study for amlodipine atorvastatin and glimepiride

Available online www.jocpr.com

Journal of Chemical and Pharmaceutical Research, 2014, 6(11):515-528

Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5

515

Simultaneous determination of Atorvastatin, Glimepiride and Amlodipine in solution and plasma matrix using HPLC/UV method

Wael Abu Dayyih*, Ala'a Al-fayez, Lina Tamimi, Eyad Mallah and Tawfiq Arafat

Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy

and Medical Sciences- University of Petra, Jordan _____________________________________________________________________________________________ ABSTRACT The aim was to develop and validate a simple, fast, reliable, selective and accurate HPLC method with UV detection for simultaneous determination of atorvastatin, glimepiride and amlodipine in a solution and plasma matrix. The method consisted of a mobile phase containing water, methanol and acetonitrile at 1.58:1:1(v/v%) buffered with triethylamine at pH8.0, a flow rate of 1.5 ml/min and a UV detector at 237 nm wavelength. Different and slight variations were introduced in the mobile phase, pH, wavelength, and column temperature to ensure method robustness in yielding good accuracy and precision. Other parameters such as sunlight sample exposure and acid degradation were evaluated on solutions containing the three drugs. A successful HPLC method was validated and developed to detect and quantify atorvastatin, glimepiride and amlodipine in a solution and plasma matrix, system and method precision were reasonable as RSD% values were below 2%, calibration curves revealed linearity in the range of 0.5-1.5 µg/ml for all the three drugs dissolved in the diluent with R2>0.99. RSD% in the linear range and for each of the calibration curve is between 0.03-0.32 for amlodipine, 0.03-0.36 for glimepiride and 0.18-0.46 for atorvastatin. Accuracy was with %recovery for each drug concentration between 98-102% which was considered as acceptable according to USP guidelines for analytical method validation. Stability tests represented reasonable stability of method for three drugs in both plasma and solutions as the % recovery was between 98-102%. Robustness was reliable as wavelength variation,pH variation, acid /base degradation, temperature changes and acetonitrile change in mobile phase showed RSD% less than 2% for all. The method was selective for the three drugs without any possible interference between excipients present in a tablet and the three drugs. Results indicated the method suitability to be used for determination of amlodipine, glimepiride and atorvastatin simultaneously in two different matrices, diluent and plasma. Key words: Atorvastatin, Glimepiride, Amlodipine, HPLC, Solution, Plasma. _____________________________________________________________________________________________

INTRODUCTION

Diabetes is a major chronic public health problem affecting people worldwide [1]. The prevalence of diabetes is projected to double globally by 2030. Poorly controlled diabetes leads to nephropathy with increased risk of renal failure, neuropathy and peripheral vascular disease with potential for loss of limbs, retinopathy with increased risk of blindness, and an increased risk of cardiovascular disease and stroke ]2 [ . The pathophysiology of type 2 diabetes mellitus (T2DM) is characterized by relative decrease in insulin secretion and/or insulin resistance, Sulfonylureas (SUs) are widely used in the management of T2DM as insulin secretagogues and are named for their common core configuration, Glimepiride(structural formula as shown in figure 1) is the

Wael Abu Dayyih et al J. Chem. Pharm. Res., 2014, 6(11):515-528 ______________________________________________________________________________

