ABOUT AUTHORS:
Ritobrata Rudra*1, Sudipta Ganguly2, Manabendra Dhua3, Binny sikdar4
1Department of pharmaceutical chemistry, Gupta College of Technological Sciences, Asansol.
2Department of Quality control, Stadmed pharmaceutical pvt ltd, Dumdum, Kolkata.
3Department of pharmaceutical chemistry, Gupta College of Technological Sciences, Asansol, W.B.
4North Bengal University, silliguri, W.B.
*hrik1990@gmail.com
ABSTRACT
Developing a single analytical method for estimation of single drug from its dosage form is a very challenging task. A simple, rapid, precise, and reliable reverse phase HPLC method was developed for the separation and estimation of Terazosin HCl in pharmaceutical dosage forms. The estimation was carried out using Inertsil ODS-C18(150 mm × 4.6 mm, 5 μm) column; mobile phase consisting of water, acetonitrile, triethylamine, and pH 6.4 adjusted by orthophsphoric acid; the flow rate of 1 mL/min and ultraviolet detection at 245 nm. Run time of this drug is 2.3 minutes. The method was validated as a final verification of method development with respect to precision, linearity, accuracy, ruggedness, and robustness. The validated method was successfully applied to the commercially available pharmaceutical dosage form, yielding very good and reproducible result.
REFERENCE ID: PHARMATUTOR-ART-1875
INTRODUCTION
Analytical chemistry is a scientific discipline that develops methods, instruments and strategies to obtain information and nature of matter. Analytical chemistry is concerned with the chemical characterization of matter and thus pharmaceutical analysis covers matter having pharmaceutical applications. Knowledge of chemical composition of many substances is important in our daily life. Analytical chemistry plays an important role in nearly all aspects of chemistry viz. agricultural, clinical, environmental, forensic, manufacturing, metallurgical and pharmaceutical chemistry1. The scientific approach which the analytical chemist applies consist of series of steps which includes understanding and defining the goal of analysis; nature of the sample; literature search; plan of action and execution2. At present several analytical methods are available for analysis viz. Spectroscopic and chromatographic, but method development & validation for estimation of terazosin HCl is done by the HPLC method.
Terazosin hydrochloride, an alpha-1-selective adrenoceptor blocking agent, is a quinazoline derivative.
Systematic (IUPAC) name:3
(RS)-Piperazine,1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-[(tetra-hydro-2 furanyl)carbonyl]-, monohydrochloride, dihydrate.
Pharmacokinetic data:3
|
Protein binding |
Extensive |
|
Metabolism |
Hepatic hydroxylation |
|
Half-life |
12 hours |
|
Excretion |
40% urine and 60% faces |
Physicochemical properties:3
|
Molecular weight |
459.93 g/mol |
|
Solubility |
Soluble in water, acetonitrile.Slightly soluble in alcohol, 0.1N hydrochloric acid. Very slightly soluble in chloroform, tetrahydrofuran. Partially soluble in acetone, hexane. |
|
PKa |
7.1 |
|
Melting point |
273°c. |
|
Appearance |
White or almost white, crystalline powder. |
Indication:3
Terazosin hydrochloride (marketed as Hytrin), an alpha-1-selective adrenoceptor blocking agent, is a quinazoline derivative which is used to treat hypertension (high blood pressure) and benign prostatic hyperplasia (enlarged prostate). It causes the blood vessels (veins and arteries) to relax and expand, improving blood flow. Terazosin also relaxes muscles in the prostate and bladder neck, making it easier to urinate.
Mode of action:3
In general, α1-adrenergic receptors mediate contraction and hypertrophic growth of smooth muscle cells. α1-Receptors are 7-transmembrane domain receptors coupled to G proteins, Gq/11. Three α1-receptor subtypes, which share approximately 75% homology in their transmembrane domains, have been identified:α1A (chromosome 8), α1B (chromosome 5), and α1D (chromosome 20). Terazosin is the first α1-receptor antagonist to demonstrate selectivity for the α1A-receptor. All three receptor subtypes appear to be involved in maintaining vascular tone. The α1A-receptor maintains basal vascular tone while the α1B-receptor mediates the vasoconstrictor effects of exogenous α1-agonists. Activation of α1-receptors activates Gq-proteins, which results in intracellular stimulation of phospholipases C, A2, and D. This results in mobilization of Ca2+ from intracellular stores, activation of mitogen-activated kinase and PI3 kinase pathways and subsequent vasoconstriction. Terozosin produces its pharmacological effects by inhibiting α1A-receptor activation. Inhibition of these receptors in the vasculature and prostate results in muscle relaxation decreased blood pressure and improved urinary outflow in symptomatic benign prostatic hyperplasia.
