High-resolution capillary electrophoresis for the determination of carbamylated albumin

Objectives Carbamylation is a non-enzymatic post-translational reaction of a primary amino group of a protein with isocyanate. The albumin carbamylation is a negative prognostic factor in chronic kidney disease (CKD) patients and induce charge difference implying an observed shift in electrophoretic mobility that can be measured through a symmetry factor (SF). Methods The Helena V8 and the Sebia Capillarys 2 systems were used for all experiments. The effect of in vitro carbamylation on the SF by spiking increasing concentrations of potassium isocyanate (KCNO) in serum of three healthy volunteers was investigated. Theoretical plate numbers (N) as a surrogate of separation efficiency were also calculated and correlations between SF and renal function biomarkers were performed on 284 patients. Results A dose-dependent impact of KCNO on the SF was observed for both methods with the Helena V8 being more sensitive. The mean N was significantly higher on the Helena V8 as compared to the Sebia Capillarys 2 (2,972 vs. 444.1, p<0.0001). The SF correlated significantly with eGFR (r=0.50, p<0.0001), creatinine (r=−0.31, p<0.0001) and urea (r=−0.34, p<0.0001) on the Helena V8. On the Sebia Capillarys 2, a significant correlation was only observed with eGFR (r=0.17, p=0.004). A better discrimination between CKD stages was also observed using the Helena V8. Conclusions Thanks to a higher mean N, the Helena V8 might offer new possibilities, including detection of carbamylated albumin through SF calculation. Further studies are still needed to confirm the interest of using this type of assays in clinical routine.

Separation of some vitamins in reversed-phase thin-layer chromatography and pressurized planar electrochromatography with eluent containing surfactant

The separation of some water- and fat-soluble vitamins via micellar systems of reversed-phase high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) was subjected to research. Hence, the influence of the mobile phase composition (surfactant and acetonitrile concentration, eluent buffer pH) on the migration distances and zone separation of some vitamins (thiamine, riboflavin, niacin, pyridoxine, cyanocobalamin, folic acid, ergocalciferol and α-tocopherol) was investigated. Our results indicated that the applied technique has an impact on the solute order. Comparing the system capacity of HPLC and PPEC (measured as height of the theoretical plate) for the mobile phase systems with and without surfactant shows differences, especially for fat-soluble vitamin. The variances and reproducibilities (% RDS) values of the vitamin are less in PPEC than in TLC. Moreover, the migration distances of water-soluble vitamins are longer than fat-soluble ones. Overall, eluent consisting of 50% acetonitrile, 18.75 mM SDS, the buffer of pH 6.99 via the PPEC technique was most appropriate for determining the investigated vitamins in the artificial mixture and the two commercially available vitamin combinations.

Quantification and Stability Indicating UPLC Method Development and Validation of Acalabrutinib in Bulk and Pharmaceutical Dosage Form

Acalabrutinib (ABN) is a Bruton tyrosine kinase inhibitor used to treat mantle cell lymphoma, chronic lymphocytic leukemia and small lymphocytic lymphoma. It has been used in the treatment of Hodgkin lymphoma, multiple myeloma and ovarian cancer. It is available in capsule dosage form, usually prescribed twice a day. The objective of the present study describes the ultra-performance liquid chromatography (UPLC) method development and validation for the estimation of the ABN in Capsule dosage form by following ICH guidelines because no methods were reported in this category. The column and Mobile phase were selected based on trial and error methods. For the estimation, the chromatogram BEH C18 (50 mm x 2.1 mm, 1.7µm) column was run through with Mobile phase 0.01N KH2PO4 : Methanol in the ratio of 50:50 at a flow rate of 0.3 ml/min. Temperature was maintained at 30°C. Optimized wavelength selected for the separation was 234nm. Retention time was achieved 1.148minute in optimized chromatogram. Theoretical plate count and tailing factor were obtained “as per recommendations of ICH limits”. The % RSD precision obtained was 0.7% and Intermediate precision obtained was 0.7% as the limit of Precision was less than 2. %Recovery obtained for marketed formulation was 99.23%. LOD, LOQ values obtained from the regression equation (y=9128.5x + 14854) of ABN were 0.18µg/ml and 0.55µg/ml respectively. Proposed method was linear over the concentration range25-150µg/ml. Different degradation studies (acid, alkali, oxidation, thermal, UV, water) were performed and all these samples passed the limits of degradation. Retention time and run time was decreased, so the method developed was simple and economical that can be adopted in regular quality control tests in Industries.

