Journal Pre-proof

HPTLC-densitometric analysis of selected antidiabetic drugs in

presence of their degradation products

Hytham M. Ahmed Analyzed and interpreted the data ,

Mahmoud A. Omar Analyzed and interpreted the data ,
Hany A. Batakoushy Performed the experimentsContributed reagentsMaterialsAnalysis tools or data ,
Mohamed A. Abdel Hamid Conceived and designed the experimentsContributed reagentsMaterialsAnalysis tools o

PII: S0026-265X(19)32412-9
DOI: https://doi.org/10.1016/j.microc.2019.104560
Reference: MICROC 104560

To appear in: Microchemical Journal

Received date: 31 August 2019

Revised date: 19 December 2019
Accepted date: 19 December 2019

Please cite this article as: Hytham M. Ahmed Analyzed and interpreted the data ,
Mahmoud A. Omar Analyzed and interpreted the data , Hany A. Batakoushy Performed the experimentsContribute
Mohamed A. Abdel Hamid Conceived and designed the experimentsContributed reagentsMaterialsAnalysis tools o
HPTLC-densitometric analysis of selected antidiabetic drugs in presence of their degradation products,
Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104560

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Highlights
 New validated HPTLC method was developed for simultaneous determination of DGF and SXG.
 The proposed method was successively applied as stability indicating assay of both drugs.
 Forced degradation studies were carried out to investigate acidic, alkaline and oxidative hydrolysis.
 HPTLC method could be applied for the studied drugs in QC-lab and in their pharmacokinetic studies.

1
HPTLC-densitometric analysis of selected antidiabetic drugs in presence of their degradation
products
Hytham M. Ahmed1, Mahmoud A. Omar2,3 , Hany A. Batakoushy1, and Mohamed A. Abdel Hamid4
1
Pharmaceutical Analysis Department, Faculty of Pharmacy, Menoufia University, Egypt.
2
Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Medinah, Saudi Arabia
3
Department of Analytical Chemistry, Faculty of Pharmacy, Minia University, Egypt.
4
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Egypt .

Abstract

In recent years, a lot of single-pill combinations (SPC) manufactured and are used as a promising choice in

diabetes treatment. However, this trend made a serious challenge to drug analysts because of the difficulty

in the analysis of two or more drugs in the presence of each other. In this study, a new validated high

performance thin-layer chromatography was developed for simultaneous determination of dapagliflozin and

saxagliptin in their pure form and dosage form and also successively applied as stability indicating assay of

both drugs. The proposed method was based on coupling high-performance thin-layer chromatography with

dual wavelength detection at 225 and 210 nm for dapagliflozin and saxagliptin, respectively.

Chromatographic separations were performed on silica gel 60-F254 aluminum plates with a mobile phase of

hexane/methanol/ethyl acetate (4:2:4, v/v/v). The retention factor values were 0.6, 0.18 and a correlation

coefficient of 0.9994 and 0.9995 for dapagliflozin and saxagliptin, respectively with a linear range of 50-

550 ng/spot for both drugs. The optimum chromatographic conditions were applied to commercial tablet

with good percent recovery values, (99.84±1.43) and (99.43±0.63) for dapagliflozin and saxagliptin,

respectively. The suggested method could be applied for the studied drugs in quality control-lab as well as

in their pharmacokinetic studies.

Keywords:

Dapagliflozin, Saxagliptin, Stability indicating, Separation, Dosage form.

2
1. Introduction

The increase in blood glucose level in diabetic patients is usually multifactorial. Therefore, it is difficult to

normalize glucose blood concentration by interfering with only one hypoglycemic mechanism. Therefore,

many single pill combinations (SPC) are used today as a promising choice in management of diabetes. That

treatment trend makes it more necessary to find suitable analytical methods for simultaneous determination

of the co-administered drugs [1]. The primary goal of any antidiabetic therapy is to get blood glucose level

in normal range without the addition of intolerable side effects, which can be accomplished by combining

drugs with different mechanisms of action. In this study, Dapagliflozin (DGF) and Saxagliptin (SXG),

