Elution Research Articles

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.

Simultaneous multi-targeted forensic toxicological screening in biological matrices by MRM-IDA-EPI mode.

The toxicologist ascertains drug assumptions in case of paediatric intoxications and death for overdose. The analytical approach consists of initially screening and consequently confirming drug positivity. We developed a toxicological screening method and validated its use comparing the results with a LC-MS/MS analysis. The method identifies 751 drugs and metabolites (704 in positive and 47 in negative mode). Chromatographic separation was achieved eluting mobile phase A (10mM ammonium formate) and B (0.05% formic acid in methanol) in gradient on Kinetex Phenyl-Hexyl (50 × 4.6mm, 2.6μm) with 0.7mL/min flow rate for 11min. Multiple Reaction Monitoring (MRM) was adopted as survey scan and, after an Information-Dependent Analysis (IDA) (threshold of 30,000 for positive and 1000 cps for negative mode), the Enhanced Product Ion (scan range: 50-700 amu) was triggered. The MS/MS spectrum generated was compared with one of the libraries for identification. Data processing was optimised through creation of rules. Sample preparation, mainly consisting of deproteinization and enzymatic hydrolysis, was set up for different matrices (blood, urine, vitreous humor, synovial fluid, cadaveric tissues and larvae). Cut-off for most analytes resulted in the lowest concentration tested. When the results from the screening and LC-MS/MS analysis were compared, an optimal percentage of agreement (100%) was assessed for all matrices. Method applicability was evaluated on real paediatric intoxications and forensic cases. In conclusion, we proposed a multi-targeted, fast, sensitive and specific MRM-IDA-EPI screening having an extensive use in different toxicological fields.

Characterization of Electrospray Ionization Complexity in Untargeted Metabolomic Studies.

The annotation of metabolites detected in LC-MS-based untargeted metabolomics studies routinely applies accurate m/z of the intact metabolite (MS1) as well as chromatographic retention time and MS/MS data. Electrospray ionization and transfer of ions through the mass spectrometer can result in the generation of multiple "features" derived from the same metabolite with different m/z values but the same retention time. The complexity of the different charged and neutral adducts, in-source fragments, and charge states has not been previously and deeply characterized. In this paper, we report the first large-scale characterization using publicly available data sets derived from different research groups, instrument manufacturers, LC assays, sample types, and ion modes. 271 m/z differences relating to different metabolite feature pairs were reported, and 209 were annotated. The results show a wide range of different features being observed with only a core 32 m/z differences reported in >50% of the data sets investigated. There were no patterns reporting specific m/z differences that were observed in relation to ion mode, instrument manufacturer, LC assay type, and mammalian sample type, although some m/z differences were related to study group (mammal, microbe, plant) and mobile phase composition. The results provide the metabolomics community with recommendations of adducts, in-source fragments, and charge states to apply in metabolite annotation workflows.

LC-MS/MS of isomeric N-and O-glycopeptides on mesoporous graphitized carbon column

BackgroundThe study of glycopeptides is associated with challenges regarding the microheterogeneity of different isomeric glycans occupying the same glycosylation sites in glycoproteins. It is immensely valuable to perform both qualitative and quantitative site-specific glycosylation analysis of glycopeptide isomers due to their link to several diseases. Achieving isomeric separation of glycopeptides is particularly challenging due to the low abundance of glycopeptides as well as inefficient ionization. Although some methods have demonstrated the isomeric separation of glycopeptides, a more efficient nanoflow-based stationary phase is needed for the isomeric separation of both N- and O-glycopeptides. ResultsIn this study, the separation of N- and O-glycopeptide isomers at 75 °C was achieved with an in-house packed 1 cm long mesoporous graphitized carbon (MGC) column. Different gradient compositions of the optimized mobile phase for separating permethylated glycans on MGC column were tested, and we observed efficient separation of N- and O-glycopeptide isomers at a gradient elution time of 120 min. After achieving the isomeric separation of sialylated glycopeptides from model glycoproteins derived from bovine fetuin, the separation of isomeric glycopeptides derived from asialofetuin, α-1 glycoprotein and human blood serum were also demonstrated. Furthermore, the developed method for the separation of isomeric N- and O-glycopeptide on MGC column showed high reproducibility over three months. We observed an average retention time shift of 1 min and consistent resolution of separated peaks throughout three months. Significance and noveltyMGC column can serve as an efficient tool for obtaining the isomeric separation of N- and O-glycopeptide from complex biological samples in future studies. This will enable a more profound understanding of the roles played by isomeric N- and O-glycopeptide in important biological processes and their correlations to various disease progressions.

