Journal of Chromatography & Separation Techniques
Open Access
ISSN: 2157-7064
ISSN: 2157-7064
Research Article - (2013) Volume 4, Issue 7
Prazosin hydrochloride is a sympatholytic alpha-adrenergic blocker used in the treatment of anxiety, hypertension, refractory pulmonary oedema and panic disorders. Rapid, efficient, cost effective and reproducible isocratic reversed phase method has been developed and validated for the determination of prazosin in active pharmaceutical ingredient, dosage formulation and human serum using mobile phase 75:25 v/v acetonitrile:water having pH 3.20 adjusted with glacial acetic acid. Mobile phase was pumped at a flow rate of 1.5 mL min−1 using gradient elution through prepacked Nuclosil, C18 (250×4.6 mm, 10 μm) column. UV detection was performed at 250 nm. Method was validated following the ICH guidelines. Calibration curve was linear in concentration range 1.0-10 μg mL−1 with correlation coefficient 0.9999, and lower limits of detection and quantitation as 3.3 and 2.2 ng mL−1 and 9.8 and 6.1 ng mL−1 in raw material and serum respectively. Recovery was found to be in the range 99-100% and precision less than 1%. Developed method was successfully applied for routine analysis of drug pharmaceutical formulations and serum and also to study the interaction of prazosin with metal essential to human body at physiological temperature (37°C).
Keywords: Prazosin; RP-HPLC; Essential metals; Interaction
Prazosin hydrochloride or 1-(4-amino-6,7-dimethoxy- 2-quinazolinyl)-4-(2-furoyl) piperazine (Figure 1) is a sympatholytic alpha-adrenergic blocker used in the treatment of anxiety, hypertension, refractory pulmonary oedema and panic disorders. It reduces peripheral resistance and blood pressure by vasodilation of peripheral vessel (by blockade of a1 - agrenergic receptors) in arterioles and veins without increasing the heart rate or significantly impairing sympathetic function [1]. The vasodilator effect may be related not only to the direct relaxant action on vascular smooth muscles but to the blockade of postsynaptic a-adrenoceptors [2]. Prazosin has favorable effects on the plasma lipids, with reductions in total serum cholesterol and low density lipoprotein cholesterol and an improved ratio of high density to low density lipoprotein cholesterol [3]. It is used in the treatment of hypertension, refractory pulmonary oedema and circulatory failure following scorpion stings [4], is also useful in treating urinary hesitancy associated with prostatic hyperplasia by blocking alpha-1 receptors, which control constriction of both the prostate and ureters. Although not a first line choice for either hypertension or prostatic hyperplasia, it is a choice for patients who present with both problems concomitantly [5]. Krystal and Davidson [6] demonstrated that prazosin is also used in the treatment of trauma nightmares and sleep disturbance in combat veterans with post-traumatic stress disorder and improves sleep quality.
Figure 1: Chemical structures of Prazosin HCl.
Prazosin could be quantitated by colorimetric [7], spectrophotometric [8,9] and high-performance liquid chromatographic techniques [10-14] with its metabolite in biological fluids. All these methods were expensive, time consuming, complex in nature and make a column more susceptible to damage. In these methods, mobile phases used were mostly buffers, which are very much hazardous for the column life and efficiency (theoretical plates). Consequently, there was still a need to develop a simple, less time consuming, economical having low levels of quantitation and detection method for the determination of prazosin in API, dosage form, human serum by RP-HPLC.
Nevertheless, many drugs behave as ligands, coordinating such biometals as iron, copper and zinc, which affect their hemostasis. Some of metal drugs are used in the treatment of metal-dependent diseases [15-17]. Arterial hypertension is being sensitive to zinc and copper concentration levels [18,19]. Our research team has developed a number of methods for the quantitation of drugs alone as well as simultaneous determination of co-administered drugs and these methods have been applied to study drug interactions. There are number of drug metal interactions reported as with lefunomide [20], halofantrine [21], enoxacin [22], sparfloxacin [23], gatifloxacin [24], gliquidone [25], diltiazem [26], atenolol [27] and so on. The availability of drugs can be affected by the concurrent ingestion of drugs containing multivalent cations. It is very important to be aware of these types of interactions. There is no information in the literature on the effect of metal ions on the availability of prazosin.
