et al. european journal of biomedical and pharmaceutical ......2017/03/17 · parmar et al.european...

Parmar et al. European Journal of Biomedical and Pharmaceutical Sciences

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668

DEVELOPMENT AND VALIDATION OF UV-SPECTROSCOPIC FIRST DERIVATIVE

AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY ANALYTICAL

METHODS FOR SIMULTANEOUS ESTIMATION OF DAPAGLIFLOZIN

PROPANEDIOL MONOHYDRATE AND SAXAGLIPTIN HYDROCHLORIDE IN

SYNTHETIC MIXTURE

Shveta H. Parmar*, Dr. Shailesh V. Luhar, Dr. Sachin B. Narkhede

SMT. B. N. B. Swaminarayan Pharmacy College, Salvav, Vapi, Gujarat, 396191.

Article Received on 17/03/2017 Article Revised on 07/04/2018 Article Accepted on 28/04/2018

INTRODUCTION

Dapagliflozin is chemically known as (2S)-propane-1,2-

diol(2S,3R,5S,6R)-2-{4-chloro-3-[(4-ethoxy phenyl)

methyl] phenyl}-6-(hydroxy methyl)oxane-3-4-5-mono

hydrate,[16,18,20]

as show in figure no.1 and it is a new

class of oral ant diabetic drugs, called Sodium Glucose

Co-Transporter 2 (SLGT2) inhibitors. These Sodium

Glucose Co-Transporters are responsible for glucose

reabsorption in the kidney. Hence inhibiting the SLGT2

have been proposed as a new strategy in the treatment of

diabetes. By suppressing the SLGT2, dapagliflozin

reduces plasma glucose concentration intern by elevating

the renal glucose excretion. it has molecular formula

502.99 gm/mol. Dapagliflozin is a white to half white

crystalline powder which is insoluble in water, and

soluble in ethanol, methanol, DMSO, dimethyl

formamide.

Saxagliptin Hydrochloride is (1R,3R)-2-[(2R)-2-amino-

2-(3-hydroxy-1-adamantyl)-acetyl]-2 azabicyclo hexane-

3-carbonitrile hydrochloride[17,19,21]

as show in figure no.

2,Saxagliptin is a competitive DPP4 inhibitor that slows

the inactivation of the incretin hormones, thereby

increasing their bloodstream concentrations and reducing

fasting and postprandial glucose concentrations in a

glucose-dependent manner in patients with type 2

diabetes mellitus. It has molecular weight of 351.870

g/mol. saxagliptin Hydrochloride is a white powder

which is soluble in methanol.

Fig. 1: Structure of Dapagliflozin.

SJIF Impact Factor 4.918 Research Article ejbps, 2018, Volume 5, Issue 5 668-681.

European Journal of Biomedical AND Pharmaceutical sciences

http://www.ejbps.com

ISSN 2349-8870

Volume: 5

Issue: 5

668-681

Year: 2018

*Corresponding Author: Shveta H. Parmar

SMT. B. N. B. Swaminarayan Pharmacy College, Salvav, Vapi, Gujarat, 396191.

ABSTRACT

In the present study two analytical Methods were developed for the estimation of Dapagliflozin propanediol and

saxagliptin Hydrochloride in Synthetic Method. where as in method A ,first derivative method based on

measurement of absorbance at two wavelengths 225 nm and 217 nm using UV visible spectrophotometer with

1cm matched quartz cells and methanol solvent were employed in this method. The Developed method obeyed

Beer’s-Lambert’s law in the concentration range of 20-100μg/ml, having correlation coefficient for Dapagliflozin

was 0.984 and 10-50 μg/ml, having correlation coefficient for Metformin hydrochloride was 0.982. In method B,

High performance thin Layer Chromatography using silica gel aluminium plate 60F254(10*10cm)as stationary

Phase and Chloroform: Ethyl acetate: methanol: ammonia (6:2:2:2 Drops) as Mobile Phase .the developed plates

scanned densiometrically using UV 210nm wavelength. The Rf value of DAPA and SAXA was found to be 0.30

and 0.54 respectively. Both the method validated for different validation parameter such as linearity, accuracy,

precision, LOD, LOQ and robustness and the result were found to be within the acceptance limit as per the

guideline of international conference on Harmonization (ICH).

KEYWORDS: Dapagliflozin propanediol, Saxagliptin Hydrochloride, first derivative method, HPTLC method,

Validation, ICH.


