Column Temperature Research Articles

An HPLC-UV Method to Assess Human Plasma 25(OH)D3.

The aim of this study was to validate an HPLC-UV method to assess vitamin D status by determining the linearity and precision of the 25-hydroxyvitamin D3 (25(OH)D3) calibration curve, the limits of detection, quantitation and robustness of the method, and its accuracy. A second stock solution of 25(OH)D3 was prepared (500 ng/mL), and working dilutions (5, 10, 20, 30, 40, and 50 ng/mL) were prepared for a calibration curve. The HPLC equipment had a UV-Vis diode-array detector and utilized an AcclaimTM 120 C18 column (5 µm, 4.6 × 250 mm) with a flow rate of 1.2 mL/min, a column temperature of 30 °C, and the standards and samples were maintained at 4 °C, with an injection volume of 100 µL. Detection of 25(OH)D3 was determined at 265 nm, with a retention time of 4.0 min. The validation was conducted according to the FDA Validation of Analytical Procedures: Guidance for Industry. Vitamin D was extracted from plasma samples using acetonitrile (ACN)-0.1% formic acid (2:1 v/v), and the percentage of recovery was calculated. The proposed method conditions gave excellent linearity (R2 = 0.9989) and the linearity coefficient was R2 > 0.99 for 25(OH)D3. The detection and quantification limits were 1.1703 ng/mL and 3.5462 ng/mL, respectively. Decreasing or increasing the reading temperature by 1 °C decreased the response units (AU) of vitamin D, 25(OH)D3. When the current flow rate decreased by 0.2 mL/min (1.0 mL/min), the retention time increased to 4.913 min, whereas an increase of 0.2 mL/min of the proposed flow rate (1.4 mL/min) decreased the retention time to 3.500 min. The percentage of recovery varied from 92.2% to 97.1%. The proposed method to quantify a vitamin D metabolite (25(OH)D3) in human plasma samples was reliable and validated.

Analytical quality by design (AQbD) based optimization of RP-UPLC method for determination of nivolumab and relatlimab in bulk and pharmaceutical dosage forms

BackgroundThe Analytical Quality by Design (AQbD) methodology extends the application of Quality by Design (QbD) principles to the management of the analytical procedure life cycle, encompassing method creation, optimization, validation, and continuous improvement. AQbD assists in creating analytical procedures that are robust, reliable, precise, and cost-efficient. Opdualag™ is a combination of Nivolumab and Relatlimab, which are antibodies that block programmed death receptor-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) receptors, used to treat advanced melanoma. This work aims to develop and validate a reversed-phase ultra-performance liquid chromatography (RP-UPLC) method using AQbD principles to determine NLB and RTB in pharmaceutical products.ResultsA central composite design (CCD) comprising three factors arranged in five distinct levels was implemented via Design-expert® software to optimize the chromatographic conditions. A mathematical model was constructed and the effects of three independent factors namely flow rate (X1), percentage of methanol in the mobile phase (X2), and temperature (X3) on responses including retention time (Y1–2), resolution factor (Y3), theoretical plates (Y4–5), and tailing factor (Y6–7) were investigated. The software determined the optimal chromatographic conditions for the separation of NLB and RTB, which were as follows: 32.80% methanol in the mobile phase, 0.272 mL/min flow rate, 29.42 °C column temperature, and 260 nm UV detection. The retention time for NLB and RTB were 1.46 and 1.88 min, respectively. The method exhibited linearity across the concentration ranges of 4–24 µg/mL for RTB and 12–72 µg/mL for NLB. The limits of detection (LOD) and limit of quantification (LOQ) for NLB and RTB, respectively, were 0.89 µg/mL, 2.69 µg/mL and 0.15 µg/mL and 0.46 µg/mL. The percentage relative standard deviation (%RSD) of intraday and interday precision for NLB and RTB was below 2. The recovery percentages for NLB and RTB were determined to be 99.57–100.43% and 99.59–100.61%, respectively. Both drugs were found to be susceptible to oxidative and photolytic degradation in forced degradation studies.ConclusionsEmploying the AQbD-based methodology, a straightforward, fast, accurate, precise, specific, and stability-indicating RP-UPLC method has been established for the quantitative analysis of NLB and its RTB in pharmaceutical formulations.

