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Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: 2231-6876

DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR THE

SIMULTANEOUS DETERMINATION OF CARVEDILOL, GLIMEPIRIDE OR

GLIBENCLAMIDE IN BINARY COMBINATIONS; AND ITS APPLICATION FOR IN VITRO

- INTERACTION STUDIES

Magda M. Ibrahim Department of Analytical Chemistry, National Organization for Drug Control and Research (NODCAR), Cairo, Egypt.

Corresponding author

Magda Mohamed Ibrahim

lecturer. Department of Analytical Chemistry,

National Organization for Drug Control and Research (NODCAR),

Cairo, Egypt. P.O. Box 29, Cairo.

35851278, +20-235855582

mag- Samaty@hotmail.com

Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical

Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ARTICLE INFO ABSTRACT

Article history

Received 08/08/2015

Available online

31/08/2015

Keywords

RP-HPLC,

Simultaneous,

Carvedilol,

Glimepiride,

Glibenclamide,

In Vitro- Interaction.

Coadministration of antihypertensive and antidiabetic drugs is quite common, as both of these

medical conditions often occur together. In the present work, a simple and accurate RP-HPLC

chromatographic method was developed for the simultaneous determination of the

antihypertensive drug carvedilol (CRV) and the hypoglycemic agent glimepiride (GMP) or

glibenclamide (GBD). This study was carried out using an isocratic reversed phase high-

performance liquid chromatographic method using a C18 column with ultraviolet detection at

220 nm. The system was operated at flow rate of 1 mL/ min using a mobile phase consisting

of 70:30 v/v methanol: 0.2M phosphate buffer (pH 3.5) and column temperature adjusted to

30 oC. The developed RP-HPLC method has been successfully applied for the estimation of

carvedilol and glimepiride or glibenclamide in their binary combination laboratory prepared

tablets with assay percent range 99.49- 99.95 % and relative standard deviation less than 1%

indicating satisfactory accuracy of the method. The method was validated according to the

ICH guidelines; the linearity range is 2-75 μg/mL for CRV and 5-300 μg/mL for each GMP

and GBD; precision, accuracy, robustness, ruggedness, specificity, LOD and LOQ were also

evaluated. The RP-HPLC method was also applied in investigating the possible in vitro drug

interactions of carvedilol and glimepiride or glibenclamide. This study was carried out at

37oC in simulated gastric juice pH 1 or simulated blood pH (pH 7.4). It was observed that

after interaction of carvedilol with glimperide or glibenclamide, the % availability values of

the two antidiabetics were decreased, by about 10 and 20% for glibenclamide and

glimepiride, respectively. In vivo pharmacological studies were recommended, which can

help with the in vitro study in the improvement of clinical efficacy of the coadministered

drugs. The developed RP-HPLC method is rapid, sensitive and accurate and can be applied in

quality control laboratories for routine determination of carvedilol, glimepiride or

glibenclamide in their binary mixtures, and also it can be applied to samples of in vivo

subjects.

Please cite this article in press as Magda Mohamed Ibrahim. Development And Validation of A RP-HPLC Method For The

Simultaneous Determination of Carvedilol, Glimepiride or Glibenclamide In Binary Combinations; And Its Application For In

Vitro - Interaction Studies. Indo American Journal of Pharm Research.2015:5(08).

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INTRODUCTION

Fixed Dose Combination (FDC) products are medicines that contain two or more active ingredients in a fixed ratio of doses

in the same formulation. FDCs may offer several advantages, such as the convenience of fewer pills a patient is required to take daily

and reduced potential for medication errors than two or more single drugs for the treatment. These combinations thus, offer simple

dosage schedule which improves patient compliance and therefore improves treatment outcomes [1]. A particular problem against

FDC is that it discourages separate titration of each active ingredient. Antihypertensive and antidiabetic drugs are commonly

prescribed. Carvedilol chemically designated as (±)-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl][2-(2-methoxyphenoxy)ethyl] amine

