Ph.D. viva voce presentation on Anti-diabetics and statins

DEVELOPMENT AND VALIDATION OF
NEW ANALYTICAL METHODS
FOR THE ESTIMATION OF
ANTI-DIABETICS AND STATINS
Ph.D. Viva-voce
By
Ms. K. Sravana Kumari, M.Pharm.,
Under the guidance of
Dr. B.Sailaja, M.Pharm., Ph.D.
Professor, IPT, SPMVV
Institute of Pharmaceutical Technology
SRI PADMAVATI MAHILA VISVAVIDYALAYAM
Tirupati, A.P.
17-09-2020
1

CONTENTS
 Introduction
 Review of Literature
 Aim and Objectives
4a. UV Spectroscopic method for Rosuvastatin Calcium
4b. Colorimetric method for Rosuvastatin Calcium
4c. Stability Indicating RP-HPLC method for Rosuvastatin Calcium
4d. UV Spectroscopic method for Pitavastatin Calcium
5a. QbD enabled Stability indicating RP-HPLC method for
Empagliflozin 2

3
5b. UV Spectroscopic method for Canagliflozin
6a. Stability indicating simultaneous RP-HPLC method for
Ertugliflozin Pidolate and Metformin Hydrochloride
6b. Stability indicating simultaneous RP-HPLC method for
Empagliflozin and Linagliptin
 In-vitro Bioanalytical RP-HPLC method for Empagliflozin
 Summary and Conclusion
 References
List of Presentations and Publications
Acknowledgements

5
In pharmaceutical industry, there is a need for the invention of suitable novel
analytical methods from time to time for testing the quality of
bulk drugs, excipients and formulations
Method development and validation is an integral part of drug discovery and
drug development
UV-Visible Spectroscopy and HPLC are the most popular techniques used for
the identification and estimation of drugs with good accuracy and precision
Simultaneous method development is useful for analysis of combination of
drugs [Rajashree Mashru et al., 2015]

6
QbD approach helps to develop a more robust and cost-effective analytical
method by studying simultaneous influence of more than one variable on
method optimization [Ahuja and Scypinski, 2013]
Bioanalytical method development is used for the estimation of drugs and their
metabolites in various biological fluids like blood, urine, plasma, serum, saliva
and cerebrospinal fluid
Worldwide, around 463 million people are suffering from type-2 Diabetes
mellitus [International Diabetes Federation, 2019]
 Type-2 Diabetes mellitus patients are at increased risk for Dyslipidemia
associated cardiovascular diseases

7
Globally, about 1/3rd of ischemic heart disease and 1/5th of cerebro-vascular
disease are due to Dyslipidemia and equates to nearly 2.6 million deaths
[WHO global estimates, (2014)]
Statins are used for treating Dyslipidemia associated cardiovascular risk
Rosuvastatin Calcium (10 mg) and Pitavastatin Calcium (4 mg), effectively
reduces LDL cholesterol and triglyceride levels [Sharma and Sharma, 2017]
Empagliflozin, Ertugliflozin and Canagliflozin (SGLT-2 inhibitors), are widely
used for controlling plasma glucose levels with decreased cardio-vascular risk
[Ralston et al., 2018]

8
Combination of Ertugliflozin with Metformin effectively reduces HbA1c levels in
chronic diabetic patients [FDA label, Segluromet, 2017]
Combination of Empagliflozin and Linagliptin [stimulates inactive beta cells]
exerts synergistic effect compared to mono-drug therapy [Katzung et al., 2015]
 There will be always a need for the development and validation of novel
analytical methods for the estimation of drugs in bulk and dosage forms in order
to deliver good quality and affordable medicines to patients

9
The present study was focused on the development and validation of novel
analytical techniques for the estimation of
Rosuvastatin Calcium by UV-Visible Spectroscopic and stability indicating
RP-HPLC methods
Pitavastatin Calcium by UV Spectroscopic method
Empagliflozin by RP-HPLC using QbD approach
Canagliflozin by UV Spectroscopic method
Simultaneous stability indicating RP-HPLC methods for Ertugliflozin and
Metformin and for Empagliflozin and Linagliptin in bulk and tablets
In-vitro bioanalytical method for Empagliflozin in human plasma by RP-HPLC

11
ESTIMATION OF ROSUVASTATIN CALCIUM IN BULK AND TABLET
FORMULATION BY UV SPECTROSCOPY
Ref Solvent
λ max
[nm]
Linearity
[μg/ml]
LOD
[μg/ml]
LOQ
[μg/ml]
Priyanka Pannure
et al., 2018
ACN:
Methanol
(6:40)
242 4-24 0.75 2.27
Singh
et al., 2018
Methanol 244 2-18 - -
Bokhare
et al., 2018
Phosphate
buffer pH 7.4
246 10-150 - -
Lahare
et al., 2014
Methanol
Absorption max: 252
AUC method : 247-257
First order derivative:
Maxima-238
Minima- 205
5-35 - -
Gupta
et al., 2008
Methanol 244 2-18
Uyar
et al., 2007
Methanol 243 1-60 0.33 1.00

12
ESTIMATION OF ROSUVASTATIN CALCIUM IN BULK AND TABLET
FORMULATION BY COLORIMETRY
Ref Reagent Solvent λ max
Linearity
[μg/ml]
LOD
[μg/ml]
LOQ
[μg/ml]
Lima
et al., 2017
Quinalizarin Methanol 579 6-15 0.9 mg/ml 3.0 mg/ml
Ramadan
et al., 2015
Bromocresol
green
Chloroform 416 0.482-24.077 0.045 0.13
Ramadan
et al., 2014
Iodine ACN 360 2.408-48.154 0.28 0.85
Dwivedi
et al., 2011
Cosneasie
Brilliant Blue R
- 595 100-500

ESTIMATION OF ROSUVASTATIN CALCIUM IN BULK AND
TABLET FORMULATION BY RP-HPLC
13
Ref Chromatographic Conditions Validation Parameters
Hassouna et al., 2017 Column : Eclipse XDB C8
(250mm x 4.6mm, 5μm)
Mobile Phase : pH 4.5 0.05M Sodium
dihydrogen phosphate
buffer : ACN (50:50 % v/v)
Flow rate : 1.2 ml/min
Wavelength : 245 nm
Diluent : Methanol
Elution : Isocratic
Inj. Vol. : 10 μl
Temperature : Ambient
Run time : 5 mins
Rt : 3.684 mins
Linearity : 5-100 µg/ml
LOD : 1.5µg/ml
LOQ : 4.56µg/ml
Sirisha et al., 2017 Column : Agilent zorbax RP C18
(150mm x 4.6mm, 5μm)
Mobile Phase : Methanol: OPA buffer
(pH 3.1) [35:65 % v/v]
Wavelength : 282 nm
Diluent : Mobile phase
Elution : Isocratic
Inj. Vol. : 50 μl
Run Time : 5 mins
Rt : 1.323 mins
Linearity : 0.5-16 µg/ml
LOD : 3.5 µg/ml
LOQ : 10.5 µg/ml

14
Suares and
Prabhakar, 2016
Column : Kromasil C18
(250mm x 4.6mm, 5μm)
Mobile phase : Buffer pH 4.8 (0.78 %
NaH2PO4) : ACN (50:50 % v/v)
Wavelength : 241nm
Diluent : Mobile phase
Elution : Isocratic
Inj. Vol. : 20 µl
Temperature : ambient
Run Time : 8 mins
Rt : 4.72 mins
LOD : 0.17 µg/ml
LOQ : 0.7 µg/ml
Ebru Cubuk
Demiralay et al.,
2016
Column : X-terra -C18
(250mm x 4.6mm, 5µm)
Mobile Phase : ACN : Water (50:50)
Wavelength : 242 nm
Diluent : ACN, Mobile Phase
Elution : Isocratic
Inj. Vol. : 20 µl
Temperature : 250C
Run time : 10 mins
Rt : 5.502 mins
LOD : 0.221 µg/ml
LOQ : 0.670 µg/ml

15
Rambabu
et al., 2015
Column : Thermo-hypersil BDS
C18 (150mm x 4.6mm, 5μm)
Mobile phase : Buffer (KH2PO4) : ACN
(40:60 v/v)
Wavelength : 243 nm
Diluent : Water : ACN (90:10)
Elution : Isocratic
Inj. Vol. : 20 µl
Temperature : 300C
Run Time : 12 mins
Rt : 3.806 mins
Linearity: 5-30 µg/ml
LOD : 0.041 µg/ml
LOQ : 0.1434 µg/ml
Prabhu
Venkatesh
et al., 2015
Column : Chiralpak IB
(250mm x 4.6mm, 5μm)
Mobile Phase : n-Heptane, 2-Propanol :
Trifluoroaceticacid (85:15:01v/v)
Wavelength : 242 nm
Diluent : HPLC water
Elution : Isocratic
Temperature : 25OC
Inj. Vol. : 10 μl
Run Time : 24 mins
Rt : 12.524 mins
LOD : -
LOQ : -

16
Usha Rani et al., 2014 Column : Eclipse XDB plus C18
(150mm x 4.6mm, 5μm)
Mobile Phase : ACN: Water (60:40)
Wavelength : 242 nm
Diluent : Mobile Phase
Elution : Isocratic
Inj. Vol. : 20 μl
Run time : 10 mins
Rt : 1.787 mins
LOD : 0.08 µg/ml
LOQ : 0.25 µg/ml
Gowrisankar et al., 2013 Column : Luna C18
(250mm × 4.6 mm, 5μm)
Mobile Phase : Triethylamine buffer
pH 4.5: ACN : Methanol
(45:25:35)
Wavelength : 248 nm
Elution : Isocratic
Inj. Vol. : 20 μl
Run Time : 11 mins
Rt : 7.18 mins
LOD : 0.2 µg/ml
LOQ : 0.55 µg/ml

17
Anup Kumar
Chakraborty et al.,
2011
Column : Phenomenex-C18
(250mm x 4.6mm, 10µm)
Mobile Phase : Buffer: ACN (55:45)
Buffer : Ammonium acetate pH 5 with
Glacial acetic acid
Wavelength : 244 nm
Elution : Isocratic
Inj. Vol. : 20 μl
Temperature : -
Run time : 10 mins
Rt : 6 mins
Linearity : 70-130 %
LOD : -
LOQ : -
Donthula et al.,
2011
Column : Luna C18
(250mm x 4.6mm, 5μm)
Mobile Phase : Buffer (pH 4.5): ACN:
Methanol (45:25:35)
Wavelength : 248 nm
Diluent : Methanol
Elution : Isocratic
Inj. Vol. : 20 μl
Temperature : -
Run Time : 20 mins
Rt : 9.9 mins
Linearity: 25-75 µg/ml
LOD : 3.5 µg/ml
LOQ : 10.5 µg/ml

18
Pandya et al., 2010 Column : Thermo hypersilC18
(100mm x 4.6mm, 5μm)
Mobile Phase : ACN : KH2PO4 (50:50, pH 3)
Wavelength : 243 nm
Diluent : Methanol, Mobile Phase
Elution : Isocratic
Run Time : 5 mins
Rt : 3.333 mins
LOD : 0.14 µg/ml
LOQ : 0.46 µg/ml
Kaila et al., 2010 Column : YMC C8
(150mm × 4.6mm, 5μm)
Mobile Phase : ACN: Water
(40:60, v/v) pH 3.5
Wavelength : 242 nm
Diluent : Water: ACN (50:50)
Elution : Isocratic
Inj. Vol. : 20 μl
Run time : 10 mins
Rt : 4.86 mins
LOD : 0.1 µg/ml
LOQ : 0.5 µg/ml

19
Lakshmana Rao and
Suneetha, 2010 Column : RP-C18 column
(100mm x 4.6 mm, 3μm)
Mobile Phase : 0.02M phosphate buffer
pH 6.8 : ACN (60:40 v/v)
Wavelength : 242 nm
Diluent : ACN, Mobile phase
Elution : Isocratic
Inj. Vol. : 20 μl
Run Time : 5 mins
Rt : 3.424 mins
LOD : 0.017 µg/ml
LOQ : 0.052 µg/ml
Hasumathi et al., 2009 Column : Phenomenex-C18
(250mm x 4.6mm, 5µm)
Mobile Phase : ACN : 0.5% Formic acid
(50:50)
Wavelength : 248 nm
Diluent : ACN
Elution : Isocratic
Inj. vol. : 20 µl
Run time : 10 mins
Rt : 6.742mins
LOD : 0.0905 µg/ml
LOQ : 0.318 µg/ml

20
ESTIMATION OF PITAVASTATIN CALCIUM IN BULK AND
TABLET FORMULATION BY UV SPECTROSCOPY
Ref Solvent
λ max
[nm]
Linearity
[μg/ml]
LOD
[μg/ml]
LOQ
[μg/ml]
Yunoos et al., 2014 0.1 N HCl 249.5 2-12 0.122 0.371
Panchal et al., 2009 Methanol 238 10-60 0.406 1.230

