Discover Chemistry
Research
Development and validation of stability indicating HPTLC method
for simultaneous estimation of Dapagliflozin and Linagliptin
Anchal Shukla1 · Usmangani Chhalotiya1 · Dimal Shah1 · Jinal Tandel1 · Heta Kachhiya1 · Mital Parmar1
Received: 22 March 2024 / Accepted: 23 May 2024
© The Author(s) 2024 OPEN
Abstract
For the treatment of Type 2 Diabetes Mellitus, Dapagliflozin selectively inhibits the sodium-glucose co-transporter-2,
while Linagliptin competitively and reversibly inhibits dipeptidyl peptidase-4 in a fixed dose combination (1:1).
Dapagliflozin and Linagliptin estimation in bulk and tablet formulation was accomplished by the development and
validation of a precise and accurate HPTLC method. The procedure used Toluene: Chloroform: Methanol: Triethylamine
(7:2:1:0.2 v/v/v) as the mobile phase and HPTLC aluminum plates pre-coated with silica gel 60 F254 as the stationary
phase used. Dapagliflozin and Linagliptin were determined to have R
­ f values of 0.23 and 0.40 correspondingly. At 224 nm,
densitometric analysis was performed in the absorbance mode. For Dapagliflozin and Linagliptin 200–1200 ng/band
were the linear ranges in which the procedure produced results. Forced degradation studies were performed on both
Dapagliflozin and Linagliptin active pharmaceutical ingredients like acid hydrolysis, base hydrolysis, chemical oxidation,
dry heat and photodegradation studies. Dapagliflozin and Linagliptin are susceptible to acid hydrolysis, base hydrolysis,
chemical oxidation while both the chemical substances are stable to dry heat and photolytic studies.
Keywords Dapagliflozin · Linagliptin · HPTLC · Validation · Forces degradation studie · Diabetes mellitus type II
1 Introduction
Dapagliflozin:(2S,3R,4R,5S,6R)-2-[4-chloro-3-[(methyl-4-ethoxyphenyl)phenyl]-6-(hydroxymethyl) oxane-3,4,5-triol is
Dapagliflozin chemical name. Its chemical formula is ­C12H25ClO6, and its weight is 408.9 g/mol (Fig. 1a). It has a pKa value
of 12.6 and a LogP value is 2.7. By blocking sodium glucose co-transporter 2 (SGLT2), which is found at the proximal
tubule site of the kidney, it works as an inhibitor. It falls under the category of anti-diabetics since it reduces blood sugar
by preventing the kidneys from reabsorbing glucose.
Linagliptin is known by the IUPAC 8-[(3R)-3-aminopiperidin-1-yl]-7-but-2-ynyl-3-methyl-1-[(4-methylquinazolin-2-yl)
methyl]purine-2,6-dione has the chemical formula ­C25H28N8O2 and a molecular weight of 472.5 g/mol (Fig. 1b). Its pKa
value is 8.6 and LogP value is 2.62. It has an anti-glycemic effect by suppressing the release of glucagon and increasing
insulin production by inhibiting dipeptidyl peptidase 4. Because the two medications work in tandem to complement
one another, using them together to treat Type 2 Diabetes is advantageous [1–5].
After a thorough literature review, it was determined that there are a number of ways to estimate the dosage and bulk
of Dapagliflozin, including UV Spectroscopy, the RP-HPLC method, and the use of analytical techniques like RP-HPLC,
HPTLC, UPLC, etc. in combination with other medications like metformin and saxagliptin. Linagliptin can also be estimated
* Usmangani Chhalotiya, usmangani.chhalotiya@cvmu.edu.in | 1Indukaka Ipcowala College of Pharmacy, A constituent college of The
CVM University, Beyond GIDC, P.B. No. 53, Vitthal Udyognagar, Gujarat 388 121, India.
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Vol.:(0123456789)
Research
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Fig. 1 a Chemical structure
of Dapagliflozin. b Chemical
structure of Linagliptin
in bulk and dosage form using a variety of techniques, including UV Spectroscopy, the RP-HPLC method, and the use of
analytical techniques like HPTLC and RP-HPLC in combination with other medications like metformin and empagliflozin
[6–28].
Extensive literature review reveals that there is no HPTLC method available for the simultaneous estimation of
Dapagliflozin and Linagliptin. Therefore, a thought of interest to develop and validate stability indication HPTLC method
for the 1:1 fixed dose combination of Dapagliflozin and Linagliptin in bulk and pharmaceutical dosage form.
