Reversed-phase Ultra-performance Liquid Research Articles

Uncommon opsin’s retinal isomer is involved in mammalian sperm thermotaxis

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts—tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

A NEW RP-UPLC METHOD FOR THE SEPARATION AND SIMULTANEOUS QUANTIFICATION OF DORZOLAMIDE HCl AND TIMOLOL MALEATE

Objective: This investigation demonstrates a stability-indicating and reliable “reverse-phase ultra-performance liquid chromatography” method to simultaneously quantify timolol maleate and dorzolamide HCl in the pharmaceutical dosage form. Methods: Successful separation was accomplished using Phenyl column (100 mm x 2.1 mm, 1.7μm) with isocratic type of elution using mobile phase containing Acetonitrile+Ammonium Formate buffer (30:70), respectively with 0.2 ml/min flow rate. The wavelength sensor was attuned at 266 nm to quantify timolol maleate and dorzolamide HCl. Results: Dorzolamide HCl and timolol maleate peaks were eluted with fine resolution at retention times 0.7 min and 1.5 min, respectively. In the 55.75-334.5 μg/ml and 6.25-37.5 μg/ml concentration ranges for dorzolamide HCl and timolol maleate, the calibration graphs were linear, with regression coefficients of 0.99997 and 0.99991, respectively. The suggested ultra-performance liquid chromatography approach has been shown as sensitive, precise, robust, accurate, specific and stability, indicating through the resolution of dorzolamide HCl and timolol maleate from its degradation-based compounds. Conclusion: The established ultra-performance liquid chromatography technique was effectively extended to the evaluation of dorzolamide HCl and timolol Maleate in the pharmaceutical dosage form and the test results appeared satisfactory.

Stability-Indicating UPLC-PDA-QDa Methodology for Carvedilol and Felodipine in Fixed-Dose Combinations Using AQbD Principles

The development of analytical procedures, in line with the recent regulatory requirements ICH Q2 (R2) and ICH Q14, is progressing, and it must be able to manage the entire life cycle of the methodology. This is also applicable to and especially challenging for combinations of drug substances and dosage form. A reliable and efficient, stability-indicating, MS-compatible, reverse-phase ultra-performance liquid chromatographic (UPLC®) method was developed for the determination of carvedilol and felodipine in a combination oral dosage form. The development of the method, performed using analytical quality by design (AQbD) principles, was in line with the future regulatory requirements. Furthermore, the fixed-dose combination dosage forms are a clear solution to the polypharmacy phenomenon in the elderly population. The main factors evaluated were the mobile phase buffer, organic modifier, column, flow, and column temperature. The optimum conditions were achieved with a Waters Acquity HSS T3 (100 × 2.1 mm i.d., 1.8 µm) column at 38 °C, using ammonium acetate buffer (5 mM, pH 4.5) (Solution A) and MeOH (Solution B) as mobile phases in gradient elution (t = 0 min, 10% B; t = 1.5 min, 10% B; t = 12.0 min, 90% B; t = 13.0 min, 10% B; t = 15.5 min, 10% B) at a flow rate of 0.2 mL/min and UV Detection of 240 and 362 nm for carvedilol (CAV) and felodipine (FLP), respectively. The linearity was demonstrated over concentration ranges of 30–650 µg/mL (R2 = 0.9984) (CAV) and 32–260 µg/mL (R2 = 0.9996) (FLP). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability, and fixed-dose combination dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with improved knowledge, leading to high-quality data, lower operation costs, and minimum regulatory risk. Furthermore, this work paves the way for providing a platform of robust analytical methods for the simultaneous quantification of innovative on-demand new dose combinations.

Isolation, characterization, identification and quantification of 6-F oxyphenisatin dipropionate, a novel illegal additive, from a fruit-flavored jelly