Development of a simultaneous LC-MS/MS analytical method for plasma: 16 antipsychotics approved in Japan and 4 drug metabolites.

The increased risk of adverse drug reactions due to the concomitant use of antipsychotics is problematic in the treatment of schizophrenia. Therefore, the simultaneous analysis of their plasma concentrations is required. In this study, we developed a simultaneous liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for analyzing plasma antipsychotics approved in Japan for therapeutic drug monitoring (TDM) applications. First, we counted the prescriptions for 16 antipsychotics and concomitant drugs used at the Tohoku University Hospital. LC-MS/MS was used for the simultaneous analysis of 16 antipsychotics and four drug metabolites. This analysis was conducted using a combination of selected reaction monitoring mode and reversed-phase chromatography. Following the examination of the MS/MS and LC conditions, an analytical method validation test was conducted. The developed method was used to analyze plasma antipsychotic levels in patients with schizophrenia. One-third of the patients received treatment with multiple antipsychotics. Under LC-MS/MS conditions, LC separation was performed using a combination of a C18 column and ammonium formate-based mobile phases with a gradient flow. The calibration curves were optimized by adjusting the ion abundance, and 11 compounds met the criteria for intra- and inter-day reproducibility tests. Some stability test results did not meet these criteria; therefore, further investigation is required. The developed method permitted the measurement of all the plasma parameters, including concentrations above the therapeutic range. Therefore, this method may be useful in the daily TDM practice of antipsychotics.

Revolutionizing HIV Treatment: Pioneering Ultra Performance Liquid Chromatography for Emtricitabine, Dolutegravir, and Tenofovir Tablet Validation

An uncomplicated and precise method has come into existence for the simultaneous quantification of emtricitabine, dolutegravir, and tenofovir in solid dosage forms. The chromatographic analysis utilized a Hibar100 column (50×2.1 mm, 2 μm) with a mobile phase of 0.1% OPA and acetonitrile in a 60:40 v/v ratio at a constant flow rate of 1.0 mL/min, maintaining a temperature of 30°C. The perfected wavelength at 260.0 nm revealed retention times of 1.951, 1.180, and 1.584 minutes for dolutegravir, emtricitabine, and tenofovir, respectively.

Multicolumn Nanoflow Liquid Chromatography with Accelerated Offline Gradient Generation for Robust and Sensitive Single-Cell Proteome Profiling.

Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.

High Throughput LC-MS/MS Method for the Quantitation of Mefenamic Acid in Rat Plasma by Protein Precipitation Technique Using 96 Well Plate Format

An advanced and validated high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach has been established to accurately determine the concentration of mefenamic acid in rat plasma. This method utilizes mefenamic acid D4 as the internal standard (ISTD). The analyte and ISTD underwent protein precipitation in a 96-well plate format and were then separated on a BDS Hypersil C8 column (3 μm, 100 x 4.6 mm) using a precise isocratic mobile phase of 2 mM ammonium formate by 0.1% formic acid and acetonitrile (30:70 v/v). The detection was performed on a triple quadrupole mass spectrometer utilizing positive ionization consideration. The approach demonstrated a concentration range of 20.659 to 20067.772 ng/mL, thru relative regaining reaching from 69.1 to 74.3%. The inter-batch precision was excellent, with a coefficient of variation (%CV) of ≤ 7.8%. Additionally, the inter-batch accuracy at four different quality control-points ranged from 97.0 to 100.4%, demonstrating high levels of accuracy. This method has been thoroughly validated and is extremely specific, accurate, and precise. It is perfectly suited for high-throughput investigation of mefenamic acid in rat plasma, making it ideal for routine analysis.