ESTIMATION OF EMPAGLIFLOZIN IN BULK AND TABLET
FORMULATION BY RP-HPLC BY APPLYING QbD APPROACH
21
Kumar
et al., 2019
Column : Enable C18
(250mm x 4.6mm, 5μm)
Mobile Phase : Methanol : Water(70:30)
Wavelength : 233 nm
Elution : Isocratic
Inj. Vol. : 20 μl
Run time : 20 mins
Rt : 6.2 mins
LOD : 0.42 µg/ml
LOQ : 1.16 µg/ml
Patil et al.,
2016
Column : Phenomenex C18
(250mm x 4.6mm, 5μm)
Mobile Phase : Methanol : Water(70:30)
Wavelength : 224 nm
Elution : Isocratic
Temperature : 400C
Inj. Vol. : -
Run time : 5 mins
Rt : 4.808 mins
LOD : 0.3589 µg/ml
LOQ : 1.0876 µg/ml

22
Shyamala et al., 2016 Column : Hypersil BDS
(250mm x 4.6mm, 5μm)
Mobile Phase : 0.1% OPA buffer :ACN
(70:30)
Wavelength : 233nm
Diluent : -
Elution : Isocratic
Temperature : 300C
Inj. Vol. : -
Run time : 7 mins
Rt : 3.23 mins
LOD : 0.068 µg/ml
LOQ : 0.207 µg/ml

23
ESTIMATION OF CANAGLIFLOZIN IN BULK AND TABLET
FORMULATION BY UV SPECTROSCOPY
Ref Solvent
λ max
[nm]
Linearity
[μg/ml]
LOD
[μg/ml]
LOQ
[μg/ml]
Singh et al., 2019 Methanol 290 5-50 0.00945 2.8639
Chinta Pooja et al., 2018 Phosphate buffer 289 20-120 - -
Kaur et al., 2015 Methanol 290 5-10 0.084 0.255

24
SIMULTANEOUS ESTIMATION OF ERTUGLIFLOZIN AND
METFORMIN IN BULK AND TABLET FORMULATION BY RP-HPLC
Parameter ERTU MET
Venkateswara Rao
et al., 2019
Column : BDS C8
(150mm x 4.6mm,5μm)
Mobile Phase : KH2PO4 buffer: ACN
(55:45)
Wavelength : 224 nm
Diluent : Water : ACN
(50:50)
Elution : Isocratic
Temperature : -
Inj. Vol. : 10 μl
Run time : 6 mins
Linearity
(µg/ml)
1.875-11.25 125-750
LOD
(µg/ml)
0.07 1.70
LOQ
(µg/ml)
0.21 5.16
Rt (mins) 3.136 2.383
Nizami et al., 2018 Column : ACE C18
(150mm x 4.6mm,5μm)
Mobile Phase : KH2PO4 buffer : ACN
(70:30)
Wavelength : PDA at 240 nm
Diluent : ACN : Water (50:50)
Elution : Isocratic
Temperature : 300C
Inj. Vol. : 10 μl
Run time : 6 mins
Linearity
(µg/ml)
3-15 12.5-125
LOD
(µg/ml)
0.43 0.74
LOQ
(µg/ml)
1.30 2.24
Rt (mins) 3.203 2.106

25
SIMULTANEOUS ESTIMATION OF EMPAGLIFLOZIN AND LINAGLIPTIN
IN BULK AND TABLET FORMULATION BY RP-HPLC
Parameter EMPA LINA
Lakshmana Rao
et al., 2019
Column : Kromasil C18
(250mm x 4.6mm, 5μm)
Mobile Phase : 0.1% OPA : ACN
(60:40 % v/v)
Flow rate : 1ml/min
Wavelength : 230 nm
Diluent : -
Elution : Isocratic
Inj. Vol. : -
Temperature : -
Run time : -
Linearity
(µg/ml)
- -
LOD
(µg/ml)
0.44 0.23
LOQ (µg/ml) 1.34 0.70
Rt (mins) 2.139 2.759
Anjali Bakshi
et al., 2018
Column : Thermo C18
(250mm x 4.6mm, 5μm)
Mobile Phase : KH2PO4 buffer pH (3.4)
: Methanol (70:30 % v/v)
Wavelength : 240 nm
Elution : Isocratic
Inj. Vol. : 10 μl
Temperature : 300C
Run time : 5 mins
Linearity
(µg/ml)
50-150 50-150
LOD (µg/ml) 0.76 0.39
LOQ (µg/ml) 2.32 1.20
Rt (mins) 3.021 3.969

26
Parameter EMPA LINA
Jayalaxmi
et al., 2018
Column : Agilent C18
(150mm x 4.6mm, 5μm)
Mobile Phase : Methanol: Phosphate
buffer pH 3 (70:30)
Wavelength : 254 nm
Elution : Isocratic
Run time : 8 mins
Linearity
(µg/ml)
10-50 20-100
LOD
(µg/ml)
2.17 0.0372
LOQ
(µg/ml)
6.60 0.1125
Rt (mins) 2.999 2.804
Sirigiri
et al., 2018
Column : Hypersil ODS 3V
(250mm x 4.6mm, 5μm)
Mobile Phase : KH2PO4 (pH 2.20) – A
Water : ACN - (5:95) - B
Wavelength : 225 nm
Diluent : 1. Water : ACN (70:30)
2. Water : ACN (50:50)
Elution : Gradient
Temperature : 300C
Inj. Vol. : 10 μl
Run time : 25 mins
Linearity
(µg/ml)
100.09-400.37 20.14-80.54
LOD
(µg/ml)
- -
LOQ
(µg/ml)
- -
Rt (mins) 8.390 5.388

27
Parameter EMPA LINA
Srinivasa rao
et al., 2017
(150mm x 4.6mm, 5μm)
Mobile Phase : Methanol : Phosphate
buffer pH 3 : 70:30
Wavelength : 254 nm
Diluent : -
Elution : Isocratic
Inj. Vol. : 10 μl
Run time : 10 mins
Linearity (µg/ml) 10-50 20-100
LOD
(µg/ml)
2.17 0.0372
LOQ (µg/ml) 6.60 0.1125
Rt (mins) 3.907 2.365
Abdel Ghany
et al., 2017
Column : X-Terra
(250mm x 4.6mm, 5μm)
Mobile Phase : 0.1% aq. formic acid
buffer (pH 3.6) :
Methanol : ACN
(40:20:40)
Wavelength : 226 nm
Diluent : Methanol
Elution : Isocratic
Temperature : 250C
Inj. Vol. : 20μl
Run time : 10 mins
Linearity (µg/ml) 4-100 2-50
LOD
(µg/ml)
0.79 0.49
LOQ (µg/ml) 2.40 1.50
Rt (mins) 3.0 1.5

28
Parameter EMPA LINA
Nazneen and
Sreedevi, 2016
Column : BDS C18
(250mm x 4.6mm, 5μm)
Mobile Phase : 0.1% Perchloric acid :
ACN (60:40)
Wavelength : 230 nm
Diluent : -
Temperature : 300C
Inj. vol. : -
Elution : Isocratic
Run time : 7 mins
30mins for degradation
Linearity (µg/ml) 25-150 12.5-75
LOD (µg/ml) 0.03 0.43
LOQ (µg/ml) 0.09 1.32
Rt (mins) 2.05 4.10
Jyothirmai et al.,
2016
Column : Inertsil ODS C18
(150mm x 4.6mm, 5μm)
Mobile Phase : Methanol : ACN : 0.1%
: OPA buffer (30:60:10)
Wavelength : 246 nm
Diluent : Methanol
Temperature : -
Inj. vol. : 20 μl
Elution : Isocratic
Run time : 10.0 mins
Linearity (µg/ml) 5-30 2.5-15
LOD (µg/ml) 0.06 0.03
LOQ (µg/ml) - -
Rt (mins) 4.16 5.73

29
Parameter EMPA LINA
Madhusudhan
et al., 2015
Column : ODS C18
(250mm x 4.6mm, 5μm)
Mobile Phase : 0.1% OPA buffer :
ACN (45:55)
Wavelength : 245 nm
Diluent : Water : ACN
(50:50)
Elution : Isocratic
Temperature : 300C
Inj. Vol. : 10 μl
Run time : 7 mins
Linearity
(µg/ml)
25-150 12.76- 76.56
LOD (µg/ml) - -
LOQ (µg/ml) - -
Rt (mins) 3.6 2.2

ESTIMATION OF EMPAGLIFLOZIN IN PLASMA BY RP-HPLC
30
Padmaja et al.,
2018
(250mm x 4.6mm, 5μm)
Mobile Phase : Methanol : ACN (50 : 50)
Extraction : Liquid-liquid extraction with
30% ethyl acetate
Wavelength : PDA detector at 270 nm
Diluent : Methanol, ACN
Temperature : 350C
Inj. Vol. : 20 μl
Run time : 10.0 mins
Rt : 8.898 mins

Aim:
To develop and validate new analytical methods for the estimation of selected
anti-diabetics and statins
Objectives:
To select anti-diabetic drugs and statin drugs
To select analytical methods to be developed
UV Spectroscopic method for Rosuvastatin Calcium
Colorimetric method for Rosuvastatin Calcium
 Stability indicating RP-HPLC method for Rosuvastatin Calcium
UV Spectroscopic method for Pitavastatin Calcium
32

33
 QbD enabled stability indicating RP-HPLC method for Empagliflozin
 UV Spectroscopic method for Canagliflozin
 Stability indicating simultaneous RP-HPLC method for Ertugliflozin and
Metformin
 Stability indicating simultaneous RP-HPLC method for Empagliflozin and
Linagliptin
 In-vitro bioanalytical RP-HPLC method for Empagliflozin in human plasma
 To validate the developed methods as per ICH Q2(R1) /USFDA guidelines

34
METHOD DEVELOPMENT
AND
VALIDATION

4a. DEVELOPMENT AND VALIDATION OF
UV SPECTROSCOPIC METHOD
FOR THE DETERMINATION OF
ROSUVASTATIN CALCIUM IN BULK AND TABLETS
 Rosuvastatin Calcium (10mg) - efficiently reduces LDL and triglycerides at
lower doses [Sharma et al., 2017]
 The methods reported for the estimation of Rosuvastatin Calcium by
UV Spectroscopy are expensive as organic solvents are used as diluent
35

36
Structure
Molecular Weight-1001.137
)Ca+2
)Ca+2
Class Statins (HMG Co A reductase inhibitors)
pKa 4.00
Log P 4.19
Solubility slightly soluble in ethanol and
sparingly soluble in water and Methanol
DRUG PROFILE: ROSUVASTATIN CALCIUM

 METHOD DEVELOPMENT [Gorog, 2011]: ROSUVASTATIN CALCIUM:
Diluent: 0.1N NaOH
 Preparation of Standard stock solution-I of Rosuvastatin Calcium
[1000μg/ml]:
37
10 mg
Rosuvastatin
Calcium
+
5ml diluent
Sonicated for
10mins
+
made up to 10ml
1000μg/ml
Rosuvastatin
Calcium
 Preparation of Standard stock solution-II of Rosuvastatin Calcium [100μg/ml]:
1.0ml standard
stock-I
Diluted to 10ml
with diluent
100μg/ml
Rosuvastatin Calcium
 Preparation of working standard solution of Rosuvastatin Calcium [4μg/ml]:
0.4 ml
standard
stock-II
4μg/ml
Rosuvastatin
Calcium
Diluted to 10ml
with diluent

UV Spectrum of Rosuvastatin Calcium [λ max 240nm]
38
RESULTS AND DISCUSSION

39
VALIDATION [ICH Q2 (R1), 2005]: ROSUVASTATIN CALCIUM
% Spiked
Fixed sample
concentration
(μg/ml)
Amount Spiked
(μg/ml)
Total Amount
recovered
(μg/ml)
Mean % recovery
± SD
%RSD
80 2 1.6 3.558 98.82± 0.18 0.18
100 2 2.0 4.101 101.01±0.54 0.54
120 2 2.4 4.332 98.45 ±0.27 0.27
PRECISION
Concentration
(μg/ml)
Intra-day
Precision
Mean ± SD
% RSD
Inter-day
Precision
Mean ± SD
% RSD
1 0.082 ± 0.000577 0.704 0.084 ± 0.0015 1.817
4 0.248 ± 0.001154 0.465 0.249 ± 0.001 0.401
6 0.360 ± 0.000577 0.160 0.358 ± 0.0015 0.418
ACCURACY

40
Concentration [x]
(μg/ml)
Absorbance* [y]
1 0.082
2 0.129
3 0.187
4 0.248
5 0.304
6 0.359
Linear Regression Equation
y = mx + c y = 0.056 x + 0.021
Slope (m) 0.056
Intercept (c) 0.021
Correlation coefficient (R2) 0.999
LOD 0.0201 μg/ml
LOQ 0.0612μg/ml
LINEARITY, LOD AND LOQ OF ROSUVASTATIN CALCIUM
*Mean of three observations

41
LINEARITY PLOT OF ROSUVASTATIN CALCIUM
y = 0.0563x + 0.0211
R² = 0.999
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 1 2 3 4 5 6 7
Absorbance(AU)
Concentration [µg/ml]
Tablet brand Label claim Amount found ± SD % Assay ± SD % RSD
Roseday 10 mg 9.890 ± 0.138 98.90 ±1.38 1.402
ASSAY OF ROSUVASTATIN CALCIUM

