Česká chromatografická škola - HPLC.cz 2021 - Den 3

První ranní přednáška byla věnována možnostem minimalizace nežádoucích adsorpcí analytů v HPLC díky technologiím systému Waters ACQUITY PREMIER

V sekci zaměřené na MS techniky jsme zabývali využití metody HILIC-MS nebo odlišným potenciálem technik UHPLC a UHPSFC. Aplikačně jsme se podívali na analýzy nízkomolekulárních alkylaminů, fytohormonů, stereoisomerních steroidů nebo genotoxických nečistot.

Druhá úterní komerční přednáška byla venováná možnostem moderních jednoduchých kvadrupólů Agilent LC/MSD – iQ v HPLC separacích, které dnes již pomalu nahrazují klasické UV-Vis nebo DAD detektory a patří ke standardní výbavě kapalinových chromatografů tak, jak je tomu již mnoho let v oblasti plynové chromatografie.

Sekce zaměřená na techniku SFC ukázala možnosti a přednosti této techniky na analýzách polárních látek, chirálních separacích boronových klasterů nebo enantioseparacích deschlorketaminu a jeho metabolitů.

Poslední úterní přednáška byla zaměřená na možnosti HPLC/UHPLC kolon na bázi core shell částic a jejich vyšší účinnosti pro zlepšení proteomických analýz.

Závěr dne již patřil prověřování a ověřování celoživotních nebo naopak v posledních dnech získaných vědomostí z oblasti separačních technik soutěživou formou různých skupin a řešením a odpovídáním na celou řadu otázek.

Kompletní program včetně abstraktů k jednotlivým přednáškám naleznete na webu konference a také níže

Úterý 21. 09. 2021

Přednáška sponzora sekce

• 9:00 – 9:10 ACQUITY PREMIER komplexní řešení aneb jak minimalizovat nežádoucí adsorpci analytů v kapalinové chromatografii (L. Matulková, Waters)

Waters Premier systémy obsahují novou inovativní technologii MaxPeak High Performance Surfaces (HPS), která účinně snižuje nespecifickou adsorpci v důsledku interakcí analytů s kovy, vše bez složitých mobilních fází a pracných metod.

Waters Premier systémy, ve spojení s Waters Premier kolonami a QuanRecovery vialkami a platíčky, jsou zárukou lepší citlivosti a reprodukovatelnosti pro analyty typu: Organické kyseliny, Organofosfáty, Oligonukleotidy, Fosfopeptidy, Kyselé glykany, Fosfolipidy a další analyty typu Lewisových bází.

5. SPOJENÍ SEPARAČNÍCH TECHNIK S MS (předseda sekce: O. Novák)

• 09:10 – 09:40 Stanovení nízkomolekulárních alkylaminů metodou HILIC-MS (M. Douša)

Stanovení nízkomolekulárních alkylaminů metodou HILIC-MS (M. Douša)

A volatile organic compounds (VOC) any organic compound having an initial boiling point less than or equal to 250° C measured at a standard atmospheric pressure of 101.3 kPa. In organic chemistry, amines are compounds and functional groups that contain a basic nitrogen atom. Amines are formally derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. The usual synthesis of metformin (MET) involves the one-pot reaction of dimethylamine (DMA) hydrochloride and 2-cyanoguanidine over heat. DMA present in MET is susceptible to oxidation yielding unsymmetrical dimethylhydrazine, which further oxidizes in contact with air or other oxidation agents to N-nitrosodimethylamine (NDMA). DMA together with other compounds is accepted to play its role as a precursor of the formation of NDMA in water and sewage. NDMA was classified by U.S. Environmental Protection Agency as a probable human carcinogen. In December 2019, the U.S. Food and Drug Administration began testing samples of MET for the content of NDMA. A sensitive and specific hydrophilic interaction chromatography (HILIC) method for the separation and determination of dimethylamine (DMA) in active pharmaceutical ingredients (APIs) and in dosage forms of metformin (MET) has been developed and validated. A feasible analytical method based on HILIC coupled with mass spectrometry detection (HILIC-MS) was established using a simple sample preparation. The separation of MET was achieved on a Cortecs HILIC column using a mixture of 10 mmol/L ammonium formate adjusted to pH 4.8 and acetonitrile (25:75, v/v) at 0.8 mL/min flow rate. The a single-quadrupole mass detector was operated in positive ion mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 46 as (M+H)+. The HILIC-MS method was validated as per International Council on Harmonization (ICH) guidelines in terms of linearity, limit of detection, limit of quantification, selectivity, accuracy, precision and intermediate precision. The method was demonstrated to be applicable for the determination of DMA in routine quality control evaluation of commercial samples of metformin of both API and dosage forms. The HILIC-MS method was developed as a simpler and faster alternative to compendial method for determination of DMA (as specific impurity F) in MET described in European Pharmacopoeia.