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.

Simultaneous Quantification of Dutasteride and Silodosin Using a Stability-Indicating RP-HPLC Approach

An alternative stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) approach remained established to quantify silodosin (SLD) and dutasteride (DTS) in the mixture, validated per International Council of Harmonization (ICH) recommendations. A Shimadzu Model LC-2030 PLUS (IND) HPLC system, 231 nm detection wavelength, and 10 minutes run duration were used to create the method. A 75:25 mobile phase of methanol and water with 0.05% OPA was used at 1-mL/min. The concentrations of SLD (8–40 μg/mL) and DTS (0.5–2.5 μg/mL) were shown to be linearly related, with R2 values of 0.9998 for SLD and 0.9993 for DTS. Both of the drugs had accuracy and precision within 2% RSD. SLD and DTS mean recoveries were 97.5 to 102%. According to ICH criteria, degradation trials with acid, alkali, oxidizing agent, and heat assessed the approach’s stability. The established approach is fast, accurate, sensitive, and precise for routine SLD and DTS analysis from bulk as well as pharmaceutical dosage forms.

Development and Validation of HPTLC for the Determination of Gefitinib HCl in Tablet Dosage Form

A simple, high-performance thin-layer chromatography (HPTLC) method of gefitinib HCl in pharmaceutical formulation was developed. Separation was done on TLC aluminum plates precoated by silica gel 60GF254 (20 × 20 cm) with 200 μm layer depth. With UV detection set to 254nm, chromatography was conceded out by the mobile phase of dichloromethane and methanol (9:1, v/v). Linearity was found to range of 50 to 300 ng/spot and, the correlation coefficient was found to be 0.99 and the Rf value was found to be 0.46. The system’s suitability was considered to ascertain the performance of ascertaining quality performance of the developed chromatographic method. The method’s accuracy, precision, and specificity were all verified against the International Council of Harmonization (ICH) standards. Application of the approach to the determination of gefitinib in tablet form was successful.

A Comprehensive Report on the HPLC-ESI-MS/MS Method Development and Validation for Quantifying Cabotegravir in K3EDTA-Human Plasma, Employing Cabotegravir-D5 as an Internal Standard

Aiming to comprehend the plasma concentration of cabotegravir, a meticulously crafted bioanalytical method, aligned with the guidelines set by the US Food and Drug Administration (FDA), was devised and validated. This involved the utilization of cabotegravir D5 as an isotopic internal standard. The mobile phase consisted of methanol and 5 mM ammonium acetate in water, in an 80:20 ratio, v/v—Zorbax SB-C18 (50 × 2.1 mm, 5 μm) served as the chromatographic column. Cabotegravir and cabotegravir D5 were identified using proton adducts at m/z 409.20/370.20 and 409.20/144.8, respectively, employing the positive mode multiple reaction monitoring. Cabotegravir exhibited linearity within concentration ranges of 2 to 1000 ng/mL (r2 = 0.999). The recovery of cabotegravir from plasma by Liquid-liquid extraction yielded an average %CV of 7.71 at four quality control levels. Intra-day precision displayed accuracy of 100, 106, 97, 97, and 103%, respectively, whereas accuracy for inter-day precision was 100, 103, 98, 100, and 100% across LLQC, LQC, MQC-1, MQC-2, and HQC levels. The stability of cabotegravir remained consistent under diverse conditions, including five freeze-thaw cycles, benchtop, autosampler, and short-term, and long-term storage. This evaluation verifies that the method aligns with predefined acceptance limits and positions it as an indispensable tool in bioanalysis, significantly expanding its clinical utility.