42
λ max 240 nm
Beer’s law limits 1-6 μg/ml
Sandell’s sensitivity
[μg/ cm2- 0.001 absorbance units]
0.012
Regression equation y = 0.056x + 0.021
Slope 0.056
Intercept 0.021
% Recovery 98.45-101.01
% RSD
Intra-day Precision 0.160-0.704
Inter-day Precision 0.401-1.817
LOD 0.0201 μg/ml
LOQ 0.0612 μg/ml
Mean % Assay
[IP, 2018 limits- 90-110 %]
98.90 ±1.38
SUMMARY OF VALIDATION OF ROSUVASTATIN CALCIUM

43
The present developed method was more sensitive and
cost-effective
 Diluent: organic solvent → 0.1N NaOH
 Linearity: [2-18 → 1-6 μg/ml]
 LOD: [0.021 μg/ml]
LOQ: [0.0612 μg/ml]

4b. DEVELOPMENT AND VALIDATION OF
COLORIMETRIC METHOD FOR THE DETERMINATION
OF ROSUVASTATIN CALCIUM IN BULK AND TABLETS
The reported methods are
Complex
Time consuming
Expensive - involves extraction of colored complex
44

METHOD DEVELOPMENT [Gorog, 2011]: ROSUVASTATIN CALCIUM
 Diluent: Methanol
 Preparation of 0.1M Ferric chloride [FeCl3] reagent
[IP, 2018]
 Preparation of 0.5% w/v Potassium ferricyanide reagent
[1000μg/ml]:
45
10 mg
Rosuvastatin
Calcium
+
5ml diluent
Sonicated for
10mins
+
made up to 10ml
with diluent
1000μg/ml

46
 Preparation of Standard stock solution-II of Rosuvastatin Calcium [100μg/ml]:
1.0ml standard
stock-I
Kept aside for
10mins
+
Diluted to
10ml
100μg/ml
Preparation of working standard solution of Rosuvastatin Calcium [4μg/ml]:
0.4ml standard
stock-II
+
1.5 ml 0.1M FeCl3
+
1.0 ml 0.5%
Potassium
ferricyanide
Diluted to 10ml
with diluent
4μg/ml

47
0
0.1
0.2
0.3
0.4
0.5
0.6
400 800
Absorbance
Wavelength
λ max OF ROSUVASTATIN CALCIUM – COLORIMETRY (635nm)

48
The probable mechanism involved in the green colored
complex formation is
 Reduction of ferric ion to ferrous ion by Rosuvastatin
Calcium
The reduced ferrous ion reacted with potassium ferri cyanide
and formed green colored Ferro-ferricyanide complex
[Vamsi Krishna and Gowrisankar, 2007]
3 Fe2+ + 2[Fe (CN) 6]3- Fe3 [Fe (CN)6]

49
VALIDATION [ICH Q2 (R1), 2005]: ROSUVASTATIN CALCIUM
% Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total Amount
recovered
(μg/ml)
Mean % Recovery
± SD
%RSD
80 4 3.2 7.21 100.09 ± 0.85 0.85
100 4 4.0 8.03 100.37 ± 0.62 0.62
120 4 4.8 8.73 99.24 ± 0.23 0.23
PRECISION
Concentration
(μg/ml)
Intra-day
Precision
Mean ± SD
% RSD
Inter-day
Precision
Mean ± SD
%RSD
4 0.2325 ± 0.0018 0.774 0.234 ± 0.0023 0.982
ACCURACY

50
Concentration [x]
(μg/ml)
Absorbance* [y]
0 0
1 0.068
2 0.124
3 0.185
4 0.237
5 0.297
6 0.357
y = mx + c
y = 0.058 x + 0.005
Slope (m) 0.058
Intercept (c) 0.005
LOD 0.041 μg/ml
LOQ 0.123 μg/ml
*Mean of three determinations

51
Calibration Curve
y = 0.0586x + 0.0052
R² = 0.9993
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 1 2 3 4 5 6 7
AbsorbanceAU
Concentration [μg/ml]
Tablet brand Label claim Amount found ±SD %Assay ± SD %RSD
Roseday 10 mg 10.05 ± 0.108 100.57 ± 1.08 1.07
ASSAY

52
λ max 635 nm
Beer’s law limits 1-6μg/ml
Sandell’s sensitivity
[μg/ cm2- 0.001 absorbance units]
0.0147
Slope 0.058
Intercept 0.005
% Recovery 99.24 – 100.34
% RSD
Intra-day Precision 0.774
Inter-day Precision 0.982
Limit of Detection 0.041 μg/ml
Limit of Quantitation 0.123 μg/ml
Mean % Assay
[IP, 2018 Limits: 90-110 %]
100.57 ±1.08

53
The developed method was simple, rapid, sensitive and cost-effective
compared to the best method reported
 Involved no extraction of colored complex
Linearity: [100-500 → 1-6 μg/ml]
 LOD: [0.041 μg/ml]
 LOQ: [0.123 μg/ml]

4c. DEVELOPMENT AND VALIDATION OF
STABILITY INDICATING RP-HPLC METHOD
FOR THE DETERMINATION OF
ROSUVASTATIN CALCIUM IN BULK AND TABLETS
The reported RP-HPLC methods are
Time consuming- Rt was more and phosphate buffer preparation
 More volume of organic solvents
54

55
Structure
Molecular Weight-1001.137
)Ca+2
)Ca+2
pKa 4.00
Log P 4.19
Solubility slightly soluble in ethanol and sparingly soluble in
water and Methanol
DRUG PROFILE: ROSUVASTATIN CALCIUM

56
10 mg
Rosuvastatin
Calcium
+
5 ml diluent
volume up to 10ml
+
filtered using 0.45µ
PTFE filter
1000µg/ml
 Preparation of Standard stock solution-II of Rosuvastatin Calcium (100µg/ml):
1.0 ml standard
stock - I
Diluted to 10ml
with diluent
100µg/ml
METHOD DEVELOPMENT: [Snyder et al.,1997] ROSUVASTATIN CALCIUM
Diluent: HPLC grade water: ACN (1:1)
(1000µg/ml):
Preparation of Working standard solution of Rosuvastatin Calcium (8µg/ml):
0.8ml stock-II
Diluted to 10ml
with diluent
8µg/ml

57
OPTIMIZED CHROMATOGRAPHIC CONDITIONS
Column Symmetry C18 (150mm x 4.6mm, 5m)
Mobile phase 0.1% OPA (pH 2.7) : ACN (55:45 % v/v)
Elution Isocratic
Flow rate 1.0 ml/min
Detector - λ max PDA (241 nm)
Column temperature 300C
Injection volume 10 μl
Diluent HPLC grade water : ACN [1:1]
Retention Time 2.872 mins
Run time 6.0 mins

58
OPTIMIZED CHROMATOGRAM OF ROSUVASTATIN CALCIUM

59
VALIDATION OF ROSUVASTATIN CALCIUM [ICH Q2 (R1), 2005]:
SPECIFICITY: Blank [HPLC grade water: ACN (1:1)]
Standard (2.915mins) Test Sample (2.902mins)

60
Stressor Condition
Acid 0.1N HCl, 24hrs, 30±20C
Alkali 0.1N NaOH, 24hrs, 30±20C
Oxidative 3.0% v/v H2O2, 24hrs, 30±20C
Thermal 50±20C on sand bath for 6 hrs
Photolytic UV Chamber for 6 hrs
Neutral HPLC grade water, 24hrs, 30±20C
FORCED DEGRADATION STUDIES [Ahuja et al., 2001, ICH Q1A (R2), ICH Q1B]

Acid degradation of Rosuvastatin Calcium
61
Alkali degradation of Rosuvastatin Calcium

Oxidative degradation of Rosuvastatin Calcium
62
Thermal degradation of Rosuvastatin Calcium

Photolytic degradation of Rosuvastatin Calcium
63
Neutral degradation of Rosuvastatin Calcium

64
DEGRADATION OF ROSUVASTATIN CALCIUM
Stressor Purity angle - Purity threshold % Assay
% degraded
[should be < 20]
Acid 0.158 < 1.148 90.73 9.27
Alkali 0.941 < 1.125 91.56 8.44
Oxidative 1.070 < 1.984 93.81 6.19
Thermal 0.847 <1.084 97.68 2.32
Photolytic 0.854 < 1.088 98.15 1.85
Neutral 0.912 < 1.094 99.41 0.59

65
80 % level 100% level
120% level
ACCURACY OF ROSUVASTATIN CALCIUM

66
%
Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total
Amount
recovered
(μg/ml)
%
Recovery
Statistical data
Mean ± SD % RSD
80 8 6.4
14.45 100.34
100.23±0.196 0.19614.40 100.00
14.45 100.34
100 8 8
16.17 101.06
101.16±0.096 0.09616.19 101.18
16.20 101.25
120 8 9.64
17.67 100.41
100.81±0.294 0.35117.76 100.92
17.79 101.10
ACCURACY

67
PRECISION OF ROSUVASTATIN CALCIUM
Injection
Intra-day Precision Inter-day Precision*
Peak area Peak area
1 947892 953130
2 948956 956338
3 932013 956588
4 932654 956115
5 933654 960602
6 943152 961183
Mean 939720.2 957326
SD 7873.8 3039.8
% RSD 0.8 0.3
*mean of two determinations

68
Level-1 Level-2
Level-3

70
% Level Concentration (x) [μg/ml] Mean Peak area*(y) [AU]
0 0 0
25 2 233476
50 4 478886
75 6 707451
100 8 956490
125 10 1184283
150 12 1407729
Linear Regression Equation y = mx + c y = 117900x + 2358
Slope (m) 117900
Intercept (c) 2358
LOD 0.013 μg/ml
LOQ 0.042 μg/ml
*mean of three determinations

71
y = 117900x + 2358.
R² = 0.999
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
0 2 4 6 8 10 12 14
PeakareaAU
Concentration [µg/ml]
STABILITY OF WORKING STANDARD SOLUTION: (30 ± 2 0C for 24 hrs)
Time [Hrs] Peak areas* % Assay* Variation
0 957286 99.89 NA
12 947905 98.91 0.98
24 940228 98.11 1.78
* Average of six determinations

72
Parameter Variation
% RSD of Peak
Area
[NMT 2.0 %]
Theoretical
plates*
[N>2000]
Tailing
factor*
<2.0
Flow rate
(1.0±0.1ml/min)
0.9 1.005 4424 1.19
1.0 0.402 4537 1.19
1.1 0.545 3804 1.20
ACN ratio in
mobile phase
[0.1 % OPA buffer :
ACN]
(45±5%)
60:40 0.802 3531 1.19
55 :45 0.402 4537 1.19
50 :50 0.406 4546 1.18
Temperature
(30±5 0C)
25 0.627 4460 1.18
30 0.402 4537 1.19
35 0.456 3695 1.23
* Mean of six determinations
ROBUSTNESS OF ROSUVASTATIN CALCIUM

73
SYSTEM SUITABILITY OF ROSUVASTATIN CALCIUM
Injection Rt [mins] Peak Area
Theoretical
Plates
N >2000
Tailing Factor
<2.0
1 2.902 955731 4423 1.18
2 2.904 956119 4606 1.20
3 2.914 955942 4369 1.21
4 2.915 960636 4310 1.20
5 2.917 949831 4746 1.19
6 2.919 959889 4771 1.20
Mean 2.911 956358 4537 1.19
S.D 3846.9
% RSD
[NMT 2.0]
0.402

74
ASSAY OF ROSUVASTATIN CALCIUM
SD- Standard deviation
Injection Rt [mins] Peak area % Assay
Amount
[mg]
1 2.902 956149 99.78 9.978
2 2.882 955931 99.76 9.976
3 2.881 955431 99.7 9.97
4 2.894 956189 99.78 9.978
5 2.902 956214 99.78 9.978
6 2.881 956018 99.76 9.976
Mean ±SD 99.76±0.39 9.976±0.03

75
Parameter Result ICH Limits
System Suitability Parameters
% RSD of peak area – 0.402 NMT 2.0
Theoretical plates- 4537 MT 2000
Tailing factor-1.19 NMT 2.0
Range [μg/ml] 2-12 -
Correlation coefficient [R2] 0.999 NLT 0.999
% Recovery 100-101.25 98-102
% RSD
Intra-day Precision 0.8 NMT 2.0
Inter-day Precision 0.3 NMT 2.0
LOD [μg/ml] 0.013 -
LOQ [μg/ml] 0.042 -
Solution stability,
% Assay difference at 24 hrs
1.78 NMT 2.0
% Assay 99.76 ± 0.39 IP, 2018: 90-110

76
The developed RP-HPLC method was simple, sensitive and
cost-effective
 Organic ratio in the mobile phase: [60 → 45%]
 Retention time: [3.806 → 2.916 mins]
 Run time: [12.0 → 6.0 mins]
 Linearity range: [5-30 → 2-12 µg/ml]
 LOD: [0.0441 → 0.013 µg/ml]
 LOQ: [0.1434 → 0.042 µg/ml]

