^{1,2}Department of Pharmaceutical Technology, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon
Email: malak.albathish@gmail.com
Received: 20 Aug 2019, Revised and Accepted: 12 Dec 2019
ABSTRAC
Objective: To develop and validate novel more sensitive analytical methods for the concurrent quantification of metformincanagliflozin and metformingliclazide in their bulk forms and in their pharmaceutical preparations.
Methods: Two methods were developed based on several chemometric assisted spectrophotometric methods and a ReversedPhase HighPerformance Liquid Chromatography (RPHPLC). The first method applies different spectrophotometric chemometric assisted methods, including ratio difference, derivative ratio and extended ratio subtraction method, while the second method describes a RPHPLC separation of metformin hydrochloridecanagliflozin and metformin hydrochloridegliclazide binary mixtures using a C18 column with a mobile phase consisting of acetonitrile: potassium dihydrogen phosphate (adjusted to pH 3) with sodium lauryl sulphate as additive in the ratio of 30:70 (%v/v) in isocratic elution mode at 1 ml/min.
Results: The proposed methods were able to quantify each of the studied drugs in their binary mixtures with high percentage recoveries in both methods. The spectrophotometric methods were able to quantify each of metformin, canagliflozin and gliclazide in the ranges of 2.020.0 μg/ml, 1.540.0 μg/ml and 2.030.0 μg/ml, respectively. The RPHPLC method produced wellresolved peaks at a retention time of 3.92, 6.92 and 9.10 min in the concentration ranges of 50.0300.0 μg/ml, 5.050.0 μg/ml and 10.0100.0 μg/ml for metformin, canagliflozin and gliclazide, respectively. The proposed methods were optimized and validated in accordance to the International Conference of Harmonisation (ICH) guidelines in terms of linearity, LOD, LOQ, precision and accuracy.
Conclusion: The developed methods were found to be sensitive and reproducible methods for the simultaneous determination of antidiabetic binary mixtures; metformin hydrochloridecanagliflozin and metformin hydrochloridegliclazide. And thus were successfully employed for the quality control analysis of the pharmaceutical formulations of the studied binary mixtures.
Keywords: Binary mixtures, MetforminCanagliflozin, MetforminGliclazide, Spectrophotometric chemometric assisted methods, RPHPLC
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an openaccess article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i2.35415. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps
Diabetes is a lifelong progressive disease characterized by high levels of blood glucose [1]. Currently, metformin is being used as firstline therapy in the treatment of diabetes mellitus type 2. However, for most patients, glycemic control is not sufficiently established by this monotherapy, leading to the requirement for combination therapy. Metformin hydrochlorideCanagliflozin is a recently introduced coformulation in the market whereas metformin hydrochloridegliclazide is one of the most used coformulation in the current practice.
Metformin (MTF) is official in both the British Pharmacopeia (BP) [2] and United State Pharmacopeia (USP) [3], gliclazide (GLIC) is official in the BP, whereas Canagliflozin (CANA) is still a novel drug in the market, and no official method is yet available in any pharmacopoeia.
Literature survey has revealed that each of the metformin hydrochloride, canagliflozin and gliclazide were determined individually as a single component in bulk, pharmaceutical formulations and biological fluids by several analytical methods. Metformin hydrochloride has been determined simultaneously with canagliflozin using RPHPLC [47] and spectrophotometry [8]. In presence of gliclazide, metformin hydrochloride has been determined by High Performance Thin Layer Chromatography (HPTLC) [9, 10], Liquid ChromatographyMass Spectrometry (LCMS) [11, 12], spectrophotometric methods [1317], and HPLC with Ultraviolet (UV) detection [1826].
Fig. 1: Structures of (A) Metformin Hydrochloride, (B) Canagliflozin, (C) Gliclazide
Spectrophotometric methods present a good option in the analysis of multicomponent pharmaceutical mixtures based on the availability, simplicity and low cost. Classical UV spectral measurement use is often limited due to the overlapping of spectral peaks unless prior separation and extraction are employed. Several UV spectrophotometric chemometric methods are used in the simultaneous analysis for resolving mixtures based on mathematical processing and manipulations of the absorption spectra [2734], offering sometimes an alternative to chromatographic methods.
This work aims to evolve new, yet more sensitive methods for concurrent quantification of MTFCANA and MTFGLIC in their bulk forms and in their pharmaceutical preparations. The idea for the comparative study using two different techniques has emerged from the need to find the most suitable analytical method in short time to be applicable in quality control labs giving the analyst the choice of the preferable detector based on its availability. The methods are based on several chemometric methods [27] including ratio difference, derivative ratio, and extended ratio subtraction methods, also a RPHPLC. Methods have been developed and followed by full validation for each method.