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newest second-generation SU and is sometimes classified as a third-generation SU because it has larger substitutions than other second-generation SUs. It was first introduced into clinical practice in Sweden. The United States Food and Drug Administration (FDA) approved glimepiride in 1995 for the treatment of T2DM as monotherapy as well as in combination with metformin or insulin ]3[ . Hypercholesterolemiais a key feature of the metabolic syndrome in humans and a risk factor for the development of cardiovascular diseases, such as myocardial infarction. Statins are widely used in the treatment of hypercholesterolemia [4]. Atorvastatin(structural formula as shown in figure 3) has been prescribed for many years and is considered as one of the most potent agents within the statin drug class, in terms of the LDL cholesterol-lowering effect [5]. Amlodipine besylate, (structural formula as shown in figure 2)is a 3rd generation dihydropyridin calcium channel blocker (CCB), is approved for the treatment of hypertension and both vasospastic and chronic stable angina [8]. The primary action of amlodipine is to inhibit calcium entry through voltage-gated transmembrane l-type channels, thus decreasing intracellular calcium concentration and inducing smooth muscle relaxation [6]. Hypertension and dyslipidemia are two of the most commonly co-occurring cardiovascular risk factors, it is suggested that a combination therapy have various advantages over monotherapy, In a multicenter trial it has been found that the number of patients who were receiving combination therapy (atorvastatin and amlodipine) achieved their blood pressure goal more than those patients receiving amlodipine and more patients receiving combination therapy achieved their LDL-C goal than patients receiving atorvastatin alone [7]. HPLC is one of the most widely used method for determination and quantification of several drugs in different dosage forms and human fluids. Triple drug combination analysis and determination were numerously carried out using validated methods of analysis such as HPLC [9-11]. Several studies have been carried out for determination of atorvastatin , amlodipine and glimepiride [ 12-20 ], while there is no specific method of analysis for determination of these three drugs simultaneously. Our research aimed to develop and validate a simple, fast, reliable, selective and accurate HPLC method with UV detection for the simultaneous determination of atorvastatin, glimepiride and amlodipine in a solution and plasma.The long term goal is to develop a drug dosage form containing the three drugs.Such combination enhances the adherence and compliance of patients to therapy and reduces cardiovascular episodes which ultimately increase health care management and reduce the overall cost of treatment risks.

Figure 1: Chemical structure of glimepiride

Figure 2: Chemical structure of amlodipine


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Figure 3: Chemical structure of atorvastatin

EXPERIMENTAL SECTION

Acetonitrile HPLC grade (Rankem), water (milli-Q) distilled water, phosphoric acid 85% (Riedel-Dehaen), triethylamine(Scharlau), methanol HPLC grade (Merck), diethyl ether (Biosolve) and ethylacetate (Acros) were used for method development. The following drugs were used to analyzed : atorvastatincalcium powder, amlodipine besylate , glimepiride CEP Powder and propylparaben powder.The following equipments were used: Column: BDS Hypersil C8 (PART NO 28205-254630) BIM (mm) 250*4.6, particle (µ) LOT: 9424, SN: 0780122T, HPLC Thermo Finnigan, Surevyor UV-VIS plus detector, Surevyor Auto sampler plus, Surevyor LC pump plus, Ultrasonic, (Elmasonic), pump for filtration of mobile phase (Vacuubrand), nylon syringe membrane filters 0.45 µm (Titan2), UV-VIS Thermo scientific, Evolution 300 UV-VIS andWhatman phase separator (silicon treated). The mobile phase was prepared by combining volumes of distilled water with methanol, acetonitrile and triethylamine. The pH was adjusted using phosphoric acid. The mobile phase was filtered through a 0.45 µm filter membrane and degassed by sonication. The diluent was prepared by adding distilled water, acetonitrile and methanol in equal volumes (1:1:1).All stock solutions of amlodipine, atorvastatin, glimepiride were prepared by dissolving 25 mg of each drug in 80 ml of diluentthen adjusted to 100 ml final solution volume.Human plasma (10 ml) was mixed with either 4 mg of glimepiride, 5 mg of amlodipine or 20 mg of atorvastatin, incubated for 30 min and then extracted usingequal mixture of ether and ethyl acetate. This is followed by adding 2 drops ofNaOH, the extraction was filtered and evaporated at low temperature (30°C). 50 ml ofdiluent were added, filtered and measured. UV-VIS scan within a range of 200-550 nm was applied for each solution of glimepiride, atorvastatin and amiodipine. A maximum absorbance was observed for each drug in a range of 230-240 nm.The effect of different chromatographic conditions on the separation of amlodipine, atorvastatin and glimepiride were studied such as pH , ion pair, composition of mobile phase and column to find out the most proper method for the determination of these drugs. Different trials of analysis were performed as indicated below.

