Electrochemical conversion of fesoterodine to one of its oxidation products was evaluated with the application of the wall-jet flow cell. A traditional, “static” mode of electrolysis was compared ...with the “dynamic” mode of cell performance. For statistical assessment of the data, experiments were planned and performed with the application of design of experiments approach, namely Taguchi L18 design. After screening phase, the experimental settings were broadened or adjusted according to the results and optimization was performed. All of the samples were electrolysed with the use of chronoamperometric method in a three electrode system. The electrolysed samples were analysed using UHPLC-PDA-QDA method. The chromatographic run was performed in gradient elution with the application of C8 column. The response was expressed as % area of the main peak found with the PDA detection method whereas QDA detector was used in positive SIM mode for structural confirmation. All data obtained for both screening and optimization were treated together and linear models were adjusted. The use of large-surface glassy carbon electrode along with pH~7 were found to be the most significant factors influencing electrochemical oxidation of fesoterodine in both modes. The major differences were identified in terms of voltage applied to the electrodes which yielded the highest amounts of oxidation product. Evolution of electrochemical methods may serve as complementary technique in stress degradation studies in pharmaceutical industry.
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•FES electrochemical oxidation was performed in the low-volume flow cell.•Static and dynamic modes of electrolysis were compared using DoE approach.•Significant factors influencing both types of electrolysis were identified.•In both modes the use of glassy carbon electrode and pH~7 promoted electrolysis.•The major difference identified between both modes was the influence of voltage.
This paper highlights the potential of electrochemical flow cells for oxidative-stress testing of active pharmaceutical ingredients using canagliflozin as a model substance. Based on design of ...experiments, we developed our method through a reduced combinatorial design, optimizing the following independent variables: cell size, electrolyte flow rate, electrolyte concentration, and electrolyte pH. Using ammonium phosphate buffer with methanol in a 50/50 vol ratio as a working electrolyte, we electrochemically oxidized samples and analyzed them by high-performance liquid chromatography, considering the following dependent variables: peak area of each impurity, peak area of canagliflozin, and the percentage of the corresponding peak areas. Our results showed that the most significant independent variables were electrolyte pH and flow rate. By data optimization, we determined the most suitable conditions for electrochemical oxidation of canagliflozin, namely 50 µm cell size, 300 mM electrolyte concentration, 0.1 mL/h electrolyte flow rate, and electrolyte pH = 4. The repeatability of the method, expressed as the relative standard deviation of the canagliflozin peak area, measured in ten separately oxidized samples, was 1.64%. For comparison purposes, we performed a degradation experiment using hydrogen peroxide, identifying five identical impurities in both cases, as confirmed by mass spectrometry. The degradation products formed when using the chemical method after 1, 3, and 7 days totaled 0.09%, 0.75%, and 3.75%, respectively, and the degradation products formed when using the electrochemical method after 3 h totaled 3.11%. Oxidation with hydrogen peroxide required 7 days, whereas electrochemical oxidation was completed in 3 h. Overall, the electrochemical method significantly saves time and reduces the consumption of active ingredients and solvents thanks to the miniaturized size of the electrochemical cell, thereby minimizing the costs of forced degradation studies.
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•Innovative electrochemical approach to oxidation study of canagliflozin.•Electrochemical oxidation is faster than hydrogen peroxide oxidation.•Identical degradation products were formed by both methods.•Different effect of pH was observed in both methods.
Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in ...coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI‐MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile–ammonium formate (10 mm, pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.
For the last decade, significant attention has been paid to the potential role of tocotrienols in prevention and therapy of breast cancer. Therefore, the aim of this study was to develop and validate ...analytical method for quantitative determination of tocotrienols (α-, β-, γ- and δ-tocotrienol) in human breast adipose tissue with the use of high performance liquid chromatography coupled with APCI-MS/MS detection. Separation of target compounds was achieved within 10min with the use of naphthylethyl Cosmosil 2.5π-NAP column with methanol/water mixture (90:10, v/v) under isocratic elution. Adipose tissue samples were obtained from breast cancer patients and women deceased as a result of accidents. Sample preparation procedure was optimized with the application of the Plackett-Burman design and included tissue homogenization with the use of isopropanol/ethanol/aqueous 0.1% FA mixture (13:3:8, v/v), centrifugation and solid phase extraction (SPE). The method was validated in terms of linearity, precision, accuracy, stability (bench top, autosampler, postpreparative, freeze and thaw stability), matrix effect (ME), recovery (RE) and process efficiency (PE). As for all four tocotrienols ME was negligible (< 15%), precision and accuracy tests were performed with the use of tocotrienols’ standard solutions within the ranges of 10.0–400.0ng/g for all four tocotrienols. As the validation requirements were met, the validated method was applied for quantitative analysis of tocotrienols in breast cancer patients.
