gradient elution
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Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 μm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 μm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50-15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
Ocrelizumab is a second generation recombinant humanized IgG1 monoclonal antibody used for the treatment of multiple sclerosis that selectively target B cells expressing the CD20 antigen. This study aimed to develop and validate a UPLC-MS/MS method for quantification of ocrelizumab in human serum, which can be used in clinical applications for therapeutic drug monitoring. The analysis of ocrelizumab was performed using a bottom-up approach on a liquid chromatography coupled to tandem mass spectrometry detection. The method involved immunoglobulin precipitation with cold methanol followed by peptide digestion with trypsin. The resulting specific peptides were separated on an Acquity UPLC BEH C18 column at 55 °C using gradient elution. The method was validated according to European Medicines Agency (EMEA) guidelines and demonstrated intra- and inter-assay precision with coefficients of variation ranging from 1.6 % to 6.1 % and accuracies between 90.2 % and 107.2 %. Stability testing, including autosampler, long-term and freeze-thaw stability, showed no more than 15 % variation. The method was successfully applied to 169 patient samples, revealing ocrelizumab concentrations ranging from 0.5 to 21.8 mg/L in patients on 6-month dosing regimen and 20.5-65.0 mg/L in 16 patients receiving an initial two-week dose of 300 mg. The newly developed UPLC-MS/MS method met all criteria for accuracy, precision and stability, confirming its suitability for clinical use in monitoring ocrelizumab levels in multiple sclerosis patients.
- MeSH
- chromatografie kapalinová metody MeSH
- humanizované monoklonální protilátky * terapeutické užití krev MeSH
- lidé MeSH
- monitorování léčiv metody MeSH
- roztroušená skleróza * farmakoterapie krev MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100 mm, 1.7 μm) using a gradient elution with an analysis time of 10 min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000 nmol/L, folic acid 2.5-250 nmol/L, α-tocopherol 0.5-100 μmol/L and all-trans-retinol 25-2500 nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.
- MeSH
- alfa-tokoferol * krev MeSH
- bariatrická chirurgie MeSH
- chromatografie kapalinová metody MeSH
- kyselina listová * krev MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- riboflavin * krev MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- vitamin A * krev MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Systemically administered antibiotics are thought to penetrate the wounds more effectively during negative pressure wound therapy (NPWT).To test this hypothesis total and free antibiotic concentrations were quantified in serum and wound exudate. METHODS: UHPLC-MS/MS methods were developed and validated for the determination of ceftazidime, cefepime, cefotaxime, cefuroxime, cefazolin, meropenem, oxacillin, piperacillin with tazobactam, clindamycin, ciprofloxacin, sulfamethoxazole/trimethoprim (cotrimoxazole), gentamicin, vancomycin, and linezolid. The unbound antibiotic fraction was obtained by ultrafiltration using a Millipore Microcon-30kda Centrifugal Filter Unit. Analysis was performed on a 1.7-μm Acquity UPLC BEH C18 2.1 × 100-mm column with a gradient elution. RESULTS: The validation was performed for serum, exudates and free fractions. For all matrices, requirements were met regarding linearity, precision, accuracy, limit of quantitation, and matrix effect. The coefficient of variation was in the range of 1.2-13.6%.and the recovery 87.6-115.6%, respectively. Among the 29 applications of antibiotics thus far, including vancomycin, clindamycin, ciprofloxacin, oxacillin, cefepime, cefotaxime, cotrimoxazole, and gentamicin, total and free antibiotic concentrations in serum and exudate were correlated. CONCLUSION: This method can accurately quantify the total and free concentrations of 16 antibiotics. Comparison of concentration ratios between serum and exudates allows for monitoring individual antibiotics' penetration capacity in patients receiving NPWT.
