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Development and validation of RP-HPLC method for estimation of brexpiprazole in its bulk and tablet dosage form using Quality by Design approach

  • Amol S. Jagdale 1 ,
  • Nilesh S. Pendbhaje 2 ,
  • Rupali V. Nirmal 1 ,
  • Poonam M. Bachhav 1 &
  • Dayandeo B. Sumbre 1  

Future Journal of Pharmaceutical Sciences volume  7 , Article number:  142 ( 2021 ) Cite this article

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A new, sensitive, suitable, clear, accurate, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of brexpiprazole in bulk drug and tablet formulation was developed and validated in this research. Surface methodology was used to optimize the data, with a three-level Box-Behnken design. Methanol concentration in the mobile phase, flow rate, and pH were chosen as the three variables. The separation was performed using an HPLC method with a UV detector and Openlab EZchrom program, as well as a Water spherisorb C 18 column (100 mm × 4.6; 5m). Acetonitrile was pumped at a flow rate of 1.0 mL/min with a 10 mM phosphate buffer balanced to a pH of 2.50.05 by diluted OPA (65:35% v/v) and detected at 216 nm.

The developed RP-HPLC method yielded a suitable retention time for brexpiprazole of 4.22 min, which was optimized using the Design Expert-12 software. The linearity of the established method was verified with a correlation coefficient (r 2 ) of 0.999 over the concentration range of 5.05–75.75 g/mL. For API and formulation, the percent assay was 99.46% and 100.91%, respectively. The percentage RSD for the method’s precision was found to be less than 2.0%. The percentage recoveries were discovered to be between 99.38 and 101.07%. 0.64 μg/mL and 1.95 μg/mL were found to be the LOD and LOQ, respectively.

The developed and validated RP-HPLC system takes less time and can be used in the industry for routine quality control/analysis of bulk drug and marketed brexpiprazole products.

Graphical abstract

thesis hplc method development and validation

Brexpiprazole is 7-[4-[4-(1-benzothiophen-4-yl) piperazin-1-yl] piperazin-1-yl] piperazin-1-yl] piperazin-1-yl] piperazin-1-yl] piperazin-1-yl] piperazin-1 butoxy] However, The USFDA approved quinolin-2 (1H)-one in 2015, and it is marketed as Rexulti, a generic name coined by Otsuka in Japan and marketed by Lundbeck in the USA for the treatment of schizophrenia as a monotherapy and as an adjunctive treatment to antidepressants in the treatment of major depressive disorder [ 1 , 2 , 3 , 4 , 5 ]. Early treatment with aripiprazole can result in problematic akathisia. Brexpiprazole may be less likely than aripiprazole to induce akathisia. This will be a big benefit, but there is not much experience with the drug yet. Brexpiprazole, like aripiprazole, is a partial agonist of the dopamine D2 receptor and has mild effects on QTc. Brexpiprazole is likely to have a wide dose range in clinical practice due to the function of CYP2D6 in its metabolism [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ].

According to a literature review, there are few publications on UV-visible spectroscopy and HPLC, but no one has used Quality by Design. To ensure process consistency throughout the product lifecycle, simple validated RP-HPLC methods for the determination of brexpiprazole in pharmaceutical dosage forms must be established using the Quality by Design (QbD) approach as per ICH Q8 (R2) guidelines [ 8 , 15 , 16 , 17 , 18 , 19 ].

Materials and reagents

Alkem Laboratories Limited, Mumbai, donated brexpiprazole (see Fig. 1 ). Merck provided HPLC grade methanol, acetonitrile, orthophosphoric acid (OPA), and analytical grade ethanol, DMF, DMSO, and HCl. Siddhi Lab provided the HPLC grade water.

figure 1

Molecular structure of brexpiprazole

Instrumentation and software

An Agilent HPLC system with DEAX02386 pump and autosampler with UV-visible detector served as the chromatographic system (DEACX16446). For data collection and processing, the chromatograms were registered using Openlab EZChrom on a Windows-based computer system. Brexpiprazole concentrations were determined using a HyPURITY C 18 (100mm × 4.6mm ID, particle size 5μ) column.