(Figure: S1) were selected as representative examples [2-6]. DGF; is chemically a (2S, 3R. 4R, 5S, 6R)-2-

[4-chloro-3-(4ethoxybenzyl) phenyl]-6-(hydroxyl methyl) tetrahydro 2H-pyran-3, 4, 5-triol). It is act as

sodium inhibitor of glucose co-transporter. SXG; ((1S, 3S, 5S)-2-[(2S)-2-amino-2-(3-hydroxy-1-adamantyl)

acetyl]-2 azabicyclo [3.1.0] hexane-3-carbonitrile). It is dipeptidyl peptidase 4-inhibitor (DPP-4). The

combination therapy of DGF and SXG, co-formulated in Qtern® tablets was shown to be superior in

lowering blood glucose when compared with either of the monotherapy regimens [7]. However, this

combination therapy leads to a big challenge in pharmaceutical and biomedical analysis area. Therefore, it is

important to get a valid analytical method suitable for the analysis of these drugs in presence of each other.

Also, the analysis should be valid in presence of their degradation products [8-11] and also in

pharmaceutical dosage form. Literature review reveals that quantification of DGF in pure form or in its

pharmaceutical dosage form. These methods included spectrophotometry [12-15], spectrofluorimetry [16],

HPLC-methods [17-22]. Also, for SXG included spectrophotometry [23-26], spectrofluorimetry [27, 28],

HPLC-methods [29-40]. On the other hand, high performance thin-layer chromatography (HPTLC) is a

promising sustainable alternative to HPLC in some analysis. As HPTLC, separations has several

advantages, it takes short time for analysis. Moreover, it requires few nanoliter injection volumes.

Furthermore, minimal use of solvent and no prior extraction steps needed compared to HPLC [41-54].

According to literature there are two HPTLC methods for simultaneous analysis of both drugs [55, 56].

However, these methods suffer from low sensitivity as they cannot be applied for stability or

3
pharmacokinetic studies of both drugs. Moreover, the reported HPTLC mobile phases contained either

chloroform, which is hepatotoxic and carcinogenic, or, acetonitrile, which is toxic and dangerous. Both

solvents must be handled with caution as they cause severe hazards and/or death. Therefore, the main

objective of the present study is to develop a sensitive eco-friendly HPTLC method for simultaneous

determination of DGF and SXG in presence of their degradation products and also in their tablets using

green mobile phase components to preclude the use of hazardous organic solvents, which lead to safe

analytical method. Hence, it can be applied for routine quality control analysis. Also, it can be employed as

a stability-indicating method and for pharmacokinetic studies of the tested drugs.

2. Experimental

2.1. Instrumentation

Chromatographic analysis were carried out on HPTLC (CAMAG), TLC scanner 3 densitometer

)Switzerland) with WINCATS software, CAMAG 100-μL syringe bandwidth 4 mm, application rate 15

s/μL, slit dimension 3 × 0.45 mm. Chromatographic tank (25 × 25 × 9 cm). Samples were applied on

aluminum plates 20 × 10 cm with 250-μm thickness pre-coated with (silica gel, 60 F254, Merck, Germany).

The system was equipped with a deuterium/halogen tungsten lamp for reflectance-absorbance mode. UV-

lamp was used with short wavelength, 254 nm (Germany).

2.2. Materials and reagents

DGF propanediol monohydrate (99.8 % purity) and SXG (99.54 % purity) were obtained from (NODCAR,

Giza, Egypt). All reagents and solvents were of analytical (HPLC-grade). Mobile phase composition:

Hexane, methanol and ethyl acetate (99 % purity) were purchased from (Sigma-Aldrich, USA). HCl, NaOH

and H2O2 (30 %) were supplied from El Nasr Chemical, Co., (Egypt). Pharmaceutical dosage forms were

obtained from local market, Farxiga® tablets (batch. No. NY924 P043383) a film-coated tablet containing

10.0 mg DGF from AstraZeneca company and Onglyza® tablets (batch No. 0J57932) labeled to contain 5.0

mg of SXG per tablet were kindly supplied by Bristol-Myers Squibb/ company. In-house formulated tablets;

composed of SXG equivalent to 5.0 mg and DGF equivalent to 10.0 mg per tablet. Tablet excipients: (tablet

core) microcrystalline cellulose, croscarmellose sodium, lactose, anhydrous magnesium stearate and dental

4
type silica (Film-coating as polymer) polyvinyl alcohol, macrogol, titanium dioxide and Talc, all were

supplied from NODCAR, Egypt.

