Scholarly article on topic 'Validated HPTLC methods for determination of some selected antihypertensive mixtures in their combined dosage forms'

Validated HPTLC methods for determination of some selected antihypertensive mixtures in their combined dosage forms Academic research paper on "Chemical sciences"

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{Carvedilol / Hydrochlorothiazide / "Amlodipine besylate" / Valsartan / HPTLC / "Pharmaceutical tablets"}

Abstract of research paper on Chemical sciences, author of scientific article — Rasha A. Shaalan, Tarek S. Belal, Fawzy A. El Yazbi, Sohila M. Elonsy

Abstract Simple and selective HPTLC methods were developed for the simultaneous determination of the antihypertensive drugs; carvedilol and hydrochlorothiazide in their binary mixture (Mixture I) and amlodipine besylate, valsartan, and hydrochlorothiazide in their combined ternary formulation (Mixture II). Effective chromatographic separation was achieved on Fluka TLC plates 20×20cm aluminum cards, 0.2mm thickness through linear ascending development. For Mixture I, the mobile phase composed of chloroform–methanol in the ratio 8:2v/v. Detection was performed at 254nm for both carvedilol and hydrochlorothiazide. For Mixture II, the mobile phase was chloroform–methanol–ammonia in the volume ratio 8:2:0.1. Detection was performed at 254nm for valsartan and hydrochlorothiazide, and at 365nm for amlodipine. Quantification was based on spectrodensitometric analysis. Analytical performance of the proposed HPTLC procedures was statistically validated with respect to linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 0.05–1.0 and 0.1–2.0μg/spot for carvedilol and hydrochlorothiazide, respectively in Mixture I, 0.1–2.0, 0.1–2.0 and 0.2–4.0μg/spot for amlodipine, hydrochlorothiazide and valsartan, respectively in Mixture II, with correlation coefficients >0.9992. The validated HPTLC methods were applied to the analysis of the cited antihypertensive drugs in their combined pharmaceutical tablets. The proposed methods confirmed peak identity and purity.

Academic research paper on topic "Validated HPTLC methods for determination of some selected antihypertensive mixtures in their combined dosage forms"

Bulletin of Faculty of Pharmacy, Cairo University (2014) 52, 225-237

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Cairo University Bulletin of Faculty of Pharmacy, Cairo University

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original article

Validated HPTLC methods for determination c^Ma*

of some selected antihypertensive mixtures in their combined dosage forms

Rasha A. Shaalan a *, Tarek S. Belal a, Fawzy A. El Yazbi a, Sohila M. Elonsy b

a Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah 21521, Alexandria, Egypt

b Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt

Received 9 April 2014; accepted 14 July 2014 Available online 14 August 2014

KEYWORDS

Carvedilol; Hydrochlorothiazide; Amlodipine besylate; Valsartan; HPTLC;

Pharmaceutical tablets

Abstract Simple and selective HPTLC methods were developed for the simultaneous determination of the antihypertensive drugs; carvedilol and hydrochlorothiazide in their binary mixture (Mixture I) and amlodipine besylate, valsartan, and hydrochlorothiazide in their combined ternary formulation (Mixture II). Effective chromatographic separation was achieved on Fluka TLC plates 20 x 20 cm aluminum cards, 0.2 mm thickness through linear ascending development. For Mixture I, the mobile phase composed of chloroform-methanol in the ratio 8:2 v/v. Detection was performed at 254 nm for both carvedilol and hydrochlorothiazide. For Mixture II, the mobile phase was chloroform-methanol-ammonia in the volume ratio 8:2:0.1. Detection was performed at 254 nm for valsartan and hydrochlorothiazide, and at 365 nm for amlodipine. Quantification was based on spectrodensitometric analysis. Analytical performance of the proposed HPTLC procedures was statistically validated with respect to linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 0.05-1.0 and 0.12.0 ig/spot for carvedilol and hydrochlorothiazide, respectively in Mixture I, 0.1-2.0, 0.1-2.0 and 0.2-4.0 ig/spot for amlodipine, hydrochlorothiazide and valsartan, respectively in Mixture II, with correlation coefficients >0.9992. The validated HPTLC methods were applied to the analysis of the cited antihypertensive drugs in their combined pharmaceutical tablets. The proposed methods confirmed peak identity and purity.

