Juraj Piešťanský

Comparison of hydrodynamically closed isotachophoresis-capillary zone electrophoresis with hydrodynamically open capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: pheniramine, its metabolite and phenylephrine in human urine.

The advanced two dimensional isotachophoresis (ITP)-capillary zone electrophoresis (CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed in this work to demonstrate analytical potentialities of this approach in the analysis of drugs in multicomponent ionic matrices. Pheniramine (PHM), phenylephrine (PHE), paracetamol (PCM) and their potential metabolic products were taken for the analysis by the ITP-CZE-ESI-QqQ technique working in hydrodynamically closed CE separation system and then a comparison with the conventional (hydrodynamically open) CZE-ESI-QqQ technique was made. The ITP-CZE-ESI-QqQ method was favorable in terms of obtainable selectivity (due to highly effective heart-cut analysis), concentration limits of detection (LOD at pgmL(-1) levels due to enhanced sample load capacity and ITP preconcentration), sample handling (on-line sample pretreatment, i.e. clean-up, preconcentration, preseparation), and, by that, possibilities for future automation and miniaturization. On the other hand, this experimental arrangement, in contrast to the CZE-ESI-QqQ arrangement supported by an electroosmotic flow, is principally limited to the analysis of uniformly (i.e. positively or negatively) charged analytes in one run without any possibilities to analyze neutral compounds (here, PCM and neutral or acidic metabolites of the drugs had to be excluded from the analysis). Hence, these general characteristics should be considered when choosing a proper analytical CE-MS approach for a given biomedical application. Here, the analytical potential of the ITP-CZE-ESI-QqQ method was demonstrated showing the real time profiles of excreted targeted drugs and metabolite (PHM, PHE, M-PHM) in human urine after the administration of one dose of Theraflu(®) to the volunteers.

Enantioselective column coupled electrophoresis employing large bore capillaries hyphenated with tandem mass spectrometry for ultra-trace determination of chiral compounds in complex real samples

A new multidimensional analytical approach for the ultra‐trace determination of target chiral compounds in unpretreated complex real samples was developed in this work. The proposed analytical system provided high orthogonality due to on‐line combination of three different methods (separation mechanisms), i.e. (1) isotachophoresis (ITP), (2) chiral capillary zone electrophoresis (chiral CZE), and (3) triple quadrupole mass spectrometry (QqQ MS). The ITP step, performed in a large bore capillary (800 μm), was utilized for the effective sample pretreatment (preconcentration and matrix clean‐up) in a large injection volume (1–10 μL) enabling to obtain as low as ca. 80 pg/mL limits of detection for the target enantiomers in urine matrices. In the chiral CZE step, the different chiral selectors (neutral, ionizable, and permanently charged cyclodextrins) and buffer systems were tested in terms of enantioselectivity and influence on the MS detection response. The performance parameters of the optimized ITP – chiral CZE‐QqQ MS method were evaluated according to the FDA guidance for bioanalytical method validation. Successful validation and application (enantioselective monitoring of renally eliminated pheniramine and its metabolite in human urine) highlighted great potential of this chiral approach in advanced enantioselective biomedical applications.

On-line column coupled isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: Varenicline and its metabolite in human urine

A new highly advanced analytical approach, based on two-dimensional column coupled CE (ITP-CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed, evaluated and applied in biomedical field in the present work. Capillary isotachophoresis (ITP) coupled on-line with capillary zone electrophoresis (CZE) used in hydrodynamically closed separation system was favorable for increasing the sample load capacity, increasing the analyte concentration, and removing the deteriorative highly conductive major matrix constituents. These factors considerably reduced the concentration limits of detection (cLOD) and external sample preparation (comparing to single column CZE), and, by that, provided favorable conditions for the mass spectrometry (enhanced signal to noise ratio, reproducibility of measurements, working life of MS). Here, the CZE-ESI combination provided more effective interfacing than ITP-ESI resulting in both a higher obtainable intensity of MS detection signal of the analyte as well as reproducibility of measurements of the analytes peak area. The optimized ITP-CZE-ESI-QqQ method was successfully evaluated as for its performance parameters (LOD, LOQ, linearity, precision, recovery/accuracy) and applied for the direct identification and ultratrace (pgmL(-1)) determination of varenicline and, in addition, identification of its targeted metabolite, 2-hydroxy-varenicline, in unpretreated/diluted human urine. This application example demonstrated the real analytical potential of this new analytical approach and, at the same time, served as currently the most effective routine clinical method for varenicline.

