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Analytical Chemistry Letters

ISSN: 2229-7928 (Print) 2230-7532 (Online) Journal homepage: http://www.tandfonline.com/loi/tacl20

A Comparative Study of Smart Spectrophotometric


Methods and a Densitometric Method for
Simultaneous Determination of Binary Mixture of
Canagliflozin and Metformin Hydrochloride

Mona S. Elshahed, Dalia Mohamed, Tamer Nasr, Nageh Aboutaleb & Ola
Zakaria

To cite this article: Mona S. Elshahed, Dalia Mohamed, Tamer Nasr, Nageh Aboutaleb & Ola
Zakaria (2018) A Comparative Study of Smart Spectrophotometric Methods and a Densitometric
Method for Simultaneous Determination of Binary Mixture of Canagliflozin and Metformin
Hydrochloride, Analytical Chemistry Letters, 8:1, 63-75, DOI: 10.1080/22297928.2017.1395295

To link to this article: https://doi.org/10.1080/22297928.2017.1395295

Published online: 15 Mar 2018.

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TACL 8 (1) 2018 pp 63 - 75 63
ISSN Print: 2229-7928
ISSN Online: 2230-7532

A Comparative Study of Smart Spectrophotometric Methods and a


Densitometric Method for Simultaneous Determination of Binary
Mixture of Canagliflozin and Metformin Hydrochloride

Mona S. Elshahed 1, Dalia Mohamed 1,3,


Tamer Nasr 2, Nageh Aboutaleb 2, Ola Zakaria 2*

Analytical Chemistry Department, Faculty of Pharmacy,


1

Helwan University, Ein Helwan, 11795, Cairo, Egypt


2
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Helwan University, Ein Helwan, 11795, Cairo, Egypt
3
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, October
University for Modern Sciences and Arts, 11787, 6 October City, Egypt
Received 03 August 2017; accepted in revised form 19 October 2017

Abstract: Simple and sensitive Spectrophotometric methods and a densitometric thin layer
chromatographic method (TLC) are developed and validated for simultaneous determination of binary mixture
of metformin hydrochloride (MET) and canagliflozin (CFZ) in combined dosage form. Three spectrophotometric
methods manipulating ratio spectra namely ratio subtraction, ratio difference and derivative ratio are used for
the determination of MET in the presence of CFZ without preliminary separation, while CFZ is directly determined
by measuring its absorbance at λmax 290 nm. The calibration curves follow Beer’s law in the concentration
range of 1 - 20 and 1 - 30 μg/mL for MET and CFZ respectively. A simple TLC method is developed where
separation is performed on TLC silica gel plates 60F254 using toluene: Methylene chloride: methanol: water:
glacial acetic acid in the volume ratio (20:15:15:1:0.25) as a mobile phase. Rf values are 0.55 ± 0.05 for CFZ and
0.21 ± 0.03 for MET, the linearity ranges are 0.3-4.5 and 0.5-5.5 μg/band for CFZ and MET respectively. The
developed methods are successfully applied for the simultaneous determination of MET and CFZ in bulk and
pharmaceutical formulation.

Keywords: Canagliflozin; metformin hydrochloride; ratio subtraction; ratio difference; derivative


ratio.

Introduction through an insulin-independent mechanism by


Canagliflozin (CFZ) is a member of new class blocking the reabsorption of glucose, causing it to
of glucose lowering agents, sodium-glucose co- be excreted in the urine. It acts upon the proximal
transporter (SGLT2) inhibitors, which got approval tubules of the kidneys and reduces the renal
by food and drug administration 1.Canagliflozin is threshold for glucose 3,4. SGLT2 inhibitors have
chemically named as (2S,3R,4R,5S,6R)-2-(3-{[5- the potential to be used not only as monotherapy
(4-fluorophenyl)thiophen-2-yl]methyl}-4- but also in combination with any of the existing
methylphenyl)-6-(hydroxymethyl)oxane-3,4,5-triol classes of glucose-lowering agents 5.
2
(Fig. 1). SGLT2 inhibition lowers blood glucose Metformin hydrochloride (MET) is a biguanide