Trial 1:

Column BDS Hypersil C8 (PART NO 28205-254630) BIM. (mm) 250*4.6, particle 5 µ

1 Solvent system (mobile phase) 950 DW, 900 ml ACN, 600 ml methanol, pH 7 Detection UV detector 237 nm Injection volume 10 µl Flow rate 1.5 ml\min Oven Temperature 25°c

Trial 2:

Column BDS Hypersil C8 (PART NO 28205-254630) BIM. (mm) 250*4.6, particle 5 µ

Solvent system (mobile phase) 950 DW, 900 ml ACN, 600 ml methanol, pH 8 Detection UV detector 237 nm Injection volume 10 µl Flow rate 1.5 ml\min Oven Temperature 25°c


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Trial 3:

Column BDS Hypersil C8 (PART NO 28205-254630) BIM. (mm) 250*4.6, particle 5 µ Solvent system (mobile phase) 950 DW, 600 ml ACN , 600 ml methanol, pH 8 Detection UV detector 237 nm Injection volume 10 µl Flow rate 1.5 ml\min Oven Temperature 25°c

Trial 4:

Column BDS HYPERSIL C8 (PART NO 28205-254630) BIM. (mm) 250*4.6particle5 µ Solvent system (mobile phase) 950 DW, 600 ml ACN, 600 ml methanol, pH 8 Detection UV detector 220 nm Injection volume 10 µl Flow rate 1.5 ml\min Oven Temperature 25°c

RESULTS AND DISCUSSION

The best absorbance profile for atorvastatin, amlodipine, and glimepiride, was between 220-240 nm. However, for assaying the three dugs simultaneously using HPLC, wavelength at 237 nm was selected for its best absorptivity for the three drugs. The best chromatographic condition for measuring atorvastatin, amlodipine and glimepiride simultaneously was based on the resolution and the retention time of each drug. It was found that mixture of mobile phase (Table 1) at pH 8.0 gave the best resolution.

Table 1: The best chromatographic conditions for simultaneous measurement of amlodipine, atorvastatin and glimepiride

Column BDS HYPERSIL C8 (PART NO 28205-254630) BIM. (mm) 250*4.6 particle Sz (u) 5

Solvent system (mobile phase) 950 D.W , 600 ml ACN , 600 ml Methanol, adjusted at 8 PH Detection UV detector 237 nm Injection volume 10 µl Flow rate 1.5 ml\min Oven Temperature 25°c Retention times* Amlodipine Glimepiride Atorvastatin

7.1-7.8 min 8.0-8.9 min 10.4-11.2 min

System precision: the procedure was applied repeatedly to multiple injections (10 injections) of the same homogenous sample. The RSD% values were below 2% for both diluent and plasma with three drugs which was indicating good system suitability according to USP guidelines as shown in table 2 and 3.

Table 2: System parameters for simultaneous measurement of diluent-containing amlodipine, atorvastatin and glimepiride

Parameters Amlo (conc) Glim (conc) Atorva (conc)

Average area of 10 injections 609675 817807 659605 RSD% 0.32 0.13 0.20 Asymmetry 1.2 1.2 1.1 Theoretical plates 8016 9255 8814 Resolution 0 5.5 10.74 Initial retention time 7.1 8.0 10.4 Final retention time 7.8 8.9 11.2

Wael Abu Dayyih et al ______________________________________________________________________________

Table 3: System parameters for simultaneous measurement of plasma

ParametersAverage area of RSD% AsymmetryTheoretical platesResolutionInitial retention timeFinal retention time

Method precision: the precision of the method was performed by analyzing a mixture of three dugs six times. The RSD values were well below 2% indicating precise method for samples in % values less than 2% indicate reasonable precision between 99-101% .

Table 4:Analytical method

Table 5: Precision of the analytical method for a plasma

Internal standard: the internal standard was chosen to be benzophenone.The retention time of benzophenone was 5.1 min in comparison to 6.2, 7.98, and 10 for amlodipine, glimepiride, and atorvastatin, respectively (Fig 4).In additiprecision of the internal standard was <2% as well as all the drugs internal standard with absence of any inappropriate interference

Figure 4: The chromatogram of a sample containing a

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System parameters for simultaneous measurement of plasma-containing amlodipine, atorvastatin and glimepiride

Parameters Amlo (conc) Glim (conc) Atorva (conc)Average area of 10 injections 552826 857718 662494

0.27 0.53 0.60 Asymmetry 1.33 1.21 1.34 Theoretical plates 7972 9530 8764 Resolution 0 3.7 5.72 Initial retention time 7.1 8.2 10.4 Final retention time 7.9 8.9 11.3

he precision of the method was performed by analyzing a mixture of three dugs six times. The RSD values were well below 2% indicating precise method for samples in diluent or plasma (Table 4 & 5), as RSD % values less than 2% indicate reasonable precision of method. In addition the recovered concentrations ranged

nalytical method precision for a sample containing one concentration of the three drugs

