Journal of Chromatographic Science 2015;53:1603– 1610
doi:10.1093/chromsci/bmv057 Advance Access publication May 24, 2015
Article
New Salting Out Stability-Indicating and Kinetic Thin Layer Chromatographic Method for
Determination of Glimepiride and Metformin HCl Binary Mixture
Yahya Abduh Salim Mohamed*, Abdel Maaboud Ismail Mohamed, Fardous Abdel-Fattah Mohamed and Sameh Abdel-Raouf Ahmed
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, B.O. 71526, Assiut, Egypt
*Author to whom correspondence should be addressed. Email: [email protected]
Received 14 July 2014; revised 5 March 2015
A simple, selective salting out and stability-indicating thin layer chromatographic (SOTLC) technique was developed for determination of
two antidiabetic drugs; glimepiride and metformin HCl in pure and in
tablets as a binary mixture. Separation was performed on silica gel 60
F254 plates using aqueous ammonium sulfate and acetonitrile (7:3, v/v)
as a mobile phase. The Rf values were 0.26 + 0.02 and 0.73 + 0.02 for
glimepiride and metformin HCl, respectively. The separated bands were
scanned at l 237 nm using CAMAG TLC scanner III. The proposed
method focusing on study of all the factors that play important role
in the mechanism of salting out process. The proposed method was
validated according to ICH guidelines and complied with USP31NF26 validation guidelines. The correlation coefficients of calibration
curves were 0.996 and 0.997 for glimepiride and metformin HCl, respectively, in the concentration range of 60–1,400 ng/band for both
drugs. The investigated drugs were also subjected to acidic, basic,
oxidative and photo-degradation and kinetic study was carried out.
Introduction
Metformin HCl (N, N-dimethylimidodicarbonimidic diamide hydrochloride) that is classified as a biguanide and glimepiride
(3-ethyl-2,5-dihydro-4-methyl-N-[2-[4-[[[[(trans-4-methyl
cyclohexyl)amino]carbonyl]amino]sulfonyl]phenyl]ethyl]-2-oxo1H-pyrrole-1-carboxamide) which classified as a second generation sulfonylurea (1, 2) are used as combined therapy in improving glycemic control (3). The chemical structures of metformin
HCl and glimepiride are shown in Figure 1 (1). Simultaneous separation of highly polar compound (metformin HCl) and less polar
oral hypoglycemic drug (glimepiride) in their binary mixture
using thin layer chromatography represents a challenge task.
There is another problem lies in the presence of metformin
HCl in high amount co-formulated with glimepiride (500:2) in
tablets that leading to measurement errors during simultaneous
UV determination of these drugs. Glimepride and metformin HCl
have been analyzed by few analytical methods; spectrophotometrically (4, 5), HPLC (6) and HPTLC (7, 8). Previously reported
spectrophotometric methods are chemometric-assisted, derivative methods and are not separation techniques. The objective
of this research was to develop a new, simple, reliable planar, salting out, stability indicating, inexpensive (the reagents and solvents available at any analytical laboratory), safe, time saving
(many samples per run and there is no need for base line stabilization with mobile phase) and thin layer chromatographic method (SOTLC) for simultaneous determination of the studied drugs
in their pure and pharmaceutical dosage forms as a binary mixture. This method also was developed to improve the sensitivity
and selectivity for separation and determination of the studied
drugs. Many research works have been carried out to study
the process of salting out thin layer chromatography and its
mechanism, one of the studies regarded salting out thin layer
chromatography SOTLC as a reversed-phase (RP) method in
which highly polar concentrated aqueous solutions of electrolytes are used as mobile phases with modified or unmodified adsorbents (9). Ammonium sulfate has an especially strong salting
out effect due to the presence of both ammonium and sulfate
ions (10). In the opinion of most authors, the separation of substances in conditions of planar salting out chromatography mostly based on nonspecific hydrophobic interactions between their
nonpolar parts and sorbents (11, 12). The theoretical bases for
this mechanism were developed under broader name “solvophobic interactions” (13). On silica with aqueous salt solutions silanol groups are mostly inhibited by specific interaction with ions
and water and the nonpolar siloxane parts become more available for nonspecific interaction (9). For this reason, nonactivated
layers were used in our experiments. Taking into account
the effect of compositions of the mobile phase. All the abovementioned studies followed the behavior of the studied substances under salting out TLC but not used for determination purposes. In our previous research, the mechanism of salting out
process of some oral hypoglycemic drugs and the factors that affecting this process have been studied. It was concluded that
SOTLC depends on many factors such as concentration of
the salt, volume fraction of both organic modifier and the aqueous salt. The effect of two-dimensional (2D) molecular descriptors such as octanol/water partition coefficient (log P o/w),
the aqueous solubility (log S) and the molar refractivity (MR)
and three-dimensional (3D) molecular descriptors such as total
hydrophobic surface area, hydrophobic volume, Van der Waals
energy and solvation energy were studied using molecular operating environment software (MOE). The process of salting out
also depends on the 3D structure of the studied drugs. It was
found that the most factors that affect on SOTLC were log P o/w,
MR and the lipophilicity of the drug (14). In this study, the less
polar compound, glimepiride separated faster (low Rf value) than
the more polar drug; metformin HCl that moved with the mobile
phase more.
