Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy(ATR-FTIR)

Method for the Determination of Dapagliflozin in Tablets

Bassel Al Sabbagh, Bontha Venkata Subrahmanya Lokesh*, Gabriel Akyirem Akowuah

Faculty of Pharmaceutical Sciences, UCSI University, No.1 Jalan Menara Gading, UCSI

Heights, 56000 Kuala Lumpur, Malaysia

* Faculty of Pharmaceutical Sciences, UCSI University, no.1 Jalan Menara Gading, UCSI Heights,

56000 Kuala Lumpur, Malaysia; Tel: +603-9101 8880, Ext:3771; E-mail:

lokeshb@ucsiuniversity.edu.my; bvslk71@yahoo.com

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Abstract

Dapagliflozin (DAPA) is a promising novel antidiabetic agent approved by the Food and Drug

Administration in 2014. There is a lack of analytical methods for routine quality control of DAPA

in pharmaceutical formulations. A validated Attenuated Total Reflectance Fourier Transform

Infrared Spectroscopy (ATR-FTIR) method is developed for the determination of DAPA in tablets.

The calibration curve is constructed on peak height location at a specific wavenumber of 1065 cm-1

with correlation coefficient of 0.997. The limit of detection(LOD) and limit of

quantification(LOQ) were 0.008(%w/w) and 0.04 (%w/w), respectively. The method was found to

be precise over a range of 10- 100%, with intra-day and inter-day precision values less than 8 and

11, respectively. The percentage of mean recovery was estimated at 99.65 ±0.74. The validated

method was used for the quantification of DAPA in tablets and percentage of labeled amount was

found to be 99.65 ±0.74. No significant interference was observed by excipients in the tablet

formulation during the spectral analysis.

Keywords:

Attenuated Total Reflectance, Fourier Transform Infrared (FT-IR) Spectroscopy, Dapagliflozin,

Determination, Tablets

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1. Introduction

Dapagliflozin (DAPA) is a well-designed, competitive, stable, reversible, highly selective and

orally active inhibitor of sodium glucose co-transporter 2 (SGLT2) [1]. The chemical structure of

DAPA, a synthetic aryl glycoside is shown in (Figure 1). DAPA is approved by Food and Drug

Administration (FDA) as SGLT2 Inhibitor [2]. DAPA has gained huge interest in pharmaceutical

and analytical fields due to its importance of enhancing and improving health of patients with Type

2 diabetes.

Figure 1. The Chemical Structure of DAPA

There are literature reports on chromatographic analysis of DAPA in pharmaceutical

formulation and in biological samples such as plasma and urine [3, 4, 5, 15]. UV-Spectroscopy

method for the analysis of DAPA using methanol as solvent and detection at 225 nm, 235 nm, and

237 nm has been reported [6]. The reported method required preparations of sample and reagent

which are time consuming in addition to the high cost due to the usage of large volume of costly

solvents and reagents. Therefore, there is a need for an alternative method which would be simple,

fast, and cost-effective for the routine analysis of DAPA in tablets. Attenuated total reflectance

Fourier transform infrared (ATR-FTIR) spectroscopy represents alternative method to achieve

these aims. ATR-FTIR is non-destructive method for quantitative analysis of samples with no or

minimal work for sample preparation. There is no reported FTIR method for quantification DAPA

in tablets. There is no official method to date for the quantification and identification of DAPA

because it has not been official in any pharmacopoeia. In this study, sensitive and simple ATR-

FTIR method was developed for the routine analysis of DAPA. The method was validated

according to the International Conference on Harmonization (ICH) guidelines [16] and

the method was also then applied for the detection and quantification of DAPA content in

commercially available tablets.

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2. Experimental

2.1 Materials and reagents

Dapagliflozin was purchased from Finetech Chemical Company (Nanjing, China) potassium

bromide, and ethanol (HPLC) were purchased from Merck (Darmstadt, Germany). Dapagliflozin

tablets were purchased from local pharmacy in Kuala Lumpur, Malaysia.