Chemicals and materials
Metformin hydrochloride was certified to contain 99.7% (Merck Sante, France), Canagliflozin was certified to contain 99.8% (Janssen Cilag, USA), and Gliclazide was certified to contain 99.3% (Servier, France).
Vokanamet^{®} tablets (Janssen Cilag, USA), labelled to contain 850 mg metformin hydrochloride and 50 mg canagliflozin. Orbizide M Forte^{®} tablets (Pharmaline, Lebanon) labelled to contain 500 mg metformin hydrochloride and 40 mg gliclazide.
All chemicals and solvents used were of HPLC grade; Methanol (SIGMAALDRICH, CHROMASOLV^{®} FOR HPLC), acetonitrile (SIGMAALDRICH CHROMASOLV^{®}FOR HPLC>99.9%), KH_{2}PO_{4} (Merck KGaA), deionized water was produced inhouse.
Instrumentation
Jasco V730 double beam UVVis Spectrophotometer interfaced to a computer programmed with Jasco Spectra Manager software was used for spectrophotometric measurements, spectral acquisition and elaboration. A pair of 1 cm quartz cells were used to measure the absorption spectra.
The RPHPLC separation was performed on an HPLC system; Jasco PU2089 series (Japan) equipped with quaternary pump, diode array detector, and a manual injector which uses a Rheodyne port sample injection valve fitted with 20 µl sample loop. Liquid separations are performed on Lichrospher 100 RP C_{18} analytical column (250 mm x 4.6 mm x 5μm). A Glassco diaphragm Vacuuum pump was used to degas and filter the mobile phase by passing through 0.5 μm pore size membrane filter at a pumping speed of 30 L/min. A 0.2 μm Minisart SRP 15 (Sartorius Stedim) syringe driven disposable filters was used to filter the samples.
Preparation of standard stock solutions
Stock solutions of MTF, CANA and GLIC standards having the concentrations of 1000 µg/ml, 1000 µg/ml, and 600 µg/ml, respectively, were prepared using methanol as a solvent.
Chemometric assisted spectrophotometric methods
Preparation of working standard solutions
Aliquots of the standard stock solutions of each drug were separately diluted with methanol to obtain working standard solutions having the concentrations of 200 µg/ml for MTF, 100 µg/ml for CANA and 60 µg/ml for GLIC.
Calibration graphs
Calibration graphs were prepared from these working standard solutions by diluting with methanol to achieve the concentration range of 220 µg/ml for MTF, 1.540 µg/ml for CANA and 230 µg/ml for GLIC. The absorption spectra were recorded at 1nm interval in the range of 200300 nm for GLIC and 200400 nm for MTF and CANA, against methanol as blank.
Synthetic mixtures
Aliquots of the working standard solutions of MTF and CANA or MTF and GLIC were transferred into a series of 10ml volumetric flasks and completed to volume with methanol to give synthetic mixtures containing 20:5 µg/ml and 15:2.5 µg/ml, respectively. The absorption spectrum for each mixture was recorded at 1nm interval in the range of 200400 nm against methanol as blank and then analysed by the developed method to assay the quantity of both MTFCANA, or MTFGLIC in the synthetic mixtures.
RPHPLC method
Chromatographic conditions
The chromatographic analysis was performed with isocratic elution using C_{18} column at a detection wavelength of 229 nm. The mobile phase used for the chromatographic separation was prepared by adding HPLC grade acetonitrile to the aqueous phase in the ratio of 30:70 %v/v. The mobile phase was pumped through the column at a flow rate of 1 ml/min and injection volume of 20 µl. Separation was carried out at ambient temperature.
The aqueous phase was prepared by weighing 1.36 g of potassium dihydrogen phosphate (KH_{2}PO_{4}) and 2.88 g of sodium lauryl sulphate and dissolving them in 1 L HPLC grade water, then adjusting its pH to a value of 3 with orthophosphoric acid.
The mobile phase was filtered and degassed for 30 min using an ultrasonic sonicator.
Calibration graphs
Calibration graphs were prepared from the standard stock solutions by diluting with methanol to achieve the concentration range of 50300 µg/ml for MTF, 550 µg/ml for CANA and 10100 µg/ml for GLIC. The above solutions were filtered and 20 µl portion of these solutions were injected and chromatographed. The standard calibration graphs were prepared by plotting the peak area values of each drug against the corresponding concentrations.
