This research was funded by the Consejer&#237;a de Universidad, Investigaci&#243;n e Innovaci&#243;n - Junta de Andaluc&#237;a (PROYEXCEL_00195) and the postdoctoral grant given by the Generalitat Valenciana and European Social Fund+ (CIAPOS/2022/049).&#160;The authors thank the &#34;Centro de Instrumentaci&#243;n Cient&#237;fica (CIC)&#34; at the University of Granada for providing access to the analytical instrumentation used in this protocol.

Enhanced Isomeric Separation of Ergot Alkaloids Using LC-IMS-MS

<ol>
	<li>Kanu, A. B., Dwivedi, P., Tam, M., Matz, L., Hill, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+mobility-mass+spectrometry.">Ion mobility-mass spectrometry.</a> J Mass Spectrom. 43 (1), 1-22 (2008).</li><li>Gabelica, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+reporting+ion+mobility+Mass+spectrometry+measurements.">Recommendations for reporting ion mobility Mass spectrometry measurements.</a> Mass Spectrom Rev. 38 (3), 291-320 (2019).</li><li>Feuerstein, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+total+ergot+alkaloids+in+wheat+flour+by+Orbitrap+mass+spectrometry.">Simultaneous determination of total ergot alkaloids in wheat flour by Orbitrap mass spectrometry.</a> Food Chem. 441, 138363 (2024).</li><li>Garc&#237;a-Juan, A., Le&#243;n, N., Armenta, S., Pardo, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+analytical+method+for+the+simultaneous+determination+of+12+ergot,+2+tropane,+and+28+pyrrolizidine+alkaloids+in+cereal-based+food+by+LC-MS/MS.">Development and validation of an analytical method for the simultaneous determination of 12 ergot, 2 tropane, and 28 pyrrolizidine alkaloids in cereal-based food by LC-MS/MS.</a> Food Res Int. 174 (Pt 1), 113614 (2023).</li><li>Carbonell-Rozas, L., Mahdjoubi, C. K., Arroyo-Manzanares, N., Garc&#237;a-Campa&#241;a, A. M., G&#225;miz-Gracia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+ergot+alkaloids+in+barley+and+wheat+from+Algeria.">Occurrence of ergot alkaloids in barley and wheat from Algeria.</a> Toxins. 13 (5), 316 (2021).</li><li>Carbonell-Rozas, L., G&#225;miz-Gracia, L., Lara, F. J., Garc&#237;a-Campa&#241;a, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+main+ergot+alkaloids+and+their+epimers+in+oat-based+functional+foods+by+ultra-high+performance+liquid+chromatography+tandem+mass+spectrometry.">Determination of the main ergot alkaloids and their epimers in oat-based functional foods by ultra-high performance liquid chromatography tandem mass spectrometry.</a> Molecules. 26 (12), 3717 (2021).</li><li>Carbonell-Rozas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+mobility-mass+spectrometry+to+extend+analytical+performance+in+the+determination+of+ergot+alkaloids+in+cereal+samples.">Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.</a> J Chromatogr A. 1682, 463502 (2022).</li><li>European Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commission+Implementing+Regulation+(EU)+2023/2782+of+14+December+2023+laying+down+the+methods+of+sampling+and+analysis+for+the+control+of+the+levels+of+mycotoxins+in+food+and+repealing+Regulation+(EC)+No+401/2006.">Commission Implementing Regulation (EU) 2023/2782 of 14 December 2023 laying down the methods of sampling and analysis for the control of the levels of mycotoxins in food and repealing Regulation (EC) No 401/2006.</a> Off J Eur Union. , (2023).</li><li>Carbonell-Rozas, L., Vander Cruyssen, L., Dall'Asta, C., Leggieri, M. 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M., Castell&#243;n-Rend&#243;n, E., G&#225;miz-Gracia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+principal+ergot+alkaloids+in+swine+feeding.">Determination of principal ergot alkaloids in swine feeding.</a> J Sci Food Agric. 101 (12), 5214-5224 (2021).</li><li>Pereira, V. L., Fernandes, J. O., Cunha, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assessment+of+three+cleanup+procedures+after+QuEChERS+extraction+for+determination+of+trichothecenes+(type+A+and+type+B)+in+processed+cereal-based+baby+foods+by+GC-MS.">Comparative assessment of three cleanup procedures after QuEChERS extraction for determination of trichothecenes (type A and type B) in processed cereal-based baby foods by GC-MS.</a> Food Chem. 182, 143-149 (2015).</li><li>Tuzimski, T., Szubartowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+development+for+selected+bisphenols+analysis+in+sweetened+condensed+milk+from+a+can+and+breast+milk+samples+by+HPLC-DAD+and+HPLC-QqQ-MS:+Comparison+of+sorbents+(Z-SEP,+Z-SEP+Plus,+PSA,+C18,+chitin+and+EMR-lipid)+for+clean-up+of+QuEChERS+extract.">Method development for selected bisphenols analysis in sweetened condensed milk from a can and breast milk samples by HPLC-DAD and HPLC-QqQ-MS: Comparison of sorbents (Z-SEP, Z-SEP Plus, PSA, C18, chitin and EMR-lipid) for clean-up of QuEChERS extract.</a> Molecules. 24 (11), 2093 (2019).</li><li>&#321;ozowicka, B., Rutkowska, E., Jankowska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+QuEChERS+modifications+on+recovery+and+matrix+effect+during+the+multi-residue+pesticide+analysis+in+soil+by+GC/MS/MS+and+GC/ECD/NPD.">Influence of QuEChERS modifications on recovery and matrix effect during the multi-residue pesticide analysis in soil by GC/MS/MS and GC/ECD/NPD.</a> Environ Sci Pollut Res. 24 (8), 7124-7138 (2017).</li><li>Schummer, C., Xandonella, I., van Nieuwenhuyse, A., Moris, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epimerization+of+ergot+alkaloids+in+feed.">Epimerization of ergot alkaloids in feed.</a> Heliyon. 6 (6), e04336 (2020).</li><li>Cherewyk, J. E., Grusie-Ogilvie, T. J., Parker, S. E., Blakley, B. R., Al-Dissi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+storage+temperature+and+time+on+ergot+alkaloid+concentrations.">The impact of storage temperature and time on ergot alkaloid concentrations.</a> Toxins. 15 (8), 497 (2023).</li><li>Silva, &. #. 1. 9. 4. ;., Mateus, A. R., Barros, S. C., Silva, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ergot+alkaloids+on+cereals+and+seeds:+Analytical+methods,+occurrence,+and+future+perspectives.">Ergot alkaloids on cereals and seeds: Analytical methods, occurrence, and future perspectives.</a> Molecules. 28 (20), 7233 (2023).</li></ol>