Sample# Amlo Glim Atorva

1 595040 814919 660871 2 594892 814942 661911 3 594446 815823 661898 4 595319 814723 661450 5 595215 815972 660945 6 594436 814986 661278

Average 594891 815228 661392 RSD% 0.06 0.07 0.07 Assay% 100.5 99.2 100.6

Precision of the analytical method for a plasma-containing one concentration of the three drugs

Sample# Amlo Glim Atorva 1 866750 1308602 4052512 2 867785 1293008 3995239 3 870363 1306931 4049627 4 870039 1301000 4039958 5 869869 1299840 4028456 6 870340 1300983 3998235

Average 869191 1301727 4027338 RSD% 0.16 0.42 0.62 Assay% 101.2 99.8 100.1

he internal standard was chosen to be benzophenone.The retention time of benzophenone was 5.1 min in comparison to 6.2, 7.98, and 10 for amlodipine, glimepiride, and atorvastatin, respectively (Fig 4).In additiprecision of the internal standard was <2% as well as all the drugs which confirms the suitability of benzophenoneas internal standard with absence of any inappropriate interference (Table 6).

The chromatogram of a sample containing a mixture of amlodipine, glimepiride, atorvastatin, and benzophenone

J. Chem. Pharm. Res., 2014, 6(11):515-528 ______________________________________________________________________________

containing amlodipine, atorvastatin and glimepiride

Atorva (conc)

he precision of the method was performed by analyzing a mixture of three dugs six times. The diluent or plasma (Table 4 & 5), as RSD

In addition the recovered concentrations ranged

oncentration of the three drugs

of the three drugs

he internal standard was chosen to be benzophenone.The retention time of benzophenone was 5.1 min in comparison to 6.2, 7.98, and 10 for amlodipine, glimepiride, and atorvastatin, respectively (Fig 4).In addition the %

which confirms the suitability of benzophenoneas

mixture of amlodipine, glimepiride, atorvastatin, and benzophenone


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Table 6: Precision of the analytical method for a sample-containing one concentration of the three drugs with benzophenone

Sample# Amlo Glim Atorva Benzophenone 1 191599 206056 166904 122837 2 189468 205903 165274 122707 3 190002 205594 165494 123205 4 191369 203834 164998 123291 5 190159 203496 164864 123337 6 191505 202469 164791 122528

Average 190684 204558 165387 122984 RSD% 0.43 0.73 0.47 0.28

Linearity and range: a series of standard samples at different concentrations of the target compounds were prepared to evaluate the linearity as illustrated in (Figures 5, 6 and 7).

.

Figure 5: Calibration curve of glimepiride

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Figure 6: Calibration curve of amlodipine

y = 21517x - 3015.

R² = 0.999

0

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250000

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350000

0.00 0.50 1.00 1.50 2.00

AU

C

Glim (µg/ml)

y = 70846x - 11352

R² = 0.998

0

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0.00 0.50 1.00 1.50 2.00

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Amlo (µg/ml)


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Figure 7: Calibration curve of atorvastatin The calibration curves revealed linearity in the range of 0.5-1.5 µg/ml for all the three drugs dissolved in the diluent with R2>0.99 whileRSD% in the linear range and for each of calibration curve was between 0.03-0.32 for amlodipine (Table 7), 0.03-0.36 for glimepiride (Table 8) and 0.18-0.46 for atorvastatin (Table 9). Values of R2

factor indicated reasonable linear performance of standard solutions of the three used drugs.