ObjectiveThis study is aimed to screen, identify and detect illegal additives from healthcare products which claim or imply to have weight-loss effects. MethodUltra-high performance liquid chromatography-quadruple-time-of-flight mass spectroscopy (UPLC-Q-TOF/MS) was employed to perform non-targeted screening of illegal additives from a total of 26 batches of healthcare products with weight-loss effects. A novel oxyphenisatin dipropionate analog was discovered in a fruit-flavored jelly that was not clearly labeled as containing added drugs. After being separated and purified by silica gel column chromatography, the analog was unambiguously characterized by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopies. The molecular structure of the analog was finally identified by comparing the spectra of the analog with those of suspected candidates prepared by de novo synthesis strategy. Thereafter, a sensitive and precise reversed phase ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method was developed and verified for the determination of the analog in 15 batches of real samples. ResultsIn the MS/MS spectra, the signal intensity of mass/charge ratios (m/z, 242 and 214) of the novel analog fragments was highly similar to that of mass/charge ratios (m/z, 224 and 196) of oxyphenisatin dipropionate fragments. Additionally, the 1D NMR spectrum of the analog was completely consistent with that of one of the suspected candidates prepared by the de novo synthesis strategy. Based on the above analysis, the structure of the analog was determined as 3,3-bis[4'-(propionyloxy)phenyl]-6-fluoro-2-oxoindoline, which was briefly named 6-F oxyphenisatin dipropionate. A developed quantitative method showed good linearity (R2 > 0.999) in a concentration range of 1.0–100 μg/mL. The limits of detection (LOD) and quantification (LOQ) for the analog was 3 mg/kg and 10 mg/kg, respectively. The average recoveries of the analog from spiked three different matrix samples in low (1 time of LOQ), medium (2 times of LOQ), and high (10 times of LOQ) concentrations were varied from 93.9 % to 107.8 % with a precision of 0.03–1.56 %. Results of quantitative analysis in 15 batches of healthcare products revealed that the content of 6-F oxyphenisatin dipropionate in a fruit-flavored jelly and a solid beverage was 118 mg/kg and 330 mg/kg, respectively. ConclusionIn terms of its structure, 6-F oxyphenisatin dipropionate replaces hydrogen atom by the fluorine atom at position 6 on the indolinone fragment in oxyphenisatin dipropionate. To our best knowledge, 6-F oxyphenisatin dipropionate has never been detected as an illegal additive in foods. Such illegal addition of the analog to foods is more concealing, thus the supervision and testing departments should attach great importance to its application in food markets.

Development and validation of a stability-indicating analytical method for simultaneous determination of sodium phenylbutyrate and taurursodiol in bulk and formulation using reverse phase ultra-performance liquid chromatography

Background Amyotrophic Lateral Sclerosis, often known as Lou Gehrig’s disease, is typically treated with a combination therapy that includes both sodium phenylbutyrate (SPB) and taurursodiol (TRS). Objectives To assess both SPB and TRS in bulk and their dosage form concurrently, a stability-indicating analytical method was developed and validated using Ultra-Performance Liquid Chromatography. Patients and methods The chromatographic separation was carried out on a Waters C18 Column, with dimensions of 150×4.6 mm i.d. and a particle size of 2 μm. A mobile phase consisting of a phosphate buffer pH 2.5 with methanol in the ratio of 45 : 65 v/v was, then delivered at a flow rate of 1 ml/min. Detection of the analytes occurred at 285 nm using a photo diode array detector. An auto sampler injected a 10 μl sample into the column, and the column was maintained at a temperature of 30°C. Results and conclusion SPB and TRS were eluted at 1.483 and 2.492 nm, respectively. Linearity was established in the range of 567–1701 μg/ml for SPB and 189–567 μg/ml for TRS. The robustness of the method was assessed by intentionally modifying parameters such as flow rate, detection wavelength, and column temperature. Furthermore, studies on forced degradation under various stress conditions, including acid, base, peroxide, heat, and ultra violet exposure, indicated the method’s capability to identify stable materials. In summary, the developed analytical approach for simultaneously determining SPB and TRS in bulk and their formulation was found to be specific, accurate, precise, and reliable.

Plasma metabolites distinguish dementia with Lewy bodies from Alzheimer's disease: a cross-sectional metabolomic analysis.