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669

Fig. 2: Structure of Saxagliptin HCl Propanediol.

Literature review indicated that numbers of analytical

methods Like UV spectroscopy, HPLC and HPTLC

method are available for estimation of Dapagliflozin

Propanediol And Saxagliptin Hydrochloride in single

dosage form and in combination with other drugs.

There is no any derivative UV-Spectroscopic and

HPTLC method have been reported for this combination

of drug So it was thought of interest to develop simple,

sensitive, specific, accurate and precise UV-

Spectroscopy (First Derivative) and HPTLC method for

estimation of Dapagliflozin Propanediol And Saxagliptin

Hydrochloride in synthetic mixture dosage form.

Nowadays, HPTLC is becoming a routine analytical

technique with numerous advantages of low operating

cost; quick conductance of several trials and need for

minimum sample clean up.

The major advantage of HPTLC is that several samples

can be run in chorus using small amount of mobile

phase; in contrast to HPLC, thus time saving and cost

effective. UV spectroscopy methods are easy to execute

and possible to perform even at smaller units for quality

control.

The present work was undertaken with an aim to

develop and validate of analytical methods as per ICH

guidelines for simultaneous estimation of Dapagliflozin

Propanediol And Saxagliptin Hydrochloride in synthetic

mixture dosage form.

MATERIALS AND METHODS

Instrumentation

Method A: The absorbance and spectral measurements

were done on a double-beam LABINDIA UV-Visible

Spectrophotometer with software UV Win. 1cm quartz

cells were used for sample handling. A digital analytical

balance was used for weighing.

Method B: Camag HPTLC system (Switzerland) with

Linomat V automatic sample applicator Camag TLC

Scanner III Camag (Muttenz, Switzerland) were used for

development of method. Pre-coated silica gel aluminum

plate 60F254, (10×10 cm; E. Merck,) were used for

separation of components.

Chemical: Dapagliflozin propanediol, Saxagliptin

Hydrochloride, is obtained as gift sample from CTX Lab.

PVT.LTD.Surat, Methanol, Ethyl Acetate, n-Hexane,

Chloroform, Glacial Acetic acid, Ammonia(RAN –KEM

LAB.) etc.

Preparation of Standard Stock Solutions

For Method A

Dapagliflozin Standard stock-I solution (1000μg/ml):

100mg of Dapagliflozin was weighed and transferred to

a 100 ml volumetric flask and dissolved in Methanol and

sonicated for about 10 min. Volume was make up to the

mark with methanol to give a solution containing

1000μg/ml .

Dapagliflozin Standard stock-II solution (200μg/ml):

Withdraw 20ml of stock-I solution was diluted to 100 ml

with Methanol to prepare 200μg/ml.

Saxagliptin Hydrochloride Standard stock-I solution

(1000μg/ml): 100mg of Saxagliptin hydrochloride was

accurately weighed and transferred to a 100ml

volumetric flask and dissolved in Methanol and



1000μg/ml Saxagliptin Hydrochloride.

Saxagliptin Hydrochloride standard stock-II solution

(100μg/ml): Withdraw 10 ml of stock-I solution was

diluted to 100 ml with Methanol to prepare 100μg/ml.

Assay of marketed formulation: 250 mg powder of

synthetic mixture was weighed accurately. Powder

equivalent to 10 mg of Dapagliflozin propanediol and 5

mg of saxagliptin hydrochloride were calculated and

added into a 100 ml volumetric flask individual

volumetric flask and volume was made up with methanol

up to100ml. From above obtained 40 µg/ml solution, 4

ml of solution was pipetted out into a 10 ml volumetric

flaks and volume was made up to 10 ml with methanol.

The resulting solution obtained was in the linearity range

and of concentration 40 µg/ml dapagliflozin propanediol

and 20µg/ml saxagliptin hydrochloride as the synthetic

mixture contained even the other drug in half amount

than dapagliflozin propanediol drug.

For Method B

Dapagliflozin Standard stock-I solution (1000μg/ml):

50 mg of Dapagliflozin was weighed and transferred to a

50 ml volumetric flask and dissolved in Methanol and



1000μg/ml Dapagliflozin. Further from the stock

solution a concentration of 500μg/ml was prepared and

was used for further analysis.