Asymmetric catalytic synthesis of chiral covalent organic framework composite (S)-DTP-COF@SiO2 for HPLC enantioseparations by normal-phase and reversed-phase chromatographicmodes.

A novel CCOF core-shell composite material (S)-DTP-COF@SiO2 was prepared via asymmetric catalytic and in situ growth strategy. The prepared (S)-DTP-COF@SiO2 was utilized as separation medium for HPLC enantioseparation using normal-phase and reversed-phase chromatographicmodes, which displays excellent chiral separation performance for alcohols, esters, ketones, and epoxides, etc. Compared with chiral commercial chromatographic columns (Chiralpak AD-H and Chiralcel OD-H columns) and some previously reported chiral CCOF@SiO2 (CC-MP CCTF@SiO2 and MDI-β-CD-modified COF@SiO2)-packed columns, there are 4, 3, 13, and 15 tested racemic compounds that could not be resolved on the Chiralpak AD-H column, Chiralcel OD-H column, CC-MP CCTF@SiO2 column, and MDI-β-CD-modified COF@SiO2 column, respectively, which indicates that the resolution effect of (S)-DTP-COF@SiO2-packed column can be complementary tothe other ones. Theeffects of the analyte mass, column temperature, and mobile phase composition on the enantiomeric separation were investigated. The chiral column exhibits good reproducibility after multiple consecutive injections. The RSDs (n = 5) of the peak area and retention time were less than 1.5% for repetitive separation of 2-methoxy-2-phenylethanol and 1-phenyl-1-pentanol. The chiral core-shell composite (S)-DTP-COF@SiO2 exhibited good enantiomeric separation performance, which not only demonstrates its potential as a novel CSP material in HPLC but also expands the range of applications for chiral COFs.

A Quality by Design (QbD) driven gradient high performance liquid chromatography method development for the simultaneous estimation of dasatinib and nilotinib in lipid nanocarriers

Tyrosine kinase inhibitors (TKIs) are effective as a targeted treatment for chronic myeloid leukemia (CML), which can selectively suppress BCR-ABL1 kinase activity. CML therapy with TKIs combination has been supported by in-vitro, in-vivo, and patient-based data where the nilotinib-dasatinib co-administration has exerted superior anticancer efficacy with greater cellular uptake, less resistance to chemotherapy, and no additive adverse events encountered. Therefore, it is essential to develop a suitable analytical method for the simultaneous estimation of these drugs in the developed novel lipid nanocarriers like liposomes. Design of Experiment (DoE) has been implemented as a tool of QbD to systematically investigate the relation between the HPLC method attributes and analytical responses, i.e., chromatographic detection, quantification, and peak properties for dasatinib and nilotinib. An Ishikawa diagram is constructed to delineate possible influencing variables to the analytical performances. Afterward, 4 factors 2 level full factorial design (FFD) was employed to model and identify the main effects and interaction effects between the factors selected after the initial risk assessment. The suggested design space for optimized chromatographic conditions by QbD analysis is linear within the selected range of drug concentrations, accurate and precise, sensitive, and robust according to the ICH guidelines. The optimal method is comprised of a 1 mL/min flow rate of mobile phase (ACN and 20 mM KH2PO4 of pH 7.00) in gradient mode at 25 °C column temperature for 20 μL sample injection volume and detection wavelength fixed at 297 nm. Most importantly, this novel HPLC method is simple and selective enough to evaluate dasatinib and nilotinib content in the lipid nanocarriers.