*Corresponding author (Ola Zakaria)


E-mail: < dr.olazakaria@yahoo.com > © 2018, Har Krishan Bhalla & Sons
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 64
anti-diabetic. It is given orally in the treatment of tometric methods were used for resolving such
type 2 diabetes mellitus. It is the drug of first choice mixtures with overlapped spectra such as; solv-
in overweight patients 6. Metformin is chemically ing two simultaneous equations (vierodet’s
known as 1, 1-Dimethylbiguanidemonohydrochlo- method) 19, the isosbestic point where the two
ride 7 (Fig. 2). The mode of action of biguanides absorbing components, X and Y, have equal ab-
is not clear. They do not stimulate insulin release sorptivity constants 20 or by using absorbance ra-
but require that some insulin be present in order tios at certain wavelengths 21. Derivative Spec-
to exert their anti diabetic effect. Possible mecha- trophotometry 22 and ratio derivative Spectropho-
nisms of action include delay in the absorption of tometry 23 are useful means of resolving two over-
glucose from the gastrointestinal tract, an increase lapped spectra and eliminating matrix interfer-
in insulin sensitivity and glucose uptake into cells, ences in the assay of binary mixtures.
and inhibition of hepatic gluconeogenesis. The The objective of the present work is to apply
major action of MET lay in increasing glucose and conduct a comparative study utilizing three
transport across the cell membrane in skeletal well established spectrophotometric methods; ra-
muscle 6. tio difference (RD), ratio subtraction (RS) and
derivative ratio (DR) for resolving overlapping
spectra of the studied binary mixture with spec-
tral interference problems without preliminary
separation steps. Another aim is to perform a new,
simple and selective densitometric -TLC method
for determination of the studied drugs in their com-
binations. The utilized methods are characterized
Fig. 1. The chemical structure of canagliflozin by being simple, accurate, and precise; with no
need for any sophisticated apparatus or computer
programs.

