HPLC Grade Acetonitrile Research Articles

Validation of Analytical Method for Vitamin A in Bioadhesive Ocular Cationic Nanoemulsion Loaded into Thermosensitive Gel Using RP-HPLC

Various test methods have been previously documented for determining vitamin A levels in different dosage forms. This study specifically examines an isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method designed for the direct extraction of vitamin A. The objective is to validate an analytical method for quantifying vitamin A in bioadhesive cationic nanoemulsions incorporated into thermosensitive gels. The method employs isocratic RP-HPLC with a YMC-Triart C18 column (L1), dimensions of 4.6 mm × 250 nm, particle size of S-5 µm, and a UV detector at λ = 265 nm. The mobile phase consists of HPLC-grade methanol, acetonitrile, and n-hexane in a ratio of 46.5:46.5:7. Validation parameters were assessed including selectivity, linearity, accuracy, precision, limit of quantification (LOQ), and limit of detection (LOD). Correlation coefficients were determined with an R2 value of 0.9995 in the concentration range of 264–396 μg/mL (w/v). Recovery percentages ranged from 99.295% to 99.878%. Repeatability and intermediate precision relative standard deviations (RSD) were found to be 0.318% and 0.254%, respectively. The LOD was established at 2.018 μg/mL, and the LOQ was determined to be 6.114 μg/mL. The results affirm cost-effective and well-suited for the accurate measurement of vitamin A levels in bioadhesive thermosensitive gel formulations.

Development and validation of stability indicating HPLC-DAD assay of adapalene, methylparaben and propylparaben in a cosmeceutical gel matrix with blueness, greenness and whiteness assessment via relative ranking: An ab initio-sub finem juxtaposed non-traditional approaches

Adapalene (ADA) is primarily used as a cosmeceutical gel in the treatment of mild-moderate acne and is also used off-label to treat keratosis pilaris as well as other skin conditions. A stability-indicating fit-for-purpose (FFP) method has been developed for co-determination of ADA, methylparaben (MPN), and propylparaben (PPN) combined in a cosmeceutical gel dosage form. This method was based on a reversed-phase RP-HPLC separation of the cited analytes. It avoided the use of tetrahydrofuran (THF), which is commonly used by both compendial and published procedures for determination of ADA, thus improving method blueness (applicability), greenness and whiteness. The method development and validation followed non-traditional eco-friendly approaches including chemical simulations and empirical modelling. The HPLC separation was performed on an RP Zorbax Eclipse XDB-C8 analytical column [150 × 4.6 mm, 5 µm (80 Å)] with gradient elution system of HPLC grade acetonitrile and 50 mM aqueous sodium dihydrogen phosphate buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA) adjusted to pH 8.0 as the mobile phase at a flow rate of 2.5 mL min−1 and column oven was set at 25 °C. EDTA played an important role for reproducibility of retention time of the three analytes especially for ADA. It controlled deprotonation of the three acidic analytes under study and hence reproduce their ionization, hydrophobicity, and hence retention time. Quantitation was achieved with the photodiode array detection (DAD) at wavelengths of 256 nm for both MPN and PPN and 312 nm for ADA. Target Measurement Uncertainty (TMU) for the three analytes was determined via two approaches; misclassification rates approach employing 100,000 Monte Carlo simulations (MCS) and in-specification probability (ISP) to ascertain fitness-for-purpose of the proposed method. Microsoft Excel workbooks were developed for both TMU calculation approaches in addition to calculation of procedure capability index (Ppk) and sigma level for each analyte and attached in the Supplementary material. Moreover, linearity of the method underwent additional assessment using the previously mentioned TMU and procedure capability determination techniques. The robustness of the method was checked using Plackett-Burman experimental design (PBD) as an empirical modelling technique which was examined thoroughly to detect some interactions. The calibration graphs for each drug were rectilinear in the range of 0.34–7.08, 0.38–7.00 and 0.08–1.75 µg mL−1 for ADA, MPN, and PPN, respectively. The stress testing revealed photostability of the three components and variable degrees of degradation in the other conditions. The proposed HPLC-DAD method was successfully validated and applied for the determination of the investigated analytes in a gel matrix. In addition, method blueness and greenness were comparatively evaluated via very recent approaches of Blue Applicability Grade Index (BAGI), Green Solvents Selection Tool (GSST), Spider Diagram for Assessment of the Green Analytical Index (SDAGI), Analytical Method Greenness Score (AMGS), Carbon Footprint (CF), Modified Complementary Green Analytical Procedure Index (ComplexMoGAPI), and Analytical GREEnness Metric Approach (AGREE). Furthermore, the method whiteness was assessed via Red Green Blue Twelve (RGB 12) model following a relative ranking approach.

