High-performance Liquid Chromatographic Assay Method Research Articles

Implementation of Quality by Design Approaches for Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Assay Method for Determination of Glycyrrhizin in Nanoformulation.

Glycyrrhizin (GL) is the principal constituent of Glycyrrhiza glabra, having antiallergic, anticancer, anti-inflammatory, and antimicrobial action. The reverse-phase high-performance liquid chromatography (RP-HPLC) analytical method was used to quantitatively estimate GL in a nanoformulation and validated as per International Conference on Harmonization Q2 (R1) standards. A stationary phase of the C18-HL reversed-phase column and a mobile phase of acetonitrile and water were used for effective elution. The chromatographic conditions of RP-HPLC were optimized utilizing a quality-by-design approach to accomplish the required chromatographic separation of GL from its nanoformulation with minimal experimental runs. Optimized RP-HPLC conditions for the assay method consist of acetonitrile (41%) and water, pH 1.8, balanced with phosphoric acid (0.1%) as a mobile phase with a flow rate of 1 mL/min. The retention time was found at 7.25 min, and method validation confirmed its sensitivity, preciseness, accuracy, and robustness.

Development and Validation of RP-HPLC Method for the Estimation of Linezolid from Bulk and Formulations

A new, simple, rapid, selective, precise and accurate isocratic reverse-phase high-performance liquid Chromatography assay method has been developed to estimate Linezolid in tablet formulations. The separation was achieved using column Inert sustain C18 (250×4.6mm, 5µ) in the mobile phase consisting of methanol: 1% Acetic acid (50:50 v/v). The flow rate was 0.80mL/min-1, and the separated Linezolid was detected using a UV detector at 249nm. Column temperature 45°C and sample temperature ambient and injection volume 20µL. The retention time of Linezolid was noted to be 7.3min. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

Convenient estimation of oxytetracycline and polymyxin B by a novel high-performance liquid chromatography method: development and validation

Aim: The aim of this research work was to develop a validated reversed-phase (RP)-high-performance liquid chromatography (HPLC) method for simultaneous estimation of oxytetracycline (OXY) and polymixin B (PMB) in fixed-dose combination. Methods: The HPLC assay method was validated on X-Bridge C18 [250 mm × 4.6 mm intradermal (i.d.), 5 μm], mobile phase consisting of aotearoa co-incidence network (ACN):water containing 0.5% (v/v) orthophosphoric acid (pH 3.5) in the ratio of 80:20 respectively. The flow rate was set at 0.9 mL/min and the column was maintained at room temperature. The RP-HPLC method was validated in terms of the calibration curve (CC), linearity and range, limit of detection (LOD), and limit of quantitation (LOQ), precision, robustness, and accuracy. Results: The method was found to be linear with a concentration range of 5–25 µg/mL. Precision results showed the developed method was found to be precise with a relative standard deviation [RSD (%)] value < 2. Accuracy showed acceptable recovery of prepared concentrations as per intracerebral hemorrhage (ICH) guidelines. Moreover, the developed method was found to be robust and rugged, as per specified ranges. The assay of these two drugs in marketed formulation, i.e., Terramycin® Ointment showed satisfactory recovery, as per ICH guidelines. The results proved that the method can be used for the routine-based estimation of OXY and PMB. Conclusions: Linear CC were obtained with a correlation coefficient (R2 > 0.99) with acceptable results of accuracy and precision.

In vitro Pharmaceutical Quality Evaluation of different Ibuprofen tablet brands available on the Republic of Kosovo Market

Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) that possesses anti-inflammatory, analgesic, and antipyretic effects and it is widely manufactured and marketed in the Republic of Kosovo. The study aims to evaluate the in vitro quality of three ibuprofen uncoated tablets 400mg formulations that are commercially most commonly used in the Republic of Kosovo markets. Ibuprofen tablets were tested for the uniformity of weight, disintegration time, friability, and analysis of the drug active content with the UV spectrophotometric and high-performance liquid chromatography (HPLC) method following comparisons with official protocols and pharmacopeia monograph. The weight uniformity testing and weight variation indicated no significant changes in the weight and variation among the analyzed brands' tablets. The friability values were <1 % and the disintegration times were between 3.87 and 10.01 min, which showed significantly variation for the first brand but all the samples meet official requirements (within 15 min for uncoated tablets). In addition, the results of the assay of chemical content using UV-VIS and HPLC analysis meet the official specification in the UV and HPLC assay methods (95-105% of active drug content). Our data indicated that the Ibuprofen tablet investigated in our study meets the in vitro quality control meets the official specifications, is chemically equivalent, and does not vary in physiochemical qualities.