Saxagliptin Hydrochloride Standard stock-I solution

(1000μg/ml): 50 mg of Saxagliptin Hydrochloride was

accurately weighed and transferred to a 100ml

volumetric flask and dissolved in Methanol and


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1000μg/ml Saxagliptin Hydrochloride. further from the

stock solution a concentration of 250μg/ml was prepared

and was used for further analysis.

Assay of marketed formulation The amount equivalent to 10 mg DAPA and 5 mg

SAXA from synthetic mixture was transferred to 100 ml

vol. flask and 50 ml methanol was added and sonicated

for 15min.

The solution was filtered through filter paper No. 41 and

diluted by methanol up to mark to get sample solution of

100 μg/ml of DAPA and 50 μg/ml of SAXA. The

quantification of DAPA and SAXA were carried out by

measuring peak area of sample solution at 210 nm and

amount of DAPA and SAXA in the sample calculated by

putting respective response into regression line equation

for DAPA and SAXA

METHOD DEVELOPMENT

Method A:

Determination of the zero crossing point

The standard solutions of dapagliflozin propanediol (100

μg/ml) and Saxagliptin Hydrochloride(50 μg/ml) were

scanned separately in the UV range of 200-400 nm. The

absorbance spectra, thus obtained were derivatized to

remove the interference of absorbing species. The two

wavelengths selected should be such that at each

wavelength the absorbance difference between the

components should be as large as possible. From the

examination of the overlay first derivative spectra of

dapagliflozin propanediol and saxagliptin hydrochloride

217 nm (λ1) and 277 nm (λ2) were selected as working

wavelengths for the first derivative spectroscopy, as at

217 nm saxagliptin hydrochloride exhibited zero

absorbance and at 225 nm dapagliflozin propanediol

showed zero absorbance.

METHOD VALIDATION

For method A The Proposed method was validated according to ICH

guidelines. The parameters assessed were linearity,

precision, accuracy, LOD and LOQ.

Linearity and Range

The linearity was determined at three levels over the

range of 20-100 μg/ml for Dapagliflozin propanediol and

10-50 μg/ml saxagliptin hydrochloride. Absorbance of

above linearity solution preparations were taken at each

concentration three times. Mean absorbance at each

concentration was calculated and Graph of absorbance

(y-axis) versus Concentration (x-axis) was plotted.

PRECISION

A. Repeatability

Standard solutions of 40, 60, 80 μg/ml dapagliflozin

propanediol and 20, 30, 40 μg/ml saxagliptin

hydrochloride were prepared and spectra were recorded.

Absorbance was measured of the same concentration

solution three times and % R.S.D. was calculated.

B. Intra-day precision



hydrochloride were analyzed three times on the same day

and % R.S.D was calculated.

C. Inter-day precision



hydrochloride were analyzed three times on three

different days and % R.S.D was calculated.

Accuracy

Recovery studies were carried out by addition of

standard drug to the sample at 3 different concentration

levels (80%, 100% and 120%) taking into consideration

percentage purity of added bulk drug samples. These

solutions were subjected to re-analysis by the proposed

method and Results are calculated.

Limit of Detection and Limits of Quantitation

Limit of Detection (L.O.D.)

From the linearity curve equation, the standard deviation

(S.D.) of the intercepts (response) was calculated. The

limit of detection (L.O.D.) of the drug was calculated by

using the following equation designated by International

Conference on Harmonization (ICH) guideline:

L.O.D. = 3.3 σ / S

Where, σ = the standard deviation of the response

S = slope of the calibration curve

Limit of Quantitation (L.O.Q.) The limit of quantitation (L.O.Q.) of the drug was

calculated by using the following equation designated by

International Conference on Harmonization (ICH)

guideline:

L.O.Q. = 10 σ / S


S = slope of the calibration curve

For method B

Method Development

Selection of wavelength for mixture

The concentration of standard mixture solution of DAPA

and SAXA ( 1000 ng/spot) were spotted in form of bands

of width 6mm using a 100µl syringe on percolated silica

Gel aluminum plate 60F254(10 *10 cm) then all plates

scan densiometrically at different wavelength like

225nm,220nm and 210nm using CAMAG TLC scanner.

Both components showed reasonable good response at

210nm. So they were detected at this analytical

wavelength.