Materials and methods
Equipment
A CAMAG TLC scanner III (Muttenz, Switzerland) provided with
linomat 5 sample automatic applicator (Muttenz, Switzerland)
and CAMAG 100 mL sample syringe (Hamilton, Bonaduz,
Switzerland) was used. TLC tank (standard type) (27.0 cm
width 26.5 cm height 7.0 cm diameter; Sigma-Aldrich Co.,
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. The chemical structures of the investigated drugs.
USA) was used. UV lamp short wavelength 254 nm (Vilber lournate
220 V 50 Hz; Marne-la-Vallee cedex, France) was used in this study.
Materials and reagents
All solvents and reagents used were of analytical grade.
Glimepiride was kindly supplied by Delta Pharma Co. (Cairo,
Egypt). Metformin HCl, methanol and ammonium sulfate were
obtained from EI-Nasr Pharmaceutical Chemical Co. (Abu-Zaabal,
Egypt). Thin layer chromatography aluminum sheets precoated
with 0.2 mm layers of silica gel 60 plates 20 20 cm were obtained from Fluka (Silica gel matrix H with fluorescent indicator
254 nm; Sigma-Aldrich, Germany). Pharmaceutical formulations
(Amarylw M 2 mg /500 mg tablets) are labeled to contain 2 mg
of glimepiride and 500 mg of metformin HCl per tablet and
were purchased from the local market.
Preparation of standard solutions
Stock solution containing 1,000 mg mL21 of standards glimepiride and metformin HCl mixture was prepared in methanol in
ratio 1:1. Working standard solutions were prepared by further
dilution of suitable volumes (0.15, 0.30, 0.60, 1.20, 2.50 and
3.50 mL of the stock solution with methanol in separated 10 mL
volumetric flasks to obtain concentrations of 15 – 350 mg mL21
corresponding to 60–1,400 ng/band for establishment of linearity
of the calibration curve).
Preparation of sample solutions
Twenty tablets finely powdered, mixed thoroughly and an accurately weighed amount equivalent to 500 mg of metformin HCl
and 2 mg of glimepiride was transferred to a 50-mL volumetric
flask to prepare a solution containing 0.040 mg mL21 of glimepiride and 10 mg mL21 of metformin HCl. The contents of the flask
were mixed with 40 mL of methanol. After 15 min of sonication, the volume was made to the mark with the same solvent.
After that the solution was filtered and the first portion of the filtrate was rejected (because it was used in washing of the filter
paper from any impurities and to ensure constant concentration
of the sample after adsorption of some amount on the filter
paper). Further dilution of the stock solution to prepare a solution containing 250 mg mL21 of metformin HCl that is corresponding to 1 mg/band, for glimepiride further dilution to
prepare a solution in concentration of 30 mg mL21 corresponding
to 120 ng/band was prepared.