Pharmacokinetics of meloxicam in laying hens after single intravenous, oral, and intramuscular administration.

The objective of this study was to determine the pharmacokinetics of meloxicam after a single intravenous (IV), intramuscular (IM), and oral (PO) dose at 1mg/kg body weight in Jing Hong laying hens. Blood samples were collected at predetermined time points. Plasma meloxicam concentrations were determined using a validated high-performance liquid chromatography (HPLC) assay method and then subjected to a non-compartmental analysis. After IV administration, meloxicam had a mean (±SD) volume of distribution at steady-state (Vdss ) of 206.50 ± 25.23 ml/kg, a terminal half-life (t1/2λ ) of 5.45 ± 0.53 h, and a total body clearance (Cl) of 26.48 ± 4.13 ml/h/kg. After PO and IM administration, meloxicam was absorbed relatively rapidly: the peak concentrations (Cmax s) of 3.04 ± 0.56 and 8.94 ± 2.31 μg/ml were observed at 3.08 and 0.80 h, respectively. After PO and IM administration, the absolute bioavailability (F) was determined as 70.13% and 125.50%, respectively. Assuming that hens shared the same analgesic threshold of meloxicam (0.5μg/ml) with humans, the plasma concentrations after three different routes (PO, IM, and IV) of administration were above this value for 16.7, 19.2, and 14.9h, respectively.

Development and Validation of New RP-HPLC Method for Estimation of Pramipexole Dihydrochloride in Bulk and Pharmaceutical Formulation

A novel high-performance liquid chromatographic assay method was developed and validated for the quantitative determination of the anti-Parkinson agent pramipexole dihydrochloride monohydrate in bulk and its tablet dosage form. In this perspective, the chromatographic separation was accomplished on Eclipse XDB-12 C18 (150 mm x 4.6 mm, 5 μm particle size) column using UV detection at 263 nm. The mobile phase consisted of distilled water: acetonitrile (10: 90 v/v), run at a flow rate of 1.0 mL/min with isocratic elution. The method was validated in accordance with ICH guidelines by evaluating the system suitability, linearity, limits of detection (LOD) and quantitation (LOQ), precision, accuracy, specificity, selectivity and short-term stability. Our findings revealed that retention time for pramipexole dihydrochloride was found to be 5.2 minutes. The linearity range was established between 6.25-225.0 μg/mL with a mean recovery of 101.26 % ± 0.56. The limits of detection and quantification were determined to be 4.18 μg/mL and 12.66 μg/mL, respectively, indicating that the method is very sensitive. Intra and inter-day precision were within acceptable limits (RSD

Evaluation of clinical relationship of serum niacin and dopamine levels in patients with fibromyalgia syndrome.

Objectives The aim of this study was to investigate the role of serum niacin and dopamine (DA) levels and their clinical importance in fibromyalgia syndrome (FMS) patients. Patients and methods Between April 2018 and October 2018, a total of 53 female patients (mean age: 38.3±5.5 years; range, 21 to 45 years) with a clinical diagnosis of FMS and 35 healthy female controls (mean age: 36.7±5.2 years; range, 25 to 44 years) were included in this cross-sectional study. The Visual Analog Scale (VAS), Beck Depression Inventory (BDI), and Fibromyalgia Impact Questionnaire (FIQ) were applied to the patients. Serum levels of niacin and DA were measured by high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) methods, respectively. Results Niacin and DA levels of the patient group were significantly lower than those of control group (p=0.003 and p=0.02, respectively). A very strong positive correlation was found between niacin and DA levels (r=0.96 p<0.001). Evaluation of the diagnostic performance of niacin and DA by the receiver operating characteristic analysis yielded an area under the curve (AUC) of 0.73 (p<0.001, 95% confidence interval [CI]: 0.62-0.85) and an AUC of 0.68 (p=0.004, 95% CI: 0.56-0.80), respectively. Conclusion Serum niacin and DA levels decrease in FMS patients in relation to the tender point numbers. It can be suggested that the levels of these two markers can be considered additional tools in the diagnosis of FMS.

