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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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&#252;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&#252;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&#38;W HP-5MS GC Column, 30 m, 0.25 mm, 0.25 &#181;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 &#181;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 &#38; Co. KG, Sprockh&#246;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 &#38; 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&#252;hlheim, Germany</td>
			<td>018131-100-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrolyzer Module for TDU</td>
			<td>Gerstel, M&#252;hlheim, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz wool</td>
			<td>Gerstel, M&#252;hlheim, Germany</td>
			<td>009970-076-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel sticks</td>
			<td>Gerstel, M&#252;hlheim, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Desorption Unit (TDU 2)</td>
			<td>Gerstel, M&#252;hlheim, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Transport adapter</td>
			<td>Gerstel, M&#252;hlheim, Germany</td>
			<td>018276-010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers for Pyrolysis tubes</td>
			<td>Gerstel, M&#252;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 ...

Watch this Scientific Journal Video about A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products at JoVE

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

Eine zweistufte Pyrolysis-Gas-Chromatographie-Methode mit Massenspektrometrische Erkennung zur Identifizierung von Tattoo-Ink-Inhaltsstoffen und gefälschten Produkten

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. ...

a-two-step-pyrolysis-gas-chromatography-method-with-mass

Research

JoVE Journal

Chemistry

10.9K Aufrufe.  German Federal Institute for Risk Assessment (BfR). Tattoo-Tinten und andere Tinten sind komplexe Mischungen von Zutaten. Unsere Methode bietet eine schnelle und einfache Möglichkeit, mehrere Komponenten gleichzeitig zu analysieren. Die Pyrolyse und dann gekoppelt mit der Gaschromatographie mit massenspektrometrischer Detektion ermöglicht die Analyse nichtflüchtiger und flüchtiger Verbindungen in zwei aufeinanderfolgenden Durchläufen.Neben der reinen Analyse von Tinteninhaltsstoffen kann diese Methode auch zur Unterscheidung von Or...

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Untargeted Metabolomics aus biologischen Quellen Mit UltraPerformance Flüssigkeits-Chromatographie-hochauflösender Massenspektrometrie (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Forschung

Chemie

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Massenspektrometrische Ansätze zur Proteinstruktur und Wechselwirkungen zu untersuchen in lyophilisierte Pulver

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

Eine einfache Methode zur Glucosinolate Extraktion und Analyse mit Hochdruck-Flüssigkeitschromatographie (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

Eine bequeme Methode für die Extraktion und Analyse mit Hochdruck-Flüssigkeitschromatographie Katecholamin Neurotransmitter und deren Metabolite

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

A Two-Step Protocol for Umpolung Functionalization of Ketones Via Enolonium Species

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. Recently, we have developed a two-step strategy for ketone enolate umpolung that has enabled the development of methods for chlorination, azidation, and amination using azoles. In addition, we have developed C-C bond&#8211;forming arylation and allylation reactions. At the heart of these methods is the preparation of the intermediate and highly reactive enolonium species prior to addition of a reactive nucleophile. This strategy is thus reminiscent of the preparation and use of metal enolates in classical synthetic chemistry. This strategy allows the use of nucleophiles that would otherwise be incompatible with the strongly oxidizing hypervalent iodine reagents. In this paper we present a detailed protocol for chlorination, azidation, N-heteroarylation, arylation, and allylation. The products include motifs prevalent in medicinally active products. This article will greatly assist others in using these methods.

A two step one-pot protocol for the umpolung of ketone enolates to enolonium species and addition of a nucleophile to the &#945;-position is described. Nucleophiles include chloride, azide, azoles, allyl-silanes, and aromatic compounds.

Ein zweistufiges Protokoll für die Umpolung Funktionalisierung von Ketonen über Enolonium Arten

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. ...

