Scholarly article on topic 'Validated HPTLC method for the simultaneous determination of alfuzosin, terazosin, prazosin, doxazosin and finasteride in pharmaceutical formulations'

Validated HPTLC method for the simultaneous determination of alfuzosin, terazosin, prazosin, doxazosin and finasteride in pharmaceutical formulations Academic research paper on "Chemical sciences"

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{Alfuzosin / Terazosin / Prazosin / Doxazosin / Finasteride / HPTLC}

Abstract of research paper on Chemical sciences, author of scientific article — Tarek S. Belal, Mohamed S. Mahrous, Magdi M. Abdel-Khalek, Hoda G. Daabees, Mona M. Khamis

Abstract Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases affecting men and it increases in both incidence and prevalence with age. This work presents a simple, sensitive and fast generic high performance thin layer chromatographic (HPTLC) method for the simultaneous determination of five drugs prescribed for the treatment of BPH. These drugs include the α1-adrenergic blockers; alfuzosin hydrochloride (ALF), terazosin hydrochloride (TER), prazosin hydrochloride (PRZ) and doxazosin mesylate (DOX) in addition to the 5α-reductase inhibitor; finasteride (FIN). The cited drugs were separated on TLC-silica plates using a mobile phase composed of methylene chloride:n-hexane:methanol (8.8:0.3:0.9, by volume). Densitometric analysis was carried out at 254nm for the α-blockers while FIN was measured at 220nm. The five drugs were detected at R f values of 0.26, 0.36, 0.45, 0.59 and 0.69 for ALF, TER, PRZ, DOX and FIN, respectively. The developed method was validated according to the International Conference on Harmonization (ICH) guidelines regarding; linearity, ranges, accuracy, precision, selectivity, robustness and limits of detection and quantification. The proposed method showed good linearity (r >0.9990) in the ranges; 30–350, 30–350, 20–200, 30–350, 200–2000ng/spot for the cited drugs, respectively. The applicability of the proposed method was verified through the analysis of laboratory-prepared mixtures and percentage recoveries between 98.27% and 101.97% were obtained. Commercial tablets were also analyzed by the developed methodology with no interference detected from the co-formulated excipients. The high sensitivity, simplicity and selectivity of the proposed method suggest its applicability for routine quality-control analysis purposes of any of the titled drugs in their pharmaceutical preparations.

Academic research paper on topic "Validated HPTLC method for the simultaneous determination of alfuzosin, terazosin, prazosin, doxazosin and finasteride in pharmaceutical formulations"

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Analytical Chemistry Research

journal homepage: www.elsevier.com/locate/ancr

Validated HPTLC method for the simultaneous determination of alfuzosin, terazosin, prazosin, doxazosin and finasteride in pharmaceutical formulations

Tarek S. Belala,*, Mohamed S. Mahrousb, Magdi M. Abdel-Khalekb, Hoda G. Daabeesc, Mona M. Khamisb,d

a Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah 21521, Alexandria, Egypt b Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah 21521, Alexandria, Egypt c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt d College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada

ARTICLE INFO ABSTRACT

Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases affecting men and it increases in both incidence and prevalence with age. This work presents a simple, sensitive and fast generic high performance thin layer chromatographic (HPTLC) method for the simultaneous determination of five drugs prescribed for the treatment of BPH. These drugs include the ^-adrenergic blockers; alfuzosin hydrochloride (ALF), terazosin hydrochloride (TER), prazosin hydrochloride (PRZ) and doxazosin mesylate (DOX) in addition to the 5a-reductase inhibitor; finasteride (FIN). The cited drugs were separated on TLC-silica plates using a mobile phase composed of methylene chloride:n-hexane:methanol (8.8:0.3:0.9, by volume). Densitometric analysis was carried out at 254 nm for the a-blockers while FIN was measured at 220 nm. The five drugs were detected at Rf values of 0.26, 0.36, 0.45, 0.59 and 0.69 for ALF, TER, PRZ, DOX and FIN, respectively. The developed method was validated according to the International Conference on Harmonization (ICH) guidelines regarding; linearity, ranges, accuracy, precision, selectivity, robustness and limits of detection and quantification. The proposed method showed good linearity (r >0.9990) in the ranges; 30-350, 30-350, 20-200, 30-350, 200-2000 ng/spot for the cited drugs, respectively. The applicability of the proposed method was verified through the analysis of laboratory-prepared mixtures and percentage recoveries between 98.27% and 101.97% were obtained. Commercial tablets were also analyzed by the developed methodology with no interference detected from the co-formulated excipients. The high sensitivity, simplicity and selectivity of the proposed method suggest its applicability for routine quality-control analysis purposes of any of the titled drugs in their pharmaceutical preparations.

© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/3XI/).

CrossMark

Keywords:

Alfuzosin

Terazosin

Prazosin

Doxazosin

Finasteride

1. Introduction

Benign prostatic hyperplasia (BPH) is a common disorder of the male urogenital tract and it is the main cause of lower urinary tract symptoms (LUTS) in older men. It affects almost 50-90% of the men aging from 50 to 85 years [1]. Compared to surgery, pharmacological intervention results in significant improvement of the symptoms with fewer, less serious and reversible side effects. Two main pharmacological classes of drugs are available; the selective a1-adrenoreceptor blockers and the 5a-reductase inhibitors. The first acts by the selective blockade of the a1-adrenoreceptors that are widely distributed in the prostatic tissues, thus inhibiting the

* Corresponding author. Tel.: +20 3 4871351; fax: +20 3 4873273. E-mail address: tbelaleg@yahoo.com (T.S. Belal).

sympathetic stimulation of the prostatic smooth muscles and relieving the urinary obstruction [1]. The alternative treatment strategy is the 5a-reductase inhibitors that exert their action by interrupting the conversion of testosterone into 5a-dihydrotestos-terone and therefore reducing the prostate volume [2]. In addition to monotherapy, several studies have proven the beneficial use of these drugs in combination for treating men at higher risk of BPH progression [1-3]. The a1-adrenoreceptor blocker family includes, but not exclusive to; alfuzosin hydrochloride (ALF), doxazosin mesylate (DOX), prazosin hydrochloride (PRZ) and terazosin hydrochloride (TER) [1,3]. All four members share a similar nucleus, namely; the 4-amino-6,7-dimethoxyquinazoline (Fig. 1). On the other hand, finasteride (FIN) is a 5a-reductase inhibitor, and it is chemically known as N-tert-butyl-3-oxo-4-aza-5a-androst-1-ene-17p-car-boxamide (Fig. 1) [3].

http://dx.doi.org/10.1016/j.ancr.2014.06.004 2214-1812/® 2014 The Authors. Published by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativec0mm0ns.0rg/licenses/by-nc-nd/3.0/).

Fig. 1. Chemical structures of ALF, TER, PRZ, DOX and FIN.

Literature review reveals the presence of numerous methods for the determination of these five drugs in different matrices. A couple of review articles were published where they provided information about the various analytical methods available for these drugs [4,5]. In this work, special emphasis will be given to analytical reports involving the use of HPTLC. Such technique was used for the quantification of ALF in plasma [6] as well as in tablets either alone [7] or in the presence of its forced degradation products [8]. DOX was also assayed in tablets using HPTLC [9,10]. In addition, HPTLC methods were reported for the simultaneous determination of the binary mixtures; ALF/solifenacin [11], DOX/ celecoxib [12] and FIN/tamsulosin [13]. Furthermore, TLC methods were applied for detection of 29 different pharmaceutical compounds including DOX, PRZ and TER in adulterated herbal remedies [14].

On the other hand, several reports investigated the simultaneous quantification of either of the selected drugs with one another or with other pharmacologically related compounds using several chromatographic methods. The use of a monolithic weak cation-exchange column was described in the HPLC concomitant determination of ALF, DOX, PRZ and TER in human plasma [15]. DOX, PRZ and TER were also simultaneously assayed using a stability-indicating HPLC-UV method [16]. In addition, PRZ and TER were concurrently determined via HPLC methods in tablets [17] and in biological fluids [18]. Moreover, HPLC with UV detection methods were presented for the specific determination of DOX, PRZ and TER in presence of their degradation products [19]. On the other hand, FIN was simultaneously determined with tamsulosin in combined dosage forms using several HPLC methods [13,20,21]. Finally, flow

injection analysis with fluorescence detection method was recently reported for determination of the four a1-blockers in pharmaceutical formulations [22].