2 Experimental
2.1 Standard API, chemicals and materials
Linagliptin API and Dapagliflozin API were supplied by a reputable pharmaceutical company with purity 99.75 and
99.40, respectively. The 5 mg tablets of Linagliptin (marketed as Linares) and Dapagliflozin (marketed as Dapagold5).
The Acetonitrile, Methanol, Toluene, Chloroform, Triethylamine were supplied by SRL Chemicals Pvt. Ltd., Mumbai, India.
2.2 Wavelength selection
Dapagliflozin and Linagliptin were freely soluble in acetonitrile and individual solutions at a concentration of 10 µg/
ml are prepared. Using a UV–Visible double beam spectrometer, both solutions were scanned from 400 to 200 nm in
wavelength. Both the drugs appreciable absorption was found to be at common wavelength 224 nm after scanning.
2.3 HPTLC system
Using a Camag Linomat 5 sample applicator (Switzerland) and a Camag 100 μl sample syringe (Hamilton, Switzerland),
the samples were applied in the form of 6 mm wide bands on pre-coated HPTLC silica gel aluminum plate 60 F254
(10 cm × 10 cm with 0.2 mm thickness, E. Merck, Germany). Densitometric scanning of the generated chromatogram
with a slit dimension of 4 × 0.45 mm was performed using the Camag TLC scanner 4.
Vol:.(1234567890)
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Research
2.4 Standard solution preparation
10 mg each of Linagliptin and Dapagliflozin were weighed, transported to separate 10-ml volumetric flasks, and swirl
to dissolved with a little amount of acetonitrile. To bring the volume up to 10 ml, acetonitrile as diluent was added. The
prepared solutions have a 1000 µg/ml concentration. A workable solution containing 100 µg/ml was prepared from this
stock solution by appropriately diluting it.
2.5 Calibration curve determination
From the working solution of 100 µg/ml the aliquots of 2, 4, 6, 8, 10, and 12 µl were applied on the HPTLC silica gel
aluminum plate 60 F254 using the Camag Linomat 5 sample applicator using 100 μl Hamilton sample syringe.
2.6 Validation
The HPTLC method was validated in accordance with the Q2 (R2) guideline of the International Council for Harmonization
of Technical Requirements for Pharmaceuticals for Human Use (ICH) [3].
2.7 Linearity
Six calibrations were conducted in the concentration range of 200 ng/ml to 1200 ng/ml to determine the linearity
of Dapagliflozin and Linagliptin in a fixed dose ratio (1:1). A straight-line equation was used to plot Peak Area v/s
Concentration on the graph to determine the calibration cure.
2.8 Precision
The degree to which values or measurements from a homogenous sample are similar is known as precision. Six injections
of the linearity range’s middle concentration (600 ng/band) were made to carry out the repeatability research.
Two investigations, intraday and interday, were carried out to achieve intermediate precision. While interday was done
on different days, intraday was done on the same day. To find the intermediate precision, three concentrations—the
lowest at 200 ng/band, the middle at 600 ng/band, and the highest at 1200 ng/band were selected. The computed Mean
of Area and %RSD was derived from the identified peak areas.
2.9 Accuracy
The degree to which the observed value and the true value agree is known as accuracy. The accuracy studies were
conducted using the usual spiking approach. Using this technique, the standard solution was spiked at 80%, 100%, and
120% concentrations in the Dapagliflozin and Linagliptin sample solution, with a concentration of 600 ng/band. There
were three injections of each spiked sample. Calculations were made for Mean Area and Recovery Percentage.
2.10 Limit of quantification and detection
The lowest concentration at which the method can consistently detect the analyte inside the matrix (instead of measuring
it) is called “limit of detection”. The term "limit of quantification" describes the smallest amount or lowest concentration
of a material that can be determined using a certain analytical method while still adhering to the predetermined degrees
of precision, accuracy, and uncertainty. The formula below can be used to calculate both.
LOD = 3.3 ×
𝜎
𝜎
and LOQ = 10 ×
s
s
Vol.:(0123456789)
Research
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
where σ is the standard deviation of y-intercepts of regression lines and S is the average slope of the calibration curves.