Experimental
Instrument
Spectrophotometric measurements were car-
ried out using Jasco (V-530) Double-beam UV-
Fig. 2. The chemical structure of metformin Vis spectrophotometer loaded with Spectra Man-
ager Program (JASCO) for spectral acquisition
MET is official in British Pharmacopeia (BP) 8 and elaboration of the data obtained. Two matched
and United States Pharmacopeia (USP) 7. 1 cm quartz cells were used. Scans were carried
CFZ is a relatively new drug, so it is not yet out in the range from 200 to 400 nm at 0.1 nm
official in any of the Pharmacopoeias. interval. Pre-coated silica gel TLC aluminum
Reviewing the literature revealed that few ana- plates F 254 ; 20 × 20 cm; (Merck KGaA,
lytical methods have been reported for determi- Darmstadt, Germany)
nation of MET and CFZ. These methods include The samples were spotted with a CAMAG
Spectrophotometric methods 9, 10, 11, LC-MS/MS micro liter syringe (100 μl) using a CAMAG
12
and capillary electrophoresis 13. High perfor- Linomat 5 auto sampler (Muttens, Switzerland).
mance liquid chromatographic methods were re- the plates were developed in CAMAG twin trough
ported for determination of mixture of both drugs glass chamber (20×10cm) and scanned using
in their binary mixture 14, 15, 16, 17, 18. CAMAG TLC densitometric scanner 3 S/N
The main problem of spectrophotometric binary 130319 in the reflectance absorbance mode at
or ternary mixture analysis is the simultaneous 236 nm and 290 nm and operated by WINCATS
determination of the compounds in the same mix- software (version 1.4.4.6337).
ture without prior separation. Several spectropho- An electronic analytical weighing balance (0.1
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 65
mg sensitivity, Shimadzu AU 220) and a sonicator dilution of the stock solution with methanol to reach
(Sonica, model 2200 MH) were used in this study. the concentration of 500 μg/mL for both CFZ and
MET.
Materials
Pure samples Procedure
Metformin hydrochloride was obtained from Construction of calibration graphs
(Sigma-Aldrich, Germany), its purity was certi- For spectrophotometric method
fied to be 100.4 % according to the official method Aliquots equivalent to 10-300 μg CFZ and 10 -
7
and Canagliflozin (99.1 %) was purchased from 200 μg MET were accurately transferred from
Beijing Huikang Boyuan Chemical Technology their working standard solutions into two sepa-
Co. Ltd. (Beijing, China) rate series of 10-mL volumetric flasks then com-
pleted to volume with methanol. The zero-order
Pharmaceutical dosage form absorption spectra (D0) for the prepared solutions
Vokanamet tablets (Janssen-Cilag international, of the two studied drugs were recorded over the
Belgium) (B.N EGZT300) labeled to contain 850 range of 200-400 nm against methanol as a blank,
mg MET and 50 mg CFZ was purchased from and stored in the computer.
online Canadian pharmacy.
Canagliflozin
Solvents Calibration graph was constructed relating the
Methanol, Methylene chloride and toluene (ana- absorbance of zero order spectra (D0) of CFZ at
lytical grade, 99.8 %) were obtained from (Sigma- 290 nm versus the corresponding concentrations
Aldrich, Germany). and regression equation was computed.
Glacial acetic acid 99.0 % was obtained from
El-Nasr Pharmaceutical Chemicals, Cairo, Egypt. Metformin
High purity distilled water was used. Ratio subtraction method (RS)
Calibration graph was constructed relating the
Standard solutions absorbance of zero order spectra of MET at 236
For spectrophotometric method nm versus the corresponding concentrations and
Standard stock solutions of CFZ and MET (100 the regression equation was computed.
μg/mL) were prepared in 100-mL volumetric
flasks, by dissolving an appropriate amount of the Ratio difference method (RD)
studied drugs in methanol and diluting to volume The stored D0 spectra of MET was divided by
with the same solvent. The stock solutions were the standard spectrum of 20 μg/mL CFZ as suit-
stable when stored in a refrigerator at 4oC for able divisor. Calibration curve was constructed
one week. by plotting the difference between the amplitudes
Standard working solutions of CFZ and MET of the resultant ratio spectra at 215 and 237 nm
were freshly prepared by dilution from the stock (ΔA= 237 - 215 nm) versus the corresponding
solutions with the same solvent to obtain a con- MET concentrations and the regression equation
centration of (10 and 50 μg/mL) for each of CFZ was computed.
and MET.
Derivative ratio method (DR)
For TLC densitometric method The stored D0 spectra of MET were divided
CFZ stock solutions (2000 μg/mL) and MET by the standard spectrum of 20 μg /mL CFZ as
stock solutions (2000 μg/mL) were prepared in suitable divisor. The first derivative of the stored
100-mL volumetric flasks, by dissolving an appro- ratio spectra (1DR) was recorded and calibration
priate amount of the studied drugs in methanol curve was constructed relating the amplitude at
and diluting to volume with the same solvent. The 245 nm to the corresponding MET concentrations
working solution for each drug was prepared by and the regression equation was computed.
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 66
For TLC densitometric method spectra of the prepared solutions were recorded
Into two separate series of 10-mL volumetric at 200-400 nm and stored in the computer. CFZ
flasks, aliquots equivalent to 0.6 - 9.0 mg CFZ concentration was determined directly after re-
and 1.0 - 11 mg MET, were accurately transferred cording the absorbance at 290 nm of D0 spectra
from their respective working solution, and then of the prepared laboratory mixtures and referring
the volume was completed with methanol. Por- to corresponding regression equation. The con-
tions were spotted as bands on HPTLC plate to centration of MET was calculated using the cor-
obtain final concentrations of 0.3-4.5 and 0.5-5.5 responding regression equation after applying the