On-line coupled capillary isotachophoresis-capillary zone electrophoresis in hydrodynamically closed separation system hyphenated with laser induced fluorescence detection

An analytical method, based on a column coupling capillary ITP and CZE in a hydrodynamically closed separation mode hyphenated with the detection in the modular arrangement, was developed in this work. Analytical possibilities of this approach are demonstrated on the direct and ultrasensitive quantitative determination of quinine (QUI) in diluted real multicomponent ionic matrices (beverages, urine). The detection cell interface, with the rectangular arrangement of the optical channels inside, connected the separation capillary with the LIF detector via optical fibers in the on‐column detection arrangement. ITP enabled the direct large volume (30 μL) injections of the diluted real matrices with an on‐line sample pretreatment (preseparation, preconcentration) so that no external sample preparation (except for the dilution) was necessary for the separation of the analyte in the multicomponent ionic matrices. Due to the ITP sample preconcentration and intrinsic sensitivity of the LIF detection, very low concentration LOD (as low as 77 pg/mL), were reached at the same time. This was ca. two orders lower than the corresponding LOD achieved by the same 2D separation system with UV absorbance detection. Compared to the single column CE‐LIF methods applied for this model analyte and matrix, this method was found to be superior in terms of concentration LOD, with acceptable selectivity and benefits of the on‐line sample preparation. A food control and bioanalytical application clearly illustrates great practical possibilities and routine use of the proposed modular ITP–CZE–LIF technique.

2D Capillary Electrophoresis Hyphenated with Spectral Detection for the Determination of Quinine in Human Urine

The possibilities of a column coupling two-dimensional capillary electrophoresis (2D CE) combined with fiber-based diode array detection (DAD) for the direct, highly reliable and ultrasensitive quantitative determination of quinine in real multicomponent ionic matrices (human urine) are demonstrated in this work. The capillary isotachophoresis (CITP) stage provided an on-line sample pretreatment (elimination of interfering matrix constituents, preseparation and preconcentration of the analyte) before the capillary zone electrophoresis (CZE) separation. Due to the large volume (30 µL) sample injection and CITP sample preconcentration, a simple absorbance photometric detection was sufficient for obtaining very low concentration limits of detection (∼8.6 ng/mL). The combination of the different separation mechanisms (CITP and CZE) resulted in enhanced separation selectivity. This enabled us to obtain a pure analyte zone in the directly injected real samples suitable for qualitative and quantitative evaluation. The spectral DAD allowed (i) characterization of the purity (i.e., spectral homogeneity) of the analyte zone; and (ii) preliminary indication of structurally related compounds (i.e., potential biodegradation products of quinine), via characteristic spectra recorded in intervals of 200-800 nm. The CITP-CZE-DAD method was characterized by favorable performance parameters that are suitable for its routine biomedical use. One of the primary benefits of the CITP-CZE-DAD method is the possibility of performing direct injections of real biological samples while avoiding external sample preparation procedures and, therefore, enhancing the reliability and applicability of analyses and the potential for method automatization and miniaturization.

Determination of curcuminoids in substances and dosage forms by cyclodextrin-mediated capillary electrophoresis with diode array detection

The present work illustrates the potential of the capillary zone electrophoresis (CZE) separation technique coupled with the on-capillary diode array detector (DAD) for highly reliable determination of curcuminoids (curcumin, CUR, demethoxycurcumin, DCUR, and bisdemethoxycurcumin, BCUR) in substances (commercially available plant extract) and pharmaceutical preparation (commercial pharmaceutical capsules) with minimal sample preparation; (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) was chosen for an anionic separation of CUR and its structural analogues (DCUR and BCUR) as an appropriate complexing agent (i) providing complete resolution of the curcuminoids and (ii) reducing adsorption of these hydrophobic analytes onto the capillary wall. DAD detection was utilised for characterisation of the composition of the separated zones via differences in the corresponding UV-VIS spectra (scanned at interval of 200–800 nm). Reference and real spectra of the analytes demonstrated that the proposed separation method was sufficiently selective to produce well-separated (i.e. spectrally homogeneous) analyte zones with no interfering compounds present. Successful validation and application of the CZE-DAD method proposed here suggest its routine use in highly effective and reliable analysis of curcuminoids in pharmaceutical samples.

HPLC-QTOF-MS Method for Identification and Determination of Bleomycin A2 and B2 Fractions

Bleomycin is a very important drug in oncology used as first-line treatment for many cancers. The development of high-performance liquid chromatography—tandem mass spectrometry method for the separation, identification, and determination of two major structurally related analogs of this antibiotic drug, namely bleomycin A2 and B2, is presented in this work. The proposed method is based on a hydrophilic interaction chromatography (HILIC) stationary phase that enables one to avoid an ion-pairing reagent in the separation system (formerly used in mobile phase for bleomycin) in order to properly separate these highly polar bleomycin analogs. The ion-pairing reagent-free HILIC separation system is suitable for coupling the chromatographic and mass spectrometry stages (for the first time for bleomycin). Some performance parameters, namely, limit of detection (ng/mL-pg/mL), limit of quantification, linearity, precision, and recovery/accuracy, were evaluated showing high reliability, selectivity, and sensitivity of the method. It was successfully applied for the quality drug control determining bleomycin A2 and B2 fractions in commercial pharmaceuticals (Bleomedac infusions). In addition, the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was able to determine an accurate molecular weight of bleomycin A2 and B2 fractions, confirming an identity/quality of the (commercial) drug. The possibilities of the HPLC-QTOF-MS method to identify bleomycin A2 and B2 in model plasma samples were also demonstrated.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300–800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL−1), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02–1.17% and 5.25–7.88%, respectively), and recovery (ranging in the interval of 90.0–93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4–491.2 ng·mL−1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

