Applying green analytical chemistry (GAC) for development of stability indicating HPLC method for determining clonazepam and its related substances in pharmaceutical formulations and calculating uncertainty

2.3.2.1 Preparation of stock solutions and standards

Stock standard solution of clonazepam, related compound A and related compound B was prepared in mobile phase at a concentration of 0.2 mg/ml. Series of dilutions at concentration levels of 4–140 μg/mL were obtained from stock solution by the appropriate dilution in mobile phase.

2.3.2.2 Sample preparation

Samples, of randomly selected clonazepam tablets, were dissolved and diluted to the appropriate volumes to get a concentration of 0.04 mg/ml using mobile phase as solvent. All samples were filtered through nylon sample filter (Whatman, 0.22 μm).

2.3.2.3 Method development and chromatographic conditions

A variety of mobile phases were investigated in the development of a stability-indicating HPLC method for the analysis of clonazepam and its related compound in pharmaceutical preparations. The suitability of mobile phase was decided on the basis of green analytical chemistry principles, selectivity, sensitivity of the assay, stability studies, and separation of clonazepam from the known related substances (A&B) beside the impurities formed during forced degradation studies. The elution was carried out on a BDS C₈ Hypersil column (250 mm × 4.6 mm, 5 μm particle size) from Thermo scientific (Massachusetts, USA). All analyses were performed at ambient temperature under isocratic conditions with a mobile phase of isopropanol: 2% sodium dodecyl sulfate (SDS): 0.05 M sodium acetate buffer (pH 3.5 ± 0.05) in ratio (20:25:55, v/v) at a flow rate of 1.5 mL/min, using DAD detector at 254 nm.

2.3.2.4 Stability indicating capabilities of the proposed method

The stability-indicating capability of the method was determined by its ability to separate the reference degradation substances (A&B) from the drug of interest in addition to subjecting reference solution of clonazepam (40 μg/mL) to forced degradation conditions by acidic, basic, oxidative, and photolytic conditions to evaluate the interferences in the quantitation of clonazepam. Standard solutions in 1 M hydrochloric acid and 1 M sodium hydroxide were used for the acidic and basic hydrolysis, respectively. Both solutions were kept at ambient temperature for 12 h. For oxidative degradation, solutions were prepared in hydrogen peroxide (3%) solution and kept at ambient temperature for 4 h. Photodegradation was induced by irradiating the neutral solution at 1.2 million lux hours for 24 h. The distance between the light source and the sample was maintained at 25 cm. These solutions were diluted with mobile phase to final concentration of 40 μg/ml and were injected into chromatographic system.

2.3.2.5 Linearity

Linearity was evaluated by determining the response of a series of dilutions of thirteen standard solutions from clonazepam and nine standard solutions of its related compounds (A&B) at a concentration range of 4–140 μg/mL and 4–64 μg/mL, respectively. Each dilution was injected in triplicate to plot the calibration curve. Slope, intercept, and regression coefficient (R²) of the calibration curves were calculated to ascertain linearity of the method.

2.3.2.6 Precision

For method repeatability, assay of authentic samples solutions was repeatedly performed six times on the same day (intra-day). For reproducibility, freshly prepared solutions at aforementioned concentration level were analyzed at different days (inter-day), and results were statistically evaluated in terms of % RSD. The authentic samples were prepared by addition of the suitable amount of standard and the related compounds (A&B) to the placebo. These samples were handled as descried under assay preparation to give the desired concentration.

2.3.2.7 Accuracy

Accuracy was calculated as the deviation of the mean from nominal concentration. To assess accuracy, freshly prepared placebo of the clonazepam pharmaceutical formulations was spiked with various amounts of clonazepam and its related compounds (A&B) to obtain the concentration levels of 30, 40 and 50 μg/ml. Each solution was injected in triplicate.

2.3.2.8 Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD is taken as the lowest concentration of an analyte in a sample that can be detected, but not necessarily quantified, under the stated conditions of the test while the LOQ is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated conditions of test. They can be calculated from the linear calibration curve where LOD is the 3 times of residual standard deviation of the regression line (at the lower concentration) divided by the slope, while LOD is ten times the division product (Shrivastava and Gupta, 2011).

2.3.2.9 Robustness

In order to check the robustness, the effect of small but deliberate variations in the chromatographic conditions was evaluated. The conditions studied were flow rate (altered by ±0.01 mL/min), and pH of buffer solution (altered by ±0.1). These chromatographic variations were evaluated for retention time, peak area, number of theoretical plate and asymmetry factor for each clonazepam and its related compound.

2.3.2.10 Selectivity and system suitability

Selectivity of this method was indicated by the absence of any excipient interference at the retention times of the peaks of clonazepam and its related compounds with an acceptable resolution in the system suitability solution. The absence of interfering peak was evaluated by injecting a blank sample consisting of diluent and placebo. The test is only valid if the resolution between clonazepam related A and clonazepam related B is more than 2 as stated in the relevant monograph in the USP 36 (USP, 2013).

2.3.2.11 Uncertainty of the method

Among the different sources of uncertainty, the uncertainty associated with calibration appears to be the most important source in the overall uncertainty (ICH guideline Q2B, 2005).

The quantification of clonazepam by was assess through the calibration curve equation (Y = aX + b), where Y is the response, a is the slope, X is the concentration of clonazepam and b is the intercept. Based on this information, the uncertainty of HPLC’s result was estimated by the following equation: $${\text{U}}_{\text{A}\%}=\text{A}\%\times t_{1-\mathrm{\alpha }/n-2}\times \sqrt{\frac{{({\sigma }_Y+{\sigma }_b)}^2}{{(Y-b)}^2}+{\left(\frac{{\sigma }_a}{a}\right)}^2}$$ where U_A% is the expanded uncertainty, t1-α/n-2 is the t-student for confidence level of 1-α and n-2 degrees of freedom (n correspond to the numbers of standard employed to obtain calibration curve), A% is the result of sample (in percentage), σY is the standard deviation of the areas obtained in chromatograms of sample, Y is the average of the areas obtained in chromatograms of sample, σb is the error for the intercept and σa is the error for the slope.

3.2.3.1 Analysis repeatability

It was evaluated by carrying out the analysis of the six homogenous solutions of same test sample and content of related substances. The determinations were carried out one after the other under conditions as similar as possible. The relative standard deviation was calculated from the results of the obtained observations (Table 3).

3.2.3.2 Intermediate precision

The intermediate precision of the method was checked by determining precision on a different instrument, analysis being performed in different laboratory on a different day. The relative standard deviation was calculated from the results of the obtained observations.

In all cases the RSD was lower than 2 (Table 3).