A NOVEL VALIDATED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC STABILITY-INDICATING ASSAY METHOD IN REVERSE PHASE FOR ESTIMATION OF HYDROXYPROGESTERONE CAPROATE IN AN INJECTABLE FORMULATION.

A high-performance liquid chromatographic stability-indicating assay method in reverse-phase has been developed and validated for the quantitative measurement of Hydroxyprogesterone corporate in injectable formulation prepared in castor oil base containing excipient benzyl benzoate as preservative. The reverse-phase method was developed by using Sunfire C18 column having dimensions (150 x 4.6 mm x 5μm) with a mobile phase[A] comprised of water, acetonitrile, and Methanol mixed in 40:30:30 v/v ratio and mobile phase [B] comprised of water, acetonitrile, and Methanol mixed in 20:40:40 v/v ratio pumped gradient program at a flow rate of 1.0 ml/min where the thermostat of the column oven controlled at 30°C and sample compartment at 5°C. The injection volume is set at 20μL. Hydroxyprogesterone corporate has a retention time of 10 minutes with UV detection at 240 nm. The detector was showing linear response in a range of 0.25 μg/ml to 150 μg/ml for Hydroxyprogesterone corporate. The guidelines of the International Conference on Harmonization on guidance for industry (2005) validation of analytical procedures, text, and methodology Q2(R1) were followed for the validation of this novel method. For quantification of Hydroxyprogesterone corporate the developed method was found to be genuinely exact precise, accurate, linear, robust, rugged, stable. and cost-effective.

A Validated specific Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography Assay Method for L-Ornithine L-Aspartate and Silymarin, and their related Substances and its Application to Dissolution Studies

The present study was aimed at the development and successive validation of a novel, simple, sensitive, and stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantitative calculation of L-ornithine L-aspartate (LOLA) and Silymarin (SL), and also their relevant substances in bulk and pharmaceutical dosage forms. The chromatographic technic was optimized using the impurity-spiked solution. The separation of all the two active components and their impurities was achieved by a chromatographic method with an Agilent Eclipse XDB-C18, 150 × 4.6 mm, 3.5 μ column, using gradient elution with mobile phase A consisting of a mixture of 0.1% orthophosphoric acid and water and acetonitrile as mobile phase B. The instrumental settings included a flow rate of 1 mL/min for both related substances and assay, a detector wavelength of 225 nm, by using a PDA detector. The established method was validated according to the current ICH requirements. The detection limit and the limit of quantification for the two active components and their related impurities were established with respect to test concentration. The calibration graphs plotted were linear with a regression coefficient R2 andgt; 0.999, indicates the linearity of the method was within the limits. Recovery studies were satisfactory and the parameters, such as, specificity, linearity, accuracy, precision, and robustness were determined as part of the method validation. Moreover, using the same method dissolution study was performed on active pharma ingredients to estimate the recovery. The obtained results were within the range of acceptance criteria. These results suggest that the developed method was found to be applicable for routine analysis for testing chromatographic purity of LOLA and SL and it can be utilized for the calculation of both active ingredients and their impurities in tablet dosage forms.

A multivariate quantification of Box-Behnken design assisted method development and validation of dextromethorphan hydrobromide and desloratadine simultaneously by reverse-phase HPLC in in-house syrup formulation.

