Validation and method development of Apixaban A research project.

Presented by-
Bhavana. G. (Y15MPh223)
II/II M. Pharm.
Dept. of Ph. Analysis
Under the guidance of-
Mrs G. Indira Priyadarshini, M.Pharm.,
Assistant Professor,
Dept. Ph. Analysis
HINDU COLLEGE OF PHARMACY
AMARAVATHI ROAD, GUNTUR – 522 002, A.P.

The main aim of this project was to develop a simple,
precise and accurate method and to validate a High
Performance Liquid Chromatography Mass
Spectrometric method for the estimation of Apixaban in
positive ion mode in human plasma using Apixaban
13C D3 as internal standard.
AIM AND OBJECTIVE

Literature survey
Procurement of the standard drugs
Development of method
Validation
Compilation of work
PLAN OF WORK

IUPAC name
DRUG PROFILE
Molecular Formula : C25H25N5O4
Drug name : Apixaban
Structure :
: 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-
4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide
Category : Anti-Coagulant

Description
Apixaban is a highly selective, orally bioavailable, and
reversible direct inhibitor of free and clot-bound factor Xa.
Apixaban has no direct effect on platelet aggregation, but by
inhibiting factor Xa, it indirectly decreases clot formation
induced by thrombin
Physicochemical properties
Physical state : White to half-white solid powder
Boiling point : 770.5 °C at 760 mmHg
Melting point : 237-238 °C
Flash Point : 419.8 °C
Gravity : 1.4 g/mL
Density : 1.42g/cm3
Solubility : DMSO and methanol

BRANDNAME ANDUSES OF APIXABAN
Apixaban systemic
Brand names: ELIQUIS
Drug class(es): Factor Xa inhibitors
Apixaban, sold under the trade name “ELIQUIS”, is
an anticoagulant for the treatment of venous thromboembolic events.
Route of administration: Orally
Dosage: “ELIQUIS” tablets are available for oral administration
in strengths of 2.5 mg and 5 mg of Apixaban.

1. Development and Validation of Stability Indicating RP-
HPLC Method on Core Shell Column for Determination of
Degradation and Process Related Impurities of Apixaban—An
Anticoagulant Drug, Vijayavitthal T. Mathad1*et.al.
The chromatographic separation was achieved on a Sigma-
Aldrich’s Ascentis Express C18 (4.6 mm × 100 mm, 2.7 µ) HPLC column
with a runtime of 40 min. Mobile phase-A and mobile phase-B were
phosphate buffer and acetonitrile respectively. The column oven
temperature was set at 35˚C and photodiode array detector was set at 225
nm.
2. Stability-Indicating High-Performance Liquid
Chromatographic Determination of Apixaban in the Presence of
Degradation Products Swarup Suresh Prabhune,* Rajendra Shankar
Jaguste, et.al.
Chromatography was performed on a 250 mm × 4.6 mm, 5 μm
C18 column with a gradient mixture of a phosphate buffer–methanol 60:40
(v/v) at 1.0 mL min-1. Ultraviolet detection of apixaban was at 220 nm.

3. Two Novel Validated RP-HPLC and UV- Spectrophotometric
methods for estimation of Apixaban in Bulk and Pharmaceutical Dosage
Forms. Rambabu. K*, Cherukuru Nagaraju, Ramasrinivas,G N Rao .
The first method was based on isocratic reverse phase liquid
chromatography using Sunfire C18, 150mm×4.6mm, 5µ and mobile phase
consists of Buffer: acetonitrile (60:40) at a flow rate 1ml/min and detection
was achieved photodiodide array detector set at 280 nm.
The second spectrophotometric method involves detection at 280 nm. The
calibration curve range between 5-50 µg/ml (R2 =0.9999).
4. Dabigatran, rivaroxaban, or apixaban versus ENOXAPARIN
for thromboprophylaxis after total hip or knee replacement: systematic
review, meta-analysis, and indirect treatment comparisons Antonio
Gómez-Outes*, Ana Isabel Terleira-Fernández.
Randomised controlled trials of rivaroxaban, dabigatran, or apixaban
compared with enoxaparin for prophylaxis against venous thromboembolism
after total hip or knee replacement.

