INTRODUCTION:

Doxazosin mesylate is one of the quinazoline composite. Doxazosin mesylate is an α-adrenoreceptar blocker used in the treatment of hypertension and urinary retention associated with benign prostatic hyper plasma^1-3. Doxazosin mesylate chemically known as 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(1,4benzodioxan-2-ylcarbonyl)piperazine methane sulfonate ,the molecular weight is 547.6 and empirical formula is C₂₃H₂₅N₅O₅ •CH₄O₃S. Reducing blood pressure diminishes the threat of fatal and nonfatal events of cardiovascular, predominantly strokes and myocardial infarctions. These welfares have been seen in skillful trials of antihypertensive medications from a extensive range of pharmacologic classes, with this drug.

Numerous analytical approaches have been described for the fortitude of Doxazosin mesylate in plasma^4-10 and formulations^11-14, as well as HPLC, LC-MS, spectrophotometry¹⁵ and HPTLC ¹⁶. Nevertheless, there is no UPLC method available for quantification of impurities in Doxazosin mesylate. Thus, the purpose of our experimentation was to develop and authorize a stability-indicating UPLC technique, which endorsed the fortitude of related substance in drug substance. This procedure is a state-of-the-art and the impurities were isolated in a short time. In attendance stayed no described approaches for Doxazosin and its related substance as per literature on UPLC.

MATERIALS AND METHODS:

Materials

Test Sample of Doxazosin mesylate and its six related impurities namely Impurity-1, 2, 3, 4, 5 and 6 (Fig.1) were acknowledged from R&D, Inogent laboratories Private Limited. Chromatographic grade Methanol, Acetonitrile ,AR grade ortho phospharic acid was obtained from Merck,. Water was arranged by using a Milli-Q system.

Doxazosin mesylate: 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(1,4benzodioxan-2-ylcarbonyl) piperazine methanesulfonate

Impurity-1 : 1-[(2RS)-2,3-dihydro-1, 4-benzodioxin-2-ylcarbonyl] piperazine

Impurity-2 : 1,4-bis (2,3-dihydro-1, 4-benzodioxin-2-ylcarbonyl) piperazine

Impurity-3: 6,7-dimethoxyquinazoline-2, 4(1H, 3H)-dione

Impurity-4: 2,4-dichloro-6, 7-dimethoxyquinazoline

Impurity-5 : 2-chloro-6, 7-dimethoxyquinazoline-4-amine mesylate

Impurity-6: 6,7-dimethoxy-2- (piperazin-1-yl) quinazolin-4-amine mesylate

Fig. 1: Structures of Doxazosin mesylate and its six impurities.

METHODS

Instrument specification

Ultra-performance liquid chromatography used for the development of analytical method and analytical procedure validation be made up of binary pumps, diode array detector from M/s. Waters, with auto injector and sampler. The UPLC was furnished with information acquision and processing software “Empower-3”.

Preparation standard solutions

A stock solution of Doxazosin mesylate was prepared by dissolving right quantity of substance in diluent. Standard solutions of mg /mL were prepared from the stock solutions for the determination of related substance. Stock solutions of impurities (combination of Impurity-1, 2, 3, 4, 5 and 6) at mg/mL were similarly arranged with the diluent.

Chromatographic Conditions

The Chromatographic separation was accomplished on an Acquity UPLC BEH C18, 100mm X 2.1 mm ID with1.7 microns with gradient elution of T/ %B 0/20, 4/20, 7/50, 10/70, 12/70, 13/20 and 15/20. Eluent-A involves of 2% H₃PO₄(Aq) and Eluent-B involves of Acetonitrile methanol and water (700:200:100 v/v) used as a eluents. The eluent were degassed and filtered over nylon membrane (0.22 micron pore size) and sonicated to use. The eluent flow rate was kept 0.3 mL/min. Column temperature was retained at 40°C, wave length was controlled at 210 nm and 2μL injection volume. The solutions of standard, test dilutions were made in the combination of acetonitrile and water in the quantity of 50:50 (v/v).

Validation of the method

Specificity

Specificity is the ability of the technique to degree of the analyte peak response in the occurrence of its approaching impurities. The specificity of the technologically advanced UPLC technique for Doxazosin mesylate was conceded in the presentation of it’s impurities viz. Impurity -1, 2, 3, 4, 5and 6. Stress experimentations were executed for Doxazosin mesylate bulk drug to afford an sign of the stability demonstrating properties and specificity of the anticipated method. Deliberate degradation was endeavored to stress circumstances of UV light ( at 254 nm), acid (0.5 N HCl), heat (105°C), oxidation (3.0 % H₂O₂), base (0.5 N NaOH), and water hydrolysis (70°C) to assess the capability of the anticipated technique to detached Doxazosin mesylate from its degradation products. For thermal and UV light study period was 7 days and for the base, acid, water hydrolysis and oxidation, kept for 24 hours. Checked the peak purity Doxazosin peak by exhausting PDA detector in above stress samples.