1604 Mohamed et al.
General procedure
About 50 mL of the mobile phase (0.025 M aq. ammonium sulfate: acetonitrile 7:3, v/v) were poured into the TLC tank
which was lined with a thick filter paper to help the chamber saturation. The tank was covered and presaturated with the vapors
of the mobile phase system for at least 30 min at room temperature (25 + 28C) before use. Samples were spotted on silica gel 60
plates (20 cm W 6 cm L with 0.2 mm thickness) in the form of
bands of length 4 mm with CAMAG microliter syringe using
CAMAG linomat 5, with a constant application rate of 150 nL s21.
The slit dimension was kept 3 0.45 mm using CAMAG TLC
scanner III in reflectance – absorbance mode. Four microliters
of the working standard or sample solutions were spotted on
the marked start edge of the TLC plate 1 cm apart from
the lower edge of the plate. The plates were then allowed to
be air dried for 5 min, then transferred to the TLC tank and developed with the specified mobile phase for 9 min (1 cm apart
from the upper edge of the plate). After development, the plates
were removed, air dried for about 5 min. The separated bands
were scanned at l max 237 nm for simultaneous determination
of glimepiride and metformin HCl using CAMAG TLC scanner III
and the obtained data were treated with winCATS software
version 1.4.4.6337.
Forced degradation
Alkaline degradation
Five milliliters of 1.0 M NaOH was added into a 25 mL- round bottomed flask containing 1 mg mL21 of each of the investigated
drugs in the binary mixture. Then the volume was completed
to the mark with methanol and heated at 758C under reflux for
9 h. The duration of heating was divided into 3 h intervals. After
each interval, 2.5 mL was taken into a 10-mL volumetric flask and
neutralized with suitable amount of 1.0 M HCl and the volume
was completed to the mark with methanol to prepare a solution
containing 250 mg mL21 (of each drug in the binary mixture).
Analysis of the intact drugs and the degradation products by
the proposed procedure specified under general assay procedure
was carried out.
Acidic degradation
Five milliliters of 0.5 M HCl was added into a 25 mL-round bottomed flask containing 1 mg mL21 of each of the investigated
drugs in the binary mixture. Then, the volume completed to
the mark with methanol and heated at 758C under reflux for
9 h. The duration of heating was divided into three intervals.
After each interval, 2.5 mL of the solution was taken and neutralized with the suitable amount of 0.5 M NaOH and the volume was
completed to the mark with methanol to prepare a solution containing 250 mg mL21 (of each drug in the binary mixture) and analyzed by the proposed assay procedure.
Oxidation degradation
Two milliliters of 30% H2O2 were introduced into a 10 mL volumetric flask containing 1 mg mL21 of each of the investigated
drugs in the binary mixture. Then the volume completed to
the mark with methanol and left at room temperature for 72 h.
After each 24 h, a volume of 2.5 mL of the sample was transferred
into the 10 mL volumetric flask and completed to the mark with
methanol to prepare a solution of concentration equivalent to
250 mg mL21 (of each drug in the binary mixture). Analysis of
the intact drugs and the degradation products by the proposed procedure specified under general assay procedure was carried out.
Photolytic degradation
For the photo-degradation study, the standard drugs (250 mg mL21
of each drug) in the binary mixture were exposed to UV light
(254 nm) in a photo-stability chamber for 15 h that was divided
into 5 h-intervals. Analysis of the intact drugs and the degradation
products by the proposed procedure specified under general assay
procedure was carried out.
Figure 2. Absorption spectra of the studied drugs mixture; metformin HCl (1,000 ng/band)
and glimepiride (1,000 ng/band), scanning using CAMAG TLC scanner III.
Effect of saturation time
Different saturation times ranged from 10 to 50 min were tested
to obtain compact separated bands of the investigated drugs. It
was found that compact bands, good resolution and maximum
absorbance when saturation time 30 min.
Results
Spectral analysis
The separated drugs were scanned in the spectrum range of
200 –400 nm. Analysis of metformin HCl and glimepiride at maximum wavelength of 237 nm has been done as shown in Figure 2.
