Current Pharmaceutical Analysis - Current Issue

Doxorubicin (DOX) causes lethal cardiotoxicity, which limits its clinical utility. The molecular mechanisms and effective strategies to combat its cardiotoxicity need further exploration. DT-010, a novel conjugate of danshensu (DSS) and tetramethylpyrazine (TMP), is considered a promising candidate for treating DOX-induced cardiotoxicity. In this study, we aimed to investigate the underlying molecular mechanisms of DOX-induced cardiotoxicity and the cardioprotective effects of DT-010.

Isobaric tags for relative and absolute quantitation (iTRAQ) in proteomics analysis was employed to analyze the differentially expressed proteins in DOX-injuried hearts. Gene ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to evaluated the potential mechanisms of DOX-induced cardiotoxicity. The effects of NCAM1 on DOX-induced cardiotoxicity in H9c2 cells, as well as the cardioprotection of DT-010 were assessed through NACM1siRNA transfection, cell viability assay, cell apoptosis staining, reactive oxygen species measurement, and western blotting.

Proteomics analysis revealed that several signaling pathways, including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, were involved in DOX-induced cardiotoxicity. NCAM1 is one of the significantly changed proteins. DT-010 treatment regulated NCAM1 protein expression. Silencing NCAM1 in DOX-treated H9c2 cells decreased cell viability, increased cell apoptosis and reactive oxygen species (ROS) generation, and attenuated the cardioprotective effects of DT-010. Furthermore, NCAM1 knockdown promoted p38 activation and inhibited the expressions of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and heme oxygenase-1 (HO-1) in DOX-treated cells.

These findings indicate a definite role of NCAM1 in DOX-induced cardiotoxicity and DT-010-exerted cardioprotection, which is mediated through the p38 and Sirt1/PGC-1α/HO-1 pathway.

A combination of empagliflozin and linagliptin in a fixed dosage was employed for treating individuals with a diagnosis of type 2 diabetes mellitus. A rapid, accurate, and sensitive liquid chromatography-tandem mass spectrometry method was devised and validated for simultaneous measuring empagliflozin and linagliptin levels in human plasma. This method provides a good analytical tool for bioequivalence and pharmacokinetic studies.

The separation was conducted employing a C8 column using a mobile phase consisting of acetonitrile (ACN, 2.5mM) and ammonium chloride (55:45). Optimal detection of the analytes and their deuterated internal standards was accomplished through electrospray ionization in the positive mode.

Validation of standard curve concentrations linearity was carried out within the ranges of 1.500 – 500.000 ng/mL for empagliflozin and 0.050 – 7.000 ng/mL for linagliptin. Both drugs showed intra-batch and inter-batch precision (CV%) of less than 3.7%. The stability of the drugs was confirmed under various storage conditions, proving suitability for routine laboratory analysis.

This validated method is appropriate for pharmacokinetic studies and large-scale analysis with high precision and accuracy.

Dorzolamide hydrochloride (DRZ) is a carbonic anhydrase inhibitor used to treat glaucoma and ocular hypertension. Drug-eluting contact lenses, such as Acuvue Theravision™ with Ketotifen, offer improved drug delivery and reduced side effects compared to eye drops. Drug-loaded nanoparticle-loaded contact lenses can sustain drug release and enhance comfort for extended wear.

High buffer concentration and low pH increase the risk of damage to silica-bonded columns. Therapeutic contact lenses face challenges related to critical lens parameters, including the estimation of drug incorporation and release due to interference of lens matrix leaching. There is currently no analytical method available for estimating DRZ in contact lenses.

The HPLC method, which was developed and validated using ICH Q2 (R1) criteria, used a C18 column (250 mm × 4.6 mm, 5 μm) as a stationary phase and methanol:water (70:30 v/v) as the mobile phase. The detecting wavelength was 253 nm. Moreover, to support the efficiency of the developed method, the marketed formulation of DRZ eye drops, drug purity, and loading in contact lenses were analysed. The method was also employed to determine the Critical Quality Attributes (CQAs) of therapeutic contact lenses and drug release and drug leaching during the sterilization process.

