Journal of Chromatographic Science, 2015, Vol. 53, No. 10, 1654–1662
doi: 10.1093/chromsci/bmv068
Advance Access Publication Date: 12 June 2015
Article
Article
Validation of RP-HPLC Method and Stress
Degradation for the Combination of Metformin
HCl, Atorvastatin Calcium and Glimepiride:
Application to Nanoparticles
Sandip Gite and Vandana Patravale*
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N.P. Marg, Matunga,
Mumbai 400019, India
*Author to whom correspondence should be addressed. Email: [email protected]
Received 7 April 2014; Revised 25 April 2015
Abstract
A stability-indicating high-performance liquid chromatography (HPLC) procedure was developed for
the determination of metformin HCl (MTH), atorvastatin calcium (AC) and glimepiride (GP) in combination and their main degradation products. The separation and quantization were achieved on a 5µm Qualisil gold, C18 column (4.6 mm × 250 mm). The mobile phase selected was phosphate buffer
( pH 2.9)–organic phase in proportion of 70:30. Organic phase consisted of methanol–acetonitrile
(90:10) at a flow rate of 1 mL/min and detection of analytes was carried out at 230 nm. The method
exhibited good linearity over the range of 10–60 µg/mL for MTH, 2–20 µg/mL for AC and 5–30 µg/mL
for GP. Square of the correlation coefficients was found to be >0.999. Various stress degradation
studies were carried out in combination as per International Conference of Harmonization (ICH)
guidelines for 4 h. The recovery and precision were determined in terms of intraday and interday precisions and expressed as relative standard deviations. These were <1 and <2%, respectively. Finally,
the applicability of the method was evaluated in nanoparticle analysis of MTH, AC and GP as well as
in stability studies of nanoformulation.
Introduction
International Conference of Harmonization (ICH) has revised parent
drug stability testing guidelines Q1A (R2) for stress testing on the drug
substance. Tests should be carried out to ascertain its inherent stability
and for supporting the suitability of the proposed analytical procedures (1). It is recommended that stress testing should be studied for
its effect on thermal, humidity, photo stability, oxidation as well as hydrolytic degradation. The aim of this study was to develop inherent
stability pattern of combination of metformin HCl (MTH), glimepiride (GP) and atorvastatin calcium (AC) as per ICH guidelines (2) and
to develop a validated stability-indicating method by reversed phase
high-performance liquid chromatography (RP-HPLC).
MTH is a biguanide antidiabetic drug, which is chemically known
as 1,1-dimethyl biguanide monohydrochloride (Figure 1a). It decreases hepatic glucose production and also intestinal absorption of glucose
and improves insulin sensitivity by increasing peripheral glucose uptake and utilization.
AC is a synthetic lipid-lowering agent, chemically known as
[R-(R*,R*)]-2-(4-fluorophenyl)β-δ-dihydroxy-5-(1-methylethyl)-3phenyl-4-[( phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid,
calcium salt (2:1) trihydrate (Figure 1b). It acts as an inhibitor to
the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase.
This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an
early and rate-limiting step in cholesterol biosynthesis (3). It is an organic acid with a pKa of 4.46, insoluble in aqueous solutions of pH 4
and below; and sparingly soluble in distilled water and pH 7.4 phosphate buffer.
GP is a medium-to-long acting sulfonyl urea, an antidiabetic drug.
It is chemically known as 1-[[ p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulfonyl]-3-(trans-4-methyl
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
1654
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Validation of RP-HPLC Method and Stress Degradation
Figure 1. Chemical structure of (a) MTH, (b) AC and (c) GP.
cyclohexyl) urea (Figure 1c). It acts by stimulating the insulin release
from functioning pancreatic cells (4).