• 09:40 – 10:00 Moderní trendy v analýze fytohormonů (A. Pěnčík)

Moderní trendy v analýze fytohormonů (A. Pěnčík)

Plant hormones (phytohormones) are signaling molecules that control physiological processes during plant growth and development as well as responses to biotic and abiotic stresses. The spatial distribution of phytohormones within organs, tissues and individual cells is essential for many developmental processes. Currently, our main interest is the organelle-specific analysis of phytohormones, which reveals their intracellular distribution and helps us to understand their homeostasis maintenance at the cellular and organelle levels. The analysis of phytohormones is a great challenge due to their various physicochemical properties and the very low concentrations in which they are present in plant tissues. Moreover, many of them form the structural isomers, and ultra-high performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS/MS) is crucial to achieve our challenging analytical goals. We have recently developed several UHPLC-MS/MS methods for targeted analysis of phytohormones, e.g. auxins (1), as well as a method for simultaneous profiling of major phytohormonal classes (2). In the last decade, instruments based on the supercritical fluid chromatography (SFC) combined with MS/MS (SFC-MS/MS) have been improved and become the additional approach to analyze low abundant compounds in complex biological matrices (3). This method utilizes supercritical carbon dioxide as a major component of the mobile phase combining the advantages of gas chromatography and liquid chromatography. It results in an excellent separation efficiency of chiral and structurally related isomers in a short time. We have developed a method for analysis of phytohormones with 1260 Infinity II SFC/HPLC Hybrid System coupled with 6495 Triple Quadrupole utilizing a unique combination of LC and SFC equipped with MS/MS detection.

• 10:00 – 10:20 Využití odlišných separačních potenciálů UHPLC a UHPSFC metod v analýze stereoisomerních steroidů (T. Gazárková)

Využití odlišných separačních potenciálů UHPLC a UHPSFC metod v analýze stereoisomerních steroidů (T. Gazárková)

The analysis of isomeric and isobaric steroids remains a troublesome analytical challenge despite many published studies using traditional gas chromatography and liquid chromatography techniques. Structural similarity resulting from a typical steroid skeleton and minor structural modifications, such as hydroxyl group position, is often multiplied even further by the common loss of one to three water molecules in MS and MS/MS spectra, leading to the creation of additional isobars. Thus, the achievement of full chromatographic separation is crucial to eliminate the observed interferences and correctly quantify the targeted analytes.

The present study aims to develop high-throughput UHPLC and UHPSFC chromatographic methods for the quantification of biogenic and synthetic steroids in mouse plasma samples. A set of 37 steroids from the C19 (androstanes), C21 (pregnanes), and synthetic steroid groups generated a total of 11 critical pairs/groups, resulting in a challenging separation of 28 analytes. The development of the UHPSFC method included an extensive screening of 19 stationary phases across the polarity range as no generic stationary phase has been discovered yet. The columns with the best separation score of isomers were selected for further optimization of the gradient program using CO₂ and various organic modifiers with additives as a mobile phase. The UHPLC screening was carried out using ten C18, aromatic, and fluorinated stationary phases, followed by detailed gradient optimization on selected C18 columns using acetonitrile and 0.1% formic acid in water as a mobile phase. The protein precipitation method was selected over supported liquid extraction for sample pretreatment of mouse plasma samples.