Precision

The precision of the technique was tested by introducing six separate measures of Doxazosin mesylate (0.5mg/ mL) spiked through 0.1% level of Impurity -1, 2, 3, 4, 5 and 6 with respect to Doxazosin mesylate analyte test concentration. Calculated the % RSD of area for each Impurity -1, 2, 3, 4, 5 and 6. The intermediate study precision of the technique was similarly assessed using dissimilar analyst and apparatus in the similar research laboratory.

Limit of Quantification (LOQ) and Limit of detection (LOD)

The LOQ and LOD was determined at a S/N ratio of 10:1 and 3:1, correspondingly, by introducing a sequence of dilute solutions with known foci. Precision experimentation was also conceded out at the LOQ by injecting six separate preparations of Impurity-1, Impurity-2, Impurity-3, Impurity-4, Impurity-5 and Impurity-6 and then deliberate the %RSD of the area of impurity peak.

Linearity

Solutions for the linearity were set by stock solution dilution to the necessary concentrations. The solutions keep on set at six deliberation levels from 0.025% to 0.15% equal of impurities specifically Impurity -1, 2, 3, 4, 5 and 6. The Y-intercept and slope of the standardization curve was deliberate.

Accuracy

The accuracy experimentation for impurities was take-out in triplicate at 150%, 100% and 50 % level with respect to 0.1% level to the Doxazosin mesylate analyte concentration. The measurements of repossessions for impurities were measured.

Robustness

To achieve the robustness of the established method, investigational circumstances were purposely changed and the resolution among Doxazosin mesylate, Impurity -1, 2, 3, 4, 5 and 6 was noted. Column temperature was wide-ranging by ± 5°C and flowrate of eluent wide-ranging from ±0.05 ml/min, while other eluent components were detained no change as indicated in chromatographic circumstances.

Solution stability and mobile phase stability

Solution constancy of Doxazosin mesylate and its related impurities in the technique was accepted out by separating spiked sample solution in resolutely covered volumetric flasks at ambient condition for 24 hrs, content of Impurity -1, 2, 3, 4, 5 and 6 were tested in test solutions.

RESULTS AND DISCUSSION:

Method development and Optimization

The foremost objective of method development was to accomplish efficient separation between Doxazosin mesylate and known impurities like Impurity-1, 2, 3, 4, 5 and 6. Impurities existed co-eluted with many phase such as C18, Phenyl, CN and C8 as well as dissimilar eluent composition. Separation was succeeded on an Acquity UPLC BEH C18, 100mm X 2.1 mm ID with 1.7 microns particles with gradient elution of T/%B 0/20,4/20,7/50,10/70,12/70,13/20 and 15/20. Eluent-A involves of Aqueous H₃PO₄ (2%) and Eluent-B involves of Acetonitrile: methanol: water in the quotient of 700: 200: 100 v/v used as a eluents. 0.3 mL / min flow rate was kept eluent , and stationary phase temperature retain at 40°C, the peak shape of the Doxazosin mesylate was establish to be symmetrical. In enhanced chromatographic situations of Doxazosin mesylate, Impurity -1, 2, 3, 4, 5 and 6 were detached with resolution over and above 2, characteristic retention times were about 7.8, 2.2, 10.3, 1.8, 5.7, 2.7 and 0.8 correspondingly (Fig 2).

The system suitability outcomes are specified in Table-1 and established UPLC technique was found to particular for Doxazosin and its six Impurities namely Impurity-1, 2, 3, 4, 5 and 6 (Fig 2).

Table-1: System suitability Results

System suitability	Doxazosin mesylate	Impurity-1	Impurity-2	Impurity-3	Impurity-4	Impurity-5	Impurity-6
R_t	7.8	2.2	10.3	1.8	5.7	2.7	0.8
RR_t	1	0.29	1.30	0.23	0.73	0.35	0.11
R_s	8.9	2.1	11.6	4.2	12.9	1.9	-
T	1.1	1.2	1.0	1.2	0.9	1.2	1.0
N	21789.6	1820.9	43728.4	1023.6	8139.0	2537.5	263.8

R_t: Retention time; T: USP tailing factor; N: Theoretical plate, R_s: Resolution; RRt: Relative retention time

a) Blank

b) Doxazosin mesylate test sample

c) Doxazosin mesylate sample spiked with all impurities

Fig.2: Illustrative chromatogram of (a) Diluent (b) Doxazosin mesylate test sample (c) Doxazosin mesylate sample spiked by impurities.

