Byla zavedena a validována jednoduchá metoda s použitím kapalinové chromatografie a tandemové hmotnostní spektrometrie (LC-MS/MS) pro stanovení kolistinu A a kolistinu B v plazmě. Extrakce proteinů z plazmy byla provedena pomocí 1 ml kazet Oasis HLB a chromatografická separace byla provedena na koloně Arion® Polar C18 (250 × 4,6 mm; 5 mm) při 35 °C. Mobilní fáze se skládala z vody obsahující 0,1 % kyseliny mravenčí a methanolu obsahujícího 0,1 % kyseliny mravenčí v poměru 40 : 60 (v/v), při průtoku 0,8 ml/minutu. Eluent byl detekován v režimu pozitivních iontů pomocí ionizace elektrosprejem s následujícími iontovými přechody m/z: kolistin A 585,55 → 101,05; kolistin B 578,5 → 101,15; a IS 602,4 → 101,1; 120,15; 86,15. Byly provedeny testy krátkodobé stability kolistinu a CMS, a to při pokojové teplotě a 37 °C, kdy se stabilita obou složek se zvyšující se teplotou snižuje. Předložený příspěvek je součástí studie Pharmacokinetics of Colistin in Critically Ill Patients With Extracorporeal Membrane Oxygenation (COL-ECMO2022), v níž budou prezentovány další výsledky.
A simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the determination of colistin A and colistin B in human plasma was developed and validated. Plasma extraction was performed using Oasis HLB 1 ml cartridges, analysis was performed using Arion® Polar C18 (250×4,6mm; 5mm) column at 35°C. Mobile phases consisted of water containing 0,1% formic acid and methanol containing 0,1% formic acid (40:60, v/v) delivered at a flow rate of 0,8 ml/minute. Eluent was detected in the positive ion mode using electrospray ionization at the following transitions of mass to charge (m/z): colistin A 585,55 → 101,05; colistin B 578,5 → 101,15; and IS 602,4 → 101,1; 120,15; 86,15. Short-term stability tests of colistin and CMS were performed, at room temperature and 37°C, where the stability of both components decreases with increasing temperature. The presented paper is part of the Pharmacokinetics of Colistin in Critically Ill Patients With Extracorporeal Membrane Oxygenation (COL-ECMO2022) study in which further results will be presented.
- MeSH
- chemické techniky analytické * metody přístrojové vybavení MeSH
- chromatografie kapalinová metody přístrojové vybavení MeSH
- hydrolýza MeSH
- kolistin * krev MeSH
- lidé MeSH
- methansulfonáty krev MeSH
- stabilita proteinů MeSH
- statistika jako téma MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinická studie MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- Diozen 500mg,
- MeSH
- chromatografie kapalinová metody přístrojové vybavení MeSH
- diosmin * analýza terapeutické užití MeSH
- flavonoidy analýza MeSH
- lidé MeSH
- schvalování léčiv MeSH
- žilní insuficience farmakoterapie MeSH
- Check Tag
- lidé MeSH
Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.
- MeSH
- amidy chemie MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- glykopeptidy chemie izolace a purifikace metabolismus MeSH
- glykosylace MeSH
- hemopexin chemie izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunoglobulin G chemie izolace a purifikace MeSH
- isomerie MeSH
- lidé MeSH
- polysacharidy chemie MeSH
- teplota MeSH
- trypsin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
A device with four parallel channels was designed and manufactured by 3D printing in titanium. A simple experimental setup allowed splitting of the mobile phase in four parallel streams, such that a single sample could be analysed four times simultaneously. The four capillary channels were filled with a monolithic stationary phase, prepared using a zwitterionic functional monomer in combination with various dimethacrylate cross-linkers. The resulting stationary phases were applicable in both reversed-phase and hydrophilic-interaction retention mechanisms. The mobile-phase composition was optimized by means of a window diagram so as to obtain the highest possible resolution of dopamine precursors and metabolites on all columns. Miniaturized electrochemical detectors with carbon fibres as working electrodes and silver micro-wires as reference electrodes were integrated in the device at the end of each column. Experimental separations were successfully compared with those predicted by a three-parameter retention model. Finally, dopamine was determined in human urine to further confirm applicability of the developed device.
The synthesis and characterization of large-bore silica-based monolithic capillary columns (0.32mm×150mm) are presented. Columns were prepared by acidic hydrolysis of a mixture containing tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (BTME) in different molar ratios in the presence of polyethylene glycol and urea. The monoliths were modified by zwitterionic monomer [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. Prepared stationary phases were evaluated by scanning electron microscopy and chromatographic separation of nucleobases and their derivatives in the HILIC mode. The best chromatographic results were obtained with the column prepared from the reaction mixture containing BTME and TMOS in a 1:4 molar ratio. The permeability of such column reached 1.68×10(-14)m(2) and the efficiency, expressed as a height equivalent of the theoretical plate, did not exceed 10.5μm for the tested compounds. The columns were successfully applied to HILIC separation of native and labeled oligosaccharides and glycans released from bovine ribonuclease B and human immunoglobulin G.
- MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- ethan analogy a deriváty chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunoglobulin G metabolismus MeSH
- lidé MeSH
- methakryláty chemie MeSH
- mikroskopie elektronová rastrovací MeSH
- oligosacharidy analýza izolace a purifikace MeSH
- organické sloučeniny křemíku chemie MeSH
- oxid křemičitý chemie MeSH
- ribonukleasy metabolismus MeSH
- skot MeSH
- trimethylsilylové sloučeniny chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.
A monolithic sulfobetaine polymethacrylate micro-column BIGDMA-MEDSA designed in our laboratory, shows dual retention mechanism: In acetonitrile-rich mobile phase, hydrophilic interactions control the retention (HILIC system), whereas in more aqueous mobile phases the column shows essentially reversed-phase behavior with major role of hydrophobic interactions. The zwitterionic polymethacrylate micro-column can be used in the first dimension of two-dimensional LC in alternating reversed-phase (RP) and HILIC modes, coupled with an alkyl-bonded core-shell or silica-based monolithic column in the second dimension, for HILIC×RP and RP×RP comprehensive two-dimensional separations. During the HILIC×RP period, a gradient of decreasing acetonitrile gradient is used for separation in the first dimension, so that at the end of the gradient the polymeric monolithic micro-column is equilibrated with a highly aqueous mobile phase and is ready for repeated sample injection, this time for separation under reversed-phase gradient conditions with increasing concentration of acetonitrile in the first dimension. The fast repeating reversed-phase gradients on a short silica-monolithic or core-shell column in the second dimension can be optimized independently of the actual running first-dimension gradient program. As the alternating HILIC and RP separations on the first-dimension zwitterionic methacrylate column are based on complementary retention mechanisms, the instrumental setup essentially represents two coupled two-dimensional systems. It is first time that such an automated dual LCxLC approach is reported. The novel system allows obtaining three-dimensional data in a relatively short time and can be applied not only to multidimensional gradient separations of flavones and related polyphenolic compounds.
- MeSH
- acetonitrily MeSH
- betain analogy a deriváty MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- chromatografie s reverzní fází přístrojové vybavení metody MeSH
- flavony izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- hydroxybenzoáty izolace a purifikace MeSH
- kyseliny polymethakrylové * MeSH
- oxid křemičitý * MeSH
- rozpouštědla MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
Cíle studie: Vývoj a implementace metody stanovení volných metanefrinů v plazmě pomocí kapalinové chromatografie s tandemovou hmotnostní detekcí (LC-MS/MS) pro rutinní použití v diagnostické laboratoři, která eliminuje omezení klasické kapalinové (HPLC) nebo plynové chromatografie (GC) a imunoanalytických metod, jako jsou náročná příprava vzorků, derivatizace a interferující substance. Typ studie: Vývoj analytické metody. Posouzení vhodnosti pro rutinní použití v diagnostické laboratoři. Název a sídlo pracoviště: Laboratoř klinické biochemie – Oddělení instrumentálních metod, Spadia lab, a.s., Dr. Slabihoudka 6232/11, 708 52 Ostrava. Materiál a metody: Vývoj a validace metody byly provedeny na LC-MS/MS systému Agilent 6490 Triple Quadrupole. Pro chromatografickou separaci byla použita kolona SeQuant®ZIC®HILIC HPLC 3,5 mm, 100Å, PEEK 100 x 2,1 mm metalfree (Merck KGaA, Německo) a gradientová eluce. Pro stanovení analytických parametrů metody byly použity lyofilizované kalibrační a kontrolní materiály z firmy Recipe Chemicals and Instruments GmbH (Německo) a Chromsystems Instruments α Chemicals GmbH (Německo). Do studie bylo zařazeno 40 pacientů s diagnózou arteriální hypertenze. Výsledky: Analytické parametry nově vyvinuté metody jsou vyhovující. Variační koeficienty (CV) preciznosti v sérii a mezilehlá preciznost se pohybovaly pod 10%. Výtěžnost, určená metodou standardního přídavku se pohybovala v rozmezí 89 až 98%. Limit stanovitelnosti pro MN byl 0,035 nmol/l, pro NMN 0,049 nmol/l. Metoda splňuje požadavky validace pro oba analyty. Bylo provedeno srovnání tří metod stanovení volných metanefrinů v plazmě: 2-MET Plasma ELISAFast Track (Labor Diagnostika Nord GmbH, Něměcko), HPLC metoda s elektrochemickou detekcí (ECD) a komerčně dodávaná diagnostická souprava FEO Kit (VUOS a.s., Česká republika) a nově vyvinutá LC-MS/MS metoda. Statistická analýza byla provedena pomocí programu MedCalc 16.1 (Medcalc Software bvba, Belgie) s využitím Passing Bablokovy regresní analýzy. Závěr: Neuroendokrinní tumory představují heterogenní skupinu onemocnění s různou klinickou symptomatologií a etiopatogenezí. Neuroendokrinní tumory, jako jsou feochromocytom nebo paragangliom, vykazují setrvalou nebo paroxyzmální nadměrnou produkci katecholaminů. Jejich 3-O-methylované metabolity, metanefrin (MN) a normetanefrin (NMN), jsou preferovány pro diferenciální diagnostiku feochromocytomu vzhledem k jejich vysoké diagnostické senzitivitě a specificitě. Prezentovaná LC-MS/MS metoda eliminuje nevýhody klasických chromatografických technik a imunoanalytických metod, přičemž zásadními aspekty jsou: jednoduchá příprava vzorku, krátká doba analýzy a požadované analytické parametry.