In multifactorial diseases, alterations in the concentration of metabolites can identify novel pathological mechanisms at the intersection between genetic and environmental influences. This study aimed to profile the plasma metabolome of patients with dementia with Lewy bodies (DLB) and Alzheimer's disease (AD), two neurodegenerative disorders for which our understanding of the pathophysiology is incomplete. In the clinical setting, DLB is often mistaken for AD, highlighting a need for accurate diagnostic biomarkers. We therefore also aimed to determine the overlapping and differentiating metabolite patterns associated with each and establish whether identification of these patterns could be leveraged as biomarkers to support clinical diagnosis. A panel of 630 metabolites (Biocrates MxP Quant 500) and a further 232 metabolism indicators (biologically informative sums and ratios calculated from measured metabolites, each indicative for a specific pathway or synthesis; MetaboINDICATOR) were analyzed in plasma from patients with probable DLB (n = 15; age 77.6 ± 8.2 years), probable AD (n = 15; 76.1 ± 6.4 years), and age-matched cognitively healthy controls (HC; n = 15; 75.2 ± 6.9 years). Metabolites were quantified using a reversed-phase ultra-performance liquid chromatography column and triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, or by using flow injection analysis in MRM mode. Data underwent multivariate (PCA analysis), univariate and receiving operator characteristic (ROC) analysis. Metabolite data were also correlated (Spearman r) with the collected clinical neuroimaging and protein biomarker data. The PCA plot separated DLB, AD and HC groups (R2 = 0.518, Q2 = 0.348). Significant alterations in 17 detected metabolite parameters were identified (q ≤ 0.05), including neurotransmitters, amino acids and glycerophospholipids. Glutamine (Glu; q = 0.045) concentrations and indicators of sphingomyelin hydroxylation (q = 0.039) distinguished AD and DLB, and these significantly correlated with semi-quantitative measurement of cardiac sympathetic denervation. The most promising biomarker differentiating AD from DLB was Glu:lysophosphatidylcholine (lysoPC a 24:0) ratio (AUC = 0.92; 95%CI 0.809-0.996; sensitivity = 0.90; specificity = 0.90). Several plasma metabolomic aberrations are shared by both DLB and AD, but a rise in plasma glutamine was specific to DLB. When measured against plasma lysoPC a C24:0, glutamine could differentiate DLB from AD, and the reproducibility of this biomarker should be investigated in larger cohorts.

The development and validation of a stability indicating Rp-UPLC method for the simultaneous estimation of clarithromycin, amoxicillin, and vonoprazan in a physical mixture

A novel technique was developed for simultaneous quantification of clarithromycin (CLA) amoxicillin (AMO), and vonoprazan (VON) in a mixture using the reverse phase ultra-performance liquid chromatography (RP-UPLC) technique and validated as per International Council for Harmonization (ICH) guidelines as there was no literature published for its estimation by UPLC. The method was developed using an acquity UPLC system from waters corporation with Hibar Bis phosphonate C18 column (100 × 2.1 mm, 2 μm) at 35°C and tunable ultra-violet detector (TUV) with detection wavelength at 210 nm, has a run time of below 3 minutes. The mobile phase proportion of 60:40 of 0.1 N monobasic potassium phosphate buffer (pH 3.8) and acetonitrile at a flow velocity of 0.2 ml/ minute was utilized. Linearity was observed for CLA, AMO, and VON between the concentration ranges of 25–150, 25–150, and 1–6 μg/ml, respectively, and R2 was 0.999 for CLA, AMO, and VON. Accuracy and precision were within 2% of the coefficient of variation (RSD) for all drugs. The observed mean percentage recoveries for the CLA, AMO, and VON were determined to be 99.74%, 99.07%, and 99.8%, respectively. The stability of the approach was assessed using degradation studies by exposing it to acid, alkali, oxidizing agent, heat, Ultra Violet (UV) light, and water as per ICH guidelines.

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of >75%.

Determination of Clorsulon and its related substances in commercial bulk drug substance batches by a rapid ion-pair UPLC method

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H2O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific.

Development and Validation of a New Reverse Phase Ultra Performance Liquid Chromatography Method for the Estimation of Related Substances of the Anticancer Drug Zanubrutinib and Characterization of its Degradants Using Liquid Chromatography Mass Spectrometry

Development and Validation of a New Reverse Phase Ultra Performance Liquid Chromatography Method for the Estimation of Related Substances of the Anticancer Drug Zanubrutinib and Characterization of its Degradants Using Liquid Chromatography Mass Spectrometry

Isolation and Structural Characterization of a major degradation product of Ambrisentan using Q-TOF-MS, NMR and FTIR

Ambrisentan, an endothelin receptor antagonist, is subjected to forced degradation under neutral hydrolytic conditions, leading to a potential unknown impurity. This neutral degradation product (DP) has been detected using reverse-phase ultra-performance liquid chromatography (UPLC), where it has eluted at 1.24 relative retention time to the Ambrisentan peak. DP was isolated using semi-preparative HPLC from the degradation mixture. Later, the structure of DP was elucidated using high-resolution mass spectrometry, 2D Nuclear magnetic resonance spectroscopy (NMR), and Fourier transformed infrared spectroscopy (FTIR) spectroscopic techniques and characterized as 2-(2,2- diphenylvinyloxy)-4,6-dimethylpyrimidine.