An Innovative RP-HPLC Method for Simultaneous Estimation of Montelukast and Ebastine in Tablet Formulations: Development, Validation, and Stability Assessment

This study aimed to develop and validate an innovative reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of montelukast sodium (MNK) and ebastine (EBA) in tablet formulations. Utilizing a Shimadzu LC2010CHT system with a Phenomenex Luna C18 column, the mobile phase consisted of ammonium acetate buffer, acetonitrile, and methanol in a 12:55:33 ratio, with a flow rate of 1.0 mL/min and detection at 250 nm. Validation followed ICH guidelines, assessing linearity, accuracy, precision, robustness, and stability under various stress conditions. Both MNK and EBA showed excellent linearity within 16 to 24 μg/mL with correlation coefficients of 0.999. Accuracy was confirmed through recovery studies showing 98 to 102% mean recoveries. Precision was demonstrated with %RSD values below 2%. The method remained robust under slight variations in conditions, and stability studies indicated minimal degradation of both drugs. The developed RP-HPLC method proved rapid, precise, and robust for the simultaneous quantification of montelukast and ebastine in tablet formulations, demonstrating practicality and reliability for pharmaceutical quality control.

Адсорбционное извлечение аминов из водных растворов

Aquatic biological resources are an important part of the ecosystems of our planet, since they are involved in economic turnover as a source of food. The composition of water, including waste water from enterprises, has a direct impact on the state of aquatic biological resources. To extract and concentrate metals from solutions, hydrometallurgical processes are used – extraction and sorption. Their implementation is possible with the use of special substances – extractants and in a number of processes – sorption materials (Solvent Impregnated Resins (SIR’s) and Levextrel’s) with a mobile phase of the extractant, combining the properties of sorbents and extractants. Despite the advantages of the latter, such as high kinetic parameters, lack of swelling and high values of mechanical strength, they are characterized by the disadvantages of extraction methods – the entrainment of the extractant into the aqueous phase in dissolved and emulsion forms. Under static conditions, the possibility of purification of aqueous solutions containing primary or tertiary amine by adsorption on carbon materials (activated carbon NWC, carbon NWC-T modified with carbon nanotubes, CS-PTFE modified with polytetrafluoroethylene), as well as on styrene-divinylbenzene nonionic copolymer Porolas-T and a natural shell rock adsorbent was studied. The kinetics of adsorption of аmines Primene-JMT and TOA from aqueous solutions by these adsorbents has been studied by the method of a limited volume of solution. Of the known pseudo-first order, pseudo-second order and Elovich models, the pseudo-second order model describes kinetic data more adequately (R2>0.99). The values of the distribution coefficients and the rate constants of adsorption of Primene-JMT and TOA are determined. A block diagram of adsorption purification of aqueous solutions from amines is proposed, the implementation of which will reduce the toxicity of waste solutions and return some of the expensive extractant to circulation.

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.

Comparative study of UV spectroscopy, RP-HPLC and HPTLC methods for quantification of antiviral drug lamivudine in tablet formulation

BackgroundIn the current study, estimation of lamivudine (LMU) by UV spectroscopy, reverse-phase HPLC (RP-HPLC) and HPTLC methods in tablet formulation was developed, and comparative studies between the methods were investigated by analytical results and statistical test analysis of variance (ANOVA) to find out best method. In the UV spectral method, LMU was quantified at 271 nm absorption maxima using methanol as the solvent. In the RP-HPLC method, the Shimadzu C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) was employed for chromatographic separation. The mobile phase used consists of methanol: water (70:30 v/v) in an isocratic mode with a 1.0 mL/min flow rate. In the HPTLC method, the chromatogram was developed on a pre-coated plate of silica gel 60 F254 with a mobile phase composition of chloroform: methanol (8:2 v/v). The quantification was performed at an absorbance mode of 271 nm by densitometry. The methods were validated according to the International Conference on Harmonization (ICH) guideline Q2 (R1). The degradation conditions were employed as per ICH guidelines Q1A(R2) and Q1B which include acid, alkaline, neutral, thermal and photostability to determine the intrinsic stability of the drug in varied environmental conditions.ResultsLMU absorption maxima was found to be 271 nm. The retention time of LMU was 3.125 min, and the total analysis time was 5 min. The Rf value of LMU was 0.49–0.62. The methods were linear within 2–12 μg/mL range. The correlation coefficient (r2) for UV, HPLC and HPTLC was 0.9980, 0.9993 and 0.9988, and percent recoveries were calculated as 98.40–100.52%, 99.27–101.18% and 98.01–100.30%, respectively, with percentage relative standard deviation (RSD) less than 2% showing that methods were precise and accurate.ConclusionDeveloped UV, RP-HPLC and HPTLC methods are free from intervention caused by excipients present in tablets and thus can be used for regular quantitative analysis of LMU in tablet formulation. Based on analytical results and statistical tests, ANOVA, it is inferred that the HPLC method is best for LMU quantification tablet formulation due to its high reproducibility, good retention time and sensitivity; it has a higher percent recovery and has less analysis time, i.e., 5 min. The degradation peaks were well separated from the LMU peak indicating stability of the HPLC method.