Objective: The development and implementation of method for the determination of free metanephrines in plasma by the liquid chromatography tandem mass spectrometry (LC-MS/MS) suitable for routine practice in diagnostic laboratory which overcomes inconveniences related with classical liquid (HPLC) or gas chromatography (GC) and immunoassay approaches, like extensive sample preparation, the step of derivatization or interfering substances. Design: Analytical method development. Assessment of suitability for routine practice in diagnostic laboratory. Settings: Department of Clinical Biochemistry – Instrumental methods section, Spadia lab, a.s., Dr. Slabihoudka 6232/11, 708 52 Ostrava Material and Methods: Method development and validation were performed on Agilent 6490 Triple Quadrupole LC-MS/MS system. Chromatographic separation was achieved on SeQuant®ZIC®HILIC HPLC 3,5 µm, 100Å, PEEK 100 x 2,1 mm metal-free HPLC Column (Merck KGaA, Germany) by gradient elution. For determination of analytical parameters lyophilised plasma calibrators (purchased from RECIPE Chemicals and Instruments GmbH, Germany) and controls (purchased from Recipe Chemicals and Instruments GmbH and Chromsystems Instruments α Chemicals GmbH, Germany) were used. The study included 40 patients with diagnosis arterial hypertension. Results: The analytical performance of the new developed method is satisfactory. The intra-assay and inter-assay coefficients of variation (CV) were below 10%. Recoveries determined by standard addition were from 89 to 98%. The limit of quantification for MN was 0.035 nmol/l, for NMN was 0.049 nmol/l. This method fulfilled requirements for validation parameters for both analytes. Comparison of three methods for quantification of free metanephrines in plasma was performed– 2-MET Plasma ELISAFast Track (Labor Diagnostika Nord GmbH, Germany), FEO Kit (VUOS a.s., Czech Republic) dedicated for HPLC-ECD systems and developed LC-MS/MS method. Statistical analysis was performed using the MedCalc 16.1 (Medcalc Software bvba, Belgium) using the Passing Bablok regression. Conclusions: Neuroendocrine tumors represent a clinically and etiologically diverse group of disorders. Neuroendocrine tumors, such as pheochromocytomas or paragangliomas, exhibit a persistent or pyroxysmal excessive catecholamines production. 3-O-methylated catecholamine metabolites, metanephrine (MN) and normetanephrine (NMN), are prioritized for diagnosis of pheochromocytoma due to their highest diagnostic sensitivity and specificity. Presented LC-MS/MS method overcomes classical HPLC and immunoassay approaches. Method provides simple step of sample preparation, short runtime and required analytical parameters
- MeSH
- časná detekce nádoru MeSH
- chromatografie kapalinová metody přístrojové vybavení MeSH
- ELISA metody MeSH
- feochromocytom diagnóza krev MeSH
- hmotnostní spektrometrie metody přístrojové vybavení MeSH
- metanefrin analýza krev MeSH
- odběr biologického vzorku metody normy MeSH
- reagenční diagnostické soupravy MeSH
V súčasnosti je často diskutovanou témou problematika zneužívania drog u mladistvých. Počet požiadaviek na vyšetrenie drog zo strany lekárov, ale aj rodičov počas posledných rokov narastá a úmerne tomu rastie aj počet pozitívnych nálezov. Popri „tradičných“ drogách bol na východnom Slovensku zaznamenaný výskyt nových psychoaktívnych látok. Cieľom tejto práce bolo informovať o výskyte dvoch syntetických látok desoxypipradrole (2-difenylmetylpiperidín, 2-DPMP) a α-pyrolidinovalerofenóne (alfa-PVP) vo východoslovenskom regióne a štatisticky zhodnotiť výsledky našich analýz za obdobie rokov 2012 – 2014. Tiež sme chceli porovnať údaje o úžívaní týchto drog s „tradičnými“ drogami ako sú marihuana, amfetamíny, opiáty a kokaín. Súbor vzoriek tvorili pacienti z psychiatrických ambulancií a z Centra pre liečbu drogových závislostí (CPLDZ) v Košiciach. Vzorky pacientov boli do laboratória posielané v rámci rutinného toxikologického vyšetrenia na zistenie prítomnosti drog a iných zneužívaných liečiv. Analýzy na zistenie prítomnosti nových psychoaktívnych látok boli robené na vysoko citlivých chromatografických prístrojoch GCMS a LCMSMS. Tieto metódy boli vybrané ako spoľahlivé na identifikáciu pôvodnej formy, ako aj metabolitov týchto látok. Viac ako 80 % chromatografických analýz (z celkového počtu 4996) realizovaných v rokoch 2012 – 2014 bolo cielených na odhalenie užívania nezákonnych látok. Z výsledkov analýz vyplýva, že drogou číslo jeden na východe Slovenska je stále marihuana. Novým fenoménom medzi drogami sú novšie psychostimulanciá, ktorých predaj je častokrát maskovaný. Vo východoslovenskom regióne bol zaznamenaný od začiatku roka 2012 výskyt dvoch psychostimulancií, a to 2-DPMP a alfa-PVP.