μg/spot for CFZ and MET respectively. corresponding manipulating steps for each
method. In case of ratio subtraction method, the
Sample loading spectra of the mixtures were divided by 20 μg/
The samples were applied to the TLC plate as mL CFZ as a divisor followed by subtraction of
bands using the specified (100-μL) TLC CAMAG the obtained constants from the division at the pla-
Linomat syringe (The band width was 5 mm and teau region. Then, the obtained spectra were
the bands were spaced at about 9 mm apart from multiplied by the spectrum of the divisor and the
each other and 15 mm from the bottom edge of absorbance at 236 nm was recorded. For deter-
the plate). The sample volume for all solutions mination of MET by RD and DR methods the
was 5 μL; each concentration was applied in trip- procedure under ratio difference method and de-
licate and allowed to air dry for 10 min. rivative ratio method was followed.
For the TLC densitometric method; six mixtures
Chromatogram development and scanning containing different ratios of the cited drugs were
The developing system was Toluene: Methyl- prepared by transferring accurate aliquots from
ene chloride: methanol: water: glacial acetic acid their working solutions into a series of 10-mL volu-
in the volume ratio (20:15:15:1:0.25). The chro- metric flasks, then the volumes were completed
matographic tank was left to be saturated with with methanol. The procedure under (Construc-
the developing system at room temperature for tion of calibration graph, For the TLC densito-
20 min then linear ascending development was metric method) was followed.
performed to a distance of approximately 70 mm
from the lower edge. The developed plates were Application to pharmaceutical dosage form
air dried and scanned using CAMAG TLC Scan- Twenty tablets were accurately weighed, pow-
ner with deuterium lamp set at 236 nm and 290 dered and homogeneously mixed. An accurate
nm. The slit dimension was kept at 4 mm × 0.45 Weight equivalent to 10 mg CFZ and 170 mg MET
mm and the scanning speed was 20 mm/s. was transferred into 100 mL volumetric flask and
The calibration curves were constructed in the dissolved in 50 mL methanol. The solution was
range of 0.3-4.5 μg/band & 0.5-5.5 μg/band for ultra sonicated for 30 min, cooled and then the
CFZ and MET, respectively, by plotting the rela- volume was completed with the same solvent and
tive peak area at 290 nm (for CFZ) and 230 nm filtered.
(for MET) against the corresponding concentra- For the spectrophotometric methods; aliquots
tion; consequently the regression equations were equivalent to 1.0 milliliter of the filtrate was trans-
calculated. ferred to 100 mL volumetric flask and the volume
was completed with the same solvent to obtain a
Application to laboratory prepared mixtures final concentration of 17 μg/mL for MET and 1
For the spectrophotometric methods, aliquots μg/mL for CFZ. The spectra of the prepared so-
were accurately transferred from the working lution were recorded at 200-400 nm and stored in
standard solutions of CFZ and MET into a series the computer and the proposed methods were ap-
of 10 ml volumetric flasks to prepare six mixtures plied for the analysis of the pharmaceutical prepa-
containing different ratios of the cited drugs. The ration solution using the procedures mentioned
volumes were completed with methanol and the under analysis of laboratory prepared mixtures.
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 67
The concentration of each drug was calculated traction, separation steps and other tedious ana-
using the corresponding regression equation for lytical process during analysis which is time con-
each method. suming. The main objective of this work is to de-
For the TLC densitometric method; aliquots velop simple, sensitive and accurate Spectropho-
equivalent to 6.0 milliliter of the filtrate was trans- tometric methods for the simultaneous determi-
ferred to 10-mL volumetric flask and the volume nation of MET and CFZ (newly approved oral
was completed with the same solvent to obtain a hypoglycemic binary mixture) in bulk and phar-
final concentration of 5.1 μg/band for MET and maceutical dosage form without prior separation.
0.3 μg/band for CFZ. The concentration of each Another goal of this work is to develop a simple
drug was calculated using the corresponding re- TLC- densitometric method that can be applied
gression equation. successfully for separation and quantification of
the studied drugs.
Results and discussion
In the last few years, many spectrophotometric Spectrophotometric Methods
methods have been developed for resolving the CFZ in the studied oral hypoglycemic binary mix-
overlapped spectra of multi component mixtures ture can be determined by direct Spectrophoto-
without prior separation of the constituent analytes. metric measurement of the absorbance at 290 nm
The developed spectrophotometric methods are of D0 spectra where there is no interference from
found to be very easy to apply, sensitive and very MET as shown in Fig. 3.
cheap methods for conducting analytical studies As shown in Fig. 3, MET D0 spectra suffer
related to quality control and routine analysis of severe overlap from the D0 spectra of CFZ so
marketed products in different laboratories. These MET cannot be measured by direct Spectropho-
methods are also capable of fast quantitative reso- tometry in the studied binary mixture. Three Spec-
lution of more than one component in a mixture trophotometric methods (ratio subtraction, ratio dif-
without any prior treatment which make them ference and derivative ratio) have been studied
preferable than expensive hyphenated analytical for the determination of MET with satisfactory
instrumentations or techniques such as liquid chro- results in the presence of CFZ. These three meth-
matography-mass spectroscopy, gas chromatog- ods are considered to be simpler and less costly
raphy mass spectroscopy which require prior ex- than other chromatographic methods.