QbD software

Design Expert® software (Design Expert trial version 12.0.10.0; State-Ease Inc., Minneapolis, MN, USA)

Preparations of solutions

Preparation of standard stock solution.

The standard solution was made by dissolving 10 mg of brexpiprazole in a 100-mL clean and dry volumetric flask, then adding approximately 70 mL of methanol to fully dissolve it and fill the flask to the mark with methanol (100 μg/mL).

Sample preparation

Ten milligrams brexpiprazole was correctly weighed and transferred to a 100-mL volumetric flask. Fifty milliliters diluent was added and sonicated to fully remove it. Using diluent, dilute the mixture by another 10 to 20 mL.

Preparation of diluted OPA

Pipette 5 mL of OPA into a 50-mL volumetric flask and top up with water to reach the desired amount. Sonicate for 5 min after thoroughly mixing.

Preparation of 1.0% OPA in water

One milliliter OPA was blown out of the solution and moved to a 100-mL volumetric flask, which was filled to the mark with water. Sonicate for 5 min after thoroughly mixing.

Preparation of 10.00 mM phosphate buffer in water

Weigh 1.36 g of OPA and dissolve it in 1000 mL of water, adjusting the pH to 2.0 (±) 0.05 with the diluted OPA solution.

Determination of detection wavelength

Between 200 and 400 nm, the standard solution was scanned. As shown in Fig. 2 , the wavelength of maximum absorption for drug was determined to be 216 nm.

figure 2

UV spectrum of brexpiprazole in methanol

Method development by QbD approach

Application of design of experiments for method optimization.

To investigate the effect of three factors on the two primary response variables, 33 randomized response surface designs with a Box-Behnken design were used with 17 trial runs. Three variables were analyzed at three levels in this design, and experimental trials were conducted in all three possible combinations. Flow rates (X1), pH (X2), and mobile phase composition (X3) were designated as independent variables, while retention time (RT), asymmetry, and theoretical plates were designated as dependent variables. The data was then entered into the Design Expert 12.0.10.0 software and evaluated using the ANOVA test. To assess the effect of flow rate, pH, and mobile phase composition on dependent variables, the results were subjected to the 3-dimensional response surface methodology. Table 1 shows the likely trial runs using 3 3 Box-Behnken designs. Table 2 shows the experimental results and selected method conditions.

Analysis of the sample

Brexpiprazole drug (api).

The drug sample solution was prepared by liquifying 10 mg of brexpiprazole API into a 100-mL volumetric flask, adding 70 mL of methanol to fully melt it by sonication, and then adjusting the volume with solvents (100g/mL). Filtered through a suitable filter, and a sufficient amount of the sample solution was discarded. Using methanol (50g/mL), dilute 5 mL of the filtrate solution to 10 mL.

Tablet formulation

Keep in mind, in the market, there is a tablet called Rexulti. However, it is not available in India. There are three doses available: 1, 2, and 4 mg. The sample preparation will be seen on a dosage of 4 mg, which is a higher dose. For preparing lab-level tablet mixture, approximately 150 mg average weight is taken into account. The formula for one tablet is shown in Table 3 .

Weigh 10 mg of brexpiprazole powder into a 100-mL flask, add about 70 mL of methanol to completely solubilize it by sonication, and complete volume up to spot with the methanol (100 g/mL). Filtered through a suitable filter, and a sufficient amount of the sample solution was discarded. Five milliliters filtrate solution was diluted to 10 mL with methanol (50 g/mL).

Control strategy

Filtration study.

Filtration experiment using centrifuged (unfiltered) sample and filtered test solution. During the filtration process, 5 mL of the aliquot sample was discarded and 0.45 m PVDF 0.45 and 0.45 m Nylon syringe filters were used.

Stability of analytical solution

A stability analysis will be carried out on both the normal and test solutions. A test sample of Rexulti tablet will be used to determine the stability of the test solution. The stability test will be carried out in a standard laboratory environment.