A stability indicating method development and validation of a rapid and sensitive RP-HPLC method for Nintedanib and its application in quantification of nanostructured lipid carriers.

Nintedanib (NTB) is a multiple tyrosine kinase inhibitor, been investigated for many disease conditions like idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD) and non-small cell lung cancer (NSCLC). NTB is available as oral capsule formulation, but its ability to detect degradants formed through oxidative, photolytic and hydrolytic processes makes it difficult to quantify. In the current work, a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated. The developed method is simple, precise, reproducible, stable and accurate. The inherent stability of NTB was evaluated using the proposed analytical method approach and force degradation studies were carried out. NTB was separated chromatographically on the Shimadzu C 18 column as stationary phase (250 ×4.6 mm, 5 µm) using an isocratic elution method with 0.1% v/v triethyl amine (TEA) in HPLC grade water and acetonitrile (ACN) in the ratio 35:65% v/v. The mobile phase was pumped at a constant flow rate of 1.0 ml/min, and the eluent was detected at 390 nm wavelength. NTB was eluted at 6.77±0.00 min of retention time (t R) with a correlation coefficient of 0.999, the developed method was linear in the concentration range of 0.5 µg/ml to 4.5 µg/ml. The recovery rate was found to be in the range of 99.391±0.468% for 1.5 µg/ml concentration. Six replicate standards were determined to have an % RSD of 0.04. The formulation excipients didn't interfere with the determination of NTB, demonstrating the specificity of the developed method. The proposed approach of the analytical method developed can be used to quantify the amount of NTB present in bulk drugs and pharmaceutical formulations.

A stability indicating method development and validation of a rapid and sensitive RP-HPLC method for Nintedanib and its application in quantification of nanostructured lipid carriers

Background: Nintedanib (NTB) is a multiple tyrosine kinase inhibitor, been investigated for many disease conditions like idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD) and non-small cell lung cancer (NSCLC). NTB is available as oral capsule formulation, but its ability to detect degradants formed through oxidative, photolytic and hydrolytic processes makes it difficult to quantify. In the current work, a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated. Methods: The developed method is simple, precise, reproducible, stable and accurate. The inherent stability of NTB was evaluated using the proposed analytical method approach and force degradation studies were carried out. NTB was separated chromatographically on the Shimadzu C18 column as stationary phase (250 ×4.6 mm, 5 µm) using an isocratic elution method with 0.1% v/v triethyl amine (TEA) in HPLC grade water and acetonitrile (ACN) in the ratio 35:65% v/v. The mobile phase was pumped at a constant flow rate of 1.0 ml/min, and the eluent was detected at 390 nm wavelength. Results: NTB was eluted at 6.77±0.00 min of retention time (tR) with a correlation coefficient of 0.999, the developed method was linear in the concentration range of 0.5 µg/ml to 4.5 µg/ml. The recovery rate was found to be in the range of 99.391±0.468% for 1.5 µg/ml concentration. Six replicate standards were determined to have an % RSD of 0.04. Conclusion: The formulation excipients didn't interfere with the determination of NTB, demonstrating the specificity of the developed method. The proposed approach of the analytical method developed can be used to quantify the amount of NTB present in bulk drugs and pharmaceutical formulations.

Development and Validation of Stability Indicating RP-HPLC Method for the Estimation of Glycopyrrolate and Neostigmine in Bulk and Injection.

A stability indicating RP-HPLC method is suggested for determination of Glycopyrrolate-Neostigmine (GLY/NEO) in bulk drugs and injection formulation. GLY/NEO were eluted from a Chromolith High Resolution RP-18e (100 mm×4.6mm) with buffer solution (pH3.0) as mobile phase A and a mixture of HPLC grade acetonitrile and water mixture (90:10) as mobile phase B. The gradient was optimized with a flowrate of 0.5mL/min and wavelength of 222nm. A complete analytical method validation was effectively carried out as per ICH Q2 (R1) guidelines. Recovery studies were performed at 50-150% level of working concentrations, and results were in the range of 99-101%. The linearity was detected in the range of LOQ to 200% of the specification limits i.e., 0.5% each for NEO and GLY, 0.01% for NEO Impurity B and 1.0% for rest of the impurities with respect to the test concentration of the respective components. For stability study, various stress conditions such as acid, base, oxidation and thermal as per ICH guidelines were studied. The high recovery and low relative standard deviation confirm the suitability of proposed method that can be employed for the routine analysis in bulk and pharmaceutical formulation.