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products and notably to generate chiral carbon centers. The crucial first step consists in the selective double hydroboration of CO2 catalyzed by an iron hydride complex. The product obtained with this 4 e- reduction is a rare bis(boryl)acetal, compound 1, which is subjected in situ to three different reactions in a second step. The first reaction concerns a condensation reaction with (diisopropyl)phenylamine affording the corresponding imine 2. In the second and third reaction, intermediate 1 reacts with triazol-5-ylidene (Enders&#39; carbene) to afford compounds 3 or 4, depending on the reaction conditions. In both compounds, C-C bonds are formed, and chiral centers are generated from CO2 as the only source of carbon. Compound 4 exhibits two chiral centers obtained in a diastereoselective manner in a formose-type mechanism. We proved that the remaining boryl fragment plays a key role in this unprecedented stereocontrol. The interest of the method stands on the reactive and versatile nature of 1, giving rise to various complex molecules from a single intermediate. The complexity of a two-step method is compensated by the overall short reaction time (2 h for the larger reaction time), and mild reaction conditions (25 &#176;C to 80 &#176;C and 1 to 3 atm of CO2).

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations are conducted in a one-pot two-step procedure for the synthesis of complex molecules. The selective 4 e- reduction of CO2 with a hydroborane reductant affords a reactive and versatile bis(boryl)acetal intermediate which is subsequently involved in condensation reaction or carbene-mediated C-C coupling generation.

Vielseitige CO 2-Transformationen in komplexe Produkte: Eine Zwei-Schritte-Strategie

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products ...

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

Mass Spectrometrie-Guided Genome Mining als Werkzeug zur Aufdeckung neuartiger Naturprodukte

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...

Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor proliferation, epilepsy, neurodegenerative diseases, and psychiatric illnesses due to their immune-modulatory, neuro-modulatory, and neurotoxic effects. The most extensively used positron emission tomography (PET) agent for mapping tryptophan metabolism, &#945;-[11C]methyl-L-tryptophan ([11C]AMT), has a short half-life of 20 min with laborious radiosynthesis procedures. An onsite cyclotron is required to radiosynthesize [11C]AMT. Only a limited number of centers produce [11C]AMT for preclinical studies and clinical investigations. Hence, the development of an alternative imaging agent that has a longer half-life, favorable in vivo kinetics, and is easy to automate is urgently needed. The utility and value of 1-(2-[18F]fluoroethyl)-L-tryptophan, a fluorine-18-labeled tryptophan analog, has been reported in preclinical applications in cell line-derived xenografts, patient-derived xenografts, and transgenic tumor models.
This paper presents a protocol for the radiosynthesis of 1-(2-[18F]fluoroethyl)-L-tryptophan using a one-pot, two-step strategy. Using this protocol, the radiotracer can be produced in a 20 &#177; 5% (decay corrected at the end of synthesis, n &#62; 20) radiochemical yield, with both radiochemical purity and enantiomeric excess of over 95%. The protocol features a small precursor amount with no more than 0.5 mL of reaction solvent in each step, low loading of potentially toxic 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222), and an environmentally benign and injectable mobile phase for purification. The protocol can be easily configured to produce 1-(2-[18F]fluoroethyl)-L-tryptophan for clinical investigation in a commercially available module.

Radiosynthesis of 1-2-[18F]Fluoroethyl-L-Tryptophan using a One-pot, Two-step Protocol

Here, we describe the radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-tryptophan, a positron emission tomography imaging agent for studying tryptophan metabolism, using a one-pot, two-step strategy in a radiochemistry synthesis system with good radiochemical yields, high enantiomeric excess, and high reliability.

Radiosynthese von 1-(2-[18F]Fluorethyl)-L-Tryptophan unter Verwendung eines Eintopf-, zweistufigen Protokolls

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor ...

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

Facettenreiche massenspektrometrische Untersuchung von Neuropeptiden in Callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Extraktion von Nicht-Protein-Aminosäuren aus Cyanobakterien für die Flüssigchromatographie-Tandem-Massenspektrometrie-Analyse

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...