4d. DEVELOPMENT AND VALIDATION OF
UV SPECTROSCOPIC METHOD FOR THE
DETERMINATION OF PITAVASTATIN CALCIUM
IN BULK AND TABLETS
Pitavastatin Calcium (4mg) - more efficiently lowers plasma LDL
cholesterol and triglycerides levels at lower doses
[Sharma et al., 2017]
 The reported methods are expensive with use of organic solvents
77

78
Structure
Molecular weight - 880.94 )Ca+2
pKa 4.13
Log P 6.13
Solubility freely soluble - pyridine, chloroform, dilute HCl,
tetrahydrofuran, soluble - ethylene glycol, sparingly
soluble - octanol, slightly soluble - methanol, water and
ethanol, practically insoluble - ACN and Diethyl ether
DRUG PROFILE: PITAVASTATIN CALCIUM

METHOD DEVELOPMENT [Gorog, 2011]: Diluent: Methanol: Distilled water (1:1)
Preparation of Standard stock solution-I of Pitavastatin Calcium [1000μg/ml]:
79
10 mg
Pitavastatin
Calcium
+
5ml diluent
Sonicated for
10mins
+
made up to 10ml
1000μg/ml
Pitavastatin Calcium
1.0ml standard
stock-I
+
5ml diluent
Diluted to 10ml
with diluent
100μg/ml
Preparation of Standard stock solution-II of Pitavastatin Calcium [100μg/ml]:
 Preparation of working standard solution of Pitavastatin Calcium [8μg/ml]:
0.8ml standard
stock-II
Diluted to 10ml
with diluent
8μg/ml

UV SPECTRUM OF PITAVASTATIN CALCIUM (λ max - 245nm)
80

81
VALIDATION [ICH Q2 (R1), 2005]: PITAVASTATIN CALCIUM
PRECISION
% Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total Amount
recovered
(μg/ml)
Mean %
Recovery ± SD
% RSD
80 8 6.4 14.38 100.11± 0.278 0.278
100 8 8.0 16.07 100.26± 0.477 0.476
120 8 9.6 17.64 100.22± 0.34 0.34
Concentration
(μg/ml)
Intra-day Precision
Mean ± SD
% RSD
Inter-day Precision
Mean ± SD
% RSD
8 0.390 ± 0.0018 0.46 0.391± 0.0034 0.86
ACCURACY

82
Concentration [x]
(μg/ml)
Absorbance* [y]
2 0.094
4 0.184
6 0.284
8 0.39
10 0.482
12 0.59
y = mx + c
y = 0.049 x – 0.005
Slope (m) 0.049
Intercept (c) -0.005
LOD 0.103 μg/ml
LOQ 0.310 μg/ml
LINEARITY, LOD AND LOQ OF PITAVASTATIN CALCIUM
* mean of three determinations

83
LINEARITY PLOT OF PITAVASTATIN CALCIUM
y = 0.048x - 0.005
R² = 0.999
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8 10 12 14
AbsorbanceAU
Formulation Label claim Amount found ± SD % Assay ± SD % RSD
Pivasta 4 mg 4.008 ± 0.037 100.24±0.945 0.942
ASSAY

84
λ max (nm) 245
Beer’s law limits (μg/ml) 2-12
Sandell’s sensitivity (μg/ cm2- 0.001 absorbance
units)
0.0212
Regression equation y = 0.048x - 0.005
Slope 0.049
Intercept 0.005
% Recovery 100.11-100.26
% RSD
LOD (μg/ml) 0.103
LOQ (μg/ml) 0.310
Mean % Assay 100.24±0.945
SUMMARY OF VALIDATION OF PITAVASTATIN CALCIUM

85
The developed method was
 Cost-effective [Methanol → Methanol: Distilled water (1:1)]
 LOD [ 0.4062 μg/ml → 0.103μg/ml]
 LOQ [1.2309 μg/ml → 0.310μg/ml]

5a. QbD ENABLED STABILITY INDICATING
RP-HPLC METHOD DEVELOPMENT AND VALIDATION
EMPAGLIFLOZIN IN BULK AND TABLETS
Empagliflozin – effectively reduces plasma glucose levels with decreased
risk of cardio-vascular deaths and heart failure hospitalizations
[Ralston et al., 2018]
No methods reported using QbD approach for EMPA
86

87
Structure
Molecular Weight - 450.91
Class SGLT-2 inhibitor
pKa 12.57
Log P 1.79
Solubility slightly soluble in ACN, ethanol, soluble in 50 %
ACN/water, sparingly soluble in methanol, very slightly
soluble in water and practically insoluble in toluene
DRUG PROFILE: EMPA

88
10 mg EMPA
+
2ml diluent,
+
Sonicated for
10 mins
volume up to
10ml
+
filtered using
0.45 µ PTFE filter
1000µg/ml
EMPA
Preparation of standard stock solution-II of EMPA [100 µg/ml]:
1ml of standard
stock-I
Diluted to 10ml
with diluent
100µg/ml EMPA
METHOD DEVELOPMENT: [Gorog, 2011]: EMPA
 Diluent: HPLC grade water: ACN (1:1)
Preparation of standard stock solution-I of EMPA [1000µg/ml]:
0.8ml of standard
stock-II 8µg/ml EMPA
Diluted to 10ml
with diluent
Preparation of working standard solution of EMPA [8µg/ml]:

89
Column Denali C18 (150mm x 4.6mm, 5m)
Mobile phase 0.1 % OPA [pH 2.7] : ACN (45:55 % v/v)
Elution Isocratic
Run time 5.0 mins
Diluent HPLC grade water: ACN [1:1]

90
METHOD OPTIMIZATION USING DESIGN EXPERT® SOFTWARE
(Trial Version 10) [Federick and Alireza, 2011]:
Step 1: Analytical target Profile [ATP]:
 ATP - bulk drug and tablet - Technique - RP-HPLC
 Goal - To develop more robust RP-HPLC method with optimum
system suitability parameters and short analysis time
 Target - To study the simultaneous influence of CMPs on CMAs
Step 2: Critical Quality Attributes:
CMPs - Buffer ratio in the mobile phase, Flow rate and Wavelength
CMAs - Retention time, Peak area and Tailing factor

91
CMP -1 level 0 + 1 level
Buffer ratio in mobile phase [% v/v] 35 45 55
Flow rate [ml/min] 0.9 1.0 1.1
Wavelength [nm] 218 223 228
CMPS AND THEIR INPUT LEVELS
Step 3: Design of Experiments using Software:
 Efficient experimental model was designed by
systematic and automated scouting of the three CMPs
For method scouting - software generated 20 trials

92
Std
Run
No.
Buffer ratio
F1/CMP1
Flow rate
[ml/min]
F2/CMP2
Wavelength [nm]
F3/CMP3
Rt [mins]
R1/CMA1
Peak area
R2/CMA2
Tailing factor
R3/CMA3
1 14 35 0.9 218 2.284 1047724 1.3
2 7 55 0.9 218 4.493 1022852 1.2
3 2 35 1.1 218 1.882 826983 1.2
4 5 55 1.1 218 3.720 778771 1.1
5 13 35 0.9 228 2.285 1015749 1.3
6 20 55 0.9 228 4.490 969482 1.2
7 6 35 1.1 228 1.873 818929 1.3
8 10 55 1.1 228 3.761 740485 1.2
9 17 28.1821[min] 1 223 1.913 1056885 1.2
10 9 61.8179[max] 1 223 7.662 992788 1.1
11 19 45 0.831821[min] 223 2.206 802203 1.2
12 12 45 1.16818 [max] 223 3.072 1227680 1.3
13 3 45 1 214.591 [min] 2.567 873994 1.2
14 18 45 1 231.409 [max] 2.564 642180 1.2
15 11 45 1 223 2.572 939959 1.2
16 4 45 1 223 2.587 933968 1.3
17 8 45 1 223 2.564 941365 1.2
18 1 45 1 223 2.568 937667 1.2
19 15 45 1 223 2.572 941063 1.2
20 16 45 1 223 2.566 941481 1.2
[F-factor, R-Response]
TRIALS PROPOSED BY SOFTWARE WITH EXPERIMENTAL RESPONSES/ CMAs

93
FIT STATISTICS FOR SELECTED CMAs/Rs
Coefficients CMA1/R1
[Retention time]
CMA2/R2
[Peak Area]
CMA3/R3
[Tailing Factor]
R2 0.9172 0.3834 0.6358
Adjusted R2 0.8427 -0.1715 0.3081
Predicted R2 0.3715 -3.6829 -0.9814
Adequate Precision 13.1692 3.6907 5.7571

94
CONTOUR PLOT SHOWING EFFECT OF AQUEOUS PHASE [CMP1/F1] AND
FLOW RATE [CMP2/F2] ON RT [CMA1/R1]

95
EFFECT OF AQUEOUS PHASE [CMP1/F 1] AND FLOW RATE [CMP2/F2] ON
PEAK AREA [CMA2/R2]

96
EFFECT OF AQUEOUS PHASE [CMP1/F1] AND FLOW RATE
[CMP2/F2] ON TAILING FACTOR [CMA3/R3]

97
Externally Studentized Residuals
Normal%Probability
Normal Plot of Residuals
-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00
1
5
10
20
30
50
70
80
90
95
99
retention time
Color points by value of
retention time:
1.873 7.662
CMA1/R1 [Rt]
NORMAL PLOT OF RESIDUALS

98
Normal%Probability
-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00
1
5
10
20
30
50
70
80
90
95
99
area
area:
642180 1.22768E+06
CMA2/R2 [Peak Area]

99
Normal%Probability
-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00
1
5
10
20
30
50
70
80
90
95
99
tailing factor
tailing factor:
1.1 1.3
CMA3/R3 [Tailing Factor]

100
Factor Level
A- Aqueous Phase [% v/v] 39.57
B- Flow rate [ml/min] 0.9761
C- Wavelength [nm] 218.51
OPTIMIZED CONDITIONS PROPOSED BY DESIGN EXPERT SOFTWARE
CMA Predicted value Observed value SD
Rt [mins] 2.04095 2.057 0.006
Peak area 938311 953531 10762
Tailing factor 1.22654 1.1 0.089
OBSERVED VALUES OF CMAs

101
OPTIMIZED CHROMATOGRAM OF EMPA [QbD APPROACH]

102
Blank [HPLC grade water: ACN (1:1)]
Standard (2.051 mins) Sample (2.063 mins)
VALIDATION: [ICH Q2 (R1), 2005]: SPECIFICITY OF EMPA

103
FORCED DEGRADATION STUDIES [Ahuja et al., 2001, ICH Q1A (R2), ICH Q1B]:
Stressor Condition
Acid 2N HCl, 30 mins, 60 ± 20C
Alkali 2N NaOH, 30 mins, 60 ± 20C
Oxidative 20 % v/v H2O2, 30 mins, 60 ± 20C
Thermal 80 ± 20C in the oven for 6hrs
Photolytic UV Chamber for 7 days
Neutral HPLC grade water, 30 mins, 60 ± 20C

104
Acid Degradation of EMPA
Alkali degradation of EMPA

105
Oxidative degradation of EMPA
Thermal degradation of EMPA

Photolytic degradation of EMPA
106
Neutral degradation of EMPA

107
DEGRADATION OF EMPA
Stressor Purity angle < Purity threshold % Assay % degraded
[< 20]
Acid 0.716 < 0.981 91.06 8.94
Alkali 0.903 < 1.349 89.31 10.69
Oxidative 0.837 < 1.137 90.46 9.54
Thermal 0.809 < 0.914 97.08 2.92
Photolytic 0.878 < 1.396 98.11 1.89
Neutral 0.658 < 0.925 99.25 0.75

ACCURACY
108
80 % level 100 % level
120 % level

109
%
Spiked
Fixed
sample
conc.
(μg/ml)
Amount
Spiked
(μg/ml)
Amount
recovered
(μg/ml)
%
Recovery
Statistical Data
Mean ± SD % RSD
80 8 6.4
14.46 100.93
100.34 ± 0.52 0.5214.40 100.04
14.40 100.03
100 8 8
15.94 99.25
99.42 ± 0.15 0.1615.96 99.45
15.96 99.55
120 8 9.6
17.57 99.72
99.60 ± 0.11 0.1117.55 99.53
17.56 99.54
ACCURACY OF EMPA

110
PRECISION OF EMPA
Injection
Peak area
Intra-day Precision Inter-day Precision*
1 892185 892994
2 894415 886254
3 896243 885649
4 893882 887612
5 894033 881625
6 896965 880253
Mean 894621 885731.2
SD 1731.7 4548.3
% RSD [NMT 2.0] 0.2 0.513

111
LINEARITY, LOD AND LOQ OF EMPA
Level-1 Level-2
Level-3

113
% Level
Concentration (x)
[μg/ml]
Mean peak area* (y)
AU
0 0 0
25 2 224755
50 4 444370
75 6 657387
100 8 896176
125 10 1088259
150 12 1316979
y = mx + c y=109460x+4373
Slope (m) 109460
Intercept (c) 4373
LOD 0.047 μg/ml
LOQ 0.142 μg/ml
LINEARITY OF EMPA