(Fig. 1a), is a nonselective beta- blocker [2] used in the treatment of mild to severe congestive heart failure (CHF) and high blood

pressure. Relative to other beta blockers, carvedilol has minimal inverse agonist activity which may decrease its potential to worsen

symptoms of heart failure[3]. Glimepiride is chemically known as 3 -ethyl-4-methyl-N-(4-[N-((1r,4r)-4-

methylcyclohexylcarbamoyl)sulfamoyl]-phenethyl)-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamide (Fig.1b). Glibenclamide also

known as glyburide is chemically defined: 5-chloro-N-(4-[N-(cyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-

methoxybenzamide(Fig.1c). Glimepiride and glibenclamide are medium to long-acting sulfonylurea -antidiabetic drugs [2]. Like all

sulfonylureas, they act as insulin secretagogue. They lower blood sugar by stimulating the release of insulin by pancreatic beta cells

and by inducing increased activity of intracellular insulin receptors [3].

Fig. 1a Carvedilol.

Fig. 1b Glimepiride.

Fig. 1c Glibenclamide.

Several analytical methods have already been developed for the determination of carvedilol individually or in combination

with the diuretic hydrochlorothiazide, they include HPLC [4-8], TLC [9,10], spectrophotometric [11-14] and electroanalytical

methods [15-17]. Various methods have been reported for the determination of glimepiride alone or in combination with other drugs,

they include HPLC [4,18-21], TLC [22-24], spectrophotometric [25-28] and electrochemical [29,30] methods. Also for glibenclamide,

many analytical methods were reported for its analysis, including HPLC [4,21, 31-33], HPTLC [34,35], spectrophotometric [36-39]

and electroanalytical [39,40] methods.

Coadministration of carvedilol with glimepiride or glibenclamide is common and clinically found to be useful to patients

[41]. In literature, no RP-HPLC methods for the simultaneous determination of CRV with GMP or GBD, were reported.

Consequently, in the present study it was aimed to estimate the antihypertensive carvedilol and the antidiabetic glimepiride or

glibenclamide simultaneously (in laboratory prepared combination mixtures) using simple, rapid and accurate RP -HPLC method.

A key goal in pharmaceutical development of dosage forms is a good understanding of the in vitro and in vivo performance of the

dosage forms, which shortens the drug development period, economizes the resources and leads to improved product quality and

clinical efficacy[42]. For this purpose, it was aimed to study the possibility of the in vitro-interaction of CRV with GMP or GBD in

their binary mixtures. This study was carried out in simulated gastric juice pH1 and simulated blood pH 7.4 at body temperature 37oC,

applying the proposed RP-HPLC technique.

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EXPERIMENTAL

Materials and reagents

Carvedilol pure material and Carvid tablets labeled to contain 12.5 mg CRV per tablet were supplied from

Kahira/Multipharma. Glimepiride and glibenclamide pure powders were obtained from Sanofi Aventis. All reagents used were of

HPLC grade. Methanol, potassium dihydrogen orthphosphate, disodium hydrogen orthphosphate, sodium dihydrogen orthophosphate,

sodium chloride and phosphoric acid 85% were purchased from Sigma-Aldrich Company. Deionized filtered water was prepared by

using Millipore purification system. Fresh working solutions were prepared daily and all solutions were filtered through 0.45 μm and

degassed using a sonicator. The mobile phase consisted of a mixture of methanol and 0.2M sodium dihydrogen orthophosphate (pH

adjusted to 3.5 using orthophosphoric acid) in 70: 30 ratio. Diluent solution (A) was prepared by mixing methanol and the phosphate

buffer pH 3.5 in the ratio 50:50.

Instrumentation and chromatographic conditions

The LC system, used for method development and validation was Agilent 1100 series liquid chromatographic RRHT (Rapid

Resolution High Throughput) system comprising of binary pumps, column oven, autosampler and UV detector. Analysis was

performed on a Zorbax Eclipse XDB-C18 of 250 × 4.6 mm id, 5-μm particle size; The mobile phase was pumped from the solvent

reservoir to the column at a flow rate of 1 ml/min for 20 min and the column temperature was maintained at 30 °C. The eluate was

monitored at 220 nm using UV detector and the injection volume was 20 μl. A Shimadzu 2450 spectrophotometer with quartz cells of

1-cm optical path length and a Jenway 3510 pH-meter with a combined glass-saturated calomel electrode were used.