2.11 Robustness
Whether or not the results remain consistent even in significantly altered settings is how one evaluates the robustness of
an analytical procedure. It was a method’s ability to function even if small changes are made. A few modest adjustments
were made to the flow rate, analytical wavelength, and mobile phase composition to assess the method’s robustness.
600 ng/band was chosen as the middle concentration for the robustness analysis. The areas acquired after the injection
were then used to calculate the mean area and percentage RSD.
2.12 Assay of synthetic mixture
An accurately weight 20 tablets of Dapagold 5 tablet of Dapagliflozin and the Linares tablet of Linagliptin were precisely
weighed and finely powdered. Weight accurately the equivalent to 5 mg in a 10 ml volumetric flask containing little
amount of acetonitrile and swirl to dissolve. The volume was made up to mark with acetonitrile to yield a 500 µg/
ml solution sonicate for 15 min. Filter it with the help of Whatman filter paper (0.45µ) and the pipette out 2 ml above
prepared solution from each of separate 10 ml volumetric flask and transfer it to another 10 ml volumetric flask to yield
solutions containing 100 µg/ml Dapagliflozin and Linagliptin. From this solution, Apply 6 µl solution on the pre-coated
silica plate using Camag Linomat 5 sample applicator and Camag 100 μl sample syringe by enabling over spotting; this
will yield a sample of 600 ng/band of Dapagliflozin and Linagliptin (1:1) solution. This sample was analysed by using
proposed developed HPTLC method. The regression equation was then used to determine how much Dapagliflozin and
Linagliptin were present in the solution.
2.13 Forced degradation study
The purpose of the forced deterioration study was to determine the intrinsic stability of both medications.
2.14 Heat induced acid hydrolysis
A 1 ml aliquot of the Dapagliflozin stock solution (1000 ng/ml) was placed in a 10 ml volumetric flask. 1 of 0.01N HCl
were added to the same flask. For two hours, the solution was heated to 70ºC. After allowing the sample to cool, it was
neutralized with 0.01 N NaOH and brought up to the required amount with Acetonitrile. 6 µl of the aforementioned
solution were spotted on a TLC plate. In a similar manner, samples were made with a stock solution of Linagliptin (1000 µg/
ml) and investigated. Additionally, a mixture of 1 ml each of Linagliptin and Dapagliflozin (1000 µg/ml) was used in the
investigation, both in the same 10 ml volumetric flask. 1 ml of 0.01N HCl were added to the same flask.
2.15 Heat induced alkali hydrolysis
In a 10 ml volumetric flask, an aliquot of 1 ml of the Dapagliflozin stock solution (1000 μg/ml) was taken. 1 ml of 0.01N
NaOH were added to the same flask. For four hours, the solution was heated to 70ºC. After allowing the sample to
cool, it was neutralized with 1 ml of 0.01 N HCl and brought up to the required amount with Acetonitrile. 6 µl of the
aforementioned solution was spotted on a TLC plate. In a similar manner, samples were made with a stock solution of
Linagliptin (1000 µg/ml) and investigated. Using an aliquot of 1 ml each of Dapagliflozin and Linagliptin (1000 µg/ml)
in the same 10 ml volumetric flask, the investigation was also conducted in combination by adding 1 ml of 0.01N NaOH,
cooling and neutraling with 1 ml of 0.01 N HCl, 6 µl of the aforementioned solution was spotted on a TLC plate.
2.16 Heat induced chemical oxidation
Aliquot of (1 ml) of stock solution (1000 μg/ml) of Dapagliflozin was taken in 10 ml volumetric flask. To the same flask,
1 ml of 3% ­H2O2 was added. The sample was heated at 70ºC for 3 h and allowed to cool and was made up to the mark with
acetonitrile, from the above solution 6 µl was spotted on TLC plate. Similarly, samples were prepared using Linagliptin
(1000 µg/ml) stock solution and study was carried out. The study was also carried out in mixture using aliquot of (1 ml) of
Dapagliflozin and Linagliptin (1000 µg/ml) in same 10 ml volumetric flask. To the same flask, 1 ml of 3% ­H2O2 was added.
Vol:.(1234567890)
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Research
The sample was heated at 70ºC for 3 h and allowed to cool and was made up to the mark with acetonitrile, from above
solution, 6 µl was spotted on TLC plate.