Estimation of Imeglimin in Pharmaceutical Tablets by RP-HPLC

Objective: This study’s primary goal is to create a novel, sensitive and comprehensive reverse phase-high performance liquid chromatography (RP-HPLC) procedure. The computation of Imeglimin’s dose and dosage is the primary goal. Method: Chemicals and impurities were separated chromatographically using a C18 (AGILENT) chromatographic column. Methanol and 0.1% OPA are present in the mobile phase in a 40:60 v/v ratio. A 240 nm ultraviolet light detector was employed for the purpose of detection. With a retention time of 4.718 minutes, the results demonstrated the effectiveness of meglimin. After adjusting the flow rate to 0.7 mL/min, there was good separation. There are several imeglimin doses planned, ranging from 10 to 50 μg/mL. 101.46 to 101.12% in yield. Results: This method’s validation has good accuracy, sensitivity, precision, linearity, specificity, and robustness, and it satisfies the requirements of the International Conference on Harmonization. Conclusion: In conclusion, imeglimin was predicted and the construction method was isolated. In conclusion, it’s feasible for daily use.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Comparative pharmacokinetics of five primary constituents in Huai-hua powder: a study on normal rats and rats with ulcerative colitis.

The goal of this research was to develop a fast, reliable, and sensitive method to simultaneously quantify five key components of Huai-hua Powder (HHP) in rat plasma with genistein served as the internal standard. Furthermore, the established method was used to perform a comparative evaluation of the pharmacokinetic properties of HHP in normal rats and rats with ulcerative colitis (UC). Chromatographic separation was conducted using an ACQUITY HSS T3 column held at a constant temperature of 35°C, with acetonitrile and a 0.1% formic acid solution in water employed as the mobile phases. Multiple-reaction monitoring facilitated MS operation in positive-negative-ion-switching mode. The method's validation demonstrated exceptional linearity (with a correlation coefficient of r ≥ 0.9970), and the validation tests, encompassing precision within and between days, accuracy, recovery, matrix effect, and stability; all met the predefined acceptable criteria. The results revealed significant variations in the pharmacokinetic characteristics of the five components between normal and UC rats, suggesting altered drug metabolism rates and extents in the latter group. These findings offer crucial scientific insights into the potential clinical application of HHP, particularly in the context of treating UC.

Stability Indicating Force Degradation Study of Nintedanib in Bulk and Pharmaceutical Dosage Form

Assay of the nintedanib drug substance can be determined using a newly designed and verified reverse-phase high-performance liquid chromatography (RP-HPLC) method, even when degradation products from forced degradation experiments are present. By using gradient elution, the mobile phase was a mixture of 70% acetonitrile and 30% water by volume. Acid, base, peroxide, thermal, and photolytic degradation were among the stress conditions that the product was subjected to. During the thermal and photolytic breakdown processes, no extra contaminants were detected. Using validation criteria such as specificity, linearity, limit of quantitation (LoQ), accuracy, precision, robustness, and ruggedness, the developed technique was validated in accordance with International Council for Harmonization (ICH) recommendations. At a concentration of 12.4 ng/mL, the LoQ value was attained. The results showed good linearity (r2 > 1.00) across doses ranging from 2 to 10 μg/mL. Verification of recovery was done by adding concentrated solutions of 5, 10, and 15 μg/mL. So, newly developed RP-HPLC technology can separate Nintedanib from its main degradation products, and it can also estimate the drug substance’s concentration.