June 28

Vicente Cebolla¹, Carmen Jarne¹, María Sancho-Albero², Luis Membrado¹, Jesús Vela³, María Savirón⁴, José-Manuel Escuín¹, Javier Galbán⁵, Pilar Martín-Duque⁶, Jesús Santamaría<sup>2

1</sup>Instituto de Carboquímica/CSIC, Miguel Luesma Castán 4, 50018, Zaragoza, Spain
²Aragon Institute of Nanoscience/Universidad de Zaragoza, Campus Rio Ebro, 50018, Zaragoza, Spain
³Analytical Chemistry Department/Universidad de Zaragoza, Campus Río Ebro, 50018, Zaragoza, Spain
⁴CEQMA/CSIC-Universidad de Zaragoza, Campus San Francisco, 50009, Zaragoza, Spain
⁵Analytical Chemistry Department/Universidad de Zaragoza, Campus San Francisco, 50009, Zaragoza, Spain
⁶Instituto Aragonés de Ciencias de la Salud/Fundación ARAID, Campus San Francisco, 50009, Zaragoza, Spain

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The current scientific paradigm to understand the role of lipids in human diseases is based on the hypothesis that some diseases have distinctive lipid profiles (biomarkers) with respect to the respective healthy status. HPTLC separation of sample, followed by densitometry and MS coupling provides a simple but powerful approach for determining lipid classes and obtaining a detailed structural elucidation of lipids present in biological extracts, allowing their exact recognition by their m/z, and confirmation by their collision-induced dissociation MS/MS data [1]. The application of HPTLC to identification in human plasma of 19 molecular species related to globotriaosylceramides (Gb3) which are considered biomarkers of Fabry disease, a lysosomal storage disorder, will be first discussed [2]. Another example is the open question of whether exosome lipids can be considered as potential cancer biomarkers. This faces our current limited knowledge of their composition. A procedure to isolate exosomes and obtain a phospholipid (PL)-class determination and identification of their molecular species will be also discussed, from embryonic murine fibroblasts (NIH-3T3 cell line), and metastatic murine skin melanoma cells with different degrees of malignancy [3]. For both examples, a detailed structural characterization of molecular species of each concerned lipid class was performed directly from the chromatographic plate, thanks to an elution-based interface, using ion-trap technology by different approaches that involve simultaneous positive and negative ESI-MS, MS/MS, or APCI.

Acknowledgements
This work was supported by DGA-FEDER (E25_20R, N&SB), AES PI21/00036, CSIC 202180E076, ISCIII PI19/01007, ERC Advanced Grant CADENCE (grant no. ERC-2016-ADG-742684)

References
[1]  V.L. Cebolla, C. Jarne, J. Vela, R. Garriga, L. Membrado, J. Galbán, J. Liq. Chromatogr. & Rel. Technol. 44 (2021) 148-170.
[2]  C. Jarne, L. Membrado, M. Savirón, J. Vela, J. Orduna, R. Garriga, J. Galbán, V.L. Cebolla, J. Chromatogr. A 1638 (2021), 461895.
[3]  M. Sancho-Albero, C. Jarne, M. Savirón, P. Martín-Duque, L. Membrado, V.L. Cebolla, J. Santamaría, Int. J. Mol. Sci. 2022, 23(3), 1150.

Eike Reich

The International Association for the Advancement of HPTLC

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The workshop will include a 30 min lecture explaining the terminology and definition of HPTLC according to USP<203> and the European Pharmacopoeia chapter 2.8.25. The concept of HPTLC fingerprints is used to illustrate how compliance of a sample with the specifications in monographs for botanical / herbal drugs can be tested in a fully cCMP compliant environment. Furthermore, the lecture will demonstrate how the quality of botanical materials can be related to HPTLC data.
The second part of the workshop will use a live experiment: the identification of Common Horse tail and limit test for adulteration with Marsh Horse tail according to the Ph.Eur. Monograph 1825, to introduce:

• the practical aspects of standardized HPTLC
• proper handling of equipment and software,
• system suitability tests,
• image and profile comparison, and
• limit tests based on image evaluation

Alice Ryckebusch

Advion Interchim Scientific, 211 bis avenue JF Kennedy, BP1140, 03103 Montluçon, France

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Liquid Chromatography is a separation technique. It can be dedicated to identify and quantify compounds present in a mixture (analytical mode). Very attractive and popular technique, when the goal is to get isolated pure product from a more or less complex mixture is a flash / preparative liquid chromatography.

There are many different purification techniques like distillation, crystallization, filtration and chromatography. They all have a goal to purify and recover samples. Purification by liquid chromatography is always a challenge and there is often a compromise to obtain desired purity, loading and throughput. To improve efficiency in delivering pure compounds, chemists may balance between purity, run time and environmental considerations. This delicate balance is often necessary for both crude and final purification.

In the workshop we will cover principles of successful purification:

• Tools for method development and method transfer from TLC or HPLC
• Parameters to consider to make a successful TLC plate
• How to choose the right size and phase of the column for the efficient separation
• Parameters for optimal injection
• Practical demonstration on a puriFlash 5.250
• Q&A part