© 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.

1. Introduction

* Corresponding author. Tel.: +20 3 4871317; fax: +20 3 4871351. E-mail address: rasha_shaalan@yahoo.com (R.A. Shaalan). Peer review under responsibility of Faculty of Pharmacy, Cairo University.

Carvedilol (CRV) (Fig. 1), chemically known as (2RS)-1-(9H-carbazol-4-yloxy)-3-[[2-(2-ethoxyphenoxy)ethyl]amino]propan-2-ol,1 is a non-cardioselective beta blocker. It has vasodilating properties, which are attributed mainly to its alpha-1 blocking

http://dx.doi.org/10.1016/j.bfopcu.2014.07.001

1110-0931 © 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.

oo y hct

/ \ HN\^N

Figure 1 Chemical structures of carvedilol (CRV), hydrochlorothiazide (HCT), amlodipine besylate (AML) and valsartan (VAL).

activity; at higher doses, calcium channel blocking activity may contribute. CRV is used in the management of hypertension, angina pectoris and as an adjunct to standard therapy in symptomatic heart failure.2 Hydrochlorothiazide (HCT) (Fig. 1), chemically known as 6-chloro-3,4-dihydro-2H-1,2,4-benzothi-adiazine-7-sulfonamide-1,1-dioxide,1 is a moderately potent diuretic. HCT is used in the treatment of hypertension either alone or with other antihypertensives. It is also used to treat edema associated with heart failure and with renal and hepatic disorders.2 The fixed dose combination of CRV and HCT has been used for the treatment of essential hypertension particularly if with the monotherapy no sufficient blood pressure lowering can be achieved.3 The simultaneous determination of CRV and HCT in their binary combination was addressed in few analytical reports. These reports proposed several spec-trophotometric4'5 and RP-HPLC with UV detection methods.4'6 A stability-indicating HPLC method was recently published.7 The fact that up till now the simultaneous HPTLC

determination of this binary mixture has been reported in the literature in only one report8 has encouraged us to develop this work.

Amlodipine besylate (AML) (Fig. 1), chemically known as 3-ethyl-5-methyl2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methylpyridine-3,5-dicarboxylate benzenesulph-onate,2 is a dihydropyridine calcium channel blocker used in the treatment of hypertension and angina pectoris.2 Valsartan (VAL) (Fig. 1), chemically known as N-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]-N-valeryl-L-valine,2 is an angiotensin II receptor antagonist used in the management of hypertension, to reduce cardiovascular mortality in myocardial infarction patients and in the management of heart failure.2 In 2009, the US Food and Drug Administration (FDA) and the European Medicines Agency approved a triple fixed-dose combination of AML, VAL and HCT. It was found that the use of this triple combination was generally more effective in reducing blood pressure and providing overall blood pressure

Table 1 Linear regression data for calibration plots of the analyzed drugs using the proposed HPTLC methods. Parameter MIX I MIX II

CRV HCT AML HCT VAL

Wavelength (nm) 254 254 365 254 254

Rf 0.62 ± 0.06 0.37 ± 0.03 0.70 ± 0.04 0.29 ± 0.03 0.07 ± 0.02

Concentration range (ig/spot) 0.05-1 0.1-2 0.1-2 0.1-2 0.2-4

Intercept (a) 86.54 259.72 415.55 336.15 785.76

Sa Sa 23.15 41.57 169.19 52.24 128.16

Slope (b) 8408.28 4492.48 6260.78 3974.91 3603.90

Sbb 45.35 40.38 127.59 55.53 53.80

RSD% of slope 0.54 0.90 2.04 1.39 1.49

r 0.9999 0.9997 0.9992 0.9992 0.9993

V Fd 50.42 88.69 253.10 108.38 241.77

34376.58 12377.49 2407.84 5123.69 4487.29

Significance F 8.01 x 10-16 4.76 x 10-14 1.03 x 10-6 1.62 x 10-12 7.44 x 10-10

LODe (ig/spot) 0.01 0.03 0.02 0.03 0.07

LOQf (ig/spot) 0.04 0.08 0.07 0.09 0.18

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.

