new study.. puplished in 2014 done by me

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Journal of Chemical and Pharmaceutical Research, 2014, 6(11):515-528
Research Article ISSN : 0975-7384
CODEN(USA) : JCPRC5
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
_____________________________________________________________________________________________
515
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

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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
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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 mlmin
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 mlmin
Oven Temperature 25°c
517

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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 mlmin
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 mlmin
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 mlmin
Oven Temperature 25°c
Retention times*
Amlodipine
Glimepiride
Atorvastatin
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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

5. Wael Abu Dayyih et al
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Table 3: System parameters for simultaneous measurement of plasma
Parameters
Average area of
RSD%
Asymmetry
Theoretical plates
Resolution
Initial retention time
Final retention time
515-528
plasma-containing amlodipine, atorvastatin and glimepiride
Amlo (conc) Glim (conc) Atorva (conc)
10injections 552826 857718 662494
0.27 0.53 0.60
1.33 1.21 1.34
7972 9530 8764
0 3.7 5.72
7.1 8.2 10.4
7.9 8.9 11.3
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
oncentration 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 additi
precision 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
J. Chem. Pharm. Res., 2014, 6(11):
519
he diluent or plasma (Table 4 & 5), as RSD
of method. In addition the recovered concentrations ranged
nalytical 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
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 addition the %
which confirms the suitability of benzophenoneas
(Table 6).
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).
y = 21517x - 3015.
R² = 0.999
AUC Amlo (μg/ml)
520
.
Figure 5: Calibration curve of glimepiride
.
Figure 6: Calibration curve of amlodipine
350000
300000
250000
200000
150000
100000
50000
0
0.00 0.50 1.00 1.50 2.00
AUC
Glim (μg/ml)
y = 70846x - 11352
R² = 0.998
1200000
1000000
800000
600000
400000
200000
0
0.00 0.50 1.00 1.50 2.00

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y = 16735x + 96769
0.00 0.50 1.00 1.50 2.00
Atorva (μg/ml)
521
.
R² = 0.999
Figure 7: Calibration curve of atorvastatin
400000
350000
300000
250000
200000
150000
100000
50000
0
AUC
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
Concentration (μg/ml) Average area RSD%
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).

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y = 10092x - 27481
R² = 0.992
522
.
Figure 8: Calibration curve of glimepiride in plasma
.
Figure 9: Calibration curve of amlodipine in plasma
140000
120000
100000
80000
60000
40000
20000
80000
70000
60000
50000
40000
30000
20000
10000
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
0
0.00 0.50 1.00 1.50 2.00
AUC
Glim (μg/ml)
y = 50347x - 6557.
R² = 0.997
0
0.00 0.50 1.00 1.50 2.00
AUC
Amlo (μg/ml)

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y = 44347x - 24152
R² = 0.996
523
.
Figure 10: Calibration curve of atorvastatin in plasma
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
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
Concentration (μg/ml) Average area RSD%
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
0
0 0.5 1 1.5 2
AUC
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
524
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
Parameters Amlo Glim Atorva
Area 787582 1271404 4040109
RSD% 0.23 0.64 0.72
Theoretical plates 4591 8125 7424
Asymmetry 1.2 1.16 1.23

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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
Parameters Amlo Glim Atorva
Area 79788 68902 240705
RSD% 0.39 0.24 0.05
Theoretical plates 11623 11583 11498
Asymmetry 1.12 1.78 1.23
Table 29: Effect of setting the temperature +3 °C on the detection parameters in plasma
Parameters Amlo Glim Atorva
Area 869891 1371725 4137533
RSD% 1.17 1.82 1.52
Theoretical plates 11700 11954 11809
Asymmetry 1.03 1.03 0.96
Table 30: Effect of setting the temperature -3 °C on the detection parameters in plasma
Amlo Glim Atorva
Area 87720 1308927 416298
RSD% 0.78 0.82 0.39
Theoretical plates 11700 11954 11809
Asymmetry 1.23 1.03 0.46
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
Parameters Amlo Glim Atorva
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
Parameters Amlo Glim Atorva
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
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J. Chem. Pharm. Res., 2014, 6(11):
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Table 34: 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
and 11.623 min (Figure 11). These two small
sensitive drug.
). peaks are indicative of amlodipine degradation since it is light
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 active
ingredients. A placebo solution was prepared by addition of 1:1:1 (water: ACN: methanol) and then analyzed in the
527
following 4 hours of sunlight exposure
Figure 12: Chromatogram of placebo excipients
515-528
detected at 9.357
ng active-ingredients.

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