LITERATURE REVIW
TERAZOSIN HCL bulk is officially determined by following methods:
1. Indian pharmacopoeia describes the potentiometric titration method for the assay of terazosin hydrochloride bulk equivalent to terazosin by dissolve in 0.01M HCl and 50ml of methanol, titrate with 0.1 M NaoH.4
2. United States Pharmacopoeia describes HPLC method using mixture of pH 3.2 Citrate buffer and Acetonitrile (1685:315) as a mobile phase for assay of Terazosin HCL bulk equivalent to terazosin.5
TERAZOSIN HCL has been used as a subject for various works. Works in the analytical field are as follows:
- Srivastava et al6 devloped a method for determination of prazosin and terazosin using another alpha one blocker tamsulosin hydrochloride as internal standard. Method was developed using Waters HPLC system equipped with the Empower software and composed of Quadruple 600E gradient pump equipped with Kromasil C18 column (250 × 4.6 mm, 5.0 μm) from Agilent Technologies using methanol as mobile phase. Method was than validated. Developed method was successfully applied for the determination of Terazosin and Prazosin in tablet formulation.
- Bharadwaz et al7developed and validated a simple and sensitive fluorimetric procedure for the estimation of terazosin HCL in tablet preparation. The method consists of measurement of fluorescence for the drug samples extracted by methanolic (0.1N) H2SO4.
- Parmar et al8developed and validated RP-UPLC method for Quantitative analysis and Content Uniformity study of Terazosin Hydrochloride dihydrate in tablets. An isocratic method for analysis of Terazosin Hydrochloride dihydrate was archived on ACQUITY UPLC BEH C18 (100*2.1) mm particle size 1.7 µ columns within shorter runtime of 6 min with a flow rate of 0.250 ml/min and using a photodiode array detector to monitor the eluent at 246 nm. The mobile phase consisted of Buffer-Acetonitrile (80:20 v/v), (Buffer: 20 mM ortho phosphoric acid in 1L water).
- Ganjali et al9 proposed potentiometric sensor for quantitative analysis of terazosin hydrochloride in pharmaceutical formulation based up on the computational study.Based on computational studies, terazosin-tetraphenyl borate was selected as a suitable ion-pair reagent in making terazosin potentiometric sensor. The wide linear range of 10-5,10-2 mol L-1, low detection limit of 7.9×10-6 mol L-1, and fast response time of ~15 s are characterizations of the proposed sensors. Validation of the method showed suitability of the sensor for application in the quality control analysis of terazosin hydrochloride in pure and pharmaceutical formulation.
- Gupta et al10 developed and validated stability indicating RP HPLC method for simultaneous determination of Prazosin, Terazosin and Doxazosin in pharmaceutical formulations. They found that the mobile phase consisting of A: ACN–diethylamine (0.05 ml), B: methanol and C: 10 ml Ammonium acetate separated these drugs effectively. Separations were carried out on a new Kromasil C18 column (250 × 4.6 mm, 5.0 μm) at 254 nm wavelength. The calibration curve was found to be linear in the range of 2–500 μg/ml. The stated method was then validated in terms of specificity, linearity, precision, and accuracy. Additionally, the proposed method reduced the duration of the analysis.
- Gupta et al11 attempted to developed an ultra violet spectroscopic method for the estimation of alpha adrenergic receptor, blocker(alfuzosin, tamsulosin, doxazosin, prazosin, terazosin). All these drugs were dissolving in the same solvent (methanol) and scanned under UV spectrophotometer, under full UV range 200-400nm. After some experiments they found that this is impossible. This was evident from their study that UV spectra were overlapping each other, except in the case of tamsulosin.
EXPERIMENTAL SECTION
MATERIALS:
1. Instruments:
Isocratic reversed phase HPLCconsisting Dual Preston pump and 2489 water UV detector and C18 column.
Computational record of that system is operated by EMPOWER software.
2. Chemicals/Reagents:
Acetonitrile, Tetrahydrofuran, Water, Orthophosphoric acid, Triethylamine. All the chemicals are HPLC graded.
