Introduction:

Prazosin hydrochloride (PRZ) is used to treat high blood pressure, anxiety, and panic disorder. PRZ is an orally administered antihypertensive drug¹ and a potent vasodialatory agent² in the quinazoline family and has been found to be value in treatment of heart failure³. It is selective α blocker relax smooth muscle in prostatic hyperplasia producing an increase in urinary flow rate and improvement in obstructive symptoms. Chemically, it is 1-(4-amino-6, 7,-dimethoxy-2-quinazolinyl)-4-(2-furanyl carbonyl) piperazine monohydrochloride and used as antihypertensive drug.

Fig. No. 1 PRZ structure

Drug is official in Merck index⁴ and USP⁵. Several techniques have been reported for the determination of PRZ which include spectrophotometric^6,7, HPLC^8-10, solid phase extraction¹¹, spectrofluorimetric¹², and ion selective electrode¹³ methods for the estimation of PRZ. However, the above mentioned methods are very complex and expensive equipment is involved. Hence, this present investigation has made to develop simple, cost-effective, selective, accurate, and rapid method for the determination of PRZ in bulk and dosage formulation by spectroscopic method.

EXPERIMENTAL:

Instrumentation:

A Bioera ultraviolet-visible double-beam spectrophotometer with 1 cm matched quartz cells was used for all the spectral measurements.

Chemicals and Reagents:

The pure sample of PRZ was gifted by Sun Pharma, Ahmadabad, Gujarat, India. AR grades of methanol were procured from Merck.

METHOD DEVELOPMENT:

Solubility Test:

Solubility test for the drug PRZ was performed by using various solvents. The solvents include methanol, dist. water, acetic acid, 0.1 N hydrochloric acid, 0.1N sodium hydroxide and chloroform. However, methanol was chosen as a solvent for developing the method.

Preparation of Stock Solution:

The standard stock solution of 1000 μg/mL of PRZ was prepared by weighing 100 mg of pure PRZ, taken in 100 mL volumetric flask and was dissolved in methanol up to the mark.

Preparation of Working Standard Solution:

1 ml of above stock solution was taken in 10 ml volumetric flask and diluted with methanol up to the mark. Further 1 ml resulting solution was taken and diluted up to 10 ml with methanol. Again 1 ml of resulting solution was taken and diluted up to 10 ml with methanol which produce final concentration of 1 μg/mL. Further dilutions were made with distilled water to obtain concentrations ranging from 1-5 μg/mL.

Selection of Wavelength:

The wavelength for the analysis of PRZ (5ppm) was selected from the UV spectrum. A wavelength of 248 nm was selected for the analysis as it is having the maximum absorbance at this particular wavelength.

Assay of PRZ Tablet

Accurately weighed and powdered 20 tablets. Drug equivalent to 50 mg of PRZ was taken and transferred to a 50 mL of volumetric flask. Then final volume was adjusted up to 50 mL with methanol. Further 1 ml of above solution was taken and diluted to 10 ml with methanol, from this solution again 1 ml was taken and again dilute up to 10 ml with methanol. Finally absorbance of this resulting solution was measured at predetermined UV wavelength 248 nm and results are tabulated in Table No. 1

Table No 1: Assay of PRZ

Brand Name	Label Claim	Assay
Prazopress XL 2.5 Alembic Pharmaceuticals	2.5 mg	99.90 %

METHOD VALIDATION:¹⁴^-16

Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its pre determined specifications and quality characteristics. The method was validated for different parameters like linearity, accuracy, precision, LOD, LOQ, robustness, ruggedness.

Linearity and Range:

Various aliquots were prepared form the stock solution ranging from 1-5 μg/ml. The samples were scanned in UV-VIS Spectrophotometer using dist. water as a blank. It was found that the selected drug showed linearity between the range 5-25 μg/ml (Table 2 and 3).

Accuracy:

Different concentrations of PRZ were prepared in methanol at different levels of concentrations i.e. at 80 %, 100% and 120%. The solutions were prepared in triplicates and the results are mentioned in Table 2 and 4.

Precision:

Precision of the method was demonstrated by intraday and intraday variation studies. In intraday variation study, 6 different solutions of same concentration that is 2μg/ml were prepared and analyzed in a day and the absorbance were noted. The result was indicated by % RSD (table no.6, and table no.7). In the intraday variation study, solutions of same concentration 4μg/ml were prepared and analyzed for three consecutive days and the absorbance's were noted. The result was indicated by % RSD (Table 2 and 5).

Ruggedness:

Ruggedness of the method was determined by analyzing same sample (different batches) by different analysts at different conditions and the respective absorbance were noted in Table No. 7.

Robustness:

Robustness of the method was determined by carrying out the analysis at two different wavelengths i.e. at (+2nm / -2nm). (Table 2 and 8)

Limit of Detection (LOD):

The limit of detection (LOD) was determined by preparing solutions of different concentrations ranging from 0.1-1μg/ml. The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantized as an exact value (Table 2).