2.3.Standard solutions

DGF and SXG standard solutions (1.0 mg mL−1) were prepared by dissolving 50.0 mg of DGF or SXG in

20.0 mL methanol. The produced volume was adjusted to 50.0 mL with same solvent. Appropriate dilutions

were made with methanol to obtain working standard solutions in the range of 25-275 µg mL−1 for each

drug. Then, two microliters were applied on plates to reach the concentration ranges of 50–550 ng/spot for

both drugs.

2.4.General procedure and chromatographic conditions

A silica gel 60 F254 HPTLC plates (0.2 mm thickness, Merck-Germany) were used in all experimental study.

2.0 μL of samples volume were applied to HPTLC aluminum plate (20 × 10 cm pieces) in the form of a

band (6 mm band width). The plate was air-dried, then developed in the HPTLC tank containing 100 mL of

mobile system. Ascending development was completed until a migration distance of 60 mm from the origin

was reached (about 6 min). The plate was removed and air-dried. After that, scanning absorbance was

measured at 225 and 210 nm.

2.5.Applications to pharmaceutical tablets

2.5.1. Formulation of in-house tablets

As the Qtern® tablets are currently unavailable in the local market. Thus, ten tablets were prepared in house

using the following quantities per tablet; SXG equivalent to 5.0 mg and DGF equivalent to 10.0 mg were

accurately weighed and mixed with 14.1 mg lactose anhydrous, 136 mg microcrystalline cellulose, 1.8 mg

silicon dioxide and 9 mg crosscarmellose sodium per tablet. The powders were blended consistently, then,

passed through the 60 Sieve followed by the addition of 1.8 mg magnesium stearate and the mixture was

blended once more. The mixture was compressed in a single-punch tablet machine (Germany).

2.5.2. Determination of the average content of the in-house formulated tablets

For the average content estimation, ten formulated in-house formulated tablets were weighted accurately,

finely grinded, and mixed well. An accurate amount equivalent to 10 mg of DGF and 5 mg SXG were

5
weighted and transferred to a 100-mL volumetric flask, dissolved in about 50 mL of methanol. The contents

of the flask were swirled, sonicated for 5 min, and then the volume was completed to the 100.0 mL with

methanol. The flask contents were mixed well and filtered. The first portion of the filtrate was rejected. The

obtained solutions were spotted on the HPTLC plates, with different volumes to give a final concentration

within the concentration range of the calibration.

2.6. Forced degradation of the studied drugs

The studied drugs solutions (1.0 mg mL−1 for each) were subjected to forced degradation under both acidic

and basic conditions. That was done by separately adding 10 mL 1 N methanolic solutions of HCl, and

NaOH to 10 mL DGF or SXG stock solution into 100-mL round bottom flasks. The mixtures were refluxed

at 70°C for 2 h. All experiments were performed in dark in order to exclude any possible degradation due to

light effect. The produced solutions were neutralized then diluted with methanol to the mark. Then it was

applied to HPTLC plates in triplicate within the concentration of calibration range. Then, the

chromatograms were obtained as described above.

For oxidative degradation study, an amount of 10 mL of 30 % H 2O2 was added to 10 mL stock drug

standard solution in a 100-mL round bottom flask. Then, the obtained solution was refluxed for 2 h to

remove excess of hydrogen peroxide. The resulted solution was diluted with methanol. Then it was applied

to HPTLC plate in triplicate within the concentration range of the calibration. Then, the chromatograms

were obtained as described above.