RP-HPLC method development and validation for novel synthesized polyethylene glycol conjugated isotretinoin prodrug

Orally administered Isotretinoin is a highly effective medication for treating cystic acne. The current study is to synthesize Isotretinoin prodrug via an ester linkage to polyethylene glycol (PEG) 4000, with DCC (dicyclohexyl carbodimide) as a coupling agent and DMAP (dimethylamino pyridine) as a catalyst. FT-IR, DSC and 1H NMR were used to characterize the synthesied prodrug. RP-HPLC method was developed and validated for prodrug. Thermo C18 (Hypersil BDS) column (250 mm × 4.6 mm, 5μm) was used for chromatographic separation with acetonitrile: water (95:05% v/v) as a mobile phase. UV detection was done at 344 nm; flow rate was 1mLmin-1 and an column temperature of 35oC. Total runtime of the analytical run was 15 min. High selectivity and good precision features were found in the method validation results, which were examined at six concentration levels. In intra- and inter-assay precision experiment, the RSD was less than 2.0%. The recovery of isotretinoin, which was examined at three different degrees of fortification, ranged from 99.06% to 100.42% and linearity was found for a range of 0. 2 to 100 μg mL-1. The calibration curve was linear over the 0.5-100 μg mL-1 concentration range. Photolytic stability of prodrug was performed under UV light. There was successfully improves the photolytic stability of Isotretinoin prodrug as compare to Isotretinoin.

Quantification of the Plasma Concentration of Vadadustat by High-Performance Liquid Chromatography with Ultraviolet Detection and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

An inexpensive, simple, and accurate plasma concentration measurement system is needed to actively conduct pharmacokinetic and pharmacodynamic analyses of vadadustat, hypoxia-inducible factor-prolyl hydroxylase inhibitor, in clinical settings. In this study, the authors aimed to develop a method for measuring vadadustat in human plasma that could be applied for therapeutic drug monitoring using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in a clinical setting. Plasma samples (100 μL) were pretreated with acetonitrile using butyl paraoxybenzoate as an internal standard. Chromatographic separation was performed on a SunShell PFP C18 column (2.6 μm, 4.6 mm × 150 mm). The mobile phase consisted of (A) 20 mM of phosphate buffer (pH 2.4) and (B) acetonitrile (60:40, v/v), delivered isocratically at a flow rate of 1 mL/min. The analytes were detected by UV absorbance at a wavelength of 220 nm, and the column temperature was 40°C. To evaluate the applicability of HPLC-UV in a clinical setting, blood samples were collected at 19 time points from 7 patients who had been taking vadadustat. The calibration curve was linear over the concentration range of 0.2-150 mcg/mL (R2 > 0.99). Intra-assay and interassay accuracy, precision, and stability met the Food and Drug Administration recommendations. The vadadustat plasma concentrations of patients analyzed using the current HPLC-UV method were almost equal to those measured using ultra-performance liquid chromatography-tandem mass spectrometry (mean difference: 0.13 mcg/mL). Large variability in the dose-adjusted plasma concentrations of vadadustat at 12 hours after administration was observed between patients (coefficient of variation = 57.6%). This HPLC-UV method is a simple, accurate quantification method for evaluating plasma concentrations in patients taking vadadustat in a clinical setting.

Fast Determination of Fat-soluble Vitamins in Pharmaceutical Preparations by High-Performance Liquid Chromatography