An innovative high-performance liquid chromatography assay method was developed and validated for quantification of dextromethorphan hydrobromide and desloratadine simultaneously in monophasic liquid formulation by preparing syrup containing 30mg/5mL of dextromethorphan hydrobromide and 1.2mg/mL of desloratadine. The chromatographic severance was executed by gradient solution A and B. The composition of buffer solution A contained 0.05M monobasic potassium, then 1mL triethylamine was added to it and the pH was adjusted to 2.3 with orthophosphoric acid. Methanol was used as solution B. The gradient elution was executed with Kromasil C8 (250mm×4.6mm) column having 1.5mL/min flow rate and 20µL injection volume with UV-estimation at 254nm for dextromethorphan hydrobromide and DES. The present research was planned according to Box-Behnken design by utilizing design expert software, using four factors such as column temperature (A), flow rate (B), mobile phase-organic phase (C), and pH (D); correspondingly the selected response variables were resolution between A and B, that is, desloratadine and methyl paraben (Y1), tailing of dextromethorphan hydrobromide (Y2), and tailing of desloratadine (Y3). The parameters such as system suitability, linearity, accuracy, precision, robustness, limit of detection, limit of quantitation, and ruggedness were analyzed to validate the developed method in accordance with current regulatory guidelines.

Stability-indicating high-performance liquid chromatographic assay methods for drugs in pharmaceutical dosage forms: Part I

A review of applications of stability-indicating high-performance liquid chromatographic (HPLC) assay methods for drugs in various pharmaceutical dosage forms is presented. A survey of articles primarily on the significant developments in this field is covered in this paper. Selected substances are grouped to integrate drugs that share similar uses or actions. Papers selected by the authors are briefly summarized, and HPLC systems developed in these papers for stability testing of pharmaceuticals are tabulated to assist readers in selecting an appropriate system for a given pharmaceutical. The information given in the table includes the drug names, materials, column types, mobile-phase compositions, detections, internal standards used and the literature references.

Stability-indicating High-Performance Liquid Chromatographic assay methods for drugs in pharmaceutical dosage forms: Part II

Stability-indicating High-Performance Liquid Chromatographic assay methods for drugs in pharmaceutical dosage forms: Part II

Characterization of Five Oxidative Degradation Impurities and One Process Impurity of Suvorexant Drug Substance by LC-MS/MS, HR-MS and 1D, 2D NMR: Validation of Suvorexant Drug Substance and Process Impurities by HPLC and UPLC.

During the oxidative (10% H2O2) degradation of suvorexant drug substance, around 1.0% of one impurity and less than 1.0% four impurities were found by a new high-performance liquid chromatography (HPLC) assay and related substance method. The mass numbers of 1.0% impurity was 469 [M+H]+, remaining four impurities were 172 [M+H]+, 467 [M+H]+, 483 [M+H]+ and 485 [M+H]+. The 469 [M+H]+, 485[M+H] and 172 [M+H]+ impurities were characterized by using the LC-MS/MS, HR-MS and 1D, 2D NMR spectroscopic data. The 172 [M+H]+ impurity was prepared synthetically and co-injected in HPLC. The retention time of synthesized 172 [M+H]+ impurity was matching with the unknown degradation impurity in HPLC. The developed mass compatible HPLC and ultra performance liquid chromatography methods were validated for drug substance and process impurities by following ICH Q2 (R1) guidelines.

Development and Validation of HPLC assay of Lycopene in Different Matrices

Lycopene is the red pigment richly found in many red -colored fruits and vegetables such as tomatoes, papaya, pink grapefruit, pink guava, and watermelon. It is an antioxidant that has various beneficent effects and protects against oxidative damage to lipids, protein, and DNA. It is a potent quencher of oxygen and most effective against cancer, cardiovascular diseases, osteoporosis, hypertension, male infertility, and neurodegenerative disease. Lycopene is synthesized by plants and microorganisms but not animals and humans. The main objective of this study is to develop and validate an HPLC assay method for the determination of lycopene in tomato, watermelon, and human blood. A simple assay method is an economical assay method, suitable for the determination of lycopene from plant and biological sources. A new accurate, specific, precise, high performance liquid chromatographic (HPLC) method has been developed and validated for the determination of lycopene in tomatoes, watermelon and blood sample according to the International Conference on Harmonization (ICH) guidelines. Acetonitrile, Methanol, Water (65%, 20%, and 15%) was used as the mobile phase at a flow rate of 0.7ml/min. using a symmetry C18 column. The effluent was spectrophotometrically monitored at 484nm. The calibration curve was linear with a correlation coefficient of 0.9994. The average recovery was found to be 102.0%. The proposed HPLC method was successfully applied to quantify the amount of lycopene in different matrices.