Capillary Electrophoresis Hyphenated with Mass Spectrometry for Determination of Inflammatory Bowel Disease Drugs in Clinical Urine Samples

Azathioprine is the main thiopurine drug used in the treatment of immune-based inflammations of gastrointestinal tract. For the purpose of therapy control and optimization, effective and reliable analytical methods for a rapid drug monitoring in biological fluids are essential. Here, we developed a separation method based on the capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) for the simultaneous determination of azathioprine and its selected metabolites (6-thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine) as well as other co-medicated drugs (mesalazine, prednisone, and allopurinol). The optimized CE-MS/MS conditions provided a very efficient and stable system for the separation and sensitive detection of these drugs in human urine matrices. The developed method was successfully applied for the assay of the targeted drugs and their selected metabolites in urine samples collected from patients suffering from inflammatory bowel disease (IBD) and receiving azathioprine therapy. The developed CE-MS/MS method, due to its reliability, short analysis time, production of complex clinical profiles, and favorable performance parameters, evaluated according to FDA guidelines for bioanalytical method validation, is proposed for routine clinical laboratories to optimize thiopurine therapy, estimate enzymatic activity, and control patient compliance with medication and co-medication.

Comparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinine

Comparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinine Comparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinine (QUI) is presented in this work. The capillary isotachophoresis (CITP) on-line coupled with capillary zone electrophoresis (CZE) and hyphenated with fibre-based spectrophotometric diode array detection (DAD) was compared with, (i) high performance liquid chromatography (HPLC) method with DAD detection, and (ii) HPLC method with fluorescence detection (FD). These methods were compared through their performance parameters and determined concentrations of QUI in beverages. The concentrations of QUI in two selected bitter drinks determined by the CITP-CZE-DAD method were in a good accordance with the HPLC-DAD and HPLC-FD methods. In addition, the electrophoretic method, as well as the chromatographic methods, was able to separate potential QUI related impurities from the QUI peak. The CITP-CZE-DAD method provided excellent performance parameters that were comparable (precision, accuracy, LOD, robustness) or even better (separation efficiency) than those ones provided by the chromatographic methods. Moreover, the effectivity of the electrophoresis method was higher when considering cost of analysis (equipment, consumption of separation systems), environmental aspects (organic vs. aqueous solvents), on-line sample pretreatment (CITP preconcentration and sample clean-up suitable also for the more complex matrices). Considering these findings, CITP-CZE-DAD was approved as a routine automatized method for the highly reliable quality food control. Porovnanie elektroforézy so spojenými kolónami s metódami kvapalinovej chromatografie v analýze potravín s obsahom chinínu Práca prezentuje porovnanie elektroforézy so spojenými kolónami s metódami kvapalinovej chromatografie v analýze potravín s obsahom chinínu (QUI). Kapilárna zónová elektroforéza (CZE) spojená on-line s kapilárnou izotachoforézou (CITP) a spektrofotometrickým detektorom s diódovým poľom (DAD) na báze optických vláken bola porovnávaná s (i) vysokoúčinnou kvapalinovou chromatografiou (HPLC) s DAD detekciou a (ii) HPLC metódou s fluorescenčnou detekciou (FD). V uvedených metódach boli porovnávané validačné parametre a stanovená koncentrácia QUI v nápojoch. Koncentrácia QUI vo dvoch vybraných horkých nápojoch stanovená CITP-CZE-DAD metódou bola v dobrej zhode s koncentráciami nameranými HPLC-DAD a HPLC-FD metódami. CITP-CZE-DAD metóda navyše dokázala, podobne ako chromatografické metódy, oddeliť potenciálne QUI príbuzné nečistoty od píku QUI. CITP-CZE-DAD metóda poskytla vynikajúce validačné parametre porovnateľné (presnosť, správnosť, LOD, robustnosť) alebo dokonca lepšie (separačná účinnosť) ako tie získané z chromatografických metód. Navyše efektivita elektroforetickej metódy bola vyššia berúc do úvahy cenu analýzy (zariadenie, spotreba separačných systémov), environmentálne aspekty (organické vs. vodné rozpúšťadlá), on-line predúpravu vzorky (CITP prekoncentrácia a vyčistenie vzorky vhodné i pre komplexnejšie matrice). Vzhľadom na získané výsledky bola CITP-CZE-DAD metóda osvedčená ako rutinná automatizovaná metóda pre vysoko spoľahlivú kontrolu kvality potravín.