Rapid and sensitive HPLC-MS/MS method for the quantification of dopamine, GABA, serotonin, glutamine and glutamate in rat brain regions after exposure to tobacco cigarettes.

Tobacco smoking is a preventable main cause of fatal diseases. Accurate measurements of the effects it has on neurotransmitters are essential in developing new strategies for smoking cessation. Moreover, measurements of neurotransmitter levels can aid in developing drugs that counteract the effects of smoking. The aim of this study is to develop and validate a fast, simultaneous and sensitive method for measuring the levels of neurotransmitters in rat brain after the exposure of tobacco cigarettes. The selected neurotransmitters include dopamine, GABA, serotonin, glutamine and glutamate. The method is based on high-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved within 3 min using a Zorbax SB C18 column (3.0× 100 mm, 1.8μm particle size). The mobile phase consisted of HPLC-grade water and acetonitrile each containing 0.3% heptafluorobutyric acid and 0.5% formic acid at gradient conditions. The linear range was 0.015-0.07, 825-7,218, 140-520, 63.42-160.75 and 38.25 × 103 to 110.35 × 103 ng/ml for dopamine, GABA, serotonin, glutamine and glutamate, respectively. Inter- and intra-run accuracy were in the range 97.82-103.37% with a precision (CV%) of ≤0.90%. The results revealed that 4weeks of cigarette exposure significantly increased neurotransmitter levels after exposure to tobacco cigarettes in various brain regions, including the hippocampus and the amygdala. This increase in neurotransmitters levels may in turn activate the nicotine dependence pathway.

Bioanalytical method development and validation of atrasentan in human plasma using verapamil as internal standard by liquid chromatography coupled with tandem mass spectrometry

A satisfactory LC-MS/MS separation and good peak symmetry for Atrasentan were obtained with Agilent, Zorbax, XDB C18 (2.1 x 50 mm ID, 5 μm), and a mobile phase containing a mixture of 5 mM Ammonium Formate buffer with 0.1% formic acid were mixed with HPLC grade Acetonitrile in the proportion of 70:30, v/v was delivered at a flow rate of 0.150 mL/min by positive ion mode (API 4000) with an injection volume of 10 µL and a run time of 3 min. Detection is performed by atmospheric pressure electrospray ionization (ESI) mass spectrometry in positive ion mode. The precursor to product ion transitions is m/z 5.11.600 to 354.04 for Atrasentan, and m/z 455.40 to 165.00 for Verapamil (Internal standard) were used for quantization. The retention time of Atrasentan and Verapamil (Internal standard) was found to be 1.68 & 0.96 min.

Bioanalytical method development and validation of atrasentan in human plasma using verapamil as internal standard by liquid chromatography coupled with tandem mass spectrometry

A satisfactory LC-MS/MS separation and good peak symmetry for Atrasentan were obtained with Agilent, Zorbax, XDB C18 (2.1 x 50 mm ID, 5 μm), and a mobile phase containing a mixture of 5 mM Ammonium Formate buffer with 0.1% formic acid were mixed with HPLC grade Acetonitrile in the proportion of 70:30, v/v was delivered at a flow rate of 0.150 mL/min by positive ion mode (API 4000) with an injection volume of 10 µL and a run time of 3 min. Detection is performed by atmospheric pressure electrospray ionization (ESI) mass spectrometry in positive ion mode. The precursor to product ion transitions is m/z 5.11.600 to 354.04 for Atrasentan, and m/z 455.40 to 165.00 for Verapamil (Internal standard) were used for quantization. The retention time of Atrasentan and Verapamil (Internal standard) was found to be 1.68 & 0.96 min.