Synthetic mixtures
Aliquots of standard stock solutions of MTF, CANA or GLIC were transferred into a series of 10ml volumetric flasks and completed to volume with methanol to give synthetic mixtures in the ratios stated in table 7. The above solutions were filtered and 20 µl portion of these solutions were injected in triplicates on three different days and chromatographed. The peak areas for each drug were used to acquire the corresponding concentration by referring to its calibration graph.
Pharmaceutical application
Ten tablets of Orbizide^{®} or Vokanamet^{®} were accurately weighed and finely powdered. Powder amount equivalent to one tablet of each, was accurately weighed, transferred to a 100mL volumetric flask and diluted to volume with HPLC grade methanol. The obtained solutions were then filtered using Whatmann No. 41 filter paper. An appropriate volume of these solutions was transferred to 10ml volumetric flask, diluted to the mark with the same solvent.
The absorption spectra of the obtained solutions were recorded at 1nm interval in the range of 200400 nm against methanol as a blank and then analyzed by the developed chemometric assisted spectrophotometric methods to assay the quantity of each drug in their coformulated pharmaceutical tablets.
In RPHPLC method, the solutions were filtered and 20 µl portion of these solutions were injected in five replicates and chromatographed. The peak areas for each drug were used to acquire the corresponding concentration by referring to its calibration graph.
Method validation
Under the optimized experimental conditions, the developed methods were validated according to the procedures described in ICH guidelines for the following parameters: Linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy [35].
Linearity, concentration range, limit of detection and limit of quantitation
Linearity was determined in the concentration range of 220 µg/ml for MTF, 1.540 µg/ml for CANA and 230 µg/ml for GLIC for the chemometric assisted spectrophotometric methods, and 50300 µg/ml for MTF, 550 µg/ml for CANA and 10100 µg/ml for GLIC for the RPHPLC method. The amount of MTF, CANA and GLIC present in the sample was computed from the corresponding calibration curve.
LOD and LOQ were calculated according to the ICH guidelines by using the following formula [35]:
Where “S_{y/x}” is the standard deviation of the regression line/residuals and “b” is the slope of the calibration curve.
Accuracy and precision
In order to assess the accuracy and precision of the proposed methods, standard solutions containing MTF, GLIC or CANA and synthetic mixtures with different ratios of MTFCANA and MTFGLIC were analysed. The precision of the proposed methods was assessed by intraday and interday variation studies using three concentrations of each for five times. During intraday studies, five sample solutions of each concentration were analysed on the same day whereas interday studies were determined by analysing five sample solutions of each concentration for three consecutive days. The accuracy of the proposed methods was assessed by analysing synthetic mixtures with different ratios. Afterwards, the mean percentage recovery and percentage relative standard deviation were calculated.
Chemometric assisted spectrophotometric methods
The spectral overlapping between the absorption spectra of MTF and CANA (fig. 2) or MTF and GLIC (fig. 3) exhibit significant interference imposed by each drug while determining the other which limits the use of conventional UV spectrophotometry for their simultaneous determination. Therefore, simultaneous determination of these drugs in binary mixture or in coformulated pharmaceutical formulations requires mathematical manipulation of the absorption data to omit the interference. Accordingly, three simple spectrophotometric methods; namely ratio difference, derivative ratio, and extended ratio subtraction methods were developed for the determination of MTFCANA and MTFGLIC in their binary mixtures.
Fig. 2: Overlain absorption spectra of MTF (16 µg/ml), CANA (5µg/ml) and their binary mixture
Fig. 3: Overlain absorption spectra of MTF (15 µg/ml), GLIC (3.5µg/ml) and their binary mixture
Ratio difference (ΔP)
As shown in fig. 2 and fig. 3, the absorption spectra of the binary mixtures composed of MTFCANA or MTFGLIC show high degree of overlap. So, to overcome this mutual interference, the absorbance ratio; a onestep correction method was adopted.
For method optimization, two factors were considered: the effect of divisor concentration for each drug and the choice of the wavelength. Selection was based on minimum noise, maximum sensitivity, high accuracy and precision. When the concentration of the divisor was varied, the ratio spectra proportionally varied with no change in the position of the peaks and troughs [27, 29].