2.17 Thermal degradation
After 4 h at 70 to 80 °C in dry heat, 10 mg each of Dapagliflozin and Linagliptin powder was transferred to two separate
volumetric flasks (10 ml each). To dissolve the powder, a few ml of acetonitrile was added to each of the flask, then more
acetonitrile was added until the desired amount was reached, yielding 1000 μg/ml of Dapagliflozin and Linagliptin. 1 ml
of the Dapagliflozin solution (1000 μg/ml) were pipetted out in 10 ml volumetric flask and made volume with acetonitrile.
6 μl of the aforementioned solution was spotted on the TLC plate. Same process was repeated for Linagliptin. Pipette out
1 ml each of the Dapagliflozin (1000 μg/ml) and Linagliptin (1000 μg/ml) solutions, then transfer them to the same flask.
The contents were then diluted with acetonitrile to produce a standard solution of a combination of medications (100 μg/
ml for Dapagliflozin and 100 μg/ml for Linagliptin). On the TLC plate, 6 μl of the aforementioned solution was observed.
2.18 Photolytic (UV light) degradation
Accurately weight 10 mg of both the active pharmaceutical ingredients individually and transfer to two separate petri
dishes. After being exposed to UV light for 24 h, add few ml of acetonitrile to in both the petri dish to dissolve the
powders of Dapagliflozin and Linagliptin. The solutions were transferred into two separate 10 ml volumetric flasks,
then more acetonitrile was added until the desired volume was reached, yielding 1000 μg/ml of Dapagliflozin and
Linagliptin. Pipette out 1 ml of aliquots from both 1000 μg/ml of Dapagliflozin and Linagliptin and makeup volume with
acetonitrile to produce solution of 100 μg/ml of Dapagliflozin and 100 µg/ml of Linagliptin. 6 μl of the Dapagliflozin
solution (100 μg/ml) and Linagliptin (100 μg/ml) were seen on the TLC plate. Pipette out 1 ml each of the Dapagliflozin
(1000 μg/ml) and Linagliptin (1000 μg/ml) solutions, then transfer them to the same flask. The contents were then diluted
with acetonitrile to produce a standard solution of a combination of medications (100 μg/ml for Dapagliflozin and 100 μg/
ml for Linagliptin). On the TLC plate, 6 μl of the aforementioned solution was observed.
The last concentration of the considerable number of solutions 100 ng/band (6 µl) was applied on the pre-coated
HPTLC plates and densitogram were recorded.
3 Results and discussion
3.1 Wavelength selection
Acetonitrile was used as a solvent to create separate solutions of Dapagliflozin and Linagliptin at a concentration of 10 µg/
ml. using a UV–Visible double beam spectrometer, both solutions were scanned from 400 to 200 nm in wavelength. The
iso-absorptive point was discovered to be 224 nm after scanning as shown in Fig. 2.
3.2 Mobile phase optimization
Toluene: Chloroform: Methanol: Triethylamine (7:2:1:0.2, V/V/V/V) was the mobile phase that generated a symmetrical
and selective peak for Dapagliflozin at Rf 0.23 and Linagliptin at Rf 0.40 as shown in Fig. 3.
Fig. 2 Overlay UV–Visible
Spectra of Dapagliflozin and
Linagliptin (10 µg/ml)
Vol.:(0123456789)
Research
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Fig. 3 Densitogram of
Dapagliflozin and Linagliptin
(600 ng/band)
3.3 Validation
The proposed developed HPTLC method was validated in accordance with the Q2 (R1) guideline of the International
Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and result and discussion
were representing as follows.
3.4 Linearity
The developed HTPLC method revealed that the correlation coefficients of 0.9978 and 0.9973 for Dapagliflozin and
Linagliptin were linear within the specified concentration range of 200–1200 ng/band. Figure 4 shows the Overlay of
the Densitogram of calibration range. The value of regression analysis is shown in Table 1.
3.5 Precision
Dapagliflozin and Linagliptin have percentage RSD value ranges for Interday precision of 0.59–1.88 and 0.39–1.48%,
respectively, and intraday precision of 0.35–0.62% and 0.51–1.81%, for Dapagliflozin and Linagliptin, respectively. When
Fig. 4 3D Overlay
densitogram of Dapagliflozin
and Linagliptin (200–1200 ng/
band)
Table 1 Regression analysis of
Dapagliflozin and Linagliptin
Vol:.(1234567890)
Parameters
Dapagliflozin
Linagliptin
Linearity range (ng/band)
Regression coefficient (­ R2)
Slope of regression equation
Standard deviation of slope
Intercept of regression
Standard deviation of intercept
200–1200
0.997
1.1499
0.0075
29.48
9.0445
200–1200
0.997
2.9126
0.0271
248.57
12.2410
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Research
intraday and interday measurements are made, the precision result demonstrates that the values are near to one another.