- MeSH
- antibakteriální látky MeSH
- cefepim MeSH
- cefotaxim MeSH
- chromatografie kapalinová metody MeSH
- ciprofloxacin MeSH
- exsudáty a transsudáty MeSH
- gentamiciny MeSH
- infekce v ráně * MeSH
- klindamycin MeSH
- kombinace léků trimethoprim a sulfamethoxazol MeSH
- lidé MeSH
- oxacilin MeSH
- sternotomie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- terapie ran pomocí řízeného podtlaku * MeSH
- vankomycin MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Efficient absorbance detection of a low-volume chromatography peak is a difficult task. In this work, an improved design of the fused silica capillary flow cell for absorbance detection in microcolumn liquid chromatography is described. The cell was fabricated from 0.15 mm I. D. fused silica capillary and silica optical fibres. Optical fibres were fully integrated into the cell design and enabled a convenient and effective connection of the cell with the light source and light detector (265 nm UV LED and photodiode in this work). Manufactured cells covered the range of physical lengths 3.1-9.9 mm (55-175 nL) and were used without any focusing optics and slits. Baseline noise was typically below 0.05 mAU and the effective optical path determined in the experiments was 83-97% of the cell's physical length. The level of stray (parasitic) light indicated by a 1% deviation from linearity at 1.7 AU was 0.08% only. The proposed cell design was found to be moderately susceptible to the refractive index change (20-35 mAU baseline change in 5-95% (v/v) gradient of acetonitrile or methanol in a mixture with water, G index up to 4 AU·s/RIU). Manufactured cells were finally applied for absorbance detection of components of test the mixture eluted off 0.3 mm I. D. microcolumn. 9.9 mm cell (175 nL) with an effective optical path of 8.9 mm exhibited contribution to the broadening of chromatography peak comparable with commercial 6 mm (80 nL) rectangular flow cell.
- MeSH
- chromatografie kapalinová MeSH
- obchod MeSH
- optická vlákna * MeSH
- oxid křemičitý * MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
Natalizumab is a humanized recombinant monoclonal IgG4 antibody used in the treatment of multiple sclerosis. Commonly used methods for natalizumab and anti-natalizumab antibodies quantification are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Measurement of therapeutic monoclonal antibodies can be challenging due to the resemblance to human plasma immunoglobulins. Recent developments in mass spectrometry enables to analyze vast variety of large protein molecules. The aim of this study was to develop a LC-MS/MS method for determining natalizumab in human serum and cerebrospinal fluid (CSF) and apply it to clinical settings. For successful quantification, it was necessary to find specific sequences of peptides in natalizumab. This immunoglobulin was treated with dithiothreitol and iodoacetamide, cleaved with trypsin into short specific peptides and determined on a UPLC-MS/MS system. An Acquity UPLC BEH C18 column at 55 °C and gradient elution was used for analysis. Intra- and interassay accuracies and precisions were tested at four concentration levels. Precision was determined by coefficients of variation and was in the range of 0.8-10.2 %, with accuracy in the range of 89.8-106.4 %. The concentration of natalizumab in patient samples ranged from 1.8 to 193.3 μg/mL. The method was validated according to the European Medicines Agency (EMA) guideline, met all acceptance criteria for accuracy and precision, and is suitable for clinical applications. In comparison to immunoassay, which can be elevated by cross-reaction with endogenous immunoglobulins, the results of developed LC-MS/MS method are more accurate and specific.
- MeSH
- chromatografie kapalinová metody MeSH
- humanizované monoklonální protilátky terapeutické užití MeSH
- lidé MeSH
- natalizumab terapeutické užití MeSH
- peptidy terapeutické užití MeSH
- roztroušená skleróza * farmakoterapie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.
Pavel Jandera was a world-leading analytical chemist who devoted his entire professional life to research in the field of high-performance liquid chromatography. During his scientific career, he worked at the Department of Analytical Chemistry at the University of Pardubice, Czech Republic. His greatest contribution to the field of liquid chromatography was the introduction of a comprehensive theory of liquid chromatography with programmed elution conditions. He was also involved in the research of gradient elution techniques in preparative chromatography, modeling of retention and selectivity in various phase systems, preparation of organic monolithic microcolumns, and, last but not least, in the development of theory and practical applications of two-dimensional liquid chromatography, mainly in the comprehensive form. In this review article, we have tried to capture the highlights of his scientific career and provide the readers with a detailed overview of Pavel Jandera's contribution to the evolution of separation sciences.
- MeSH
- chromatografie kapalinová * metody MeSH
- lidé MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.
- MeSH
- Barrettův syndrom * metabolismus MeSH
- chromatografie kapalinová MeSH
- formiáty MeSH
- glycin analýza MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- methanol analýza MeSH
- sliny chemie MeSH
- taurin analýza MeSH
- žlučové kyseliny a soli analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.
- MeSH
- androgeny MeSH
- androstendion * MeSH
- chromatografie kapalinová metody MeSH
- estron MeSH
- lidé MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