The authors have no conflicts of interest to disclose.

The successful use of this protocol is based on the optimization of the extraction procedure, previously carried out by Carbonell-Rozas et al.17, who implemented the use of an extraction solvent effective enough to extract EAs from complex food matrices such as barley and wheat, and a clean-up that provided relatively low SSE values. The choice of extraction solvent represents a critical step considering the chemical characteristics of the analytes and the lability of EAs to decomposition and epim...

Ion mobility mass spectrometry (IMS) is becoming a growingly used analytical technique, often presented as an additional separation dimension integrated into traditional liquid/gas chromatography (LC/GC) coupled to MS workflows. IMS consists of the separation of molecules along a mobility cell, filled with a buffer gas, under an electric field and at atmospheric pressure1. Depending on the mass-to-charge ratio (m/z) and the geometrical conformation, an ionized molecule will interact with the buffer gas as it moves across the mobility cell, which is reflected in the ion mobility (K) parameter2 and calculated through the following equation:
<img alt="Equation 1" src="/files/ftp_upload/67484/67484eq01v2.jpg" style="margin: auto;" />
where D represents the total drift length, td is&#160;the total drift time, and E is&#160;the electric field. Therefore, K is measured in m2&#160;V&#8722;1&#160;s&#8722;1, although for practical reasons it is often expressed as cm2 V&#8722;1 s&#8722;1. The intrinsic capability to move across the mobility cell can be measured by the drift time and later converted to the so-called collision cross section (CCS) value, which is a highly reproducible parameter for each molecule independently of the IMS instrument3. The CCS can be derived from the mobility following this equation:
<img alt="Equation 2" src="/files/ftp_upload/67484/67484eq02v2.jpg" style="margin: auto;" />
q being the charge of the ion; N the buffer gas number density; &#181; the reduced mass of the collision partners buffer gas-ion; kB&#160;the Boltzmann constant; and T the buffer gas temperature. Therefore, IMS provides additional information complementary to the analytical data resulting from chromatography and MS analyses.
The implementation of IMS in LC-MS platforms has been shown to increase the reliability of analytical determinations, especially when working with compounds that are at trace concentrations. Several studies have reported that LC-IMS-MS methods improve the quality of mass spectra by reducing background noise, which ultimately affects the sensitivity of the method, and reduces the rate of false positives and negatives provided by multi-residue LC-MS methodologies4,5,6. Further, the reproducibility of CCS values allows the comparison not only between different instruments using the same technology, but also between different ion mobility technologies, namely traveling wave ion mobility spectrometry (TWIMS), trapped ion mobility spectrometry (TIMS), and drift tube ion mobility spectrometry (DTIMS)2,7, which are the most frequently used systems1. Thus, a remarkable consequence of the potential of CCS as an identification parameter lies in the possibility of building CCS libraries, reflected in its applicability in metabolomics studies8. Nonetheless, one of the most powerful features of IMS is the ability to separate isomeric and isobaric compounds that may not be sufficiently resolved by LC-MS methods. This may be the case when working with large sets of analytes of interest in complex matrices, which is a common situation in environmental and food analysis. In this context, LC-IMS-MS methods have been proposed for the monitoring of pesticides and, to a lesser extent, veterinary drugs and mycotoxins in food9.
Due to their high resolving power and selectivity, LC/GC-IMS-MS platforms emerge as the most useful tools to address some of the current challenges in food safety, especially those related to isomeric mixtures. The health concern related to isomeric mixtures as food contaminants has been reflected in the current European legislation, which, for instance, limits the maximum concentration of six main ergot alkaloids (EAs) and their corresponding six epimers in several food products10.
