Development And Validation of RP-HPLC Method for Transdermal Patch of Buspirone Hydrochloride

Anxiety disorders are the most prevalent psychiatric disorders with a current worldwide prevalence of 7.3 % [4.8 %-10.9 %] among them, specific phobias are the most common, with a prevalence of 10.3 %, then panic disorder (with or without agoraphobia) is the next most common with a prevalence of 6.0 %, followed by social phobia (2.7 %) and generalized anxiety disorder (2.2 %). Evidence is lacking as to whether these disorders have become more prevalent in recent decades. Generally speaking, women are more prone to develop emotional disorders with an onset at adolescence; they are 1.5 to 2 timesmore likely than men to have an anxiety disorder. Buspirone hcl is classified in the azapirone drug class. It has a strong affinity for serotonin 5HT1a receptors, where it acts as a partial agonist, which some researchers believe produces the preponderance of clinical effects. It also has a weak affinity for serotonin 5HT2 receptors and acts as a weak antagonist on dopamine D2 auto receptors. There is no effect on Benzodiazepine GABA receptors.

MATERIALS AND METHODS:

Table: 1 Chemical and Reagents Used

Table: 2 Instruments Used

Table 3 Preparation of Standard and Sample Solutions

Table: 4 Chromatographic Conditions

Table: 5 Method Validation Parameters

Identification of Drugs

Identification by Melting Point Determination

Melting point of Buspirone HCL has been determined. The melting points of the compounds were taken by open capillary method.

Table 6 Melting Point of Drugs

Identification by UV Spectroscopy.

Buspirone HCL

Fig. 1 Structure of Buspirone HCL

• Buspirone HCL standard solution was scanned in the UV-visible range (200–400 nm).
• The maximum absorbance was observed at 210 nm, which was selected as the detection wavelength.

Solution Stability

The solubility of Buspirone HCL was practically determined by weighing 100 mg of the drug and dissolving it in 100 mL of various solvents in separate volumetric flasks. The solutions were shaken at room temperature for a few minutes until equilibrium was reached. The solubility classification was based on the relative amount of solvent required to dissolve the drug.

Table 3 Solubility Table

Development and Optimization of RP-HPLC Method

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Buspirone HCL was scanned between 200-400 nm against diluents. Absorbance maxima Buspirone HCL detected at 240 nm. Chromatogram was taken at 210 nm, drug give good peak height and shape. So, 210 nm was selected for estimation of Buspirone HCL formulation. The UV overlain spectrum for Buspirone HCL shown in fig No.7.7

Selection of Chromatographic Conditions

Proper selection of the HPLC method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight, pKa and solubility. RP-HPLC was selected for the initial separation based on literature survey and its simplicity and suitability. To optimize the chromatographic conditions the effect of chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. Finally, the chromatographic condition was chosen that give the best resolution, symmetry and capacity factor for estimation of both drugs.

Selection of Column

For RP-HPLC Method, various columns are available but based on literature survey C-18 (id 4.6 x 250 mm, 5 µm) was selected over the other columns.

Preparation of Solution

Preparation of Mobile Phase

HPLC method was followed by isocratic elution technique. Mobile phase comprised of Acetonitrile: Methanol(55:45 v/v%) ratio because it elutes both drugs peak efficiently in short time with satisfactory resolution, tailing factor and theoretical plates.

Preparation of Standard Stock Solution:

Accurately weighed quantity of Buspirone HCL 10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL.Withdraw 0.4 ml from Stock Solution and make up to 10 ml with to get 4 μg/mL.

Chromatographic condition

The chromatographic separation of Buspirone HCL was achieved on C-18 (id 4.6 x 250 mm, 5 µm) by using mobile phase composed of Acetonitrile: Methanol(55:45 v/v%), at flow rate 1.0 ml/min with run time of 10 minutes. Detection of drug was carried out at 210 nm by using diluent as mobile phase.

Method of validation

As per ICH guideline (Q2R1), the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

Specificity

The analytical method for specificity was evaluated by injecting the following solutions. Diluent was prepared and inject into the HPLC system in triplicate. Sample solution was prepared with appropriate levels of excipients as a placebo sample and inject into the HPLC system in triplicate for all the dosage strengths. Placebo was prepared by mixing all excipients without active ingredients. Standard and sample solutions were prepared for assay (100% Conc.) and inject into the HPLC system in triplicate.

Linearity and Range

Preparation of Solution for linearity studies: For the purpose of linearity, accurately weighed amount of Buspirone HCL (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Buspirone HCL. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 2, 4, 6, 8 and 10 µg/ml of Buspirone HCL respectively.

Precision

Repeatability

Prepared standard working solution of mixtures having concentration of Buspirone HCL (4 μg/ml) was injected at volume of 20 μL into column by employing optimized chromatographic conditions. Each standard mixture was injected 5 time and peak area was monitored. Each concentration was monitored for repeatability by RSD. Intra-day and Inter-day Precision

• Method precision was determined by performing intraday and inter day precision.
• Mixture that represents overall range (Buspirone HCL = 2, 6 and 10 µg/ml) was analyzed on same day at different time interval for intraday precision.
• Mixture that represents overall range (Buspirone HCL = 2, 6 and 10 µg/ml) was analyzed on different days for inter-day precision.