To the best of our knowledge, we could not find any articles in the literature describing the simultaneous determination of the five selected drugs by any analytical methodology. Moreover, no reports could be found for the simultaneous determination of the four structurally related a1-blockers using HPTLC. This encouraged us to investigate the development of a generic, simple and selective HPTLC procedure applicable for the routine quality control analysis of any of the designated drugs in their pure or tablets dosage forms.

2. Experimental

2.1. Materials and reagents

ALF was kindly provided by Amriya Pharmaceutical Industries (Alexandria, Egypt), while, DOX was a gratuity from the Egyptian International Pharmaceutical Industries Co., EIPICO (10th of Ramadan City, Egypt). PRZ and TER were supplied by Pfizer Egypt S.A.E. (Cairo, Egypt) and European Egyptian Pharmaceutical Industries (Alexandria, Egypt), respectively. FIN was generously donated by Adwia Co. S.A.E. (10th of Ramadan City, Egypt). HPLC grade methanol was purchased from Lab-Scan Analytical Sciences (Gliwice, Poland). Methylene chloride (CDH, India), HPLC grade n-hexane (Carlo Erba, France), absolute ethanol (BDH Laboratory Suppliers, Poole, England) and ethyl acetate (Chemajet Chemical Company,

Egypt) were used. All other chemicals and solvents used were of analytical grade.

Xatral® SR tablets labeled to contain ALF 5 mg (B.N. 517901 [B]) are manufactured by Amriya Pharmaceutical Industries (Alexandria, Egypt) under license of Laboratoires Synthelabo, Synthelabo groupe, Le Plessis Robinson, France. Cardura® tablets (B.N. 2202) labeled to contain DOX 4 mg and Minipress® tablets (B.N. 1762) containing PRZ 2 mg are manufactured by Pfizer Egypt S.A.E. (Cairo, Egypt) under authority of Pfizer Inc, USA. Itrin® tablets (B.N. 06939/3J) labeled to contain TER 5 mg are manufactured by Kahira Pharmaceuticals & Chemical Industries Company (Cairo, Egypt) under license from Abbott laboratories (Abbott Park, IL, USA). Proscar® tablets labeled to contain FIN 5 mg (B.N. 310510) are manufactured by MSD (Merck Sharp & Dome, Whitehouse Station, NJ, USA).

For the construction of calibration curves, serial portions of the previous stock solutions were separately transferred into 5 sets of 10 mL volumetric flasks to produce working standard solutions of concentrations 6-70, 6-70, 4-40, 6-70 and 40-400 ig/mL for ALF, TER, PRZ, DOX and FIN, respectively. All flasks were completed to volume with methanol. From these working standard solutions, 5-iL portions were spotted as bands on HPTLC plates to obtain final concentrations of 30-350, 30-350, 20-200, 30-350 and 200-2000 ng/spot for ALF, TER, PRZ, DOX and FIN, respectively. Sample spotting as well as plate development and scanning were performed as mentioned under Instrumentation. For each drug, the obtained peak areas were correlated to the corresponding concentrations and the regression equation was developed.

2.4. Application of the proposed method

2.2. Instrumentation

The precoated TLC silica gel aluminum plates 60F254 (20 x 15 cm, 200 im thickness) used in the study were purchased from Fluka (Buchs, Switzerland). Samples were spotted on plates in the form of 5 mm-wide bands using a Camag microliter syringe under nitrogen stream with the aid of Camag Linomat IV sample applicator (Switzerland). Sample volume was fixed at 5 iL and applied at a rate of 0.15 iL/s and at a distance of 4 mm apart. Following sample application, plate development was achieved in the linear ascending mode using 20 mL of the mobile phase consisting of methylene chloride, n-hexane and methanol (8.8:0.3:0.9, by volume). A Camag twin trough glass chamber (20 x 20 cm) pre-satu-rated with the mobile phase for 15 min at room temperature was used for development. The length of the chromatogram run was 135 ± 2 mm. Subsequent to development, the plates were air dried and scanned densitometrically in the absorbance mode at 254 nm for ALF, TER, PRZ and DOX determination and at 220 nm for the determination of FIN using Camag TLC scanner III operated by CATS software (V 3.15). The slit dimensions were adjusted at 4 x 0.45 mm, and a scanning speed of 20 mm/s was employed. The deuterium lamp was used as the radiation source.