• 10:20 – 10:40 Strategie k vývoji metod pro stanovení genotoxických nečistot (J. Jireš)

Strategie k vývoji metod pro stanovení genotoxických nečistot (J. Jireš)

Availability of safe human drugs is, without any doubt, a key component in the public healthcare of any developed country. One of the main reasons for drug recalls by Food and Drug Administration (FDA) and European Medicines Agency (EMA) was the content of one or more genotoxic impurities in the final product. Recently, organic azides and N-nitrosamines were triggering the most genotoxicity alerts. Limiting the content of such genotoxic impurities in active pharmaceutical ingredients (APIs) and drugs forms (DFs) is crucial for the safety profile of the product and the solution always requires a multidisciplinary approach.

Such an approach was demonstrated during the process optimization study with the goal of decreasing the N-Nitrosodimethylamine (NDMA) content in the final pharmaceutical products containing metformin. The entire study was conducted in the environment of the pharmaceutical company Zentiva, k.s. and in compliance with the guideline M7(R1) of International Council for Harmonisation. The analytical method utilises an instrument consisting of high-performance liquid chromatograph coupled with a tandem mass spectrometer. The mass spectrometer was operated with atmospherical pressure chemical ionization (APCI) in positive ion mode. The method was calibrated using an isotopically labelled internal standard. Validation of the method was performed according to the guideline Q2(R1) ICH. Furthermore, the performance of the method was evaluated during an interlaboratory comparison using a regression analysis. Key factors influencing the formation of NDMA in metformin containing products were formulated according to the results of the performed process optimization study. NDMA content was determined in 469 samples of metformin API and FCT during the study. A hypothesis explaining the mechanism of NDMA formation in the studied products was formulated.

Another method for the determination of genotoxic azide impurities in sartans was developed. The method utilises a very efficient liquid chromatograph Waters Acquity I-Class coupled with a highly sensitive tandem mass spectrometer Xevo TQ-XS. The method allows for accurate quantification of both impurities GTI-azide-1 and GTI-azide-2 at levels below 1/10th of the specification limit, which is crucial in the context of pharmaceutical analysis. The limit of quantification was determined to be 0.033 ppm and 0.025 ppm for GTI-azide-1 and GTI-azide-2, respectively.

• 10:40 – 11:10 Přestávka na kávu a diskuse u plakátových sdělení

Přednáška sponzora sekce

• 11:10 – 11:20 Agilent LC/MSD – iQ pro Vaši laboratoř (J. Kovář, HPST)

Agilent LC/MSD – iQ pro Vaši laboratoř (J. Kovář, HPST)

Nejste MS expert? Nevadí! Agilent InfinityLab LC/MSD iQ je detektor, který je navržen tak, aby jste i s minimálními vědomostmi o hmotnostní detekci získali maximum informací o vašem analytu.

A jaké jsou výhody LC/MSD iQ při srovnání s DAD detektorem? Možnost monitorovat chemické reakce bez nutnosti nástřiku standardů, jednoduchá detekce/zjištění koelujících látek, lepší specificita pro látky špatně detekovatelné UV detektorem nebo výrazně větší citlivost při detekci nečistot o nízké koncentraci.

6. SUPERKRITICKÁ FLUIDNÍ CHROMATOGRAFIE (předseda sekce: P. Bednář)

• 11:20 – 11:50 Superkritická fluidní chromatografie polárních látek (K. Lemr)

Superkritická fluidní chromatografie polárních látek (K. Lemr)

Modern ultra-high performance supercritical fluid chromatography (UHPSFC) attracts attention as a useful alternative to gas chromatography and high-performance liquid chromatography. Although SFC was considered to be useful for apolar and mildly polar compounds, UHPSFC is also successfully applied for the separation of polar and even ionic analytes (1). To elute strongly retained polar compounds in an appropriate time and to reach a reasonable peak shape, the mobile phase is modified using mixtures of alcohols with water and additives such as ammonium salts. Although the mobile phase is not in a supercritical state after some modifications, the term SFC is still used. The proper setting of a gradient and wide range of stationary phase chemistries are also useful for the separation of polar analytes. Many examples of polar compound separation can be found in the literature (1), some examples are discussed in details (2-4).