5. Dabigatran, Rivaroxaban, or Apixaban versus WARFARIN in
Patients with Nonvalvular Atrial Fibrillation: A Systematic Review and
Meta-Analysis of SubgroupsAntonio Gómez-Outes, Ana Isabel Terleira-
Fernández.
New oral anticoagulants (NOAC; rivaroxaban, dabigatran, apixaban)
have become available as an alternative to warfarin anticoagulation in non-
valvular atrial fibrillation. Relative risks (RR) and 95% confidence intervals
(CI) were estimated using a random effect meta-analysis.
6. Representativeness of the Dabigatran, Apixaban and
Rivaroxaban clinical trial populations to real-world atrial fibrillation
patients in the United Kingdom: A cross-sectional analysis using the
General Practice Research Database. Sally Lee*, Brigitta U
Monz, Andreas Clemens.
Three oral anticoagulants have reported study results for stroke
prevention in patients with atrial fibrillation (AF) (dabigatran etexilate,
rivaroxaban and apixaban); all demonstrated superiority or non-inferiority
compared with warfarin (RE-LY, ARISTOTLE and ROCKET-AF)

7. Economic Evaluation of Warfarin, Dabigatran, Rivaroxaban,
and Apixaban for Stroke Prevention in Atrial Fibrillation
Gunhild Hagen*, Marianne Klemp.
Atrial fibrillation is a major risk factor for stroke, which causes
thousands of deaths and sequelae. It is recommended that atrial fibrillation
patients at medium or high risk of stroke use an oral anticoagulant to reduce
the risk of stroke. In the past few years, three new oral anticoagulants
(NOACs), dabigatran, rivaroxaban, and apixaban, have been introduced in
competition to the older oral anticoagulant warfarin.
8. A Systematic Literature Review on the Cost-Effectiveness of
Apixaban for Stroke Prevention in Non-valvular Atrial Fibrillation
Carme Pinyol + 6.
Different cost-effectiveness analyses suggest that Apixaban is a
cost-effective therapeutic option according to the WTP thresholds used in
countries where cost-effectiveness analyses, were performed.

9. Meta-Analysis on Risk of Bleeding With Apixaban in Patients
With Renal Impairment. Ranjan Pathak*, MD, Anil Pandit, MDPress,
Paras Karmacharya.
Apixaban is a novel oral anticoagulant which is approved for the
management of atrial fibrillation and venous thromboembolism prophylaxis.
There have been concerns regarding bleeding risks with Apixaban in
patients with renal impairment.

Apixaban Stock Solution:
Weighed about 5.0000 mg of Apixaban working standard and
transferred to a 5 ml clean glass volumetric flask, dissolved in
methanol and made up the volume with the same to produce a
solution of 1.000 mg/ml. The stock solution was stored in
refrigerator at 2 - 8 °C and used for maximum of 4 days.
Apixaban 13CD3 Stock Solution (Internal Standard):
Weighed about 2.0000 mg of Apixaban 13CD3 transferred to a 2
ml volumetric flask, dissolved in HPLC grade methanol and made
up the volume with the same to produce a solution of 1.000 mg/ml.
The stock solution was stored in refrigerator at 2 - 8 °C and used for
maximum of 4 days.

 Mobile Phase (v/v/v):
A mixture of HPLC grade Acetonitrile and methanol and
5mM ammonium formate in 0.1% formic acid buffer in the volume
ratio of 20:60:20 as mobile phase. The mobile phase was sonicated
in an ultra sonicator for 5 minutes. The mobile phase was used
within a period of 7 days and stored at room temperature (20±5ºC).
 Diluent (60% methanol, v/v):
A mixture of HPLC grade methanol and Milli-Q water or HPLC
grade water in the volume ratio of 60:40 as diluent. The solution
was sonicated in an ultra sonicator for 5 minutes. The solvent was
used within a period of 7 days and stored at room temperature
(20±5ºC).