MTF was determined in MTFCANA binary mixture, where the recorded spectra of standard MTF solutions and that of its mixtures with CANA were divided by the absorption spectrum of CANA having the concentration of 2 µg/ml. The ratio spectra of different MTF standard solutions were obtained. The peak to trough amplitudes between 233211 nm on the established ratio spectra were proportional to MTF concentration. Consequently, the regression equation was derived (fig. 4).
Fig. 4: Ratio spectra of MTF (16 µg/ml), CANA (5µg/ml) and their binary mixture where the divisor is CANA (2µg/ml)
CANA was determined in MTFCANA binary mixture, where the recorded spectra of standard CANA solutions and that of its mixtures with MTF were divided by the absorption spectrum of MTF having the concentration of 18 µg/ml. The ratio spectra of different CANA standard solutions were obtained. The peak to trough amplitudes between 278295 nm on the established ratio spectra were proportional to CANA concentration. Consequently, the regression equation was derived (fig. 5).
Fig. 5: Ratio spectra of MTF (16 µg/ml), CANA (5µg/ml) and their binary mixture where the divisor is MTF (18µg/ml)
MTF was determined in MTFGLIC binary mixture, where the recorded spectra of standard MTF solutions and that of its mixtures with GLIC were divided by the absorption spectrum of GLIC having the concentration of 4.5µg/ml. The ratio spectra of different MTF standard solutions were obtained. The peak to trough amplitudes between 248222 nm on the established ratio spectra were proportional to MTF concentration. Consequently, the regression equation was derived (fig. 6).
Fig. 6: Ratio spectra of MTF (15 µg/ml), GLIC (3.5µg/ml) and their binary mixture where the divisor is GLIC (4.5 µg/ml)
GLIC was determined in MTFGLIC binary mixture, where the recorded spectra of standard GLIC solutions and that of its mixtures with MTF were divided by the absorption spectrum of MTF having the concentration of 18 µg/ml. The ratio spectra of different GLIC standard solutions were obtained. The peak to trough amplitudes between 223248 nm on the established ratio spectra were proportional to GLIC concentration. Consequently, the regression equation was derived (fig. 7).
Fig. 7: Ratio spectra of MTF (15 µg/ml), GLIC (3.5µg/ml) and their binary mixture where the divisor is MTF (18 µg/ml)
First derivative ratio D_{1}R
In this method, two parameters were studied and optimized to obtain good selectivity and high sensitivity: wavelength interval (Δλ) and divisor concentration [27, 3133].
The influence of Δλ was investigated to obtain the optimum wavelength intervals in terms of maximum resolution and sensitivity whereby the Δλ value affects the shape and the position of the peaks to be analysed as well as the zerocrossing point of the other component in the mixture. The D_{1} curves were traced at 8 nm interval for MTFCANA and at 6 nm for MTFGLIC, where other Δλ values gave poor resolution.
The concentration of MTF was determined at 244 nm for both binary mixtures in the corresponding D_{1} curves obtained from the ratio spectra where the divisors were CANA (2 µg/ml) and GLIC (4.5 µg/ml), respectively. The concentration in the mixture was derived from the calibration curve of the D_{1}R of standard MTF (fig. 8 and fig. 9).
Fig. 8: D_{1} of the ratio spectra of MTF (16 µg/ml), CANA (5µg/ml) and their binary mixture where the divisor is CANA (2µg/ml)
Fig. 9: D_{1} of the ratio spectra of MTF (15 µg/ml), GLIC (3.5µg/ml) and their binary mixture where the divisor is GLIC (4.5 µg/ml)
The concentration of CANA was determined at 320 nm in MTFCANA binary mixture in the corresponding D_{1} curves obtained from the ratio spectra where the divisor was MTF (18 µg/ml). The concentration in the mixture was derived from the calibration curve of the D_{1}R of standard CANA (fig. 10).
The concentration of GLIC was determined at 240 nm in MTFGLIC binary mixture in the corresponding D_{1} curves obtained from the ratio spectra where the divisor was MTF (18 µg/ml). The concentration in the mixture was derived from the calibration curve of the D_{1}R of standard GLIC (fig. 11).
Fig. 10: D_{1} of the ratio spectra of MTF (16 µg/ml), CANA (5µg/ml) and their binary mixture where the divisor is MTF (18µg/ml)
Fig. 11D_{1} of the Ratio spectra of MTF (15 µg/ml), GLIC (3.5µg/ml) and their binary mixture where the divisor is MTF (18 µg/ml)
Extended ratio subtraction method (EXRSM)
The proposed method is based on that MTF has a more extended spectrum than CANA and GLIC as shown in fig. 2 and fig. 3.