EAs constitute a family of toxic secondary metabolites produced by a wide range of fungi, mainly of the family Clavicipitaceae (e.g., Claviceps purpurea, the most important EA producer due to its wide host range), but also Trichocomaceae, which can parasitize the seed head of living plants (such as rye, barley, wheat, and oat) at the time of flowering11,12. Under specific conditions, especially temperature and water activity, Claviceps fungi can produce EAs that accumulate in fruiting bodies, known as sclerotia or ergot, in the host crop. To a certain extent, EAs can withstand the processing of the raw material until reaching the final product; therefore, breaking into the food chain. Ingestion of contaminated food can lead to EA intoxication, known as ergotism, which presents with acute symptoms such as abdominal pain, vomiting, burning sensation of the skin, insomnia, and hallucinations13. To reduce the impact of EAs on human health, the European Commission set maximum levels (MLs) in several foods for the sum of the main EAs: the R-epimers ergometrine (Em), ergotamine (Et), ergosine (Es), ergocristine (Ecr), ergokryptine (Ekr), and ergocornine (Eco) and their corresponding S-epimers: ergometrinine (Emn), ergosinine (Esn), ergotaminine (Etn), ergocorninine (Econ), ergokryptinine (Ekrn), and ergocristinine (Ecrn). These compounds can epimerize from R to S forms and vice versa, especially under exposure to strong light, prolonged storage, or contact with some solvents at high or low pH 12. Although the proportion of R and S forms may vary under different conditions, the EFSA CONTAM Panel reported a higher occurrence of R forms than S forms after reviewing available literature on EAs in food products14. Hence, the MLs vary depending on several factors, such as the susceptibility of the crop, degree of processing, or frequency of consumption. In the EU framework, MLs for milled products of barley, wheat, spelt, and oat have been set at 50 or 150 &#181;g/kg (depending on the ash content lower or higher than 900 mg/100 g, respectively), whereas cereals intended directly for human consumption are subject to an ML of 150 &#181;g/kg, except for cereal-based baby food, in which the ML is reduced to 20 &#181;g/kg10.
This stringent legislation requires analytical methodologies sensitive enough to determine trace concentration&#160;(&#181;g/kg) levels&#160;while properly identifying regulated EAs and their corresponding epimers, as both forms, R- and S-isomers, can be found together in contaminated samples. This task represents a major challenge since each toxin-epimer pair shares the same exact mass and fragmentation pattern. In addition, a proper chromatographic separation between both compounds may be complex. Therefore, well-optimized LC gradients are required to avoid misquantification when EA epimers co-occur in food samples. Although several studies have reported LC-MS methods for unambiguous determination of EAs15,16,17,18, the chromatographic method must be studied extensively to achieve adequate separation of the chromatographic peaks to unequivocally identify EAs. However, this is not usually feasible for multi-class methods in which contaminants belonging to different chemical families are simultaneously determined. In this context, a recent study conducted by Carbonell-Rozas, Hern&#225;ndez-Mesa, et al.19 reported an LC-IMS-MS method for the quantification of EAs in wheat and barley samples, using two different TWIMS instruments that provided reproducible CCS values and low limits of quantification (LOQs) to detect any non-compliance in accordance with current legislation. Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for mycotoxin identification. This protocol is designed to clearly explain the benefits of implementing IMS in food safety analysis, taking as an example the determination of EAs in cereals. This protocol addresses the sample treatment based on a QuEChERS procedure, sample analysis by LC-TIMS-MS, and IMS data extraction and interpretation.