Table 2 shows the summary of validation parameters of proposed HPTLC method for Dapagliflozin and Linagliptin.
3.6 Accuracy
The accuracy studies were conducted using the usual spiking approach. The regression equation was used to compute
the percentage recovery of Linagliptin and Dapagliflozin. The percentage recovery of Dapagliflozin and Linagliptin was
found to be 98.82–101.44% and 98.35–101.01%, respectively. The results fall between the range of 98 to 102% which
indicates that the procedure is deemed accurate.
3.7 Limit of detection and limit of quantification
The results indicate that the approach is very sensitive for both detection and quantification. Specifically, the LOD and
LOQ for Dapagliflozin were determined to be 25.80 ng/band and 72.22 ng/band, while the corresponding values for
Linagliptin were 13.09 ng/band and 42.14 ng/band respectively.
3.8 Robustness
Some deliberate modifications were made to the test parameters to carry out the robustness investigation. The
modifications included a minor shift in the analytical wavelength from 224 to 223 nm and 225 nm, a change in saturation
time from 40 to 35 min and 45 min, and a change in the composition of the mobile phase from toluene: chloroform:
methanol: Triethylamine (7:2:1:0.2) to 7.2:2:0.8:0.2 and 6.8:2:1.2:0.2. The percentage RSD was found to be less than 2%
which demonstrates that Dapagliflozin and Linagliptin may be assessed using this method with minor adjustments to
the optimized chromatographic conditions are shown in Tables 3 and 4.
3.9 Assay of synthetic mixture
The recommended methodology was utilized to examine the commercially available tablet formulations of Dapagliflozin
and Linagliptin and the percentage amount of drug was found to be 100.7% and 99.8%. Figure 5 displays the overlay
densitogram, selectivity, and specificity of the Linagliptin standard and formulation along with the Dapagliflozin standard.
Peak purity spectra of Dapagliflozin and Linagliptin in calibration curve is shown in Fig. 6.
Table 2 Summary of validation parameters of proposed HPTLC method for Dapagliflozin and Linagliptin
Parameters
Dapagliflozin
Linagliptin
Acceptance criteria
Linearity range (ng/band)
Retention factor (Rf )
Detection limit (ng/band)
Quantitation limit (ng/band)
Accuracy (% Recovery)
Specificity
Robustness
200–1200
0.23
25.80
72.22
98.82–101.44
Specific
Robust
200–1200
0.40
13.09
42.14
98.35–101.01
Specific
Robust
–
–
–
–
> 98% < 102%
–
% RSD value < 2%
0.59–1.88
0.35–0.62
0.39–1.48
0.51–1.81
%RSD value < 2%
%RSD value < 2%
1.88
100.7
1.65
99.8
%RSD value < 2%
> 98% to < 102%
Precision (%RSD)
Interday precision (n = 3)
Intraday precision (n = 3)
Repeatability study (%RSD)
Repeatability (n = 6)
Assay (% Recovery)
RSD is Relative Standard Deviation and “n” is number of determinations
Vol.:(0123456789)
Research
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Table 3 Robustness study for Dapagliflozin (600 ng/band) by proposed HPTLC method
Parameters
Optimized condition
Change in condition
Rf
Mean area ± SD (n = 3)
% RSD
Change in wavelength
224 nm
Change in saturation time
40 min
Change in mobile phase composition
7:2:1:0.2 v/v/v/v
223 nm
225 nm
35 min
45 min
7.2:2:0.8:0.2 v/v/v/v
6.8:2:1.2:0.2 v/v/v/v
0.20
0.20
0.18
0.21
0.25
0.23
747.5667 ± 6.07
808.0667 ± 7.03
7.031595 ± 1.90
1075.3 ± 7.35
938.7 ± 3.54
1131.4 ± 20.58
0.81
0.87
0.40
0.68
0.37
0.81
Table 4 Robustness study for Linagliptin (600 ng/band) by proposed HTPLC method
Parameters
Optimized condition
Change in condition
Rf
Mean area ± SD (n = 3)
% RSD
Change in Wavelength
224 nm
Change in Saturation time
40 min
Change in Mobile Phase Composition
7:2:1:0.2 v/v/v/v
223 nm
225 nm
35 min
45 min
7.2:2:0.8:0.2 v/v/v/v
6.8: 2:1.2:0.2 v/v/v/v
0.38
0.38
0.35
0.35
0.45
0.43
1749.767 ± 17.89
1770.733 ± 27.22
1967.667 ± 20.57
2553.867 ± 16.76
2674.733 ± 10.36
2048.667 ± 18.50
1.02
1.53
0.04
0.65
0.38
0.90
Fig. 5 Overlay Densitogram
of standard and synthetic
mixture of Dapagliflozin and
Linagliptin (600 ng/band)
3.10 Forced degradation
Dapagliflozin and Linagliptin were degraded in acid hydrolysis with 0.01 N HCl at 70ºC for 2 h. Figure 7 represent
densitogram of combined drug under the acid degradation study.