Limit of Quantification (LOQ):

The LOQ is the concentration that can be quantized reliably with a specified level of accuracy and precision. The LOQ was calculated using the formula involving standard deviation of response and slope of calibration curve (Table 2).

RESULTS AND DISCUSSION:

The developed method was found to be precise as the %RSD values for intra-day and inter-day were found to be less than 2%. Good recoveries (98% to 101%) of the drug were obtained at each level of concentration, indicating that the method was accurate. The method was also found to be specific indicated by the % recoveries ranging from 99.8% to 101.2%. The method was also found to be robust and rugged as indicated by the % RSD values which are less than 2%. The results of Assay showed that the amount of drug was in good agreement with the label claim of the formulation as indicated by % recovery (99.90 %). Summary of validation parameters of proposed spectrophotometrically method is shown in table 2.

Table No. 2 Validation Summery

Parameters	Results
Linearity indicated by correlation coefficient	0.999
Intraday Precision indicated by %RSD	1.312
Interday Precision indicated by %RSD	0.250
Accuracy indicated by % RSD	0.05-0.13
Range	1-5 μg/ml
Linear regression equation	y = 0.428x
Limit of Detection	1.18 μg/ml
Limit of Quantification	4.02 μg/ml
Robustness indicated by %RSD	0.03-0.025
Assay indicated by % Recovery	99.90 %

VALIDATION:

Linearity:

Table No. 3 Linearity of PRZ

Cocⁿ(μg/ml)	Absorbance	Calculation
1	0.443	SD= 0.667 RSD=0.5183 Slope=0.4223 C.C. =0.999 Intercept=-0.021
2	0.865
3	1.293
4	1.706
5	2.134

Fig. No. 3 Linearity curve of PRZ

Accuracy:

Table No.4 Accuracy of PRZ

Sample	Absorbance	Statistical Results
		Mean	SD	%RSD
S1 – 80%	1.703	1.7053	0.0022	0.13%
S2 – 80%	1.709
S3 – 80%	1.704
S4 – 100%	2.136	2.1376	0.0022	0.05%
S5 – 100%	2.139
S6 – 100%	2.138
S7 – 120%	2.556	2.559	0.0018	0.07%
S8 – 120%	2.560
S9 – 120%	2.561

Precision

Table No 5: Intraday precision

Sr. No.	Conc.( μg/ml)	Abs
1	2	0.867
2	2	0.877
3	2	0.869
4	2	0.887
5	2	0.853
6	2	0.866
Avg.		0.869
SD		0.011
%RSD		1.312

Table No 6 : Interday precision

Sr. No.	Concn. (ppm)	Day –I	Day-II	Day III
1	4	1.706	1.712	1.711
2	4	1.710	1.712	1.699
3	4	1.709	1.699	1.699
4	4	1.706	1.698	1.701
5	4	1.706	1.701	1.703
6	4	1.709	1.706	1.706
Mean		1.709	1.706	1.706
SD		0.001	0.006	0.004
%RSD		0.108	0.370	0.273

Ruggedness:

Table No. 7 Ruggedness of PRZ

Sr. No.	Parameter	Set I	Set II
1	System	Bioera	Shimadzu 1700
2	Sample	Batch No-X	Batch. No- Y
3	Day	Thursday	Mona
4	Time	10:00 am	3:00pm
5	Lab	Analysis	Chemistry
6	Analyst	73/12	76/12
7	Sample	5 μg/ml	5 μg/ml
8	Assay	99.77%	99.87%

Robustness:

Table No. 8 Robustness of PRZ

Sr. No.	Cocⁿ(μg/ml)	Wavelength	Absorbance	Calculations
1	5	250 nm	2.010	Mean = 2.016 S. D. = 0.0007 %RSD = 0.03
			2.011
			2.011
2	5	246 nm	1.998	Mean = 1.997 S. D. = 0.0005 %RSD = 0.025
			1.997
			1.997

Limit of detection (LOD) and limit of quantification (LOQ):

Detection and quantification limits were taken as the lowest concentration used in the construction of the calibration curves. LOD and LOQ were determined by the formula based on the standard deviation of the response and slope. (Table No.2)

LOD = 3 × s/S

and

LOQ = 10 × s/S.

Where, “s” is the standard deviation of the intercept and S is the slope.

CONCLUSION:

The proposed method development and validation of UV-Vis Spectrophotometrically method was to determine PRZ. The developed method was validated in distilled water according to ICH guideline and shown to be accurate, precise and cost effective. It do not require expensive or sophisticated and chemicals in contrast with chromatographic method. It can be used for the routine Q. C. analysis and quantification of the drug in the formulations.

ACKNOWLEDGEMENT:

The authors are wish to thanks Principal and Management of Shivnagar Vidya Prasarak Mandals College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune and Mr. Prabhatkumar Jain, Scan Research Bioanalytical Laboratories, Bhopal, for providing required lab facilities with enthusiastic environment.

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Received on 06.11.2015 Modified on 12.11.2015

Research J. Science and Tech. 8(1): Jan.– Mar. 2016; Page 01-04

DOI: 10.5958/2349-2988.2016.00001.2