Therefore, we attempted to develop a fast and reproducible method for the determination of prazosin in bulk drug and studied in-vitro interactions of prazosin with essential and trace elements, which may be either present in low concentrations in human body or may be ingested as a result of multiple drug therapy. Applicability of method was demonstrated by determining the studied drug in pharmaceutical formulations n serum without interference of excipients or endogenous components of serum.
Materials and reagents
Pharmaceutical grade prazosin was a kind gift from Pfizer Pakistan Limited and it dosage formulation, Minipress® 2 mg were purchased from local pharmacy, the expiry of which was not less than 2 years at the time of study. Analytical grade acetonitrile, methanol and glacial acetic acid were purchased from Merck (Darmstadt, Germany). Double distilled de-ionized water was used throughout. All other reagents used for metal interaction were of analytical grade and obtained from BDH laboratories (BDH Chemical Ltd., Poole, U.K.).
Instrumentation and chromatographic conditions
Liquid chromatographic system consisted of Shimadzu model LC- 10AT VP gradient pump, SPD 10AVP, variable wavelength UV-Visible detector and CBM 102 communication bus module (integrator). Analysis were performed on Nuclosil C18 (250×4.6 mm) reversedphase column at ambient temperature using mobile phase 75:25 v/v acetonitrile:water having pH 3.20 adjusted with glacial acetic acid. Flow rate was set at 1.5 mL min-1 with detection wavelength 250 nm. Samples were introduced through rheodyne injector valve with a 20 μL sample loop. In addition, mobile phase was filtered through 0.45 μm membrane filter paper and degassed by sonicator.
Preparation of stock solutions
10 mg prazosin was dissolved in methanol in 100 mL volumetric flask to get the final concentration of 100 μg mL-1. This stock solution was further diluted to prepare working solutions of required concentrations.
Analytical procedure
Aliquots of prazosin solution were accordingly diluted with 75:25 v/v acetonitrile: water diluent to working standard solutions of concentration range 1-10 μmL-1 of drug. 20 μL of these solutions were injected into the LC system (n=5). Before analysis, all the solutions were filtered through a 0.45 μm vacuumed filter and degassed by sonicator.
Pharmaceutical formulation
20 tablets of Minipress® (2 mg) were accurately weighed and finally powdered. Amount of powder equivalent to 10 mg of API was transferred into a 100 mL volumetric flask with methanol and dissolved by sonication for 20 minutes. The flask was filled to mark and the resulting solution was filtered. Method was followed as describe under analytical procedure.
Drug serum solution
Blood samples from a healthy volunteer (age 24 at Fatmid Foundation, Karachi, Pakistan) were collected in evacuated glass tubes through an indwelling cannula placed in the forearm veins or directly from vein. The blood was then slightly shaken and centrifuged at 10,000 rpm for 15 minutes, plasma was separated. To 1.0 mL plasma, 9.0 mL of acetonitrile was added, mixture was vortexed for one minute and then centrifuged for 10 minutes at 10,000 rpm. Obtained supernatant was filtered through 0.45 μm-membrane filter. Serum thus obtained was used for analysis [28]. Stock solution was spiked with serum to prepare working solutions in concentration range 1-10 μgmL-1 and analyzed. These serum solutions were stored at -20°C for pending analysis and for inter day variation studies.
Prazosin–metal ion interaction studies
Equimolar solutions of prazosin (100 μg mL-1) and metal salt solutions (100 μg mL-1) were mixed into a reaction flask. These reaction mixtures were allowed to react at 37°C in water bath for 60 minutes with constant stirring. Resulting solutions were filtered through 0.45 μ membrane filter and then introduced in LC system and chromatographed.