Table-2: Results of forced degradation

Stress conditions	Time (h)	Purity	Total Impurities	Peak Purity
Normal		99.96%	0.04%	Pass
Acid hydrolysis	24	99.43%	0.57%	Pass
Base hydrolysis	24	99.48%	0.52%	Pass
Oxidation(3%H2O2)	24	99.92%	0.08%	Pass
Water, 70°C	24	99.94%	0.06%	Pass
Thermal (105°C)	7 days	99.99%	0.01%	Pass
UV Visible	7 days	99.99%	0.01%	Pass

VALIDATION OF METHOD

Results of forced degradation studies

Degradation was not identified in Doxazosin mesylate sample after exposed to stress circumstances corresponding to light, thermal, UV and Oxidation. Doxazosin mesylate was degraded to unknown impurity under hydrolysis of base and acid. Peak purity test outcomes inveterate that the Doxazosin peak is homogeneous and unadulterated in all the stress samples. Doxazosin mesylate is unpretentious in the demonstration of total impurities and its degradation crops endorse the stability representing power of the technique. Forced degradation studies summary mentioned in Table 2.

Method Precision

%RSD for the area of Impurity -1, 2, 3, 4, 5 and 6 in method precision testing was within 4.34% conforming precision of the technique. (table-3)

Limit of detection (LOD) and Limit of Quantification (LOQ):

The detection limit of altogether impurities specifically Impurity -1, 2, 3, 4, 5and 6 were achieved 0.008%, 0.0075%, 0.0072%, 0.069%, 0.00731% and 0.0073% for 2 μL injection volume. Quantification limit of all impurities namely Impurity -1, 2, 3, 4, 5 and 6 are 0.025%, 0.024%, 0.024%,0.02%,0.025% and 0.026% for 2μL injection volume. The precision at quantification level concentrations for Impurity -1, 2, 3, 4, 5 and 6 were underneath 3.4%.

Linearity

Calibration plot for Linearity in the related substances technique was attained over the calibration ranges tested i.e. 0.025%, 0.05%, 0.075%, 0.10%, 0.125% and 0.15 % of Impurity -1, 2, 3, 4, 5 and 6. The correlation coefficient accomplished over and beyond 0.999. The above results shows that an outstanding correlation existed amongst the peaks are and the concentrations of Impurity-1-6. The regression information is available in table 4.

Accuracy

The % recoveries of six impurities in Doxazosin mesylate samples at 0.05%, 0.10% and 0.15% level were ranged from 92.9-107%. The % recovery values for impurities are available in Table 5.

Robustness

In all the considered varied chromatographic circumstances (temperature and flow) the resolution amongst critical pair, i.e. Impurity-3 and Impurity-1 and Impurity-1 and Impurity-5 was over and above 2.0. The tailing factor for Doxazosin mesylate and impurities was below 1.4.Theoretical plates for Doxazosin mesylate is more than 3000.

Solution stability and Mobile phase stability

% RSD of related substances of Doxazosin mesylate for the duration of solution stability trials, No notable change were seen in the content of impurities specifically Impurity -1, 2, 3, 4, 5 and 6 throughout the solution steadiness and mobile phase steadiness experimentations when accomplished exhausting the technique. The solution steadiness and eluent steadiness experimentation statistics recommends that the sample solution and eluent used throughout the method is determination were unwavering for 48 hrs.

Table-3: Method Precision Results

Name	Impurity-1	Impurity-2	Impurity-3	Impurity-4	Impurity-5	Impurity-6
%RSD	1.74	3.29	2.32	2.90	2.61	4.34

Table-4: Linearity data

Name	Impurity-1	Impurity-2	Impurity-3	Impurity-4	Impurity-5	Impurity-6
Linearity
Y Intercept	-10.5	321	325	-1479	-199	3423
Slope	22006	35588	22972	27580	24217	13645
r	0.9992	0.999	0.9991	0.9999	0.9993	0.999

Table-5: Accuracy data

Level	Impurity-1	Impurity-2	Impurity-3	Impurity-4	Impurity-5	Impurity-6
0.05%	95.9-97.6	102.2-103.6	96.5-100.9	98.5-103.3	96.9-98.1	98.7-102.7
0.10%	98.7-100.9	99-102.6	98-101.4	98.8-100.6	98.7-102	95-102.2
0.15%	97.9-99.8	92-94.6	97.2-98.7	100.2-107	97.3-99.7	96.4-101

CONCLUSION:

The UPLC procedure established for measureable and related substance determination of Doxazosin mesylate is accurate, linear, rapid, specific and precise. The technique was wholly validated presenting adequate information for validation considerations tested. The established procedure is stability representative and can be suitably for quality control to regulate this technique in their regular Doxazosin mesylate commercial samples and similarly stability samples analysis. The UPLC procedure is a state-of-the-art and the impurities were detached in a shorter time.

ACKNOWLEDGMENT:

The authors wish to acknowledge the organization of M/s. Inogent Laboratories Pvt Ltd. for allowing to carry out the contemporaneous work. The authors also wish to acknowledge the associates of Analytical Research & Development and Process Research & Development division for sympathetic this work.

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Received on 13.12.2016 Modified on 05.01.2017

Asian J. Research Chem. 2017; 10(2):95-100.

DOI: 10.5958/0974-4150.2017.00014.1