Jaroslav Galba

Quantitative analysis of phenylalanine, tyrosine, tryptophan and kynurenine in rat model for tauopathies by ultra-high performance liquid chromatography with fluorescence and mass spectrometry detection.

We developed and validated a simple and sensitive ultra-high performance liquid chromatography (UHPLC) method for the analysis of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm×50 mm, 1.8 μm particle size) using a 4 min ammonium acetate (pH 5) gradient and detected by fluorescence and positive ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. The procedure was validated in compliance with the FDA guideline. The limits of quantification (LOQ) were 0.3 μM for Kyn and from 1.5 to 3 μM for Phe, Tyr, Trp. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 86-108%. The inter-day precision (n=5 days), expressed as % RSD, was in the range 1-13%. The benefit of using UHPLC is a short analysis period and thus, a very good sample throughput. Using this method, we analyzed plasma samples and detected significant changes of Kyn and Phe in transgenic rat model for tauopathies.

Multidrug analysis of pharmaceutical and urine matrices by on-line coupled capillary electrophoresis and triple quadrupole mass spectrometry

The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion-quantifier product ion-qualifier product ion) provided their selective determination at maximum S/N. The proposed CE-MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per-oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.

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.

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.

A novel liquid chromatography/mass spectrometry method for determination of neurotransmitters in brain tissue: Application to human tauopathies

Neurotransmitters, small molecules widely distributed in the central nervous system are essential in transmitting electrical signals across neurons via chemical communication. Dysregulation of these chemical signaling molecules is linked to numerous neurological diseases including tauopathies. In this study, a precise and reliable liquid chromatography method was established with tandem mass spectrometry detection for the simultaneous determination of aspartic acid, asparagine, glutamic acid, glutamine, γ-aminobutyric acid, N-acetyl-l-aspartic acid, pyroglutamic acid, acetylcholine and choline in human brain tissue. The method was successfully applied to the analysis of human brain tissues from three different tauopathies; corticobasal degeneration, progressive supranuclear palsy and parkinsonism-dementia complex of Guam. Neurotransmitters were analyzed on ultra-high performance chromatography (UHPLC) using an ethylene bridged hybrid amide column coupled with tandem mass spectrometry (MS/MS). Identification and quantification of neurotransmitters was carried out by ESI+ mass spectrometry detection. We optimized sample preparation to achieve simple and fast extraction of all nine analytes. Our method exhibited an excellent linearity for all analytes (all coefficients of determination >0.99), with inter-day and intra-day precision yielding relative standard deviations 3.2%-11.2% and an accuracy was in range of 92.6%-104.3%. The present study, using the above method, is the first to demonstrate significant alterations of brain neurotransmitters caused by pathological processes in the brain tissues of patient with three different tauopathies.

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.

Determination of Evans blue as a blood–brain barrier integrity tracer in plasma and brain tissue by UHPLC/UV method

ABSTRACT Blood–brain barrier changes are an integral part of many neurodegenerative diseases. Evans blue is an intravital dye that binds to albumin and can therefore be used to monitor extravasation of this plasma protein across blood–brain barrier in animal models of neurodegeneration. To monitor extravasation of albumin across blood–brain barrier, we developed and validated an ultrahigh-performance liquid chromatography method for the analysis of Evans blue in rat plasma and brain samples. Analyte was separated on ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm) using a 5-min gradient run and detected by a UV detector. The limits of quantification (LOQ) were 10 µg/mL in plasma and 0.5 µg/g in brain samples. The limits of detection (LOD) were 1 µg/mL in plasma and 0.015 µg/g in brain samples. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 91–105%. The inter-day precision was in the range of 1.3–8%. The benefits of using UPLC are selectivity, short analysis period, and thus, a very good sample throughput. Using this method, we analyzed albumin extravasation across blood–brain barrier in transgenic rat model for tauopathy SHR-72 and age-matched control animals. GRAPHICAL ABSTRACT

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.