2.2 Instrumentation

Infrared spectra were obtained with Nicolet™ iS5 FTIR spectrometer (Thermo Scientific,

Madison, Wisconsin, USA) with iD5 ATR accessory featuring a diamond crystal. The spectra

were collected against the diamond window background controlled by OMNIC software for

spectra collection and TQ Analyst software for Data processing (Thermo scientific, USA). The

instrument is equipped with iD5 ATR accessory featuring a top plate diamond crystal with a fixed

angel of incidence of 42°.

2.3 FTIR-ATR method development

All spectra were recorded at 4 cm-1 resolution with average of 16 scans in the range of 4000

– 600 cm-1. In addition, a scan of the air background was always applied before scan of sample. An

analytical balance with 0.01 mg readability and minimum weight of 2 mg in fine range was used.

All measurements were taken at ambient temperature and samples were stored in a desiccator

when not be used. Ethanol (95%) was used to clean the diamond crystal intermittently between

each scan. DAPA tablets 5 mg strength were used for the quantitative determination of DAPA. IR

grade KBr was used to dilute the standard. A series of concentrations of DAPA standard ranging

from 10-100% w/w were prepared by diluting with potassium bromide. Different amounts of

DAPA were weighed and mixed with weighed amount pure KBr, to get a total weight of 0.1 mg of

each working standard. The mixing of KBr and DAPA standard was done in FTIR mortar till

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homogenization. The mixture was transferred to Eppendorf tube then mixed by using vortex for 5

min. DAPA working standards were used to plot a standard calibration curve with six points

ranging from 10-100 % w/w. Each working standard was applied on the ATR-FTIR to scan for its

spectrum in the range of 4000 to 600 cm-1. A background scan of air was done prior to each

scanning of the working standards. All spectra were recorded at 4 cm-1 resolution with average of

16 scans per spectrum. The diamond crystal of the ATR accessory was cleaned using ethanol

before each application of the working standards. The calibration curve was generated through the

TQ analyst software following the Beer’s Law and utilizing the FTIR spectra of the working

standards. Different wavenumbers ranging from 2970 to 600 cm-1 were selected and two types of

regions including fixed height location and peak area were used in order to select the most suitable

location and peak area, respectively to generate two calibration curves of which their R2 is higher

than 0.99. The data for the calculated versus the actual measurements of the standards was used for

the linear regression analysis [7].

2.4. Validation of the FTIR-ATR method

The proposed method was validated according to the ICH guidelines [8]. The parameters

like linearity, sensitivity, precision, and accuracy were studied and evaluated as per guidelines.

2.4.1 Linearity

Five different standard concentrations at 10, 20, 40, 80, 100% w/w of DAPA standard were

used to plot a standard calibration curve. The calibration curve was generated through the TQ

analyst software following the Beer’s law. Different wavenumbers ranging from 2970 to 600 cm-1

were selected. Fixed height location were used in order to select the most suitable peak at 1065 cm-

1 to generate the calibration curve. The data for the calculated versus the actual measurement of the

standards was used for the linear regression analysis.

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2.4.2 Sensitivity

The sensitivity of the method was determined from the calibration curve with respect to

limit of detection (LOD) and limit of quantification (LOQ) as described by Boyd and Kirkwood

[9]. The LOQ is based on the standard deviation of the response and the slope. The LOD is

determined when the peak height for the sample is triple to that of peak to peak noise from

surrounding region in the spectra. LOQ and LOD were calculated by the equations below.

LOQ= 10 x SD/S

LOD = 3.3 x SD/S

where SD is the standard deviation and S is the slope of the calibration curve.

2.4.3 Precision

The precision of the FTIR system was evaluated by the intra-day (repeatability) and inter-day

(reproducibility) by using the following quality control (QC) standards: 10% w/w (low), 40 and 60

%w/w (medium) and 100% w/w (high) concentrations of pure DAPA with KBr. Intra- day

precision was evaluated by measuring the QC standards in three replicates each on the same day.

For the inter-day precision, three replicates of the QC standards were measured over three

consecutive days. The relative standard deviation (%RSD) of the selected fixed location height

was used to express the precision.