This method begins by applying the Ratio subtraction method (RSM). It is done by dividing the zero order absorption spectra of the studied mixtures (A) by an absorption spectrum of carefully chosen concentration (A °). Different concentrations of CANA and GLIC were tested, it was found that 2 µg/ml CANA or 4.5 µg/ml GLIC gave minimum noise, smoother ratio spectra and maximum sensitivity. Dividing the mixture absorption spectra by 2 µg/ml standard CANA or 4.5 µg/ml standard GLIC, produces new spectra, that represent (A_{CANA}/A_{CANA} °+A_{CANA}/A_{CANA} °) or (A_{GLIC}/A_{GLIC} °+A_{MTF}/A_{GLIC} °), where (A_{CANA}/A_{CANA} °) or (A_{GLIC}/A_{GLIC} °) have constant values. Then, subtraction of these constant values in the plateau region (270300 nm) in MTFCANA binary mixture or (265280 nm) in MTFGLIC binary mixture, followed by multiplication of the obtained spectra by the divisor spectrum, the original absorption spectrum of MTF is obtained and can be used directly for MTF determination at 237 nm from the corresponding regression equation [27,34].
(A_{MTF}+A_{CANA})/A_{CANA} °
A_{MTF}/A_{CANA} °_{+}A_{CANA}/A_{CANA} ° = A_{MTF}/A_{CANA} °_{+}constant.
A_{MTF}/A_{CANA} °+constantconstant
A_{MTF}/A_{CANA} ° * A_{CANA} °
A_{MTF}
(A_{MTF}+A_{GLIC})/A_{GLIC} °
A_{MTF}/A_{GLIC} °_{+}A_{GLIC}/A_{GLIC} ° = A_{MTF}/A_{GLIC} °_{+}constant.
A_{MTF}/A_{GLIC} °+constantconstant
A_{MTF}/A_{GLIC} ° * A_{GLIC} °
A_{MTF}
The determination of CANA or GLIC could be done by the EXRSM. The spectrum of MTF obtained from the RSM method and the absorption spectra of the mixtures, are divided by the absorption spectrum of standard MTF having the concentration of 18 µg/ml; producing a new spectrum that represents (A_{CANA}/A_{MTF} °+A_{CANA}/A_{MTF} °) or (A_{GLIC}/A_{MTF} °+A_{MTF}/A_{MTF} °) and the constant (A_{MTF}/A_{MTF} °) in the plateau region (210250 nm). Subtracting the above produced constant spectrum (A_{MTF}/A_{MTF} °) from (A_{CANA}/A_{MTF} °+A_{CANA}/A_{MTF} °) or (A_{GLIC}/A_{MTF} °+A_{MTF}/A_{MTF} °) followed by multiplication by the divisor A_{MET} °_{,} will generate new spectra. These spectra could be used for the direct determination of CANA at 290 nm and GLIC at 228 nm and calculate their concentrations from the corresponding regression equation.
(A_{MTF}+A_{CANA})/A_{MTF} °
A_{MTF}/A_{MTF} °_{+}A_{CANA}/A_{MTF} ° = A_{CANA}/A_{MTF} °_{+}constant.
A_{CANA}/A_{MTF} °+constantconstant
A_{CANA}/A_{MTF} ° * A_{MTF} °
A_{CANA}
(A_{MTF}+A_{GLIC})/A_{MTF} °
A_{MTF}/A_{MTF} °_{+}A_{GLIC}/A_{MTF} ° = A_{GLIC}/A_{MTF} °_{+}constant.
A_{GLIC}/A_{MTF} °+constantconstant
A_{GLIC}/A_{MTF} ° * A_{MTF} °
A_{GLIC}
RPHPLC method
RPHPLC method was developed to provide an adequate method for the simultaneous determination of MTFCANA and MTFGLIC in the synthetic mixtures and in pharmaceutical formulations. The most important attribute is to achieve sufficient resolution with acceptable peak symmetry in a RPHPLC method at reasonable time. So, preliminary investigations aimed to maximize the resolution and sensitivity of the method. The parameters were assessed and optimized as stated in Table 1.
Several mobile phases were tried using various ratios of different aqueous and organic modifiers. The best separation was obtained using a mobile phase composed of acetonitrile: phosphate buffer (70:30 %v/v) at pH 3 with 0.01 M sodium lauryl sulfate. Separation was done at wavelength of 229 nm and at a flow rate of 1 ml/min. Fig. 12 and 13, show the separation of MTF at 3.9 min, CANA at 9.1 min, and GLIC at 6.9 min.