All tracks @ 254 nm

0 80 0 0

Figure 2 3D chromatogram of 0.1, 0.2, 0.5, and 1 ig/spot CRV and 0.1, 0.2, 0.5, and 1 ig/spot HCT duplicate spot for each concentration detected at 254 nm.

control than the dual combination therapies regardless of age, race, gender, ethnicity, or hypertension severity.9'10 Few reports can be found in the scientific literature for the simultaneous determination of AML, VAL and HCT. These reports presented spectrophotometry,11-14 HPTLC14-16 and several HPLC methods.14-20

This work includes HPTLC determination of the two antihypertensive mixtures namely; CRV-HCT binary mixture and AML-HCT-VAL ternary mixture. The objective of the work was directed toward the development, validation and application of simple and easy to use methods for routine determination of the studied drugs in their formulations. As

Figure 3 3D chromatogram of 0.1, 0.2, 0.5, 1, 1.50, and 2 ig/spot AML, 0.1, 0.2, 0.5, 1, 1.50, and 2 ig/spot HCT and 0.4, 0.8, 1, 2, 3, and 4 ig/spot VAL duplicate spot for each concentration at (A) 254 nm and (B) 365 nm.

method validation is an important requirement in analytical method development, the proposed method has been validated following the guidelines of the USP and ICH.

2. Experimental

2.1. Instrumentation

CAMAG Linomat TLC-Applicator. A CAMAG Linomat syringe (100 iL) was used for the application of solutions onto silica gel plates. The plates were developed in CAMAG twin

trough chromatographic tanks (15 x 20 x 30 cm), and scanned densitometrically using CAMAG TLC-Scanner 3 (Version 4.06), Supported with deuterium, and tungsten lamp and interfaced to an IBM computer loaded with CAMAG-TLC-Soft-ware (CATS).

2.2. Materials and reagents

CRV was kindly supplied by Chemipharm Pharmaceutical Industries, 6th October City, Egypt. HCT was kindly donated by Pharco Pharmaceuticals Co., Alexandria, Egypt. AML was

Table 2 Precision and accuracy for determination of the analyzed drugs in bulk form using the proposed HPTLC methods. Drug CRV HCT

Nominal value (ig/spot) Within-day

Found ± SDa (ig/spot)

RSD(%)b

Er(%)c

Between-day

Found ± SDa (ig/spot)

RSD(%)b

Er(%)c

0.2022 ± 0.0035

0.2034 ± 0.0014

0.5006 ± 0.0055 1.10 0.12

0.5001 ± 0.0044

0.9954 ± 0.0020

0.9948 ± 0.0061

0.2027 ± 0.0039

0.2027 ± 0.0032

0.5099 ± 0.0054 1.06 1.98

0.5039 ± 0.0070

0.9940 ± 0.0014 0.14 -0.6

0.9983 ± 0.0031

Mix II

Nominal value (ig/spot) 0.2

Within-day

Found ± SDa (ig/spot) 0.2027 ± 0.0026 0.4966 ± 0.0026 1.5003 ± 0.0073 0.2036 ± 0.0017 0.5095 ± 0.0019 1.0079 ± 0.0017 0.8082 ± 0.0091 1.5096 ± 0.0102 3.9901 ± 0.0052

1.28 0.52 0.49 0.83 0.37 0.17 1.13 0.68 0.13

1.35 -0.68 0.02 1.8 1.9 0.79 1.03 0.64 -0.25

RSD(%)b Er(%)c

Between-day

Found ± SDa (ig/spot) 0.2036 ± 0.0035 0.4987 ± 0.0057 1.5092 ± 0.0071 0.1994 ± 0.0024 0.509 ± 0.0055 1.0061 ± 0.0141 0.8015 ± 0.0069 1.5095 ± 0.0042 3.9908 ± 0.0184

1.2 -0.3

RSD(%)b

Er(%)c

1.72 1.8

0.47 0.61

1.08 1.8

1.4 0.61

0.86 0.19

0.28 0.63

0.46 -0.23

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

Figure 4 Purity spectra between 200 and 400 nm for CRV (A), HCT (B), AML (C), VAL (D).

kindly supplied by Pfizer Egypt S.A.E., Cairo, Egypt, and Sentmenat, Spain), HPLC-grade methanol and chloroform VAL was kindly provided by Novartis Pharma S.A.E., Cairo, (Sigma-aldrich Chemie GmbH, Buchs, Switzerland) were used. Egypt. HPLC-grade acetonitrile (Scharlau Chemie S.A., Pharmaceutical preparations containing the binary mixture are

Table 3 Determination of laboratory-prepared mixtures of the analyzed drugs in bulk form using the proposed HPTLC methods.