Method development and optimization
Various methods have been developed for the quantitation of prazosin [7-14] in pharmaceutical formulation and biological fluids; here we report a simple, efficient and least time consuming LC-UV method for its determination with detection limits up to nanogram level. Some chromatographic parameters such as column type, mobile phase, wave length and conditioning time were investigated to obtain a suitable peak for prazosine within an acceptable time. Preliminary experiments were performed to select the column most suitable for our purpose. Initially Nuclosil, C18 (250×4.6 mm, 10 μm) column was used for quantitation at ambient temperature. This column provides an efficient and reproducible peak area and good shape peak. Different ratios of acetonitrile and water were taken as the starting test solvents for mobile phase considering peak parameters ease of separation and cost, but the best composition was 75:25 v/v acetonitrile: water to have good resolution. The lower percentage of acetonitrile in mobile phase results in peak tailing and peak splitting. The pH effect was studied from 2.5 to 4.5, it was observed that well resolved and sharp peak obtained when pH was adjusted to 3.20 ± 0.02. Glacial acetic acid was used for pH adjustment for its inertness towards column packing. The optimum wavelength for prazosin detection was established using two UV absorbance scans over the range of 190 to 400 nm, one scan of methanol and the second of the analytes in the methanol. It was shown that 242– 258 nm is the optimal wave length to maximize the signal. Solution of 10 μg mL-1 at the wavelength 242-258 nm was also analyzed and it was observed that at 250 nm, maximum area was obtained; therefore 250 nm wavelength was selected for study. Chromatographic conditions were optimized with respect to specificity, reproducibility and time of analysis. The method has been successful for the determination of prazosin hydrochloride in concentration, as low as 100 ng mL-1, with retention time of only 4.227 minutes. After the method was developed and optimized it was validated. Representative chromatogram of prazosin in API is given in Figure 2.
Figure 2: A Representative chromatogram of prazosin in raw material.
Method validation
After establishing the optimal conditions for separation, linearity, precision, accuracy and recovery, system suitability, limit of detection and limit of quantitation were determined. The validation was conducted according to the ICH guidelines (1997) [29] and USP (2007) [30].
System suitability testing
A system suitability test was developed for the routine application of the assay method to determine the accuracy and precision of the system by making six injections of a solution containing 10 μg mL-1 of prazosin. All peaks were well resolved and the precision was acceptable. System suitability of the method was studied through method development by calculating theoretical plates, peak symmetry factor and tailing factor. Resolution and repeatability favorable for the system are summarized in Table 1.
| Parameters | Minimum | Maximum | Average | %RSD |
|---|---|---|---|---|
| Retention time (Rt in minutes) | 4.224 | 4.228 | 4.225 | 0.046 |
| Capacity factors (K’) | 0.000 | 0.000 | 0.000 | 0.000 |
| Theoretical plates (N) | 2404.70 | 2414.62 | 2410.54 | 0.159 |
| Tailing factor (T) | 1.440 | 1.470 | 1.457 | 1.103 |
| Peak area | 2555760 | 2577184 | 2563731 | 0.383 |
Table 1: System suitability parameters.
Linearity and range
Linearity of method was tested in order to demonstrate a proportional relationship of response verses analyte concentration. It was studied at six concentration level in the range 1-10 μg mL-1 (n=3). The regression equation was found linear by plotting peak area verses prazosin concentration, correlation coefficient obtained for API, tablet and serum obtained for the regression line was greater than 0.999. Table 2 represents the regression data including, correlation coefficient, slope, intercept, standard error and standard error estimate.
| R2 | Std Era | Std Er Estb | Intercept | Slope | LOD ng mL-1 | LOQ ng mL-1 | |
|---|---|---|---|---|---|---|---|
| API | 0.99992 | 5964.87 | 8367.29 | -1840.93 | 256586 | 3.3 | 9.8 |
| Minipress | 0.99992 | 6015.37 | 8438.13 | -3265.90 | 256740 | 3.0 | 7.0 |
| Serum | 0.99994 | 5195.46 | 7287.99 | -3829.48 | 256871 | 2.2 | 6.1 |
astandard error, bstandard error estimate
Table 2: Regression statistics of the proposed method with LOD, LOQ.