METHOD:
1. Selection of Mobile Phase:
According to the theory there are so many considerations can be taken into account to select mobile phase.
On the basis of absorbance maxima and solubility of this drug and also literature review
Acetonitrile, Methanol, Water, Buffer and some mobile phase modifiers (mentioned above) were selected. Various compositions in different ratio was tried to get the best peak. The maximum absorption of terazosin was found at 245 nm.
2. Development of method:
This includes setting initial chromatographic conditions, method optimization, performing system suitability test, detection of impurities of API according to the principle of ICH guide line sample was placed under unstressed condition like acid, base, oxidation.22, 23,24
Initially the conditions were chosen like below,
1.Water:Acetonitrile:Tetrahyrofuran= 60:30:10(pH 6.4by dilute orthophosphoric acid) with C18 column
But then in result the retention time was found after 10 minutes and peak is asymmetric.
2.Water:Acetonitrile:Tetrahydrofuran=40:50:10 (pH 6.4 by orthophosphoric acid) with C18 column.
Resulting the retention time was found in between 2-3 min and peak is asymmetric and system suitability parameters were found out of acceptance limit.
3.Water:acetonitrile=40:60 (pH 6.4 by orthophosphoric acid) with C18 column.
Resulting the retention time was found in between 2-3 min and system suitability parameters were found out of acceptance limit.
4.Water:Acetonitrile:triethylamine=40:60:0.2(pH 6.4 by orthophosphoric acid) with C18 column.
After that mobile phase modifier was introduced and ultimately the solvent and ratio which was used with acceptable system suitability parameter and good analysis time was found between2.2-2.5 and no tailing effect, peak is symmetric.
3. Optimization of method:
Weakness of the method was indentified in this step and method was optimized through experimental design.
4. Validation:12,13,14,15
Method validation should be treated as a “final verification” of the method performance and should not be used as a part of method development.
Method validation was completed to ensure that analytical methodology was accurate, reproducible and rugged over the specific range.
ANALYTICAL METHOD PROCEDURE:
METHOD A:
Mobile phase preparation:Mixture of Water and Acetonitrile and triethylamine was prepared in ratio of 40:60:0.2, pH adjusted to 6.4 with orthophosphoric acid.
METHOD B:
Preparation of Stander solution:About 5.0 mg of terazosin hydrochloride equivalent to terazosin 4.296 mg was diluted to 25 ml volumetric flask with mobile phase.(conc-171.84mcg/ml) ………(1)
From (1) 1ml was taken and diluted to 10 ml volumetric flask with mobile phase.(conc-17.184mcg/ml)
METHOD C:
Preparation of sample solution:Weight accurately quantity of the Powdered tablet containing about 2 mg terazosin and was transfer in to 10 ml of volumetric flask & made up volume with mobile phase shake vigorously for 15 min…………(2)
From (2) 1 ml was taken and dilute with mobile phase and volume was adjusted with 10 ml with mobile phase.
CHROMATOGRAPHIC CONDITION:
Column-RP, c18, (150x4.6nm) particle size-5μm
Mobile phase-water: acetonitrile: triethylamine=40:60:0.2
pH – adjusted 6.4 by dilute orthophosphoric acid.
Flow rate-1ml/min
Injection volume-20μL
Uv detection-245nm
PROCEDURE:
Equal volume (20µl) of blank, standard and sample preparation was injected and peak responses for TERAZOSIN HCl were recorded and content was calculated. The relative standard deviation for standard preparation should be not more than 2.0%, the peak tailing is not more than 2.
ACCURACY OF STANDARD INJECTIONS:
Five (5) replicate injection of the working standard solution at concentration of 17.184mcg/ml as described in METHOD B were made.
Preparation of sample solution of different concentration minimum three concentrations and three determinations.
Terazosin HCl sample was also prepared at same proportion as in the tablet (50%-150% 0f nominal concentration).
Preparation of 50% test solution
Content of terazosin hydrochloride IP equivalent to terazosin found in tablet -2.12mg 2.12 mg terazosin sample was transferred in to 10 ml volumetric flask and dissolved with mobile phase and volume was adjusted up to 10 ml with mobile phase……(1)
From (1) 1 ml was taken and volume was adjusted up to 10 ml with mobile phase. From standard solution 0.1ml (17.184mcg) was taken and added to the sample solution. Filter with 0.45 μm before injection in to the HPLC.