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•A novel breast adipose tissue sample pretreatment method development.•Method validation for quantitative determination of tocotrienols in the adipose tissue.•Separation of target compounds within 10min.•Quantification of tocotrienols with the use of APCI-MS/MS detection.
Rationale
Tocopherols and tocotrienols are chemical compounds insusceptible to the ionization process under atmospheric pressure conditions. Therefore, the selection of the optimal ion source ...settings for their quantification requires special attention. The aim of this study was to analyse the influence of the APCI source parameters on the response of tocochromanols and two related compounds.
Methods
Standard solutions of target compounds were injected on the high‐performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI‐MS/MS) system separately and analysed with 30 randomly selected ion source settings. The obtained responses were modelled by multivariate linear regression with least absolute shrinkage and selection operator. The developed models were used to choose the best APCI conditions.
Results
Multivariate linear models were built for eight tocochromanols, trolox and BHT. The APCI settings derived from the models did not increase the peak areas obtained for T and T3 during the ionization process. Ionization conditions based on models for trolox and BHT improved analytical responses by 12–36% and 4–32%, respectively. The application of the ion source settings optimal for trolox and BHT to tocochromanols did not result in better analytical responses.
Conclusions
The ionization pattern of tocochromanols in the APCI source is problematic and should be further investigated. Modelling methodology for response improvement presented in this study can be applied in similar studies.
Tocopherols and tocotrienols have been extensively studied owing to their anticancer potential, especially against breast cancer. Therefore, the aim of this study was to quantitatively determine ...tocochromanols in human breast adipose tissue with the use of HPLC–FLD. The sample preparation procedure included homogenization and solvent extraction with isopropanol–ethanol–0.1% formic acid mixture prior to solid‐phase extraction. After implementation of central composite design, satisfactory separation of all eight target compounds was achieved within 10.5 min. Chromatographic runs were carried out with the use of a naphthylethyl chromatographic column with methanol–water mixture (89:11, v/v) as the mobile phase. Fluorescence detection of tocochromanols was performed with excitation and emission wavelengths 298 and 330 nm, respectively. The method was validated in terms of linearity, carryover, recovery, precision, accuracy and stability. Extraction yield was also determined for accurate evaluation of vitamin E content in human breast adipose tissue samples. Finally, concentrations of particular tocochromanols compounds were assessed in human breast adipose tissue samples obtained from 99 patients, including women with breast cancer, healthy volunteers and deceased women who had died as a result of accidents. The raw data was transformed according to the newly developed equation for accurate estimation of the concentrations of tocochromanols in breast adipose tissue samples. Results obtained in the study indicated that the proposed analytical assay could be useful in breast cancer research.
The purpose of this work was to develop and validate a rapid and robust LC-MS/MS method for the determination of dexmedetomidine (DEX) in plasma, suitable for analysis of a large number of samples.
...Systematic approach, Design of Experiments, was applied to optimize ESI source parameters and to evaluate method robustness, therefore, a rapid, stable and cost-effective assay was developed. The method was validated according to US FDA guidelines. LLOQ was determined at 5 pg/ml. The assay was linear over the examined concentration range (5-2500 pg/ml),
Experimental design approach was applied for optimization of ESI source parameters and evaluation of method robustness. The method was validated according to the US FDA guidelines. LLOQ was determined at 5 pg/ml. The assay was linear over the examined concentration range (R
> 0.98). The accuracies, intra- and interday precisions were less than 15%. The stability data confirmed reliable behavior of DEX under tested conditions.
Application of Design of Experiments approach allowed for fast and efficient analytical method development and validation as well as for reduced usage of chemicals necessary for regular method optimization. The proposed technique was applied to determination of DEX pharmacokinetics in pediatric patients undergoing long-term sedation in the intensive care unit.