Development and Validation of a RP-UHPLC Method for estimation of Gliclazide loaded Microsphere

Objective: The objective of the current work is to develop and validate a simple yet effective, efficient and reproducible RP-UHPLC method for the determination of Gilcazide loaded in a microsphere formulation. Methods: The chromatographic analysis was carried out on Thermo Scientific (Dionex Ultra 3000plus) UHPLC using a stainless steel YMC C8 column (25cm × 4mm) packed with endcapped octylsilane bonded to porous silica (4µm) in isocratic elution mode using phosphate buffer pH 3.4 and HPLC grade acetonitrile in the ratio of 20: 80 (v/v) as eluent. The flow rate was adjusted as 1ml/min and eluent was detected at 230 nm. The retention time was observed at 1.9 min. Results: The linear dynamic range for the developed UHPLC method was found in the concentration range of 2-10µg/ml and percentage recovery was in the range of 94%-98%. Drug Entrapment Efficiency Data of Gliclazide loaded microsphere was found to be 93.72% Conclusion: In this RP–UHPLC method, the linearity was within the range of 2–10μg/mL and the validation parameters were within the limits was performed as per ICH guidelines. The % Drug entrapment Efficiency of the Gliclazide loaded microsphere was found to be 93.72 % which was satisfactory.

Validation of the HPLC\u2013PDA method for detection of eluxadoline and rifaximin in rat plasma and application in a pharmacokinetic study

BackgroundA precise, simple, accurate, and quick HPLC–PDA method for the determination of eluxadoline and rifaximin in rat plasma was developed and validated in this study. In this method, Loperamide hydrochloride was used as the internal standard and plasma samples were prepared using a liquid–liquid extraction technique for which acetonitrile was a solvent. An Agilent Symmetry C8 column (5 µm, 250 mm × 4.6 mm) at 283 nm and isocratic elution using HPLC grade acetonitrile and 7 mM TEA (pH 2.5) with a ratio of (40: 60 v/v) was used as a mobile phase and the flow rate employed was 1 mL min−1. A satisfactory chromatographic separation was accomplished.ResultsAn HPLC–PDA method for the determination of eluxadoline and rifaximin with retention times of 3.06 and 7.82 min, respectively, was developed. The calibration curves appear linear for both eluxadoline and rifaximin in the range of 5–200 ng mL−1 and 10–400 ng mL−1, and the corresponding correlation coefficient values were found to be 0.9999 and 0.9998 respectively. Lower limits of quantification (LLOQ) for eluxadoline and rifaximin were evaluated to be 5.0 ng mL−1 and 10.0 ng mL−1, respectively. The accuracy and precision results in all validation experiments were within the acceptance limits of FDA guidelines.ConclusionThe developed HPLC–PDA approach was fully validated to meet the USFDA guidelines for bioanalytical method validation in terms of precision, accuracy, and stability. The presented approach could be beneficial for the determination of ELX and RFX in rat plasma, according to validation parameters. This is one of the efficient method to study the pharmacokinetics of ELX and RFX in rats.Graphical abstract

Investigation of an artificial solution degradant of linagliptin: An undesired linagliptin urea derivative generates in sample preparation of linagliptin tablet treated by sonication in acetonitrile containing diluent

During the related substances testing method development for linagliptin tablet, an unknown peak was observed in HPLC chromatograms with a level exceeding the identification threshold. By using a strategy that combines LC-PDA/UV-MSn with mechanism-based stress studies, the unknown peak was rapidly identified as linagliptin urea, a solution degradant that is caused by the reaction between the API and hydrocyanic acid with sonication treatment to accelerate dissolution of the drug substance in sample preparation of linagliptin tablets, and hydrocyanic acid is a known impurity in HPLC grade acetonitrile and acetonitrile is used as part of diluent. The mechanism of the solution degradation chemistry was verified by stressing linagliptin API with trimethylsilyl cyanide (TMSCN, which can give off HCN slowly in the presence of water) treated with sonication in the sample preparation. Further investigation found that when the sonication treatment was replaced by vortex vibration in the process of the sample preparation, the RRT 1.28 species was decreased to below the level of the detection limit (0.02%). The structure of this impurity was further confirmed through the synthesis of the impurity and subsequent structure characterization by 1D and 2D NMR. Due to the presence of trace amount of HCN in HPLC grade acetonitrile, these types of solution degradation would likely occur in analysis of pharmaceutical finished products containing APIs with primary and secondary amine moieties drug product during sample preparations, particularly when sonication treatment is used to accelerate dissolution of drug substance from the finished drug product. In the GMP quality control laboratories, such events may trigger undesirable out-of-specification (OOS) events. Hence, the results of this paper can help to prevent these events from happening in the first place or resolve these OOS events in GMP laboratories.