Preparation of 100% test solution
Content of terazosin hydrochloride IP equivalent to terazosin found in tablet -2.12mg 2.12 mg terazosin sample was transferred in to 10ml volumetric flask and dissolved with mobile phase and volume was adjusted up to 10 ml with mobile phase……(2)
From (2) 1 ml was taken and volume was adjusted up to 10 ml with mobile phase. From standard solution 0.2ml (34.37mcg) was taken and added to the sample solution. Filter with 0.45 μm before injection in to the HPLC.
Preparation of 150% test solution
Content of terazosin hydrochloride IP equivalent to terazosin found in tablet -2.12mg 2.12 mg terazosin sample was transferred in to 10 ml volumetric flask and dissolved with mobile phase volume was adjusted up to 10 ml with mobile phase……(3)
From (3) 1 ml was taken and volume was adjusted up to 10 ml with mobile phase. From standard solution 0.3ml (51.55mcg) was taken and added to the sample solution. Filter with 0.45 μm before injection in to the HPLC.
PRECISION:
System Repeatability:
Five replicate injection of the standard solution as described in the Method B were made and the relative standard deviation (RSD) of the peak area was calculated.
Method Repeatability:
The full HPLC method as described in Method- A, Method- B and Method- C was carried out on the product. The method repeated six (6) times and the Relative Standard Deviation was calculated.
Intermediate Precision:
The full method as described in Method A, B and C was carried out on the product for the 2 mg tablet formulation. The method was repeated five times by a second analyst on a different HPLC instrument. The average assay and the relative standard deviation (RSD) were calculated.
Inter day Precision:
The full HPLC method as described in Method A, Method B and Method C was carried out on the product. The method repeated six (6) times and the Relative Standard Deviation was calculated.
LINEARITY:
Standard solution was prepared at 10 to30 µg/ml, equal volume of each concentration was injected and area obtained for each analysis was plotted over respective concentrations. Then slope and coefficient of regression was calculated. If the value of regression coefficient is found to be 1 or near to 1 then the result shows its acceptance. Linear regression analysis demonstrates acceptability of the method for quantitative analysis over this range.
Preparation of Standard solution for linearity
Preparation of 50% solution
5.00mg Terazosin HCL was dilute in to 25ml volumetric flask with mobile phase……(1)
From (1) 0.5 ml was taken & volume was adjusted up to 10 ml in to volumetric flask with mobile phase.
Preparation of 75% solution
5.00mg Terazosin HCL was dilute in to 25ml volumetric flask with mobile phase……(2)
From (2) 0.75 ml was taken & volume was adjusted up to 10 ml in to volumetric flask with mobile phase.
Preparation of 100% solution
5.00mg Terazosin HCL was dilute in to 25ml volumetric flask with mobile phase……(3)
From (3) 1.0 ml was taken & volume was adjusted up to 10 ml in to volumetric flask with mobile phase.
Preparation of 125% solution
5.00mg Terazosin HCL was dilute in to 25ml volumetric flask with mobile phase……(4)
From (4) 1.25 ml was taken & volume was adjusted up to 10 ml in to volumetric flask with mobile phase.
Preparation of 150% solution
5.00mg Terazosin HCL was dilute in to 25ml volumetric flask with mobile phase……(5)
From (5) 1.50 ml was taken & volume was adjusted up to 10 ml in to volumetric flask with mobile phase.
DETECTION LIMIT (LOD)15:
Detection limit was determined based on the Standard Deviation of the Response and the Slope.
The detection limit (DL) may be expressed as:
DL = 3σ/S
Where σ = the standard deviation of the response
S = the slope of the calibration curve.
QUANTITATION LIMIT (LOD) 15:
The quantitation limit (QL) may be expressed as:
QL = 10σ/S
Where σ = the standard deviation of the response.
S = the slope of the calibration curve.
ROBUSTNESS:
It should show the reliability of analysis with respect to deliberate variation in method parameters.
Flow rate 0.8 ml/min:
Flow rate was decreased from 1ml/min to 0.8 ml/min and the assay was completed as shown in Method A, B and C.
Flow rate 1.2 ml/min:
Flow rate was increased from 1ml/min to 1.2 ml/min and the assay was completed as shown in Method A, B and C.