Arterial stiffening is a hallmark of early vascular aging (EVA) syndrome and an independent predictor of cardiovascular morbidity and mortality. In this case-control study we sought to identify ...plasma metabolites associated with EVA syndrome in the setting of hypertension. An untargeted metabolomic approach was used to identify plasma metabolites in an age-, BMI-, and sex-matched groups of EVA (
= 79) and non-EVA (
= 73) individuals with hypertension. After raw data processing and filtration, 497 putative compounds were characterized, out of which 4 were identified as lysophosphaditylcholines (LPCs) LPC (18:2), LPC (16:0), LPC (18:0), and LPC (18:1). A main finding of this study shows that identified LPCs were independently associated with EVA status. Although LPCs have been shown previously to be positively associated with inflammation and atherosclerosis, we observed that hypertensive individuals characterized by 4 down-regulated LPCs had 3.8 times higher risk of EVA compared to those with higher LPC levels (OR = 3.8, 95% CI 1.7-8.5,
< 0.001). Our results provide new insights into a metabolomic phenotype of vascular aging and warrants further investigation of negative association of LPCs with EVA status. This study suggests that LPCs are potential candidates to be considered for further evaluation and validation as predictors of EVA in patients with hypertension.
The aim of this study was to evaluate the nailfold videocapillaroscopic examination results from patients with pseudoexfoliative glaucoma (XFG) and to assess the relationship between the results of ...this examination and the patient's clinical status in the XFG group.
The studied group consisted of 39 Caucasian patients with XFG and 32 patients in a control group. The patients were classified into two subgroups: the hypertensive pseudoexfoliative glaucoma (hXFG) subgroup and the normotensive pseudoexfoliative glaucoma (nXFG) subgroup. The nailfold videocapillaroscopy (NVC) was performed on all participants. The results of each NVC were classified as having a normal or abnormal pattern.
There was no statistical difference between the results of an abnormal NVC pattern in the study group vs. the control group (
= 0.8773). Microhemorrhages were shown in 30.0% of patients with nXFG vs. the control group (6.25%) (
= 0.0520). Microhemorrhages tended to be more frequent in the XFG group (
= 0.1221). A prevalent number of tortuous capillaries was observed in hXFG patients with advanced glaucomatous neuropathy. Dilatation in the capillaries and microbleedings were observed in the group of patients with lower IOP values. Tortuosity in the capillaries was significantly more frequent in PEXG patients (XFG vs. control:
= 0.0386). No relationships between the results of NVC and age, c/d, BCVA, time of treatment, and visual field defect were found.
Specific features of NVC examination differentiate nXFG from hXFG patients. Some capillaroscopic features may correlate with the patient's clinical status of XFG.
The purpose of this study was to evaluate the influence of polymorphisms of the eNOS gene on the clinical status of patients with normal and high tension glaucoma.
266 Polish Caucasian patients with ...primary open angle glaucoma were studied. Of the 266, 156 had normal tension glaucoma (NTG) and 110 high tension glaucoma (HTG). DNA material was isolated from peripheral venous blood using commercial kits. Real-time PCR reaction was used to amplify the promoter site of the endothelial nitric oxide synthase (eNOS) gene, including the single nucleotide polymorphism (SNP) site T-786C and part of the 7th exon of eNOS, including G894T SNP. Genotypes were determined with TaqMan SNP Genotyping Assays.
There were no significant differences in frequencies of the allelic variants of both polymorphisms. In G894T SNP, however, the wild GG form was more common in the HTG group. The SNP of the eNOS gene did not significantly influence the progression rate in either of the groups studied. There were no differences in variants of the eNOS gene regarding the necessity for and success of surgery and the progression of the disease. In the NTG group, no statistical correlation was observed between G894T, T786C polymorphism variants, and risk factors such as optic disc haemorrhages, optic disc notches, and peripapillary atrophy. Mean diastolic and systolic pressure during the day and night were lowest in NTG patients with the CC variant of the T786C polymorphism. No statistical correlation was observed between the G894T and T786C polymorphisms and capillaroscopic examination results.
Genotype frequencies are similar for both the eNOS G894T and T-786C polymorphisms in NTG and HTG patients. These polymorphisms do not correlate with risk factors and do not influence the state of the capillary system in NTG patients. Systolic blood pressure is lower in NTG patients with mutated alleles of both polymorphisms.
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