Preparation of solutions

Standard solutions and calibration Graphs

Stock Standard solutions (1.0 mg ml-1

) of carvedilol, glimepiride and glibenclamide drugs were prepared by dissolving

appropriate amounts of each in methanol. Aliquots of the stock solutions of the drugs were diluted with solution (A) to yield standard

solutions over a concentration range of 2-75 μg/mL for CRV and 5-300 μg/mL for GMP or GBD.

Laboratory prepared combination (binary) tablets solutions

Ten tablets of carvid (carvedilol 12.5mg/tablet) were weighed finely powdered and thoroughly mixed. To the weight of the

powdered tablets equivalent to 25mg carvedilol, 8mg glimepiride or 10mg glibenclamide were added. The mixture was transferred

into 50 ml volumetric flasks containing few milliliters of methanol sonicated for about 15 minutes then filtered and the residue was

washed with methanol and washings were added to the filtrate. Then dilution to volume (50 ml) was made with methanol to reach a

final concentration of 500 g/mL carvedilol and 160g/mL glimepiride or 200g/mL glibenbenclamide.

1.5 ml of solution mixture was diluted to 10 ml with solution A (six replicates), injected and compared to a similarly prepared

solution of a mixture of reference standards of carvedilol and glimepiride or glibenclamide (instead of tablets powder).

Solutions for interaction studies

Preparation of simulating gastric juice pH 1

Simulated gastric juice pH 1 was prepared by diluting concentrated HCl with de-ionized water to obtain 0.1 N HCl.

Preparation of simulating blood pH (buffer pH 7.4)

Buffer of pH 7.4 was prepared by dissolving 0.6 gm of potassium dihydrogen o-phosphate, 6.4 g of disodium hydrogen o-

phosphate and 5.85 g of sodium chloride in sufficient deionized water to produce 1000 mL and the pH was adjusted.

Procedure for interaction Amount equivalent to 25 mg of carvedilol and 12.5 mg of glimepiride or glibenclamide were separately dissolved in 10 ml

methanol. 5 ml of each solution was diluted to 25 ml with simulated gastric juice p H 1 or simulated blood pH 7.4. The concentration

of carvedilol was 500 μgmL−1

and of glimeperide or glibenclamide was 250 μgmL−1

.

Equal volumes of carvedilol: glimepiride or glibenclamide (1:1 v/v) were mixed into a reaction flask to produce the final

concentration of CRV (250 μgmL−1

) and GMP or GBD (125 μgmL−1

) and kept at 37oC in water bath with constant stirring. 2 mL of

this mixture was withdrawn immediately (at zero time) and periodically at the interval of 30 minutes for 2 hours, completed to 10 ml

with diluent solution A to record its chromatogram. Peak areas were recorded and compared with standard solutions (calibration

graphs) from which degree of interactions were evaluated.

The effect of simulated gastric juice and blood pH 7.4 was also studied individually on each drug by repeating the whole procedure on

the drug alone without mixing and recording the % availability.

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RESULTS AND DISCUSSION

Optimization of Chromatographic Conditions

The developed HPLC method has been applied for simultaneous determination of carvedilol and glimepiride or

glibenclamide. The UV absorption spectra of the three drugs standard solutions showed that carvedilol and glimepiride or

glibenclamide absorbed appreciably at 220 nm; so, detection at this wavelength was selected for method development. First trials

showed that working solutions diluted with methanol, resulted in carvedilol peak splitting and glimepiride or glibenclamide peak

tailing. On dilution with mobile phase of the ratio 70: 30 v/v (MeOH: 0.2 M phosphate buffer, pH 3.5), the peaks shapes were

improved but dilution with solution A (50: 50, MeOH: buffer) resulted in good peak shape for all drugs.