There are a few reported liquid chromatography–mass spectrometry (LC–MS) methods which are available for analysis of antidiabetic
drugs and its metabolites in human plasma and urine (5–7). Several
methods have been developed individually and for combined dosage
forms in human plasma for aforementioned drugs (8–10). Even
though various methods were reported in the literature for estimation
of MTH, GP and pioglitazone alone and in combination with other
drugs (11–14). As per reports till date there is no information available
on the stability behavior of these drugs under hydrolytic, oxidative,
thermal and photo degradative conditions. To the best of our knowledge, this is the first report of a stability-indicating method for the determination of MTH, AC, GP in combination as well as its degradative
products.
LC–GC Chromatography Solutions Pvt. Ltd, Mumbai, India. This
column can also be purchased from Agilent Technologies Inc., USA
( part number: US603-25). The mobile phase employed comprised solvent A: phosphate buffer ( pH 2.9) (70%) and solvent B: organic phase
(30%). The organic phase consists of methanol–acetonitrile (90:10).
Prior to use, buffer was filtered through a 0.45 µm filter membrane.
Mobile phase was pumped through the column at the flow rate of
1.0 mL/min. The injection volume was 20 µL. The analytes were analyzed at a single wavelength of 230 nm for MTH, AC, GP and their
associated degradation products.
Preparation of phosphate buffer
Phosphate buffer solution (0.2 M) was prepared by dissolving 27.22 g
of monobasic potassium phosphate in 1,000 mL of water, and pH was
adjusted to 2.9 with O-phosphoric acid.
Experimental and methods
Materials and reagents
MTH, AC and GP were obtained as a gift sample from Arti drugs Ltd,
Mumbai, India; Cadila Pharmaceuticals Ltd, Ahmadabad, India and
Cipla Ltd, Mumbai, India, respectively. HPLC grade acetonitrile,
methanol, potassium dihydrogen phosphate and ortho-phosphoric
acid were purchased from S.D. Fine Chemicals (Mumbai, India). Furthermore, 0.45 µm membranes were purchased from Pall Life Sciences. All other chemicals used were of analytical grade unless otherwise
indicated. High purity water using Milli Q Plus purification system
(Millipore, Bedford, MA, USA) was used for the preparation of mobile
phase.
Preparation of stock and standard solutions
Stock solution of MTH, AC and GP (100 µg/mL) was prepared by dissolving accurately weighed 10 mg each of MTH, AC and GP in mobile
phase using 100 mL volumetric flasks separately. Standard solutions
were prepared by dilution of the stock solution with mobile phase
to give solutions containing MTH in the concentration range of 10–
60 µg/mL, AC in the concentration range of 5–30 µg/mL and GP in
the range of 2–20 µg/mL.
Forced degradation studies
Instrumentation
Effect of acid and base hydrolysis
The HPLC system consisted of Agilent 1100 modules (G1310A Isocratic Pump with solvent container, G1314A VW Detector with standard flow cell and G1328A Manual Injector). The output signal was
monitored and processed using an Agilent single G2220AA 2D-Value
Solution ChemStation. Chromatographic separation was achieved on
a 5 µm, Qualisil gold, C18 column (4.6 mm × 250 mm) procured from
Sample solution containing 1 mL aliquot of MTH, AC and GP was
transferred into a 10-mL amber volumetric flask, then mixed with
1 mL of 1 M HCl as well as 1 M NaOH separately and left to stand
for 4 h at 80 ± 2°C. Both the samples were injected in triplicate after
the neutralizing procedure and chromatogram was run as described
previously.
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Gite and Patravale
Figure 2. Chromatogram of (a) MTH, (b) AC and (c) GP at λmax 230 nm (20 µg/mL each).
Effect of oxidation
Sample solution containing 1 mL aliquot of MTH, AC and GP was
transferred into a 10-mL amber volumetric flask, then mixed with
1 mL of 1% (v/v) hydrogen peroxide and left to stand for 4 h at 80°
C ± 2°C. The final solutions were injected in triplicate and chromatogram was run as described previously.