2.3. General procedure

Methanolic stock solutions of ALF, TER and DOX were separately prepared at a concentration of 250 ig/mL. PRZ 200 ig/mL and FIN 2000 ig/mL stock solutions were also prepared in methnol. Stock solutions were kept refrigerated at 4 °C and protected from light.

Table 1

Regression and statistical parameters for the determination of ALF, DOX, PRZ, TER and FIN using the proposed HPTLC method.

2.4.1. Analysis of laboratory prepared mixtures

Aliquots from the stock solutions were transferred into 10 mL volumetric flasks to produce synthetic mixtures containing the 5 drugs at different concentrations, and the flasks were completed to volume with methanol. From these mixture solutions, 5-p.L portions were spotted on HPTLC plates to obtain final concentrations within the specified linearity ranges (Table 1). Spot application, plate development and scanning were performed as mentioned under Instrumentation. The peak area of each drug in every mixture was recorded and the recovered concentration was calculated from the corresponding regression equation.

2.4.2. Analysis of tablets

For the analysis of each drug in its single component pharmaceutical preparation, twenty tablets were accurately weighed, finely powdered and thoroughly mixed. For every preparation, a quantity of the powdered tablets was precisely weighed so as to contain 12.5 mg of either ALF, TER or DOX, 10 mg of PRZ or 50 mg of FIN. The measured weight was extracted with 25 mL methanol with the aid of vortex mixing for 10 min. The obtained extract was filtered into a 50 mL volumetric flask and the residue was washed with 2 x 10 mL portions of methanol. The washings were combined with the corresponding extract solution and the volume was completed to mark with methanol. The produced tablet-extract solutions were labeled to contain 250 ig/mL ALF, 250 ig/mL DOX, 250 ig/mL TER, 200 ig/mL PRZ or 1000 ig/mL FIN. Accurate volumes from the aforementioned extract solutions were separately transferred into 10 mL volumetric flasks to prepare working sample solutions and the flasks were completed to

Parameter ALF TER PRZ DOX FIN

Wavelength (nm) 254 254 254 254 220

Concentration range (ng/spot) 30-350 30-350 20-200 30-350 200-2000

Intercept (a) 28.79 60.39 29.07 96.74 45.84

S a 33.86 39.24 40.07 45.32 67.56

Slope (b) 14.23 13.24 28.63 15.64 3.68

Sbb 0.24 0.18 0.33 0.23 0.06

RSD% of the slope (Sb%) 1.69 1.36 1.15 1.47 1.63

Correlation coefficient (r) 0.9994 0.9996 0.9997 0.9996 0.9995

c Sy/x 47.45 55.74 55.37 70.37 82.71

Fd 3575 5453 7489 4681 4228

Significance F 4.7 x 10-7 2.0 x 10-7 1.1 x 10-7 2.7 x 10-7 3.4 x 10-7

LODe (ng/spot) 7.85 9.78 4.62 9.56 60.58

LOQf (ng/spot) 23.80 29.64 14.00 28.98 183.59

a Standard deviation of the intercept. b Standard deviation of the slope. c Standard deviation of residuals.

d Variance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). e Limit of detection. f Limit of quantification.

Fig. 2. Mixture of ALF 100 ng/spot, TER 100 ng/spot, PRZ 200 ng/spot, DOX 200 ng/spot and FIN 500 ng/spot at (a) 254 nm and (b) 220 nm.

volume with methanol. From these working sample solutions, 5-p.L portions were spotted on HPTLC plates to obtain final concentrations within the specified linearity ranges (Table 1). Samples were analyzed as mentioned under Instrumentation and General Procedure and the found concentrations were calculated from similarly prepared external standard solutions. For standard addition assay, sample solutions were spiked with aliquots of standard solutions of the corresponding drugs to obtain total concentrations within the previously specified ranges then treated as under General Procedure. Recovered concentrations were calculated by comparing the analyte response with the increment response attained after addition of the standard.