For obtaining good separation of CRV, GMP or GBD, optimization procedure of the chromatographic conditions was

focused on the analytical column, the mobile phase composition and the separation temperature. C18 analytical column presented a

good stability in a wide pH range. Therefore, we selected a Zorbax Eclipse XDB - C18 column (250 mm × 4.6 mm i.d., 5μm particle

size) because with shorter columns (150 mm) peaks were not well separated. For optimization of mobile phase, different proportions

of methanol and 0.2 M phosphate buffer (pH 3.5) were tried. At methanol to buffer proportion, 80: 20 v/v, peaks of carvedilol and

glimepiride or glibenclamide were early observed but were not well separated and the symmetry of the peaks was poor, with tailing.

At 65:35 v/v proportion, good separation was obtained but GBD and GMP were highly retained and their separation were observed at

longer retention time of 10 and 15 min; respectively. The mobile phase composition of 70:30 v/v was shown to have good resolution,

with no tailing and with acceptable elution times at about 1.9, 8.7 and 6.4 min for CRV, GMP and GBD; respectively (Fig. 2a &2b).

In order to understand the influence of temperature on the separation, a temperature test on the chromatographic column was

carried out by varying the temperature between 25 and 40 °C in 5 °C steps. As was expected, with increase in temperature of

chromatographic column, retention times slightly decreased and became too close. The best results were achieved at 30 °C with

adequate retentions and resolution. Finally, satisfactory separation, with high symmetric peaks and acceptable retention time was

obtained with mobile phase consisting of methanol: 0.2 M phosphate buffer (pH 3.5) in the ratio 70:30 v/v; with flow rate of 1 ml/min,

column temperature of 30oC and UV detector adjusted to 220 nm.

Fig. 2a: Chromatogram obtained for standard mixture 75 g/mL carvedilol and 24 g/mL glimepiride

(ret time 1.909 and 8.732 min, respectively).

Fig. 2b: Chromatogram obtained for standard mixture 75 g/mL carvedilol and 30 g/mL glibenclamide

(ret time 1.900 and 6.345 min, respectively)

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Method validation

Validation Procedure

After satisfactory development of the method, it was subjected to method validation as per ICH guidelines [43] with respect

to validation parameters: linearity, detection (LOD) and quantification (LOQ) limits, intra and inter-day precision, ruggedness,

robustness, accuracy, and specificity.

Linearity Calibration curve representing the relation between the concentrations of drug versus the peak area were constructed by

taking appropriate aliquots of the standard stock solutions of the drug in different 10 ml volumetric flasks and the solutions were

made up to volume with solution (A) and subjected to regression analysis using the least square method. These standard solutions

were analyzed in triplicates. Results show linear relationship in the range of 2-75 μg/mL for CRV, 5-300 μg/mL for each GMP and

GBD. The regression equations were computed and found to be:

y = -50 +130.75 x r=0.9991 (CRV)

y= -20 +57.20 x r= 0.9998 ( GMP)

y= 80 + 76.15 x r= 0.9996 (GBD)

Where y is the integrated peak area at 220 nm and x is the concentration of the drug in μg mL-1

and r is the correlation

coefficient. The results are collected in table 1.

Detection and quantification limits Sensitivity was determined by establishing the limit of detection (LOD) and limit of quantification (LOQ). The limit of

detection (LOD) represents the concentration of analyte that would yield a signal-to-noise ratio of 3:1 and the limit of quantification

(LOQ) represents the concentration of analyte that would yield a signal-to-noise ratio of 10:1. LOD and LOQ were calculated

according to ICH guidelines [43] using following formula: LOD= 3.3(SD)/S and LOQ= 10(SD)/S, where SD=standard deviation of

response (peak area) and S= average of the slope of the calibration curve. Results are tabulated in table 1 and conclude that the method

is sensitive for the simultaneous determination of CRV and GMP or GBD.

Table 1: Analytical data for the calibration graphs for the determination of CRV, GMP and GBD using the proposed RP-

HPLC method.