Nominal value (ig/ Found ± SDa (ig/spot) RSD(%)b Er(%)c

CRV HCT CRV HCT CRV HCT CRV HCT

0.20 0.40 0.2017 ± 0.0029 0.4043 ± 0.0022 1.44 0.54 0.85 1.08

0.20 0.20 0.2020 ± 0.0013 0.2024 ± 0.0037 0.64 1.83 1 1.20

0.40 0.20 0.3996 ± 0.0013 0.1999 ± 0.0037 0.33 1.85 -0.1 -0.05

0.60 0.20 0.6014 ± 0.0035 0.1970 ± 0.0030 0.58 1.52 0.23 -1.50

0.80 0.20 0.7961 ± 0.0033 0.1966 ± 0.0027 0.41 1.37 -0.49 -1.70

1.00 0.20 0.9959 ± 0.0011 0.2030 ± 0.0007 0.11 0.34 -0.41 1.50

Nominal value (ig/ Found ± SDa (ig/spot) RSD(%)b Er(%)c

AML HCT VAL AML HCT VAL AML HCT VAL AML HCT VAL

0.20 0.20 4.00 0.2002 ± 0.0018 0.2001 ± 0.0031 3.9972 ± 0.0132 0.90 1.55 0.33 0.10 0.05 -0.07

0.10 0.30 3.20 0.1011 ± 0.0019 0.2983 ± 0.0020 3.1955 ± 0.0075 1.88 0.67 0.23 1.10 -0.57 -0.14

1.00 1.00 1.00 1.0015 ± 0.0025 0.9975 ± 0.0092 1.0066 ± 0.0117 0.25 0.92 1.16 0.15 -0.25 0.66

0.25 0.25 1.50 0.2514 ± 0.0001 0.2506 ± 0.0027 1.5072 ± 0.0104 0.04 1.08 0.69 0.56 0.24 0.48

0.25 1.5 0.25 0.2506 ± 0.0013 1.5031 ± 0.0109 0.2515 ± 0.0016 0.52 0.73 0.65 0.24 0.21 0.60

1.50 0.25 0.25 1.4983 ± 0.0162 0.2503 ± 0.0022 0.2499 ± 0.0019 1.08 0.88 0.75 -0.11 0.12 -0.05

a Mean ± standard deviation for five determinations.

b % Relative standard deviation.

c % Relative error.

Co-Dilatrol tablets (Chemipharm Pharmaceutical Industries S.A.E., 6th October City, Egypt, Batch No. 110872A) and Co-Dilatrend tablets (Roche Austria GmbH, Wien, Austria, Batch No. M5015M1) labeled to contain 25 mg CRV and 12.5 mg HCT per tablet. Pharmaceutical preparation containing the three drugs is Exforge HCT tablets labeled to contain 10 mg AML, 320 mg VAL and 25 mg HCT (Novartis Pharma, Switzerland). Analytical grade of ortho-phosphoric acid, hydrochloric acid, ammonia and high purity distilled water was used. Aluminum cards (20 x 20 cm), 0.2 mm thickness with fluorescent indicator at 254 nm Fluka Chemie GmbH, and Sigma-aldrich Chemie GmbH, Buchs, Switzerland were used.