3. Results and discussion

3.1. Optimization of the mobile phase and the detection wavelength

Preliminary trials were directed towards the separation of all five drugs on 10 cm TLC plates. Mobile phases composed of: [ethyl

acetate and ethanol], [ethyl acetate and methanol], [methylene chloride and ethanol], [methylene chloride and methanol], [methylene chloride and isopropanol], [toluene and ethanol] or [toluene and methanol], in different ratios, failed to resolve the investigated drugs. Although that those containing ethyl acetate provided slightly better resolutions, still, in all cases, the co-elution of ALF with TER and/or DOX with FIN was observed. Further increase in solvent polarity resulted in the co-elution of FIN with the solvent front. Therefore, it was clear that the use of 15-cm TLC plates for development is inevitable. Initially, mobile phases composed of different ratios of ethyl acetate with either ethanol or methanol were tried and the following elution order was observed; ALF, TER, PRZ, DOX and finally FIN. The ethyl acetate/methanol mixture was slightly better in terms of resolution, however, spots were broad and diffuse and those of DOX and FIN overlap. As the ablockers bear a basic center, ammonia seemed as an appropriate additive to this mobile phase in order to overcome spot tailing as well as to enhance peak shape and resolution. In general, the [ethyl acetate:methanol:ammonia] system resulted in reversing the order of elution to become as follows: TER, ALF, PRZ, FIN and finally

DOX in terms of increasing Rf values. A wide range of ratios of this system was tried and all resulted in the constant co-elution of FIN with either PRZ or DOX as well as relatively large Rf values. In fact, the addition of ammonia had a negative impact of compounds' resolution with only slight improvement in peak shape, therefore, its use in the ethyl acetate: methanol system was excluded. Afterwards, trials were directed towards enhancing the resolution of the aforementioned system through adding a non-polar solvent such as n-hexane or toluene in different ratios and it was clear that the former solvent produced better results. Finally, a mobile phase composed of ethyl acetate:n-hexane:methanol [6:3:1, by volume] resulted in adequate resolution between all five compounds, however, the broad and diffuse peak shapes were still unacceptable. Gradual introduction of methylene chloride on the expense of both ethyl acetate and n-hexane resulted in a significant improvement in peak shape without affecting resolution and this effect was more pronounced by decreasing the ethyl acetate content of this quaternary mixture. Indeed, the total replacement of ethyl acetate with methylene chloride while maintaining the other 2 components provided the best and the simplest developing system in terms of resolution, peak shape and symmetry. Finally, the developing system was adjusted by using [methylene chloride:n-hex-ane:methanol] in the ratio [8.8:0.3:0.9, by volume] and the spots of ALF, TER, PRZ, DOX and FIN were observed at Rf values equivalent to 0.26 ±0.02, 0.36 ±0.02, 0.45 ±0.02, 0.59 ±0.02 and 0.69 ± 0.02, respectively. The chamber was saturated with the mobile phase at room temperature for 15 min prior to development. The optimized procedure resulted in well-defined spots with reproducible Rf values (Fig. 2).

Pertaining to the choice of the detection wavelength, each plate was scanned twice for the simultaneous determination of all 5 drugs in mixture. Due to the structural similarity between ALF, TER, PRZ and DOX, they as well share similar absorption characteristics, where they considerably absorb UV-light in the range of 247-257 nm (Fig. 3). Therefore, the universal wavelength (254 nm) was selected for determination of the a-blockers concurrently. On the other hand, FIN is a steroidal compound with weak UV absorption (Fig. 3). It exhibits considerable absorption only in the short UV region (below 230 nm), consequently, its wavelength of detection was optimized at 220 nm in order to enhance the sen-

sitivity of its quantification without being affected by the high background noise seen at lower wavelengths.

3.2. Method validation

3.2.1. Linearity and ranges

Serial dilutions of the studied drugs over the concentration ranges stated in Table 1 were prepared in triplicates and analyzed as previously described. The average peak areas of each drug were plotted versus the corresponding concentrations and the calibration curve was constructed using the least squares regression model. From the values gathered in Table 1, good linearity can be verified from the close to unity correlation coefficient (r p 0.9994) and the low values of the relative standard deviation of the slope (Sb% < 1.7%). The standard deviation of the residuals is another important statistical tool to assess method linearity. In the developed method, low values of Sy/x were obtained which designate the negligible difference between the calculated and the found y-values and hence, the closeness of the experimental points to the best fitted regression line. Furthermore, the regression equations showed high F values which indicate steeper regression lines, and low significant F values which confirm the less scattered experimental points around the regression line.

3.2.2. Limits of detection and quantification

Limits of detection (LOD) and quantification (LOQ) were determined according the ICH recommendations [23]. LOD was calculated from 3.3S/b while LOQ was computed from 10S/b, where S is the standard deviation of the intercept of the calibration curve, and b is the slope. As seen from values collected in Table 1, high method sensitivity could be inferred from the low values of LOD that were found to be 7.85, 9.78, 4.62, 9.56 and 60.58 ng/spot for ALF, TER, PRZ, DOX and FIN respectively.