Photochemical stability of DGF and SXG were also studied by exposure of 10 mL of drug stock solution to

direct sun light for 3 days (from 8:00 am to 4:00 pm at 25 °C) which was then diluted to the mark with

methanol, and the chromatographic procedure was followed. For UV degradation study, an amount of 10

mL of methanolic drug solution was exposed to UV-radiations at 254 nm for 24 h. The resulted solution was

diluted to the mark with methanol, and the chromatographic procedure was followed.

3. Results and Discussion

3.1. Effect of experimental variable

6
The proposed analytical technique has been described for separation and simultaneous determination of

DGF and SXG at 25 ᵒC and room humidity. The absorption spectra of the studied drugs were scanned in

the range from 200 to 400 nm (Figure: S2). As a result; 210 and 225 nm wavelengths were chosen as

best for SXG and DGF, respectively.

Different developing systems of variable compositions and ratios were tried to obtain the optimum condition.

The most suitable mobile phase, hexane/ethyl acetate/methanol (4:4:2, v/v/v), was used. To produce

good Rf values; the used chamber was saturated with the used developing system for 25 min before use.

Then, linear ascending development was performed. Plates were developed over a distance of 8 cm and

then air-dried. The produced bands were compact and sharp (Figure: 1, S3). The separated bands were

detected under a UV lamp at the selected wavelengths. Rf values were 0.65 and 0.18 for DGF and SXG,

respectively.

3.2. Method Validation

The proposed method was considered due to ICH-guidelines [57] included; linearity range, accuracy,

precision, LOD and LOQ.

Linearity was assessed by construction of calibration curves of six concentrations (six replicates for each

concentration) in the range of 50–550 ng/spot for DGF and SXG. The concentrations of the studied drugs

were plotted versus corresponding peak areas to produce the calibration graphs.

System suitability parameters of the proposed HPTLC method were calculated showing good resolution,

selectivity and capacity factor with high value of the resulted correlation coefficient as shown in Table 1.

To estimate accuracy; five concentrations of DGF and SXG in the linear range (50-550 ng/spot) were

examined. For each concentration, three replicates were investigated and the resulted data were presented as

percent recovery ± RSD as shown in Table 2. The obtained results indicated good accuracy of HPTLC

method. To examine intra and inter-day precision; three replicate determination of three different

concentrations for the studied drugs (100, 300 and 500 ng/spot) were examined on the same day and three

times on the successive day (Table S1). The obtained results proved that excellent precision of the method.

7
LOD and LOQ were calculated to evaluate method sensitivity as the following expression; LOD as 3.3 σ / S

while LOQ as 10 σ / S, where (S) mean the slope of calibration curve and (σ) mean the standard deviation of

intercept. The resulted values were 16.70 and 50.12 ng/spot for DGF also, 14.25 and 42.74 ng/spot for SXG,

respectively indicated a good sensitivity.

In robustness, small variations in the experimental parameters lead to insignificant change in Rf values

and/or the peak areas (Table 3). Therefore, the proposed method is robust.

To evaluate specificity of the proposed method, the studied drugs were determined in presence of their

degradation products. The obtained chromatograms (Figure: S2) indicated that there is no interferences at

the Rf values of DGF and SXG. Moreover, selectivity of the suggested method was tested by comparison of

the chromatograms of blank versus dosage form (Figure: S3). The obtained results proved that good

selectivity and specificity of the method. Furthermore, the proposed method results were compared with

those of a reported method (Table S2). The comparison indicated that the proposed method was much better

than the reported in different aspects.

3.3. Forced Degradation of DGF and SXG

The chromatogram of acid degradation samples in (Table S3) showed four different peaks with different R f

values (0.25, 0.29, 0.44 and 0.64) and five different peaks with different R f values (0.07, 0.13, 0.39, 0.48

and 0.63) for different acid degradation products for DGF and SXG, respectively. While the chromatogram

of alkaline degradation samples showed seven different peaks with different R f values (0.07, 0.19, 0.31,

0.33, 0.43, 0.46 and 0.65) and seven different peaks (0.06, 0.10, 0.23, 0.30, 0.48 and 0.54) for DGF and

SXG, respectively as shown in (Figure: S4).