2.4.4 Accuracy

The accuracy of the FTIR method was evaluated by calculating the percentage recovery of

DAPA standard. One DAPA tablet was grounded to fine powder. The powder of the tablet was

then spiked with accurately weighed amounts of DAPA. The tablet powder was spiked with 10, 40

and 100 %w/w of DAPA standards, respectively. The FTIR spectrum of each spiked sample was

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collected three times and the accuracy was expressed as the mean percentage recovery of three

replicates for each spiked sample.

2.4.5 Quantification of DAPA based on FTIR method

After the validation of the FTIR method, the quantification of the DAPA in tablet was

carried out based on the calibration curve. DAPA content in the tablets was determined by

grinding the tablet and directly applying it on the ATR accessory to get the corresponding FTIR

spectrum. The quantification was carried out in triplicate and the results were expressed as mean ±

standard deviation.

3.0 Results and discussion

3.1. Method Development

During the development of the ATR-FTIR method, the spectra of the standard DAPA and

DAPA tablets were obtained initially as a primary step in the qualification and quantification

process. The FTIR spectra were scanned from 4000- 600 cm -1. The ATR-FTIR spectrum of pure

DAPA standard and DAPA tablet are shown in Figures 2 and Figure 3, respectively. The IR

spectra showed peaks corresponding to the functional groups in the chemical structure that could

be used for qualitative and quantification analysis. The FTIR spectra showed a hydroxyl group O-

H that range from 3570 - 3100 cm-1, C-H stretch group was found between 2800 -3000 cm-1,

alkane CH3 that range from 1375- 1450 cm-1, alkene C=C in benzene rings that ranging from 1475-

1600 cm-1. Interesting to note that, DAPA has two types of ether C-O groups from cyclic ether and

phenyl alkyl ether. The cyclic ether C-O group is indicated by the peaks between 1130-1110 cm-1,

and Phenyl alkyl ether C-O at 1040 cm-1 were clearly observed [10]. Hydroxide, alkane, and

alkenes functional groups could be identified in almost any organic substance hence the peaks

corresponding to these functional groups were not considered for quantification purpose in this

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research to avoid any interference of similar peaks from the excipient in the tablet. In this research

the C-O (stretch) from ether and alcohol groups was selected for qualitative and quantitative

analysis. The FTIR spectrum of the tablet also showed C-O (stretch) peaks corresponding to the

ether and alcohol groups from 1300 to 1000 cm-1.

A set of five working standards with concentration from 10 to 100% w/w was used for the

generation of the calibration curve. The calibration was performed by TQ analyst software based

on Beer’s law. The fixed height location of the peaks was used for the generation of the calibration

curve by scanning the major peaks from 2970 to 600 cm-1 in order to get the highest correlation

coefficient with a value more than 0.99. TQ analyst calibration of DAPA based on fixed height

location of the peaks from 2970 to 600 cm-1 of the calibration standards showed higher r2 value >

0.9972 for the peak at 1065 cm-1 hence this peak was selected for method validation and

quantification of DAPA in tablets. Figure 4a shows overlay spectra of the calibration standards

(10, 40 and 100 % w/w). The calibration curve of calculated concentration against actual

concentration is shown in Figure 4b.

3.2. Method validation

Table 1 shows the results of validation parameters for ATR-FTIR analysis of DAPA in

tablets at 1065 cm-1. The calibration curve gave correlation coefficient of 0.9972 which indicates

good linearity. The method was considered sensitive with the calculated LOD and LOQ values of

0.008%w/w and 0.04 %w/w, respectively. The calculated % RSD values for intra-day precision and

inter-day precision were 8% and 11% indicating good repeatability of the proposed method.

Excellent mean recovery percent value of 99.65±0.74% were obtained, which indicated high

accuracy of the proposed method.

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3.3. Quantification of DAPA in tablets

The validated proposed ATR-FTIR method was used for the quantification of DAPA

tablets. FTIR spectra of the tablets were collected using OMNIC software. The DAPA

concentration was calculated using the regression equation from the calibration curve, using TQ

analyst software. Table 2 shows the results of quantification of three DAPA tablets. The mean

percentage of labeled amount was found to be 99.65% with RSD of 0.74%, and the tablet

excipients were showed no interference with the analysis. The results of the proposed FTIR

method was comparable to reported HPLC techniques [11, 12].