Table 1: Optimized chromatographic conditions for the separation of MTFCANA and MTFGLIC
Parameter  Condition 
Mobile Phase  Acetonitrile: phosphate buffer (70:30) at pH 3 with 0.01 M sodium lauryl sulfate 
Stationary Phase  C18 column (250 mm x 4.6 mm x 5μm) 
Flow rate (ml/min)  1 
Run time (min)  10 
Volume of injection (μl)  20 
Detection wavelength (nm)  229 
Table 2: HPLC system suitability parameters for the determination of MTFCANA and MTFGLIC using the proposed method
Retention time t_{R} 
Capacity factor k’ 
Resolution R_{s} 
Column efficiency N 
Tailing factor T 
Selectivity α 

Metformin  3.92  1.04    2230  1.407   
Canagliflozin  6.917  2.60  5.5  3265  1.303  2.5 
Gliclazide  9.10  3.74  12.3  3702  1.244  3.60 
Fig. 12: HPLC chromatogram of a 20 µl injection of a standard mixture of 250 µg/ml MTF and 50 µg/ml CANA using the optimized chromatographic conditions
Fig. 13: HPLC chromatogram of a 20 µl injection of a standard mixture of 250 µg/ml MTF and 40 µg/ml GLIC using the optimized chromatographic conditions
Table 3: Assay parameters for the determination of MTFCANA and MTFGLIC in presence of each other using the applied spectrophotometric methods
Metformin  
Parameters  A  ΔP  D_{1}R  
Conc. Range (µg/ml)  2.020.0  
λ or λ _{range(nm)}  237  228  248222  233211  244  244 
Δλ _{(nm)}  6  8  
r  0.9993  0.9993  0.9993  0.9994  0.9996  0.9989 
S _{y/x}  0.0149  0.0119  0.29543  2.68213  0.02027  0.23625 
a (intercept)  0.0262  0.002  0.19663  1.14748  0.02569  0.16933 
b (slope)  0.0905  0.0687  2.13804  15.4521  0.18266  1.02883 
S_{a}  0.0103  0.0083  0.31418  1.65844  0.02156  0.13769 
S_{b}  0.0008  0.0007  0.02279  0.13509  0.00156  0.01162 
LOD (µg/ml)  0.54  0.57  0.46  0.57  0.37  0.76 
LOQ (µg/ml)  1.65  1.73  1.38  1.74  1.11  2.30 
Gliclazide  
Parameters  A  ΔP  D_{1}R  
Conc. Range (µg/ml)  2.030.0  
λ or λ _{range(nm)}  228  237  223248  240  
Δλ _{(nm)}  
r  0.9998  0.9983  0.9997  0.9996  
S _{y/x}  0.00732  0.00407  0.00711  0.00046  
a (intercept)  0.00717  0.00537  0.00092  0.00069  
b (slope)  0.04354  0.02551  0.03703  0.00204  
S_{a}  0.00456  0.00253  0.00381  0.00025  
S_{b}  0.00027  0.00015  0.00024  0.00002  
LOD (µg/ml)  0.55  0.53  0.63  0.75  
LOQ (µg/ml)  1.68  1.60  1.92  2.28  
Canagliflozin  
Parameters  A  ΔP  D_{1}R  
Conc. Range (µg/ml)  1.540.0  
λ or λ _{range(nm)}  290  237  278295  320  
Δλ _{(nm)}  8  
r  0.9999  0.9987  0.9998  0.9999  
S _{y/x}  0.00560  0.00564  0.71436  0.01384  
a (intercept)  0.00169  0.00858  0.70414  0.02059  
b (slope)  0.04180  0.00958  2.96632  0.36026  
S_{a}  0.00317  0.00320  0.31916  0.00618  
S_{b}  0.00015  0.00015  0.01739  0.00034  
LOD (µg/ml)  0.44  1.94  0.79  0.13  
LOQ (µg/ml)  1.34  5.89  2.41  0.38 
Table 4: Assay parameters for the determination of MTF, GLIC and CANA using the proposed RPHPLC method
Parameter  MTF  GLIC  CANA 
Concentration range (µg/ml)  50300  10100  550 
Regression equation  
Intercept (a)  1680665.31  102139  44163.13 
Slope (b)  99378.45  39954.37  15229.25 
Correlation coefficient (r)  0.998  0.999  0.999 
S_{a}  459559.95  33158.89  1645.24 
S_{b}  2360.08  484.32  55.82 
S_{y/x}  493646.57  27074.12  1966.22 
LOD (µg/ml)  16.39  2.24  0.43 
LOQ (µg/ml)  49.67  6.78  1.29 
System suitability
In accordance to Food and Drug Administration (FDA) guidance, system suitability tests are of the essentials in any liquid chromatographic method [36]. Under the optimized chromatographic conditions, system suitability parameters including capacity factor (k’), resolution (R_{s}), column efficiency (N), tailing factor (T), repeatability in terms of Relative Standard Deviation (RSD), and selectivity (α) were performed and listed in table 2. All were satisfactory pointing out the selectivity and efficiency of the method for separation of the binary mixtures MTFCANA and MTFGLIC.