Characterization of pneumococcal serotype 7F in vaccine conjugation

Streptococcus pneumoniae is a highly invasive bacterial pathogen that can cause a range of illnesses. Pneumococcal capsular polysaccharides (CPS) are the main virulence factors that causes invasive pneumococcal disease (IPD). Pneumococcal CPS serotype 7F along with a few other serotypes is more invasive and likely to cause IPD. Therefore, 7F is a target for pneumococcal vaccine development, and is included in the two recently approved multi-valent pneumococcal conjugated vaccines, i.e. VAXNEUVANCE and PREVNAR 20.To support process and development of our 15-valent pneumococcal conjugated vaccine (PCV15), chromatographic methods have been developed for 7F polysaccharide and conjugate characterization. A size-exclusion chromatography (SEC) method with UV, light scattering and refractive index detections was employed for concentration, size and conformation analysis. A reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was used for analysis of conjugate monosaccharide composition and degree of conjugation. The collective information obtained by these chromatographic analysis provided insights into the pneumococcal conjugate and conjugation process.Graphical

Improved Stability-Indicating RP-UPLC Method for the Levamisole Hydrochloride Assay and Estimation of Its Related Compounds.

Levamisole hydrochloride (LVM) is an anthelmintic drug substance with immunomodulatory and anticancer activities. LVM has also found usage as a cutting agent in street cocaine. This study was aimed to develop and validate an alternative and improved stability-indicating reversed-phase ultraperformance liquid chromatography (RP-UPLC) method for the determination of LVM and the estimation of its related compounds. The UPLC method for the assay was optimized in terms of organic solvents consumed, pH, column temperature, and flow rate. Determination of LVM and its related compounds was performed using a gradient elution on a Waters ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm i.d., 130 Å). The column temperature was maintained at 35°C. Mobile phase A was composed of aqueous 5 mM ammonium hydroxide, and mobile phase B was composed of acetonitrile. All the analytes were monitored by UV detection at 215 nm with a flow rate of 0.7 mL/min. The total runtime of the method with column equilibration is 4.0 min. The developed method met all the acceptance criteria of the current International Council for Harmonization [ICH Q2 (R1)] guidelines. The method was tested in terms of specificity, linearity (R2 > 0.999), limit of detection (LOD; 0.06 μg/mL), limit of quantitation (LOQ; 0.2 μg/mL), accuracy, precision, and robustness. With a short analysis time (<2.5 min) and reduced consumption of organic solvents, the proposed method is considered a greener alternative to conventional chromatographic methods. An alternative and improved UPLC method was successfully developed and validated in accordance with the ICH guidelines for the determination of LVM and the estimation of its related compounds. Due to its high degree of selectivity, speed, and accuracy, the developed method can significantly benefit the end-users with laboratory efficiency and throughput during routine analysis of production batches and stability monitoring of LVM-related drug products.

UHPLC-MS-MS Determination of THC, CBD, and their Metabolites in Whole Blood of Light Cannabis Smokers.

"Light cannabis" is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration lower than 0.2% and variable cannabidiol (CBD) content. In this study, we aimed to assess the time-courses of THC and metabolites (11-nor-9-carboxy-THC and 11-hydroxy-THC) and CBD and metabolites (CBD-7-oic acid, 7-hydroxy-CBD, 6α-hydroxy-CBD, and 6β-hydroxy-CBD) in whole blood of 10 healthy participants after smoking one or four light cannabis cigarettes (0.16% THC and 5.8% CBD). Blood samples were collected 0.5 to 4 h after administration. Blood analysis was performed by reversed-phase ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) in multiple reaction monitoring mode, after glucuronide hydrolysis and liquid-liquid extraction in basic and acidic conditions. The method was validated following the most recent guidelines in toxicology: the method was linear, accurate, precise, and sensitive (lower limits of quantification ranged from 0.005 to 0.01 ng/mL); carryover, matrix effect, recovery, process efficiency, and dilution integrity were also assessed. As previously reported, the main metabolites of THC were THC-COOH, then 11-OH-THC, and the main metabolites of CBD were 7-OH-CBD, then 7-COOH-CBD. The time of first collection, which likely occurred after the maximal concentration of most of the analytes, and the short monitoring time, up to 4 h after smoking, limited the evaluation of the pharmacokinetic parameters.