Table 7: RSD% for each of the calibration points of amlodipine

Concentration (µg/ml) Average area RSD% 0.5 237782 0.03 0.75 429626 0.05 1.0 590728 0.23 1.25 756765 0.21 1.5 959793 0.32

Table 8: RSD% for each of the calibration points of glimepiride

Concentration (µg/ml) Average area RSD%

0.5 105574 0.04 0.75 157045 0.03

1 210638 0.06 1.25 268943 0.25 1.5 318593 0.39

Table 9: RSD% for each of the calibration points of atorvastatin


0.5 182163 0.21 0.75 220562 0.39 1.0 264521 0.26 1.25 303502 0.18 1.5 349891 0.46

Calibrations in plasma linearity ranges were similar in the range of 0.5-1.5 µg/ml for glimepiride and amlodipine (Figure 8 and 9). For atorvastatin, the linearity range was from 0.75-1.5 µg/ml (Figure 10). The R2value for all three were >0.99 which performs acceptable linearity , all %RSD values were less than 1% which confirms proper validity of results (Table 10, 11 and12).

y = 16735x + 96769

R² = 0.999

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Figure 8: Calibration curve of glimepiride in plasma

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Figure 9: Calibration curve of amlodipine in plasma

Table 10: RSD% for all calibration points of amlodipine in plasma

Concentration (µg/ml) Average area RSD% 0.5 19351 0.33 0.75 31296 0.68 1.0 42674 0.39 1.25 55392 0.26 1.5 70237 0.49

y = 10092x - 27481

R² = 0.992

0

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0.00 0.50 1.00 1.50 2.00

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Glim (µg/ml)

y = 50347x - 6557.

R² = 0.997

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Figure 10: Calibration curve of atorvastatin in plasma

Table 11: RSD% for all calibration points of glimepiride in plasma

Concentration (µg/ml) Average area RSD% 0.5 27021.8 0.21 0.75 45489.4 0.31

1 69592.9 0.53 1.25 98347.1 0.71 1.5 126742.5 0.49

Table 12:RSD% for all calibration points of atorvastatin in plasma


0.75 99691.3 0.27 1 190761.8 0.52

1.25 309356.5 0.35 1.5 429724.3 0.18

Accuracy: three samples at three different concentrations levels 0.5, 1, and 1.5 µg/ml were analyzed and calculated from a standard curve. The %recovery is calculated for each drug concentration and was found to be between 98-102%, reasonable recovery% indicated accepted accuracy of method (Table 13, 14 and15).

Table 13: Accuracy for the determination of amlodipine

Concentration (µg/ml) Average area RSD% %Recovery 0.5 1208005 0.26 98.4 1.0 251066 0.08 99.7 1.5 360482 0.38 99.3

Table 14: Accuracy for the determination of glimepiride

Concentration (µg/ml) Average area RSD% Assay%

0.5 35652 0.62 99.2 1.0 69049 0.47 100.2 1.5 117549 0.71 100.5

y = 44347x - 24152

R² = 0.996

0

50000

100000

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250000

300000

350000

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450000

500000

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Atorva (µg/ml)


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Table 15: Accuracy for the determination of atorvastatin

Concentration (µg/ml) Average area RSD% Assay% 0.5 63782 0.44 101.5 1.0 126694 0.19 100.3 1.5 229061 0.52 98.7

Stability of drugs in analytical solution: concentrations determination of the analytical solutions (standard and sample) stored for 24 hours at 4°C and room temperature in comparison to freshly prepared standard solutions were carried out. The % recovery was between 98-102% in all stored conditions in two different matrices; diluent and plasma which indicated reliable stability of these drugs (Table 16 and 17).

Table 16: Stability for amlodipine, glimepiride and atorvastatin in diluent solution

Drug Time and Temp. Assay% RSD

Amlodipine Standard fresh sample 100.6 0.04 24 hours at 4C 101.1 0.52 24 hours at 25C 101.9 0.11

Glimepiride Standard fresh sample 99.3 0.10 24 hours at 4C 100.2 0.41 24 hours at 25C 98.4 0.13

Atorvastatin Standard fresh sample 100.4 0.05 24 hours at 4C 100.6 0.22 24 hours at 25C 99.3 0.32

Table 17: Stability for amlodipine, glimepiride and atorvastatin in plasma

Drug Time and Temp. Assay% RSD

Amlodipine Standard fresh sample 100.9 0.05 24 hours at 4C 101.6 0.77 24 hours at 25C 99.9 0.11

Glimepiride Standard fresh sample 100.3 0.05 24 hours at 4C 101.6 0.41 24 hours at 25C 99.2 0.16

Atorvastatin Standard fresh sample 99.2 0.40 24 hours at 4C 98.5 0.38 24 hours at 25C 99.0 0.06

Robustness: slight variations (± 3) in wavelength have been made. The RSD% was less than 2% indicating slight change in wavelength did not affect the detection parameters of the assay which emphasized the robustness of method against such changes. (Table 18-21).