• 11:50 – 12:10 Chirální separace boronových klasterů pomocí superkritické fluidní chromatografie (O. Horáček)

Chirální separace boronových klasterů pomocí superkritické fluidní chromatografie (O. Horáček)

Boron clusters are artificial three-dimensional structures, which exhibit unique physicochemical properties. Carboranes as a subgroup of boron cluster compounds were derived by substituting BH units for CH units. The similar steric volume occupied by a rotating phenyl ring and an icosahedral carborane cage has led to the extensive research of carborane moieties as new pharmacophores. The carborane moiety has already substituted phenyl groups of some conventional pharmaceuticals, e.g., aspirin (Figure 1), tamoxifen, penicillin, lidocaine, celecoxib, etc., and their activity has been tested. (1)

Figure 1. Asborin. The carborane analogue of aspirin.

Chirality of carboranes is caused by introducing endo-/exoskeletal substituents, which impair the symmetry of the cage. Hence, it is vital to evaluate analytical methods for chiral separation of boron clusters concerning their potential use in pharmacy. Although the successful chiral separations of neutral, zwitterionic, and recently anionic carboranes were achieved by HPLC (2), no chiral separations of these species have been carried out in supercritical fluid chromatography so far.

Supercritical fluid chromatography is generally accepted as the most widely used and the most versatile technique for chiral separations in the pharmaceutical industry, thanks to the more straightforward and faster chiral method development and faster chiral separation methods than in HPLC. (3) Hence, supercritical fluid chromatography was employed to assess the chiral separations of the carboranes. Firstly, the chiral screening method in gradient elution was performed on nine polysaccharide-based columns. Secondly, the successful enantioseparations obtained in chiral screening were subsequently optimized in isocratic elution to achieve the baseline separation of the carboranes in the shortest possible time.

• 12:10 – 12:30 Enantioseparace deschlorketaminu a jeho metabolitů superkritickou fluidní chromatografií: lehký úkol? (N. Kolderová)

Enantioseparace deschlorketaminu a jeho metabolitů superkritickou fluidní chromatografií: lehký úkol? (N. Kolderová)

Deschloroketamine (DCK) is classified as a new psychoactive substance that belongs among dissociative anesthetics. It is being abused instead of ketamine with which it shares many attributes. Unlike ketamine, there is lack of information about metabolism, toxicity or the difference in the effects of the DCK enantiomers (1).

To achieve facile monitoring of enantiomers of DCK and its metabolites in biological samples, we decided to develop a chiral separation method using mass spectrometry (MS) detection. As an MS compatible mobile phase, supercritical CO₂ with various organic modifiers has been chosen. However, enantioseparation achieved with an optimized method was diminished by excessive peak distortion of DCK, which rendered its quantitation impossible. We found that the peak distortion is caused by a chemical reaction producing a stable carbamic acid salt. To suppress its formation, ethansulfonic acid can be used, however, then enantioseparation of metabolites is not achieved. Thus, currently, two methods of chiral separation shall be used to fully determine the composition of a DCK-containing biological sample.

• 12:30 – 14:00 Oběd

Přednáška sponzora workshopu

• 14:00 – 14:10 Využití vyšší účinnosti core shell částic ke zlepšení proteomických analýz (E. Kosovic, Phenomenex)

Využití vyšší účinnosti core shell částic ke zlepšení proteomických analýz (E. Kosovic, Phenomenex)

7. SOUTĚŽNÍ WORKSHOP

• 14:10 – 17:00 Aneb, OTESTUJTE SI VAŠE VĚDOMOSTI V OBLASTI SEPARAČNÍCH METOD (L. Nováková, M. Douša, F. Švec)

SOUTĚŽNÍ WORKSHOP. Aneb, OTESTUJTE SI VAŠE VĚDOMOSTI V OBLASTI SEPARAČNÍCH METOD (L. Nováková, M. Douša, F. Švec)

SOUTĚŽNÍ WORKSHOP. Aneb, OTESTUJTE SI VAŠE VĚDOMOSTI V OBLASTI SEPARAČNÍCH METOD (L. Nováková, M. Douša, F. Švec)

• 19:00 Raut s živou hudbou