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Selection of mobile phase

Preliminary trials First of all prewashing of TLC plate was done using

methanol and activated in hot air oven for 5 min at 60°

C. The standard stock solution of DAPA and SAXA

(1000ng/band)) were spotted separately on TLC plate by

glass capillary tube and allowed it to dry for 4 to 5 min at

room temperature. The mobile phase as displayed in

table 5.4 was taken in CAMAG glass Chamber and

allowed it to saturate for 20 min The optimize mobile

phase was selected after number of trails using different

reagents The optimize mobile phase consisting of

mixture of “Chloroform: Ethyl Acetate:

Methanol: Ammonia (6.0:2.0:2.0:0.2 v/v/v)”

Table 1: HPTLC Mobile Phase Optimization.

Trial Mobile Phase

Ratio (%V/V) Rf value Observation

Figure

No.

DAPA SAXA

For individual component

1 CHCl3:Methanol (9:1) %v/v 0.46 0.16 Two peak observed but peak shape

& baseline was not proper 7.28

2 Toluene: Methanol (9:1) %v/v 0.17 0.10 peak observed but peak area &

baseline was not proper 7.29

3 n-Hexane: Methanol (9:1) %v/v 0 0 No peak observed 7.30

4 CHCl3:Methanol (8:2) %v/v 0.67 0.35

Good separation but baseline not

proper & SAXA has peak

broadening

7.31

5 CHCl3:Ethyl Acetate: Methanol

(7:2:1) %v/vv 0.28 0.10

Two peak observed but baseline was

not proper & less Rf value 7.32

6 CHCl3: Ethyl Acetate: Methanol(7:1:2)

%v/v/v 0.63 0.27

Two sharp peak appear & good Rf

value but less peak area 7.33

7 CHCl3:Methanol:

Ammonia (8:2:1Drops) %v/v/v 0.78 0.60

Two peak observed but Rf value too

high near to solvent front 7.34

8 CHCl3:Methanol:GAA (8:2:1drop)

%v/v/v 0.72 0.23

Peak broadening of saxa & DAPA

found High Rf value 7.35

For mixture of DAPA and SAXA solution

9 CHCl3: Ethyl Acetate :Methanol (7:1:2)

%v/v/v 0.85 0.56

Rf value of DAPA found High &

peak of SAXA not sharp 7.36

10 CHCl3: Ethyl Acetate :Methanol:

Ammonia (7:2:1:2 drop)%v/v/v 0.10 0.20

Two sharp peak observed but less Rf

value 7.37

11 CHCl3:Ethyl Acetate :Methanol:

Ammonia (6:2:2:2 drops)%v/v/v 0.30 0.54

Two Sharp Peak With Good

Separation ,No Any Tailing 7.38

METHOD VALIDATION

Linearity

Calibration curves were plotted over the concentration

range of 500– 4000 ng/band and 250 –2000 ng/band for

DAPA and SAXA respectively. Accurately prepared

mixed standard solutions of DAPA (1, 2, 3, 4, 5, 6,7, and

8 µl ) and SAXA (1, 2, 3, 4, 5, 6,7, and 8 µl) were

applied to the plate. The calibration curves were

constructed by plotting peak areas (Y- axis) against the

concentrations (X- axis).The correlation coefficient and

equation and the calibration plot in FIG 7.45 and For

dapagliflozin and Saxagliptin respectively.

Precision

The precision of the analytical method was studies by

analysis of multiple sample of homogeneous sample .The

precision is expressed as standard deviation or relative

standard deviation.

(a) Intraday Precision

The intraday precision of the proposed method was

determined by analyzing mixed standard solution of

DAPA and SAXA at 3 different concentrations (1000,

2000 and 3000 ng/band for DAPA; 500, 1000 and 1500

ng/band for SAXA) 3 times on the same day. The results

are reported in terms of relative standard deviation

(%RSD).

(b) Interday Precision

The interday precision of the proposed method was

determined by analyzing mixed standard solution of

DAPA and SAXA at 3 different concentrations (1000,

2000 and 3000 ng/band for DAPA; 500, 1000 and 1500

ng/band for SAXA) 3 times on different days. The

results are reported in terms of relative standard

deviation (%RSD).

The repeatability was carried out by repeated scanning

and measuring the peak area of DAPA (2000 ng/band)

and SAXA (1000 ng/band) (n = 6) without altering the

parameters of the proposed Method. The results are

reported in terms of relative standard deviation (% RSD).


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Limit of Detection (LOD) & Limit of Quantitation

(LOQ)

They were calculated by following equation

LOD = 3.3 X σ/S

LOQ = 10 X σ/S


S = slope of the calibration curve.