MASS TUNING
+Q1: 0.301 min from Sample 1 (TuneSampleID) of MT20160328131755.wiff (Turbo Spray), C... Max. 2.0e7 cps.
300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
m/z, amu
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e6
7.0e6
7.5e6
8.0e6
8.5e6
9.0e6
9.5e6
1.0e7
1.1e7
1.1e7
1.2e7
1.2e7
1.3e7
1.3e7
1.4e7
1.4e7
1.5e7
1.5e7
1.6e7
1.6e7
1.7e7
1.7e7
1.8e7
1.8e7
1.9e7
1.9e7
2.0e7
2.0e7
Intensity,cps
433.2
403.2
463.2
460.2
493.3
417.3
304.2 373.1 387.2
442.4
472.3
327.1
330.4
447.3
358.4
305.2
321.1
461.3
477.2
382.3332.4
365.2
359.3 412.3
434.1 464.1356.9316.3
405.2
388.4 435.2
331.5322.1 371.2 421.2391.2
352.2 494.3345.1 374.2 409.2301.2 413.3 470.4357.4 473.2451.3333.4 404.2353.2 385.3 496.7440.3341.3 430.3369.2 415.3
315.2 482.4311.1 453.2 481.2424.2361.0 393.2343.1 381.3 400.3376.6 431.2335.2 436.3318.2 483.3474.3
347.2 491.2459.3399.2312.3 414.4398.3389.4
336.9
Analyte: Apixaban ANACIPHER CLINICAL RESEARCH Printing Date: 28/03/16
Printing Time: 13:19:06
WorkStation: BE-MS-04
Verified By:
Mass tuning of APIXABAN

Mass tuning of APIXABAN 13C-D3.

Method Development
 During method development we are performed three methods
 Liquid – liquid extraction
 Protein precipitation method
 Solid phase extraction method

Method -1 ( LLE method )
Placed in auto sampler for analysis
Vertex for 30 secs and loaded to vials
Allowed to dryness + 500 µl mobile phase for reconstitution
Placed in turbo evaporater at 450
Transferred in to dry test tube
Separate the 4 ml of supernant liquid
Centrifuge for 20 mins at 4 degrees (4000 rpm)
Add 5 ml of 10mM ammonium formate buffer in 0.1% formic acid (w/v)shake using
reciprocating pump for 20 mins
250 ml of plasma + 25 µl of internal standard( test tube)

 Column: Zodiac, C18, 50 x 4.6 mm, 3.0 µm (Make: Zodiac
Life Sciences)
 Buffer:10mM Ammonium formate
 Mobile Phase: HPLC grade acetonitrile: methanol: 10mM
ammonium formate in 0.1% formic acid (30:40:30, v/v/v)
 Diluent: HPLC grade methanol: HPLC grade water (60:40).
 Conclusion: We observed that base line is not proper.
so this method is not suitable.

Method -2 ( SPE) optimization method
200 µL of the plasma +20 µL of internal
standard & vortexed
500 µL of 1% Formic acid buffer, v/v added and
vortexed.
The sample mixture will be loaded onto Starata X-
33µM polymeric sorbent cartridges (Pre
conditioning with 1 ml methanol and 1 ml HPLC
grade water)
Extraction cartridge will be washed with 1 mL
of 5mM ammonium formate in 0.1% formic
acid and followed by 1.0ml of HPLC grade
water
The sample will eluted with 1 ml of mobile
phase

Chromatographic conditions
• Buffer : 1% Formic acid buffer, v/v)
• Mobile phase : HPLC grade acetonitrile: methanol: 5mM
Ammonium Formate in 0.1% formic acid (20:60:20, v/v/v)
• Column : Zodiac, C18, 50 x 4.6 mm, 3.0 µm (Make:
Zodiac Life Sciences)
Conclusion: The batch was within acceptance criteria peak shape
and response was good.