Optimization of the proposed method conditions
Effect of mobile phase compositions
After many trials, it was found that aq. ammonium sulfate is necessary for movement of metformin HCl. Different solvents
(organic modifiers) were tested (acetonitrile, methanol and
ethanol) to obtain good separation and resolution but good separation was obtained when using acetonitrile, so different ratios
of the mobile phase compositions (aq. ammonium sulfate
0.025 M:acetonitrile) in ratios 5:5, 6:4, 7:3 and 8:2 were tested.
Compact bands as well as good separation and resolution with
considerable Rf values (0.26 + 0.02 and 0.73 + 0.02 for glimepiride and metformin HCl, respectively) were obtained when
the ratio of aq. ammonium sulfate:acetonitrile was 7:3 (v/v),
respectively (Figure 3). Resolution (Rs) between the two peaks
of the studied drugs was 2.58 as calculated.
Effect of ammonium sulfate concentration
Different concentrations of ammonium sulfate in the range of
0.0125 – 0.2000 M were prepared in distilled water and tested
with acetonitrile as mobile phase in the ratio of 7:3 (v/v).
The peak area of the studied drugs increased with increasing
the concentration of ammonium sulfate till concentration of
0.025 M, then decreased. So the concentration of 0.025 M aq. ammonium sulfate was selected for all the subsequent work.
Validation of the proposed SOTLC method
The proposed method was validated according to ICH guidelines
(15) and complied with USP 31-NF26 validation guidelines (16).
All results were expressed as percentages. For the statistical analysis, Excel 2003 (Microsoft Office) was used. A 5% significance
level was selected. The developed method was validated for
the following parameters.
Calibration and linearity
The linearity of the method was in the range 60–1,400 ng/band
for both glimepiride and metformin HCl. Good correlations were
obtained between drug concentration and the peak area, the correlation coefficients for glimepiride and metformin HCl were
0.996 and 0.997, respectively (Figure 4).
The limit of detection and limit of quantitation
The limit of detection (LOD) and limit of quantitation (LOQ) for
the investigated drugs were calculated using the formula
LOD or LOQ ¼
kSDa
;
b
ð1Þ
where k ¼ 3.3 for LOD and 10 for LOQ, SDa is the standard deviation of the intercept and b is the slope (16). The LOD and LOQ
were experimentally verified by diluting known concentration of
reference solution until the average responses were 3.3 or 10
times the standard deviation of the responses for six replicate determinations. LODs were 12.17 and 12.83 ng/band and LOQs
were 36.89 and 38.87 ng/band for glimepiride and metformin
HCl, respectively, which indicated high sensitivity of the proposed SOTLC method.
Determination of Glimepiride and Metformin HCl Binary Mixture 1605
Figure 3. Densitograms showing the effect of the mobile phase compositions on the separation of (1) glimepiride (1,000 ng/band), (2) metformin HCl (1,000 ng/band) in binary
mixture in ratios; (a) 5:5, (b) 6:4, (c) 7:3, (d) 8:2 of 0.025 M aq. ammonium sulfate:acetonitrile, respectively, and scanning at l 237 nm using CAMAG TLC scanner III.
Accuracy
The accuracy of the developed analytical procedure expresses
the closeness of agreement between the value which is accepted
either as conventional true value or an accepted reference value
and the value found. This is sometimes termed trueness. Method
accuracy was determined by addition of known amounts of standard glimepiride and metformin HCl (240.0 and 400.0 ng/band)
to a sample solution (Amarylw M 2 mg /500 mg tablets) of known
concentration (320.0 ng/band) and four replicate measurements
were done then calculating the recovery percentages. The results of standard addition method revealed a good accuracy and
recovery percentages ranged from 99.3 to 100.1% and from 97.1
to 101.3% for glimepiride and metformin HCl, respectively.
Precision
The precision expresses the closeness of agreement between
a series of measurements obtained from multiple sampling of
the same homogeneous sample under the prescribed conditions.