Stability of Insulin Detemir Injection in Different Primary Packaging Systems at Room Temperature

Purpose: To assess the stability of insulin detemir at controlled room temperature (RT) at 25°C in different packaging systems over 7 days. Methods: The degradation characteristics of insulin detemir were determined based on the assay results in different packaging systems (pinhole glass vial, closed glass vial, glass syringe, and plastic syringe) at RT using a reverse-phase high-performance liquid chromatography (HPLC) assay method for insulin injection. Each packaging system was compared to insulin detemir stored in the original packaged closed glass vial at 2°C to 8°C. Results: Insulin detemir stored in a closed glass vial and a glass syringe showed minor degradation at the end of day 7 (98.96% ± 1.49% and 99.78% ± 0.10%, respectively). Insulin detemir stored in plastic syringe decreased to 94.90% ± 2.50% by day 3 and to 93.52% ± 0.29% by day 7. Storage in pin-hole glass vial showed an increase in the assay (152.13% ± 0.12%) by day 7. Conclusion: Stability studies in different packaging systems demonstrated that insulin detemir remained stable for at least 7 days in a closed glass vial or glass syringe, but for only 3 days in a plastic syringe at RT. This study will allow pharmacists in the hospital setting to deliver patient-specific insulin doses into an insulin syringe with confidence in the stability.

RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

A “Large-N” Content Uniformity Process Analytical Technology (PAT) Method for Phenytoin Sodium Tablets

Accurate assessment of tablet content uniformity is critical for narrow therapeutic index drugs such as phenytoin sodium. This work presents a near-infrared (NIR)-based analytical method for rapid prediction of content uniformity based on a large number of phenytoin sodium formulation tablets. Calibration tablets were generated through an integrated experimental design by varying formulation and process parameters, and scale of manufacturing. A partial least squares model for individual tablet content was developed based on tablet NIR spectra. The tablet content was obtained from a modified United States Pharmacopeia phenytoin sodium high-performance liquid chromatography assay method. The partial least squares model with 4 latent variables explained 92% of the composition variability and yielded a root mean square error of prediction of 0.48% w/w. The resultant NIR model successfully assayed the composition of tablets manufactured at the pilot scale. For one such batch, bootstrapping was applied to calculate the confidence intervals on the mean, acceptance value, and relative SD for different sample sizes, n = 10, 30, and 100. As the bootstrap sample size increased, the confidence interval on the mean, acceptance value, and relative SD became narrower and symmetric. Such a ‘large N’ NIR-based process analytical technology method can increase reliability of quality assessments in solid dosage manufacturing.

Pharmacokinetics and Bioavailability Study of a Prednisolone Tablet as a Single Oral Dose in Bangladeshi Healthy Volunteers.

The aim of the study was to assess the pharmacokinetic and bioavailability of 2 formulations of 5-mg prednisolone tablets, reference product (Teva UK Limited) and Pred (Eskayef Bangladesh Ltd) as test product. The open-label, randomized, 2-way crossover studies were conducted on 14 healthy subjects. Participants were assigned to receive both products as a single dose (20 mg formulations, 4 × 5 mg tablets) followed by a 2 weeks’ washout period. Following oral administration, samples were obtained at various time intervals and analyzed for prednisolone concentrations using a validated high-performance liquid chromatography assay method with ultraviolet detection. The obtained values for test and reference products were 683.00 ± 94.54 ng/mL and 635.16 ± 125.57 ng/mL for Cmax; 2716.54 ± 196.28 ng·h/mL and 2780.5 ± 119.73 ng·h/mL for AUC0-12; 3284.36 ± 138.12 ng·h/mL and 3317.96 ± 133.95 ng·h/mL for AUC0-∞, respectively. From the paired Student t test, no significant differences between 2 formulations were observed (P > .05). The 90% confidence intervals of Cmax, AUC0-12, and AUC0-∞ were found to be 99.0% to 100.9%, 99.4% to 100.5%, and 99.9% to 101.3%, respectively. Finally, it can be concluded that Pred (Test) of Eskayef Bangladesh Ltd and prednisolone (Reference) of Teva UK Limited are bioequivalent and interchangeable.