2.3. General procedure and construction of calibration graphs

2.3.1. Preparation of stock and working solutions

Mix I: CRV 1000 ig/mL and HCT 1000 ig/mL stock solutions were prepared in HPLC-grade methanol. The final working solutions were prepared by diluting appropriate aliquots of the stock solutions with HPLC grade methanol into two different sets of 10-mL volumetric flasks to reach the concentration ranges of 5-100 and 10-200 ig/mL for CRV and HCT, respectively. For Mix II: AML (1000 ig/mL), HCT (1000 ig/mL) and VAL (2000 ig/mL) stock solutions were prepared in HPLC-grade methanol. The final working solutions were prepared by diluting appropriate aliquots of the stock solutions with HPLC grade methanol into three different sets of 10mL volumetric flasks to reach the concentration ranges of 10-200, 20-400 and 10-200 ig/mL for AML, HCT and VAL, respectively.

2.3.2. Sample loading

The plates were first activated at 90 °C for 30 min, the solutions were then applied to the marked start edge of the TLC plate at a height of 15 mm from the lower edge of the plate using the specified TLC CAMAG linomat syringe. The sample volume for all solutions was 10 iL and spotting was performed in the form of bands of 6-mm width and the spots were kept at a constant distance of 7 mm from each other to avoid edge effect. Each solution was applied in duplicate and allowed to air-dry for 5 min.

2.3.3. Chromatogram development and scanning

The mobile phase used was chloroform-methanol in the volume ratio 8:2 v/v for mix I, while for Mix II the mobile phase consists of chloroform-methanol-ammonia 8:2:0.1 v/v. The TLC chamber was first rinsed with the mobile phase, and then 15 mL of the mobile phase was poured in the chamber. The chamber was covered with a lid and saturated for 30 min at room temperature (25 ± 2 °C). The sample-loaded TLC plates were transferred to the chamber; Linear ascending development of plates was carried out. Chromatogram run was kept up to 80 mm. Spectrodensitometric analysis of the separated components was carried out using Camag TLC Scanner using deuterium/tungsten lamps set at 254 nm for both mixtures except for AML the lamp was set at 365 nm. The slit dimension used was 4 x 0.45 mm and sensitivity was kept at auto mode. Scanning speed was 20 mm/s.

2.3.4. Data processing

The dried plates were densitometrically scanned at the appropriate wavelengths. The TLC chromatogram was captured by

the scanner and loaded into the CAMAG TLC software. Integration of the chromatogram was carried out using Planar chromatography manager-win CATS (CAMAG).

In the software, the series of spots were selected as tracks. Each track was evaluated based on the position of the spot and its peak area. Quantity calibration was then performed by pre assignment of the concentration of the investigated drugs.

2.3.5. Preparation of sample solutions

MIX I: Ten tablets of each brand (Co-Dilatrol and Co-Dila-trend) were weighed and average weight was calculated. An amount of powdered tablets equivalent to 25 mg of CRV and 12.5 mg of HCT from each brand was transferred into two 100-mL volumetric flasks with the aid of 50 mL methanol, the solutions were stirred for 10 min then filtered into 100-mL calibrated flasks. The residues were washed with 2 x 10 mL methanol and washings were added to the filtrates and diluted to final volume with methanol. MIX II: Ten Exforge HCT® tablets were weighed and finely powdered. HPLC-grade methanol (30 mL) was added to a quantity of the powdered tablets equivalent to 5 mg AML, 12.5 mg HCT, and 16 0 mg VAL, the solution was stirred for 10 min then filtered into a 50-mL calibrated flask. The residue was washed with 2 x 5 mL methanol and washings were added to the filtrate and diluted to final volume with methanol. Aliquots of the tablet solutions were diluted with methanol to obtain final concentrations within the specified ranges then treated as under General Procedure and recovered concentrations were calculated from the corresponding calibration graphs. For standard addition assay, sample solutions were spiked with aliquots of standard solutions of each drug 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.

2.4. Method validation

The proposed HPTLC methods were validated according to the guidelines of the International Conference on Harmonization (ICH).21 The linearities of the methods for the analyzed drugs were checked between 0.05-1.0 ig/spot for CRV, 0.12.0 ig/spot for HCT (in both mixtures), and AML, and 0.24.0 ig/spot for VAL, finally concentration was plotted against the peak area.

2.4.1. Accuracy

Accuracy, as recovery, was determined by the standard addition method. Preanalyzed samples of the analyzed drugs (0.5 ig/spot) were spiked with extra standard solutions (0%, 50%, 100%, and 150%), and the mixtures were reanalyzed. Percentage recovery and relative standard deviation (RSD, %) were calculated for each concentration level.

