeted compounds, which significantly increases the characterization power for a wide range of applications. Examples are presented to evidence the performance for the classification and comparison of chromatographic patterns from sample sets analyzed under similar conditions.

This protocol presents an approach to fingerprint and explore multi-dimensional data collected by comprehensive two-dimensional gas chromatography coupled to mass spectrometry. Dedicated pattern recognition algorithms (template matching) are applied to explore the chemical information encrypted in the extra-virgin olive oil volatile fraction (i.e., volatilome).

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass ...

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 ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Neuroscience

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

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.

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.

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

Harvesting the Orthogonality of LC and TIMS&#45;TOF MS&#47;MS to Screen Fentanyl Analogs

Fentanyl Analog Screening using LC-TIMS-TOF MS/MS

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States. Most recent cases of fentanyl-related overdose are linked to illicitly manufactured fentanyl and its associated extreme potency. In the present work, we describe a high-throughput analytical protocol for the screening of fentanyl analogs. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry allow for the separation and assignment of hundreds of fentanyl analogs from a single sample in a single scan. The described approach takes advantage of the recent development of data-dependent acquisition and data-independent acquisition using parallel accumulation in the mobility trap followed by sequential fragmentation using collision-induced dissociation. The fentanyl analogs are confidently assigned based on their retention time, mobility, and MS fragmentation pattern.

Author Spotlight: An Efficient Methodology to Confidently Differentiate and Characterize Fentanyl Analogs 

A method for the screening of fentanyl analogs based on their retention time, mobility, and mass spectrometry fragmentation pattern.

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its ...

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

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