Investigation of an Artificial Solution Degradant of Linagliptin: An Undesired Linagliptin Urea Derivative Generate in Sample Preparation of Linagliptin Treat by Sonication in Acetonitrile Containing Diluent

During the related substances testing method development for linagliptin tablet, an unknown peak was observed in HPLC chromatograms with a level exceeding the identification threshold. By using a strategy that combines LC-PDA/UV-MSn with mechanism-based stress studies, the unknown peak was rapidly identified as linagliptin urea, a solution degradant that is caused by the reaction between the API and hydrocyanic acid with sonication treatment to accelerate dissolution of the drug substance in sample preparation of linagliptin tablets, and hydrocyanic acid is a known impurity in HPLC grade acetonitrile and acetonitrile is used as part of diluent. The mechanism of the solution degradation chemistry was verified by stressing linagliptin API with trimethylsilyl cyanide (TMSCN, which can give off HCN slowly in the presence of water) treated with sonication in the sample preparation. Further investigation found that when the sonication treatment was replaced by vortex vibration in the process of the sample preparation, the RRT 1.21 species was decreased to below the level of the detection limit (0.02%). The structure of this impurity was further confirmed through the synthesis of the impurity and subsequent structure characterization by 1D and 2D NMR. Due to the presence of trace amount of HCN in HPLC grade acetonitrile, these types of solution degradation would likely occur in analysis of pharmaceutical finished products containing APIs with primary and secondary amine moieties drug product during sample preparations, particularly when sonication treatment is used to accelerate dissolution of drug substance from the finished drug product. In the GMP quality control laboratories, such events may trigger undesirable out-of-specification (OOS) events. Hence, the results of this paper can help to prevent these events from happening in the first place or resolve these OOS events in GMP laboratories.

Anticancer Activity Assay of Nano-Fractional Compounds that Purified from Soil Actinomycetes

Background and objectives: Cancer remains a global problem of health, and has been recorded as one of the causes of death after heart disease Natural products from plants, the environment and microorganisms are leveraged for the purpose of fighting cancer. Actinobacteria have been recognized as main sources of bioactive natural products as early as in the 1950s, for which about half of the secondary metabolites revealed, including enzymes, antibiotics, immunosuppressive, and anti-tumour agents Materials and methods:The methods of this study included isolated and identification of bacteria from soil samples and identified by morphology characters and biochemical test. Subjected extract of actinomycetes to HPLC purification then collected purified fractions then analyzed by GC-mass. After the fractions were mixed with liposome nanoparticals which tested activity on HT29 colon cancer cell line. Results: The results of identification of bacterial isolates showed the colonies growing on a SNA medium were morphologically identified where the colonies were well-growth and had a gray color, not producing dyes in the medium. The results of the biochemical tests indicated that isolates were amylase, catalase, and gelatinase producing isolates and non-lipase producing, non H2S production and consuming urea, while the carbon consumption test indicated the isolates' ability to consume starch, glucose and sucrose respectively. While The results of preparative HPLC revealed that 4 fractions analytical HPLC with (50 % HPLC-grade acetonitrile) at 254 nm and cycling up was employed to increase the separation efficiency. The chemical composition of the HPLC fractions using GC-MS showed the identification of many components example ( Hexadeconic acid , Octadecanoic acid,ethyl ester and Fumaric acid). The results of In vitro anti-tumor cytotoxicity showed that all four nano purified fractions were applied on HT 29 colon cancer cells and exhibited significantly differences compared with control.

Potential Assessment of UGT2B17 Inhibition by Salicylic Acid in Human Supersomes In Vitro.