A Representative Chromatogram of an Aqueous Standard
and Internal Standard Mixture of Apixaban
Sample Name: "AQM(Zodiac 4.6*100mm,C18,µm,5mMAA:ACN15:85)" Sample ID: "" File: "001.wiff"
Peak Name: "Canagliflozin" Mass(es): "462.200/267.100 Da"
Comment: "" Annotation: ""
Sample Index: 1
Sample Type: Unknown
Concentration: N/A
Calculated Conc: 2393.615 ng/mL
Acq. Date: 03/08/2016
Acq. Time: 14:47:12
Modified: No
Proc. Algorithm: Analyst Classic
Bunching Factor: 1
Noise Threshold: 25.00 cps
Area Threshold: 250.00 cps
,Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
Exp. Peak Ratio: 5.00
Exp. Adj. Ratio: 4.00
Exp. Val. Ratio: 3.00
RT Window: 30.0 sec
Expected RT: 1.51 min
Use Relative RT: No
Int. Type: Base To Base
Retention Time: 1.27 min
Area: 4341331 counts
Height: 9.37e+005 cps
Start Time: 1.13 min
End Time: 1.56 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
Intensity,cps
1.27
Peak Name: "Canagliflozin D4(IS)" Mass(es): "466.000/267.100 Da"
Sample Index: 1
Concentration: 1.00 ng/mL
Calculated Conc: N/A
Acq. Date: 03/08/2016
Acq. Time: 14:47:12
Modified: No
Bunching Factor: 1
,Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
End Time: 1.51 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
Intensity,cps
1.26
Sample Name: "BLANK(250µL sample,0.5ml RC with MP,TBME)" Sample ID: "" File: "001.wiff"
Sample Index: 2
Sample Type: Double Blank
Acq. Date: 03/08/2016
Acq. Time: 14:49:54
Modified: No
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
Intensity,cps
1.82
1.09 1.43
1.25 1.300.24 0.82 1.160.65
Sample Index: 2
Acq. Date: 03/08/2016
Acq. Time: 14:49:54
Modified: No
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0
5
10
15
20
25
30
35
40
45
Intensity,cps
0.40
0.81
1.08
0.27
1.291.231.18
0.21
0.02
0.08 0.14 1.49
0.54
0.70 0.99
0.91
1.53

By LLE-Method 1 By SPE -Method 2
By changing Concentrations
of mobile phases and
compositions, several peaks
are obtained.
HPLC grade HPLC grade acetonitrile: methanol:
10mM ammonium formate in 0.1% formic acid
(30:40:30, v/v/v)
HPLC grade acetonitrile: methanol: 5mM
Ammonium Formate in 0.1% formic acid
(20:60:20, v/v/v)
Sample Name: "LQC-178" Sample ID: "" File: "001.wiff"
Peak Name: "Febuxostat" Mass(es): "317.1/261.0 amu"
Sample Index: 4
Sample Type: Standard
Acq. Date: 6/6/2016
Acq. Time: 3:17:38 PM
Modified: No
Bunching Factor: 1
Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
Area: 16248 counts
End Time: 0.984 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4400
4600
4800
5000
5200
5400
5600
5800
6000
6200
6400
6600
Intensity,cps
Peak Name: "Febuxostat D9(IS)" Mass(es): "326.1/262.0 amu"
Sample Index: 4
Acq. Date: 6/6/2016
Modified: No
Bunching Factor: 1
Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
Area: 261000 counts
End Time: 0.970 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e4
1.0e5
1.1e5
1.1e5
1.2e5
1.2e5
1.3e5
1.3e5
1.4e5
1.4e5
1.5e5
1.5e5
1.6e5
1.6e5
1.7e5
1.7e5
1.8e5
1.8e5
1.9e5
Intensity,cps
0.93
Analyte Name: Gliclazide ANACIPHER CLINICAL RESEARCH Printing Date: 30/09/16
Peak Name: "Febuxostat" Mass(es): "317.1/261.0 amu"
Sample Index: 16
Acq. Date: 6/7/2016
Modified: No
Bunching Factor: 1
Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
Area: 10621 counts
End Time: 1.12 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
Intensity,cps
Peak Name: "Febuxostat D9(IS)" Mass(es): "326.1/262.0 amu"
Sample Index: 16
Acq. Date: 6/7/2016
Modified: No
Bunching Factor: 1
Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
Area: 613342 counts
End Time: 1.22 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
Intensity,cps
0.96
Sample Index: 1
Concentration: N/A
Acq. Date: 03/08/2016
Acq. Time: 14:47:12
Modified: No
Bunching Factor: 1
,Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
End Time: 1.56 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
Intensity,cps
1.27
Sample Index: 1
Acq. Date: 03/08/2016
Acq. Time: 14:47:12
Modified: No
Bunching Factor: 1
,Num. Smooths: 5
Sep. Width: 0.20
Sep. Height: 1.00
RT Window: 30.0 sec
Use Relative RT: No
End Time: 1.51 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
Intensity,cps
1.26
Sample Index: 2
Acq. Date: 03/08/2016
Acq. Time: 14:49:54
Modified: No
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
Intensity,cps
1.82
1.09 1.43
1.25 1.300.24 0.82 1.160.65
Sample Index: 2
Acq. Date: 03/08/2016
Acq. Time: 14:49:54
Modified: No
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
0
5
10
15
20
25
30
35
40
45
Intensity,cps
0.40
0.81
1.08
0.27
1.291.231.18
0.21
0.02
0.08 0.14 1.49
0.54
0.70 0.99
0.91
1.53
Optimized
Chromatogram