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	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>VWR</td>
			<td>83640.32</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber glass tubes 4 mL</td>
			<td>VWR</td>
			<td>548-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber glass tubes 12 mL</td>
			<td>VWR</td>
			<td>548-0903</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber vials 1.5 mL</td>
			<td>Agilent</td>
			<td>5190-9063</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium carbonate</td>
			<td>Fluka</td>
			<td>9716</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance BAS 31&#160;</td>
			<td>Boeco</td>
			<td>4400519</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance CP 323 S&#160;</td>
			<td>Sartorius</td>
			<td>23-84182</td>
			<td></td>
		</tr>
		<tr>
			<td>C18</td>
			<td>Supelco</td>
			<td>52604-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>VWR</td>
			<td>525-1082</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL</td>
			<td>VWR</td>
			<td>525-0155</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Universal 320 R</td>
			<td>Hettich</td>
			<td>1406</td>
			<td></td>
		</tr>
		<tr>
			<td>Compass HyStar&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td>Acquisition software</td>
		</tr>
		<tr>
			<td>DataAnalysis</td>
			<td>Bruker</td>
			<td></td>
			<td>Qualitative software</td>
		</tr>
		<tr>
			<td>Elute PLUS&#160;UHPLC</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EVA EC-S evaporator</td>
			<td>VLM</td>
			<td>V830.012.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid GR for analysis ACS, Reag. Ph Eur</td>
			<td>Merck</td>
			<td>100264</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinder TitanMill300</td>
			<td>Cecotec</td>
			<td>1559</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>83638.32</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q water purification system (18.2 M&#937; cm)</td>
			<td>Millipore</td>
			<td>ZD5211584</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips 1- 5 mL</td>
			<td>Labortecnic</td>
			<td>162005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips 100 - 1000 &#181;L</td>
			<td>Labortecnic</td>
			<td>1622222</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips 5 - 200 &#181;L</td>
			<td>Labortecnic</td>
			<td>162001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette Transferpette S variable, DE-M 10 - 100 &#181;L</td>
			<td>BRAND</td>
			<td>704774</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette Transferpette S variable, DE-M 100 - 1000 &#181;L</td>
			<td>BRAND</td>
			<td>704780</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette Transferpette S variable, DE-M 500 - 5000 &#181;L</td>
			<td>BRAND</td>
			<td>704782</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 2 mL</td>
			<td>VWR</td>
			<td>613-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter 13 mm, 0.22&#181;m</td>
			<td>Phenomenex</td>
			<td>AF-8-7707-12</td>
			<td></td>
		</tr>
		<tr>
			<td>TASQ</td>
			<td>Bruker</td>
			<td></td>
			<td>Quantitative software</td>
		</tr>
		<tr>
			<td>timsTOFPro2 IM-HRMS</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Scientific Industries</td>
			<td>15547335</td>
			<td></td>
		</tr>
		<tr>
			<td>Zorbax Eclipse Plus RRHD C18 column (50&#160;x&#160;2.1 mm, 1.8 &#181;m particle size)</td>
			<td>Agilent&#160;</td>
			<td>959757-902</td>
			<td></td>
		</tr>
		<tr>
			<td>Z-Sep+</td>
			<td>Supelco</td>
			<td>55299-U</td>
			<td>Zirconia-based sorbent</td>
		</tr>
		<tr>
			<td>Ergot alkaloids</td>
			<td></td>
			<td></td>
			<td>CAS registry sorbent</td>
		</tr>
		<tr>
			<td>Ergocornine (Eco)</td>
			<td>Techno Spec</td>
			<td>E178</td>
			<td>564-36-3</td>
		</tr>
		<tr>
			<td>Ergocorninine (Econ)</td>
			<td>Techno Spec</td>
			<td>E130</td>
			<td>564-37-4</td>
		</tr>
		<tr>
			<td>Ergocristine (Ecr)</td>
			<td>Techno Spec</td>
			<td>E180</td>
			<td>511-08-0</td>
		</tr>
		<tr>
			<td>Ergocristinine (Ecrn)</td>
			<td>Techno Spec</td>
			<td>E188</td>
			<td>511-07-9</td>
		</tr>
		<tr>
			<td>Ergokryptine (Ekr)</td>
			<td>Techno Spec</td>
			<td>E198</td>
			<td>511-09-1</td>
		</tr>
		<tr>
			<td>Ergopkryptinine (Ekrn)</td>
			<td>Techno Spec</td>
			<td>E190</td>
			<td>511-10-4</td>
		</tr>
		<tr>
			<td>Ergometrine (Em)</td>
			<td>Romer Labs</td>
			<td>&#34;002067&#34;</td>
			<td>60-79-7</td>
		</tr>
		<tr>
			<td>Ergometrinine (Emn)</td>
			<td>Romer Labs</td>
			<td>LMY-090-5ML</td>
			<td>479-00-5</td>
		</tr>
		<tr>
			<td>Ergosine (Es)</td>
			<td>Techno Spec</td>
			<td>E184</td>
			<td>561-94-4</td>
		</tr>
		<tr>
			<td>Ergosinine (Esn)</td>
			<td>Techno Spec</td>
			<td>E194</td>
			<td>596-88-3</td>
		</tr>
		<tr>
			<td>Ergotamine (Et)</td>
			<td>Romer Labs</td>
			<td>&#34;002069&#34;</td>
			<td>113-15-5</td>
		</tr>
		<tr>
			<td>Ergotaminine (Etn)</td>
			<td>Romer Labs</td>
			<td>&#34;002075&#34;</td>
			<td>639-81-6</td>
		</tr>
	</tbody>
</table>