STABILITY INDICATING STUDY:
FORCED DEGRADATION OF TERAZOSIN HCl STANDARD AND SAMPLE:
Stress testing of the drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures used. The nature of the stress testing will depend on the individual drug substance and the type of drug product involved.
Acid Hydrolysis:
1. Sample solution of Terazosin HCl was prepared as in Method C. An amount equivalent to 2 mg of Terazosin was weighed and kept into 20 ml of volumetric flask,after that 1 ml of 0.1 (N) HCl was added. The solution was allowed to stand for 18 hours. After that 1 ml of 0.1 (N) NaOH was added, Volume was adjusted with the mobile phase and sonicated before injection into HPLC.
2. Standard solution was prepared as in Method B, an amount of 5 mg Terazosin HCl was weighed into 250 ml volumetric flask. 1 ml of 0.1 (N) HCl was added to it and the solution was allowed to stand for 18 hours. After that 1 ml of 0.1 (N) NaOH was added, volume was adjusted with mobile phase sonicated for 5 minutes before injection into HPLC.
Base Hydrolysis:
1. Sample solution of Terazosin HCl was prepared as in Method C. An amount equivalent to 2 mg of Terazosin was weighed and kept into 20 ml of volumetric flask, 1 ml of 0.1 (N) NaoH was added and the solution was allowed to stand for 18 hours. After that 0.1 (N) HCl added and volume was adjusted by mobile phase and sonicated before injection into HPLC.
2. Standard solution was prepared as in Method B, an amount of 5 mg Terazosin HCl was weighed into 250 ml volumetric flask.. 1 ml of 0.1 (N) NaoH was added and the solution was allowed to stand for next 18 hours. After that added 0.1 (N) of HCl was added to it and volume was adjusted by mobile phase and sonicated before injecting into HPLC system.
Oxidation:
1. Sample solution of Terazosin HCl was prepared as in Method C. An amount equivalent to 2 mg of Terazosin was weighed and kept into 20 ml of volumetric flask, and 10 ml of30% H2O2was added and allowed to stand for 6 hours then the volume was made up by the mobile phase sonicated before injecting into HPLC system.
2. Standard solution was prepared as in Method B, an amount of 5 mg Terazosin HCl was weighed into 250 ml volumetric flask and 2 ml of 30% H2O2 was added and allowed to stand for 6 hours then volume was made up with mobile phase and sonicated before injecting into HPLC system.
RESULTS & DISCUSSIONS
SYSTEM SUITABILITY PARAMETERS:
RESULT:
|
PARAMETRES |
MEAN |
S.D |
R.S.D % |
|
Theoretical Plates |
103.72 |
4.56 |
0.044 |
|
Resolution |
------ |
------ |
------ |
|
Tailing Factor |
1.055 |
0.00548 |
0.519 |
|
Capacity Factor |
2.3575 |
0.0131 |
0.555 |
DISCUSSION
All the parameters checked throughout the experiment and obtained results are within acceptance limit.
ACCURACY:
RESULT:
|
Serial number |
Standard peak area |
|
1. |
3277847 |
|
2. |
3289571 |
|
3. |
3269468 |
|
4. |
3268233 |
|
5. |
3284241 |
|
Mean |
3277872 |
|
Standard Deviation |
9232.671 |
|
% Relative Standard Deviation |
0.281 |
Limit not more than - 2.0% (RSD)
For 50% sample solution
|
Sample 0.1 |
Observed Area |
|
Sample A |
4407831 |
|
Sample B |
4453353 |
|
Sample C |
4422131 |
|
INITIAL Con OF THE SAMPLE SOLU |
STANDARD SOLUTION CONTAINING 171.84 mcg/ml ADDED TO THE SAMPLE SOLUTION |
