- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.
Para Red (PR) and Sudan dyes have been illegally used as colorants to adulterate certain foods by enhancing their red/orange colour. In addition, they are toxic and carcinogenic. This work presents the development of a simple flow injection chromatographic method combined with chemometric tools to perform the determination of PR, Sudan I (SI) and Sudan II (SII) in food samples. The flow chromatographic system consisted of a low-pressure manifold coupled to a reverse phase monolithic column. A Partial Least Square (PLS) model was applied to resolve overlapped absorption spectra registered for each dye at the corresponding retention time. The relative errors of calibration (RMSECV, %) were 0.49, 0.85 and 0.23, and the relative errors of prediction (RMSEP, %) were 1.12, 0.75 and 0.33 for PR, SI and SII, respectively. The residual predictive deviation (RPD) values obtained were higher than 3.00 for all analytes. The method was successfully applied to quantify the dyes in six different commercial spices samples. The results were compared with the HPLC reference method concluding that there were no significant differences at the studied confidence level (α = 0.05). The proposed method can be used to rapidly determine the analytes in a simple, reliable, low-cost and environmentally-friendly manner.Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05299-8.
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.
A fully automated sequential injection system was tested in terms of its application in liberation testing, and capabilities and limitations were discussed for clotrimazole liberation from three semisolid formulations. An evaluation based on kinetic profiles obtained in short and longer sampling intervals and steady-state flux values were applied as traditional methods. The obtained clotrimazole liberation profile was faster in the case of Delcore and slower for Clotrimazol AL and Canesten cream commercial formulations. The steady-state flux values for the tested formulations were 52 µg cm-2 h-1 for Canesten, 35 µg cm-2 h-1 for Clotrimazol AL, and 7.2 µg cm-2 h-1 for Delcore measured in 4 min sampling intervals. A simplified approach for the evaluation of the initial rate based on the gradient between the second and third sampling points was used for the first time and was found to correspond well with the results of the conventional methods. A comparison based on the ratio of the steady-state flux and the initial rate values for Canesten and Clotrimazol AL proved the similarity of the obtained results. The proposed alternative was successfully implemented for the comparison of short-term kinetic profiles. Consequently, a faster and simpler approach for dissolution/liberation testing can be used.
A double-stage Lab-In-Syringe automated extraction procedure coupled online to HPLC for the determination of four sulfonamides in urine has been developed. Our method is based on homogeneous liquid-liquid extraction at pH 3 using water-miscible acetonitrile with induction of phase separation by the addition of a saturated solution of kosmotropic salts MgSO4 and NaCl. The procedure allowed extraction of the moderately polar model analytes and the use of a solvent that is compatible with the used separation technique. The automated sample preparation system based on the stirring-assisted Lab-In-Syringe approach was coupled on-line with HPLC-UV for the subsequent separation of the sulfonamide antibiotics. To improve both preconcentration factor and extract cleanup, the analytes were trapped at pH 10 in an anion-exchange resin cartridge integrated into the HPLC injection loop thus achieving a double-stage sample clean-up. Analytes were eluted using an acidic HPLC mobile phase in gradient elution mode. Running the analytes separation and the two-step preparation of the following sample in parallel reduced the total time of analysis to mere 13.5 min. Limits of detection ranged from 5.0 to 7.5 μg/L with linear working ranges of 50-5000 μg/L (r2 > 0.9997) and RSD ≤ 5% (n = 6) at a concentration level of 50 μg/L. Average recovery values were 102.7 ± 7.4% after spiking of urine with sulfonamides at concentrations of 2.5 and 5 mg/L followed by 5 times dilution. To the best of our knowledge, the use of Lab-In-Syringe for the automation of coupled homogeneous liquid-liquid extraction and SPE for preparation of the complex matrices suitable for separation techniques is here presented for the first time.
The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.
- MeSH
- aerosoly chemie MeSH
- antioxidancia chemie MeSH
- chromatografie metody MeSH
- elektrochemie metody MeSH
- fenol chemie MeSH
- fenoly analýza MeSH
- kalibrace MeSH
- limita detekce MeSH
- Malus metabolismus MeSH
- potravinářská technologie MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