Table 18: Effect of changing the wavelength detection by -3nm on the detection parameters in diluents

Parameters Amlo Glim Atrova Area 97408 162058 125750 RSD% 0.23 0.56 0.41 Theoretical plates 2600 4694 4723 Asymmetry 1.76 1.15 1.08

Table 19: Effect of changing the wavelength detection by +3nm on the detection parameters in diluents

Parameters Amlo Glim Atorva

Area 103622 153252 133343 RSD% 0.82 0.61 0.52 Theoretical plates 2352 4089 4152 Asymmetry 1.9 1.19 1.17

Table 20: Effect of changing the wavelength detection by -3nm on the detection parameters in plasma




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Table 21:Effect of changing the wavelength detection by +3nm on the detection parameters in plasma

Parameters Amlo Glim Atorva Area 790964 1234503 4015214 RSD% 0.29 0.42 0.50 Theoretical plates 4524 7978 7290 Asymmetry 1.22 1.18 1.24

Slight change in pH of the diluent was made. The assay% and RSD% were close to 100% and <1%, respectively indicating slight pH change has no effect on detection parameters of amlodipine, glimepiride or atorvastatin (Table 22 and .23) .In plasma, however, the RSD% of glimepiride was more than 2% indicating that pH changes affected its measurement and reduced the precision of the its assay (Table 24 and 25).

Table 22: Effect of changing the pH -0.2 on the detection parameters in diluents

Amlo Glim Atorva Assay% 100.1 100.9 99.2 RSD% 0.29 0.62 0.12

Table 23: Effect of changing the pH +0.2 on the detection parameters in diluents

Amlo Glim Atorva Assay% 101.1 99.0 99.6 RSD% 0.26 0.58 0.09

Table 24: Effect of changing the pH -0.2 on the detection parameters in plasma

Parameter Amlo Glim Atorva Assay% 100.9 98.3 99.1 RSD% 1.26 2.12 0.64

Table 25: Effect of changing the pH +0.2 on the detection parameters in plasma

Parameter Amlo Glim Atorva Assay% 101.5 98.4 100.1 RSD% 1.17 2.57 0.52

Acid or base degradation was tested on the detection parameters of the assay. A volume of 1N HCL or 1N hydroxide was added to solution of amlodipine, glimepiride and atorvastatin for 30 min and then analysis was performed. The results showed that %RSD values were below 1% (Table 26 and 27) .

Table 26: Effect of acid on the detection parameters ofamlodipine, atorvastatin, and glimepiride

Parameter Amlo Glim Atrova Area 536094 755657 318543 RSD% 0.77 0.39 0.43 Theoretical plates 8316 9293 8856 Asymmetry 1.20 1.10 1.05 Resolution 0.00 5.52 5.56

Table 27: Effect of base on the detection parameters of amlodipine, atorvastatin, and glimepiride

Parameter Amlo Glim Atorva

Area 604181 811560 715518 RSD% 0.01 0.23 0.21 Theoretical plates 8037 9187 8703 Asymmetry 1.19 1.11 1.11 Resolution 3.79 5.48 5.54

Changing the setting of instrument temperature was also tested on the method parameters. The results were obtained by comparing the standard solution tested under ± 3 Celsius change of 25°C.The RSD% values of either higher or lower temperatures were less than 2% (Table 27, 28) . Similarly, RSD% values were less 2% when drugs prepared in plasma (Table 29, 30).


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Table 27: Effect of setting the temperature +3 °C on the detection parameters

Parameters Amlo Glim Atorva Area 79475 68673 240660 RSD% 0.13 0.18 0.43 Theoretical plates 10572 11517 11763 Asymmetry 1.22 1.53 0.92

Table 28: Effect of setting the temperature -3 °C on the detection parameters



Table 29: Effect of setting the temperature +3 °C on the detection parameters in plasma



Table 30: Effect of setting the temperature -3 °C on the detection parameters in plasma

Amlo Glim Atorva


The effect of 5% change of the ACN in the mobile phase was evaluated on the detection parameters of drug containing solutions and in plasma. The RSD% values were less than 2% which confirms reasonable robustness against this change in mobile phase composition (Table 31-34) .