Figure 3. Zero order absorption spectra of 10 μg/ml MET (λ max 236 nm)
and 10 μg/mL CFZ (λ max 290 nm) using methanol as a blank
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 68
Ratio subtraction method (RS) as shown in Fig. 4. The constant value is calcu-
This method 20 is used for the determination of lated from the plateau region and then subtracted
one component (MET) in the studied binary mix- from the ratio spectra to get the spectra shown in
ture in the presence of the interfering extended Fig. 5. The obtained spectra are then multiplied
spectrum of other component (CFZ).MET is de- by the divisor where, the original D0 spectra of
termined by dividing the D0 spectra of mixture by pure MET are recovered as indicated in Fig. 6
a carefully chosen known concentration of CFZ and they are used for determination of MET at its
(20 μg/ml) as a divisor to obtain new ratio spectra λmax (236 nm). The concentration can be calcu-

Figure 4. Ratio spectra of laboratory prepared mixtures of MET and


CFZ, using 20 μg/mL of CFZ as a divisor and methanol as a blank

Figure 5. Ratio spectra of laboratory prepared mixtures of MET and CFZ using 20 μg/mL
of CFZ as a divisor and methanol as a blank after subtraction of the constant

Figure 6. The zero order absorption spectra of MET obtained by the proposed RS method
for the analysis of laboratory prepared mixtures after multiplication by the divisor
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 69
lated using the regression equation (obtained by and 237 nm as shown in Fig. 7.The concentration
plotting the absorbance values of the D0 spectra of MET was calculated using the regression equa-
of MET at 236 nm against its concentration) with tion obtained from the linear relationship between
optimum accuracy and precision. the amplitude difference at the two selected wave-
lengths and the concentration of the drug.
Ratio difference method (RD)
This method was introduced by Lofty et Hegazy Derivative ratio method (DR)
24
. The basic principle of this method is that the According to the theory of derivative ratio
amplitude difference between two points on the method 28, the absorption spectrum of the mixture
ratio spectra of a mixture is directly proportional is divided by the absorption spectrum of a stan-
to the concentration of the component of interest dard solution of one of the components, and the
with independence of the interfering component. first derivative of the ratio spectrum (1DR) is ob-
It eliminates derivative steps and therefore the tained. The advantages of the DR spectra method
signal to noise ratio is enhanced 25, 26, 27. The only over the zero-crossing derivative method is that
requirement for determination of MET by RD easy measurement on the separate peaks can be
method was the contribution of the two compo- carried out and there is no need to work only at
nents at the two selected wavelengths (215 nm zero-crossing point as in case of derivative meth-
and 237 nm) where the ratio spectra of the inter- ods. It was difficult to measure MET using dif-
fering component (CFZ) showed the same am- ferent orders of derivative spectrophotometry in
plitudes (constant) whereas ratio spectra of the terms of sensitivity and recovery percent. Thus
component of interest (MET) showed significant the DR spectrophotometry was used instead. MET
difference in these two amplitude values at these is determined by dividing its D0 absorption spec-
two selected wavelengths with concentration. trum by a suitable divisor of CFZ (20 μg/ml) which
RD method requires critical choice of a suit- gave minimum noise, smoother ratio spectra and
able divisor as well as the wavelengths at which maximum sensitivity. The first derivative of the
amplitude values are recorded. The chosen wave- obtained spectrum is recorded (Fig. 8 and Fig. 9)
lengths should be checked to ensure that they ex- and the concentration of MET is calculated using
hibit significant difference in absorbance and show the regression equation resulted from the relation-
good linearity. Different concentrations of divisor ship between the Amplitude of 1DR spectrum at
(CFZ) and the wavelengths were tested and the 245 nm and the concentration of the drug.
best results were obtained by dividing the mixture
spectrum by 20 μg/ml CFZ as a divisor and mea- Optimization of TLC densitometric method
suring the amplitude difference between 215 nm This method has offered a simple way to sepa-