Chromatographic Conditions:
o Column : Zodiac, C18, 50 x 4.6 mm, 3.0 µm (Make: Zodiac Life
Sciences)
o Mobile phase : HPLC grade acetonitrile: methanol: 5mM
ammonium formate in 0.1% formic acid (20:60:20, v/v)
o Rinsing solution : HPLC grade methanol: Milli-Q/HPLC grade
water (60:40, v/v)
o Flow rate : 1.000 mL/minute (With Splitter)
o Sample cooler temperature : 10°C
o Injection volume : 10 µL
o Column oven temperature : 35°C
o Retention time : Apixaban 0.74 ± 0.3 minutes
Apixaban 13C D3 0.74 ± 0.3 minutes
o Run Time :2.00 minutes

Representative chromatograms of aqueous standard analytes and
internal standard mixture, blank plasma, blank plasma with
internal standard, LLOQ QC sample, LQC sample, MQC1 sample,
MQC2 sample, HQC sample, regression analysis and extracted
blank plasma sample analyzed for Apixaban and are given below:

Tests Acceptance criteria
Carry over test Response of blank <20%of avg. drug response of LLOQ
and Is must be <5% of avg. of LLOQ
Matrix Selectivity RT of interfering peaks ≤ 20% of mean drug response in
LLOQ & IS ≤ 5% of mean IS in LLOQ
Analyte Selectivity ‖
Sensitivity Accuracy of LLOQ must be ±20% of normal value &
precision must be ≤20%. at-least 67% of LLOQ must be
±20% of normal value
Matrix Effect IS should be ≤ 15% and mean matrix factor should be in
between 0.85-1.15 at QC
Linearity Response of blank ≤20%of avg. drug response of LLOQ
and Is must be <5% of avg. of LLOQ & r² is > 0.98

Cont…
Tests Acceptance Criteria
Precision & Accuracy Response of blank ≤20%of avg. drug
response of LLOQ and Is must be <5%
of avg. of LLOQ
RT of interfering peaks ≤ 20% of mean
drug response in LLOQ & IS ≤ 5% of
mean IS in LLOQ for accuracy.
Ruggedness ‖
Dilution Integrity %CV & IS should be ≤15% for QC
Recovery ‖
Stability % stability should be in between 90 &
110 & %CV is ≤ 10%.

1. Carry Over Test
Prepared suitable extracted matrix blank (COT BLANK)+ COT
ULOQ, COT LLOQ of CC range. The prepared samples were injected
in the following sequence:
1. (COT LLOQ) 2. (COT ULOQ)
3. (COT BLANK) 4. (COT ULOQ)
9. (COT BLANK) 10.(COT ULOQ)
11.(COT BLANK) 12.(COT ULOQ)
13.(COT BLANK)

Calculations:
The results demonstrate that there was no carryover effect
of analyte and internal standard.
% of drug carry over = 𝐷𝑟𝑢𝑔 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑖𝑛 𝐶𝑂𝑇 𝐵𝐿𝐴𝑁𝐾 𝑋 100
Average drug response in COT LLOQ
% of internal std. carry over = 𝐼𝑛𝑡𝑒𝑟𝑛𝑎𝑙 𝑠𝑡𝑑. 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑖𝑛 𝐶𝑂𝑇 𝐵𝐿𝐴𝑁𝐾 𝑋 100
Average internal std. response in COT LLOQ
A Representative Chromatogram of Extracted blank plasma
sample analysed for Carryover Test