Parameters Carvedilol Glimepiride Glibenclamide

Linearity range

(μg/mL)

2-75 5-300 5-300

Regression equation

(Y)(a)

Slope (b) 130.75 57.20 76.15

Intercept (a) -50 -20 80

Correlation coefficient (r) 0.9991 0.9998 0.9996

LOD (μg mL-1

) 0.25 0.69 0.46

LOQ (μg mL-1

) 0.76 2.1 1.4 a

Y= bx+a where Y is the integrated peak area at 220 nm and x is the concentration

of the drug in μg mL -1

Precision

The precision of the proposed methods was investigated with respect to intraday and interday precision. The intraday

precision (repeatability) was evaluated by assaying freshly prepared drug solutions in six replicates at concentrations 25, 50 and 75 μg

mL-1

of carvedilol and 50, 100 and 150 μg/mL for each glimepiride and glibenclamide; these solutions were analysed daily. The

specified chromatographic conditions were followed as described above. The interday precision (reproducibility) was calculated from

assaying freshly prepared solution of drug (with the same formentioned concentrations) over a period of three days (intermediate

precision). The relative standard deviation (RSD) was less than 2% indicating high degree of precision of the proposed method (Table

2).

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Table 2: Precision data for the determination of CRV, GMP and GBD by the proposed HPLC method.

Drug Intra-day precision

Inter-day precision

Conc added

(μg/mL)

Conc found(μg/mL)

± SD RSD %

Conc added

(μg/mL)

Conc found(μg/mL)

±SD RSD %

CRV 25

50

75

24.95± 0.32

1.28

50.01± 0.67

1.33

74.98±0.55

0.73

25

50

75

25.03± 0.41

1.63

50.04± 0.62

1.24

74.99± 0.77

1.03

GMP 50

100

150

49.93± 0.41

0.82

99.98±0.44

0.44

150.10±0.75

0.50

50

100

150

50.06± 0.39

0.78

100.11±0.71

0.71

149.97±0.98

0.65

GBD 50

100

150

50.02±0.57

0.40

100.10±0.64

0.64

149.99±0.83

0.55

50

100

150

49.95±0.65

1.30

100.20±0.79

0.79

150.13±0.66

0.44

*Results of six replicates.

Ruggedness and robustness

Ruggedness of the method was determined by carrying out the analysis by two different analysts and the respective peak

areas were noted. The result was indicated by % RSD, values were found less than 2%, thus indicating the ruggedness of the method.

The robustness of the method shows the reliability of an analytical method with respect to small, but deliberate variations in

method performance parameters. Small deliberate variations of the experimental conditions for HPLC were applied in order to

determine the effect on resolution. Changes in mobile-phase composition (±2%), flow rate (±0.1ml/min) or buffer pH (±0.2 pH unit)

did not affect significantly the chromatographic method illustrating the robustness of the method.

Accuracy (standard addition method)

The accuracy of the method was determined by calculating recoveries of the drug by the standard addition method. For this

reason, a known amount of the drug standard solution within the range of linearity was added to the mixture of sample solution

previously analysed: 50 μg ml-1

CRV and 30 μg ml-1

GMP ( mixture I) or 50 μg ml-1

CRV and 30 μg ml-1

GBD ( mixture II). The

solutions prepared in six replicates were analyzed and concentration of added standards were calculated each time from the

corresponding regression equation, the mean recovery percentages (Table 3) were found to be 100.02 % and 99.93% for CRV and

GMP, respectively (mixture I) ; and 100.53% and 100.00% for CRV and GBD, respectively (mixture II); with mean % RSD less than

1%. The results indicate the method enables highly accurate simultaneous determination of carvedilol and glimepiride or

glibenclamide in their binary mixtures.

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Table 3: Determination of Carvedilol, Glimepiride and Glibenclamide in laboratory prepared binary mixtures by standard

addition method (Accuracy).