The developed HPLC method shows Rt for DRZ at 2.881 minutes with good linearity (r2 > 0.998) between 2-32µg/mL, precision (RSD < 2%), accuracy (Recovery > 99.5%), sensitivity, and specificity for quantifying DRZ in marketed formulations and therapeutic contact lenses. The developed method is devoid of any buffer or modifier in the mobile phase, making it safer for the stationary phase. This method mitigates the interference of lens matrix leaching, which induces an overestimation of DRZ. All the result for therapeutic contact lenses was found to be closely aligned with theoretically expected results, confirming the reliability of the developed HPLC method for therapeutic contact lenses.

This method is specific, accurate, and precise for quantifying DRZ in commercial formulations and newly developed therapeutic contact lenses. It effectively evaluates the critical quality attributes of these lenses, demonstrating their reliability for assessing their performance and ensuring quality in therapeutic applications.

Rapid tablet or capsule dissolution requires the tablet to disintegrate and dissolve at a higher rate, enhancing drug dissolution and bioavailability. Suitable disintegrants have shown an appreciable rate of disintegration or dissolution. Using factorial design for formulation to improve bioavailability is a key focus in pharmaceutical research to enhance dissolution.

Azelnidipine (Azp) tablets were formulated with Hydroxypropyl β-cyclodextrin (HβCD), β-cyclodextrin (βCD), and Kolliphor HS15 (HS15) to enhance solubility. A 2³ factorial design optimized the formulation, focusing on disintegration time (DT) and time for 90% dissolution (T90). Eight formulations (F1-F8) were prepared using the kneading method. Tablets were evaluated for physical properties, drug content, friability, dissolution, and drug-excipient interactions (FTIR and DSC). The optimal formulation (F9) was determined via desirability analysis.

Tablets showed acceptable Carr's index (CI), Hausner ratio (HR), and Angle of Repose (AR). Increasing βCD concentration decreased DT, enhancing water absorption and faster dissolution. βCD tablets had the lowest DT among the formulations, with F4 showing the best disintegration. Higher HS15 concentration also reduced DT, with F8 achieving the highest drug release (T90%) within 60 minutes. R² values ranged from 0.922 to 0.994, indicating high predictability. The optimal formulation had a desirability of 1.0, consisting of 3.523 mg HS15, 28.4 mg βCD, and 1.49 mg βCD, with a DT of 102 ± 1.13 seconds and 98% dissolution. FTIR and DSC confirmed no drug–excipient interactions.

Optimized super disintegrant concentrations and wet granulation techniques resulted in tablets with strong mechanical properties, rapid disintegration, and consistent drug content. Future research and in vivo studies should explore additional excipient combinations.

It is very important to assess the effects of NaOCl and ClO2 on dentine deproteination because these solutions are in contact with dentine during endodontic treatment and may affect the physical and chemical structure of dentine.

This study aimed to analyze the effects of sodium-hypochlorite (NaOCl) and chlorine- dioxide (ClO2) on the chemical structure of human dentine by Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy.

Fifteen human maxillary incisor roots were cut longitudinally into two parallel dentin discs being approximately 4x4x2 mm in size. 30 samples were randomly allocated to 6 groups treated with 5.25% NaOCl or 0.014% ClO2 (1, 5, or 10 minutes); self-control was used. The effect of solutions on the organic and inorganic components of the radicular dentine surface was analyzed using the amide:phosphate ratio and carbonate:phosphate ratio. The intragroup differences (paired t-test) and intergroup differences (one-way analysis of variance and Tukey’s posthoc test) were analyzed.

The intragroup comparisons showed the amide:phosphate ratio to be higher at all times at ClO2. NaOCl caused a decrease in the amide:phosphate ratio at 10 minutes (p<0.05). The intergroup comparison showed that NaOCl caused a greater decrease in amide:phosphate at all times compared to ClO2 (p<0.05). All comparisons demonstrated no significant difference in the carbonate:phosphate ratio (p>0.05).