TOTAL CONCEN |
TOTAL CONCENTRATION OF THE SOLUTION mcg/ml |
CONCENTRATION FOUND IN THE SOLUTION mcg/ml |
AVARAGE VALUE |
% OF RECOVERY |
|
212 |
0.1ml (17.184) |
212+17.184= 229.18 |
22.918 |
23.10 |
|
|
|
212 |
0.1ml (17.184) |
212+17.184= 229.18 |
22.918 |
23.34 |
23.20 |
101.25% |
|
212 |
0.1ml (17.184) |
212+17.184= 229.18 |
22.918 |
23.18 |
|
|
For 100% sample solution:
|
Sample 0.2ml |
Observed area |
|
Sample A |
4680735 |
|
Sample B |
4709938 |
|
Sample C |
4600291 |
|
INITIAL Con OF THE SAMPLE SOLU |
STANDARD SOLUTION CONTAINING 171.84 mcg/ml ADDED TO THE SAMPLE SOLUTION |
TOTAL CONCEN |
TOTAL CONCENTRATION OF THE SOLUTION mcg/ml |
CONCENTRATION FOUND IN THE SOLUTION mcg/ml |
AVARAGE VALUE |
% OF RECOVERY |
|
212 |
0.2ml (34.368) |
212+34.368= 246.37 |
24.637 |
24.53 |
|
|
|
212 |
0.2ml (34.368) |
212+34.368= 246.37 |
24.637 |
24.69 |
24.44 |
99.21% |
|
212 |
0.2ml (34.368) |
212+34.368= 246.37 |
24.637 |
24.11 |
|
|
For 150% sample solution
|
Sample 0.3ml |
Observed Area |
|
Sample A |
5015230 |
|
Sample B |
5092449 |
|
Sample C |
5066241 |
|
INITIAL Con OF THE SAMPLE SOLUTION mcg/ml |
STANDARD SOLUTION CONTAINING 171.84 mcg/ml ADDED TO THE SAMPLE SOLUTION |
TOTAL CONCENTRATION OF THE SOLUTION mcg |
TOTAL CONCENTRATION OF THE SOLUTION mcg/ml |
AVARAGE VALUE |
% OF RECOVERY |
|
212 |
0.3ml (51.552) |
212+51.552= 263.55 |
26.355 |
|
|
|
212 |
0.3ml (51.552) |
212+51.552= 263.55 |
26.355 |
26.51 |
100% |
|
212 |
0.3ml (51.552) |
212+51.552= 263.55 |
26.355 |
|
|
DISCUSSION:}
RSD found here not more than 2.0 % and % of recovery with in acceptance limit. So that method is Accurate.
PRECISION:
RESULT:
System repeatability:
System repeatability:
|
Serial number |
Observed Peak Area |
|
1 |
3277847 |
|
2 |
3289571 |
|
3 |
3269468 |
|
4 |
3224208 |
|
5 |
3227486 |
|
MEAN |
3257716 |
|
Standard Deviation (SD) |
29978.10 |
|
%Relative Standard Deviation (RSD) |
0.92 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
174mg |
3916214 |
2.05 |
102.8% |
2.5 |
Method repeatability:
|
Serial number |
Observed Peak Area |
|
1 |
3265819 |
|
2 |
3244835 |
|
3 |
3243692 |
|
4 |
3276201 |
|
5 |
3248821 |
|
MEAN |
3255873.6 |
|
Standard Deviation (SD) |
14423.03 |
|
Relative Standard Deviation (RSD) |
0.44 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
173.9mg |
3888621 |
1.99 |
99.69 |
-0.5 |
Intermediate Precision:
|
Serial number |
Observed Peak area |
|
1 |
3265881 |
|
2 |
3298715 |
|
3 |
3279320 |
|
4 |
3287946 |
|
5 |
3265881 |
|
MEAN |
3279548.6 |
|
Standard Deviation (SD) |
14243.64 |
|
Relative Standard Deviation (RSD) |
.43 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
173.4mg |
3980421 |
2.03 |
101.5 |
1.5 |
Inter day Precision:
|
Serial number |
Observed Peak area |
|
1 |
3269271 |
|
2 |
3260191 |
|
3 |
3268811 |
|
4 |
3258931 |
|
5 |
3266324 |
|
MEAN |
3264705.6 |
|
Standard Deviation (SD) |
4848.81 |
|
Relative Standard Deviation (RSD) |
0.14 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
174.02mg |
3905721 |
2.008 |
100.4 |
0.42 |
- DISCUSSION:
RSD and percentage deviation are in range. So the method is précised.
LINEARITY
Linearity was observed over 10-30 mcg/ml.
The observed area of corresponds to concentration.