Effect of photo degradation
Photolytic degradation was studied by placing a 20 µg/mL solution of
MTH, AC and GP (diluted with mobile phase) in a clear volumetric
flask and exposing it to direct sunlight for 4 h. The resultant solution
was injected in triplicate, and the chromatogram was run as described
previously.
Heat-induced degradation
One milliliter aliquot of a sample solution containing MTH, AC
and GP was transferred to a 10-mL amber volumetric flask and
then heated for 4 h at 80 ± 2°C. The resultant solution was injected in triplicate, and the chromatogram was run as described
previously.
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Validation of RP-HPLC Method and Stress Degradation
Figure 3. Chromatogram of MTH, AC and GP combination at λmax 230 nm (20 µg/mL each).
Method validation
Linearity and range
The working standard solutions of 100 µg/mL were prepared and further diluted with mobile phase so as to obtain a mixture of MTH, AC
and GP in the range of 10–60, 5–30 and 2–20 µg/mL, respectively.
The calibration curves were drawn by plotting the peak areas of the
drug against the corresponding concentration. The slope and Y intercept of the calibration curve were calculated.
Recovery and precision
Recovery and precision determination was carried out at a concentration of 5, 15 and 30 µg/mL for AC, at 10, 30 and 60 µg/mL for MTH
and at 2, 8 and 20 µg/mL for GP. At each level of the amount, six determinations were performed and both intra- and interday variation
were expressed in terms of % relative standard deviation (% RSD).
Limit of detection and limit of quantification
To estimate the limit of detection (LOD) and limit of Quantification
(LOQ), mobile phase was injected six times following the same method as explained above. The LOD and LOQ for all actives were estimated at a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively.
Solution stability
The solution stability of all actives was carried out by leaving a spiked
sample (20 µg/mL) solution in a tightly capped volumetric flask at
room temperature for 12 h. Content of MTH, AC and GP was determined at 1, 2, 4, 6 and 12 h intervals by following the procedure as
described previously.
Application of the developed RP-HPLC method for
nanoformulation evaluation
Analysis of MTH, AC and GP from polymeric
nanoparticles
To determine the content of MTH, AC and GP in spray-dried polymeric nanoparticles (label claim: 10 mg each of MTH, AC and GP
per 120 mg of spray-dried polymeric nanoparticles), 60 mg nanoparticles equivalent to 5 mg of MTH, AC and GP were accurately weighed
and transferred to a volumetric flask containing 50 mL mobile phase.
To ensure complete extraction of the drug, it was further sonicated for
10 min and then volume was made up to 100 mL. The resulting solution was filtered through 0.45 µm membrane and analyzed for drug
content. The filtrate containing 15 µg/mL of the drug was injected
and the chromatogram was run as previously described. The analysis
was repeated in six individual steps and the possibility of excipient interference in the analysis was studied.
Ruggedness and robustness test
As recommended in the ICH guidelines, a robustness assessment was
performed during the development of the analytical procedure (15).
The ruggedness (16) of the method is assessed by comparison of the
intra- and interday precision results for MTH, AC and GP. This was
performed on two different instruments by two analysts in the same
laboratory. In addition, the robustness of the method was investigated
under a variety of conditions including changes of flow rate,
analyst, column temperature, composition of buffer and detector
wavelength (17).
Results
The separation and quantification were achieved on a 5 µm, Qualisil gold, C18 column (4.6 mm × 250 mm). Initially, the ratio of
buffer–organic phase was optimized by carrying out variations in
the proportion of both phases. When only methanol was used as
an organic phase, all three peaks were resolved but there was no reproducibility in the peak area. So acetonitrile was introduced in the
organic phase, initially in concentration of 5% and then increased
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Gite and Patravale
Figure 4. (a) Chromatogram of acid (0.1 N HCl, heated for 4 h at 80°C) treated MTH, AC and GP (20 µg/mL each). (b) Chromatogram of base (0.1 N NaOH, heated for
4 h at 80°C) treated MTH, AC and GP (20 µg/mL each).
up to 10%. The optimized mobile phase was phosphate buffer ( pH
2.9)–organic phase in proportion of 70:30. Organic phase consisted
of methanol–acetonitrile (90:10), set at a flow rate of 1 mL/min and
detection of analytes was carried out at 230 nm. The flow rate of
1 mL/min was selected for further studies after several preliminary
investigatory chromatographic runs. Under the described experimental conditions, well-defined peaks were obtained by fixing the
run time of 10 min and all peaks were free from tailing (Figures 2a–c and 3).