Specificity

Specificity is a procedure to detect quantitatively the

analyte in the presence of components that may be

expected to be present in the sample matrix. Specificity

of developed method was established by spiking of

DAPA and SAXA in hypothetical placebo (i.e. might be

expected to be present) and expressing that analyte was

not interfered from Excipients.

The measurement of specificity was perform by

comparing standard sample. The spots for DAPA and

SAXA in sample can be proven by comparison of sample

Rf and spectra to the standard spectra and Rf.

Accuracy (% Recovery study)

The accuracy of the proposed method was determined by

standard addition method by calculating the percentage

recoveries of all three drugs. The accuracy was evaluated

in triplicates, at three different concentrations levels i.e.

50, 100 and 150 % of the active ingredients, by adding

different concentration of Dapagliflozin (DAPA) and

Saxagliptin (SAXA) standard to the known amount of

sample and calculating the recovery and % RSD for all

the drugs.

Recovery studies were carried out by spiking three

different amount of DAPA standard (500 ng/band, 1000

μg/band, 1500 ng/band) to the dosage form (1000

ng/band) ,and SAXA standard (250 ng/band, 500μg/band

and 750 ng/band) to the dosage form (500 ng/band) by

standard addition method.

Robustness

The robustness of the method was evaluated by varying

method parameters such as saturation time (25 min and

35 min); and changing the composition of the mobile

phase Each parameter was varied at a time. It was

assessed by using the three replicates of one standard

concentration (1000μg/band of DAPA, and 500ng/band

of SAXA) and calculating the values of mean area and %

RSD.

The robustness of the method was established by

making deliberate minor variations in the following

method parameters

a) mobile phase Ratio (% v/v)

1. (CHCl3: Ethyl Acetate: MeOH: Ammonia (6.5 :1.5 :

2:2 drops)

2. (CHCl3: Ethyl Acetate: MeOH: Ammonia (5.5 :2.5 :

2:2 drops)

b) Saturation time of development chamber {8 min0.2),

12 min (+0.2)}

RESULTS AND DISCUSSION

Method A: First Derivative method

Selection of Detection Wavelength

Figure 3: Selection of Detection Wavelength.

Figure 4: Overlain first order derivative spectra of

Dapagliflozin propanediol.

Figure 5: Overlain first order derivative spectra

spectra of saxagliptin Hydrochloride.

Figure 6: Overlain first order derivative spectra of

Dapagliflozin propanediol and Saxagliptin

hydrochloride.


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Calibration Curve

Results of calibration reading of Dapagliflozin

propanediol at 217 nm ( ZCP of SAXA)

Table 2: Calibration data of Dapagliflozin

propanediol at 217 nm.

Conc. (µg/ml) Mean Abs. ± S.D.

0 0

20 0.006 ± 0.0005

40 0.014 ± 0.0005

60 0.023 ± 0.0005

80 0.034 ± 0.0005

100 0.041 ± 0.0005

Figure 7: Calibration curve of Dapagliflozin

proanediol at 217 nm.

Results of calibration reading of Saxagliptin

hydrochloride at 225 nm (ZCP of DAPA)

Table 3: Calibration data of Saxagliptin

hydrochloride at 225 nm.

Conc. (µg/ml) Mean Abs. ± S.D.

0 0

10 0.007 ± 0.0005

20 0.017 ±0.0005

30 0.024 ± 0.0005

40 0.032 ± 0.0005

50 0.041 ± 0.0005

Figure 8: Calibration curve of Saxagliptin

hydrochloride at 225 nm.

Assay Result of Marketed Formulation

Table 4: Assay result of Marketed formulation.

Drug

Actual conc.

Of Drug

(μg/ml)

Amt. Of

Drug Found

(μg/ml)

% of

Drug

found

Avg. Of

% Drug

found

SD %RSD

Dapagliflozin

Propanediol

40 41.7 97

101.08 1.31 1.125 40 40.1 99.25

40 39.5 100.125

Saxagliptin

hydrochloride

20 20.07 100.35

100.63 1.18

1.178

20 20.03 99.65

20 19.8 100.3

METHOD VALIDATION

Precision

A. Repeatability Study

Table 5: Repeatability data for Dapagliflozin propanediol and Saxagliptin hydrochloride.

Conc.

(µg/ml)

Dapagliflozin at 217 nm Conc.

(µg/ml)

Saxagliptin at 225 nm

Mean Abs. ± S.D.

(n = 3) % R.S.D.

Mean Abs. ± S.D.

