Implementation of QbD Strategy in RP-HPLC Method Development and Validation for Lasmiditan in Pharmaceutical Formulation

Fig.no.2 UV Spectrum of Lasmiditan

2.5. Development of method

The chromatographic conditions were optimized to obtain good peak parameters such as a good peak shape, the lowest tailing factor, a short retention time, and a high theoretical plate number. Initially, mobile phases consisting of various buffer systems were investigated, but the required system compatibility characteristics could not be achieved. Different types (X-Terra C18 (250×4.6 mm×5μm), Extend C18 (250 ×4.6 mm, 5μm), Synergy Hydro C18 (250× 4.6 mm×4μm) and Luna C18 (250×4.6mm×5μm) and different lengths of columns (X-Terra C18 (150× 4.6 mm ,5μm), Extend C18 (150×4.6mm, 5μm), Synergy Hydro C18 (150×4.6 mm, 4μm) and Luna C18 (150×4.6mm,5μm) were tested but showed poor system compatibility parameters. Good peak parameters were obtained using an Sunfire C18 Column, (4.1 x 250mm, 5.0µm) column.

Different ratios of water/methanol, water/ acetonitrile, and 0.01N KH₂PO₄/acetonitrile mixtures were tested as mobile phases. Initially, acetonitrile and 0.1% OPA (60/40, V/V) were used as mobile phase, which resulted in a very long analysis time. Under these conditions, the sample solution was injected to detect both impurities that may interfere with the analyte peak and the presence of drug matrix components that may remain longer on the column under the specified conditions. Furthermore, the sample solutions were injected sequentially into the system with an analysis time of 10 min and it was observed that no impurities were carried over from one analysis to the next. Therefore, the analysis time was set to 10 min. Furthermore, the column temperature was chosen as 30°C due to its many advantages such as high column efficiency, low column pressure favorable peak shape, and cost-effectiveness.

The spectral pattern of Lasmiditan was comprehensively investigated using different solvents (ultrapure water, ethanol, methanol) for spectrophotometric analysis. Since the standard solutions of Lasmiditan have maximum absorbance at 254 nm wavelength, the absorbance values of the standard and sample solutions were measured at this wavelength.

2.5 Development by QbD Approach

The method was optimized using central composite design (CCD). The initial trials are needed to optimize the final method. Organic concentration, Flow rate and column temperature were needed to be optimized. So, CCD was used to optimize these parameters which were varied over three level (high, mid and low). different ranges of four parameters ranging from 55-65%, Aqueous solvent, column temperature 27°C and 33°C and 0.9-1.1ml/min flow rate respectively were taken and counter and 3D surface plot showing the effect of each parameter on retention Time, theoretical plates and asymmetry (CQA) were generated. A desirability function applied to the optimized conditions to predict retention time, asymmetry, theoretical plates, and peak area.

Table no. 2.  Design summary of CCD

Table no. 3. Central composite experimental design matrix with response

Statistical Analysis and Final Optimization

The responses obtained after carrying out the above trial runs were fed back to Design Expert software and plots like 3D-response surface plots and Graph plots were plotted. These graphs demonstrated how important procedure factors affected the chosen quality criteria. The analysis of these plots was used to estimate which method parameter gave the most acceptable responses. Thus, the method's final critical method parameters and optimal chromatographic conditions were established in light of these observations. Additionally, the significance of each method parameter chosen for the study was determined using a statistical analysis tool like ANOVA for each response using the p-value (probability).

2.6 Validation of the Optimized Method:

According to ICH guidelines method validation can be defined as   “Establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics”.

Validation of analytical procedures was performed for Lasmiditan using the following parameters.

Specificity: The following solutions will be made and injected (peak purity double-checked) to show the accuracy of the procedure.

1. Blank (methanol 100% as a diluent)

2. Standard solution

3. Sample solution

4. Placebo treatments

Linearity: The method's linearity was examined at six distinct Lasmiditan concentrations ranging from 12.5µg/ml to 75µg/ml to produce the calibration curve, peak area was plotted on the x axis against concentration. The regression line equation and correlation coefficient values were computed.

Accuracy (% Recovery): The accuracy of the method was confirmed by a recovery study from marketed formulation at 3 levels of standard addition. The percentage recovery of Lasmiditan was found out.

Precision: The precision is reported in terms of relative standard deviation (RSD). There are three levels of precision: repeatability, reproducibility and intermediate precision. It takes place using a sample API.