Method validation
Under the optimized experimental conditions, the developed methods were validated according to the procedures described in ICH guidelines [35].
Linearity, concentration range, limit of detection and limit of quantitation
Under the described experimental conditions, linearity was established over the concentration ranges stated in Tables 3 and 4; the graphs were obtained by plotting the ratio difference (ΔP), and D_{1} of the ratio spectra or the peak area each versus concentration of MTF, CANA or GLIC in case of chemometric methods and RPHPLC method, respectively.
The values of the correlation coefficient (r), intercept (a), slope (b) and standard deviation of residuals (S_{y/x}) showed good linearity of the calibration graphs and the conformity with Beer’s law. The low LOD and LOQ values obtained confirm the sensitivity of the proposed methods (tables 3 and 4).
Accuracy and precision
The percentage recoveries were obtained for each mixture by applying the ΔP, D_{1}R, RSM and RPHPLC methods (Tables 5, 6 and 7). The spectral interferences in the determination of MTFCANA or MTFGLIC in the presence of each other using the proposed methods were corrected. Good percentage recoveries were obtained. These applied methods indicate their potential for correction of spectral interferences in the determination of MTFCANA and MTFGLIC binary mixtures.
Table 5: Intraday and interday precision for the simultaneous determination of MTFCANA, and MTFGLIC in presence of each other in synthetic mixture using the proposed spectrophotometric methods
Analytical Method  MTF: GLIC µg/ml 
Intraday precision, mean recovery±SD %RSD, %error 
Interday precision, mean recovery±SD %RSD, %error 

MTF  GLIC  MTF  GLIC  
ΔP  15:2.5  99.32±1.15 1.16 0.68 
98.99±0.37 0.38 1.01 
99.10±1.44 1.45 0.90 
99.04±4.47 4.52 0.96 
D_{1}R  15:2.5  99.51±0.88 0.88 0.49 
100.39±0.35 0.35 0.39 
99.34±1.14 1.15 0.66 
100.39±1.21 1.20 0.39 
RSM  15:2.5  99.57±0.29 0.30 0.43 
100.38±0.75 0.75 0.38 
99.51±1.94 1.95 0.49 
100.57±2.19 2.18 0.57 
Analytical Method  MTF: CANA µg/ml 
Intraday precision, mean recovery±SD %RSD, %error 
Interday precision, mean recovery±SD %RSD, %error 

MTF  CANA  MTF  CANA  
ΔP  20:5  101.28±0.07 0.07 1.28 
100.73±1.87 1.86 0.73 
101.35±0.31 0.31 1.35 
99.34±2.12 2.13 0.66 
D1R  20:5  100.03±0.29 0.29 0.03 
100.95±1.01 1.00 0.95 
100.34±0.45 0.45 0.34 
101.32±1.65 1.63 1.32 
RSM  20:5  100.77±0.11 0.11 0.77 
104.65±1.71 1.63 4.65 
100.88±0.36 0.36 0.88 
103.06±2.08 2.01 3.06 
mean±SD for five determinations, Relative standard deviation, Relative error
Method application
The validated spectrophotometric chemometric and RPHPLC methods were successfully applied for the determination of the binary mixtures MTFCANA and MTFGLIC in their pharmaceutical formulations Orbizide^{®} and Vokanamet^{®}. High percentage recoveries were obtained with low % RSD which indicate high accuracy and precision in the determination of the studied mixtures in their commercial tablets without the interference of the common excipients in the determination. The results obtained are summarized in table 8 and compared statistically by means of Student’s ttest for accuracy and Ftest for precision at 95% confidence level.