Dapagliflozin and Linagliptin were significantly degraded in alkali degradation using 0.01 N NaOH at 70 ºC for 4 h.
Figure 8 displays the densitogram of combined drugs in alkali degradation study.
Dapagliflozin and Linagliptin were somewhat vulnerable to oxidative stress conditions. Figure 9 displays the
densitogram of during chemical oxidative breakdown.
Dapagliflozin and Linagliptin were more prone to thermal degradation conditions. Figure 10 displays the mixture’s
densitogram during thermal degradation.
Dapagliflozin and Linagliptin were found to be slightly vulnerable to photo degradation conditions. Figure 11
displays the mixture’s densitogram in UV photolytic degradation.
With no interference, all of the degradant peaks could be clearly separated from the drug peak, and a detector was
used to measure the peak purity. For the analysis of stability samples, the suggested approach is applicable which
Vol:.(1234567890)
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Research
Fig. 6 Peak purity spectra of
Dapagliflozin and Linagliptin
in calibration curve
Fig. 7 Combined densitogram
Dapagliflozin (600 ng/band)
and Linagliptin (600 ng/band)
in 0.01 N HCl reflux for 2 h at
70 ºC
Fig. 8 Combined densitogram
Dapagliflozin (600 ng/band)
and Linagliptin (600 ng/band)
in 0.01 N NaOH reflux for 4 h
at 70 ºC
Fig. 9 Combined densitogram
of Dapagliflozin (600 ng/
band) and Linagliptin (600 ng/
band) in 3% H
­ 2O2 reflux for 3 h
at 70–80 ºC
Vol.:(0123456789)
Research
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Fig. 10 Combined
densitogram of Dapagliflozin
(600 ng/band) and Linagliptin
(600 ng/band) in dry heat for
4 h at 70–80 ºC
Fig. 11 Combined
Densitogram of Dapagliflozin
(600 ng/band) and Linagliptin
(600 ng/band) in UV exposure
for 24 h
Table 5 Result of force degradation studies by proposed HPTLC method
Condition
Time(hr)
%Amount of
%Amount of
Dapagliflozin degradant Linagliptin degradant
Rf value of degradants of Rf value of
Dapagliflozin
degradants of
Linagliptin
Acid 0.01 N HCl
Base 0.01N NaOH
Oxidation 3% ­H2O2
Dry ­Heat*
Photo ­Degradation#
2
4
3
4
24
18.91
16.96
15.54
20
12.45
0.03
0,22
0.05,0.25
0.28
0.03
*
19.21
19.66
7.05
17.58
6.61
0.46, 0.77
0.30
0.42
0.52
0.59
Drugs heat for 4 h at 70–80 °C in Hot air oven, # Exposed to UV light for 24 h
represent the Dapagliflozin and Linagliptin are susceptible to acid, alkali, chemical oxidative, dry heat and photolytic
stress conditions. Table 5 represent the results of forced degradation studies by proposed HPTLC method.
4 Conclusion
The proposed developed and validated stability indicating HPTLC method for simultaneous estimation of Dapagliflozin
and Linagliptin in bulk and pharmaceutical dosage form represent a major advance in quality assurance. The value of
percentage (%) relative standard deviation represent the reliability, reproducibility, and suitability for routine quantitative
analysis. Under the forced degradation study, Dapagliflozin and Linagliptin was susceptible to acid and base hydrolysis,
chemical oxidation, dry heat, and photolytic degradation stress conditions. In which, degradant peaks of Dapagliflozin
and Linagliptin were clearly separated from the parent drug peak, and a detector was used to measure the peak purity
Vol:.(1234567890)
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
Research
which helps to determine the intrinsic behavior of molecule under different stress conditions as well as helps to determine
the self-life of drugs.