(n = 3) % R.S.D.

40 0.014 ± 0.0005 4.02 20 0.016 ± 0.00005 3.53

60 0.023 ± 0.0005 2.47 30 0.025 ± 0.0005 2.27

80 0.034 ± 0.0005 1.66 40 0.031 ± 0.0005 1.82

B. Intraday

Table 6: Intraday data for Dapagliflozin propanediol and Saxagliptin.

Conc. (µg/ml)

Dapagliflozin propanediol Conc.

(µg/ml)

Saxagliptin hydrochloride

Mean Abs. ± S.D.

(n = 3) % R.S.D.

Mean Abs. ± S.D.

(n = 3) % R.S.D.

40 0.014 ± 0.0005 4.028 20 0.017 ± 0.0005 3.330

60 0.023± 0.0005 2.432 30 0.025 ± 0.0005 2.249

80 0.034±0.0005 1.681 40 0.032 ± 0.0005 1.767


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C. Interday

Table 7: Interday data for Dapagliflozin propanediol and Saxagliptin hydrochloride.

Conc. (µg/ml)

Dapagliflozin propanediol

Conc. (µg/ml)

Saxagliptin hydrochloride

Mean Abs. ± S.D.

(n = 3) %R.S.D.

Mean Abs. ± S.D.

(n = 3) %R.S.D.

40 0.013±0.00005 4.22 20 0.016±0.0005 3.46

60 0.023±0.0005 4.16 30 0.025±0.0005 2.27

80 0.034±0.0005 2.85 40 0.031±0.0005 2.24

ACCURACY

Table 8: Accuracy data for Dapagliflozin propanediol.

%

Recovery

Target

Conc.

Spiked

Conc.

Final

Conc.

Conc.

Obtained %recovery

%Mean recovery

±SD (n=3)

80 %

40 32 72 71.5 101.75

99.62±0.94 40 32 72 71.2 99.75

40 32 72 72.5 97.55

100 %

40 40 80 80.3 99.36

101.29±0.93 40 40 80 81 98.88

40 40 80 81.8 100.69

120%

40 48 88 87.2 100.37

100.41±1.57 40 48 88 88 101.25

40 48 88 89.9 102.25

Table 9: Accuracy data for Saxagliptin hydrochloride.

%

Revery

Target

Conc.

Spiked

Conc.

Final

Conc.

Conc.

obtained

%

Assay

% Mean recovery

±SD (n=3)

80 %

20 16 36 35.89 35.89

98.97±1.52 20 16 36 35.98 35

20 16 36 36.2 36

100 %

20 20 40 39.96 40.06

100.15±0.15 20 20 40 40.12 40.12

20 20 40 39.97 40

120%

20 24 44 43.97 43.97

100.96±1.54 20 24 .44 44.21 45.21

20 24 44 44.1 44.1

Limit of Detection and Limits of Quantitation

Table 10: L.O.D. and L.O.Q data for Dapagliflozin propanediol and Saxagliptin Hydrochloride

Drugs L.O.D. (μg/ml) L.O.Q. (μg/ml)

Dapagliflozin propanediol 6.41 19.44

Saxagliptin Hydrochloride 2.46 7.47

For Method B: HPTLC method

Selection detection wavelength

Standard Solution Of DAPA &SAXA (1000ng/spot)

Were Applied To Silica Gel 60GF254 By Means Of

Applicator And Plate Was Developed In A Twin Trough

Chamber. Scanning Was Performed In The Reflectance-

absorption Mode Using UV-detector In The Range Of

200-300nm.Both DAPA and SAXA Showed Reasonable

Good Response At 225nm and 215nm.

Dapagliflozin

at 225nm Saxagliptin at

215nm


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Figure 9: wavelength selection for individual

Dapagliflozin propanediol and Saxagliptin

hydrochloride.

The concentration of standard mixture solution of DAPA

and SAXA (1000 ng/band) were spotted in form of bands

of width 6mm using a 100µl syringe on precoated silica

Gel aluminum plate 60F254

(10 *10 cm) then all plates

scan densiometrically at different wavelength using

CAMAG TLC scanner.

Fig 10: standard mixture solution of DAPA and

SAXA (1000 ng/band)scan at 225nm.

Fig 11: Standard mixture solution of DAPA and

SAXA (1000 ng/band) scan at 220nm.

Fig 12: standard mixture solution of DAPA and

SAXA (1000 ng/band) scan at 210nm.