ATR accessory employs the phenomenon of total internal reflection. The IR beam entering

the crystal undergoes total internal reflection when the angle of incidence (θi) at the interface

between the sample and crystal is greater than the critical angle (θc). The critical angle, described

by Snell’s law is a function of the refractive indices of the two surfaces. Total internal reflection of

the beam to occur only when the refractive index of the crystal¿) is higher than the refractive index

of the sample (n2) [13]. In this study, diamond crystal with refractive index (n1=2.4) was used,

which is higher than the refractive index of DAPA (n2=1.61). Hence, DAPA can be successfully

analyzed using diamond ATR crystal. The main limitation of using diamond crystal is the intrinsic

absorption from approx. 2300 cm-1 to 1800 cm-1 [14]. However, this region of IR spectrum was not

used for the quantification hence, this limitation did not affect the results.

Attenuated Total Reflection (ATR) accessory provide a better option for quantification of

solid samples. It eliminates problems associated with pellet formation, and avoids the interference

caused by solvents. It involves only mixing the sample with small amount of KBr (or without

dilution with KBr) and directly spreading a small amount of the powder on the internal reflection

element (IRE). It is quick and non-destructive to substances in terms of quantification. The use of

ATR accessory with FTIR provides advantages over the widely used transmission accessory with

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regards to handling solid samples. The analysis of solid samples using transmission FTIR involves

formation of KBr discs which has some limitations during quantification process because it is

difficult to achieve consistent thickness of the discs used with different concentrations. Moreover,

the use of mull and film methods in IR analysis of solids require the dilution of analyte with liquid

paraffin and volatile solvent, respectively. This will lead to the scattering of the IR beam and thus

affecting the transmission. On the other hand, ATR accessory eliminates the formation of disc as it

only requires mixing the analyte with small amount of KBr and then applying it directly on the

instrument. Furthermore, it avoids diluting the solid samples with solvents and thus excludes the

presence of interferences in the FTIR spectrum as a result of peaks from the solvent.

4.0 Conclusion

The FTIR method developed for DAPA was sensitive for the quantitative determination of

DAPA in tablet formulation. The technique is simple, fast and excludes the usage of solvents thus

reducing the cost of overall analysis. The ATR-FTIR method provides technique that can be used

for routine analysis of DAPA in pharmaceutical products.

Conflicts of interest

The contributing authors have no conflict of interest.

Acknowledgement

This work was conducted and performed under the support of Faculty of Pharmaceutical Sciences,

UCSI University, Kuala Lumpur, Malaysia.

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References

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Table 1. Validation Parameters for ATR-FTIR analysis of Dapagliflozin in tablets

Validation parameters Fixed location height (1065 cm-1)

Linear range (% w/w) 10 – 100

Correlation coefficient (r) 0.9972

Regression equation (y = a + bc)

Slope (b)

Intercept (a)

0.182

32.5

Limit of detection (% w/w) 0.008

Limit of quantification (% w/w) 0.04

Intraday Precision (n=5, % RSD)

10 %w/w

40 %w/w

100 %w/w

7.65

6.81

4.91

Interday Precision (n=5, % RSD)

10 %w/w

40 %w/w

100 %w/w

10.24

8.48

7.49

Recovery (n= 3)

Mean

% RSD

96.15

3.59

RSD = Relative standard deviation; % RSD = [(S.D./mean) × 100].

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Table 2. Results of quantification of Dapagliflozin tablets

Sample Dapagliflozin labeled(mg/tablet) Percentage of labeled amount* (%)

1 5 99.6

2 5 92.70

3 5 96.16

Mean ± SD = 96.15 ± 3.45

*percentage of labeled amount = (amount found/ amount labeled) X 100

Figure 1. Chemical Structure of Dapagliflozin.

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Figure 2. ATR-FTIR Spectrum of Dapagliflozin standard

Figure 3. ATR-FTIR Spectrum of Dapagliflozin tablets

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Figure 4. TQ analyst calibration of Dapagliflozin based on fixed height location at 1065 cm-1

(A) Overlay ATR-FTIR spectra of the calibration standards (10, 40, and 100 % w/w), (B)

The calibration curve of calculated concentration against actual concentration

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(A)

(B)