Forced degradation of MTH, AC and GP
The chromatograms of the samples of MTH, AC and GP subjected to
various forced degradation conditions showed well-separated peaks of
the actives and the degradation products at different retention times.
However, in some conditions the actives did not show separate peaks
of the degradation products, rather a decrease in height and area of the
peak was observed. The peaks of the degradation products were identified and compared with that of the standard solution and were found
to be well resolved from the peaks of the actives (Figures 4a, b, 5a, b
and 6a).
Acid degradation
The chromatogram for acidic degradation of MTH, AC and GP
showed degradation of ∼0.5, 81.55 and 13.06%, respectively (Figure 4a). The chromatogram of AC presented two additional peaks
at 6.4 and 7.2 min, but only a small change in peak area and a
decrease in peak height were observed in the case of MTH
and GP.
Base degradation
The chromatogram for basic degradation of the actives showed degradation of ∼42.75 ± 0.78%, 2.29 ± 0.88% and 8.52 ± 0.19% for
MTH, AC and GP, respectively. The chromatogram of AC exhibited
one additional peak at 2.75 min (Figure 4b).
Oxidative degradation studies
The chromatograms of MTH, AC and GP showed 0.9, 18.57
and 14.15% degradation, respectively, in response to oxidative
degradation and one additional peak at 3.96 min (Figure 5a) for
MTH.
Validation of RP-HPLC Method and Stress Degradation
1659
Figure 5. (a) Chromatogram of H2O2 (3 % v/v) treated MTH, AC and GP (20 µg/mL each). (b) Chromatogram of photochemical degradation of MTH, AC and GP (20 µg/
mL each).
Photo degradation
Photo degradation resulted in degradation of 16.65 ± 0.47%, 18.57 ±
0.97% and 1.2 ± 0.09% for MTH, AC and GP, respectively (Figure 5b).
Heat degradation
MTH, AC and GP were evinced at 16.65 ± 0.47%, 18.57 ± 0.97%
and 1.2 ± 0.09%, respectively (Figure 6a).
adequate for the analysis to be performed, the parameters for combination of three APIs were evaluated and results are reported in
Table I.
Linearity and range
Linearity of the standard curve was established by least squares linear
regression. The developed standard curve was linear over the concentration range of 10–60 µg/mL for MTH (n = 5), 5–30 µg/mL for AC(n
= 5) and 2–20 µg/mL for GP (n = 5).
Method development
The developed method was validated, as described below, for the following parameters: system suitability, linearity, LOD, LOQ, ruggedness, robustness and recovery-precision.
System suitability
As the system suitability test is an integral part of chromatographic
method development and it is used to verify that the system is
Recovery and precision
The recovery of an analytical method is the closeness of the test results
to the true value. It has been determined by application of the analytical procedure to recovery studies, where low medium and high concentrations were analyzed. The results of recovery studies are shown in
Table II. The precision of an analytical procedure expresses the degree
of scatter between a series of measurements obtained from multiple
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Gite and Patravale
Figure 6. (a) Chromatogram of heat degradation product of MTH, AC and GP (20 µg/mL each). (b) Chromatogram of nanoparticles of MTH, AC and GP (drug
equivalent to 20 µg/mL, of nanoparticles).
Table I System Suitability Parameters
Parameters
MTH
AC
GP
Number of theoretical plates/meter
RT
Resolution
Symmetry
4,356.1
2.6
3.15
0.866
7,894.2
6.15
1.45
0.881
6,894.1
7.77
–
0.867
sampling of the same homogeneous sample under the prescribed conditions. The intra- and interday precision results obtained are shown
in Table III.