The chromatogram of oxidative degradation samples showed three different peaks with different R f values

(0.30, 0.55 and 0.69) for DGF and three different peaks with different R f values (0.07, 0.13 and 0.45) for

SXG as shown in (Figure: S4).

The chromatogram of daylight degradation samples showed two different peaks with different R f values for

each DGF and SXG (0.09 and 0.74), (0.09 and 0.66) respectively. Insignificant degradations of the DGF and

SXG in daylight were produced and the obtained results are shown in Table S2. The chromatogram of UV

8
degradation samples showed two different peaks with different R f values (0.09 and 0.68) for DGF and (0.09

and 0.36) for SXG. The suggested degradations pathway of DGF and SXG was observed as shown in

(Figure 3). The obtained degradation product of the studied drugs after heating at 70°C was found as 20 %

with 1 N HCL, 89 % with 1 N NaOH, 6 % with H2O2 and stable with UV-lamp as shown in Table S2.

3.4. Kinetic degradation

In this study, kinetic of DGF and SXG in alkaline hydrolysis were performed at different interval times; 10,

20, 30, 40, 50, 60, 70 min. It was found that, a decrease in the concentration of studied drugs with increasing

time. The logarithmic of remained drug concentration versus time was plotted indicated a linearity with

good correlation coefficient (Figure S5). The expression of rate constant (K), half‐ life (t1/2) and shelf‐ life

(t90) were calculated according to the following equations [58]:

Log [Ct] = log [C0] − Kt /2.303 (1) t1/2 = 0.693/k (2) t90 = 0.105/k (3)

Where K is the rate constant, [C0] is the concentration of DGF or SXG at time t= 0 and [Ct] are their

concentration at time t. Under alkaline hydrolysis conditions at 1 N NaOH, where the K value was found to

be the highest compared with acidic and oxidative degradations. While, t 1/2 and t90 at alkaline conditions

were found to be lower than those of other conditions.

4. Applications

4.1. Application to tablets dosage form

The proposed HPTLC method was successfully evaluated to determine DGF and SXG in the in-house

prepared tablets as shown in Table S4. The average recovery values of prepared tablets were 99.89 % with

RSD of 1.43 % and 99.43 % with RSD of 0.63 %, for DGF and SXG respectively, compared to the reported

method. Both t and F tests indicated a good level of precision. The proposed HPTLC method is suitable for

application in quality control laboratories.

5. Conclusion

In the present work, an efficient HPTLC method with dual wavelength was developed for simultaneous

estimation of DGF and SXG in the presence of degradation products. The proposed method was selective

for analysis of the cited drugs in pharmaceutical formulations and in the presence of degradation products.

9
 Two wavelengths were used, 225 and 210 nm, for DGF and SXG, respectively to obtain high sensitivity in

linear range of 50-550 ng/spot for both drugs. The obtained Rf values were 0.65, 0.18. F values were 3592.25

and 4940.12 with significance F of 4.64×10−7 and 2.45×10−7 for DGF and SXG, respectively.

As the method separates the studied drugs from their different degradation products, it can be employed as a

stability-indicating method. The optimum chromatographic conditions were applied to commercial tablet

with good percent recovery values, (99.84±1.43) and (99.43±0.63) for DGF and SXG, respectively.

Author contribution statement

Mohamed Abdel-Hamid: Conceived and designed the experiments; Contributed reagents, materials,

analysis tools or data; Wrote the paper.

Hany A. Batakoushy: Performed the experiments; Contributed reagents, materials, analysis tools or data.

Mahmoud A. Omar and Hytham M. Ahmed: Analyzed and interpreted the data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-

profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Acknowledgements

The authors are grateful for NODCAR for their support during this research study.

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List of Figure captions

13
 Figure 1: Two-dimensional
HPTLC-chromatogram of DGF and SXG standard solution (300 ng/spot).

Figure 2: Atypical (3D) chromatogram for the calibration of DGF and SXG.

14
Figure 3: The suggested degradations pathway of DGF and SXG

15
Figure S1: The chemical structure of (a) dapagliflozin (DGF) and (b) saxagliptin (SXG).

Figure S2: Two and three-dimensional HPTLC-chromatogram for overlay spectra of DGF and SXG in

presence of degradation products on stationary material.