The solution will be held in a brightly lit laboratory for 12 to 24 h before being analyzed. The discrepancy between the test solution’s results at each stability time point and the original will be calculated for the test solution stability analysis. The discrepancy between the effects of the stability time point and the original will be calculated in a standard solution stability analysis.

Method validation

The developed method for estimating brexpiprazole was validated for the following parameters using ICH Q2 (R1) guidelines [ 20 , 21 , 22 , 23 , 24 , 25 ].

Specificity

To demonstrate the method’s precision, the following solutions will be prepared and injected (double-checked the peak purity).

Blank (methanol as a diluent)

Brexpiprazole standard solution

Brexpiprazole sample solution

Placebo treatments

Linearity and range

The statistical treatment of test results obtained by examination of samples with analyte concentrations around the claimed spectrum determines the analytical method’s linearity. As a function of analyte concentration, the region is graphically plotted. Curve fitting percentages are measured.

Accuracy (%recovery)

The accuracy will be tested in the range of 50 to 150% of the working concentration of 4 mg strength. Every occurs solution will be prepared in triplicate. A placebo will be included in the experiment. For each study, the percent recovery was determined.

There are two levels of precision: repeatability and intermediate precision. It is carried out on a sample API.

Repeatability (intraday precision)

Intermediate precision (interday precision)

The API test sample was created from scratch. As shown below, these samples were injected under various chromatographic conditions.

Flow rate changes (20% of the total)

A change in wavelength (3 nm)

± 2°C increase in column oven temperature

The limit of detection (LOD) and limit of quantification (LOQ) were calculated separately using the following equations based on the standard deviation of the y-intercept and the slope of the calibration curve, respectively.

Optimization of mobile phase

Methanol: water (70:30), acetonitrile: water (70:30), acetonitrile: 1% OPA in water (80:20), and acetonitrile: 10 mM phosphate buffer were among the mobile phases that were optimised, shown in Fig. 3 . Acetonitrile: 10 mM phosphate buffer adjusted pH 2.5 by OPA (80:20), acetonitrile: 10 mM phosphate buffer adjusted pH 2.5 by OPA (65:35), acetonitrile: 10 mM phosphate buffer adjusted pH 2.5 by OPA (65:35). As a result, chromatographic conditions in trial were used for process validation, as shown in Fig. 4 : Acetonitrile: 10 mM phosphate buffer modified pH 2.5 by OPA (65:35) provides a better peak, lower retention time, and tailing factor. Typical chromatogram of optimized run is shown in Fig. 5 . Analytical data for typical chromatogram of optimized run are shown in Table 4 .

figure 3

Solutions for optimized run

figure 4

Typical chromatogram obtained from optimized mobile phase

figure 5

Typical chromatogram of optimized run

Optimization of various parameters for analysis of brexpiprazole using HPLC (by using central composite design)

Design summary for optimization is given in Table 5 . Obtained solution for optimized formulation is given in Table 6 .

System suitability test (SST)

It was observed from the data tabulated that the method complies with system suitability parameters. Hence, it can be concluded that the system suitability parameter meets the requirement of method validation. Typical chromatogram of SST for brexpiprazole is shown in Fig. 6 . Analytical data of system suitability test are given in Table 7 .

figure 6

Typical chromatogram of SST for brexpiprazole

Filter test

Both filters PVDF and Nylon pass the criteria for filter study; hence, both filters can be used because %absolute difference is NMT 2.0, and it follows acceptance criteria. Analytical data of filter test are given in tabular form in Table 8 . Typical chromatogram of unfiltered sample, sample filtered through 0.45 μ PVDF filter, and sample filtered through 0.45 μ Nylon filter is shown in Figs. 7 , 8 , and 9 respectively.

figure 7

Typical chromatogram of unfiltered sample

figure 8

Typical chromatogram of sample filtered through 0.45μ PVDF filter

figure 9

Typical chromatogram of sample filtered through 0.45μ Nylon filter

Solution stability

Both standard solution and sample solution were found stable for 24 h; hence, prepared solution can be used up to 24 h. (User can check solution stability even after 24 h if he/she wants to inject solution after 24 h.) Analytical data are given in Table 9 .