Development of a rapid and validated stability-indicating UPLC-PDA method for concurrent quantification of impurity profiling and an assay of ipratropium bromide and salbutamol sulfate in inhalation dosage form

The objective of the proposed work was to develop a rapid and new reverse phase ultra-performance liquid chromatographic (RP-UPLC) method for the simultaneous quantification of related impurities of ipratropium bromide and salbutamol sulfate in the combined inhalation dosage form. Herein, the chromatographic separation was achieved on Acquity BEH C18 (100mm×2.1mm, 1.7μm) column by following gradient elution of solvent A as 2mM potassium dihydrogen phosphate with 0.025% of 1-pentane sulphonic acid sodium salt (pH 3.0 buffer) and solvent B as pH 3.0 buffer, acetonitrile and methanol in the ratio of (32:50:18, v/v/v) at a flow rate of 0.3mL/min. The samples were detected and quantified at 220nm. To prove the stability-indicating potential of the method, forced degradation studies were performed using acidic, basic, oxidative, thermal, and photolytic conditions. After sufficient exposure, the resultant solutions were injected and found that all degradants and impurities formed during stress studies were well separated from each other and from the main peak compounds. The performance of the method was validated according to the present ICH Q2 (R1) guidelines. The method has good linearity (r≥0.999) and consistent recoveries were obtained with a range of 91.3-108.8% for all compounds. The % RSD obtained for the precision experiments was less than 5% and also there is a good sensitivity (LOQ≤0.5μg/mL) for all compounds. The intended method proved its applicability and that it can be beneficial to pharmaceutical industries for quick quantification of related impurities and assay in quality control department for analysis of ipratropium bromide and salbutamol sulfate inhalation dosage form.

A Novel Stability Indicating Reversed Phase Ultra Performance Liquid Chromatography Method for Estimation of Asciminib its Bulk and Tablet Formulation

The key objective of the proposed study was to creat and validate an economical, perceptive, specific, reliable and simple reversed phase ultra-performance liquid chromatography (RP-UPLC) method with better response was anticipated for the investigation of asciminib bulk powder and its tablet form. UPLC system (WATERS, Model-2695) connected with PDA (Model-2996) detector was opted to made the present method. To separate asciminib proficiently, method conditions such as C18 column (50 x 4.6 mm, 1.7 m), mobile system of trifluoro aceticacid (TFA) in water (0.1%) and acetonitrile in 75:25 v/v, 0.3 mL/min flow rate and 260 nm wavelength were used as optimized chromatographic conditions. The anticipated method was validated by the ICH specifications. The retention time of asciminib was noticed at 0.925 minute with good efficiency, respectively. Linearity was noticed for concentrations ranging fom 5 to 30 μg/mL of asciminib with R2 value of 0.999. The %RSD of both system and method precision was assessed in the range of 0.49 to 0.86. The percentage recovery of asciminib was in the range of 100.06–100.6%. The logarithm of the odds (LoD) and limit of quantitation (LoQ) of asciminib determined to be 0.23 and 0.69 μg/mL correspondingly. The results confirmed that the projected technique was economical, simple, responsive and precise. Exploration of asciminib under various FD environments confirms the stability representing the nature of the stated method. The proposed UPLC method is extremely valuable in the separation and estimation of asciminib.

Sensitive and selective bioscreening of the most commonly used coronavirus disease drug, Favipiravir, and its co-administered therapeutic, Meropenem, in human plasma.

Favipiravir and Meropenem have been concurrently used as directly acting antiviral and antibiotic agents for the treatment of coronavirus disease in human plasma. Accurate and specific reversed phase ultra-performance liquid chromatographic and high-performance thin-layer chromatographic methods were developed and validated for the first time analysis of this combination in spiked human plasma using Cefepime as an internal standard. In the developed ultra-high-performance liquid chromatography method, separation was performed on a BEH C18 column with a mixture of ACN and 0.05 M potassium dihydrogen orthophosphate (pH = 3) in a ratio of 10:90 (v/v) as an eluate. Scanning of the separated peaks was at 298 nm. The developed method showed high sensitivity, and the drugs showed linearity in the range of 5-70 μg/ml for Favipiravir and 2-50 μg/ml for Meropenem. The proposed high-performance thin-layer chromatographic method included the separation using a mixture of ethyl acetate:methanol:deionized water:formic acid (5:4:1.5:0.3, by volume), then spots detection at 300 nm. Methods were investigated for greenness using the eco-scale and national environmental method index tools and were validated according to food and drug administration guidelines. Methods can be applied for bio-analysis and therapeutic drug monitoring studies.