Figure 7. Ratio spectra of MET (1-20 μg/mL) using 20 μg/mL of CFZ as a divisor and
methanol as a blank showing the two selected wavelengths (237 nm and 215 nm)
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 70

Figure 8. First derivative of ratio spectra of MET (1-20 μg/mL)


using 20 μg/mL of CFZ as a divisor and methanol as a blank

Figure 9. First derivative of ratio spectra of different laboratory prepared mixtures


of MET and CFZ using 20 μg/mL of CFZ as a divisor and methanol as a blank
rate and quantify MET and CFZ directly on TLC Rf values (0.55 ± 0.05 for CFZ, 0.21 ± 0.03 for
plates by measuring the optical density of the sepa- MET) and without tailing of the separated bands
rated bands. Different mobile phases were tried (Fig. 10).
to improve the separation of the studied drugs, The optimum band width was 5 mm and the
initially a mixture of methanol, Methylene chlo- inter-space between bands was 9 mm. Slit di-
ride and ammonia was used but development of mensions of the scanning light beam should en-
mobile phase was time consuming (4 hours). By sure complete coverage of band dimensions on
addition of toluene, development time became ac- the scanned track without interference of adja-
ceptable but Rf for MET was less than 0.1. By cent bands. 4 mm × 0.45 mm proved to be the
substituting ammonia with formic acid, Rf for CFZ slit dimension of choice which provides highest
was very close to solvent front. There was no sensitivity. Detection at 236 nm for MET and at
improvement by substituting the methanol with any 290 nm for CFZ was suitable providing good
of the following solvents, ethyl acetate, isopro- sensitivity with minimum noise. The proposed
panol or acetone. Finally; sharp and symmetric TLC method has the advantages of requiring
peaks were obtained by using toluene: Methylene simple developing systems with no pH adjust-
chloride: methanol: water: glacial acetic acid in ments, additionally; several samples can be run
the volume ratio (20:15:15:1:0.25) as a mobile simultane-ously with a small amount of the mo-
phase where good separation was obtained bet- bile phase, thus offering shorter time and lower
ween the studied drugs in binary mixture with good cost for analysis.
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 71
800
AU
700

600

500

400

300

200

100

0
0.20 0.40 0.60 0.80 1.00 Rf
Figure 10. 2D diagram showing an example of separated mix of 0.3 μg/band CFZ (Rf 0.55),
5.1 μg/band MET (Rf 0.21) and dosage form by the proposed TLC densitometric method
Method validation obtained from the corresponding regression equa-
Validation of the proposed methods was per- tions. Good accuracy of the proposed methods is
formed according to ICH guidelines 29. represented by satisfactory percentage recover-
ies (mean) and small percent relative error as
Linearity shown in Table 1.
The linearity of the proposed methods was
evaluated by analyzing six concentrations of CFZ Precision
and MET in the ranges 1-30 μg/mL and 1-20 μg/ Precision of the proposed method was proved
mL for spectrophotometric methods and in the by low values of relative standard deviation as
ranges 0.3-4.5 μg/band and 0.5-5.5 μg/band for shown in Table 1.
TLC- densitometric method respectively. Each
concentration was repeated three times. The lin- Repeatability
earity of the calibration curves were validated by The repeatability (intra-day precision) was
the high value of correlation coefficients , small evaluated through replicate analysis of three dif-
values of % RSD, % Err, and residual standard ferent concentrations within the linearity range of
deviation which point out to the low scattering of pure MET and CFZ using the proposed methods.
the points around the calibration curve. The ana- Each concentration was measured three times on
lytical data of the calibration curves are summa- a single day. The relative standard deviations were
rized in Table 1. calculated as shown in Table 1.