Blank and placebo solution are not having interference at R.T. of brexpiprazole. Peak purity for both standard as well as sample was within limits. Sample solution exhibits the same R.T. as that of standard solution. Hence, developed chromatographic method passed the criteria for specificity. Result of specificity is given in Table 10 .

%Recovery was found well within acceptance range (98.00 to 102.0%) at all three levels. Result and statistical data of accuracy are given in Table 11 .

%RSD for 12 samples (precision and intermediate precision samples) NMT 2.0%. The %RSD of method precision is 0.53 and 0.495. Therefore, the HPLC method for the determination of brexpiprazole is precise. Analytical data of both precision of brexpiprazole is given in Table 12 .

From the calibration curve, we had to conclude that brexpiprazole shows linear response in the range of 5.05–75.75 μg/mL. The regression value was found well within the limit. Result and statistical data of linearity of brexpiprazole are given in Table 13 . Linearity graph of brexpiprazole is shown in Fig. 10 .

figure 10

Linearity graph of brexpiprazole

Based on the calibration curve, we can deduce that brexpiprazole has a linear response in the 5.05–75.75 g/mL range. The regression value was discovered to be well within the acceptable range. Data for calibration curve of brexpiprazole is shown in Table 14 .

It may be calculated based on the standard deviation (SD) of the response and slope of the curve (S). Result of detection limit is given in Table 15 . Calibration curve of brexpiprazole for LOD and LOQ is given in Fig. 11 .

figure 11

Calibration curve of brexpiprazole for LOD and LOQ

The robustness of an analytical method is determined by analysis of aliquots from homogenous lots by differing physical parameters that may differ but are still within the specified parameters of the assay. Analytical interpretation is given in Table 16 .

The aim of this project was to create a simple, reliable, precise, and appropriate RP-HPLC system using the Quality by Design (QbD) approach. DOE results, including ANOVA, diagnostic graphs, and model graphs, were examined for each factor. The effect of each factor on the response result was investigated in this result.

In terms of analytical method creation and validation, the results of all system suitability parameters were appropriate within the limits specified by applying ICH (Q2 R1) guidelines, indicating that the system is functioning properly and can provide accurate and precise results. The established method’s analysis results were validated in terms of linearity, accuracy, precision, and robustness, as well as the detection and quantification limits.

The developed method has many advantages, according to Mondal et al., including reproducibility of findings, rapid interpretation, easy sample preparation, and improved selectivity and sensitivity. The developed method can be used for routine research in the pharmaceutical industry for the bulk drug brexpiprazole as well as the pharmaceutical dosage type since it is stable and reproducible and takes less time [ 10 ].

According to the above experimental results, this newly developed method for estimating brexpiprazole was found to be simple, precise, and accurate, with a shorter retention time that makes it more acceptable and cost effective, and it can be effectively applied for routine analysis in research institutions, quality control departments in industries, and approved testing laboratories.

Availability of data and materials

Data and material are available upon request.

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Abbreviations

Reversed-phase high-performance liquid chromatography

Percentage recovery

Limit of detection

Limit of quantification

Quality by Design

United States Food and Drug Administration

International Council for Harmonization of Technical Requirement for Pharmaceutical for Human Use

Orthophosphoric acid

Dichloromethane

Dimethyl sulfoxide

Hydrochloric acid

Polyvinylidene fluoride

Analysis of variance

Active pharmaceutical ingredient

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Acknowledgements

Authors express their sincere gratitude to N.D.M.V.P. College of Pharmacy, Nashik, and Sanjivani College of Pharmaceutical Education and Research, Kopargaon, for continuous motivation, support, and guidance for research activity and for providing all required facilities to accomplish the entitled work.