Background: Vitamins are needed for the healthy functioning of the body. When these vitamins are not in sufficient amounts in the body, they are usually taken externally with pharmaceutical preparations. Taking vitamins into the body in the right amounts is possible by analyzing the amounts in pharmaceutical preparations with validated methods. Methods: In this study, a fast, simple, easily applicable, and selective normal-phase HPLC method was developed for the simultaneous determination of fat-soluble A, D3, E, and K1 vitamins in pharmaceutical preparations from syrup and tablets. Separation of the vitamins was carried out on a Zorbax CN column (250 x 4.6 mm, 5 µm) using a mixture of hexane-isopropyl alcohol (98:2, v/v) at 30°C column temperature and 1 ml/min flow rate. The detection wavelength is 280 nm. The developed method has been validated according to ICH Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2(R1) rules. Results: Calibration graphs are linear over the range of 10-1000 µg ml-1 , 1-50 µg ml-1 , 10-2000 µg ml-1 , 0.5-20 µg ml-1, and the limit of detection values were found to be 1.496, 0.280, 1.388 and 0.040 µg ml-1 for A, D3, E and K1 vitamins, respectively. Relative standard deviation values, which express within-day and between-day repeatability, were found below 2.54%. Average recovery values were also found at about 100.28, 101.46, 100.65, and 100.29% for A, D3, E, and K1 vitamins, respectively Conclusion: The developed and validated method was successfully applied to the simultaneous analysis of fat-soluble vitamins A, D3, E, and K1 in pharmaceutical preparations in syrup and tablet form.

Quality control method for phenolic acid components in abandoned stems and leaves of Artemisia selengensis based on UPLC fingerprint combined with quantitative analysis of multi-components by single marker (QAMS)

This study established an ultra-performance liquid chromatography(UPLC) fingerprint of abandoned stems and leaves of Artemisia selengensis and quantitative analysis of multi-components by single marker(QAMS) for five phenolic acid components. Waters Acquity UPLC BEH C_(18) chromatography column(2.1 mm×100 mm, 1.7 μm) was used. The gradient elution was carried out with the mobile phase composed of 0.1% phosphoric acid water and acetonitrile at a flow rate of 0.3 mL·min~(-1) and a column temperature at 30 ℃. The detection wavelength was 330 nm, and the injection volume was 2 μL. Similarity evaluation and cluster analysis were conducted on the fingerprint data, and 15 common components in 13 batches of abandoned stems and leaves of A. selengensis were identified. The relative correction factors of ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C were calculated using chlorogenic acid as the internal reference. The QAMS for determining five components in the abandoned stems and leaves of A. selengensis was established. At the same time, the content of these five components was determined using the external standard method(ESM), and the results showed that there were no significant differences in their content determined by the QAMS and the ESM. The results indicated that the content of phenolic acid components in the abandoned stems and leaves of A. selengensis from different varieties and different origins had obvious differences. In addition, the content of phenolic acid components in the abandoned stems and leaves of lignified A. selengensis was significantly higher than that of non-lignified A. selengensis. In summary, QAMS established in this study can be quickly, accurately, and economically used to determine the content of five phenolic acid components in abandoned stems and leaves of A. selengensis, laying a foundation for the resource development and utilization of abandoned stems and leaves of A. selengensis.

Identification of chemical components in Qinggu San reference sample of classial prescription based on UHPLC-Q/Orbitrap HRMS combined with molecular network technology

The method of ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UHPLC-Q/Orbitrap HRMS)combined with molecular network was developed in this study for rapidly analyzing the chemical components of the Qinggu San reference sample of classical prescription. Firstly, an ACQUITY UPLC BEH Shield RP_(18) column(2.1 mm×100 mm, 1.7 μm)was used, and acetonitrile and 0.1% formic acid were taken as the mobile phases for gradient elution. The flow rate was 0.4 mL·min~(-1), and the column temperature was 30 ℃. Under these conditions, the mass spectrum data were collected in both positive and negative ion modes of the heated electrospray ionization source. Subsequently, the mass spectrum data of the Qinggu San reference sample were uploaded to the Global Natural Products Social Molecular Network(GNPS)platform for calculation and analysis, and a visual molecular network was built with Cytoscape 3.8.2 software. On this basis, the chemical components of the Qinggu San reference sample were identified by fragmentation regularity of standard compounds, retention time, accurate relative molecular weight of HR-MS, characteristic fragment ions information, literature, and databases. Finally, a total of 105 chemical components were identified and speculated in the Qinggu San reference sample, including 19 iridoid glycosides, 23 flavonoids, 15 phenylpropanoids, 11 triterpene saponins, and 37 other components. Meanwhile, two of these components are potential new compounds. The method used in this study not only achieved rapid and accurate identification of chemical components in the Qinggu San reference sample and provided a scie-ntific basis for the study of pharmacological substances and quality control of Qinggu San compound preparations but also provided a refe-rence for the rapid identification of chemical components in traditional Chinese medicine compound preparations.