114
y = 109460x + 4373.
R² = 0.999
0
200000
400000
600000
800000
1000000
1200000
1400000
0 2 4 6 8 10 12 14
PeakAreaAU
STABILITY OF STANDARD SOLUTION (30±20C for 24 hrs)
Time [Hrs] Peak areas* % Assay* Variation
0 896756 99.93 -
12 888120 98.96 0.97
24 880700 98.14 1.79
LINEARITY PLOT OF EMPA

115
ROBUSTNESS OF EMPA
Parameter Variation
% RSD of peak
area [NMT 2.0]
Theoretical
plates*
[N>2000]
Tailing
factor*
<2.0
Flow rate
[0.9761 ± 1
ml/min]
0.8761 0.549 5405 1.13
0.9761 0.279 5431 1.16
1.0761 0.740 5239 1.14
ACN ratio in
Mobile phase
Buffer: ACN
[60.43 ± 5% v/v]
44.57 : 55.43 0.841 5775 1.11
39.57 : 60.43 0.279 5431 1.16
34.57 : 65.43 0.583 5365 1.14
Temperature
[30 ± 50C]
25 1.062 6044 1.12
30 0.279 5431 1.16
35 1.802 5666 1.14
*Mean of six determinations

116
SYSTEM SUITABILITY OF EMPA
Injection Rt [mins] Peak Area
Theoretical Plates
N > 2000
Tailing
Factor
< 2.0
1 2.049 892431 5256 1.17
2 2.048 898403 5522 1.15
3 2.038 893051 5532 1.14
4 2.051 896632 5705 1.14
5 2.058 895224 5400 1.16
6 2.055 897993 5169 1.18
Mean 2.05 895622 5431 1.16
SD 2503
% RSD
[< 2.0 %]
0.279

117
ASSAY OF EMPA
Injection Rt [mins] Peak area % Assay
Amount
[mg]
1 2.062 892563 99.46 9.946
2 2.058 891489 99.34 9.934
3 2.058 890210 99.2 9.92
4 2.054 893518 99.57 9.957
5 2.055 894314 99.65 9.965
6 2.062 891210 99.31 9.931
Mean ± SD 99.42±0.17 9.942 ± 0.17

118
Parameter Result ICH Limits
System Suitability Parameters
% RSD of peak area – 0.279 NMT 2.0
Theoretical plates- 5431 MT 2000
Tailing factor-1.16 NMT 2.0
Range [μg/ml] 2-12 -
Correlation coefficient [R2] 0.999 NLT 0.999
% Recovery 99.25-100.93
% RSD
Intra-day precision 0.21 NMT 1.0
Inter-day precision 0.513 NMT 2.0
LOD [μg/ml] 0.047 -
LOQ [μg/ml] 0.142 -
% Assay difference at 24 hrs 1.79 NMT 2.0
% Assay 99.42±0.17 98-102
SUMMARY OF VALIDATION OF EMPA

119
Preliminary trial runs were performed for the identification of CMPs/Fs:
 On initial optimization of the method,
 0.1 % OPA (pH 2.7): ACN [45:55 % v/v] - ideal mobile phase
 Increase in % aqueous ratio [% OPA buffer] in the mobile phase - resulted in
increase in Rt- CMP1/F1, change in flow rate affects the Rt -
CMP2/F2, Wavelength - CMP3/F3
 Multivariate DoE approach using Central composite design - selected to
evaluate the simultaneous effect of selected CMPs/Fs on the selected CMAs/Rs
On the evaluation of the statistical data
 Quadratic model was the best-fit model for the central composite design

120
 The R2 - signifies the percentage of variation for a dependent variable
(response) by independent variables (factors)
 In the present study, the R2 - 0.9172 for CMA1/R1
- 0.3834 for CMA2/R2
- 0.6358 for CMA3/R3
 Difference between the adjusted R2 and predicted R2 - more than 0.2 –
reflects large block effect of CMA1/R1 using the model
 Negative Predicted R² for CMA2/R2 and CMA3/R3 - overall mean is a better
predictor of the response
 The adequate precision for CMA1/R1 (13.16) and CMA2/R2 (5.75) were
greater than 4.0 - adequate signal and the model was suitable for navigating
design space

121
 From the contour plot 1, increase in % Aqueous ratio with constant Flow rate
and Wavelength increased the Rt
 Contour plot 2 , decrease in Flow rate with constant % Aqueous ratio and
Wavelength has effect on Peak area
 Contour plot 3, increase in Wavelength with constant
Peak area didn’t show significant effect on Retention time, Peak area and
Tailing factor
 The normal plot of residuals for the three selected CMAs reflects that the data
points were distributed normally along the straight line and the error was
distributed equally across each individual point

122
The equation used for predictions about the CMAs/Rs for the given levels of each
CMP:
RT = +2.58 + 1.30 x A-0.0629 x B + 0.0018 x C-0.0860 x AB + 0.0058 x AC +
0.0043 x BC + 0.7248 x A2-0.0348 x B2-0.0608 x C2
Area = + 9.402E + 05 – 22376.52 x A - 12819.56 x B - 38189.48 x C - 6939.63 x
AB - 6453.38 x AC + 4875.62 x BC + 24013.40 x A2 + 20514.99 x B2 - 70296.79
x C2
Tailing factor = + 1.22 – 0.0416 x A - 0.0023 x B + 0.00146 x C + 0.0000 x AB +
0.0000 x AC + 0.0001 x BC - 0.0181 x A2 - 0.01738 x B2 - 0.0004 x C2
Where A = Aqueous phase
B = Flow rate
C = Wavelength

123
 Central composite design was used to evaluate the simultaneous effect
of % Aqueous ratio in the mobile phase , Flow rate and Wavelength
(CMPs) on Rt, Peak area and Tailing factor (CMAs)
 Sum of squares (-0.0860): The simultaneous decrease in % Aqueous
ratio in the mobile phase and Flow rate results in better retention time
 Sum of squares: Indicates that the simultaneous change in Flow rate and
Wavelength, there was no change in Rt (+0.000115) and Tailing factor
(-0.0001) and less effect on Peak area (-0.0182)

124
 Sum of squares: Indicates that the simultaneous change in
% Buffer ratio in the mobile phase and Wavelength, there was no
change in Rt (+0.0085), Peak area and Tailing factor
 No significant effect of wavelength on CMAs
 % Buffer ratio in the mobile phase and Flow rate influenced Rt
The interactive effects of CMPs on tailing factor was insignificant

125
COMPARISON AMONG CMPs AND CMAs OF INITIAL OPTIMIZED METHOD,
DOE DATAAND FINAL OPTIMIZED METHOD
CMP/F
CMPs
CMA/R
CMAs
Initial
optimized
method
Final optimized
Method (DOE Data)
Initial
optimized
method
DOE
data*
Final
optimized
method
Mobile
Phase
[% v/v]
0.1% OPA :
ACN
(45:55)
0.1% OPA : ACN
(39.57:60.43)
Rt [Mins] 2.629 2.04 2.05
Flow Rate
[ml/min]
1.0 0.961 Peak area 1028275 938311 953531
Wavelength
[nm]
223 218.5 Tailing factor 1.2 1.22 1.1
Comparison between reported non-QbD best method and present QbD method
 Run time: 6.0 mins → 5.0 mins
 Retention time: 4.81mins → 2.051mins
 LOD : 0.3589 µg/ml → 0.047 µg/ml
 LOQ: 1.0876 µg/ml → 0.142 µg/ml

UV SPECTROSCOPIC METHOD
FOR THE ESTIMATION OF CANAGLIFLOZIN
IN BULK AND TABLETS
Canagliflozin- reduced risk of cardio-vascular deaths and heart failure
hospitalizations in addition to the management of increased plasma
glucose levels [Ralston et al., 2018]
 The UV Spectroscopic methods reported - expensive
126

127
Structure
Molecular Weight – 453.53
½ H2O
Class SGLT-2 inhibitor
pKa 12.57
Log P 3.44
Solubility soluble in organic solvents like ethanol, methanol,
acetone and THF and insoluble in water
DRUG PROFILE: CANAGLIFLOZIN

METHOD DEVELOPMENT [Gorog, 2011]: CANAGLIFLOZIN
Diluent : Methanol and distilled water (1:1)
 Preparation of Standard stock solution-I of Canagliflozin [1000μg/ml]:
128
10 mg
Canagliflozin
+
5ml diluent
Sonicated for
10mins
+
made up to
10ml
1000μg/ml
Canagliflozin
 Preparation of Standard stock solution-II of Canagliflozin [100μg/ml]:
1.0ml standard
stock-I
Diluted to 10ml
with diluent
100μg/ml
Canagliflozin
 Preparation of working standard solution of Canagliflozin [40μg/ml]:
4.0ml standard
stock-II
Diluted to 10ml
with diluent
40μg/ml Canagliflozin

129
UV SPECTRUM OF CANAGLIFLOZIN [λ max – 224nm]

130
VALIDATION [ICH Q2 (R1), 2005]: CANAGLIFLOZIN
% Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Mean
Amount
recovered
(μg/ml)
Mean %
Recovery ± SD
% RSD
80 40 32 72.07 100.09 ± 0.55 0.55
100 40 40 79.86 99.82±0.37 0.37
120 40 98 87.91 99.89±0.59 0.60
ACCURACY
Concentration
(μg/ml)
Intra-day
Precision
Mean ± SD
% RSD
Inter-day Precision
Mean ± SD
% RSD
40 0.201±0.0015 0.75 0.2021 ± 0.0017 0.84
PRECISION

131
Concentration (μg/ml) Absorbance*
0 0
10 0.051
20 0.104
30 0.152
40 0.202
50 0.252
60 0.302
y = mx + c
y = 0.005x + 0.001
Slope (m) 0.005
Intercept (c) 0.001
LOD 0.33 μg / ml
LOQ 1.00 μg / ml
LINEARITY, LOD AND LOQ OF CANAGLIFLOZIN
*Average of three determinations

132
y = 0.0051x + 0.0016
R² = 0.9999
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 10 20 30 40 50 60 70
AbsorbanceAU
Concentration (μg/ml)
LINEARITY PLOT OF CANAGLIFLOZIN
Tablet Brand Label claim Amount found ± SD % Assay ± SD
Invokana 100 mg 99.79 ± 1.17 99.79 ± 1.17
ASSAY OF CANAGLIFLOZIN

133
λ max 224 nm
Beer’s law limits 10-60 μg/ml
Sandell’s sensitivity (μg/ cm2- 0.001
absorbance units)
0.196
Slope 0.005
Intercept 0.001
% Recovery 99.82-100.09
% RSD
LOD 0.33 μg/ml
LOQ 1.00 μg/ml
Mean % Assay 99.19 ± 1.17
SUMMARY OF VALIDATION OF CANAGLIFLOZIN

134
The developed UV Spectroscopic method was
simple and sensitive
 Linearity : 10-60 μg/ml
 LOD : 0.33 μg/ml
 LOQ : 1.00 μg/ml

6a. DEVELOPMENT AND VALIDATION OF
STABILITY INDICATING RP-HPLC METHOD FOR THE
SIMULTANEOUS DETERMINATION OF
ERTUGLIFLOZIN PIDOLATE AND METFORMIN HYDROCHLORIDE
IN BULK AND TABLETS
 ERTU and MET combination - efficiently reduces elevated HbA1c
levels in type-2 diabetic patients [FDA label, Segluromet, 2017]
The RP-HPLC methods reported were
complex
involved high volumes of organic solvent in the mobile phase
135

136
ERTU MET
Structure
.
HCl
Class SGLT-2 inhibitor Biguanide anti-diabetic
Molecular weight 566 165.62
pKa 11.98 12.33
Log P 2.21 -0.92
Solubility soluble in ethanol, sparingly
soluble in ethyl acetate, ACN
and very slightly soluble in water
freely soluble in water, sparingly
soluble in alcohol, practically
insoluble in acetone and
dichloromethane
DRUG PROFILE:

137
3.75 mg ERTU
+
250 mg MET
+
10ml diluent
volume up to 100ml
+
filtered using 0.45 µ
PTFE filter
37.5µg/ml ERTU
2500 µg/ml MET
Preparation of working standard solution of ERTU [3.75µg/ml] and MET [250µg/ml]:
1ml of standard
stock
volume upto 10ml
with diluent
3.75µg/ml ERTU
250µg/ml MET
METHOD DEVELOPMENT: [Snyder et al., 1997]: ERTU AND MET
Preparation of standard stock solution of ERTU [37.5µg/ml] and MET
[2500µg/ml]:

138
Column Kromasil C18 (150mm x4.6 mm, 5 µm)
Mobile phase 0.1% OPA [pH 2.7]: ACN (65:35 %v/v)
Elution Isocratic
Detector and λ max PDA and 224.0 nm
Column temperature 30 ± 20C
Injection volume 10 µl
Diluent HPLC grade water : ACN [1:1]
Retention time
MET- 2.170 mins
ERTU- 2.929 mins
Run time 6.00 mins