• Repeatability (Intraday precision)

• Intermediate precision (Inter day precision)

Limits of Detection and Quantitation: Limits of detection (LOD) and limit of quantitation (LOQ) were determined from the signal-to-noise ratio. The detection limit was referred to as the lowest level of concentration resulting in a peak area of three times the baseline noise. The quantitation limit was referred to as the lowest possible concentration that provided a peak area with a signal-to-noise ratio higher than ten.

LOD = 3.3 × δ/S

LOQ = 10× δ/S

Robustness: To demonstrate the robustness of the procedure, the following optimized conditions were slightly varied.

• Acetonitrile and 0.1% OPA (60:40%v/v) of mobile phase
• 0.98 ml/min of flow rate.
• 30? of temperature.

2.7 Application of analytical methods to commercial formulations

Preparation of Sample stock solutions: 10 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (1000µg/ml of Lasmiditan)

Preparation of Sample working solution: 0.5ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (50µg/ml of Lasmiditan)

3. RESULTS AND DISCUSSION

3.1. Determination of the wavelength

Standard solutions prepared using ultrapure water were scanned in a spectrophotometer device in the 200–400 nm wavelength range. The maximum absorption wavelength of Lasmiditan was determined as 254 nm.

3.2. Development of methods

The conditions of the developed analytical methods are given below. Sunfire C18 Column, (4.1 x 250mm, 5.0µm) at a constant temperature of 30? was used for separation in the chromatographic method. The mobile phase contained Acetonitrile and 0.1% OPA (60:40%v/v). At a flow rate of 0.98min/ml, isocratic elution was carried out and 254 nm was chosen for detection. Spectrophotometric method; the spectral pattern of Lasmiditan was extensively analyzed. To determine the wavelength at which Lasmiditan standard solutions maximally absorbed UV light, the standard solutions were scanned in the 200–400 nm wavelength range. A wavelength of 254 nm was selected for detection.

3.3 Statistical Analysis of Experimental Data by Design-expert Software:

In order to understand the results, contour plots and 3D plot were generated after processing all data using the Design Expert® software (Fig.3). It shows the two-dimensional contour plot as a function of Column temperature, Flow rate and organic ratio. Based on the color code, the working region can be easily identified. Retention time maps represent the value of the retention time, with warm “red” colors indicating larger retention time, cold “blue” colors lower and light green to yellow color represent intermediate retention time.

Table no. 4 ANNOVA for CCD

Fig.no.3 2D and 3D contour plots of retention time as a function of Column temperature, Flow rate and organic ratio.

Fig.no.4 Overall desirability of final method

3.4 Method Validation

The analytical methods developed for the quantification of Lasmiditan in oral formulations were validated for “selectivity, system suitability, linearity, precision, sensitivity, robustness, and specificity” according to ICH Q2 (R1) guidelines. Standard, sample, and mobile phase solutions were injected into the chromatographic system to evaluate the selectivity of the chromatographic method.

Table no.5 final optimized HPLC chromatographic conditions

Fig.no.5 final optimized chromatogram

3.4.1 Linearity:

Six linear concentration of Lasmiditan (12.5-75µg/ml) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Lasmiditan was y = 16896x + 4262. Correlation coefficient obtained was 0.999 and Linearity plot was shown in Fig 8.

Fig.no.6 Linearity Plot

3.4.2. Precision: 

Results of precision are shown in Table No. 5, and it shows a standard deviation of less than 2, which indicates that the proposed method is precise.

Table no.6 Precision Studies

3.4.3 Accuracy:

Three Concentrations of 50%, 100%, 150% are Injected in a triplicate manner and %Recovery was calculated as 99.87%.

Table no.7 Accuracy studies

3.4.4 Specificity

It was tested for the interference of any degraded substance, and as shown in the chromatogram (Fig. no. 7,8,9) there is no other peak.

Fig.no.7 blank Chromatogram

Fig.no.8  Placebo Chromatogram

Fig.no.9 Standard chromatogram

3.4.5 Robustness

The results of robustness studies by varying mobile phase composition, flow rate and column temperature are shown in (Table no.8). RSD less than 2 indicated that the method is robust.

Table no.8 Robustness Studies

3.4.6. LOD :

Detection limit of the Lasmiditan in this method was found to be 0.1µg/ml.

Fig.no.10 LOD Chromatogram of Lasmiditan

3.4.7. LOQ :

Quantification limit of the Lasmiditan this method was found to be 0.32µg/ml.

Fig.no.11 LOQ Chromatogram of Lasmiditan

3.5 ASSAY OF MARKETED FORMULATION

The results of the assay are shown in Table No.9. The percent RSD was 0.47 which is within limits.

Table no.9 Assay of Formulation