Area of Analyte Area of Internal Standard
COT-LLOQ
1 10218 1186003
2 9615 1112944
3 9393 1107467
4 8584 1065641
5 8963 1055050
6 9197 1070333
Mean 9328.3 1099573.0
Analyte Internal Standard
COT-Blank
Area
response
% Carry over Area response % Carry over
1 832 8.92 0 0.00
2 861 9.23 0 0.00
3 770 8.25 0 0.00
4 0 0.00 0 0.00
5 0 0.00 0 0.00
6 266 2.85 0 0.00
Carry Over Test Data for Apixaban

2. Matrix Selectivity
Plasma samples (from 8 individuals)+Aq. Mixture of drug + IS are
injected to check the retention time.
3. Analyte selectivity
Interference at analyte retention time caused due to internal standard
will be evaluated by injecting six replicates of matrix blank with
internal standard.

4. Sensitivity
Sensitivity will be determined in terms of LLOQ, ‘Lower Limit of
Quantification’ where the response of LLOQ must be at least five times
greater than the response of interference in blank matrix at the retention
time.
5. Matrix Effect
The matrix effect will be determined at two concentration levels (LQC
and HQC concentrations) in eight replicates each for analyte along with
internal standard.
IS normalized Matrix Factor = Peak response area ratio in presence of matrix ions
Mean peak response area ratio in absence of matrix ions
6. Linearity
A regression equation with a weighting factor of 1/(concentration
ratio) 2 of drug to IS concentration
.

A Representative Calibration Curve for Regression Analysis of
Apixaban

7. Precision and Accuracy
This includes-
• A medium standard (MQC2) dilution with internal standard
• Blank matrix
• Blank matrix with internal standard
• Spiked calibration standards
• RS (Mobile phase)
• Six LLOQ quality control samples
• Six low quality control samples
• Six middle (MQC1 & MQC2) quality control samples
• Six high quality control samples

Nominal Concentration (ng/mL)
LLOQ QC LQC MQC1 MQC2 HQC
QC# 1.031 % Accuracy 3.031 % Accuracy 45.238 % Accuracy 150.792 % Accuracy 226.754 % Accuracy
1 0.831 80.62 2.954 97.47 46.726 103.29 157.025 104.13 223.162 98.42
2 1.065 103.31 2.922 96.41 48.058 106.23 156.852 104.02 226.721 99.99
3 1.104 107.05 2.949 97.31 46.900 103.67 156.411 103.73 229.623 101.27
4 1.080 104.79 3.031 100.00 46.511 102.81 157.808 104.65 226.639 99.95
5 1.053 102.09 2.908 95.93 47.272 104.50 154.423 102.41 226.704 99.98
6 0.992 96.23 2.997 98.88 47.248 104.44 157.749 104.61 228.155 100.62
Mean 1.0208 2.9602 47.1192 156.7113 226.8340
S.D. 0.10027 0.04629 0.54660 1.24292 2.14825
C.V.% 9.82 1.56 1.16 0.79 0.95
% Nominal 99.01 97.66 104.16 103.93 100.04
N 6 6 6 6 6
7 0.867 84.13 2.962 97.72 47.000 103.90 155.579 103.17 225.480 99.44
8 0.957 92.84 2.912 96.06 46.360 102.48 156.749 103.95 226.651 99.95
9 0.970 94.11 2.996 98.84 47.261 104.47 155.969 103.43 225.837 99.60
10 0.911 88.33 2.897 95.57 47.454 104.90 154.125 102.21 226.411 99.85
11 0.928 89.97 2.919 96.30 46.818 103.49 156.583 103.84 228.312 100.69
12 0.892 86.48 2.998 98.91 46.618 103.05 156.742 103.95 224.344 98.94
Mean 0.9208 2.9473 46.9185 155.9578 226.1725
S.D. 0.03899 0.04413 0.40572 1.01337 1.32656
C.V.% 4.23 1.50 0.86 0.65 0.59
% Nominal 89.31 97.24 103.71 103.43 99.74
N 6 6 6 6 6
13 0.848 82.23 2.931 96.71 46.644 103.11 156.776 103.97 226.071 99.70
14 1.089 105.64 2.929 96.63 47.325 104.61 154.756 102.63 226.612 99.94
15 1.088 105.54 2.871 94.73 47.817 105.70 157.391 104.38 226.460 99.87
16 1.052 102.00 2.849 94.00 47.487 104.97 157.111 104.19 227.851 100.48
17 1.091 105.82 2.853 94.14 47.508 105.02 156.909 104.06 228.176 100.63
18 1.118 108.46 2.841 93.74 47.219 104.38 156.723 103.93 227.284 100.23
Mean 1.0477 2.8790 47.3333 156.6110 227.0757
S.D. 0.10005 0.04071 0.39405 0.94106 0.83170
C.V.% 9.55 1.41 0.83 0.60 0.37
% Nominal 101.62 94.99 104.63 103.86 100.14
Within batch response.