Considering the results of this study, it is recommended to avoid prolonged exposure to minimize NaOCl-induced dentine deproteination. It should also be taken into account that ClO2 increases the amide:phosphate ratio in radicular dentine, and this effect is advantageous in clinical use for collagen structure, contrary to the negative impact of NaOCl.

Developing a simple HPLC method requires an expansive array of literary evidence and experimental routines to perceive the nature of a drug and eventually determine the specific mobile phase and column to be used for attaining better results.

The study aimed to develop and optimize a new, simplified, robust, and sensitive method for the determination of cilostazol in tablets by high-performance liquid chromatography using a Box Behnken design.

The chromatographic separation was carried out on an ODS C18 (4.6 X 250mm and 5µm) column with acetonitrile and methanol (25:75% v/v) at an effluent flow rate of 1 mL/min and detected at 257 nm.

The method was found to be linear in the concentration range of 10-50 µg/mL, and the correlation coefficient was found to be 0.988, and the recovery of cilostazol was 98.16%. The optimized method validated as per ICH Q2A guidelines was found to be accurate, precise, robust, and stable.

This research thus throws light on the implementation of statistical multivariate analysis techniques used for drug analysis.

Pirtobrutinib is a novel non-covalent BTK inhibitor used to treat adult patients with relapsed/refractory mantle cell lymphoma and B-cell leukemias. The mechanism of action involves binding and inhibition of Bruton's tyrosine kinase (BTK).

The main goal of the current work was to create a selective liquid chromatographic method (RP-HPLC) that is easy to use, accurate and exact for quantifying Pirtobrutinib and its degradation products, thereby seeking insight into the drug’s degradation behaviour.

Using an isocratic mode and a 50:50 mixture of acetonitrile and buffer solution (0.1% orthophosphoric acid) as the mobile phase, a High Performance Liquid Chromatographic System with a PDA detector and an X-Bridge Phenyl column (150 x 4.6mm, 3.5µm) at a flow rate of 1.0 ml/min was able to achieve good chromatographic separation. At 219 nm, the detection was carried out. A retention time of 2.271 minutes was discovered. To ascertain the drug's degrading properties and stability, forced degradation tests were carried out, leading to the development of the RPHPLC method. The chemical structures of the degradation products were clarified and their fragmentation mechanisms were suggested using LC-MS.

The suggested approach demonstrated a linearity within the 25-150% (2.5 to 15μg/mL) concentration range, with a correlation coefficient of 0.9999. The precision of the system (measured by % RSD=0.49) and the method (measured by % RSD=0.86) were all within the acceptable limits set by ICH guidelines, with % RSDs less than 1% and less than 2% for system precision and method precision, respectively. The LOD was 0.3μg/mL, and the LOQ was1μg/mL. Pirtobrutinib underwent rigorous tests for forced degradation under the specified conditions outlined in ICH Q1 (R2) guidelines. Pirtobrutinib was primarily broken down in acidic, alkaline, peroxide, and thermal conditions. It was found to remain stable under reduction, photolytic, and hydrolytic conditions. Through LC-MS analysis, the chemical structures of the resulting degradation by-products were identified, along with the proposed pathways of their fragmentation.

The current study gives insight into Pirtobrutinib’s degradation behaviour. Pirtobrutinib was stable in reduction, photolytic, and hydrolytic conditions but degraded more readily in acidic, alkaline, peroxide, and thermal environments. The degradation products were characterized as 4-carbamoyl-5-chloro-3(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-4,5-dihydro-1H-pyrazol-5-aminium (acid impurity, DP1), N-(4-(4,5-diamino-1H-pyrazol-3-yl)benzyl)-5-fluoro-2-hydroxybenzamide (alkali impurity, DP2), 5-amino-3-(4-((s-fluro-2-methoxybenzamido)methyl))-4,5dihydro-1H-pyrazol-5-aminium (peroxide impurity, DP3) and N-(4-(4-acetyl-5-amino-4,5-dihydro-1H-pyrazol-3-yl)benzyl)-5-fluoro-2-methoxybenzamide compound with λ¹-oxidane (1:1) (thermal impurity, DP4) and their fragmentation pathways were proposed. This study presents the first ever reported method for the quantification of Pirtobrutinib. It ensures precise quantitation of Pirtobrutinib, a new Bruton's Tyrosine Kinase inhibitor, and its degradation products. The method's enhanced sensitivity and regulatory compliance ensure its consistent use in the quantification of Pirtobrutinib.