A NEW VALIDATED SIMULTANEOUS REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ASSAY METHOD FOR ESTIMATION OF TWO FLAVONES (BAICALEIN AND CHRYSIN) IN API DRUGS

Objective: The scope of the present work is a reversed-phase high-performance liquid chromatography (RP-HPLC) assay method developed for the simultaneous determination of baicalein and Chrysin in API drugs.Methods: The RP-HPLC method employed a Hypersil BDS C18 reversed-phase column (150×4.6 mm×5.0 μm) with an isocratic mixture of water and acetonitrile in the ratio of 55:45; v/v as the mobile phase. The column temperature was kept at 25°C. The flow rate was 1.0 mL/min, and detection was by means of a photodiode array detector at wavelength of 268 nm and validated for various parameters such as specificity, system suitability, method precision, linearity, accuracy, solution stability, ruggedness, and robustness.Results: The system suitability parameters were evaluated from standard chromatograms by calculating the % relative standard deviation (RSD) from six replicate injections for baicalein and Chrysin peak areas. The % RSD for the peak area of the principal peak from six replicate injections of each standard solution was &lt;2.0%. The linearity and correlation coefficient of baicalein and Chrysin was found to be 0.999. Method precision was performed, and % RSD from six different preparations for baicalein and Chrysin were found to be &lt;2.0. The % recovery for each level was found to be the range of 98.0–102%. In ruggedness and robustness, the % RSD of the peak area of all peaks for six replicate injections should be not more than 2.0.Conclusion: Hence, the chromatographic assay method developed for baicalein and chrysin is said to be rapid, simple, specific, sensitive, precise, accurate, and reliable that can be effectively applied for routine analysis in research institutions, quality control department in industries, approved testing laboratories, bio-pharmaceutics, and bio-equivalence studies and in clinical pharmacokinetic studies.

A NEW VALIDATED SIMULTANEOUS REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ASSAY METHOD FOR ESTIMATION OF TWO FLAVONES (BAICALEIN AND CHRYSIN) IN API DRUGS

Objective: The scope of the present work is a reversed-phase high-performance liquid chromatography (RP-HPLC) assay method developed for the simultaneous determination of baicalein and Chrysin in API drugs.Methods: The RP-HPLC method employed a Hypersil BDS C18 reversed-phase column (150×4.6 mm×5.0 μm) with an isocratic mixture of water and acetonitrile in the ratio of 55:45; v/v as the mobile phase. The column temperature was kept at 25°C. The flow rate was 1.0 mL/min, and detection was by means of a photodiode array detector at wavelength of 268 nm and validated for various parameters such as specificity, system suitability, method precision, linearity, accuracy, solution stability, ruggedness, and robustness.Results: The system suitability parameters were evaluated from standard chromatograms by calculating the % relative standard deviation (RSD) from six replicate injections for baicalein and Chrysin peak areas. The % RSD for the peak area of the principal peak from six replicate injections of each standard solution was &lt;2.0%. The linearity and correlation coefficient of baicalein and Chrysin was found to be 0.999. Method precision was performed, and % RSD from six different preparations for baicalein and Chrysin were found to be &lt;2.0. The % recovery for each level was found to be the range of 98.0–102%. In ruggedness and robustness, the % RSD of the peak area of all peaks for six replicate injections should be not more than 2.0.Conclusion: Hence, the chromatographic assay method developed for baicalein and chrysin is said to be rapid, simple, specific, sensitive, precise, accurate, and reliable that can be effectively applied for routine analysis in research institutions, quality control department in industries, approved testing laboratories, bio-pharmaceutics, and bio-equivalence studies and in clinical pharmacokinetic studies.