- RESULT
|
Serial number |
Preparation |
Concentration mcg/ml |
Area |
Average Area
|
|
1. |
Sample A |
0 |
0 |
|
|
2. |
Sample B |
0 |
0 |
0 |
|
3. |
Sample A |
10 |
1524089 |
|
|
4. |
Sample B |
10 |
1521268 |
1522678.5 |
|
5. |
Sample A |
15 |
2272763 |
|
|
6. |
Sample B |
15 |
2267163 |
2269964.5 |
|
7. |
Sample A |
20 |
3037755 |
|
|
8. |
Sample B |
20 |
3030452 |
3034103.5 |
|
9. |
Sample A |
25 |
3751534 |
|
|
10 |
Sample B |
25 |
3746165 |
3748849.5 |
|
11. |
Sample A |
30 |
4526522 |
4526522.0 |
Correlation coefficient=0.999
Y=150468.43x+9212.43
- DISCUSSION:
R2 Value was found to be 0.999.So from the above result linearity was established over the range of 10 to 30 mcg /ml of standard drug.
DETECTION LIMIT (LOD):
- RESULT
|
Serial no |
Observed Peak Area |
|
1. |
3219959 |
|
2. |
3242715 |
|
3. |
3247943 |
|
4. |
3227946 |
|
5. |
3223337 |
|
MEAN |
3232380 |
|
Standard Deviation |
12295.69 |
|
Relative Standard Deviation (RSD) |
0.38 |
Calculation:
LOD = 3σ/S
(3 x 12295.68/188104.0)= 0.196
DISCUSSION:
Detection limit of Terazosin HCL is 0.196µg/ml.
Quantitation Limit (LOQ):
- RESULT
|
Serial no |
Observed Peak Area |
|
1. |
3219959 |
|
2. |
3242715 |
|
3. |
3247943 |
|
4. |
3227946 |
|
5. |
3223337 |
|
MEAN |
3232380 |
|
Standard Deviation |
12295.69 |
|
%Relative Standard Deviation (RSD) |
0.38 |
Calculation:
LOQ = 10σ/S
(10 x 12295.69)/188104.0= 0.653
- DISCUSSION:
Quantitation limit of Terazosin HCl is 0.653µg/ml.
ROBUSTNESS:
- RESULT
0.8 ml/min:
|
Serial no |
Observed Peak Area |
|
1. |
3416328 |
|
2. |
3369020 |
|
3. |
3399272 |
|
4. |
3401325 |
|
5. |
3378902 |
|
MEAN |
3392969.4 |
|
Standard Deviation |
18885.36 |
|
%Relative Standard Deviation (RSD) |
0.55 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
175.88mg |
4203815 |
2.03 |
101.6 |
1.5 |
|
TerazosinHCl |
174.01mg |
4175593 |
2.05 |
102.5 |
2.5 |
1.2 ml/min:
|
Serial no |
Observed Peak Area |
|
1. |
3069820 |
|
2. |
3133179 |
|
3. |
3119941 |
|
4. |
3072663 |
|
5. |
3189441 |
|
MEAN |
3117008 |
|
Standard Deviation |
49269.23 |
|
%Relative Standard Deviation (RSD) |
1.5 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
%in deviation |
|
TerazosinHCl |
174.08mg |
3898441 |
2.07 |
103 |
3.5 |
|
TerazosinHCl |
174.88mg |
3857904 |
2.04 |
102 |
2.0 |
- DISCUSSION:
This method passes the acceptable criteria of Robustness for change in flow rate.
STABILITY INDICATING STUDY:
Acid Hydrolysis:
- RESULT:
|
Serial number |
Observed Peak area |
|
1 |
3243561 |
|
2 |
3214865 |
|
3 |
3156885 |
|
4 |
3258931 |
|
5 |
3236324 |
|
MEAN |
3222113 |
|
Standard Deviation (SD) |
39764.18 |
|
%Relative Standard Deviation (RSD) |
1.2 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
174.02mg |
3811274 |
2.008 |
100 |
0.4 |
|
TerazosinHCl |
174.02mg |
3751277 |
1.977 |
98.5 |
-1.15 |
- DISCUSSION:
RSD and percentage deviation are in range and chromatograms show no significant degradation.
Base Hydrolysis:
- RESULT:
|
Serial number |
Observed Peak area |
|
1 |
3368903 |
|
2 |
3394987 |
|
3 |
3360483 |
|
4 |
3244692 |
|
5 |
3299012 |
|
MEAN |
3333615 |
|
Standard Deviation (SD) |
60906.33 |
|
%Relative Standard Deviation (RSD) |
1.8 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
%in deviation |
|
TerazosinHCl |
166.01mg |
3732842 |
1.98 |
99 |
-1 |
|
TerazosinHCl |
166.01mg |
3745829 |
1.98 |
99 |
-1 |
- DISCUSSION:
RSD and percentage deviation are in range and chromatograms show no significant degradation.
