Stability Indicating Assay Method Development and Validation of HPTLC Method for Estimation of Prochlorperazine maleate in Bulk and Tablet Dosage Form

Prochlorperazine Maleate ^[4-10] chemically known as Bis ((2Z)-but-2-enedioic acid); 2-chloro-10- [3-(4- methylpiperazin-1-yl) propyl]-10H-phenothiazine, falls under category Antiemetic and Antipsychotic Tranquilizer. It is used as a first-generation antipsychotic drug that is used for the treatment of severe nausea and vomiting, as well as short-term management of psychotic disorders Fig.1. Chemical Structure of Prochlorperazine maleate Fig.1. Chemical Structure of Prochlorperazine maleate such as generalized non-psychotic anxiety and schizophrenia. The drug Prochlorperazine maleate was approved by FDA in 10^th June1999.

Fig.1. Chemical Structure of Prochlorperazine maleate

Experimental:

Materials:

Drug Prochlorperazine was obtained from Mehta Pharmaceutical Industries, Virar, Maharashtra as Gratis sample.

Instrument:

The samples were applied in the form of bands of 6 mm width with a CAMAG 100 µl sample syringe using Linomat V automatic sample applicator on pre-coated HPTLC silica gel aluminium plate 60 F₂₅₄ (10 cm × 10 cm with 0.2 mm thickness, E. Merck, Germany). CAMAG TLC scanner 4 was used for the densitometric scanning of the developed chromatogram having slit dimension 4 × 0.45 mm. All the drug and chemicals were weighed on Mettler Toledo electronic balance (ML204).

Preparation of Solutions:

Preparation of standard stock solution (1000µg/ml): For the preparation of stock solution 10mg of drug sample was weighed accurately and transferred in 10ml of volumetric flask and ethanol was added up to the mark to obtain appropriate concentration. The solution was prepared and used as standard stock solution. Preparation of working standard solution (100µg/ml): For the preparation of working stock solution, the aliquot of 1ml from stock solution was taken and diluted to 10ml with ethanol in 10ml of volumetric flask. The sample solution prepared was of 100µg/ml. Preparation of working standard solution (600µg/ml): 6ml of the Prochlorperazine maleate stock solution was transferred to a 10ml volumetric flask and diluted with ethanol to the desired concentration. Preparation of stock solution (Tablet dosage form): The powdered tablet equivalent to 10mg of drug was accurately weighed and diluted with the solvent in 10ml volumetric flask to prepare 1000µg/ml stock solution.

Chromatographic Condition:

Samples of various concentrations were spotted using a micro-syringe and a Camag Linomat V on a pre-coated silica gel aluminium Plate 60 F₂₅₄ in the shape of bands with a band width of 6 mm. Linear ascending development was carried out in a twin trough glass chamber. The mobile phase consisted of Methanol: n-butanol: triethylamine (9: 0.9: 0.1 v/v/v). The optimized chamber saturation time before chromatographic development was 20 min at room temperature. The length of the chromatographic run was 7 cm. Densitometric scanning was performed using a Camag TLC scanner 4 with the winCATS software. All measurements were made in the absorbance mode at 258 nm, with a slit dimension of 4 × 0.45 mm, scanning speed of 20 mm/s. The radiation source was deuterium lamp discharging a continuous UV spectrum between 190 and 400 nm.

Method Validation

Method validation was done as per ICH guideline Q2(R1) for which parameters like Linearity, Accuracy, Precision, Limit of detection, Limit of Quantitation, Specificity and Robustness study were performed.

Linearity:

The linearity was tested by spotting 2, 4, 6, 8, 10µl of the standard solution containing 100µg/ml of Prochlorperazine maleate. In the 200-1000ng/band concentration range, Prochlorperazine maleate's linearity responses were assessed. Following six independent chromatographic runs of the sample solution, the mean peak area of Prochlorperazine maleate was determined. The calibration curves were created by plotting the peak area versus the concentration.

Accuracy:

Accuracy was established by computing the drug recovery using the conventional addition method at three distinct drug concentration levels (80%, 100%, and 120%). Standard was made according to procedure. To acquire a final concentration, range of 360, 400, and 440ng/band for Prochlorperazine Maleate, 1µl (200ng/band of Prochlorperazine Maleate) of solution containing Prochlorperazine Maleate was spotted on a TLC plate along with a standard calibration curve. The plate was processed, dried, and subjected to a photometric analysis. Using the associated calibration curve calculations, the amount of medication was determined. Each level's amount detected, recovery percentage, and mean recovery were computed and documented

Precision: The precision study was performed by repeating the analysis of Prochlorperazine maleate at different time intervals like within the same day (Intraday precision) and on other day (Inter-day precision) and % RSD was calculated.

Limit of Detection and Limit of Quantitation: LOD and LOQ of the drug were derived by calculating the signal-to-noise ratio using the following equation as per the ICH guideline.