Accuracy/recovery studies
The accuracy of an analytical method is determined by how close the test results obtained by that method come to the true value. It can be determined by recovery studies, where a known amount of standard is spiked in the sample to be analyzed [31]. The results of accuracy studies are shown in Table 3 and it is evident that the method is accurate within the desired range.
| Conc. | % Recovery | Recovered conc. (μg mL-1) | %RSD | |||
|---|---|---|---|---|---|---|
| mg mL-1 | Minipress | Serum | Minipress | Serum | Minipress | Serum |
| 1 | 100.10 | 99.95 | 1.00 | 1.00 | 0.27 | 0.09 |
| 2 | 99.59 | 99.25 | 1.99 | 1.98 | 0.05 | 0.18 |
| 3 | 99.98 | 99.98 | 2.99 | 3.00 | 0.20 | 0.20 |
| 4 | 99.70 | 99.87 | 3.98 | 3.99 | 0.01 | 0.05 |
| 5 | 99.99 | 99.99 | 5.00 | 4.99 | 0.24 | 0.19 |
| 10 | 100.02 | 100.04 | 10.00 | 10.00 | 0.62 | 0.52 |
Table 3: Method accuracy from recovery assays for the studied analytes.
Precision
Precision of method, reported as %RSD, was estimated by measuring repeatability (intra-day assay) on six replicate injection at concentration of 10 μg mL-1, and intermediate precision (inter-day variation) was studied for two days using six solutions in the concentration range 1, 2, 3, 4, 5 and 10 g mL-1 (n=2). All the results given in Table 4 are within the acceptance criteria ranged from 0.01 % to 0.62%.
| Added (μg mL−1) | Intra-day | Inter-day | ||||
|---|---|---|---|---|---|---|
| Found | %RSD | %Rec | Found | %RSD | %Rec | |
| 1.0 | 1.00 | 0.27 | 100.10 | 1.00 | 0.09 | 99.95 |
| 2.0 | 1.99 | 0.05 | 99.59 | 1.98 | 0.18 | 99.25 |
| 3.0 | 2.99 | 0.20 | 99.98 | 3.00 | 0.20 | 99.98 |
| 4.0 | 3.98 | 0.01 | 99.70 | 3.99 | 0.01 | 99.87 |
| 5.0 | 5.00 | 0.24 | 99.99 | 4.99 | 0.19 | 99.99 |
| 10.0 | 10.00 | 0.62 | 100.02 | 10.00 | 0.52 | 100.04 |
Table 4: Intra and inter-day precision and accuracy of prazosin.
Lower limits of detection and quantitation
Lower limit of detection (LLOD) and quantitation (LLOQ) are the concentrations that give signal to noise ratio of 3:1 or 10:1 respectively which can be detected and verified by the relation of standard deviation of response (SD) to the slope of calibration curves (S):
LLOD=3.3 SD/S
LLOQ=10 SD/S
The LLOD was 3.3, 3.0 and 2.2 ng mL-1 and LLOQ was 9.8, 7.0 and 6.1 ng mL-1 for API, serum and dosage form respectively (Table 2).
Specificity
Specificity is the ability of a method to discriminate between analyte of interest and other components that are present in sample [31,32]. It was demonstrated by assaying the studied drug in pharmaceutical formulation and human serum. Figure 3 represents no additional peaks due to excipients or endogenous serum components at the same retention time of analyte, which confirms the specificity of method.