Glucuronidation is a Phase 2 metabolic pathway responsible for the metabolism and excretion of testosterone to a conjugate testosterone glucuronide. Bioavailability and the rate of anabolic steroid testosterone metabolism can be affected upon UGT glucuronidation enzyme alteration. However, there is a lack of information about the in vitro potential assessment of UGT2B17 inhibition by salicylic acid. The purpose of this study is to investigate if UGT2B17 enzyme activity is inhibited by salicylic acid. A UGT2B17 assay was developed and validated by HPLC using a C18 reversed phase column (SUPELCO 25 cm × 4.6 mm, 5 μm) at 246 nm using a gradient elution mobile phase system: (A) phosphate buffer (0.01 M) at pH = 3.8, (B) HPLC grade acetonitrile and (C) HPLC grade methanol. The UGT2B17 metabolite (testosterone glucuronide) was quantified using human UGT2B17 supersomes by a validated HPLC method. The type of inhibition was determined by Lineweaver–Burk plots. These were constructed from the in vitro inhibition of salicylic acid at different concentration levels. The UGT2B17 assay showed good linearity (R2 > 0.99), acceptable recovery and accuracy (80–120%), good reproducibility and acceptable inter and intra-assay precision (<15%), low detection (6.42 and 2.76 μM) and quantitation limit values (19.46 and 8.38 μM) for testosterone and testosterone glucuronide respectively, according to ICH guidelines. Testosterone and testosterone glucuronide were found to be stable up to 72 h in normal laboratory conditions. Our investigational study showed that salicylic acid uncompetitively inhibited UGT2B17 enzyme activity. Thus, drugs that are substrates for the UGT2B17 enzyme have negligible potential effect of causing interaction with salicylic acid in humans.

Development and Validation of RP-HPLC Method for the Estimation of Clomiphene Citrate in Pharmaceutical Dosage Form

A simple, specific, precise, and accurate RP-HPLC method has been developed and validated for the estimation of Clomiphene citrate in bulk and pharmaceutical dosage form using C18 column Shimadzu (250mm × 4.5mm × 5μm) with a mobile phase consisting of 900mL of HPLC grade methanol and 100mL of HPLC grade acetonitrile. The mobile phase was sonicated for 10 min and filtered through a 0.45μm membrane filter at a flow rate of 1.0mL/min. The detection was carried out at 295nm and retention time of Clomiphene citrate was found to be 3.44 min. Linearity was observed in the concentration range of 10–50μg/mL (coefficient of determination R2=0.999) with regression equation y =20321x + 60021. The method was validated as per ICH guidlines.

Development and Validation of a HPLC Method for Determination of Oxadiargyl Residues in Vegetables and Soil

A standardized and validated method for the estimation of residues of oxadiargyl in green onion, cabbage and soil is reported. The substrates were extracted in acetonitrile, the extracts diluted with brine solution, partitioned in dichloromethane, cleaned up using activated charcoal and dried over anhydrous sodium sulphate. Final clear extracts were concentrated under vacuum and reconstituted in HPLC grade acetonitrile, and residues estimated by using high performance liquid chromatography equipped with PDA detector and C18 column. Acetonitrile was used as mobile phase at flow rate of 0.3 mL min−1. The average recoveries were over 85 per cent for oxadiargyl at different spiking levels. The precision (repeatability) and reproducibility) in different matrices ranged from 3.01 to 5.21 and 2.43 to 3.07 per cent, respectively. The limit of quantification was worked out to be 0.05 mg kg−1. The analytical method was validated in terms of parameters including selectivity, linearity, precision and accuracy.

Development and validation of a stability-indicating RP-HPLC method for the identification, determination of related substances and assay of dimethyl fumarate in drug substance

The objective of current study was to develop and validate a short, economical, accurate, precise stability-indicating RP-HPLC method for identification, quantitation of related substances (fumaric acid and mono methyl fumarate) and assay of dimethyl fumarate (DMF) drug substance. The RP-HPLC method was developed by using liquid chromatography (waters 2695 with PDA detector & Agilent 1200 with DAD) with Symmetry C18 column. Pharmaceutical grade of high pure materials of DMF, MMF, FA and HPLC grade water, acetonitrile and orthophosphoric acid were used for this study. The mobile phase consists of 0.1% of ortho-phosphoric acid in water: acetonitrile (55:45% v/v). The developed method was validated according to ICH guidelines. To prove the stability indicating potential, stress studies performed using acid, base, peroxide and thermal. After sufficient exposure, these solutions were injected in to HPLC and found that all degradants formed during stress study were well separated from the main peak and resolution between all impurities was more than ICH requirements. A simple, short and stability indicating RP-HPLC method was developed and validated for simultaneous estimation of DMF and its related substances in drug substance. Based on literature survey it was evident that many methods were published for determination of DMF individually or its related substances, however short run time methods were not available for simultaneous estimation of DMF and its related substances. The intended method would support to industries for quick quantitation of DMF and its related substances without compromising quality parameters like precision and accuracy.