LOQ and LOD
The detection limit and quantification limit were calculated by
the method as described previously. The S/N of 3:1 and 10:1
were considered LOD and LOQ, respectively. The LOD for
MTH, AC and GP was observed to be 0.73, 1.1 and 0.39 ppm,
respectively. Similarly, the LOQ was calculated for MTH,
AC and GP and it was observed to be 2.37, 3.33 and
1.17 ppm, respectively.
Ruggedness and robustness test
Ruggedness and robustness study results are shown in Table IV.
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Validation of RP-HPLC Method and Stress Degradation
Table II Recovery Studies
Drug
MTH
Amount of drug
added (µg/mL)
Amount of drug
remained (µg/mL)
8
10
12
AC
7.98
9.95
12.02
GP
% of drug
recovered
%
RSD
Amount of drug
remained (µg/mL)
99.75
99.50
100.16
0.95
1.10
1.24
7.89
10.04
11.95
Table III Precision Studies
Amount of drug
added (µg/mL)
MTH
8
10
12
AC
8
10
12
GP
8
10
12
Repeatability
Amount of drug
found (µg/mL)
Intermediate precision
%
RSD
Amount of drug
found (µg/mL)
%
RSD
8.03
9.68
11.84
0.784
0.895
0.154
8.00
9.59
12.05
0.687
0.541
0.743
7.91
9.79
12.08
0.541
0.984
0.126
7.76
10.09
11.88
1.05
0.571
0.341
8.10
9.94
11.90
0.654
0.451
1.02
8.07
10.11
12.15
0.914
0.841
0.167
Solution stability
The % of RSD for MTH, GP and AC concentration during solution
stability experiments was within 1%. There was no significant change
observed for the chromatograms of standard solution and the experimental solution. Further, the absence of degradation peaks confirmed
that the sample is stable in solvent used during the assay for 24 h.
Application of the developed RP-HPLC method for nanoformulation
evaluation
HPLC chromatogram was obtained from nanoparticle sample, excluding the formation of the degradation products. There was no interference from the excipients commonly present in the formulations
(Figure 6b). The percent drug content for MTH, AC and GP was
found to be 101.05, 99.94 and 100.78%, respectively, with a %
RSD of <1.
Discussion
The development of a stability-indicating assay method for a combination of MTH, AC and GP; since it is a popularly used combination
of drugs and moreover there are no reports recounting an analytical
method to identify the degradation products that might arise from
exposure of this combination of drugs to different environmental
conditions. Various mobiles phases and mobile phase compositions
were evaluated at the beginning of the development to arrive at an
optimized mobile phase composition which yields suitable peaks
for all the three drugs with proper resolution. There are some reports
which illustrate various mobile-phase compositions for separation of
MTH, AC and GP combinations and as an individual entity. Raja
et al. has described a method for separation of MTH, pioglitazone
and GP where the authors employed mobile phase system comprising
% of drug
recovered
%
RSD
Amount of drug
remained (µg/mL)
98.62
100.40
99.58
1.47
1.56
0.78
8.07
9.87
12.10
% of drug
recovered
%
RSD
100.87
98.70
100.83
1.13
1.81
1.63
of methanol and phosphate buffer ( pH 3) in the ratio of 75:25 (v/v).