Currently, one of the most discussed topic is the problem of drug abuse in adolescents. The number of requests for investigation of drugs by doctors, as well as parents increases over the years and is growing in proportion to the number of positive findings. In addition to „traditional“ drugs, the emergence of new psychoactive substances has been recorded in eastern Slovakia. The aim of this work is to provide information on the presence of two synthetic substances desoxypipradrol (2-diphenylmethylpiperidine, 2-DPMP) and α-pyrrolidinovalerophenone (alpha-PVP) in the eastern region of Slovakia and statistically evaluate the results of our analysis for the period 2012-2014, and compare data on the use of these drugs with „traditional“ drugs such as marijuana, amphetamines, opiates and cocaine. Samples were collected from psychiatric clinics and the Center for Treatment of Drug Addiction (CTDD) in Košice. Patient samples were sent to our laboratory under routine toxicological testing for the presence of drugs and other abused drugs. The presence of new psychoactive substances was determined using a highly sensitive chromatography techniques – GCMS and LCMSMS. These methods were selected as reliable for the identification of the original form of the drugs and their metabolites. More than 80 % chromatographic analyses (of total 4996) were performed for the purpose of detection of illicit substances over the years 2012 to 2014. Analysis has shown that the number one drug in eastern Slovakia is marijuana. A new phenomenon among new drugs are stimulants, the sale of which is often masked. Since the beginning of 2012 the incidence of two psychostimulants, 2-DPMP and alpha-PVP has been observed.
- Klíčová slova
- desoxypipradrol, alfa-pyrolidinovalerofenon, toxikologická analýza, tradiční drogy,
- MeSH
- chromatografie kapalinová metody normy přístrojové vybavení MeSH
- chromatografie plynová metody normy přístrojové vybavení MeSH
- hmotnostní spektrometrie metody normy přístrojové vybavení MeSH
- lidé MeSH
- mladiství MeSH
- odhalování abúzu drog metody přístrojové vybavení využití MeSH
- piperidiny * aplikace a dávkování izolace a purifikace moč škodlivé účinky MeSH
- poruchy spojené s užíváním psychoaktivních látek * diagnóza klasifikace prevence a kontrola terapie MeSH
- uživatelé drog * klasifikace statistika a číselné údaje MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Slovenská republika MeSH
The history of liquid chromatography started more than a century ago and miniaturization and automation are two leading trends in this field. Nanocolumn liquid chromatography (nano LC) and largely synonymous capillary liquid chromatography (capillary LC) are the most recent results of this process where miniaturization of column dimensions and sorbent particle size play crucial role. Very interesting results achieved in the research of extremely miniaturized LC columns at the end of the last century lacked distinctive raison d'être and only advances in mass spectrometry brought a real breakthrough. Configuration of nano LC-electrospray ionization mass spectrometry (LC-ESI-MS) has become a basic tool in bioanalytical chemistry, especially in proteomics. This review discusses and summarizes past and current trends in the realization of nano liquid chromatography (nano LC) platforms. Special attention is given to the mobile phase delivery under nanoflow rates (isocratic, gradient) and sample injection to the nanocolumn. Available detection techniques applied in nano LC separations are also briefly discussed. We followed up the key themes from the original scientific reports over gradual improvements up to the contemporary commercial solutions.