Optimization of catalyst service cycle and start-up considering the reactor-distillation-HEN integration and climate

Considering the catalyst deactivation and seasonal temperature variation, a systematic method is proposed for clarifying the dynamic variation of system operating parameters and targeting the optimal catalyst regeneration cycle and start-up date. Relationships between catalyst activity, reactor inlet and outlet temperatures, reactor heat load, and running time are deduced, as well as that between column temperatures, pressures, energy demands, processing capacity, and production month. Diagrams are constructed to illustrate the parameter variations. With the energy consumption and processing capacity combined, an evaluation index is introduced for integrating the reactor-separator-heat exchanger network (HEN) and optimizing the production start-up date. The proposed method is intuitive and efficient and can be used for system design, guiding production, or fault diagnosis. A benzene alkylation process is studied; the optimal regeneration cycle of the ZSM-5 catalyst is 11 months, and the best start-up date is August, saving 2.32 × 105 kgce (kg standard coal) per production cycle.

Robust U-HPLC Method Development of Desonide and its Application to In Vitro Release Testing (IVRT) of Topical Cream Products

Background: The study focuses on establishing In Vitro Release Testing (IVRT) parameters for Desonide cream, following the guidelines of the Topical Classification System (TCS), to assess the bioequivalence between the Reference Listed Drug (RLD) and test. Aim: This study aimed to develop a reliable IVRT method using Franz diffusion cells. An environmentally friendly U-HPLC method was created to analyze Desonide in the samples. Objectives: To evaluate the drug release in Desonide products in accordance with SUPAC guidance, quantify the drug concentration using an analytical method, as per bioanalytical method validation guidelines, and ensure that the results meet the acceptance criteria. Linearity was established from 0.50 μg/mL to 40 μg/mL with acceptable regression values. Precision was confirmed three times, with an average % RSD of below 15% for 3 sets of 6QC level sample preparations. Stability tests demonstrated Desonide stability in receptor fluid (LLOQ and ULOQ) for 72 hours at 2-8°C and 25°C. Autosampler stability at LQC and HQC levels was proven at 25°C for 72 hours. Additionally, the stock solution remained stable at both 25°C and 2-8°C for 72 hours. Methods: The study involved evaluating the dosing regimen, release medium, and membrane while optimizing the U-HPLC method based on three variables including column temperature, mobile phase composition, and flow rate. After experimentation, it was determined that Nylon membrane and 0.9% NaCl: Methanol release media (70:30 v/v) with 1000 mg dose were used to maximize the release profile of desonide. Results: The created explanatory strategy is precise, delicate, and exact for measuring Desonide, with satisfactory Limits of Location LOD and Lower Limits of Measurement LLOQ measured at 0.15 and 0.50 ng /mL, respectively. The Regression coefficient r2 was identified to be 0.9996. The degree of Desonide measurement lessening was considered palatable, basically since the recuperation was underneath 30.00, additionally due to the favourable linear relationship watched within the Desonide discharge rates amid the IVRT study. Conclusion: All three generic products analyzed were found to be equivalent to the RLD, meeting for "sameness" outlined in the FDA's SUPAC-SS guidance. A novel U-HPLC method was developed for Desonide, covering the range from 0.5 to 40 μg/ml, with intra and inter-day variability below 2% RSD. Additional characterizations were established, and the stability of Desonide was successfully determined.