Oxidation:
- RESULT:
|
Serial number |
Observed Peak area |
|
1 |
3019959 |
|
2 |
3042715 |
|
3 |
3147943 |
|
4 |
3069923 |
|
5 |
3112459 |
|
MEAN |
3078600 |
|
%Standard Deviation (SD) |
51857.19 |
|
%Relative Standard Deviation (RSD) |
1.6 |
|
Active Content |
Wt. of Sample |
Area of Sample |
Assay |
Assay % |
% in deviation |
|
TerazosinHCl |
175.99mg |
3756514 |
2.04 |
102 |
2 |
|
TerazosinHCl |
175.99mg |
3769372 |
2.04 |
102 |
2 |
DISCUSSION:
RSD and percentage deviation are in range and chromatograms show no significant degradation.
CONCLUSION
R2 Value was found to 0.999 and the above result is linear within the concentration range 10 to 30 mcg /ml of standard drug. The method is linear over a wide range, economical and utilizes a mobile phase which can be easily prepared. RSD found here not more than 2.0 % and % of recovery with in acceptance limit. So that method is Accurate. RSD of limit of detection, limit of quantitation of the method is 0.38. Limit of detection of this method is 0.196 mcg/ml & limit of quantitation found 0.653 mcg/ml. The result of limit of detection & quantitation found within the acceptance limit. All methods are suitable for quantitation of Terazosin in bulk drug and in pharmaceutical dosage form. This method passes the acceptable criteria of Robustness for change in flow rate. % of RSD for the inter day precision is less than 1% so that proposed method is highly precise. From the above stability indicating study it was seen that the product Terazosin HCl was free from any degradation in Acid, Base and Oxidation condition. Result of accuracy, linearity, limit of detection, limit of quantitation, robustness, precision , specificity lies within acceptance limit, so the proposed method is validated. Based up on the results we can conclude that this method can be used for routine analysis for terazosin hydrochloride in bulk as well as tablet formulation.
REFERENCES
1. Anjeneyulu, Y;Chandrasekhar, K.;Manikam, V.A text book of Analytical Chemistry; Pharma Book Syndicate: Hyderabad, 2005, 3.
2. Settal, F.A.Handbook of Instrumental Techniques of Analytical Chemistry; Pharma Book Syndicate: Hyderabad, 2004, 19.
3. Takara.;K, Sakaeda.;T, Kakumoto.;M, Okumura.;K, Ohonishi.;N, Yokoyama.;T, Effect of alpha adrenoceptor antagonist teraziosin doxazosin on MDR1-mediated multidrug resistanceand transcellular transport, 2009, 17.
4. Indian pharmacopoeia, Govt of India Ministry of Health & Family Welfare, Published by The Indian Pharmacopoeia Commission, Ghaziabad, 2010, vol3.
5. United State Pharmacopoeia, National Formulary 2008, vol 3.
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8. Dabhi Batuk, Pandya.D, Parma.A, jadej.Y, Patel.M, Jebaliya.H, Karia.D, Shah.A, Inventi Rapid: Pharm Analysis & Quality Assurance. 2012 ,12.
9. M.D Ganjali.R, Faridbod.F, Larijani.B, Riahi.S, Hosseini.M, Nasli-Esfahani.N, Norouzi.P, Int. J. Electrochem. Sci,2010, 5
10. Shrivastava.A, B.Gupta, ScientiaPharmaceutica, 2012, 23
11. Shrivastava .A, B.Gupta , Journal of Pharmaceutical Negative Result, 2011, 2.
12. ICH Q1D: Braketing and marketing designs for stability testing of new drug substance and products, International Conference on Harmonization, Genava, 2002.
13. ICH Q1B: Photo stability testing of New Drug Substance and products, International Conference on Harmonization, Genava, 1998.
14. ICH Q1C: Stability testing of New Dosage Forms, International Conference on Harmonization, Genava, 1998.
15. ICH Q2B: Validation of analytical procedure: methodology, International Conference on Harmonization, Genava, 1996.
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