Table 11: selection of wavelength.

Wavelenght Scan At AREA

DAPA SAXA

225nm 14533 15870

220nm 15157 17979

210nm 15337 18921

Conclusion: good peak area at 210nm

Selection of wavelength for analysis of mixture

sample

Selection of Mobile Phase

The optimize mobile phase was selected after number of

trails using different reagents .The optimize mobile

phase consisting of mixture of “Chloroform: Ethyl

Acetate: Methanol: Ammonia (6.0:2.0:2.0:0.2 v/v/v)”

Figure 23: mobile phase: CHCl3: Ethyl Acetate:

Methanol: Ammonia (6:2:2:2 drops) v/v/v.

Assay of Dapagliflozin and saxagliptin in marketed

tablet formulation


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Figure 24: Chromatogram of Synthetic Mixturer of Dapagliflozin and saxagliptin in (CHCl3: Ethyl.

Acetate: MeOH: ammoniam (6:2:2:2 Drops) %v/v/v) at 210 nm

Assay Result of Marketed Formulation

Table 12: Assay result of Marketed formulation.

Drug Actual conc. Of

Drug (ng/band)

Amt. Of Drug

Found (ng/band)

% of Drug

found

Avg. Of %

Drug found SD %RSD

Dapagliflozin

1000 999.7 99.97

99.94

0.1334

0.1335 1000 1000.6 100.06

1000 998 99.80

Saxagliptin

500 449.9 99.98

100.38 0.8676 0.8643 500 501.8 101.38

500 448.9 99.79

VALIDATION OF THE PROPOSED METHOD

Linearity and range

Calibration graphs were plotted using peak areas of

standard drug v/s concentration of standard drug

solutions. From the determination it can be concluded

that the linear correlation was achieved between 500-

4000 (ng/band) & 500-2000 (ng/band) for DAPA &

SAXA respectively. The calibration curves for DAPA &

SAXA.

Figure 25: 3-D Chromatogram for calibration Curve

of dapagliflozin and saxagliptin.

Linearity for dapagliflozin

Table 13: Linearity for Dapagliflozin.

Conc.

(ng/band)

Area. Mean ± S.D (n=3) %

RSD

0 0 0

500 1035.20± 3.43 0.33

1000 1533.96±3.40 0.22

1500 2333.16±4.47 0.19

2000 2864.70±3.23 0.11

2500 3455.60±1.42 0.04

3000 4046.50±2.42 0.05

3500 4690.23±1.03 0.02

4000 5360.20±2.15 0.04

Figure 26: Calibration curve of dapagliflozin

propanediol.


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Linearity for saxagliptin

Table 14: Linearity for saxagliptin.

Conc.

(ng/band) Area. Mean ± S.D (n=3)

%

RSD

0 0 0

250 582± 3.60 0.62

500 1095.6.9±3.77 0.34

750 1684.8±1.70 0.10

1000 2065.5±3.45 0.16

1250 2675.7±3.45 0.12

1500 3078±4.11 0.13

1750 3581.6±3.43 0.09

2000 4083.1±1.51 0.03

Figure 27: Calibration curve of Saxagliptin

hydrochloride.

Statistical data of dapagliflozin propanediol and

saxagliptin hydrochloride

Table 15: Statistical data of dapagliflozin propanediol

and saxagliptin hydrochloride.

Parameters

Result

Dapagliflozin

propanediol

Saxagliptin

hydrochloride

Linearity

Range

(ng/band)

500-4000 250-2000

Slope 0.052 0.919

Intercept 32.56 116.47

Rf value 0.31 0.54

Correlation

Coefficient

(r2

)

0.9957 0.9969

Precision

A. Repeatability Study

Table 16: Repeatability data for dapagliflozin and

saxagliptin.

Drug Concentration

(ng/band)

Abs. Mean ±

S.D (n=6)

%

R.S.D

DAPA 2000 2865.71±1.89 0.06

SAXA 1000 2065.5±1.85 0.08

B. Intraday

Table 17: Intraday data for dapagliflozin and saxagliptin.

Conc. (ng/band)

Dapagliflozin

Conc. (ng/spot)

Saxagliptin

Mean Area ±S.D.

(n = 3) %R.S.D.

Mean Area ± S.D.

(n = 3) %R.S.D.