Table 6: System accuracy and precision data for the determination of MTF, GLIC and CANA using the proposed RPHPLC method
t_{R}  Amount taken(µg/ml)  Amount found(µg/ml)  ^{a}% RSD  ^{b}% Recovery  
MTF  3.92±0.02  100  98.82  1.24  98.82 
200  198.46  1.07  99.23  
250  245.38  0.44  98.15  
GLIC  9.10±0.33  25  25.05  1.22  100.20 
40  40.03  0.90  100.07  
100  100.42  0.40  100.42  
CANA  6.917±0.12  5  4.93  1.33  98.60 
10  10.12  1.1  101.20  
50  49.99  1.73  99.98 
^{a}Relative standard deviation, ^{b}Mean for five determinations
Table 7: Accuracy and precision data for the simultaneous determination of MTFGLIC and MTFCANA in synthetic mixtures using the proposed RPHPLC method
Mixture  Nominal value (µg/ml)  Statistic parameters, ^{a}Mean recovery±SD, ^{b}%RSD, ^{c}%Error  
Interday  Intraday  
MTF: GLIC  MTF  GLIC  MTF  GLIC  MTF  GLIC 
250  15  100.36±1.91 1.90 0.36 
101.53±0.31 0.31 1.53 
99.01±1.09 1.10 0.99 
101.31±0.99 0.98 1.31 

250  30  101.10±1.64 1.62 1.10 
99.64±0.12 0.12 0.35 
99.94±1.95 1.96 0.06 
99.72±1.68 1.68 0.28 

250  40  101.76±1.05 0.99 1.76 
100.82±1.69 1.68 0.82 
101.05±1.09 1.08 1.05 
99.62±1.88 1.89 0.38 

MTF: CANA  MTF  CANA  MTF  CANA  MTF  CANA 
250  10  99.58±1.09 1.10 0.42 
99.59±1.37 1.38 0.41 
100.36±1.73 1.72 0.36 
100.57±1.68 1.67 0.57 

250  15  99.76±0.18 0.18 0.24 
100.53±0.41 0.41 0.53 
99.63±0.61 0.61 0.37 
100.24±0.79 0.79 0.24 

250  45  100.52±0.34 0.34 0.52 
99.96±0.60 0.60 0.04 
100.28±0.36 0.36 0.28 
99.54±0.89 0.90 0.46 
^{a}mean±SD for three determinations, ^{b}Relative standard deviation, ^{c}Relative error
Table 8: Assay results for MTFGLIC, and MTFCANA in commercial tablets using the proposed spectrophotometric methods
Drug  ^{a}Mean recovery±SD, ^{b}%RSD, ^{c}%Error  
Analytical method  ΔP  
Orbizide^{®}  MTF **ttest **Ftest 
99.30±2.02 2.04 0.70 0.234635 0.498552 
GLIC **ttest **Ftest 
100.70±3.56 3.54 0.70 0.349676 0.404388 

Vokanamet^{®}  MTF **ttest **Ftest 
99.18±0.20 0.20 0.82 0.000823 0.074935 
CANA **ttest **Ftest 
99.70±1.71 1.71 0.30 0.126203 0.092205 
^{a}mean±SD for five determinations, ^{b}Relative standard deviation, ^{c}Relative error, ^{**}Theoretical values of tand Ftest at P=0.05 are 2.13 and 5.05 respectively
In conclusion, the present work represents an approach for the assay of the MTFCANA and MTFGLIC antidiabetic binary mixtures. The developed chemometric methods depend on the absorption spectra of the drugs. The use of ratio spectra can magnify the prediction ability of the usual spectrophotometric techniques, where the treatment of absorbance ratio generates signals for the mixtures depending only on one analyte and eliminating the interfering effect of other analytes in the mixture. Such methods are considered as highly sensitive, low cost green analytical techniques, where most of the work is done in front of the computer, thus it applies neither sophisticated instrument nor prior separation steps. Accordingly, such methods can be employed for the quality control of these drugs in pharmaceutical companies. Although the developed RPHPLC is not more sensitive than the applied chemometric methods, but it can give an advantage for future plans for the analysis of the same mixtures in biological fluids.
Nil
Malak Y. Al Bathish designed and performed the experiments and the measurements. Azza A. Gazy was involved in planning and supervised the work. Malak Y. Al Bathish and Azza A. Gazy processed the experimental data along with the calculations, designed the fig. and interpreted the results. Malak Y. Al Bathish, Azza A. Gazy and Marwa K. El Jamal revised the results and drafted the manuscript.
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