Acknowledgements The authors are extremely appreciative Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar, SICART and
The Charutar Vidya Mandal University, Vallabh Vidyanagar for providing necessary facilities to carry out research work.
Author contributions All authors associated with this research work declared that there is no conflict of interest for publication of work. All
authors have read and approved the manuscript. The contribution of each authors are mentioned below: Anchal Shukla: She is post graduate
student and above work has been carried out by her as dissertation work. Usmangani Chhalotiya: He is a mentor of Ms. Anchal Shukla and
under his noble guidance proposed method has been developed and validated as per ICH guideline. He is also giving training for ease of
operation sophisticated reverse phase liquid chromatography instrument and involved in interpretation of data. Dimal Shah: Through his good
relationship with pharmaceutical industry we have received all active pharmaceutical ingredients and he is having sound technical knowledge
Waters HPLC software system. Jinal Tandel: under her noble guidance student can understand the concept of mobile phase selection and
how to optimize chromatographic conditions. Heta Kachhiya: nder her noble guidance student can understand the concept of mobile phase
selection and how to optimize chromatographic conditions. She is also involved in interpretation of data. Mital Parmar: She is a co-mentor of
Anchal Patel helping hand throughout cited research work.
Data availability The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary
Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable
request.
Declarations
Competing interests All authors disclose any financial and personal relationships with other people or organizations that could inappropriately
influence (bias) their work. All authors associated with this research work declared that there is no conflict of interest for publication of work.
This research did not receive any specific grant from any funding agencies in the public, commercial, or not-for-profit sectors.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation,
distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://c​ reati​ vecom
​ mons.o
​ rg/l​ icens​ es/b
​ y/4.0
​ /.
References
1. Skoog DA, West DM, Hollar FJ, Crouch. Fundamentals of analytical chemistry. 8th ed. Philadelphia: Saunders College publishing; 2004. p.
973–92.
2. Sethi PD. HPTLC quantitative analysis of drugs in pharmaceutical formulation. 6th ed. New Delhi: Central Book Services Publishers and
Distributors; 2011. p. 1–72.
3. ICH Guidelines Q2 (R2). Validation of Analytical Procedure: Text and Methodology. Geneva, Switzerland, March, 2022.
4. Pubchem, Dapagliflozin, https://​pubch​em.​ncbi.​nlm.​nih.​gov/​compo​und/​Dapag​liflo​zin Accessed 13 Sept 2023.
5. Pubchem, Linagliptin, https://​pubch​em.​ncbi.​nlm.​nih.​gov/​compo​und/​Linag​liptin Accessed 13 Sept 2023.
6. Park J, Kim J, Noh JH, Kim K, Chung H, Kang M, Park J. Pharmacokinetics of a fixed-dose combination product of dapagliflozin and
linagliptin and its comparison with co-administration of individual tablets in healthy humans. Pharmaceutics. 2022;14(3):591.
7. Mante GV, Gupta KR, Apte AT. Estimation of Dapagliflozin from its tablet formulation by UV-Spectrometry. Pharma Methods.
2017;8(2):102–7.
8. Mante GV, Hemke AT, Umekar MJ. RP-HPLC methods for estimation of Dapagliflozin from its tablet. Int J Chem Tech Res. 2018;11(01):242–8.
9. Urooj A, Sundar PS, Vasanthi R, Raja LA, Dutt KR, Rao KNV, Hamana H. Development and validation of RP-HPLC method for simultaneous
estimation of Dapagliflozin and metformin in bulk and in synthetic mixture. World J Pharm Pharm Sci. 2017;6(7):2135–50.
10. Manoharan G, Ismaiel AM, Ahmed Z. Stability indicating RP-HPLC method development for simultaneous determination of dapagliflozin
in raw and tablet formulation. Chem Res J. 2017;3(2):159–64.
11. Godge RK, Shinde GS, Joshi S. Simultaneous estimation and validation of Dapagliflozin and Saxagliptin in bulk drug and dosage form by
RP-HPLC. Res J Sci Tech. 2019;11(1):59–63.