detection of regulated ergot alkaloids in food matrices by liquid chromatography-trapped ion mobility spectrometry-time-of-flight mass spectrometry

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

First, working standard solutions were injected into the LC-IMS-MS instrument to obtain all the identification features (i.e., retention time, CCS, and mass spectra) of each EA analyzed here. Since the identification parameters, except the exact mass, were initially unknown, the acquisition method was based on a two-scan event, starting with a full scan of the entire mass spectrum followed by a bbCID. The retrospective way of approaching this study is enabled by the Q-TOF high-resolution mass spectrometer, which acquires...

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

detection-regulated-ergot-alkaloids-food-matrices-liquid

Research

JoVE Journal

Chemistry

438 Views.  University of Granada. This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915. 

Video: Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

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.

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

Analyzing the Photo-oxidation of 2-propanol at Indoor Air Level Concentrations Using Field Asymmetric Ion Mobility Spectrometry

We demonstrate a versatile protocol to be used for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) volatile organic carbons (VOCs), illustrating this with a titanium dioxide based catalyst, and the VOC 2-propanol. The protocol takes advantage of field asymmetric ion mobility spectroscopy (FAIMS), an analysis tool that is capable of continuously identifying and monitoring the concentration of VOCs such as 2-propanol and acetone at the ppb level. The continuous nature of FAIMS allows detailed kinetic analysis, and long-term reactions, offering a significant advantage over gas chromatography, a batch process traditionally used in air purification characterization. The use of FAIMS in photocatalytic air purification has only recently been used for the first time, and with the protocol illustrated here, the flexibility in allowing alternative VOCs and photocatalysts to be tested using comparable protocols offers a unique system to elucidate photocatalytic air purification reactions at low concentrations.

A protocol for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) model volatile organic carbons such as 2-propanol is described.

We demonstrate a versatile protocol to be used for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) volatile ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial resolution (~1 &#181;m). Furthermore, it describes how to obtain realistic three-dimensional (3D) distributions of segregated impurities/dopants in solid state materials. Direct 3D depth profile reconstruction is often difficult to achieve due to SIMS-related measurement artifacts. Presented here is a method to identify and solve this challenge. Three major issues are discussed, including the i) nonuniformity of the detector being compensated by flat-field correction; ii) vacuum background contribution (parasitic oxygen counts from residual gases present in the analysis chamber) being estimated and subtracted; and iii)&#160;performance of all steps within a stable timespan of the primary ion source. Wet chemical etching is used to reveal the position and types of dislocation in a material, then the SIMS result is superimposed on images obtained via scanning electron microscopy (SEM). Thus, the position of agglomerated impurities can be related to the position of certain defects. The method is fast and does not require sophisticated sample preparation stage; however, it requires a high-quality, stable ion source, and the entire measurement must be performed quickly to avoid deterioration of the primary beam parameters.

The presented method describes how to identify and solve measurement artifacts related to secondary ion mass spectrometry as well as obtain realistic 3D distributions of impurities/dopants in solid state materials.

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

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.

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 ...