Microchemical Journal 154 (2020) 104560

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

HPTLC-densitometric analysis of selected antidiabetic drugs in presence of T

their degradation products
Hytham M. Ahmeda, , Mahmoud A. Omarb,c, , Hany A. Batakoushya, Mohamed A. Abdel Hamidd
⁎ ⁎

a
Pharmaceutical Analysis Department, Faculty of Pharmacy, Menoufia University, Shebin Elkoum, Menoufia, Egypt
b
Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Medinah, Saudi Arabia
c
Department of Analytical Chemistry, Faculty of Pharmacy, Minia University, Egypt
d
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Egypt

ARTICLE INFO ABSTRACT

Keywords: In recent years, a lot of single-pill combinations (SPC) manufactured and are used as a promising choice in
Dapagliflozin diabetes treatment. However, this trend made a serious challenge to drug analysts because of the difficulty in the
Saxagliptin analysis of two or more drugs in the presence of each other. In this study, a new validated high performance thin-
Stability indicating layer chromatography was developed for simultaneous determination of dapagliflozin and saxagliptin in their
Separation
pure form and dosage form and also successively applied as stability indicating assay of both drugs. The pro-
Dosage form
posed method was based on coupling high-performance thin-layer chromatography with dual wavelength de-
tection 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 da-
pagliflozin 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.

1. Introduction The combination therapy of DGF and SXG, co-formulated in Qtern®

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 si-
multaneous determination of the co-administered drugs [1]. The pri-
mary 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).
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 ana-
lytical method suitable for the analysis of these drugs in presence of
each other. Also, the analysis should be valid in presence of their de-
gradation 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 spectro-
photometry [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 alter-
native 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

⁎
Corresponding authors.
E-mail addresses: hmaahmed@menofia.edu.eg (H.M. Ahmed), momar71g@mu.edu.eg (M.A. Omar).

https://doi.org/10.1016/j.microc.2019.104560
Received 31 August 2019; Received in revised form 19 December 2019; Accepted 19 December 2019
Available online 23 December 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
H.M. Ahmed, et al.
both drugs [55, 56]. However, these methods suffer from low sensitivity
as they cannot be applied for stability or pharmacokinetic studies of
both drugs. Moreover, the reported HPTLC mobile phases contained
either chloroform, which is hepatotoxic and carcinogenic, or, acetoni-
trile, 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 or-
ganic 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 re-
flectance-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) la-
beled 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, croscarmel-
lose sodium, lactose, anhydrous magnesium stearate and dental 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 dilu-
tions 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 vo-
lume 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
2
Microchemical Journal 154 (2020) 104560
60 mm from the origin was reached (about 6 min). The plate was re-
moved 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 con-
sistently, 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 for-
mulated tablets were weighted accurately, finely grinded, and mixed
well. An accurate amount equivalent to 10 mg of DGF and 5 mg SXG
were 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 fil-
tered. The first portion of the filtrate was rejected. The obtained solu-
tions 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% H2O2
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 re-
move 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 ex-
posure 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
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
H.M. Ahmed, et al. Microchemical Journal 154 (2020) 104560

Table 1
Quantitative parameters and regression analysis data of the proposed method
for the studied drugs.
Parameter Method
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
Determination coefficient (r2) 0.9989 0.9991
SD of residual (Sy/x) 132.25 10.967
F value 3592.25 4940.12
Significance F 4.64×10−7 2.45×10−7
Limit of detection (ng/spot) 16.70 14.25
Limit of quantitation (ng/spot) 50.12 42.74

Fig. 1. Two-dimensional HPTLC-chromatogram of DGF and SXG standard so-

lution (300 ng/spot).
3.2. Method validation

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 de-
velopment was performed. Plates were developed over a distance of
8 cm and then air-dried. The produced bands were compact and sharp
(Fig. 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.
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,