3.2.3. Precision and accuracy

The accuracy, intra-day and inter-day precisions of the proposed method were evaluated according to the ICH recommendations [23]. For each drug; method repeatability was tested through the analysis of standard solutions prepared in triplicates at 3 concentration levels within the same day. On the other hand, the intermediate precision was examined by analyzing standard solutions

Fig. 3. Absorption spectra of ALF (

), TER (-), PRZ (

[ nm ]

,, DOX (-) and FIN (-

Table 2

Precision and accuracy for the determination of ALF, DOX, PRZ, TER and FIN in bulk form using the proposed HPTLC method.

Drug Nominal value (ng/spot) Within-day Between-day

Found ± SDa (ng/spot) RSD(%)b Er(%)c Found ± SDa (ng/spot) RSD(%)b Er(%)c

ALF 80 79.49 ± 0.90 1.13 -0.64 79.82 ±1.00 1.25 -0.22

200 203.73 ± 0.94 0.46 1.87 199.89 ±3.45 1.73 -0.05

300 300.82 ±1.86 0.62 0.27 300.00 ± 5.09 1.70 0.00

TER 80 81.01 ±1.10 1.36 1.26 78.43 ±1.23 1.57 -1.96

200 203.72 ±1.60 0.79 1.87 197.95 ±3.34 1.69 -1.02

300 296.57 ± 2.87 0.97 -1.14 300.03 ±4.10 1.37 0.01

PRZ 50 50.14 ± 0.63 1.26 0.28 50.74 ± 0.69 1.36 1.48

100 99.29 ±1.24 1.25 -0.71 100.72 ±1.76 1.75 0.72

150 150.96 ±1.05 0.70 0.64 149.14 ±1.70 1.14 -0.57

DOX 80 79.98 ± 0.83 1.04 -0.03 81.15 ±1.14 1.41 1.44

200 200.28 ±1.51 0.75 0.14 199.31 ±2.07 1.04 -0.35

300 301.67 ±2.12 0.70 0.56 304.74 ± 2.74 0.90 1.58

FIN 500 499.91 ± 4.63 0.93 -0.02 494.96 ± 9.56 1.93 -1.01

1000 1012.10 ±10.01 0.99 1.21 1002.99 ±12.17 1.21 0.30

2000 2015.30 ±27.88 1.38 0.77 1975.11 ±29.40 1.49 -1.24

a Mean ± standard deviation for 3 determinations. b % Relative standard deviation. c % Relative error.

Table 3

Determination of ALF, DOX, PRZ, TER and FIN in laboratory-prepared mixtures by the proposed HPTLC method.