Nominal Concentration (ng/mL)
LLOQ QC LQC MQC1 MQC2 HQC
QC# 1.031
%
Accuracy
3.031
%
Accuracy
45.238
%
Accuracy
150.792
%
Accuracy
226.754
%
Accuracy
1 0.831 80.62 2.954 97.47 46.726 103.29 157.025 104.13 223.162 98.42
2 1.065 103.31 2.922 96.41 48.058 106.23 156.852 104.02 226.721 99.99
3 1.104 107.05 2.949 97.31 46.900 103.67 156.411 103.73 229.623 101.27
4 1.080 104.79 3.031 100.00 46.511 102.81 157.808 104.65 226.639 99.95
5 1.053 102.09 2.908 95.93 47.272 104.50 154.423 102.41 226.704 99.98
6 0.992 96.23 2.997 98.88 47.248 104.44 157.749 104.61 228.155 100.62
7 0.867 84.13 2.962 97.72 47.000 103.90 155.579 103.17 225.480 99.44
8 0.957 92.84 2.912 96.06 46.360 102.48 156.749 103.95 226.651 99.95
9 0.970 94.11 2.996 98.84 47.261 104.47 155.969 103.43 225.837 99.60
10 0.911 88.33 2.897 95.57 47.454 104.90 154.125 102.21 226.411 99.85
11 0.928 89.97 2.919 96.30 46.818 103.49 156.583 103.84 228.312 100.69
12 0.892 86.48 2.998 98.91 46.618 103.05 156.742 103.95 224.344 98.94
Mean 0.9708 2.9538 47.0188 156.3346 226.5033
S.D. 0.08938 0.04364 0.47075 1.15058 1.73694
C.V.% 9.21 1.48 1.00 0.74 0.77
%
Nominal
94.16 97.45 103.94 103.68 99.89
N 12 12 12 12 12
Intraday Precision and Accuracy for Apixaban

8. Recovery
Mean absolute % recovery of drug =
Mean Peak Area Response of analyte in extracted samples at LQC, MQC2 and HQC x 100
Mean Peak Area Response of analyte in non-extracted samples at LQC, MQC2 and HQC
Mean absolute % recovery of internal standard =
Mean Peak Area Response of internal standard in extracted samples x 100
Mean Peak Area Response of internal standard in non-extracted samples
Finally, calculate the overall recovery for the analyte.
Overall Absolute % Recovery = Average of absolute %
recoveries of LQC, MQC2 and HQC.
9. Dilution Integrity
12 sets of QCs spiked with about 1.7 and 3.4 times the
concentration of the highest standard (ULOQ) will be prepared
and stored in the deep freezer along with bulk spiked samples.

PARAMETER MEAN SD %CV N
Analyte Area IS Area
Matrix
selectivity 8776.5
ULOQ(60.00)
LLOQ(8776.5
1032475.2
138145.0
0.00
- - 12
Sensitivity 0.9302 0.10814 11.63 6
Matrix
Effect
{Absence of
matrix ions.}
[Presence of
matrix ions.]
LQC(0.02200)
HQC(1.59655)
LQC(1.013)
HQC(1.000)
LQC(0.0132)
HQC(0.0106)
LQC(1.30)
HQC(1.06)
LQC(12)
HQC(12)
Linearity r²=0.99 - - 6
Recovery 98.63 4.736 8.36 3