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

3
H.M. Ahmed, et al. Microchemical Journal 154 (2020) 104560

Table 2 3.3. Forced degradation of dDGF and SXG

Evaluation of the accuracy of the proposed HPTLC-method.
Sample Method The chromatogram of acid degradation samples in (Table S3)
number DGF SXG showed four different peaks with different Rf values (0.25, 0.29, 0.44
Taken Found a % Recovery Found a % Recovery and 0.64) and five different peaks with different Rf values (0.07, 0.13,
(ng/spot) (ng/spot) (ng/spot) 0.39, 0.48 and 0.63) for different acid degradation products for DGF
1 100 99.16 99.16 102.31 102.31
and SXG, respectively. While the chromatogram of alkaline degradation
2 200 204.32 102.15 197.72 98.86 samples showed seven different peaks with different Rf values (0.07,
3 300 296.86 98.95 303.39 101.13 0.19, 0.31, 0.33, 0.43, 0.46 and 0.65) and seven different peaks (0.06,
4 400 404.94 101.24 414.19 103.55 0.10, 0.23, 0.30, 0.48 and 0.54) for DGF and SXG, respectively as
5 500 502.94 100.59 507.55 101.51
shown in (Figure: S4).
Mean 100.42 101.47
SD 1.36 1.73 The chromatogram of oxidative degradation samples showed three
RSD 1.35 1.70 different peaks with different Rf values (0.30, 0.55 and 0.69) for DGF
and three different peaks with different Rf values (0.07, 0.13 and 0.45)
a
Mean of three replicate measurement, SD: standard deviation, RSD: relative for SXG as shown in (Figure: S4).
standard deviation. The chromatogram of daylight degradation samples showed two
different peaks with different Rf values for each DGF and SXG (0.09 and
three replicates were investigated and the resulted data were presented 0.74), (0.09 and 0.66) respectively. Insignificant degradations of the
as percent recovery ± RSD as shown in Table 2. The obtained results DGF and SXG in daylight were produced and the obtained results are
indicated good accuracy of HPTLC method. To examine intra and inter- shown in Table S2. The chromatogram of UV degradation samples
day precision; three replicate determination of three different con- showed two different peaks with different Rf values (0.09 and 0.68) for
centrations for the studied drugs (100, 300 and 500 ng/spot) were DGF and (0.09 and 0.36) for SXG. The suggested degradations pathway
examined on the same day and three times on the successive day (Table of DGF and SXG was observed as shown in (Fig. 3). The obtained de-
S1). The obtained results proved that excellent precision of the method. gradation product of the studied drugs after heating at 70 °C was found
LOD and LOQ were calculated to evaluate method sensitivity as the as 20% with 1 N HCL, 89% with 1 N NaOH, 6% with H2O2 and stable
following expression; LOD as 3.3 σ / S while LOQ as 10 σ / S, where (S) with UV-lamp as shown in Table S2.
mean the slope of calibration curve and (σ) mean the standard devia-
tion of intercept. The resulted values were 16.70 and 50.12 ng/spot for
3.4. Kinetic degradation
DGF also, 14.25 and 42.74 ng/spot for SXG, respectively indicated a
good sensitivity.
In this study, kinetic of DGF and SXG in alkaline hydrolysis were
In robustness, small variations in the experimental parameters lead
performed at different interval times; 10, 20, 30, 40, 50, 60, 70 min. It
to insignificant change in Rf values and/or the peak areas (Table 3).
was found that, a decrease in the concentration of studied drugs with
Therefore, the proposed method is robust.
increasing time. The logarithmic of remained drug concentration versus
To evaluate specificity of the proposed method, the studied drugs
time was plotted indicated a linearity with good correlation coefficient
were determined in presence of their degradation products. The ob-
(Figure S5). The expression of rate constant (K), half‐life (t1/2) and
tained chromatograms (Figure: S2) indicated that there is no inter-
shelf‐life (t90) were calculated according to the following equations
ferences at the Rf values of DGF and SXG. Moreover, selectivity of the
[58]:
suggested method was tested by comparison of the chromatograms of
blank versus dosage form (Figure: S3). The obtained results proved that Log [Ct ] = log[C0] Kt /2.303 (1)
good selectivity and specificity of the method. Furthermore, the pro-
posed method results were compared with those of a reported method t1/2 = 0.639/k (2)
(Table S2). The comparison indicated that the proposed method was
t90 = 0.105/k (3)
much better than the reported in different aspects.
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 degrada-
Table 3
tions. While, t1/2 and t90 at alkaline conditions were found to be lower
Robustness of the proposed method for the studied drugs.
than those of other conditions.
Variation Method
DGF Conc. (300 ng/ SXG Conc. (300 ng/
spot) spot) 4. Applications
% Recovery ± SD % Recovery ± SD
4.1. Application to tablets dosage form
No variation (Optimum condition) 100.24 ± 0.73 101.27 ± 0.82
Mobile system composition (hexane/ethyl acetate/methanol, 4: 4: 2, v/v/v)
Hexane +5% 100.40 ± 1.23 101.00 ± 0.74 The proposed HPTLC method was successfully evaluated to de-
−5% 97.18 ± 1.51 100.70 ± 0.79 termine DGF and SXG in the in-house prepared tablets as shown in
Ethyl acetate +5% 99.30 ± 0.78 101.22 ± 1.13 Table S4. The average recovery values of prepared tablets were 99.89%
−5% 98.40 ± 1.72 99.68 ± 2.12 with RSD of 1.43% and 99.43% with RSD of 0.63%, for DGF and SXG
Methanol +5% 101.00 ± 0.33 99.82 ± 1.55
−5% 100.65 ± 0.89 98.63 ± 0.83
respectively, compared to the reported method. Both t and F tests in-
Saturation time +5 min. 99.50 ± 1.63 100.10 ± 0.63 dicated a good level of precision. The proposed HPTLC method is sui-
−5 min. 102.00 ± 0.29 99.40 ± 0.54 table for application in quality control laboratories.
Excitation +5 nm 99.89 ± 0.83 101.20 ± 0.43
wavelength −5 nm 103.01 ± 1.14 100.66 ± 1.71
5. Conclusion
Optimum condition: Mobile phase composition (hexane/ethyl acetate/me-
thanol, 4: 4: 2, v/v/v), saturation time: 30 min. and excitation wavelength: 225, In the present work, an efficient HPTLC method with dual wave-
210 nm for DGF, SXG, respectively. length was developed for simultaneous estimation of DGF and SXG in

4
H.M. Ahmed, et al.
Fig. 3. The suggested degradations pathway of DGF and SXG.
the presence of degradation products. The proposed method was se-
lective for analysis of the cited drugs in pharmaceutical formulations
and in the presence of degradation products. 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 ob-
tained 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
5
Microchemical Journal 154 (2020) 104560
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.
Funding statement
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
H.M. Ahmed, et al.
Additional information
No additional information is available for this paper.
CRediT authorship contribution statement
Hytham M. Ahmed: Conceptualization, Methodology, Project ad-
ministration, Supervision, Validation, Visualization, Writing - original
draft, Writing - review & editing. Mahmoud A. Omar:
Conceptualization, Methodology, Project administration, Supervision.
Hany A. Batakoushy: Data curation, Formal analysis, Investigation,
Software, Validation, Writing - original draft, Writing - review &
editing. Mohamed A. Abdel Hamid: Conceptualization, Investigation,
Methodology, Supervision, Validation, Visualization.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors are grateful for NODCAR for their support during this
research study.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.microc.2019.104560.
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