A Review on Lightning and its effects on Trees

Lightning in mother’s nature show, which cannot be controlled. Lightning contacts the earth about more than 8 million times a day. Due to so many strikes, the probability of damage to the plants and trees is very high. The total available energy for an average lightning stroke is of the order of 108 Joule. Of the total available energy about 85-90% is dissipated in resistive part of the air column, which appears as heat energy to raise the temperature of column so high, that shock waves are produced due to expansion of the column and strong thunder is produced utilizing almost whole of this heat energy. These heat and shock waves generated during lightning process can damage or even kill the trees instantaneously. Lightning may cause wilting, yellowing, premature fruit drop and other severe injuries to trees. Also, the probability of a tree being struck by lightning depends on the tree size and morphology. This paper seeks to present a review on lightning phenomena, its effects on trees and also provides a brief information how difference among trees can influence lightning effect.

Theoretical Investigation of Dual-Mode Gradient Elution Chromatography Considering Simultaneous Variations in the Column Temperature and Solvent Composition

Theoretical Investigation of Dual-Mode Gradient Elution Chromatography Considering Simultaneous Variations in the Column Temperature and Solvent Composition

Development of UPLC–MS/MS method for the simultaneous quantification of valproic acid and phenytoin in human plasma and application to study pharmacokinetic interaction in epilepsy patients

Valproic acid and phenytoin are two prevalent antiepileptic medications known for their narrow indices and propensity for cardiovascular and respiratory system toxicity. Therefore, therapeutic drug monitoring (TDM) of valproic acid (VAL) and phenytoin (PHE) concentrations in patient plasma is extremely beneficial for improving clinical choices, avoiding adverse reactions, and optimizing treatment for individual patients. In this study, a rapid and sensitive ultra-performance liquid chromatographic tandem mass spectrometer (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of valproic acid (VAL) and phenytoin (PHE) in human plasma. Negative electron spray ionization (ESI-) mode with selective ion recording (SIR) was employed to determine the transitions of m/z 142.98 and m/z 250.93 for VAL and PHE, respectively. The internal standard (IS) betamethasone (BETA) was ionized using positive electron spray ionization (ESI+) and detected by multi-reaction monitoring (MRM) mode to obtain precursor ions and specific fragment ions for quantification, and the MRM transition was chosen to be m/z 393.17 → 355.16. The separation was performed using a Phenomenex Synergi Hydro-RP (4 μm, 250 × 4.6 mm, I.D.) with an isocratic mobile phase consisting of acetonitrile – water (75:25, v/v) at a flow rate of 0.8 mL/min. The column temperature was maintained at 25 °C. The lower limit of quantification of VAL and PHE was 3.6 μg/mL and 0.72 μg/mL, respectively, which resulted in a recovery of more than 85 % for most analytes. According to US-FDA bioanalytical technique validation, the specificity, intra- and inter-day precision and accuracy, matrix effect, carryover, dilution, and stability of all analytes were within acceptable ranges. This analytical method was successful in evaluating the levels of valproic acid and phenytoin in human plasma from epileptic patients.

Study on UPLC–MS/MS Method for Four Compositions of Cangzhu

Background: The legal quality control of Cangzhu is used the single component as an evaluation indicator, which would not be enough to reflect the authenticity of quality. It is necessary to select several typical chemical components as the basis for quality control of medicinal materials and decoction pieces to reflect the content of effective ingredients. Purpose: Based on UPLC-MS/MS, the study aimed to build the content determination method of atractyloidin, atractylidin A, atractylenolide I, and atractylenolide III in Cangzhu from different sources. Materials and Methods: Agilent SB-C18 (2.1 × 100 µm, 1.8 µm) was used to be the stationary phase. The mobile phase system was formed by water-formic acid (1000:1) and acetonitrile-formic acid (1000:1) in gradient elution. The column temperature set at 40℃, and the flow rate was 0.3 mL min–1. Results: The contents of atractyloidin, atractylidin A, atractylenolide I, and atractylenolide III had good linearity in a certain range (r2 ≥ 0. 9990). The RSD of the precision, stability and repetitive test results were less than 3.00%. In addition, the content of the four components were some different among different patches of Cangzhu. Conclusion: The method was simple, rapid and highly sensitive, which was suitable for the determination of atractyloidin, atractylidin A, atractylenolide I, and atractylenolide III in Cangzhu.