LOD = 3.3 × ????/S

LOQ = 10 × ????/S

where, ???? = standard deviation of the response

            S = slope of the calibration curve

Specificity:

Prochlorperazine maleate was taken at a concentration of 600ng/band. By comparing the R_f and spectra of the marketed formulation to pure API, the band's specificity was verified. By contrasting the spectra at the peak start (S), peak apex (M), and peak end (E) positions of the band, the 258nm wavelength for identifying the peak purity of Prochlorperazine maleate was determined. (Fig 6)

Robustness:

The impact of slight, intentional changes to the analytical conditions on the drug peak regions was investigated. Change in wavelength and change in chamber saturation duration (5 min) and an evaluation of %RSD was calculated.

Analysis of Prochlorperazine maleate in marketed formulation:

To determine the content of Prochlorperazine maleate from Tablet (SANOTILL tab with label claim 5mg), a quantity of tablet powder equivalent to 10mg was transferred to 10ml volumetric flask containing ethanol and sonicated for 5-10mins, solution was filtered using Wattmann filter paper, from this 1ml volume was taken and transferred into 10ml volumetric flask and diluted to mark with ethanol to obtain working standard solution of Prochlorperazine maleate 100µg/ml. 6μl of this solution was applied on TLC plate followed by development and scanning at 258nm. The analysis was repeated for 3 times.

Forced Degradation Study:

Acid degradation:

The powdered tablet equivalent to 10mg was weighed and transferred in 10ml volumetric flask and dissolved in ethanol to obtain 1000µg/ml, the solution then sonicated for 15 mins and filtered using Whatmann filter paper (0.45µm). 1ml aliquot from this solution was taken among three separate 10ml volumetric flasks and mixed with 0.3ml each of 0.5N, 1N, and 2N HCl and set aside for 72 hours at 80^o C reflux. The flask solution was then adjusted to the proper concentration using ethanol and neutralised with 0.5N, 1N, and 2N NaOH, respectively. A chromatogram was created using 6µl of the resultant solution (600ng/band) and recorded at 258 nm. The quantity of drug affected acid was calculated. 

Base degradation:

The powdered tablet equivalent to 10mg was weighed and transferred in 10ml volumetric flask and dissolved in ethanol to obtain 1000µg/ml, the solution then sonicated for 15 mins and filtered using Whattman filter paper (0.45µm). 1ml aliquot from this solution was taken among three separate 10ml volumetric flasks and mixed with 0.3ml each of 0.5N, 1N, and 2N NaOH and set aside for 72 hours at 80^o C reflux. The flask solution was then adjusted to the proper concentration using ethanol and neutralised with 0.5N, 1N, and 2N HCl, respectively. A chromatogram was created using 6µl of the resultant solution (600ng/band) and recorded at 258 nm. The quantity of drug affected by bases was calculated.

Oxidative Degradation:

The powdered tablet equivalent to 10mg was weighed and transferred in 10ml volumetric flask and dissolved in ethanol to obtain 1000µg/ml. the solution then sonicated for 15 mins and filtered using Whatmann filter paper (0.45µm). 1ml aliquot from this solution was taken among three separate 10ml volumetric flasks and mixed with 1%, 3% H₂O₂ solution were added in each flask respectively and allowed to set for 48 hrs at room temperature. After the completion of 48 hrs the chromatogram was obtained by spotting 6µl solution from each flask at 258nm. The quantity of drug affected by the peroxides was calculated.

Photolytic Degradation:

The powdered tablet equivalent to 10mg was weighed was allowed to degrade under UV light at short wavelength for 48 hours. After completion of 48 hours 1mg of drug was dissolved in ethanol using 10ml volumetric flask (100µg/ml). From this solution chromatogram was taken by spotting 6µl at 258nm. The quantity of drug affected by UV light was calculated.

Thermal Degradation:

The powdered tablet equivalent to 10mg was weighed and allowed to degrade in hot air oven at 80^o C for 48 hours. After completion of 48 hours, 1mg of drug was dissolved in ethanol using 10ml volumetric flask (100µg/ml). From this solution chromatogram was taken by spotting 6µl at 258nm. The quantity of drug affected by temperature was calculated. 

RESULTS AND DISCUSSION

Selection of Mobile phase:

Methanol: n-butanol: Triethyl amine (9: 0.9: 0.1) v/v/v respectively were mixed properly in twin trough chamber and the chamber was then saturated with this mobile phase.

Wavelength Selection:

In the present study drug solutions of 10 µg/ml Prochlorperazine maleate was prepared in ethanol and scanned in the UV region of 200-400 nm. The overlain spectrums were taken and the wavelength at which the drug showed good absorbance was selected as a detection wavelength (258 nm). (Fig 2.)

Method Optimization:

The chromatographic conditions were optimized in order to develop an HPTLC method for the simultaneous estimation of Prochlorperazine maleate. (Fig 3.)