2.4.2. Precision

Method repeatability was obtained from RSD values by repeating the assay three times on the same day for inter-day precision. Intermediate precision was assessed by the assay of two sets on different days (inter-day precision). Intra- and

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inter-day variations by the proposed HPTLC methods were carried out at three different concentration levels.

2.4.3. Robustness of the method

Robustness of the proposed TLC-densitometric methods was determined to evaluate the influence of small deliberate changes in the chromatographic conditions during determination of the cited drugs.

2.4.4. Limits of detection and quantification

Limit of detection (LOD) and limit of quantification (LOQ) were determined by the signal to noise ratio method. The LOD is defined as the concentration that has a signal-to-noise ratio of 3:1, while for LOQ the ratio is considered to be 10:1. The minimum amount detected under the described chromato-graphic conditions used was estimated.

2.4.5. Specificity

Specificity of the proposed TLC-densitometric methods was confirmed by analyzing and comparing the RF values and spectra of the spots for the analyzed drugs in the samples with those of the standards.

2.4.6. Quantification of analyzed drugs in pharmaceutical formulations and prepared mixtures of standard compounds

The test samples obtained from extraction of tablets or prepared from standards in a concentration similar to that present in formulations were applied, and chromatograms were obtained under the same conditions as for the analysis of standard drugs. The area of the peak corresponding to the Rf value of the drug standards was recorded. Recovered concentrations were calculated from corresponding external standards. For standard addition assay, sample solutions were spiked with ali-quots of standard solutions of the 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 chromatographic conditions

Chromatographic separation studies were carried out on the standard solutions of CRV, HCT, AML, and VAL. Initially, spots of standard solutions were applied on the plates. Plates were developed by linear ascending development using neat solvents like toluene, benzene, methanol, chloroform, ethyl acetate, and acetone. Based on the results of these initial trials, binary and ternary mixtures of solvents were tried to achieve optimum resolution between the mentioned drugs in their respective binary and ternary mixtures under investigation. Accurate, precise, and reproducible results for simultaneous determination of CRV and HCT in MIX I were obtained; good separation of both drugs (Rf values of CRV 0.62 ± 0.06 and HCT 0.37 ± 0.03, respectively) with good symmetrical peaks was obtained using the mobile phase, chlo-roform:methanol (8:2, v/v). The separated spots of both drugs were scanned at 254 nm. For Mix II, good separation of the three drugs was obtained using the mobile phase, chloro-form-methanol-ammonia 8:2:0.1 v/v. Acidification of the mobile phase using acetic acid was tried as well as alkaliniza-tion using ammonia. Acidification of the mobile phase using acetic acid causes overlapping spots, while the use of ammonia gives good resolved and compact spots. The concentration of ammonia in the mobile phase was optimized and changed from 0.1 to 1.0 mL and it was found that 0.1 mL is quite enough. Detection of spots was then performed at 254 nm for HCT and VAL and at 365 nm for AML, where the latter drug shows a broad absorption band suitable for its measurement. Other chromatographic conditions like chamber saturation time, run length, sample application rate and volume, sample application positions, distance between tracks were optimized to

Figure 5 Densitogram of Co-Dilatrend® tablet containing 0.25 ig/spot CRV and 0.125 ig/spot HCT.

give reproducible Rf values, better resolution, and symmetrical peak shape for the studied drugs. The optimized chamber saturation time for the mobile phase was found as 30 min at room temperature (25 ± 2 °C). The length of the chromatogram run was approximately 80 mm. The spots appeared more compact and the peak shape was more symmetrical when the TLC plates were pre-treated and activated at 90 °C for 30 min. After development the plates were dried in air for 5 min. Slit dimension was 4 x 0.45 mm and the scanning speed was 20 mm/s. Densitometric measurements were performed with a CAMAG TLC Scanner 3 in the absorbance mode operated by CAMAG TLC software. The optimized chromatographic conditions gave compact spots for the cited drugs at the specific Rf values mentioned in Table 1.