A Green Liquid Chromatography Method for Simultaneous Quantification of Caffeine and its Three Major Metabolites in Urine, Drinks, and Herbal Products

ABSTRACTA green reversed‐phase liquid chromatography method was developed to determine caffeine (CF), theobromine (TB), theophylline (TP), and paraxanthine (PX). The stationary phase was a silica‐based C18 monolithic column (100 × 3.0 mm2 i.d.), eluted with a gradient from 6% to 10% (v/v) of ethanol in water, at a flow rate of 0.75 mL/min (35°C) and UV detection at 272 nm. The compounds were separated in less than 6 min, with a total time of 10 min for column reconditioning. The effect of gradient composition, flow rate, injection volume, and column temperature was evaluated in the separation efficiency, especially regarding the critical pair PX/TP, for which the best resolution was 1.37. The linear range was between 0.5 and 100 µg/mL. The LODs were 0.065, 0.057, 0.040, and 0.044 mg/mL for TP, TB, PX, and CF, respectively. The method was applied to green tea and yerba mate infusions, soft drinks, energy drinks, and soluble coffee. Urine samples were analyzed before and after solid‐phase extraction. The accuracy was evaluated by the spiking/recovery strategy standing in the 79.6% to 116.4% range. The separation performance and the mobile phase consumption were compared to those of another method using a gradient of methanol in water.

Development and optimization of high-performance extraction chromatography method for separation of rare earth elements

The effectiveness of commonly used extractants for chromatographic separation of rare earth elements (REEs) was compared. Columns loaded with similar molar concentrations of tributyl phosphate (TBP), di-(2-ethylhexyl) phosphoric acid (HDEHP), and N-Methyl-N, N, N-tri-octyl-ammonium chloride (Aliquat-336), with mineral acid as eluent were evaluated. Retention factors were determined, and separation efficiency was assessed based on the resolution data of the REEs acquired under the same elution conditions for each column. HDEHP demonstrated the best separation efficiency for the entire REE series (mean Rs = 2.76), followed by TBP (mean Rs = 1.52), while Aliquat-336 exhibited the lowest performance (mean Rs = 1.42). The HDEHP-coated column was then used to optimize the extraction chromatographic separation of the REEs. The primary challenge was to completely elute the heavy REEs (Tb – Lu) while maintaining adequate separation of the light REEs (La – Gd) within a reasonably short time. The stepwise gradient elution procedure improved the resolution between adjacent REEs, allowing the complete separation of the entire REE series within 25 minutes. Better separation efficiency for light REEs was achieved at higher column temperatures and a mobile phase flow rate of 1.5 mL/min in the tested domain of 20–60 °C, and 0.5–2.0 mL/min, respectively, resulting in plate heights (H) ranging from 0.011 to 0.027 mm.

Concepts for a New Rapid and Simple HPLC Method for Simultaneous Determination of Rosuvastatin and Perindopril in Dosage Forms