Method Validation:

Linearity: Linear regression data for the calibration plot revealed good linear relationship between area and concentration over the ranges 200-1000 ng/band. The linear equation for calibration plot was y = 5.2086x + 183.59 with correlation coefficient (R²) being 0.9991. (Table 2 and 3) (Fig 4.)

Acceptance Criteria:

The correlation coefficient value should not be less than 0.995 over the working range.

Accuracy: When used to evaluate the recovery after spiking with three concentrations of standard, 80%, 100% and 120%, the proposed method showed percentage recovery rates between 99.47- 101.29.

Acceptance Criteria

Individual % Recovery and Mean % Recovery at each level should be between 98 and  102%, with %RSD not to exceed 2.0.

Precision: The %RSD of Prochlorperazine maleate was found to be 1.802 which was within the limit (<2). This indicates that the developed method has good repeatability. The % RSD of Prochlorperazine maleate was found to be 1.09- 1.75(Intra-day precision) and 0.559- 0.699 (Inter day precision). This indicates the method is precise.

Acceptance criteria

RSD for six preparations should not be more than 2.0%. The mean assay result obtained in the intermediate precision study and that of the method precision study should not deviate in absolute terms by more than 2%. Overall RSD of method precision and intermediate precision should not be more than 2.0%

Limit of detection and Limit of Quantitation:  The LOD and LOQ of the developed method were found to be 35.48 and 107.53 ng/band, respectively, for Prochlorperazine maleate.

Acceptance criteria

LOQ should be 3 times higher than LOD.

Robustness: Acceptance criteria: %RSD value for three replicate injections of standard injection should not be more than 2.0%. 

Forced Degradation:

Acid Degradation: Prochlorperazine maleate was found sensitive to Acidic conditions. After 72hrs at 80ºC with reflux. Two degradant products were found having %degradation 14.58, 7.20 and 14.04, 6.85 for API and Formulation respectively.

Base Degradation: Prochlorperazine maleate was found sensitive to Basic conditions. After 72hrs at 80ºC with reflux. Two degradant products were found having % degradation 8.19, 9.04 and 8.05, 9.20 for API and Formulation respectively.

Oxidative degradation: Prochlorperazine maleate was found sensitive to Oxidative conditions. After 48 hrs at room temperature one degradant product was found having % degradation 19.80, 20.24 for API and Formulation.

Thermal degradation: Prochlorperazine maleate was found sensitive to Temperature. After 48 hrs at 80ºC. Two degradant products were found having % degradation 14.25, 15.17 & 14.89, 15.61 for API and Formulation respectively.

Photolytic (light control) degradation: Prochlorperazine maleate was found sensitive to Photolytic conditions. After 48 hrs at short wavelength one degradant product was found having % degradation 14.41, 14.80 for API and Formulation respectively.

Photolytic (light control) degradation: Prochlorperazine maleate was found sensitive to Photolytic conditions. After 48 hrs at short wavelength one degradant product was found having % degradation 16.06, 18.37 for API and Formulation respectively.

Tables And Figures:

Table 2. Statistical data for Prochlorperazine maleate by HPTLC method

Table 3. Data derived from Accuracy experiment

Table 4. Method Precision Data

Table 9. Summary of validation parameters

Table 11. Optimized condition of forced degradation for Prochlorperazine maleate

Fig.2 Spectrum of the drug sample at (λ_max = 258 nm)

Fig.3 Photograph of developed plate of HPTLC plate of Prochlorperazine maleate

Fig.4 3-D densitogram of calibration curve for Prochlorperazine maleate

Fig 5. Calibration curve of Prochlorperazine maleate at 258 nm

Fig 6. Overlaying spectrum of Prochlorperazine maleate API and Formulation (λ_max = 258nm)

Fig 7. Densitogram of API of Prochlorperazine maleate for Acid degradation (1N HCl)

Fig 8. Densitogram of Formulation of Prochlorperazine maleate for Acid degradation (1N HCl)

Fig 9. Densitogram of API of Prochlorperazine maleate for Base degradation (0.5N NaOH)

Fig 10. Densitogram of Formulation of Prochlorperazine maleate for Base degradation (0.5N NaOH)

Fig 11. Densitogram of API of Prochlorperazine maleate for oxidative degradation (3% H₂O₂)

Fig 12. Densitogram of Formulation of Prochlorperazine maleate for Oxidative degradation (3% H2O2)

Fig 13. Densitogram of API of Prochlorperazine maleate for Thermal degradation (70º-80ºC)

Fig 14. Densitogram of Formulation of Prochlorperazine maleate for Thermal degradation (70º-80ºC)

Fig 15. Densitogram of API of Prochlorperazine maleate for Photolytic (light control) degradation

Fig 16. Densitogram of formulation of Prochlorperazine maleate for Photolytic (light control) degradation

Fig 17. Densitogram of API of Prochlorperazine maleate for Photolytic (Dark control) degradation

Fig 18. Densitogram of formulation of Prochlorperazine maleate for Photolytic (Dark control) degradation