Since the International Olympic Committee (IOC) was established, sports doping control analyses have revealed a high rate of positive cases for cannabinoids. Cannabinoids were banned in all sports where they were used in-competition as per the Prohibited List published annually. Further, it was also included in the threshold drug category. Consequently, developing a reliable method for urine Cannabinoids metabolite quantification plays a pivotal role in sports dope testing.

This work aimed to develop and validate a reliable, cost-effective, robust gas chromatography-tandem mass spectrometry method for detecting (−)-11-nor-9-Carboxy-Δ9-THC component in human urine samples, in compliance with ICH and WADA guidelines.

The sample preparation was done by enzymatic hydrolysis for deconjugation, further proceeded with solid phase extraction (SPE), liquid-liquid extraction (LLE), and using an XAD2 column, and N-methyl trimethylsilyl trifluoroacetamide (MSTFA) for derivatization.

The linearity was obtained in a range of 50–300 ng/mL, and the correlation coefficient was found to be higher than 0.99. Throughout the entire validation study, the difference in Retention Time (RT) for the analyte, including the Internal Standard (IS), was shown to be less than 1.0%. The quantification limit (LOQ) was calculated at a level of 50 ng/mL in human urine samples for the 3 most abundant ion transitions. The detection limit (LOD) was established at 4 ng/mL.

The accuracy, precision, linearity, recovery, quantification limit, and selectivity by GC-MS/MS technique were found acceptable and well satisfactory while following the ICH guidelines. The developed method has been proven to be fit for purpose in accordance with the enforced Guidelines of WADA.

Gardeniae fructus (Zhi-Zi) is the dry ripe fruit of the plant Gardenia jasminoides Ellis (Rubiaceae), which can be used as both food and medicine. Geniposide, a key constituent of Gardeniae fructus, serves as an indicator component for evaluating the quality of Gardeniae fructus. Traditionally, the quantification of geniposide in Gardeniae fructus is achieved through High-performance Liquid Chromatography (HPLC)-based methods.

The present study aimed to introduce a rapid approach to quantifying geniposide content in Gardeniae fructus along with validating its effectiveness.

The experiments involved finding a suitable deuterium solvent, Internal Standard (IS), specific peak, and Nuclear Magnetic Resonance (NMR) parameters for quantitation, and validating specificity, accuracy, precision, and stability.

The results have indicated that methanol-d4 as a solvent has facilitated excellent signal separation in the proton Nuclear Magnetic Resonance (¹H NMR) spectroscopy, with trimethyl 1,3,5-benzenetricarboxylate emerging as the ideal IS. The specific signal at δ 7.45, corresponding to H-3 in geniposide, has been identified as the optimal peak for integration. The application of distinctive signals from the ¹H NMR spectroscopy has allowed for the precise quantification of geniposide in Gardeniae fructus.

This study has suggested using ¹H NMR to quantify geniposide in Gardeniae fructus to be a viable alternative to HPLC-based methods, providing a suitable approach for quality control of Gardeniae fructus.

Vildagliptin, Dapagliflozin and Metformin Hydrochloride are anti- hyperglycemic agents. Anti-hyperglycemic agents are drugs that stimulate insulin secretion and lower the glucose level in the blood. They were investigated as a medication for Diabetes Mellitus (DM). Various studies have reported the HPLC, HPTLC, LC/MS methods for the estimation of individuals of VILD, DAPA and METF. However, until date stability indicating HPLC analysis method has not been reported for the estimation of VILD, DAPA and METF in combined glucagon secretion in a glucose-dependent manner.

The objective of present work was to develop and validate the stability of RP-HPLC method for the estimation of Vildagliptin (VILD), Dapagliflozin (DAPA) and Metformin Hydrochloride (METF) in combined dosage form.