ABSTRACTSimultaneous determination of a tandem of drugs like perindopril and rosuvastatin with differences in water and organic solvent solubilities, hydrophobicity, polarities, and UV‐absorbing possibilities between them necessitates careful selection of chromatographic conditions. The four specified and different bonded reversed‐phase columns were tested with running mobile phases containing various composition contents concepts; the concept of using diluted trifluoroacetic acid in mobile phase composition with acetonitrile enabled simultaneous acidification and weak ion‐pairing with protonized molecules of analytes, yielding appropriate, short, reasonable run times and resolutions of analytes, satisfying UV‐transparency, and good peak symmetries. These facts especially stand for the peak of perindopril, as a member of the group of proline‐containing moiety in its structure, often and easily prone to peak‐tailing, skewing, or splitting on the high‐performance liquid chromatography chromatograms, which is usually solved by using alkyl‐sulfonate base and ion pairs on higher concentrations of buffering salts with low pH values and with column temperatures increasing by 50°C–60°C. Linearity range was 0.002/004–0.5 mg/mL. The score for method AGREE was 0.73–0.77. The method development approaches, with all columns employing our concepts in mobile phases and experimental conditions, can cover the complete needs and demands of quality control departments and laboratories as simple high‐throughput analysis methods.

Validation of Commercial Formulation of Difenoconazole Using HPLC Equipped with Dad

Effective, selective, precise, and accurate liquid chromatographic analytical methods for the determination of difenoconazole fungicide in the formulation have been optimized and validated to meet the accreditation requirements, the performance characteristics of the analytical method were validated which is also one of the basic requirements of ISO Standard 17025:2017, for the rapid determination of difenoconazole by HPLC-DAD. The used method involves the extraction of the substances by sonication of the sample with acetonitrile, followed by dilution in acetonitrile, and direct injection on a liquid chromatography system. For the analysis LC system from Agilent Technologies 1290 Infinity II was used. Good separation was achieved on a Luna C18 column (3µm, 100°A, 3x150mm) with a guard column (C18 4x2.0mm I.D), an isocratic mobile elution consisting of acetonitrile and water acidified 0.075% with formic acid (85:15, v/v), at a flow rate of 0.7 ml/minute and UV detection at 220 nm. The column temperature was 25⁰C, injected volume was 1 μL. The analysis duration was 10 minutes (the retention time of difenoconazole and 4 – Hydroxybenzoic acid – methyl ester was 1.846 and 1.135 minutes, respectively. The linearity within the concentration range of 750-125 µg mL-1, with the internal standard at a concentration of 250 µg mL-1 with an average correlation coefficient (R2) of 0.9998. We have considered precision, repeatability, and selectivity in the validation.

Development and Validation of a High-Performance Liquid Chromatography Coupled With Ultraviolet Detection Method for Quantification of Bictegravir in Human Plasma.

To establish a method for determining the bictegravir (BIC) concentration in human plasma using high-performance liquid chromatography coupled with ultraviolet detection. The analysis was performed on a CLC-octadecylsilane column (150 × 6.0 mm, 5 μm) using a mixture of phosphate buffer and acetonitrile (62:38, v/v) as the mobile phase at the flow rate of 1.4 mL/min. The column temperature was maintained at 40°C. Using triamcinolone acetonide as the internal standard, 100 μL of plasma sample was extracted by methyl tert-butyl ether, followed by evaporating under nitrogen stream, redissolving with 100 μL mobile phase, and injection of 20-40 μL of supernatant into the chromatographic system. Ultraviolet detection was performed at 260 nm, and the total run time for each sample was 14 minutes. The method exhibited good linearity within the range from 0.10 to 10.0 mcg/mL (r = 0.9995, n = 5). The intraday and interday relative standard deviations for low-, medium-, and high-concentration quality control samples (0.20, 4.00, 8.00 mcg/mL) and the lower limit of quantification (0.10 mcg/mL) were 1.31%-6.20% (n = 10) and 1.18%-2.87% (n = 5), respectively. The intraday and interday accuracies were 100.53%-102.32% and 97.96%-103.84%, respectively. The extraction recovery rates ranged from 80.00% to 88.09% (n = 3). The stability tests showed that the BIC concentration changed by <15%. This study successfully established a high-performance liquid chromatography coupled with ultraviolet detection method for determining plasma BIC concentrations. This method is simple, selective, sensitive, and accurate, making it suitable for clinical monitoring and pharmacokinetic studies of BIC.