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Amol S. Jagdale, Rupali V. Nirmal, Poonam M. Bachhav & Dayandeo B. Sumbre

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R.N. contributed to literature survey and performed practical work and thesis typing, N.P. supported in performing experimental work, A.S. had contributed to guide the whole work, and P.B. and D.S. contributed to editing in thesis typing. The authors read and approved the final manuscript and approve the submission.

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Jagdale, A.S., Pendbhaje, N.S., Nirmal, R.V. et al. Development and validation of RP-HPLC method for estimation of brexpiprazole in its bulk and tablet dosage form using Quality by Design approach. Futur J Pharm Sci 7 , 142 (2021). https://doi.org/10.1186/s43094-021-00293-5

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Development and validation of a new HPLC analytical method for the determination of diclofenac in tablets

A new selective and sensitive high-performance liquid chromatography (HPLC) method was developed for the quantification of diclofenac sodium (DS) in pharmaceutical dosage form using lidocaine as internal standard (IS). Chromatographic separation was achieved on a symmetry C18 column (4.6 mm × 150 mm, 3 μm spherical particles) using 0.05 M orthophosporic (pH 2.0) 35% and acetonitrile as 65%, as the mobile phase at a flow rate of 2.0 mL/min and monitored at 210 nm. The run time was 2 min.

The method was validated to fulfill International Conference on Harmonisation (ICH) requirements and this validation included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness. The calibration curve was linear over the concentration range from 10 to 200 µg/ml, and lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations. Diclofenac was unstable at room temperature it showed more than 25% loss after 24 h. While, DS is very stable at refrigerator 4 °C auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at −35 ± 2 °C.

All results were acceptable and this confirmed that the method is suitable for its intended use in routine quality control and assay of drugs.

1. Introduction

Diclofenac sodium [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide] is one of the analgesic-antipyretic-nonsteroidal anti-inflammatory drug. Diclofenac is a widely used for the treatment of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis osteoarthritis, musculoskeletal injuries, and post surgery analgesia in human and veterinary medicine ( Bhattacharya et al., 2013 ). The molecule is practically water insoluble, but it is readily absorbed from the gastrointestinal tract as the salt form.

Various analytical techniques have been reported for the quantification of diclofenac sodium (DS) in different matrices. High-pressure liquid chromatography detection (HPLC) is the most common used method for the determination of DS in biological sample or dosage forms ( Bhattacharya et al., 2013 , Klimeš et al., 2001 , Yan et al., 2014 , Vieira et al., 2016 , Mahdi et al., 2016 , Cordery et al., 2017 , Khan et al., 2016 , Pireddu et al., 2015 , Aldwaikat and Alarjah, 2015 , Hegazy et al., 2015 , Kasperek, 2008 , Basusarkar, 2011 , Nivsarkar et al., 2015 , Korodi et al., 2012 , Chaudhary et al., 2011 , Kubala et al., 1993 , Mulgund et al., 2009 , Gaudiano et al., 2003 ).

Analytical methods keep on updating with time as per the requirements so as to develop a simple, reliable, cost effective, reproducible and above all a method bearing a high level of accuracy and precision.

Our study aimed to develop a rapid, robust, selective, sensitive, and precise HPLC method for the determination of DS. The assay method was validated using by USP 26 ( United States Pharmacopeial Convention, 2003 )or by the ICH guidelines ( CPMP/ICH/381/95, 1994 ). The linearity, accuracy, precision, specificity, limit of detection (LOD), and limit of quantification (LOQ) and used for in determination of drug content of the DS in different pharmaceutical commercial products.

2. Materials and methods

2.1. materials.

Diclofenac was a gift from Spimaco (Riyadh, Saudi Arabia). All other reagents and chemicals were of HPLC analytical grade, and were used as received. Water was deionized and purified using a Milli-Q Reagent Grade water system (Millipore Cor), poration, Bedford, MX 01730, USA).

To determine the content of Diclofenac Sodium in conventional tablet available in Saudi market, randomly select the following products (Brand name: voltaren®, Clofen®, voltaic®, Rapidus® and Rofenac® label claim: 50 mg Diclofenac per tablet).