Table 31: Effect of 5% increase of ACN in the mobile phase on the detection parameters

Parameters Amlo Glim Atorva Area 644997 799565 685560 RSD% 0.09 0.62 0.25 Theoretical plates 8152 9283 9014 Asymmetry 1.28 1.15 1.14 Resolution 0 0 0

Table 32: Effect of 5% decrease of ACN in the mobile phase on the detection parameters


Area 637153 802193 657064 RSD% 0.25 0.11 0.02 Theoretical plates 8001 9359 9283 Asymmetry 1.26 1.14 1.12 Resolution 0 0 0

Table 33: Effect of 5% increase of ACN in the mobile phase on the detection parameters in plasma


Area 790886 1207760 3861363 RSD% 0.12 0.42 0.05 Theoretical plates 3731 7470 5903 Asymmetry 1.38 1.25 1.40 Resolution 0 1.5 2.7

Wael Abu Dayyih et al ______________________________________________________________________________

Table 34: Effect of 5% decrease of ACN in the mobile phase on the detection parameters in plasma

When samples were exposed to sunlight for four hours and then analyzed, two small peaks were detecteand 11.623 min (Figure 11). These two small sensitive drug.

Figure 11: Chromatogram of amlodipine, glimepiride, and atorvastatin followi

Selectivity: a standard, sample, solvent and a placebo solution were injected into column according to the parameters stated under the developed method. It was found that there is no interference between the analyte and both the solvent or placebo. Placebo effect: a placebo solution prepared based on the excipients present in a tablet without having any activeingredients. A placebo solution was prepared by addition of 1:1:1 (water: ACN: methanol) and then analyzed in the

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Effect of 5% decrease of ACN in the mobile phase on the detection parameters in plasma

Parameters Amlo Glim Atorva Area 838034 1404787 4330478 RSD% 0.19 0.28 0.45 Theoretical plates 3673 7589 7311 Asymmetry 1.38 1.45 1.34 Resolution 0 1.3 2.9

When samples were exposed to sunlight for four hours and then analyzed, two small peaks were detecte). These two small peaks are indicative of amlodipine degradation since it is light

Chromatogram of amlodipine, glimepiride, and atorvastatin following 4 hours of sunlight exposure

Figure 12: Chromatogram of placebo excipients

standard, sample, solvent and a placebo solution were injected into column according to the parameters stated under the developed method. It was found that there is no interference between the analyte and

Placebo effect: a placebo solution prepared based on the excipients present in a tablet without having any activeingredients. A placebo solution was prepared by addition of 1:1:1 (water: ACN: methanol) and then analyzed in the

J. Chem. Pharm. Res., 2014, 6(11):515-528 ______________________________________________________________________________

Effect of 5% decrease of ACN in the mobile phase on the detection parameters in plasma

When samples were exposed to sunlight for four hours and then analyzed, two small peaks were detected at 9.357 peaks are indicative of amlodipine degradation since it is light

ng 4 hours of sunlight exposure

standard, sample, solvent and a placebo solution were injected into column according to the parameters stated under the developed method. It was found that there is no interference between the analyte and

Placebo effect: a placebo solution prepared based on the excipients present in a tablet without having any active-ingredients. A placebo solution was prepared by addition of 1:1:1 (water: ACN: methanol) and then analyzed in the


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analytical system. No peaks were detected indicating no interference between the excipients and the active ingredients would result (Figure 12).

CONCLUSION

A suitable method for determination of amlodipine, glimepiride and atorvastatin simultaneously in two different matrices, diluent and plasma was developed and validated. Such method is robust enough and not affected by slight variations in the wavelength, pH, acid or base degradation and temperature.Even though the resolution, retention times and peak heights were good enough but did change in different conditions. However, future studies involving the pharmacokinetics and plasma bioavailability of the three drugs following oral administration of a drug dosage form containing; amlodipine, atorvastatin, and glimepiride is needed with mass spectrometry in order to detect the plasma levels (ng/ml) of such drugs. Acknowledgements The authors would like to thank Faculty of Pharmacy and Medical Sciences-University of Petra and Dar Al Dawa Pharma (DAD).

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study for amlodipine atorvastatin and glimepiride

Health & Medicine

Transcript of study for amlodipine atorvastatin and glimepiride