Drug Initial amount

(μg/mL)

Amount added

(μg/mL)

Amount *recovered

(μg/mL)

*Recovery%

± SD

RSD %

Carvedilol (50μg/mL) + Glimepiride (30μg/mL)

Mixture (I)

Carvedilol 50 25 24.97 99.88±0.39

0.39

50 50.10 100.20±0.45

0.45

75 74.98 99.97±0.20

0.20

Mean recovery %±RSD 100.02±0.35

Glimepiride 30 15 14.93 99.53± 0.42

0.42

30 29.96

99.86± 0.72

0.73

50 50.20 100.40 ±0.68

0.68

Mean recovery %±RSD 99.93±0.60

Carvedilol (50μg/mL) + Glibenclamide (30μg/mL)

Mixture (II)

Carvedilol 50 25 25.15 100.60± 0.56

0.56

50 50.24 100.48±0.77

0.77

75 74.99 99.98±0.42

0.42

Mean recovery %±RSD 100.53±0.58

Glibenclamide 30 15 15.08 100.53±0.71

0.71

30 29.91 99.70± 0.83

0.83

50 49.89 99.78±0.55

0.55

Mean recovery %±RSD 100.00± 0.70

*Results of six replicates.

Analytical application and specificity

The developed and validated chromatographic method was successfully applied for simultaneous determination of carvedilol

and glimepiride or glibenclamide in their laboratory prepared tablets combinations. The CRV, GMP or GBD contents of tablets was

determined using the calibration curve or regression equation method.

The average % amount ranges from 99.49 % to 99.95 % with relative standard deviation less than 1 % indicating satisfactory

accuracy and precision of the method (Table 4). The obtained mean assay results were in good agreement with the corresponding

labeled amount and with the certified assay results (99.86 to 100.10 %) obtained by official USP methods [4].

The specificity of the method was investigated by observing any interference encountered from the common tablet

excipients. It was observed that single peaks for CRV, GMP or GBD were obtained under optimized conditions (Fig. 3a&3b). These

excipients, thus did not interfere with the proposed method which indicates good specificity of the method.

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Fig 3a: Chromatogram obtained for prepared tablet mixture: 75 g/mL carvedilol and 24 g/mL glimepiride

(ret time 1.915 and 8.752 min, respectively)

Fig 3b: Chromatogram obtained for prepared tablet mixture: 75 g/Ml carvedilol and 30 g/mL glibenclamide

(ret time 1.890and 6.204 min, respectively)

Table 4: Determination of carvedilol, glimepiride and glibenclamide in binary combination tablets by the proposed HPLC

method.

Tablet mixture Drug Taken

amount

(μg/mL)

*Found amount

(μg/mL)

* recovery %±SD

RSD%

Carvid (12.5mg CRV/tablet)+

glimepiride (4mg/tablet)

CRV 75 74.87 99.82±0.71

0.71

GMP 24 23.99 99.95± 0.89

0.89

Carvid (12.5mg CRV /tablet)+

Glibenclamide

(5mg/tablet)

CRV 75 74.62 99.49±0.92

0.92

GBD 30 29.89 99.63±0.41

0.41

* Mean of six determinations.

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In vitro - interaction studies by the proposed RP-HPLC

Single-drug therapy remains the preferred way to begin treatment of a certain desease. However, combination drug therapy

can be used initially or added gradually if the therapeutic responses with monotherapy are not suitable [44]. Due to the clinical

importance of coadministration of carvedilol with glimepiride or glibenclamide [41], a vitro-interaction study was conducted to

investigate the availabilities of their binary combinations. The combined CRV and GMP or GBD were subjected to simulated gastric

juice of pH1 and simulated blood pH 7.4. Their behaviors were quantitated by the developed HPLC method, the quantitations were

examined by calculating the % assay of CRV and GMP or GBD before and after their binary mixing. It was observed that

availabilities of the hpoglycemic drugs espescially glimepiride were decreased in presence of carvedilol. In simulated gastric juice

pH1 and at blood pH 7.4; only 77.52 % and 82.50% of GMP were available, respectively ( at the end of the experiment after 2 hours).