RP-HPLC method development and validation for the quantification of Efonidipine hydrochloride in HME processed solid dispersions

BackgroundEfonidipine hydrochloride (EFO) is a poorly water-soluble drug and, hence, has poor bioavailability. Solid dispersions (SDs) of EFO using Eudragit EPO were prepared using hot-melt extrusion (HME) for the first time. The current study aims at developing a simple RP-HPLC method to quantify EFO in the developed SDs.ResultsThe chromatographic separation was carried out on an Agilent Eclipsed XDB-C18 column (4.6 × 250 mm), packed with 5 μm particles. The optimized mobile phase consisted of HPLC grade acetonitrile and 0.020 mol/L KH2PO4 (pH 2.5) buffer in the ratio of 85:15 v/v with a flow rate optimized at 1.2 ml/min. The developed method was validated for system suitability, linearity, accuracy, precision, and robustness. The linearity results showed an excellent linear relationship between the drug concentration and peak area, indicating the peak area is directly proportional to the analyte concentration within a specific range and an excellent correlation coefficient of 0.9998. Intermediate precision and repeatability confirmed that the method provides precise results with %RSD value less than 2% for EFO. The assay results of the developed formulations were in the acceptable range with RSD less than 2%. The enhanced drug dissolution from the Eudragit EPO carrier with 10% Citric Acid (CA) is attributed to the conversion of the drug from crystalline to amorphous form, and microenvironmental acidic pH provided by CA.ConclusionIn a nutshell, the developed RP-HPLC method showed excellent ability to differentiate the formulations and highlights the role of the polymer and the plasticizer.Graphical abstract

Single-step extraction for simultaneous quantification of desvenlafaxine and alprazolam in human spiked plasma by RP-HPLC.

Desvenlafaxine (DES) and Alprazolam (ALP) are the drugs commonly prescribed together for the treatment of Major Depressive Disorders (MDD). A literature survey revealed, there is no method for the simultaneous determination of these two drugs. The purpose of this research was to develop and validate a simple, accurate, precise, robust, and isocratic RP–HPLC method for simultaneous determination of DES and ALP in human spiked plasma using UV-detector in short analysis time. The method utilized Hypersil BDS C18 (250 mm×4.6 mm, 5 μm) through an isocratic mode of elution using HPLC grade acetonitrile and 0.02M KH2PO4 buffer (65:35) and 0.1% Tri Fluoro Acetic acid (TFA) with pH 4.00 adjusted with 1M KOH. The flow rate was 1.00 mLmin-1 and elution of the drugs was monitored at 230nm. The elution time of DES and ALP was 4.011 and 5.182 minutes respectively. The method was linear for the concentration range 10–150 μgmL-1 for DES and 5.0–75.0 μgmL-1 for ALP. According to the validation results, the method is sensitive with Limit of Detection (LOD) 4.740 μgmL-1 and Limit of Quantification (LOQ) of 14.365 μgmL-1 for DES and LOD 1.891 μgmL-1 & LOQ 5.730 μgmL-1 for ALP. The reproducibility of results with minute deliberate variations in method parameters has proven that the method is robust. The data from stability studies show a non-significant change in drugs solutions for 2 months. The optimized method was validated as per International Conference for Harmonisation (ICH) Q2(R1) guidelines. This method can be used for the estimation of DES and ALP in plasma and can evaluate pharmacokinetic parameters of both drugs simultaneously.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING METHOD FOR IPRATROPIUM BROMIDE BY USING RP-HPLC

A simple stability indicating RP-HPLC method was validated for determination of ipratropium bromide in the bulk drug. The drug was resolved using HPLC Column (Kromasil ODS 150 x 4.6 mm C18 column) with mobile phase of HPLC grade acetonitrile:potassium di-hydrogen phosphate buffer (60:40 V/V) at a flow rate of 1 ml/min. The retention time of ipratropium bromide was 3.7 min with UV detection at 254 nm. The method was validated with respect to linearity, sensitivity, accuracy, precision and robustness as per the International Conference on Harmonization (ICH) guidelines. The method was specific and it was observed that no interference with diluents. The linearity was established over the concentration range of 20-120 μg/ml with correlation coefficients (r2) 0.9958 for ipratropium bromide. The mean recovery was found to be in the range of 99.8% for ipratropium bromide. The % R.S.D. values for intraday precision study and inter-day study were &lt;1.0%, confirming that the method was sufficiently precise. The drug was subjected to forced degradation under different conditions. The drug was degraded more in oxidative condition (28.89%) then alkaline condition (26.39%) then acidic condition (13.42%). There was no degradation seen under the thermal conditions.