Accuracy Reproducibility (Intermediate precision)


The accuracy of the results was checked by The previous procedures were repeated on three
applying the proposed methods for determination consecutive days for the analysis of the three cho-
of different concentrations of pure MET and CFZ sen concentrations. The relative standard devia-
within the linearity range .The concentrations were tions were calculated as shown in Table 1.
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 72
Table 1. Regression and validation parameters of the proposed
methods for determination of CFZ and MET in pure form

Spectrophotometric TLC densitometric


methods method
Parameter Direct MET CFZ MET
spectrophotometric RS RD DR
determination of CFZ

Wavelength (nm) 290 236 237-215 245 290 236


Calibration range 1-30 1-20 0.3-4.5 0.5-5.5
(μg/mL) (μg/mL) (μg/band) (μg/band)
Slope 0.04 0.09 0.53 0.04 7235 5014
Intercept 0.001 0.01 -0.04 -0.002 -207.56 -68.88
Correlation coefficient 1 0.9999 0.9999 1 0.9997 0.9997
S y/x 0.002 0.004 0.05 0.002 1.62 1.21
Mean 100.22 100.43 100.16 100.71 99.8 100.08
±SD 1.44 0.82 0.68 1.01 1.47 1.1
RSD% 1.43 0.82 0.68 1.01 1.47 1.1
% Er 0.59 0.33 0.28 0.41 0.6 0.45
LOD (μg/mL) 0.18 0.13 0.27 0.13 0.0007 0.0007
LOQ (μg/mL) 0.59 0.44 0.91 0.42 0.002 0.002
Repeatability (RSD %) a,b 0.63 0.77 0.39 0.4 0.56 0.49
Intermediate precision a,b 0.65 0.36 0.15 0.61 0.57 0.47
(RSD %)

a
Average of three experiments
b
Relative standard deviation of three concentrations of each drug (5, 10, 15 μg/mL) for spectrophotometric
methods , (1.5, 2.5, 3.5 μg/band) for TLC- densitometric method
LOQ and LOD percentages with accepted standard deviations
The limit of quantification (LOQ) was deter- were obtained in all cases. Satisfactory results
mined by establishing the lowest concentration that are shown in Table 2.
can be measured according to ICH Q2(R1) rec-
ommendations, below which the calibration graph Statistical analysis
is non linear, (LOQ=10 σ/S where S is the slope Results obtained by the proposed methods for
and σ is the standard deviation of the intercept of the determination of pure samples of CFZ and
regression line of the calibration curve). The limit MET were statistically compared to those obtained
of detection (LOD) was determined by evaluat- by the reported HPLC method 15. The values of
ing the lowest concentration of the analyte that Student’s t-test and variance ratio F-test 30 were
can be readily detected (LOD=3.3 σ/S). The re- calculated. The results show no significant dif-
sults of LOD and LOQ of the studied drug by the ferences between calculated and tabulated val-
proposed method are abridged in Table 1. ues at P = 0.05, indicating that the proposed meth-
ods are as accurate and precise as the respective
Specificity (Selectivity) reported method (Table 3).
Specificity of the methods was achieved by the
analysis of different laboratory prepared mixtures Application of the proposed methods
containing both drugs MET and CFZ in different The proposed spectrophotometric and TLC-
ratios within the linearity range. Good recovery densitometric methods were successfully applied
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 73
Table 2. Analysis of laboratory prepared mixtures
of the studied drugs by the proposed methods