Figure 3: Representative chromatogram of prazosin in pharmaceutical formulation and in human serum.
Ruggedness
Ruggedness of proposed method was evaluated for two days in two different labs with two different instruments i.e HEC Lab, Department of Chemistry, University of Karachi, and the Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi. The instruments in both laboratories were Shimadzu model LC 10AT VP. %RSD and %recovery obtained are tabulated in Table 5 which was found within acceptable limits and proved the ruggedness of method.
| Parameter | % RSD | % Rec |
|---|---|---|
| Flow rate (1.49 mL min-1) | 0.461 | 99.62 |
| Acetonitrile:water (80:20 v/v) | 0.201 | 99.24 |
| pH (3.3) | 0.124 | 99.85 |
Table 5: Robustness data for prazosin.
Recovery in pharmaceutical formulation and human serum
Recovery of the proposed method was evaluated by determining the percent recovery tests of prazosin in pharmaceutical formulation and human serum by spiking a known amount of standard solution. Assay was carried out in triplicates. The obtained percent recovery values in the range 99.59-100.10 and 99.25-100.04 in pharmaceutical formulation and human serum respectively clearly demonstrates that no significant difference in the outcomes obtained from the assay of API. The results are tabulated in Table 3.
Drug–metal interaction studies
The amine group of prazosin is electron rich and thus interact with metals salts resulting in the formation of charge transfer complex [32]. Interaction was signified by measuring the area under curve (AUC), % drug availability and considerable drift in retention time from 4.721 min. Figure 4 clearly indicates the slight difference in retention time. The complexation occurred with ferric, cobalt, zinc, chromium, manganese, cadmium, calcium, nickel and copper, data is presented in Table 6. In all the cases percent availability of prazosin decreased indicating the complexation, except in case of iron, which showed availability of prazosin up to 132.57%. However, in case of magnesium 99.79% drug was available which demonstrate negative reaction.
Figure 4: Chromatograms showing prazosin–metal interactions.
| Drug/metal ion | Retention time | AUCa (mean) | % availability |
|---|---|---|---|
| PRZ | 4.721 | 1150214.5 | 100.00 |
| PRZ + Mg(II) | 4.800 | 1147752.5 | 99.79 |
| PRZ + Ca(II) | 4.790 | 1047694.0 | 91.09 |
| PRZ + Cr(III) | 4.768 | 1001878.5 | 87.10 |
| PRZ + Mn(II) | 4.739 | 1029312.0 | 89.49 |
| PRZ + Fe(III) | 4.712 | 1524870.0 | 132.57 |
| PRZ + Co(II) | 4.764 | 913505.0 | 79.42 |
| PRZ + Ni(II) | 4.731 | 1072859.0 | 93.27 |
| PRZ + Cu(II) | 4.767 | 1099120.5 | 95.56 |
| PRZ + Zn(II) | 4.722 | 937667.5 | 81.52 |
| PRZ + Cd(II) | 4.772 | 1034287.5 | 89.92 |
aarea under curve
Table 6: Quantitation of prazosin-metal ion complexes.
A simple, rapid, accurate, precise, low cost and least time consuming RP-HPLC method for the quantitative analysis of prazosin in API, pharmaceutical dosage formulations and human serum has been developed and validated. The intra-run and inter-run variability and accuracy results were found in acceptable limits. Simplicity of the method, shorter run time, economical nature and low limits of detection and quantitation makes the method superior to the other reported HPLC methods. The method could be applied routine analysis of studied drug in clinical labs, forensic medicine and to study drug interactions. The method has been applied to study prazosin-metal interactions and the results reveal that the presence of metals ion has a more pronounced effect on availability of drug and complexation is the main factor responsible for decreased or increased availability of prazosin. Therefore, a concomitant administration of prazosin with metals does not seem advisable, in case, it is necessary to administer the drug with multivitamins. Moreover, a proper time interval should be maintained.