Figure S3: Two-dimensional HPTLC-chromatogram of DGF and SXG mixture (300 ng/spot).

Figure S4: Two-dimensional HPTLC-chromatogram of degradation products (a) DGF-acid degradation, (b)

DGF-alkaline degradation, (c) DGF-oxidative degradation, (d) SXG-acid degradation, (e) SXG-alkaline

degradation and (f) SXG-oxidative degradation.

Figure S5: Kinetic plots for basic hydrolysis of DGF and SXG (300 ng/spot).

List of Tables

16
Table 1: Quantitative parameters and regression analysis data of the proposed method for the studied drugs.
Method
Parameter
DGF SXG
Wavelength 225 210
Rf 0.65 0.18
Selectivity (α) K2/K1 8.43
Capacity factor K=(1-Rf)/Rf 0.54 4.55
Linearity Range (ng/spot) 50-550 50-550
Intercept (a) -650.25 347.03
SD of Intercept (Sa) 102.91 8.534
Slope (b) 20.32 1.976
SD of Slope (Sb) 0.3391 0.028
Correlation coefficient (r) 0.9994 0.9995
2
Determination coefficient (r ) 0.9989 0.9991
SD of residual (S y/x) 132.25 10.967
F value 3592.25 4940.12
−7
Significance F 4.64×10 2.45×10−7
Limit of detection (ng/spot) 16.70 14.25
Limit of quantitation (ng/spot) 50.12 42.74

Table 2: Evaluation of the accuracy of the proposed HPTLC-method.

Method
DGF SXG
Sample number a a
Taken Found % Found %
(ng/spot) (ng/spot) Recovery (ng/spot) Recovery
1 100 99.16 99.16 102.31 102.31
2 200 204.32 102.15 197.72 98.86
3 300 296.86 98.95 303.39 101.13
4 400 404.94 101.24 414.19 103.55
5 500 502.94 100.59 507.55 101.51
Mean 100.42 101.47
SD 1.36 1.73
RSD 1.35 1.70
a
Mean of three replicate measurement, SD: standard deviation, RSD: relative standard deviation.

17
 Table 3: Robustness of the proposed method for the studied drugs.
Method
DGF Conc. SXG Conc.
Variation (300 ng/spot) (300 ng/spot)
% %
Recovery ± SD Recovery ± SD
No variation
100.24 ± 0.73 101.27 ± 0.82
(Optimum condition)
 Mobile system composition (hexane/ethyl acetate/methanol, 4: 4: 2, v/v/v)
Hexane +5% 100.40 ± 1.23 101.00 ± 0.74
-5% 97.18 ± 1.51 100.70 ± 0.79
Ethyl acetate +5% 99.30 ± 0.78 101.22 ± 1.13
-5% 98.40 ± 1.72 99.68 ± 2.12
Methanol +5% 101.00 ± 0.33 99.82 ± 1.55
-5% 100.65 ± 0.89 98.63 ± 0.83

 Saturation time +5 min. 99.50 ± 1.63 100.10 ± 0.63 100.40

-5 min. 102.00 ± 0.29 99.40 ± 0.54 100.40

 Excitation wavelength +5 nm 99.89 ± 0.83 101.20 ± 0.43 100.40

-5 nm 103.01 ± 1.14 100.66 ± 1.71

Optimum condition: Mobile phase composition (hexane/ethyl acetate/methanol, 4: 4: 2, v/v/v), saturation time: 30min. and excitation
wavelength: 225, 210 nm for DGF, SXG, respectively.

Table S1: Precision data for the determination of the cited drugs by the proposed HPTLC-method.

Table S2: Comparison between the proposed HPTLC and reported method.

Table S3: Different stress conditions for degradation of the studied drugs.

Table S4: Analysis of tablets of estimated drugs by the proposed and reported method [15].

18
19
Graphical abstract

Atypical two and three-dimensional HPTLC-chromatogram for dapagliflozin and saxagliptin determination

20