139
OPTIMIZED CHROMATOGRAM OF ERTU AND MET

VALIDATION [ICH Q2 (R1), 2005]: ERTU AND MET: SPECIFICITY
140
Blank [HPLC grade water: ACN (1:1)]
Standard
(MET 2.170mins and ERTU 2.929 mins)
Sample
(MET 2.168mins and ERTU 2.827mins)

FORCED DEGRADATION STUDIES [Ahuja et al., 2001, ICH Q1A (R2), ICH Q1B]:
141
Stressor Condition
Acid 2N HCl, 30 mins, 60±20C
Alkali 2N NaOH, 30 mins, 60±20C
Oxidative 20% v/v H2O2, 30 mins, 60±20C
Thermal In the oven at 105±20C for 6 hrs
Light UV Chamber for 7 days
Neutral HPLC grade water, 6 hrs, 60±20C

142
Acid Degradation of ERTU and MET

143
Alkali Degradation of ERTU and MET

144
Oxidative Degradation of ERTU and MET

145
Thermal degradation of ERTU and MET

146
Photolytic degradation of ERTU and MET

147
Neutral degradation of ERTU and MET

FORCED DEGRADATION OF ERTU AND MET
148
Stressor
ERTU MET
Purity Angle-
Purity Threshold
% Assay
%
Degraded
[<20]
Purity Angle-
Purity Threshold
%
Assay
% Degraded
[<20]
Acid 1.493< 1.966 92.45 7.55 1.448 < 1.801 92.29 7.71
Alkali 1.766< 2.146 94.56 5.44 1.402 < 1.752 93.07 6.93
Oxidative 1.693 < 2.130 95.42 4.58 1.348 < 2.270 94.47 5.53
Thermal 1.936 < 2.341 96.86 3.14 2.753 < 2.927 97.07 2.93
Photolytic 1.659 < 2.009 98.30 1.70 2.489 < 2.786 98.21 1.79
Neutral 1.780 < 2.134 98.98 1.02 2.358 < 2.915 99.10 0.90

149
ACCURACY
80% level 100% level
120% level

150
ACCURACY OF ERTU
% Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total amount
recovered
(μg/ml)
% Recovery
Statistical Data
Mean ± SD % RSD
80 3.75 3.0
6.73 99.27
100.04 ± 0.306 0.3066.77 100.29
6.76 100.14
100 3.75 3.75
7.51 100.13
99.99 ± 0.135 0.1357.49 99.86
7.50 100.00
120 3.75 4.50
8.22 99.63
99.87 ± 0.64 0.6048.30 100.60
8.20 99.39

151
%
Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total Amount
recovered
(μg/ml)
% Recovery
Statistical Data
Mean ± SD % RSD
80 250 200
450.31 100.16
99.7 ± 0.54 0.54448.22 99.11
449.76 99.88
100 250 250
499.72 99.89
99.91 ± 0.57 0.57497.04 99.35
501.23 100.49
120 250 300
550.57 100.19
100.65 ± 0.47 0.47
553.38 101.13
551.95 100.65
ACCURACY OF MET

152
PRECISION OF ERTU AND MET
Injection
Intra-day Precision Inter-day Precision
Peak Area Peak Area*
ERTU MET ERTU MET
1 97558 5494807 96427 5519305
2 97857 5513074 97898 5518566
3 98282 5480884 98136 5521823
4 98143 5512058 97249 5547798
5 98046 5483072 98243 5586469
6 97987 5542631 97334 5560570
Mean 97978.8 5504421 97547.8 5539089
SD 251.1 23218 684.56 31980.6
% RSD [<2.0] 0.25 0.42 0.70 0.56

LINEARITY, LOD AND LOQ OF ERTU AND MET
153
Level-1 Level-2
Level-3

155
% Level Concentration [x] (μg/ml) Mean Peak area* [y] (AU)
0 0 0
25 0.9375 24802
50 1.875 49541
75 2.8125 74657
100 3.75 99130
125 4.6875 125714
150 5.625 145645
Linear Regression Equation (y = mx + c) y = 26223x + 460.9
Slope (m) 26223
Intercept (c) 460.9
LOD 0.025 μg/ml
LOQ 0.076 μg/ml
LINEARITY, LOD AND LOQ OF ERTU

156
% Level Concentration [x] (μg/ml) Mean Peak area* [y] (AU)
0 0 0
25 62.5 1374565
50 125 2780573
75 187.5 4158469
100 250 5566797
125 312.5 6877296
150 375 8152632
Linear Regression Equation (y = mx + c) y = 21857x + 31878
Slope (m) 21857
Intercept (c) 31878
LOD 0.87 μg/ml
LOQ 2.63 μg/ml
LINEARITY, LOD AND LOQ OF MET

157
LINEARITY PLOT OF ERTU
y = 26223x + 460.94
R² = 0.9993
0
20000
40000
60000
80000
100000
120000
140000
160000
0 1 2 3 4 5 6
PeakAreaAU
LINEARITY PLOT OF MET
y = 21857x + 31878
R² = 0.9997
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
0 50 100 150 200 250 300 350 400
PeakAreaAU

158
STABILITY OF STANDARD SOLUTION: 30 ± 20C for 24 hrs
ERTU
Time
[Hrs]
Peak areas* % Assay* Variation
0 98132 99.62 NA
12 97376 98.86 0.76
24 96418 97.88 1.74
Time
[Hrs]
0 5559487 99.96 NA
12 5509688 99.13 0.83
24 5475626 98.45 1.51
MET

159
Parameter
Modified
condition
% RSD of Peak
area [NMT 2.0 %]
Theoretical
Plates* N
[>2000]
Tailing factor*
[<2.0] Rs*
[>2.0]
ERTU MET ERTU MET ERTU MET
Flow rate
(1.0± 0.1 ml/min)
0.9 0.893 1.209 12959 10911 1.17 1.17 7.96
1.0 0.531 0.382 11679 9947 1.23 1.33 7.21
1.1 0.949 1.113 11564 9463 1.21 1.16 7.63
ACN ratio in
Mobile phase
Buffer: ACN
(35 ± 5 % v/v)
70 : 30 1.377 1.039 11949 11751 1.26 1.15 9.2
65 : 35 0.531 0.382 11679 9947 1.23 1.33 7.21
60 : 40 1.360 1.293 12741 9485 1.22 1.14 6.78
Temperature
(30 ± 50C)
25 0.912 1.278 13396 9470 1.14 1.20 7.03
30 0.531 0.382 11679 9947 1.23 1.33 7.21
35 0.574 0.993 13396 9516 1.21 1.17 7.01
* Mean of six determinations
ROBUSTNESS OF ERTU AND MET

160
SYSTEM SUITABILITY OF ERTU AND MET
Injection
ERTU MET
Rs > 2.0Rt
[mins]
Peak Area
Theoretical
Plates
N > 2000
Tailing
Factor
< 2.0
Rt
[mins]
Peak
Area
Theoretical
Plates
N >2000
Tailing
Factor
< 2.0
1 2.913 97891 10960 1.31 2.170 5524816 11033 1.24 5.9
2 2.926 97643 11512 1.20 2.171 5566109 11319 1.24 6.8
3 2.926 98719 11296 1.34 2.177 5536275 11623 1.15 7.2
4 2.929 98905 11897 1.19 2.186 5564500 8425 1.24 7.8
5 2.929 98824 11716 1.13 2.206 5581534 8208 1.38 7.7
6 2.932 98424 12694 1.23 2.297 5564584 9073 1.25 7.9
Mean ±
SD
2.925
98401±
523.3
11679 1.23 2.201
5556303
±
21265.4
9947 1.25 7.21
% RSD
[<2.0]
0.531 0.382

161
ASSAY OF ERTU AND MET
Injection
ERTU MET
Rt
[Mins]
Peak area % Assay
Amount
[mg]
Rt
[mins]
Peak area % Assay Amount [mg]
1 2.932 98132 99.63 7.47 2.177 5552565 99.83 499.15
2 2.929 97653 99.14 7.43 2.174 5531564 99.46 497.30
3 2.923 97956 99.45 7.46 2.176 5520126 99.25 496.25
4 2.933 97896 99.39 7.45 2.173 5529625 99.42 497.10
5 2.934 97698 99.19 7.44 2.174 5526486 99.36 496.99
6 2.933 97621 99.11 7.43 2.173 5536125 99.54 497.70
Mean ±
SD
99.31±0.21 7.45 ± 0.16 99.48±0.20 497.4 ± 0.97

162
Parameter
Result
ICH Limits
MET ERTU
System
suitability
parameters
% RSD 0.382 0.531 NLT 2.0
Theoretical plates 9947 11679 MT 2000
Tailing Factor 1.25 1.23 NMT 2.0
Range [μg/ml] 62.5-375 0.9375-5.625 -
Linearity [R2] 0.999 0.999 NLT 0.999
% Recovery 99.11-101.13 99.27-100.60 98-102
% RSD
Intra-day Precision 0.42 0.25 NMT 1.0
Inter-day precision 0.56 0.70 NMT 2.0
LOD [μg/ml] 0.87 0.025 -
LOQ [μg/ml] 2.63 0.076 -
% Assay difference at 24 hrs 1.51 1.74 NMT 2.0
% Assay 99.48 99.31 -
SUMMARY OF VALIDATION OF ERTU AND MET

163
The developed stability indicating RP-HPLC method was
 Simple and more sensitive
 Retention time : MET – 2.383 → 2.170mins
ERTU – 3.136 → 2.929mins
 Linearity range : MET – 125-750 → 62.5- 375μg/ml
ERTU – 1.875 - 11.25 → 0.9375 – 5.625μg/ml
 LOD : MET – 1.7 → 0.87μg/ml
ERTU – 0.07 → 0.025μg/ml
LOQ : MET – 5.16 → 2.63μg/ml
ERTU – 0.21 → 0.076μg/ml

STABILITY INDICATING ANALYTICAL METHOD
FOR THE SIMULTANEOUS DETERMINATION OF
EMPAGLIFLOZIN AND LINAGLIPTIN IN
BULK AND TABLETS BY RP-HPLC
SGLT-2 and DPP-4 inhibitors combination exerts
 Synergistic effect
Better glycemic control in diabetic patients compared to mono-drug
therapy [Katzung et al., 2015]
The reported RP-HPLC methods
Involve use of high volume of organic solvents
164

165
EMPA LINA
Structure
Class SGLT-2 inhibitor DPP-4 inhibitor
Molecular Weight 450.91 472.54
pKa 12.57 9.86
Log P 1.79 2.8
Solubility slightly soluble in ACN, ethanol,
soluble in 50 % ACN/water,
sparingly soluble in methanol,
very slightly soluble in water and
practically insoluble in toluene
very slightly soluble in water, soluble
in methanol, sparingly soluble in
ethanol, very slightly soluble in
isopropanol, and acetone
DRUG PROFILE: EMPAAND LINA

166
10mg EMPA
+
5 mg LINA
+
5 ml diluent,
sonicated for
10 mins
volume upto 10ml
+
filtered using 0.45 µ
PTFE filter
100µg/ml EMPA
50 µg/ml LINA
Diluted to 10 ml with
diluent
Preparation of standard stock solution-II of EMPA [100µg/ml] and LINA [50µg/ml]:
1ml standard
stock -I
1000µg/ml EMPA
500 µg/ml LINA
METHOD DEVELOPMENT: [Snyder et al., 1997] EMPAAND LINA
Diluent: HPLC grade water and Methanol [1:1]
Preparation of standard stock solution-I of EMPA [1000µg/ml] and LINA
[500µg/ml]:
Preparation of Working standard solution EMPA [10 µg/ml] and LINA [5 µg/ml]
diluent
10 µg/ml EMPA
5 µg/ml LINA
1ml standard
stock -II

167
Column Ascentis C18 (150mm x4.6 mm, 5µm)
Mobile phase 0.1 % OPA [pH 2.7] : Methanol (35:65 % v/v)
Elution Isocratic
Detector and λ max PDA and 220.0 nm
Column temperature 30°C
Diluent HPLC grade water : Methanol [1:1]
Retention time (mins)
EMPA- 2.218
LINA- 2.531
Run time 5.00 mins

168
OPTIMIZED CHROMATOGRAM OF EMPA AND LINA

VALIDATION [ICH Q2 (R1), 2005]: EMPAAND LINA: SPECIFICITY
169
Blank [HPLC grade water: Methanol (1:1)]
Standard
EMPA [2.218 mins) and LINA (2.531 mins)
Sample
EMPA (2.226 mins) and LINA (2.544 mins)

170
FORCED DEGRADATION STUDIES [Ahuja et al., 2001, ICH Q1A (R2), ICH Q3]:
Stressor Condition
Acid 2N HCl, 30 mins, 60±20C
Alkali 2N NaOH, 30 mins, 60±20C
Oxidative 20 %v/v H2O2, 30 mins, 60±20C
Thermal In oven at 105±20C for 6 hrs
Light UV Chamber for 7 days
Neutral HPLC grade water, 30 mins, 60±20C