Synthetic mix Drug Nominal value (ng/spot) Found ± SDa (ng/spot) RSD(%)b Er(%)c

1 ALF 200 202.72 ±3.15 1.55 1.36

TER 200 197.76 ±0.95 0.48 -1.12

PRZ 200 196.24 ±1.60 0.82 -1.88

DOX 200 199.79 ±3.74 1.87 -0.11

FIN 200 202.36 ± 4.74 2.34 1.18

2 ALF 200 198.19 ±1.91 0.96 -0.91

TER 200 201.77 ±2.16 1.07 0.89

PRZ 200 199.69 ±3.55 1.78 -0.16

DOX 200 200.69 ± 4.36 2.17 0.35

FIN 400 392.04 ± 4.53 1.16 -1.99

3 ALF 200 199.28 ±2.23 1.12 -0.36

TER 200 198.54 ±2.43 1.22 -0.73

PRZ 100 100.26 ±1.20 1.20 0.26

DOX 100 101.92 ±1.23 1.21 1.92

FIN 1000 994.91 ±17.84 1.79 -0.51

4 ALF 300 300.17 ±3.15 1.05 0.06

TER 300 302.46 ± 3.48 1.15 0.82

PRZ 60 59.68 ± 0.38 0.64 -0.53

DOX 60 59.91 ±1.15 1.92 -0.15

FIN 600 605.00 ± 3.68 0.61 0.83

5 ALF 100 99.55 ±1.67 1.68 -0.45

TER 100 101.97 ±1.98 1.94 1.97

PRZ 200 196.54 ±4.36 2.22 -1.73

DOX 200 198.14 ±2.98 1.50 -0.93

FIN 500 493.89 ± 7.49 1.52 -1.22

6 ALF 100 99.17 ±1.24 1.25 -0.83

TER 200 201.48 ±3.63 1.80 0.74

PRZ 200 203.94 ± 5.61 2.75 1.97

DOX 100 99.29 ±1.26 1.27 -0.71

FIN 500 498.23 ± 3.67 0.74 -0.35

7 ALF 300 296.34 ± 3.90 1.32 -1.22

TER 60 59.80 ± 0.59 0.99 -0.33

PRZ 60 60.59 ±1.23 2.03 0.98

DOX 300 300.94 ± 5.92 1.97 0.31

FIN 300 297.12 ±5.67 1.91 -0.96

a Mean ± standard deviation for five determinations. b % Relative standard deviation. c % Relative error.

prepared at the same concentration levels repeated over 3 days. Table 2 compiles the obtained percentage relative standard deviation (RSD%) values; which in all cases were less than 2%. Accordingly such low values of RSD% can indicate the satisfactory level of precision of the proposed method. In addition, the method can be deemed accurate as shown by the recovered concentrations and the values of percentage relative error (Er%) that did not exceed ±2%.

3.2.4. Selectivity and specificity

The selectivity of the proposed method for the simultaneous determination of the cited drugs was assessed through the analysis of laboratory prepared synthetic mixtures. These were prepared in order to contain a combination of the 5 drugs at different ratios within their linearity ranges mentioned in Table 1. The recovered concentration for each drug was further calculated from the corresponding regression equation. As seen in Table 3, the acceptable

Fig. 4. Purity assessment of standard and tablet solutions of (a) ALF, (b) TER, (c) PRZ, (d) DOX and (e) FIN.

values of the found concentration, percentage relative standard deviation RSD% (62.75) and percentage relative error Er% (within ±2%) confirm the accuracy, precision and selectivity of the developed method. In addition, it can be inferred that the proposed method is adequately capable of resolving and simultaneously quantifying the investigated drugs when present over a wide range of ratios within solutions.

The specificity of the method was also ascertained through the peak purity profiling performed by the Wincats® software. Following the recording of the UV absorption spectrum at several points across each peak by the TLC scanner, the software is capable of evaluating the purity of the peaks through 2 main steps. At first, the correlation coefficient (rsm) between the spectra extracted at peak start and peak maximum and the correlation coefficient (re m) between the spectra extracted at peak end and peak maximum are calculated. Secondly the software interprets mathematically the significance of the correlation values and gives a decision about the purity of the designated peak [24]. Following plate scanning, drug spots from both standard and tablet sample solutions were further analyzed for purity. As seen in Fig. 4, spots were declared pure as the spectra extracted at different points across the absorption spectra were superimposed on one another and the calculated correlation coefficients values were not less than 0.9991.

3.2.5. Robustness

Robustness of the proposed method was evaluated through investigating its ability to remain unaffected by small but deliberate

variations in method conditions [23]. Accordingly, the wavelength of detection was varied by a value of ±2 nm. Another parameter is the mobile phase total volume that was changed by ±5 mL, and additionally, the individual components that were alternatively altered by ±0.2 mL. Times of saturation and development were also varied and their effect on quantification was investigated. Finally, variation of the source of TLC plates was tested (Fluka or Merck). In all these experiments, the variation in conditions did not have any significant effect on the separation or quantification of the studied drugs. The found percentage recoveries were between 96.96% and 102.14%. In addition, the relative standard deviation of peak areas did not exceed 3% thus confirming the adequate level of method robustness (Table 4).

3.2.6. Stability of solutions

The stability of working solutions of the five drugs in the diluting solvent (methanol) was examined and no chromatographic changes were observed within 24 h at room temperature. Chro-matographic parameters including Rf values and peak areas of the drugs remained almost unchanged and no significant degradation was observed during this period. Also, the stock solutions prepared in methanol were stable for at least two weeks when kept refrigerated at 4 °C and protected from light.

4. Analysis of tablets

In order to further assess the applicability of the proposed method, commercially available tablets containing the investigated

Table 4

Robustness of the proposed HPTLC method.