Intra-day precision was determined by replicate analysis (n ¼ 6) of
standard solutions of the binary mixture at three concentration
levels (120, 500 and 1,000 ng/band) covering the low, medium
and higher ranges of the calibration curve. The inter-day precision
was carried out by repeating the analysis of standard solutions of
the binary mixture at three concentration levels (120, 500 and
1,000 ng/band) covering the low, medium and higher ranges of
the calibration curve over a period of three consecutive working
days. The overall precision of the method was expressed as relative
1606 Mohamed et al.
standard deviations (RSD). A good precision and repeatability of
the proposed method was obtained where the % RSD 1.5 and
1.7 for glimepiride and metformin HCl, respectively. The recovery
percentages were in the range 98.2 – 101.3% which makes it
adequate for application in the quality control laboratories.
Robustness
Robustness of an analytical procedure is a measure of its capacity
to remain unaffected by small, but deliberate variations in
method parameters and provides an indication of its reliability
during normal usage. The studied parameters were the volume
of the mobile phase compositions, aq. ammonium sulfate and
acetonitrile (+0.7 and +0.3 mL respectively), time of saturation
(+3 min), time of development (+1 min) and concentration of
ammonium sulfate (+0.0025 M). It was found that slight variation of these variables didn’t significantly affect the performance
of the proposed method. The recovery percentages obtained by
application of the proposed procedure were 99.8 and 100.8% for
glimepiride and metformin HCl, respectively, whereas by slight
variation of the proposed method variables the recovery percentages were in the range 98.0 to 101.6%. So the proposed method
could be considered robust and reliable during the normal usage.
Selectivity
The selectivity of an assay is a measure of the extent to which this
method can determine a particular compound in the analyzed
matrices without interference from matrix components, other
Figure 4. (a) 3D graph representing calibration of standard binary mixture of glimepiride (Rf ¼ 0.26 + 0.02) and metformin HCl (Rf ¼ 0.73 + 0.02) in concentrations of 60, 120, 240,
480,1,000 and 1,400 ng/band, each concentration in three replicates and scanning at lmax 237 nm, (b) the calibration plot for SOTLC analysis of glimepiride and (c) the calibration plot
for SOTLC analysis of metformin HCl correlating the concentration of the drug (ng/band) with peak area.
drugs or degradation products. It is obvious from the previous results that there is no interference in application of the proposed
SOTLC in separation and determination of the studied drugs in
their binary mixture since a good resolution and separation
between the peaks of the investigated drugs was obtained by calculating the Rs that was 2.58 and there is no interference in the
presence of dosage forms (tablets) additives and excipients during application of the standard addition method. The selectivity
Determination of Glimepiride and Metformin HCl Binary Mixture 1607
and specificity of the proposed separation method was evaluated
also by study of peak purity and identity analysis which can be
used to demonstrate that an observed chromatographic peak is
attributable to a single component (17). This was done by comparison of the Rf values of the pure substances as binary mixture
with their Rf values as binary mixture in tablets. In this study,
maximum Rf values were 0.26 + 0.02 and 0.73 + 0.02 for authentic glimepiride and metformin HCl; respectively, while in tablets
were 0.27 + 0.01 and 0.72 + 0.01 for glimepiride and metformin
HCl, respectively. After alkaline degradation Rf values were
0.28 + 0.01, 0.56 + 0.01 and 0.70 + 0.01 for glimepiride,
D1(degradation product) and metformin HCl, respectively. The
peak purity and identity test of the proposed method also studied
by comparison of correlation coefficient (r) along the matched
spectra of the intact studied drugs and the spectra of the studied
drugs after alkaline degradation in the presence of degradation
products. CAMAG TLC scanar III is equipped with proprietary
winCATS software version 1.4.4.6337 that will perform these calculations needed for peak purity and identity tests. This carried
out by calculation of correlation between wavelength and the absorbance from start to maximum r (s,m) and from maximum to
the end r (m,e) for the matched spectra of both intact drugs and
drugs under alkaline degradation. The results of peak purity and
identity test are as shown in Table I. The closeness of values to 1
indicated that the peaks are attributable to the studied compounds. Therefore, the proposed method is considered selective
for simultaneous determination of the studied drugs in their
binary mixture.