2.2. Liquid chromatography conditions

The HPLC system consisted of Waters 1525 binary pump Separation module (Waters, USA) fitted with C 18 column (300 mm × 4.6 mm). The autosampler injection system (Waters 2707) used was a 10 µl sample loop. A Millipore Swinnex type filter (pore size = 0.45 µm) was obtained from Millipore (Bangalore, India). A Waters HPLC system equipped with a Waters 484 variable UV absorbance detector and a Waters 2707 plus autosampler was used. Waters 515 solvent delivery system was used to operate the gradient flow through a symmetry C 18 column (4.6 × 75 mm, 3.5 μm spherical particles). with 0.05 M orthophosporic pH 2.0 and acetonitrile as 35 and 65%, respectively as a mobile phase at a flow rate of 2.0 mL/min and the run time was 2 min. Degassing was achieved via filtration through a 0.45 μm Millipore membrane filter and sonication for 10 min. The injection volume was 20 µl and detection was at 210 nm. The HPLC system was operated at 25 °C. Data were collected with a Breeze Chromatography Manager Data Collection System. A daily standard calibration curve (6 standards ranging from 10 to 200 µg/ml was prepared to determine the unknown DS concentration.

2.3. Preparation of stock solutions

The stock standard solution of DS was prepared in methanol at a concentration of 0.5 mg/mL and stored in 4.0 mL glass vials in a refrigerator at 4 °C. Different working standard solutions of DS(10–200 µg/ml) were prepared by diluting of the above mentioned stock solution in pure methanol and were stored at 4 °C.

2.4. Validation of diclofenac HPLC assay

The RP-HPLC method for DS assay was validated in term of accuracy, reproducibility, linearity, specificity, LOD, LOQ, and robustness according to ICH Harmonized Tripartite Guidelines. Three standard calibration curves were prepared at different times (at least three months) to evaluate the linearity, precision, accuracy and stability.

2.4.1. Specificity

The specificity of the HPLC method was evaluated to ensure that there was no interference from the excipients present in the formulations. The specificity was studied by injecting the excipients.

2.4.2. System specificity

The system suitability was assessed by six replicate analyses of DS at a concentration of 20 μg/ml. The acceptance criterion was ±2% for the percent relative standard deviation (% RSD) for the peak area and retention times for DS.

2.4.3. Linearity and range

Linearity is the ability to obtain test results that are directly proportional to the concentration of the analyte. Linearity was determined by three injections of seven different DS concentrations (10, 20, 80, 120, 160 and 200 µg/ml). The average peak areas were plotted against concentrations. Then linearity was evaluated using the calibration curve to calculate coefficient of correlation, slope and intercept. In general, a value of correlation coefficient (r 2 ) > 0.998 is considered as the evidence of an acceptable fit for the data to the regression line

2.4.4. Accuracy

The accuracy of an analytical method expresses the nearness between the expected value and the value found. It is obtained by calculating the percent recovery (R%) of the analyte recovered. In this case, to evaluate the accuracy of the developed method, successive analysis (n = 3) for three different concentrations (200 ng/ml, 120 ng/ml and 20 µg/ml) of standard DS solution were performed using the developed method. The data of the experiment were statistically analyzed using the formula [% Recovery = (Recovered conc. /Injected conc.) × 100] to study the recovery and validity of the developed method. The mean recovery should be within 90–110% to be accepted.

2.4.5. Precision

Precision of a measurable technique is the degree of agreement among individual tests, when the technique is applied repetitively to analyze multiple replicates in three different occasions. The intraday precision was assessed by analyzing the calibration curves of six replicates of different concentrations of DS within the same day. The inter-day precision was determined by analyzing of six replicates of different concentrations of DS on three different days. The total precision of the method was expressed as the relative standard deviation (%RSD). In the current method development and validation protocol, precision was determined by six replicate analyses at a concentration of 120 µg/mL of standard DS solution using the developed method and % RSD ≤ 2% was accepted.