The composition, pH and flow rate of the mobile phase were altered
to optimize the separation conditions using standard samples of the
MTH, AC and GP. The final mobile phase composition in the current
research comprised phosphate buffer ( pH 2.9)–organic phase (methanol–acetonitrile, 90:10) in the ratio of 70:30 at a flow rate of 1 mL/
min and detection of analytes was carried out at 230 nm. The linearity results were within the acceptable limits (R 2, correlation coefficient is 0.999). The tailing factor for the peak was <2% with good
resolution between the three peaks. Stress testing showed that
MTH, AC and GP degraded via acid, base, oxidation, heat and photochemical degradation, with formation of two or one degradation
product. This may be caused by intramolecular abridgment of
three molecules (18). Thus, care should be taken in the manufacturing process and during storage of this product in order to prevent
degradation, for the reason that drug degradation could attenuate
the therapeutic activity and safety. The chromatograms of the samples of the degradation studies showed that the method was able to
separate MTH, AC and GP from its degradation products unlike any
of the earlier described analytical methods for this combination. The
suitability results of the chromatographic system (number of theoretical plates per meter, RT, resolution and symmetry) was demonstrated by comparing the obtained parameter values, which are found to
be within the acceptance criteria of the CDER guidance document
(19). The LOD values 0.79, 1.10 and 0.39 µg /mL for MTH, AC
and GP, respectively, and the LOQ values 2.37, 3.33 and 1.17 µg
/mL, respectively, for MTH, AC and GP, indicated that this method
is suitable for application of nanoparticle formulation. The recovery
results at all the three concentration levels, i.e. 5, 15, 30 µg/mL for
AC, 10, 30, 60 µg/mL for MTH and 2, 8, 20 µg/mL for GP, are within the acceptable limit (recovery ranged from 98 to 100%) at interday and intraday variation, which indicated that the method is highly
accurate.
The precision study results showed low values of % RSD (<2) for
inter and intraday variation, which suggested an excellent precision of
the method. The ruggedness and robustness test results are within the
acceptable limits (flow rate, analyst, column temperature, composition
of buffer and detector wavelength), which indicates that the method is
highly robust. As the system suitability test is an integral part of chromatographic method development and it is used to verify that the system is adequate for the analysis to be performed, the parameters for
combination of three APIs were evaluated.
The chromatographic system was found to be suitable for the evaluation of the drug content of this combination from the nanoparticle
formulation. In the chromatogram of the MTH, AC and GP combination of the samples extracted from nanoparticles, there was no interference from the excipients commonly present in formulation. The low
% RSD value indicated the suitability of this method for routine analysis of combination of MTH, AC and GP in pharmaceutical dosage
forms as well as nanoparticulate formulations.
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Gite and Patravale
Table IV Robustness and Ruggedness Studies
Sr. no.
Parameter
1
Flow rate mL/min
2
Wavelength
3
Analyst
4
Column temperature
5
Composition of buffer
Level
0.8
1.0
1.2
226
228
230
Analyst-1
Analyst-2
Analyst-3
25
30
35
24
25
26
RT (min)
As USP
MTH
AC
GP
MTH
AC
GP
2.70
2.68
2.65
2.72
2.68
2.63
2.71
2.68
2.67
2.73
2.68
2.67
2.72
2.68
2.69
5.83
5.82
5.80
5.86
5.82
5.77
5.85
5.82
5.79
5.84
5.82
5.80
5.86
5.82
5.84
7.61
7.57
7.54
7.59
7.57
7.53
7.60
7.57
7.69
7.59
7.57
7.55
7.60
7.57
7.62
0.845
0.966
1.245
1.412
0.966
1.164
0.874
0.966
1.415
1.112
0.966
1.345
0.984
0.966
0.995
1.114
0.881
1.125
0.974
0.881
1.318
1.452
0.881
0.856
0.891
0.881
1.467
0.937
0.881
1.24
0.949
0.867
0.843
1.354
0.867
0.934
0.978
0.867
0.997
0.945
0.867
0.997
0.864
0.867
0.913
Conclusion
The developed HPLC method is precise, specific, accurate and stability
indicating for the determination of MTH, AC and GP in combination.
The results of statistical analysis also proves that the method is reproducible and specific for the analysis of MTH, AC and GP. The method
is versatile for analysis of formulations such as nanoparticles of MTH,
AC and GP.
Acknowledgements
The authors are grateful to University Grants Commission and AICTE for the
financial assistance provided for the research work.
Conflict of interest statement. None declared.
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