System Suitability Parameters

Solution of Buspirone HCL (4 μg.ml-1) was injected 3 times for determination of System suitability parameters which includes Retention time (Rt), Tailing factor (Tf), Resolution (Rs) and number of theoretical plates. System suitability parameters for selected concentration were determined by C.V.

Accuracy

Accuracy of the analytical method has been performed by spiking of sample with the standard. Spiking of the placebo was performed at 50,100 and 150 % of the target concentration

Limit of detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ):

LOQ = 10 σ/s and LOD = 3.3 σ/s

Where, σ = the standard deviation of the response.

S = the slope of the calibration curve

Robustness

Following parameters were altered one by one for determination of robustness of the method and their effect was observed by comparing with the standard preparation. Mobile phase flowrate (± 0.1 mL/min), optimized flowrate was 1.0 mL/min. Mobile phase composition (± 2 mL), in optimized ratio 2 determinations of Buspirone HCL = 2 µg/mL for each alteration were carried out and RSD was measured

Assay

Sample preparation

Label claim

(Total contents 30grams-Equivalent to 2%w/w of Buspirone HCL). Squeeze all the contents in beaker and extract all the quantity with 100 ml methanol. Filter the phase, if necessary, that gives the solution containing 6000 µg/ml of Buspirone HCL. Dilute 50 µl of previous solution to 10 ml with mobile phase to give solution containing 300 µg/ml of Buspirone HCL.

Test solution:

Withdraw 100µl from above filtrate in 10 mL volumetric flask; make up the volume with mobile phase, which contain Buspirone HCL = 3 µg/ml. Inject the above solution for 3 times under optimized conditions.

RESULT AND DISCUSSION

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Buspirone HCL was scanned between 200-400 nm against diluents. Absorbance maxima of Buspirone HCL have detected at 210 nm. Chromatogram was taken at 219 nm, drug give good peak height and shape. So, 210 nm was selected for estimation of Buspirone HCL in formulation.

Selection of Mobile phase

Trail 1

• Column: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Acetonitrile:Water 30:70v/v)
• Detection: 210 nm
• Flow rate:1 ml/min
• Run Time: 10 minutes
• Observations: No peak detected.

Fig 3 Trial 1: Chromatogram of Buspirone HCL (4 µg,ml-1)

Trail 2

• Column: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Acetonitrile: Methanol (50:50v/v)
• Detection: 210 nm
• Flow rate: 1 ml/min
• Run Time: 10 minutes
• Observations: Peaks detected and separated, but broad peaks observe.

Fig 4 Trial 2: Chromatogram of Buspirone HCL(4 µg,ml-1)

Trial 3

• Column: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Acetonitrile: Methanol (55:45 v/v)
• Detection: 210 nm
• Flow rate:1 ml/min
• Run Time: 10 minutes
• Observations: Good peak with Adequate solution was observed.

Fig 5 Trial 3: Chromatogram of Buspirone HCL(4 µg,ml-1)

Chromatographic conditions for optimized mobile phase trial

• Stationary phase: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Acetonitrile: Methanol (55:45 v/v)
• Detection: 210 nm
• Flow rate:1 ml/min
• Run Time: 10 minutes
• Detector: UV detector
• Injection volume: 20 μl
• Column Temperature: 40ºC
• Mode: Isocretic

Fig 6: Optimized mobile phase trial for optimized chromatogram of Std. Buspirone HCL:7.215 min

Fig 7: Chromatogram of blank Acetonitrile: Methanol (55:45 v/v)

Method Validation

Linearity

For the purpose of linearity, accurately weighed amount of Buspirone HCL (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Buspirone HCL. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 20, 40, 60, 80 and 100µg/ml of Buspirone HCL

Table 11 Linearity data for Buspirone HCL

Fig 9: Overlain Linearity Spectra of Buspirone HCL

Fig 12: Calibration curve of Buspirone HCL

Table 12 Linearity results for Buspirone HCL

Precision

Repeatability

The data for repeatability for Buspirone HCL is shown in table 7.13. The % R.S.D For Repeatability data was found to be 1.10 % for Buspirone HCL.

Table 13 Repeatability data for Buspirone HCL

Inter-day precision

The data for interday precision for Ranitidine and Ondansetron is shown in table 7.14. The % R.S.D for intraday precision was found to be 0.27-1.50 % % for Buspirone HCL

Table 14 Inter-day precision data for estimation of Buspirone HCL

Intra -day precision

The data for intra-day precision for Buspirone HCL is shown in table 7.14. The % R.S.D for intraday precision was found to be 0.11-0.53 % for Buspirone HCL.

Table 15 Intra-day precision data for estimation of Buspirone HCL

Accuracy

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Buspirone HCL was found to be 99.48- 99.78%. The results are shown in table.7.17-7.17.

Table 16 Recovery data for Buspirone HCL

LOD and LOQ

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Table 18 LOD and LOQ Limit for Buspirone HCL

Selectivity

There is no interference in the mixture.

Robustness

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 7.19. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Table 19 Robustness data for Buspirone HCL

Analysis of marketed product

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Table 20 Analysis of marketed formulations

SUMMARY OF METHOD VALIDATION

Table 21 Summary of validation parameter of RP-HPLC method