2.4.6. Limit of detection and limit of quantification

LOD is the lowest concentration in a sample that can be detected, but not necessarily quantified under the stated experimental conditions. LOQ is the lowest concentration of analyte that can be determined with acceptable precision and accuracy. These two parameters were calculated using the formula LOD = 3.3 × SD/S and LOQ = 10 × SD/S, where SD = standard deviation of response (peak area) and S = slope of the calibration curve.

2.4.7. Robustness

The robustness of an analytical procedure is the measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions.

2.4.8. Analysis of a marketed formulation

To determine the content of Diclofenac Sodium in conventional tablet (Brand name: voltaren®, Clofen®, voltaic®, Rapidus® and Rofenac® label claim: 50 mg Diclofenac per tablet), twenty tablets were weighed, their mean weight determined and finely powdered. The weight of the tablet triturate equivalent to 50 mg of Diclofenac Sodium was transferred into a 50 mL volumetric flask containing 30 mL methanol, sonicated for 30 min and diluted up to 50 mL with methanol. The resulting solution was centrifuged at 3000 rpm for 5 min and the drug content of the supernatant was determined (1000 μg/mL). Supernatant was taken and after suitable dilution the sample solution was then filtered using 0.45-µm filter (Millipore, Milford, MA). The above stock solution was further diluted to get sample solution of 50 μg/mL. A 20 μl volume of sample solution was injected into HPLC, six times, under the conditions described above. The peak areas were measured at 210 nm and concentrations in the samples were determined using multilevel calibration developed on the same HPLC system under the same conditions using linear regression equation.

2.5. Data and statistical analysis

In vitro results were expressed as mean ± SD of at least three replicates. The HPLC results of DS were calculated using linear regression without weighting, according to the equation: Y = 0.0225x + 0.8858, where Y is the area under the peak (AUP) ratio of the drug and X is the concentration of DICNA. The % RSD was calculated for all values. Student’s t -test was used to inspect the concentration difference at each day and one-way analysis of variance (ANOVA) was used to assess the reproducibility of the assay using IBMSPSS Statistics 21. The level of confidence was 95%.

3.1. Development of HPLC method

In Fig. 1 , chromatogram A represents the blank mobile phase, and chromatogram B represents DS with an average retention time of 1.345 ± 0.127 min and with no interfering peaks. This is an indication of the specificity of the developed HPLC method. The retention time was comparable with the shorter published data for DS.

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HPLC chromatograms of mobile phase spike with 50 µg/ml of lidocaine (1) (chromatogram A) and HPLC chromatograms of mobile phase containing 10 µg/ml DS(2) and 50 µg/ml of lidocaine (1) (chromatogram B).

3.1.1. System suitability

The system suitability was assessed by six replicate analyses of DS at a concentration of 20 µg/ml. The acceptance criterion was ±2% for the percent relative standard deviation (% RSD) for the peak area and retention times for DS ( Table 1 ).

System suitability study.

3.2. Specificity

The specificity of the method was monitored by analyzing the placebo and standard solution. No peak was detected close to the retention time of DS, which proved the high degree of specificity of the method ( Fig. 1 ).

3.3. Linearity, limit of quantification, limit of detection

Linear relationship (r > 0.999) was observed between AUP of DS and the corresponding concentrations over 10–200 µg/mL ( Fig. 2 ). The mean linear regression equation of the peak area ratios (Y) versus drug concentrations (X) of DS was typically of the form Y = (b ± S.D.) X ± (a ± S.D.) and it was Y = 0.0222 X − 0.9196 for DICNA. The LOQ of this assay was 3.9 ng/mL with a corresponding relative standard deviation of 4.8 and 4.0%. The LOD was 13.1 ng/mL at a signal-to-noise ratio of >3.

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Standered calibration curve of Diclofenac ranging from 10 to 200 µg/ml in mobile phse. Each point represent the mean of 6 determination.