Process Intensification of Chemical Exchange Method for Boron Isotope Separation Using Micro-Channel Distillation Technology

A micro-channel distillation device was used for the process intensification method to separate boron isotopes, 10B and 11B. Three-dimensional (3D) printing technology was introduced to manufacture the micro-channel device, which used the chemical exchange method with anisole as the donor to separate the boron isotopes. This device was tested in total reflux mode, and the height of an equivalent theoretical plate of the micro-channel distillation equipment was reduced to 1.56 cm. The accurate control of pressure and temperature, as well as the flow rate of the complex, were factors that affected separation ability. Thus, for process intensification, this micro-channel distillation device can be operated horizontally and connected in series into similar modules to effectively improve separation efficiency and reduce the size of the equipment.

Development and Validation of RP-HPLC Chromatographic Dissolution Method for the Simultaneous Estimation of Ramipril and Hydrochlorothiazide from Solid Dosage Formulation

The present study describes the dissolution method development and validation of Ramipril and Hydrochlorothiazide in tablet dosage form by HPLC Method. A simple, rapid, selective, reproducible and isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated as per ICH guidelines. Analysis was performed on a Thermo, Sunniest C8 (150 mm x 4.6 mm, 5 µm) with the mobile phase consisting of mixing 500 mL of buffer solution and 500 mL of acetonitrile at a flow rate of 1.0mL/min. UV detection was performed at 210nm and the Run time for Ramipril and Hydrochlorothiazide were 10 minutes. The calibration curve was linear (correlation coefficient = 1.000) in the selected range for both analytes. The optimized dissolution conditions include the USP Type 1 (Basket) rotation rate of 100 rpm and 750 mL of 0.1 N Hydrochloric acid as dissolution medium, at 37.0 ± 0.5°C. The method was validated for precision, linearity, specificity, accuracy, limit of quantitation and ruggedness. The system suitability parameters, such as theoretical plate, tailing factor and relative standard deviation (RSD) between six standard replicates were well within the limits. The stability result shows that the drug is stable in the prescribed dissolution medium.

Characterization of linearly coupled capillaries with various inner diameters in the context of capillary electrophoresis

As a result of continuous instrumental progress, capillary electrophoresis has become an established separation technique. However, the choice of the suitable capillary inner diameter is sometimes difficult due to different instrumental requirements concerning injection, separation, or detection. To overcome this problem, we assembled two capillaries with different inner diameters, meaning that the inner diameter of the capillary at the injection side was different from that at the detection side. Since this was a rather uncommon approach, we focused on the associated effects in this proof-of-concept study. For the experiments, a non-aqueous model system was used, consisting of an acetonitrile-based background electrolyte and the two ferrocene derivates, ferrocenemethanol and decamethylferrocene. Using capillary flow injection analysis hyphenated to capacitively coupled contactless conductivity detection, it could be shown that fragmented capillaries of the same inner diameter had slightly lower volume flow rates than non-fragmented capillaries. With non-aqueous capillary electrophoresis hyphenated to UV detection, it was found that the coupling of capillaries with different inner diameter had a much stronger effect on the capillary electrophoresis flow than combinations with the same inner diameter. Additionally, if the inner diameter of the second capillary was larger than the inner diameter of the first capillary, a higher theoretical plate number and an increased sensitivity were found. Furthermore, it was found that there was no significant peak tailing introduced by the coupling. Graphic abstract

Estimation Of Glucose And Galactose Content In A Natural Polysaccharide Isolated From Tubers Of Colocasia Esculenta Using A Validated Rp-Hplc Method

Background: Now a days, the natural polysaccharides are preferred over synthetic polymers because they are biocompatible, cheap, and easily available than the synthetic ones. The polysaccharide obtained from the tuber of the Colocasia esculenta tubers was reported to exhibit many pharmaceutical applications as an excipient. Objective: The application of any polymer in formulating various dosage form depends upon their qualitative and quantitative chemical composition. Therefore, in the current research work, the quantitative composition of the polysaccharide isolated from Colocasia esculenta was determined by validated HPLC method. Methods: RP-HPLC analysis was performed on an Agilent, Zorbax Eclipse XDB C8 column (250 mm × 4.6 mm, 5 μm) with the mobile phase consisting of citrate buffer PH 5.5: Tetrahydrofuran: Acetonitrile (87.1:11.9:1 v/v) at a flow rate of 0.6 mL/min. UV detection was performed at 307 nm. The method was validated for various parameters as per ICH guidelines. Results: The calibration curve constructed for Glucose and Galactose was found linear with correlation coefficient of 0.997 and 0.994 respectively. The system suitability parameters, such as theoretical plate, tailing factor and relative standard deviation (RSD), were well within the limits. Solution stability data indicated that the solution was stable for 24 hours at 25°C. The water-soluble polysaccharide from Colocasia esculenta was found to contain glucose and galactose in the ratio 8:1. Conclusion: The validated RP-HPLC method was found to be specific, linear, precise, and accurate and can be successfully used for simultaneous estimation of glucose and galactose content in the polysaccharides.