Novel RP-UPLC Method Development and Validation for Simultaneous Quantification of Emtricitabine, Tenofovir and Efavirenz in Bulk and Tablet Dosage Forms

Objective: The objective of this work is to develop a precise, accurate and validated reverse phase ultra-performance liquid chromatographic technique for effective simultaneous determination of Emtricitabine, Tenofovir and Efavirenz in bulk and tablet formulation. Method: Separation of the selected drugs was optimized after several trials including changing mobile phase and its composition, stationary phase, flow rate, column temperature, etc. Finally the separation of drugs was achieved on BEH C18 column using a mixture of methanol and phosphate buffer having pH 3.5 in the ratio of 65:35 v/v as mobile phase with flow rate of 0.3 ml/min and the analytes were detected at a wavelength of 260 nm. Results: The developed method was validated by determining the parameters like linearity, system suitability, recovery, precision, specificity, robustness, ruggedness, LOD, and LOQ as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The system suitability parameters were within the limits, retention times (Rt) for Emtricitabine, Tenofovir and Efavirenz were found to be 0.432, 0.671, and 2.772 min respectively. The method showed linearity between the concentration range of 10-50 μg/ml for Emtricitabine (r2= 0.9987), 15-75 μg/ml for Tenofovir (r2= 0.9983) and 30-150 μg/ml for Efavirenz (r2= 0.9982). The percentage recovery results at 50%, 100% and 150% of Emtricitabine, Tenofovir and Efavirenz were found to be in the range of 99.45 % - 100.15 %. Since there was no interference due to excipients and mobile phase, the method was found to be specific. The assay results of the combined tablet dosage form by the developed method were identified as in good agreement with the acceptance limit. Conclusions: The current method was proved to be effective for routine simultaneous determination of Emtricitabine, Tenofovir and Efavirenz in bulk and tablet formulation.

Development and Validation of new UPLC Method for the Simultaneous estimation of Darunavir, Dolutegravir and Ritonavir in Combined Tablet Dosage form

A rapid, simple, sensitive and selective analytical method was developed by using reverse phase Ultra performance liquid chromatographic technique for the simultaneous estimation of darunavir, dolutegravir and ritonavir in combined tablet dosage form. The developed method is superior in technology to conventional high performance liquid chromatography with respect to speed, resolution, solvent consumption, time, and cost of analysis. Elution time for the separation was 3 min and ultra violet detection was carried out at 225 nm. Efficient separation was achieved on BEH C18 (50 x 2.1mm, 1.7 ) UPLC column using phosphate buffer and acetonitrile as organic solvent in a linear isocratic program. Resolution between darunavir, dolutegravir and ritonavir was found to be more than 3.5. The active pharmaceutical ingredient was extracted from tablet dosage from using a mixture of water and acetonitrile used as diluent. The calibration graphs were linear for darunavir, dolutegravir and ritonavir in the range of 100-600 g/ml, 12.5-75 g/ml and 6.25-37.5 g/ml respectively. The mean percentage recoveries for darunavir, dolutegravir and ritonavir were found to be in the range of 99.13 and 99.59 and 99.72 % respectively. Developed UPLC method was validated as per International Conference on Harmonization specifications for method validation. This method can be successfully employed for simultaneous estimation of darunavir, dolutegravir and ritonavir in bulk drugs and formulations.

STABILITY INDICATING METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF PREGABALIN AND ETORICOXIB IN BULK AND TABLET DOSAGE FORM BY UPLC

A simple, accurate and precise reverse phase ultra-performance liquid chromatography method was developed and validated for simultaneous estimation of Pregabalin and Etoricoxib in formulations as well as in pure form. The developed method was shown acceptable separation of pregabalin and Etoricoxib. The retention times for Pregabalin and Etoricoxib were 1.134 min. &amp; 1.535 min. respectively. The limits of detection and quantification were obtained from regression equations and observed to be 0.85ppm and 0.54ppm for Pregabalin, 2.58 and 1.62ppm for Etoricoxib, respectively. Degradation studies of the tablet dosage form under stress conditions of acid, base, oxidation , temperature, water, UV light indicated that the method was particular with no interferences from any of the potential impurities that could form over the shelf life. The new developed RP-UPLC method which was sensitive, accurate stability indicating and fast, for active pharmaceutical ingredients and dosage forms. Key words: Pregabalin, Etoricoxib, Simultaneous, Reverse Phase - UPLC