In this research work, High Performance Liquid Chromatography (HPLC) was used for validation in the chromatography. In the HPLC, Methanol and Potassium Dihydrogen phosphate buffer (85:15 v/v) were used as mobile phases. HPLC analysis was performed at 230 nm. The method was validated with different parameters such as linearity, precision, accuracy, specificity, and robustness, limit of detection (LOD) and limit of quantitation (LOQ). The RF values of VILD, DAPA and METF were 6.24 ± 0.05 min, 8.34 ± 0.05 min. and 3.68 ± 0.05 min, respectively. The accuracies of drug recovery were 0.6398%,0.3680%, 0.6398% and over the range of 1–4 µg/ml, 20-80 µg/ml and 15-85 µg/ml of VILD, DAPA and METF, respectively.

Degradation products found in stress conditions did not interfere with the detection of VILD, DAPA and METF; therefore, the proposed technique can be considered stable. Significant degradation products of VILD, DAPA and METF were obtained in an acid at METF at 2.05 ± 0.05 min. (MDP-1) and degradation product for oxidation of METF at 2.11 ± 0.05 min. (MDP-2). When alkaline hydrolysis was conducted, photolytic and thermal conditions were applied alone or in combination with METF, no degradation product of VILD, DAPA and METF was found.

The developed and validation method was satisfactorily applied for the analysis of pharmaceutical preparations and proved specific and accurate for quality control of the cited drugs in their combined dosage form.

The aim of this study is to develop a sensitive, specific, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method and validate it to determine the Roflumilast in rat plasma.

This study was aimed at developing a simple HPLC method for the detection of Roflumilast in rat plasma after ingestion of nanoparticles into the rat. A bioanalytical method was developed and validated.

The drug sample (Roflumilast) was eluted isocratically using a mobile phase of Ammonium acetate buffer (pH = 7.0), acetonitrile, and methanol (20:40:40, v/v/v) made up the mobile phase on a Phenomenex C-18 (150mm x 4.68mm) pore size 5-micron analytical column. The drug was quantitatively determined at a UV wavelength of 254 nm using a standard calibration curve spanning the range of 1.5, 3.0, 4.5, 6.0, 7.5 μg/mL^-1.

The results showed that the limits of quantitation (LOQ) and detection (LOD) were 0.18 μg/ml and 0.54μg/ml, respectively. For both the intra-day and inter-day analysis, relative standard deviation (% RSD) was less than 2%, and the drug’s percentage accuracy was between 96% and 98%.

As per the established protocols, the developed method was quantitatively assessed for its intended use in terms of specificity, linearity, accuracy, precision, robustness, and sensitivity analysis. Roflumilast-loaded nanoparticles were effectively used to determine the drug entrapment efficiency using the currently validated RP-HPLC method. Besides, highly efficient drug extraction from plasma was attained, and mobile phase composition was suitable to the pharmacokinetic study of Roflumilast-loaded nanoparticles in rats.

Imatinib is a first-line medicine for chronic myeloid leukemia (CML) and gastrointestinal mesenchymal stromal tumors (GIST). Co-administration of nifedipine may lead to drug-drug interactions that affect the clinical efficacy of imatinib. Imatinib and nifedipine are substrates for the cytochrome enzyme CYP3A4. This study aimed to research the pharmacokinetic effect of nifedipine on imatinib and its metabolism N-desmethyl imatinib in rats.

Twenty healthy SD rats were randomly divided into two groups. The control group was administered imatinib by gavage for 14 days, and the experimental group was co-administered imatinib and nifedipine by gavage for 14 days. The plasma concentrations of imatinib and N-desmethyl imatinib in rats were determined by ultra-performance liquid chromatography-mass spectrometry.

The MRT0-∞ and Tmax of imatinib in the experimental group differed significantly from the control group after a single dose (P < 0.05, 95% CI). Tmax and t1/2z of imatinib and AUC0-∞ and Tmax of N-desmethyl imatinib were also obviously different between the two groups after multiple doses (P < 0.05, 95% CI).

The study showed that nifedipine might inhibit the imatinib metabolism after single- dose administration, but nifedipine did not significantly impact imatinib metabolism after multiple-dose administration.