3.2. Validation of the proposed method

Validation of the proposed methods was carried out in accordance with the International Conference on Harmonization (ICH) guidelines (2005).21

3.2.1. Linearity and concentration ranges Linear correlations were obtained between peak area and concentration for each drug in the ranges specified in Table 1. 3D chromatograms for the linearity sets prepared for the analyzed drugs were presented as double spots in Figs. 2 and 3. Table 1 presents the performance data and statistical parameters including linear regression equations, concentration ranges, correlation coefficients, standard deviations of the intercept

Trick 9. ID: Stindird5

Figure 6 Densitogram of Exforge-HCT® tablet containing 0.1 ig/spot AML, 0.25 ig/spot HCT, and 3.2 ig/spot VAL at (A) 254 nm and (B) 365 nm.

(Sa), slope (Sb), and standard deviations of residuals (Sy/x). Linearity of the calibration plots was confirmed by the high value of the correlation coefficients (P 0.9992). In addition, linearity can be further evaluated by calculation of the RSD% of the slope (Sb%) values, which did not exceed 2.04%.

3.2.2. Detection and quantification limits

The sensitivity of the analytical methods was evaluated by determining the LOD and LOQ. The LOD is defined as the concentration that has a signal-to-noise ratio of 3:1, while for LOQ the ratio is considered to be 10:1. The minimum amount detected under the described chromatographic conditions used was estimated and listed in Table 1. The tabulated values indicate that the methods have sufficient sensitivity.

3.2.3. Accuracy and precision

The intra-day and inter-day precision of the proposed methods were determined by estimating the corresponding responses three times on the same day and on three different days for three different concentrations. Testing of the intra-day and inter-day precision in quantification of the drugs showed that the RSD% values were always less than 2% (Table 2) which confirm the high repeatability and intermediate precision of the developed methods. Additionally, the Er% values were less than 2%, which confirms the accuracy of the proposed methods.

3.2.4. Selectivity and specificity

The peak purity of analyzed drugs was assessed by comparing their respective spectra at peak start, apex and end positions of the peak (Fig. 4A-D). A good correlation (r value more than

0.999) was obtained for all drugs. Acceptable peak purity and correlation values suggest no interference in the quantification of the five analyzed drugs in sample solutions. This proves that the methods are specific. Method selectivity was further examined by preparing several laboratory-prepared mixtures of the mentioned drugs at various concentrations within the linearity ranges mentioned in Table 1. The laboratory-prepared mixtures were analyzed according to the proposed procedure. The recovery values, RSD%, and Er% values shown in Table 3 were satisfactory thus validating the selectivity of the methods.

3.2.5. Robustness

The robustness of the methods was studied by performing assays of cited drugs in pure form, and in tablet formulations. The parameters of the optimized methods were deliberately varied [plate source (Fluka or Merck), plate activation time in the range of 30-50 min, plate activation temperature, chamber saturation time from 15 to 45 min, and time elapsed until scan], changes in the responses of the analyzed drugs were noted and the assay values were calculated in the changed parameters (Table 4). The methods proved to be robust, because the assay and system suitability values in the changed parameters were within the accepted range.

3.2.6. Stability of solutions

The stability of standard working solutions as well as sample solutions in methanol was examined, and no chromatographic changes were observed within 24 h at room temperature. Also, the stock solutions prepared in HPLC-grade methanol were stable for at least two weeks when kept at 4 °C. Rf and spot

Table 5 Application of the proposed HPTLC methods for determination of the analyzed drugs in commercial pharmaceutical tablets. Co-Dilatrend® tablets Results for external Standard Reference method7 Standard addition

CRV HCT CRV HCT CRV HCT

% Recovery ± SDa 99.81 ± 0.50 100.13 ± 0.58 100.10 ± 0.66 99.92 ± 0.24 100.35 ± 0.60 100.11 ± 0.99

RSD%b 0.50 0.58 0.66 0.24 0.6 0.99

t 0.78 0.75

F 1.71 5.82

Co-Dilatrol® tablets Results for external standard Reference method7 Standard addition

CRV HCT CRV HCT CRV HCT

% Recovery ± SDa 99.84 ± 0.49 100.12 ± 0.59 99.56 ± 0.53 99.10 ± 1.13 99.75 ± 0.31 99.89 ± 0.54