171
Acid Degradation of EMPA and LINA

172
Alkali degradation of EMPA and LINA

Oxidative degradation of EMPA and LINA
173

174
Thermal degradation of EMPA and LINA

175
Photolytic degradation of EMPA and LINA

176
Neutral degradation of EMPA and LINA

177
Stressor
EMPA LINA
Purity Angle -
Purity Threshold
%
Assay
%
Degraded
[<20]
Purity Angle -
Purity Threshold
%
Assay
%
Degraded
[<20]
Acid 0.179 < 0.352 93.88 6.12 0.108 < 0.313 95.05 4.95
Alkali 0.179 < 2.648 93.40 6.60 0.988 < 1.142 94.65 5.35
Oxidative 0.199 < 0.743 93.85 6.15 0.278 < 0.357 92.62 7.38
Thermal 0.328 < 0.411 97.56 2.44 0.274< 0.298 96.32 3.68
Photolytic 0.478 < 0.569 98.77 1.23 0.275 < 0.318 98.57 1.43
Neutral 0.395 < 0.410 99.29 0.71 0.289< 0.320 99.42 0.58
FORCED DEGRADATION OF EMPAAND LINA

178
ACCURACY
80% level 100% level
120% level

179
% Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total Amount
recovered
(μg/ml)
%
Recovery
Statistical Data
Mean ± SD % RSD
80 10 8.0
18.06 100.78
100.59±0.265 0.26318.02 100.29
18.06 100.71
100 10 10
20.18 100.90
100.30±0.556 0.55319.96 99.80
20.04 100.21
120 10 12
22.04 100.20
99.80±0.520 0.5221.83 99.21
21.99 99.98
ACCURACY OF EMPA

180
%
Spiked
Fixed sample
concentration
(μg/ml)
Amount
Spiked
(μg/ml)
Total
Amount
recovered
(μg/ml)
% Recovery
Statistical Data
Mean ± SD % RSD
80 5.0 4.0
8.96 99.07
99.02±0.126 0.1278.95 98.88
8.96 99.12
100 5.0 5.0
9.99 99.82
99.64±0.162 0.1629.97 99.51
9.98 99.58
120 5.0 6.0
10.98 99.64
99.99±0.327 0.32711.00 100.04
11.02 100.29
ACCURACY OF LINA

181
PRECISION OF EMPAAND LINA
Injection
Intra-day Precision Inter-day Precision
Peak area Peak area*
EMPA LINA EMPA LINA
1 664012 371986 662885 375212
2 661089 371272 667110 373903
3 660789 371586 665238 370561
4 662598 371690 661401 374428
5 664236 372963 669054 373830
6 662359 372016 661814 371197
Mean 662514 371919 664584 373189
SD 1431 580.6 3081.6 1866.7
% RSD [< 2.0] 0.216 0.156 0.5 0.5

LINEARITY, LOD AND LOQ OF EMPAAND LINA
182
Level-1 Level-2
Level-3

184
% Level Concentration (x) [μg/ml] Mean peak area* (y) [AU]
0 0 0
25 2.5 163870
50 5.0 326856
75 7.5 455834
100 10.0 647690
125 12.5 796507
150 15 957091
Linear Regression Equation y = mx + c y = 63677x + 687.3
Slope (m) 63677
Intercept (c) 687.3
LOD 0.04 μg/ml
LOQ 0.13 μg/ml
LINEARITY OF EMPA

185
LINEARITY OF LINA
% Level Concentration (x) [μg/ml] Mean peak area*(y) [AU]
0 0 0
25 1.25 88606
50 2.50 175935
75 3.75 252744
100 5.00 351847
125 6.25 434506
150 7.50 517806
Linear Regression Equation y = mx + c y = 69175x + 799.5
Slope (m) 69175
Intercept (c) 799.5
LOD 0.02 μg/ml
LOQ 0.07 μg/ml

186
LINEARITY PLOT OF EMPA
y = 63677x + 687.38
R² = 0.999
0
200000
400000
600000
800000
1000000
1200000
0 2 4 6 8 10 12 14 16
PeakAreaAU
y = 69175x + 799.57
R² = 0.9995
0
100000
200000
300000
400000
500000
600000
0 1 2 3 4 5 6 7 8
PeakAreaAU
LINEARITY PLOT OF LINA

187
STABILITY OF STANDARD SOLUTION: 30 ±20C for 24 hrs
EMPA
Time
[Hrs]
0 666641 100.04 -
12 661647 99.29 0.75
24 657292 98.64 1.4
*Average of six determinations
LINA
Time
[Hrs]
Peak areas % Assay* Variation
0 374719 100.07 -
12 371708 99.27 0.8
24 368030 98.29 1.78
*Average of six determinations

188
Parameter
Modified
condition
% RSD of Peak area N* [>2000]
Tailing Factor*
[<2.0] Rs
[>2.0]
EMPA LINA EMPA LINA EMPA LINA
Flow rate
(0.8 ± 0.1ml/min)
0.7 0.518 0.892 13746 9506 1.35 1.31 3.23
0.8 0.613 0.577 12600 8928 1.29 1.34 3.33
0.9 1.019 1.409 8430 7148 1.28 1.37 2.68
Methanol :
0.1% OPA
buffer
(65 ± 5% v/v)
60 : 40 1.170 0.610 8546 7231 1.26 1.36 3.48
65 : 35 0.613 0.577 12600 8928 1.29 1.34 3.33
70 : 30 0.932 0.778 8931 8124 1.28 1.33 2.11
Temperature
(30±50C)
25 0.913 0.980 9621 8136 1.32 1.32 2.65
30 0.613 0.577 12600 8928 1.29 1.34 3.33
35 0.575 0.686 9098 7586 1.26 1.29 2.61
ROBUSTNESS OF EMPAAND LINA
*Mean of six determinations

189
SYSTEM SUITABILITY OF EMPAAND LINA
Injection
EMPA LINA
Rs [> 2.0]Rt
[mins]
Peak
Area
Theoretical
Plates
N [>2000]
Tailing
Factor
[< 2.0]
Rt [mins] Peak Area
Theoretical
Plates
N [>2000]
Tailing
factor
[< 2.0]
1 2.221 659494 12393 1.34 2.535 373303 9349 1.33 3.3
2 2.221 661554 12085 1.32 2.540 370806 8945 1.32 3.3
3 2.222 670414 12633 1.34 2.542 375348 8216 1.35 3.4
4 2.223 664401 13115 1.34 2.544 376969 9303 1.32 3.3
5 2.224 666030 13282 1.18 2.544 372942 8251 1.36 3.4
6 2.226 668190 12089 1.20 2.545 372811 9507 1.33 3.3
Mean ±
SD
2.222±
0.0019
665014 ±
4076.6
12600 1.29 2.541±0.037
373697±
2158.5
8928 1.33 3.33
% RSD
[< 2.0]
0.613 0.577

190
ASSAY OF EMPAAND LINA
Injection
EMPA LINA
Peak area % Assay Amount
[mg]
Peak area % Assay Amount
[mg]
1 661911 99.33 9.933
371923 99.33 4.96
2 665542 99.88 9.988
372364 99.44 4.972
3 662310 99.39 9.939
371756 99.28 4.964
4 669312 100.45 10.045
372584 99.50 4.975
5 662423 99.41 9.941
371489 99.21 4.960
6 666502 100.02 10.002
372186 99.40 4.97
Mean ± SD 99.74±0.45 9.975 ± 0.045 99.36±0.107 4.97 ± 0.006

191
Parameter
Result
ICH Limits
EMPA LINA
System
suitability
Parameters
% RSD 0.613 0.577 -
Theoretical plates 12600 8928 MT 2000
Tailing factor 1.29 1.33 NMT 2.0
Resolution - 3.33 NLT 2.0
Range [μg/ml] 2.5-15 1.25-7.5 -
Linearity [R2] 0.999 0.999 NLT 0.999
% Recovery 99.21-100.90 98.88-100.29 98-102
% RSD
Intra-day Precision 0.216 0.156 NMT 2.0
Inter-day Precision 0.5 0.5 NMT 2.0
LOD [μg/ml] 0.04 0.02 -
LOQ [μg/ml] 0.13 0.07 -
Solution Stability
% Assay difference at 24 hrs
1.4 1.78 NMT 2.0
% Assay 99.74 99.36 -
SUMMARY OF VALIDATION PARAMETERS

192
The present developed RP-HPLC method was
 More sensitive
 Retention time : EMPA- 3.907 → 2.218mins
 Run time : 10.0 → 5.0 mins
 Linearity : EMPA – 10-50 → 2.5-15μg/ml
LINA – 20-100 → 1.25-7.5μg/ml
 LOD : EMPA - 2.17 → 0.04μg/ml
LINA – 0.0372 → 0.02μg/ml
 LOQ : EMPA – 6.60 → 0.13μg/ml
LINA – 0.112 → 0.07μg/ml

193
7. DEVELOPMENT AND VALIDATION OF
IN-VITRO BIOANALYTICAL RP-HPLC METHOD
EMPAGLIFLOZIN BULK DRUG
The reported in-vitro bioanalytical method by RP-HPLC is
 Expensive – used only organic solvents as mobile phase and
diluent
 More run time

194
EMPA Dapagliflozin
(Internal Standard)
Structure
Molecular Weight 450.91 502.98
Class SGLT-2 inhibitor SGLT-2 inhibitor
pKa 12.57 12.57
Log P 1.79 2.52
Solubility Slightly soluble in ACN, ethanol,
soluble in 50 % ACN/water,
sparingly soluble in methanol, very
slightly soluble in water and
practically insoluble in toluene
soluble in ethanol, DMSO and DMF
DRUG PROFILE: EMPAAND DAPAGLIFLOZIN

195
METHOD DEVELOPMENT:
Buffer [0.01N KH2PO4]: (IP, 2018)
Mobile phase [Buffer: ACN (65:35 % v/v)]:
Preparation of EMPA stock solution [75μg/ml]:
7.5 mg EMPA
+
20ml diluent
Sonicated for 10mins
+
made up to 100ml
filtered using 0.45μ
PTFE filter
75μg/ml

196
Preparation of Internal Standard (IS) stock solution-I [100μg/ml]:
1.0 ml of stock-I
100μg/ml
filtered through
0.45μ PTFE filter
Sonicated for 10mins
+
made up to 100ml
10 mg IS
+
10ml diluent
diluent 10μg/ml
Preparation of Internal Standard (IS) stock solution-II [10μg/ml]

197
Vol. of stock
[ml]
Final Vol. [ml]
Concentration
[μg/ml]
EMPA
Standard Solutions-
Codes
0.05 10.0 0.375 EMPA S 1
0.1 10.0 0.75 EMPA S 2
0.15 10.0 1.125 EMPA S 3
0.4 10.0 3.0 EMPA S 4
1.0 10.0 7.5 EMPA S 5
1.2 10.0 9.0 EMPA S 6
1.6 10.0 12.0 EMPA S 7
2.0 10.0 15.0 EMPA S 8
PREPARATION OF CALIBRATION CURVE STANDARD EMPA SOLUTIONS:

198
EXTRACTION PROCEDURE:
plasma with
anti-coagulant,
K2EDTA
+
stored at -70°C
vortexed for
2 mins
+
1.0 ml Ethyl
acetate
vortexed for
2 mins
+
0.5 ml IS
stock-II
0.75 ml blank
plasma
+
0.25 ml EMPA
standard stock
thawed on
water bath
at 30±20C
vortexed for
2 mins
+
Left aside for
separation
centrifuged
in Cooling
centrifuge at
8000 rpm for
10 mins
separated
organic layer
evaporated to
dryness

199
PREPARATION OF PLASMA SOLUTIONS OF EMPA [37.5-1500 ng/ml]:
0.25 ml each
standard
solution of
EMPA
+
0.75 ml plasma
0.5ml IS stock-II
+
1.0 ml ethyl
acetate
37.5-1500 ng/ml
Preparation of plasma spiked QC samples of EMPA:
0.25 ml each EMPA
standard solution
+
0.75 ml plasma
0.5 ml IS stock-II
+
1.0 ml ethyl acetate
37.5 ng/ml (LLOQ)
112.5 ng/ml (LQC)
750 ng/ml (MQC)
1200 ng/ml (HQC)
1500 ng/ml (ULOQ)

200
Column Phenomenex C18 (250mm x 4.6 mm, 5m)
Mobile phase 0.01N KH2PO4 buffer (pH 3.0) : ACN (65:35 % v/v)
Elution Isocratic
Diluent HPLC grade water: ACN [1:1]
Run time 6.0 mins
OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR EMPA

201
Blank (Plasma)
Optimized Chromatogram of EMPA in Plasma

202
VALIDATION [USFDA 2018]: EMPA
Blank (Plasma)
LINEARITY
Zero Calibrator (Plasma + IS)
Level - 1 [37.5ng/ml] Level – 2 [75ng/ml]

203
Level – 3 [112.5ng/ml] Level – 4 [300ng/ml]
Level – 6 [900ng/ml]Level – 5 [750ng/ml]

204
Level – 8 [1200ng/ml]Level – 7 [1500ng/ml]
Concentration [ng/ml] Peak area of IS Peak area of EMPA
Peak area of
EMPA/IS
37.5 86452 1452 0.0168
75 86543 2904 0.0336
112.5 86259 4457 0.0517
300 86892 12618 0.1452
750 86501 29047 0.3358
900 86432 34856 0.4038
1200 86535 46475 0.5371
1500 86446 58894 0.6813
LINEARITY OF EMPA IN PLASMA