12. Reddy P, Sivagami B, Chandrasekar R, Niranjan M. A highly validated RP-HPLC method development for simultaneous estimation of
Dapagliflozin and Saxagliptin in tablet dosage form. Int J Pharm Sci Res. 2018;10(5):372–8.
13. Madhvi S, Prameela R. Development and validation of a method for simultaneous determination of Dapagliflozin and Saxagliptin in a
formulation by RP-UPLC. World J Pharm Res. 2017;6(12):901–16.
14. Abdelrahman AE, Maher HM, Alzoman AN. HPTLC method for determination of metformin Hydrochloride, Saxagliptin and Dapagliflozin
in pharmaceuticals. Current Anal Chem. 2020;16(5):609–19.
15. Nasser M, Salama I, Samia M. Compartitive HPLC and HPTLC study for simultaneous determination of Dapagliflozin and Metformin
Hydrochloride in bulk and pharmaceutical formulation. J Planar Chromat. 2016;31(6):469–76.
Vol.:(0123456789)
Research
Discover Chemistry
(2024) 1:4
| https://doi.org/10.1007/s44371-024-00002-0
16. Vijaya SK, Anusha A. Sudhakar “UV-Spectrometry method for estimation of linagliptin in bulk and pharmaceutical formulations.” Asian J
Res Chem. 2016;9(1):47–50.
17. Rajbanshi JC, Alam M, Hossain S, Islam S, Rouf AS. Development and validation of a RP-HPLC method for quantitative analysis of linagliptin
in bulk and dosage forms. Dhaka Univ J Pharm Sci. 2018;17(2):175–82.
18. Varaprasad C, Asif M, Ramakrishna K. RP-HPLC Method for simultaneous estimation of Metformin and Linagliptin in tablet dosage Form.
Rasayan J Chem. 2015;8(4):426–32.
19. Shirisha S, Haque MA, Sireesha D, Bakshi V, Harshini. Development and validation of RP-HPLC Method for simultaneous estimation of
Metformin and Linagliptin in combined pharmaceutical dosage form. Int J Pharma Res Health Sci. 2014;2(6):491–5.
20. Donepudi S, Achanta S. Validated HPLC-UV method for simultaneous estimation of Linagliptin and Empagliflozin in human plasma. Int
J Applied Pharm. 2018;10(3):56–61.
21. Sivagami B, Purushotham A, Sikdar P, Chandrasekar R, Babu M, Babu M. A validated method for the simultaneous estimation of Linagliptin
and Metformin in tablet dosage forms by RP-HPLC. Res J Pharmacy and Tech. 2020;13(3):1266–70.
22. Naga R, Parvathi P, Kumar JNS. Development and Validation of RP-HPLC method for the simultaneous estimation of Linagliptin,
Empagliflozin and Metformin in solid Dosage form. Asian J Pharm Anal. 2020;10(3):117–24.
23. Rao KV, Gorla R, Sreenivasulu R, Sreenivas N, Kaleemullah T, Sharma HK, Korupoulu RB. A simple and sensitive stability-indicating HPTLC
assay method for the determination of Linagliptin. Pharmacore. 2014;5(5):693–700.
24. Jillala S, Balekari U, Ciddi V. Development and validation of stability indicating HPTLC method for simultaneous determination of
Linagliptin and Metformin. Int J Pharmacy Pharm Sci. 2016;8(1):112–5.
25. Bhole RP, Wankhede SB, Pandey M. Stability indicating HPTLC method for simultaneous estimation of Empagliflozin and Linagliptin in
pharmaceutical formulation. Anal Chem Lett. 2017;7(1):76–85.
26. Sathya S, Siva K. Analytical method development and validation of Dapagliflozin tablets by RP-HPLC. Ymer. 2023;22(04):923–41.
27. Kant R, Babu R, Kapoor G, Bhutani R. Optimization of a single HPLC-PDA method for quantifying Metformin, Gliclazide, Pioglitazone,
Dapagliflozin, Empagliflozin, Saxagliptin, Linagliptin and Teneligliptin using central composite design. Bioorg Chem. 2019;2019(91):103111.
28. Shukla A, Chhalotiya UK, Shah D, Tandel J, Kachhiya H, Parmar M. Simultaneous estimation of dapagliflozin and linagliptin using reverse
phase-HPLC with photo diode array (PDA). J Chem Metrol. 2024. https://​doi.​org/​10.​25135/​jcm.​104.​2401.​3020.
Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Vol:.(1234567890)