1000 1534.9± 2.12 0.13 500 1095.6±3 0.27

2000 2865.13±2.91 0.10 1000 2065.5±3.25 0.15

3000 4043.6±1.9 0.13 1500 3076.3± 3.93 0.12

C. Interday

Table 18: Interday data for dapagliflozin and saxagliptin.

Conc. (ng/band)

Dapagliptin

Conc. (ng/spot)

Saxagliptin

Mean Area ± S.D.

(n = 3) %R.S.D.

Mean Area ± S.D.

(n = 3) %R.S.D.

1000 1533.9 ±1.35 0.08 500 1095.6 ±1.52 0.13

2000 2864.1 ±1.51 0.05 1000 2063.5 ± 1.66 0.08

3000 4043.6 ±1.9 0.04 1500 3073.0 ±2.11 0.06


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Accuracy

Table 19: Accuracy data for dapagliflozin.

%Recovery Target Conc.

(ng/band)

Spiked Conc.

(ng/band) Final Conc. (ng/band) Conc. Obtained(ng/band) %recovery

0 %

1000 0 1000 992.5 99.25

1000 0 1000 1000.5 100.5

1000 0 1000 993.9 99.39

50 %

1000 500 1500 1493 99.57

1000 500 1500 1491.36 99.42

1000 500 1500 1504 100.27

100 %

100000 1000 2000 1998.91 99.94

1000 1000 2000 1991.9 99.59

1000 1000 2000 2005.8 100.27

150%

1000 1500 2500 2472.2 98.58

1000 1500 2500 2532.28 101.28

1000 1500 2500 2501.31 100.05

Table 20: Accuracy data for saxagliptin.

%Recovery Target Conc.

(ng/band)

Spiked Conc.

(ng/band)

Final Conc.

(ng/band)

Conc. Obtained

(ng/band) %Assay

0 %

500 0 500 498.6 99.6

500 0 500 495.0 99

500 0 500 501.5 100.3

50 %

500 250 750 746.9 99.58

500 250 750 738.24 98.43

500 250 750 754 100.61

100 %

500 500 1000 989.1 98.91

500 500 1000 1018.34 101.83

500 500 1000 980.5 98.06

150%

500 750 1250 1252.59 100.17

500 750 1250 1235.38 98.81

500 750 1250 1250.44 100.03

Specificity

The specificity of the method was ascertained by

analyzing standard drugs and sample of DAPA and

SAXA. The results suggested that proposed method is

specific, the excipients present in the formulation does

not affect the result. The chromatogram taken by running

with mobile phase, diluent, Dapaglflozin Api and

Saxagliptin Api market formulation.

Fig. 28: 3- D Chromatogram of specificity.

Robustness

Table 21: Robustness data for dapagliflozin.

Sr.

No.

dapagliflozin (2000 (ng/band))

Saturation Time Mobile phase

(+0.2 Unit) (-0.2 unit) (+2.0 %) (-2.0%)

1 1545 1543 1540.93 1535.6

2 1546.5 1542 1540.50 1536.8

3 1550 1540.3 1538.6 1534.2

SD 2.56 1.36 1.23 0.80

Mean 1547.16 1541.76 1540.01 1535.13

%

RSD 0.16 0.08 0.08 0.05

Table 22: Robustness data for saxagliptin.

Sr.

No.

Saxagliptin (1000 (ng/band))

Saturation Time Mobile phase

(+0.2 unit) (-0.2 unit) (+2.0%) (-2.0%)

1 1096 1095.3 1095.63 1093

2 1096.3 1093.6 1096.2 1092.5

3 1094.3 1092.6 1098 1094

SD 1.07 1.36 1.23 0.76

Mean 1095.53 1093.83 1096.61 1093.16

%

RSD 0.09 0.12 0.11 0.06


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L.O.D. AND L.O.Q

Table 23: L.O.D. and L.O.Q. data for dapagliflozin

and saxagliptin.

Parameter Dapagliflozin Saxagliptin

L.O.D. 159.95 82.26

L.O.Q. 484.71 249.29

SUMMARY AND CONCLUSION

The proposed derivative ratio spectrophotometric, TLC

and HPLC methods are simple, precise, accurate, and

sensitive. These methods have wider range with good

accuracy and precision. They can be used for the routine

analysis of both drugs in pharmaceutical formulations.

AKNOWLEDGEMENT

The authors are thankful to CTX LABORATORY

PVT.LTD, SURAT, Gujarat, for providing the

Saxagliptin Hydrochloride and Dapagliflozin API as a

gift sample.

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