Spectrophotometric methods TLC densitometric method


Conc. in CFZ MET Conc. in CFZ MET
μg/mL RS RD DR μg/band
CFZ : MET CFZ : MET

1:10 98.87 101.70 99.75 98.18 0.45:4.5 99.82 99.14


1:5 100.09 99.99 99.56 101.47 1:5 99.02 101.00
10:10 100.41 100.12 99.99 100.56 2.5:2.5 99.52 101.08
5:10 100.87 98.80 100.78 101.21 1.5:3 100.02 100.35
10:5 100.44 101.21 100.96 100.63 3:1.5 100.91 101.18
1:17* 101.61 100.08 100.49 99.81 0.3:5.1* 100.52 99.89
Mean** 100.38 100.32 100.26 100.31 Mean** 99.97 100.44
RSD % 0.90 1.02 0.57 1.19 RSD % 0.68 0.81
% ER 0.37 0.42 0.23 0.49 % ER 0.28 0.33
Variance 0.82 1.04 0.32 1.42 Variance 0.46 0.65

* Ratio present in the dosage form


** Average of three experiments

Table 3. Statistical comparison between the results obtained by the proposed methods
and reported method 15 for the determination of CFZ and MET in pure form

Item CFZ MET


290nm TLC Reported RS RD DR TLC Reported
densito- method 15 densito- method 15
metric metric

Mean 100.22 99.80 100.14 100.43 100.16 100.71 100.08 100.04


RSD % 1.44 1.47 1.24 0.82 0.68 1.01 1.10 0.69
Variance 2.07 2.16 1.55 0.68 0.47 1.03 1.20 0.48
N 6 6 6 6 6 6 6 6
t-test * 0.10 0.43 0.89 0.31 1.35 0.09
F-test ** 1.34 1.40 1.42 1.02 2.16 2.53

* The corresponding tabulated value of t -test equals to 2.23 at p = 0.05 30


** The corresponding tabulated value of F-test equals to 7.146 at p = 0.05 30
for the determination of MET and CFZ in their Conclusion
combined pharmaceutical preparation (Vokanamet The data of the results given by the proposed
tablets). The results were compared with those spectrophotometric and densitometric methods are
of a reported HPLC method 15 as shown in table indicative for good sensitivity, accuracy, simplic-
4. No significant difference between the perfor- ity and reasonable selectivity. The proposed meth-
mance of our work and the reported method re- ods are cost effective, less time consuming and
garding accuracy and Precision as indicated by do not require elaborate treatment associated with
Student’s t-test and variance ratio F-test 30 re- chromatographic methods so they can be used
spectively. for routine analysis of MET and CFZ in their bulk
Mona S. Elshahed et al., / TACL 8 (1) 2018 63 - 75 74
Table 4. Statistical comparison between the results obtained by the
proposed methods and reported method 15 for the determination
of CFZ and MET in pharmaceutical dosage form

CFZ MET
Item 290 nm TLC Reported RS RD DR TLC Reported
densito- method 15 densito- method 15
metric metric

Mean 101.43 100.60 101.28 99.15 98.88 98.95 99.76 99.28


RSD % 0.40 1.07 0.23 0.83 0.45 0.69 0.38 0.33
Variance 0.16 1.15 0.06 0.67 0.19 0.47 0.15 0.11
N 3 3 3 3 3 3 3 3
t-test * 0.56 1.07 0.25 1.27 0.75 1.69
F-test ** 2.99 20.89 6.44 1.85 4.45 1.39

* The corresponding tabulated value of t equals to 2.78 at p = 0.05 30


** The corresponding tabulated value of F equals to 39 at p = 0.05 30
and marketed dosage forms. Furthermore the pro- are also suitable and valid for application in labo-
posed methods can be easily applied in quality ratories lacking liquid chromatographic instru-
control laboratories without prior separation; they ments.

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