3.4. Precision and accuracy

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out 6 times and the percentage recovery and % relative standard deviation was calculated. From the data obtained, recoveries of standard drugs were found to be accurate ( Table 2 ). The %CV of interday and intraday precision obtained was less than 7% for the drugs as recommended by ICH guidelines

Inter- and Intra-day statistics.

3.5. Recovery, accuracy, and precision

Within-day precision and accuracy of the method were determined from replicate analysis (n = 6) of DStest standards at concentrations within the linear range of the assay for each drug ( Table 2 ). The reproducibility of the assay was evaluated by comparing the linear regressions of three standard plots prepared on three different days over a 3-week period. The mean correlation coefficient was >0.999 with % R.S.D. of the slopes of the three lines beign 4.3%. ANOVA of the data indicated no significant difference (p > 0.05) in the slopes, intra- and inter-day, of the calibration curves. The results confirmed the reproducibility of the assay method. The mean percentage recovery of 10–200 µg/mL DS was 95.2 ± 4.9%.

3.6. LOD and LOQ

Signal-to-noise ratios of 3:1 and 10:1 were obtained for the LOD and LOQ respectively. The LOD and LOQ were found to be 2 μg/mL and 4 μg/mL for Diclofenac Sodium.

3.7. Robustness

The robustness of the method was studied by deliberate changes in the method like alteration in pH of the mobile phase, percentage organic content, changes in the wavelength. It was observed that there was no marked changes in the chromatograms demonstrate that the HPLC methods have developed are robust.

3.8. Stability studies

Fig. 3 shows that DS was stable in the processed samples held in the autosampler at 4° and −35 °C for three weeks. There were no evident changes observed in the elution profile and chromatographic reports. All the values of %CV were within the range provided in FDA guidelines (i.e., less than3%), indicating the fact that the developed method is stable ( Fig. 1 ). However, the samples lost 26.3% (RSD of 6.7%) of its nominal concentration within 3 days if stored in the autosampler at room temperature. Therefore, it is not recommended to keep DS in the autosampler for longer than overnight to ensure reproducibility of the assay. The freeze–thaw temperature cycles did not significantly (p > 0.05) affect the stability of DS in first cycle, after 72 h, with the mean calculated values within 2.3% of the nominal concentration, while after the third cycle, 11.8% of DS was lost with an RSD of 5.6%. Unexpectedly, exposing DS to drastic conditions revealed that DS is stable in both 2 M HCl and 1 N NaOH solutions even after boiling, losing only 4.3% and 5.2% respectively (RSD of 5.3 and 6.6%) of its nominal value.

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stability of diclofenac in different temperature for three weeks (n = 6)

3.9. Analysis of a diclofenac in the dosage form

Experimental results of the amount of Diclofenac Sodium in the selected commercial tablets, expressed as a percentage of label claim were in good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present. The drug content was found to be 99.93% for Diclofenac Sodium five different lots of Diclofenac Sodium tablets were analyzed using the proposed procedures as shown in Table 3 .

Analysis of commercial formulation Diclofenac Sodium (50 mg).

4. Discussion

Many studies reviewed the use of C 18 for separation of the drug using acetonitrile as the main solvent. These HPLC methods reported in have several disadvantages, including unsatisfactory separation times, poor resolution, complicated solvent mixtures with gradient elution, and long analysis times. The aim of this study was to develop and validate a new simple and rapid analytical method for DS with short run time. The chromatographic runtime is also short. Therefore, the developed analytical method can be reliably employed as an assay method for pharmaceutical study of any dosage form containing DS.

5. Conclusions

A simple, rapid and sensitive analytical method was developed and validated for the analysis for DS. The chromatographic runtime was also short.

Statistical analysis proves that the method is suitable for the analysis of Diclofenac Sodium in pharmaceutical formulation without any interference from the excipients. It may be extended to study the degradation kinetics of Diclofenac Sodium and also for its estimation in plasma and other biological fluids.

Acknowledgement

The authors gratefully acknowledge Female Center for Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia for their support.

Conflict of interest

The authors have no conflict of interests to disclose other than what has been acknowledged above.

Peer review under responsibility of King Saud University.

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