For glibenclamide, in simulated gastric juice and at blood pH 7.4, its availabilities were less affected by the presence of carvedilol (

about 90 % of GBD was available after 2 hours in the two mediums). At all pH, neither glimepiride nor glibenclamide exerted, any

effect on carvedilol, but it was observed that pH 7.4 decreases the availability of the antihypertensive alone and not due the presence

of any of the hypoglycemic drugs. The percentage of in vitro availabilities of carvedilol, glimepiride and glibenclamide before and

after interaction are presented in table 5. According to these results a slight increase in the antidiabetic drugs doses can be suggested,

but additional pharmacological in vivo-studies are required. The in vivo study combined with the proposed in vitro one can help to

improve the quality and clinical efficacy of the binary combinations, which was an important interest of our study.

Table 5: Percent availability of Carvedilol, glimepiride or glibenclamide in simulated gastric juice pH1 and simulated blood

pH 7.4.

Sample

% availabilities

Time/min

0 30 60 90 120

Simulated gastric juice pH 1

Before mixing

Carvedilol 99.95 99.92 99.93 99.93 99.96

Glimepiride 101.01 98.95 95.32 95.22 95.30

Glibenclamide 99.98 99.97 99.95 99.93 99.97

After mixing

Carvedilol

(CRV+GMP)

99.94 99.95 99.94 99.97 99.95

Glimepiride

(CRV+GMP)

99.91 87.12 82.00 79.12 77.52

Carvedilol

(CRV+GBD)

99.96 99.96 99.94 99.97 99.96

Glibenclamide

(CRV+GBD)

100.10 93.80 91.95 90.05 90.11

Simulated Blood pH 7.4

Before mixing

Carvedilol 99.98 97.14 94.00 87.00 76.90

Glimepiride 99.93 98.88 95.45 95.33 95.00

Glibenclamide 99.98 99.97 99.97 99.93 99.95

After mixing

Carvedilol

(CRV+GMP)

99.99 93.81 89.50 85.00 75.30

Glimepiride

(CRV+GMP)

99.97 93.70 89.20 87.70 82.50

Carvedilol

(CRV+GBD)

99.95 97.90 91.3 82.30 76.00

Glibenclamide

(CRV+GBD)

99.99 99.94 95.83 92.40 90.60

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CONCLUSION

Due to the clinical importance of coadministration of the antihypertensive carvedilol with one of the antidiabetic drugs

glimepiride or glibenclamide, a RP-HPLC method for their simultaneous determination has been developed. It was rapid, precise,

accurate showing good percent recovery as well as low LOD and LOQ values, showing sensitivity of the method. It could be applied

to the analysis of CRV, GMP or GBD simultaneously in pure forms or in binary combination tablets. The good validation criteria of

the proposed method allow its use in quality control laboratories. The proposed method was utilized for the monitoring of in vitro -

interaction studies of carvedilol and glimepiride or glibenclamide in presence of each other, in simulated gastric juice of pH1 and

simulated blood pH 7.4. On the basis of the interaction results, it is proposed that if carvedilol and one of the hypoglycemic drugs are

coadministered, the availability of the antidiabetic drugs especially glimepiride may be decreased. It can be suggested that the dosages

of the two hypoglycemic drugs should be increased to improve the efficacy of the binary combinations, but more in vivo-

pharmacological interaction studies are recommended to make any conclusive remark. Due to its simplicity, rapidity and accuracy, the

developed HPLC method could facilitate such in vivo studies.

ACKNOWLEDGEMENT

The author is grateful to Department of Analytical Chemistry, National Organization for Drug Control and Research, for

providing the facilities and sincere help.

CONFLICT OF INTERESTS None.

Abbreviations

RP-HPLC: Reversed Phase -High performance Liquid Chromatography

CRV: Carvedilol.

GMP: Glimepiride

GBD: Glibenclamide

FDC: Fixed dose combination

TLC: Thin Layer Chromatography

HPTLC: High Performance Thin Layer Chromatography

LC: Liquid Chromatography

ICH: International Conference of Harmonization

LOD: Limit of Detection

LOQ: Limit of Quantification

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