205
y = 0.00045x + 0.00148
R² = 0.99968
0.0000
0.1000
0.2000
0.3000
0.4000
0.5000
0.6000
0.7000
0.8000
0 200 400 600 800 1000 1200 1400 1600
PeakarearatioofEMPA/IS
Concentration [ng/ml]
LINEARITY PLOT OF EMPA IN PLASMA
Concentration
[ng/ml]
Concentration
Mean ± SD*
[ng/ml]
Intra-day
Accuracy*
[%]
Intra-day
Precision*
[% CV]
Concentration
Mean ± SD*
[ng/ml]
Inter-day
Accuracy*
[%]
Inter-day
Precision*
[% CV]
LLOQ
37.5
37.23 ± 3.65 99.27 9.82 36.89 ± 2.64 99.90 7.15
LQC
112.5
112.18 ± 4.99 99.72 4.45 112.18 ± 5.40 99.72 4.82
MQC
750
748.54 ± 6.61 99.81 0.88 748.70 ± 4.03 99.83 0.54
HQC
1200
1200.56 ± 5.29 100.05 0.44 1198.74± 5.76 99.90 0.48
*mean of five determinations
ACCURACY AND PRECISION OF EMPA IN PLASMA

206
Concentration
[ng/ml]
Concentration
[ng/ml]
Accuracy
[%]
LLOQ
37.5
37.52 100.053
37.98 101.28
38.2 101.86
38.3 102.13
37.82 100.85
37.89 101.04
Mean ± SD
37.95 ± 0.279 101.20 ± 0.744
% CV
0.735 0.735
SENSITIVITY OF EMPA IN PLASMA

207
S. No
HQC (1200 ng/ml) LQC (112.50 ng/ml)
Fresh solution
(ng/ml)
Stored solution
( ng/ml)
Fresh solution
( ng/ml)
Stored
solution
(ng/ml)
1. 1206.890 1198.930 110.169 108.185
2. 1204.851 1194.860 109.184 109.201
3. 1202.841 1196.841 111.192 106.168
4. 1198.912 1201.913 112.166 111.176
5. 1194.914 1205.911 114.178 115.194
6. 1196.894 1203.964 113.182 114.188
Mean ± SD 1200.88 ± 4.714 1200.40 ± 4.263 111.68 ± 1.870 110.69 ± 3.513
% CV 0.39 0.36 1.67 3.17
% Mean
Accuracy
100.07 100.03 99.27 98.39
STABILITY OF EMPA IN PLASMA [-28 ± 50C]

208
S. No
HQC (1200 ng/ml) LQC (112.50 ng/ml)
Fresh solution Stored solution Fresh solution Stored solution
1. 1198.884 1206.920 108.173 111.167
2. 1199.920 1204.901 110.184 108.182
3. 1201.900 1205.879 109.166 109.176
4. 1200.932 1199.865 113.194 112.199
5. 1204.892 1196.924 114.200 115.191
6. 1203.880 1194.896 115.169 114.188
Mean ± SD 1201.73 ± 2.310 1201.56 ± 5.05 111.68 ± 2.89 111.68 ± 2.74
% CV 0.19 0.42 2.59 2.46
% Mean
Accuracy
100.14 100.13 99.27 99.27
STABILITY OF EMPA IN PLASMA [-80 ± 50C]

209
Analyte EMPA
Internal standard Dapagliflozin
Method RP-HPLC
Biological matrix Human plasma
Anti-coagulant K2 EDTA
Extraction method Liquid-liquid extraction
Linearity range 37.5-1500 ng/ml
Correlation Coefficient [R2] 0.99968
QC concentrations [ng/ml] LLOQ- 37.5
LQC- 112.5
MQC- 750
HQC- 1200
ULOQC- 1500
Accuracy
[% recovery]
Intra-day 99.27-100.05
Inter-day 99.72-99.90
Precision
[% CV]
Intra-day 0.44-9.82
Inter-day 0.48-7.15
Long term stability at -28± 50C HQC- 100.03
LQC- 98.39
Long term stability at -80± 50C HQC- 100.13
LQC- 99.27
Sensitivity
LLOQ
Accuracy 101.2
% CV 0.735
SUMMARY OF VALIDATION

210
The developed in-vitro bioanalytical method for the estimation of
EMPA in human plasma was
 Simple, rapid and cost-effective
 Mobile Phase [ACN: Methanol (50:50 % v/v) →0.01N KH2PO4
buffer (pH 3.0): ACN (65:35 % v/v)]
 Retention time [8.898 → 4.390 mins]
 Run time [10.0 → 6.0 mins]
 Linearity [50-150 → 37.5-1500 ng/ml]

212
 Methods developed – 9: Drugs selected – 7
 Statin drugs – 2 : Rosuvastatin Calcium and Pitavastatin Calcium
 Anti-diabetic drugs – 5: Empagliflozin, Canagliflozin,
Ertugliflozin, Metformin and Linagliptin
UV SPECTROSCOPIC METHODS COLORIMETRY
Diluent/
parameter
ROS-Ca PIT- Ca CANA ROS-Ca
Diluent 0.1 N NaOH
Methanol :
Distilled water
(1:1)
Methanol :
Distilled
water (1:1)
Methanol
Linearity
range, LOD,
LOQ
↓ ↓ ↓ ↓
SUMMARY

213
Mobile Phase/
Parameter
ROS-Ca
Simultaneous estimation
ERTU MET EMPA LINA
Mobile Phase 0.1% OPA (pH 2.7) : ACN
(55:45 % v/v)
0.1% OPA (pH 2.7) :
ACN (65:35 % v/v)
0.1% OPA (pH 2.7) :
Methanol (35:65 % v/v)
Retention time, Run
time, Linearity range,
LOD, LOQ
↓ ↓ ↓
RP-HPLC METHODS
Mobile Phase/
Parameter
EMPA
QbD Approach Bioanalytical
Mobile phase 0.1% OPA (pH 2.7) : ACN (39.57:60.43 % v/v) 0.01N KH2PO4 buffer (pH 3.0) :
ACN (65:35 % v/v)
Retention time, Run
Time, Linearity range
↓ [LOD, LOQ] ↓

214
CONCLUSION
The methods designed and developed being simple, sensitive
and cost-effective may be useful for the estimation of the
selected statins and anti-diabetic drugs in API and tablet
dosage form in academic institutions, research centers, small
scale industries and drug testing labs

215
Ahuja S, and Scypinski S (2013): Handbook of Modern Pharmaceutical Analysis,
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Dwivedi J, Singhvi I, Vaya R, Kapadiya N, Mehta A, Jain V, Mahatma OP (2011):
Quantitative estimation of Rosuvastatin Calcium from tablet formulation by colorimetric
method using Cosneasie Brilliant Blue R Dye, Inventi Rapid - Pharm Analysis and Quality
Assurance, 198(11).
FDA Label: Merck & Co. Inc. SEGLUROMET™ (Ertugliflozin and Metformin
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https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/209806s000lbl.pdf
Goodman L, Gilman A, Brunton L, and Chabner B, Knollmann B (2011): Goodman and
Gilman's the Pharmacological Basis of Therapeutics, 12th Edn: New York, McGraw-Hill, pp
892-1266
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216
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potential side effects, Acta Cardiologica Sinica, 32(6): 631
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Pharmacology, Edinburgh, Churchill Livingstone, 7th Edn, pp 377-383

220
Sharma HL and Sharma KK (2017): Principles of Pharmacology, 3rd Edn, Hyderabad, Paras
Medical Publisher, pp 334-336
Snyder LR, Kirkland JJ and Glajch JL (1997): Practical HPLC method development. John
Wiley and Sons: 1-438
The Expert Panel, (2002): Third report of the National Cholesterol Education Program
(NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in
Adults (Adult Treatment Panel III), Final Report, Circulation, 106, 3143-3421
USFDA Guidance for Industry (May 2018): U.S. Department of Health and Human
Services, Food and Drug Administration, Food and Drug Administration: Center for Drug
Evaluation and Research (CDER): Center for Veterinary Medicine (CVM):
Biopharmaceutics: pp 1-37

Presentations
1. Presented a poster entitled “Analytical Method Development and Validation
for the Estimation of Rosuvastatin Calcium in Raw Material and Tablet
Formulation by UV spectroscopy and RP-HPLC” in UGC Sponsored
National Conference (IPTCON-2017) on “Current Challenges In Drug
Discovery and Development” at SPMVV, Tirupati on 6th and 7th Feb, 2017
2. Presented a poster entitled “Analytical Method Development and Validation
for the Estimation of Canagliflozin in Bulk and Formulation by UV
Spectroscopic Method”, in DST CURIE Sponsored National Conference
(IPTCON-2018) on “Innovative Research Trends in Drug Discovery” at
SPMVV, Tirupati on 21, 22 March 2018
221

Publications
1. Published a paper entitled “Analytical Method Development and Validation
for Estimation of Dipeptidyl Peptidase-4 Inhibitors: A Review” B.Sailaja and
K.Sravana Kumari. International Journal of Current Research in Chemistry
and Pharmaceutical Sciences, 2(4), 2015, 83–98
2. Published a paper entitled “Analytical Method Development and Validation for
the Estimation of Rosuvastatin Calcium in Raw Material and Tablet
Formulation by UV Spectrometric Method” B.Sailaja and K.Sravana Kumari.
Saudi Journal of Medical and Pharmaceutical Sciences, 2(1), 2016, 7-11
222

223
3. Published a paper entitled “Stability-indicating Method Development and
Validation for the Estimation of Rosuvastatin Calcium in Bulk and Tablet
Formulation by Reverse-phase High-performance Liquid Chromatography”
B.Sailaja and K.Sravana Kumari, Asian Journal of Pharmaceutical and
Clinical Research, 12(8), 2019, 251-256
Paper accepted for Publication:
4. “Development and Validation of Stability Indicating RP-HPLC Method for
the Simultaneous Determination of Ertugliflozin Pidolate and Metformin
Hydrochloride in bulk and tablets”
B.Sailaja and K.Sravana Kumari, Future Journal of Pharmaceutical Sciences

224
I express my sincere thanks to Prof. R. Nagaraju, Dean (In charge), for his kind support
and encouragement
It is my privilege to thank Prof. G. Rajitha, BOS Chairperson, Institute of Pharmaceutical
Technology, SPMVV for her moral support during my Ph.D. work
I express my sincere thanks to Prof. Y. Indira Muzib, Head of the Department, Institute of
Pharmaceutical Technology, SPMVV for her support during the work
I take this opportunity to thank Prof. M. Ajitha, External examiner and other evaluators for
their valuable suggestions
I take this opportunity to express my gratitude to my research supervisor
Prof. B. Sailaja, for her guidance, valuable suggestions, incredible support, patience and
persistent creative encouragement through out my research work
ACKNOWLEDGEMENTS

225
I owe a great debt of gratitude to Prof. K.V.S.R.G. Prasad,
Prof. K.Bharathi, Prof. Santh Rani Thakur,
Prof. B. Jeevana Jyothi, Prof. M. Vidyavathi, Prof. A. Sreedevi, Prof. S. Joshna Rani,
Associate Professors, Dr. K. Madhavi, Dr. B. Ramya Kuber and Dr.K.Swathi, Assistant
Professors, Dr. Shaheen Begum and
Dr. D. Sujatha for their kind support throughout my Ph.D. course
I take this opportunity to thank Laurus labs, Hyderabad, Apotex Pharma, Bangalore,
Micro labs, Bangalore, Hetero drugs, Hyderabad, Natco Pharma, Hyderabad, Ajanta
Pharma, Mumbai for providing me the gift samples of APIs
I express my sincere thanks to Dr. K. S. Murali Krishna, Principal,
Marri Laxman Reddy Institute of Pharmacy, Hyderabad for providing the facilities
required for Ph.D. work and for his support and encouragement

226
I am thankful to Dr. D. Dachinamoorthi, Principal, QIS College of Pharmacy, Ongole
for his support and encouragement
I am grateful and sincerely thankful to Dr. Gummalla Pitchaiah, Head of the
Department, QIS College of Pharmacy, Ongole for providing facilities for Ph.D. work
and encouragement
I am thankful to Dr. P. Sreenivasa Prasanna, Principal, Malineni Lakshmaiah College
of Pharmacy, Singarayakonda for providing the facilities required for Ph.D. work
My special thanks go to my friends Mrs. E. Pushpa Latha,
Mrs. K. Veditha, Mrs. S. Bhargavi for their help during my Ph.D. work

227
I express my heartfelt love and affection to my father, late
Mr. K. Anjaneyulu, mother, Mrs. K. Sujatha and brother, Mr. K. Siva Teja for their
constant encouragement and moral support without which this work would have not been
completed
I take this opportunity to express my profound sense of gratitude and respect to all those
who helped me directly and indirectly in completion of my Ph.D.

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