Application of the proposed SOTLC method for analysis of
Amarylw M tablets
The proposed method was applied successfully for the determination of the studied drugs binary mixture in the pharmaceutical
dosage form (Amarylw M 2 mg/500 mg tablets). Three replicate
measurements were made; the results obtained were validated by
comparison with previously reported methods (18, 19). Found
percentages for glimepiride and metformin HCl in tables were
99.9 and 98.5% for both drugs, respectively, by application of
the proposed method whereas the found percentages that obtained after application of reported methods were 99.2 and
99.5% for glimepiride and metformin HCl, respectively. No significant difference was found by applying t- and F-tests at 95% confidence level (significance level a 5%) indicating good accuracy,
precision and suitability of the proposed method for simultaneous determination of the investigated drugs in their pharmaceutical dosage forms.
Discussion
Stability-indicating assay and kinetic study of
glimepiride and metformin HCl binary mixture using
the proposed salting out TLC method
Alkaline degradation
The separated drugs and degradation products were scanned in
the spectrum range 200– 400 nm. It was found that glimepiride,
metformin HCl and their degradation products have the same
maximum absorbance wavelength of l 237 nm that has been
chosen for scanning. On exposure to alkaline solution metformin
HCl decomposes to form ammonia and dimethyl amine (20). The
studied binary mixture has undergone to alkaline degradation
and the degradant gave well-separated bands from the bands of
the pure intact drugs (Figure 5). Glimepiride undergoes to alkaline degradation to form degradant D1 (1-[4-(2-Aminoethyl)
phenylsulfonyl]-3-trans-(4-methylcyclohexyl)urea) (21) with Rf
0.56 + 0.01, while metformin HCl degraded to ammonia and
dimethyl amine that cannot be detected and did not give clear
bands, so the decrease in the absorbance of the intact drug has
been measured, the chemical structures of the degradation products of metformin HCl and glimepiride binary mixture are shown
in Scheme 1. The results of the kinetic study of alkaline degradation of the investigated binary mixture are shown in Table II.
Alkaline degradation of glimepiride obeys zero-order reaction
rate while degradation of metformin HCl obeys the pseudo-firstorder reaction rate. The reaction rate of degradation of glimepiride and t1/2 were calculated using the formulae (22)
k0 ¼
dx
;
dt
ð2Þ
t1=2 ¼
a
;
2k0
ð3Þ
where k0 is the reaction rate of zero order. dx is the difference
in drug concentration, dt is the difference in time, t 1/2 is
the half-life and a is the initial concentration of the studied
drug. It was found that the reaction rate 3.48% hour21 while
t1/2 was 14.50 h.
Table I
Peak Purity and Identity Test for Evaluation of Selectivity and Specificity of the Proposed Method for
Simultaneous Determination of Glimepiride and Metformin HCl Obtained by winCATS Software
Drug
r (s, m)a
r (m, e)b
Intact glimepiride
Glimepiride after alkaline degradation
Intact metformin HCl
Metformin after alkaline degradation
0.9978
0.9965
0.9960
0.9982
0.9974
0.9978
0.9952
0.9953
a
Correlation coefficient from start to maximum position of the spectrum.
Correlation coefficient from maximum to end position of the spectrum.
b
1608 Mohamed et al.
Figure 5. Densitogram of alkaline degradation of glimepiride (1,000 ng/band) and
metformin HCl (1,000 ng/band) binary mixture using 1.0 M NaOH and heating for
6 h at 758C under reflux.
Scheme 1. Alkaline degradation products of glimepiride and metformin HCl binary mixture.