Method Development and Validation for Quantitative Analysis of Anti-Histamine Promethazine Hydrochloride by RP-UPLC

The quantitative analysis method for the quantitative analysis of the anti-histaminic drug Promethazine Hydrochloride (PMZ•HCl) is stated by a straightforward, smooth, reliable and reverse step of the ultra-performing liquid chromatographic method (RP-UPLC). Following ICH quidelinesQ2(R1), the RP-UPLC method has been developed and checked. The uniform solution of 3.4% KH2PO4 solution in water, 7.0 pH with dilute KOH, ACN, andMeOH in ratio of 40:40:20, used as a mobile phase. The flow of 0.6 mL/minusing photo diode array detector / UV detector by with wavelength of 254 nm and runtime 3 min. This gives linearty from 80-120 % with correlation coefficient of 0.99988. Repeatability and intermediate precision shows relative standard deviation (percent RSD) of 0.52, 0.24 and a overall RSD of 0.43. Robustness studies show no indicative changes in SST requirements, like asymmetry factor, theoretical plate & percentage relative standard deviation. These criteria's values are well within their acceptability limit. The degradation of promethazine under different stress conditions has been studied and shows that all known impurities and degradants are well separated from promethazine peak.This RP-UPLC is descriptive and accurate.

Development and Validation of Stability-Indicating UPLC-TUV Method for Simultaneous Estimation of Darunavir and Ritonavir in Bulk and Tablet Dosage Form

A straightforward, explicit, accurate and monetary ultra-performance liquid chromatographic with tunable ultraviolet indicator (UPLC-TUV) strategy was produced for the simultaneous estimation of Darunavir and Ritonavir in bulk and tablet dosage form. The Separation was accomplished on a BEH C18 section (4.6 mm X 50 mm, 5 µm) at a frequency of 270 nm, utilizing a mobile phase acetonitrile and water (50:50 V/V) in an isocratic elution mode at a stream pace of 0.3 mL/min. The maintenance time for Darunavir and Ritonavir was discovered to be 0.739 min and 0.401 min, individually. The proposed strategy was validated for precision, linearity range, accuracy, roughness, and constrained degradation concentrates according to ICH rules. The adjustment bends of Darunavir and Ritonavir were linear over the scope of 100-600 µg/mL and 12.5 to 75 µg/mL. The LOD's were discovered to be 1.93 and 0.03 for Darunavir and Ritonavir, separately. The LOQ's were discovered to be 5.84 for Darunavir and 0.08 for Ritonavir. The strategy was discovered to be precise and stability-showing has no meddling pinnacles of debases and excipient were noticed. The created technique was appropriate for quality-control labs for quantitative examination of both in bulk and joined dose structure. The created strategy is new and better in innovation when contrasted than the announced techniques and less maintenance time, high theoretical plate check and structure of the mobile phase with great division, precise and stability-showing has no meddling pinnacles of debases and excipient were noticed. The technique was appropriate for quality-control research centers for quantitative examination of both in bulk and consolidated measurements structure.

One-pot Synthesis of Polyethylene glycol-based Polymeric Monolithic Stationary Phases for Capillary Ion Chromatography

Polyethylene glycol-based polymeric monolithic stationary phases (capillary columns of 0.32 mm I.D.) were successfully prepared via one-pot-single-step reaction and were use in ion chromatography for the separation of several common inorganic anions. It should be noted that the prepared polyethylene glycol-monoliths were able to retain and separate the anions even though there were no ion-exchange sites. The retention mechanism was found to be based on the eluent cations trapped among the polyethylene glycol chains and worked as the anion-exchange sites. Several parameters such as the reaction conditions, monomer:porogen ratios, type of eluents, etc., were investigated. The relative standard deviations obtained for the retention times and signal intensities were less than 5 and 10% respectively. Theoretical plate numbers obtained for the separation of these anions were calculated to be in the range of 700-4300 plates for capillary columns of 10 cm in length.