STABILITIES
• Room Temperature Standard Stock Solution Stability (20 ± 5 C)
• Room Temperature Spiking Solution Stability (20 ± 5 C)
• Refrigerated Stock Solution Stability at 2 - 8 °C
• Freeze-thaw Stability
• Bench Top Stability
• Short-term Plasma Samples Stability at -20 C
• Wet Extract Stability at 20±5 °C
• Auto sampler Stability
• Re Injection Stability
• Whole blood stability

A Representative Chromatogram of Blank Plasma Sample of
Apixaban

A Representative Chromatogram of an Aqueous Standard and IS
mixture of Apixaban

A Representative Chromatogram of Blank Plasma with IS of
Apixaban

A Representative Chromatogram of LLOQ QC Sample of Apixaban

A Representative Chromatogram of LQC Sample of Apixaban

A Representative Chromatogram of MQC1 Sample of Apixaban

A Representative Chromatogram of MQC2 Sample of Apixaban

A Representative Chromatogram of HQC Sample of Apixaban

Validation
Run
Analysis Date Testing performed Data Reported Comments
1 25/11/16 Carryover Test Yes No carryover effect(BE-MS-05)
2 25/11/16 Selectivity Yes No significant interference
3 25/11/16 Matrix effect Yes No matrix effect
4
26/11/16 Stock solution stability at room temperature
(20±5°C)
Yes Established 23 hours 13 min
5 26/11/16
Precision and accuracy batch-1 and
Sensitivity (Determination of the Lower
Limit of Quantitation)
Yes Results met the acceptance criteria
6 26/11/16
Precision and accuracy batch-2 and Dilution
integrity
7 28/11/16 Recovery Yes Results met the acceptance criteria
8 28/11/16
concomitant drug effect
9 28/11/16 Reinjection stability Yes Established 52 hours 40 min
10 29/11/16 Precision and accuracy batch-4 Yes Results met the acceptance criteria
11 29/11/16 Carryover Test Yes No carryover effect(BE-MS-04)
12 29/11/16
Ruggedness
13 30/11/16 Run size evaluation Yes Results met the acceptance criteria
14 31/11/16
Stock solution stability in refrigerator (at 2-
8°C)
Yes Established 5 days 21 hours
15 31/11/16
Freeze-thaw stability, Bench top stability,
short term plasma samples stability at -
20±5°C, wet extract stability and auto
sampler stability
Yes
Established Freeze-thaw stability IV
cycles, Bench top stability 17 hours
33 min, short term plasma samples
stability at -20±5°C for 5 days 11
hours, wet extract stability 68 hours
57 min and auto-sampler stability
72 hours 38 min
16 31/11/16 Drug stability in whole blood Yes
Established 3 hours 27 min at room
temperature (20±5°C)
Validation Run Analysis Summary

The results of selectivity, carryover test, matrix effect, sensitivity,
linearity, precision and accuracy, stabilities, recovery, dilution integrity,
run size evaluation presented in this report are within the acceptance
range for Bioanalytical batch acceptance criteria The analytical method
described above is valid for the estimation of Apixaban in human
plasma over a range of 1.022 ng/mL to 301.296 ng/mL with the
detection of Apixaban m/z – 460.20 (parent) and 77.10 (product) and
IS Apixaban 13CD3 m/z – 464.10 (parent) and 78.10 (product) for BE-
MS-05 and Apixaban m/z – 460.20 (parent) and 77.20 (product) and
IS Apixaban 13CD3 m/z – 464.20 (parent) and 78.20 (product) for BE-
MS-04 in Positive ion mode

I wish to express my sincere thanks and gratitude to the
Faculty and Management of Hindu College of Pharmacy for
their valuable suggestions and support and providing an
opportunity to carryout my project work at
“ANACIPHER CLINICAL RESEARCH ORGANISATION”,
Hyderabad.

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• Howard Hill., Development of bioanalysis: a short history.
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and Application, 1st ed., John Willey & Sons, Chichester,
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• Journal of pharmacy and pharmaceutical sciences,volume8,
issue9,2016
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no: 2328-0182Current Issue Volume No : 1 Issue No : 1

Validation and method development of Apixaban A research project.

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