Table II
Concentration Remained Intact and Percentage After Alkaline Degradation of Glimepiride and
Metformin HCl Binary Mixture
Table III
Concentration Remained Intact and Percentage After Acidic Degradation of Glimepiride and
Metformin HCl Binary Mixture Using the Proposed SOTLC Method
Time
(h)
Glimepiride
Time (h) Glimepiride
Content remained
intact (ng/band)a
Drug
concentration (%)
Content remained
intact (ng/band)a
Drug
concentration (%)
0.0
3.0
6.0
9.0
1008.4
894.9
805.0
715.0
100.8
89.5
80.5
71.5
998.4
544.0
349.9
161.1
99.8
54.4
35.0
16.1
Metformin HCl
0.0
3.0
6.0
9.0
Metformin HCl
Content remained
intact (ng/band)a
Drug
concentration (%)
Content remained
intact (ng/band)a
Drug
concentration (%)
1008.4
787.7
573.4
327.0
100.8
78.8
57.3
32.7
998.4
826.5
665.7
490.1
99.8
82.6
66.6
49.0
Following the general assay procedure after degradation procedure and using 1,000 ng/band of
the intact and degraded standard binary mixture of the studied drugs.
a
Average of three determinations.
Following the general assay procedure after degradation procedure and using 1,000 ng/band of the
intact and degraded standard binary mixture of the studied drugs.
a
Average of three determinations.
Whereas the reaction rate and t1/2 for degradation of metformin
HCl were calculated using the formulae (22):
Stability of glimepiride and metformin HCl binary
mixture on exposure to UV light at 254 nm
Glimepiride and metformin HCl were degraded when exposed to
UV light at 254 nm for 9 h. The photolytic degradation of glimepiride and metformin HCl binary mixture was of zero order and
the rates were 1.73% h21, 0.83% h21 while t1/2 were 29.08 and
60.40 h for glimepiride and metformin HCl, respectively.
log a
2:303
;
k1 ¼
ax
t
t1=2 ¼
0:693
;
k1
ð4Þ
ð5Þ
where k1 is reaction rate of pseudo-first order, x is the degraded
concentration of the studied drug and t is the time. It was found
that the reaction rate was 0.24 h21 and t1/2 was 2.91 h.
Acidic degradation
Acidic degradation of glimepiride and metformin HCl was of
zero-order degradation reaction but did not give new bands.
The reaction rates were 7.39 and 5.64% hour21 while t1/2 were
6.82 and 8.85 h for glimepiride and metformin HCl, respectively.
The kinetic results of the acidic degradation of the studied binary
mixture are shown in Table III.
Oxidative degradation
The separated drugs and degradation products were scanned in
the spectrum range 200 – 400 nm. It was found that glimepiride,
metformin HCl and the oxidative degradation products have
the same maximum absorbance wavelength at 237 nm that
was chosen for scanning. The reduction in absorbance at l
237 nm was measured. Oxidation rate of glimepiride and metformin HCl of zero order during successive three days. The reaction rate of glimepiride was 17.25% (24 h)21 while t1/2 was
70.08 h (3 days) while the reaction rate of metformin HCl
(k 0) was 2.47 (24 h)21 and t 1/2 was 484.08 h (20.17 days)
(Table IV).
Determination of Glimepiride and Metformin HCl Binary Mixture 1609
Table IV
Concentration Remained Intact and Percentage After Oxidation of Glimepiride and Metformin HCl
Binary Mixture Using the Proposed SOTLC Method
Time (h)
Content remained intact
(ng/band)a
Recovery (%)
Glimepiride
Metformin HCl
Glimepiride
Metformin HCl
0.0
24.0
48.0
72.0
1008.4
848.3
647.2
477.9
998.4
975.7
944.7
924.2
100.8
84.8
64.7
47.8
99.8
97.6
94.5
92.4
Following the general assay procedure after degradation procedure and using 1,000 ng/band of
the intact and degraded standard binary mixture of the studied drugs.
a
Average of three determinations.
Stability of the studied binary mixture in methanol solution
The binary mixture was stable at ambient temperature in
methanol up to 6 days.
Conclusion
The developed salting out thin layer chromatographic method
for determination of glimepiride and metformin HCl describes
a new simple, rapid, selective and sensitive method for the analysis of the studied drugs as binary mixture. The method is reliable
for the accurate determination of these drugs in bulk, tablets and
could be recommended for the routine use in quality control laboratories. This is a typical stability-indicating assay, established according to recommendations of the ICH guidelines. The method can be
used to determine the purity of the studied drugs in mixtures available from different sources by detecting any related impurities.
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