RSD%b 0.49 0.59 0.53 1.14 0.31 0.54

t 0.86 1.80

F 1.17 3.61

Results for external standard Reference method18 Standard addition

Exforge AML HCT VAL AML HCT VAL AML HCT VAL

tablets

% Recovery 99.55 ± 0.63 100.01 ± 0.51 99.32 ± 0.91 100.28 ± 1.12 99.67 ± 0.93 100.56 ± 1.10 100.26 ± 0.78 100.01 ± 0.72 99.41 ± 0.56

± SDa

RSD%b 0.63 0.51 0.92 1.12 0.93 1.09 0.78 0.72 0.56

t 1.26 0.71 1.93

F 3.23 3.23 1.45

Theoretical values for t and F at P = 0.05 are 2.31 and 6.39, respectively. a Mean ± standard deviation for five determinations. b % Relative standard deviation.

areas of the drugs remained unchanged and no significant degradation was observed during these periods.

3.3. Assay of tablet dosage forms

The developed HPTLC procedure was applied for the assay of commercial drug combinations in different tablet brands. The active ingredients eluted at their specific Rf values. No interfering peaks were observed from any of the inactive ingredients or the dosage form matrix (Figs. 5 and 6). Recoveries were calculated using both external standard and standard addition methods. The assay results revealed satisfactory accuracy and precision as indicated from % recovery, SD, and RSD% values (Table 5). The obtained results by the proposed methods were also statistically compared to those obtained by the RP-HPLC reference methods7,18 using the Student's t- and the variance ratio F-tests. In both tests, the calculated values did not exceed the theoretical ones at the 95% confidence level which indicated that there were no significant differences between the recoveries obtained from the developed method and those of the reference method (Table 5). It is evident from these results that the proposed methods are applicable for the analysis of the cited active pharmaceuticals in their combined commercial formulations with minimum sample preparation and satisfactory level of selectivity, accuracy, and precision.

4. Conclusion

The developed HPTLC technique is precise, specific, and accurate. The methods were simple and rapid, allowing a high sample throughput necessary for quality control routine analysis with an added advantage of low solvent consumption. The proposed methods were validated as per ICH guidelines, and statistical analysis proves that the methods are repeatable and selective for the analysis of the drugs in bulk form and in pharmaceutical formulations without any interference from the excipients. The standard deviations and RSD% calculated for the methods are good, indicating a high degree of precision of the methods. The results of the recovery studies performed show a high degree of accuracy of the proposed methods. The methods can be used to determine the purity of the drugs available from various sources by detecting the related impurities and their respective Rf values. Moreover, the proposed methods have the advantages of simplicity and convenience.

To the best of our knowledge, the binary mixture of CRV and HCT has been assayed by the TLC method in one recently published report.8 Comparing our proposed method with this report revealed that the proposed method is far more sensitive than the reported method from the point of view of concentration range, LOD and LOQ. Also the mobile phase used in the proposed method is a simpler binary solvent mixture. Two different tablet brands were assayed in this work to validate the applicability of the proposed TLC method for the analysis of pharmaceutical formulations. The precision was studied at three different concentration levels covering the linearity range and not only one concentration. Detailed statistical parameters were provided as discussed in the ''Linearity and concentration ranges'' and presented in Table 1. Comparing our proposed method for the ternary mixture with the few HPTLC reports found in the literature published in sound reputable journals

reveals that our proposed method is of comparable or even better sensitivity and wider concentration ranges for determining the three drugs. The LOD of the proposed method are less than those reported in the literature. The mobile phase used in the study consisting of a binary mixture of chloroform and methanol alkalinized with ammonia (0.1%) is considered more simple and environment friendly than the mobile phases used in the reported methods using ternary and quaternary solvent combinations including ethyl acetate and methanol in addition to chloroform, toluene and n-butyl acetate. Obviously, the described HPTLC methods offer selectivity advantage over the previously published spectrophotometric non-separation methods. Moreover, the proposed methods do not require elaborate treatment or sophisticated experimental setup usually associated with HPLC methods of analysis.

5. Conflict of interest

None declared. References

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