Parameter Drug Peak area ± SD RSD%

Detection wavelength, ±2 nm ALF 1422 ± 29 2.04

TER 1384 ±22 1.59

PRZ 2259 ± 31 1.37

DOX 1651 ±18 1.09

FIN 2576 ± 66 2.56

Mobile phase volume, 20 ± 5 mL ALF 1437 ± 25 1.74

TER 1409 ± 41 2.91

PRZ 2263 ± 48 2.12

DOX 1643 ±19 1.16

FIN 2578 ± 76 2.95

Mobile phase composition (methylene ALF 1446 ±13 0.90

chloride: n-hexane:methanol) TER 1395 ±35 2.51

(17.6 ±0.2:0.6 ±0.2:1.8 ±0.2) PRZ 2250 ± 34 1.51

DOX 1616 ±10 0.62

FIN 2597 ± 49 1.89

Time from development to scan 10, 20, 30 min ALF 1443 ± 16 1.11

TER 1369 ±35 2.56

PRZ 2256 ± 49 2.17

DOX 1669 ±24 1.44

FIN 2562 ± 34 1.33

Time of saturation, 15, 30, 45 min ALF 1432 ± 32 2.24

TER 1363 ±40 2.94

PRZ 2333 ± 61 2.62

DOX 1671 ±38 2.27

FIN 2637 ± 37 1.40

Plate source (Fluka, Merck) ALF 1455 ± 32 2.20

TER 1420 ±7 0.49

PRZ 2313 ±49 2.12

DOX 1640 ±41 2.50

FIN 2695 ± 64 2.38

Table 5

Determination of ALF, DOX, PRZ, TRZ and FIN in commercial tablets by the proposed HPTLC method.

External Reference Standard

standard method addition

ALF in Xatral® %Recovery ± SDa RSD% t F

TER in Itrin® %Recovery ± SDa RSD% t F

PRZ in Minipress® %Recovery ± SDa RSD% t F

DOX in Cardura® %Recovery ± SDa RSD% t F

FIN in Proscar® %Recovery ± SDa RSD% t F

a Mean ± standard deviation for five determinations. Theoretical values for t and F are 2.31 and 6.39, respectively.

drugs were analyzed as previously described. Results obtained were precise, accurate and in good agreement with the label claim as displayed by the adequate values of percentage recoveries, standard

deviation and RSD(%) (Table 5). HPLC-UV reference methods were also applied for the quantification of TER, PRZ and DOX [16] as well as the determination of ALF [7] and FIN [25]. Results obtained from the proposed method as well as the reference ones were statistically compared with one another using the Student's t-test for accuracy and the variance ratio F-test for precision. Calculated t and F values were less than the tabulated ones for each drug representing the insignificant difference between the reported and the proposed method at the 95% confidence level. The standard addition technique was also applied by spiking tablet sample solutions with known volumes of the corresponding standard solution to obtain total concentrations within the specified concentration ranges and the recovery of each drug was then calculated. The good recoveries obtained using both external standard and standard addition methods suggest that there was no interference from the co-formulated inactive ingredients (Table 5). It can be concluded that the satisfactory analytical performance of the method fortifies its aptness for the routine analysis of any of the selected drugs in quality control units.

5. Conclusion

To the best of our knowledge, the literature contains no records of a generic method that allows the simultaneous determination of alfuzosin, doxazosin, prazosin, terazosin and finasteride by any analytical methodology. HPTLC exhibits several advantages over HPLC and consequently it is sometimes favored over it. HPTLC consumes by far less amount of solvents and therefore can be regarded as more economic and environment friendly. In addition, it is a fast method of analysis allowing the simultaneous processing of large number of samples with no memory effect. Furthermore, it allows the detection of all compounds even those strongly retained on baseline. Indeed, HPTLC does not require elaborate treatment or the sophisticated experimental setup usually associated with HPLC methods of analysis. This work describes a simple, sensitive and robust HPTLC method for the simultaneous determination of the aforementioned drugs in their bulk form. The proposed method met the ICH validation acceptance criteria concerning; linearity, ranges, precision and accuracy. The selectivity of the proposed method was evaluated through the analysis of several laboratory prepared mixtures at different ratios within the linearity ranges of the drugs. In addition, the applicability of the proposed method to real life situations was assessed through the analysis of commercially available tablets and satisfactory results were obtained in comparison to other reported methods.

Conflicts of interest

The authors declared no conflicts of interest.

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