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CHAPTER-XI

DEVELOPMENT AND VALIDATION

OF BIOANALYTICAL METHOD

FOR THE DETERMINATION OF

DEFERASIROX IN HUMAN PLASMA

BY LC-MS/MS

Chapter-XI Deferasirox

33

11.1 DRUG PROFILE

Deferasirox is an oral iron chelator widely approved for the treatment of transfusional

chronic iron overload. In patients with transfusion-dependent anemias, toxic and

potentially lethal levels of iron accumulate over time. Humans are unable to actively

eliminate iron from the body, once it has been acquired. Toxic and eventually lethal

levels of iron accumulate as a result of repeated transfusions or because of excessive

dietary iron uptake in anemias and hereditary hemochromatosis. The harmful effects

of chronic iron overload can lead to damage of the liver, heart, and endocrine glands,

resulting in organ compromise and death (Felix Waldmeier et al.). Its main use is to

reduce chronic iron overload in patients who are receiving long term blood

transfusions for conditions such as beta-thalassemia and other chronic anemias

(Choudhry VP et al. and Yang LP et al.). It is the first oral medication approved

for this purpose.

Deferasirox works in treating iron toxicity by binding trivalent (ferric) iron and

forming a stable complex which is eliminated via the kidneys. Two molecules of

deferasirox are capable of binding to 1 atom of iron. Its low molecular weight and

high lipophilicity allows the drug to be taken orally unlike desferoxamine which has

to be administered by intravenous infusion. Deferasirox is highly (~99%) protein

bound almost exclusively to serum albumin. CYP450-catalyzed (oxidative)

metabolism of deferasirox appears to be minor in humans (about 8%).

Glucuronidation is the main metabolic pathway for deferasirox, with subsequent

biliary excretion. Deferasirox and metabolites are primarily (84% of the dose)

excreted in the feces. Renal excretion of deferasirox and metabolites is minimal (8%

of the administered dose). The mean elimination half-life ranged from 8 to 16 hours

following oral administration.

Drug Properties:

Name : Deferasirox

IUPAC name : 4-[3,5-Bis (2-hydroxyphenyl)-1H-1,2,4-triazol-1yl]-

benzoic acid

Chemical formula : C21H15N3O4


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Molecular weight : 373.4

Description : White to slightly yellow powder

pKa : 4.55, 0.19

logP : 4.74

Melting Point : 116-117 °C

Solubility : Methanol

Chemical Structure:

Figure 11.1 Chemical Structure of Deferasirox

Figure 11.2 Chemical Structure of Deferasirox D4 (Internal standard)


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11.2 LITERATURE REVIEW AND AIM OF THE WORK

11.2.1 LITERATURE REVIEW

At the time of initiation of method development, literature survey was performed to

study the reported methods. Few methods are reported using different analytical

techniques. Methods for the determination of deferasirox and its impurities in

pharmaceutical formulations were reported by using HPLC (Kalyana Chakravarthy V

et al., Mubeen Ahmad Khan et al., Padmaja N et al., Sambasivarao Vattikuti et al. and

Sampath Saravanan et al.,).

Bioanalytical methods for the determination of Deferasirox from plasma were

reported by using HPLC (Felix Waldmeier et al. and Silvia De Francia et al.) and

liquid chromatography coupled to mass spectroscopy (Chauzit E et al. and Felix

Waldmeier et al.) and hydrophilic interaction liquid chromatography coupled to mass

spectroscopy (Pligoropoulou H et al.). Deferasirox determination using dried blood

spot analysis using liquid chromatography mass spectroscopy (Ramakrishna Nirogi et

al.) was also reported.

11.2.2 AIM OF THE WORK

As very few bioanalytical methods with LC-MS/MS detection are reported for the

determination of deferasirox in human plasma and even the reported methods

established the higher lower limit of quantifications of 0.5 µg/ml. (Chauzit E et al.,)

There was need to for a simple bioanalytical method with the lower limit of

quantification of about 0.2µg/ml considering the dosage required for the

bioequivalence and pharmacokinetic evaluations. The aim of the work was to develop

and validate an LC-MS/MS method for the determination of deferasirox in human

plasma using labeled internal standard. The method was validated for the linearity

range required for the bioequivalence and pharmacokinetic studies.

11.3 MATERIAL AND METHODS

11.3.1 INSTRUMENTATION

LC-MS/MS system configuration included Shimadzu HPLC coupled with sciex API

3000 mass spectrometer. Discovery C18 (50x4.6mm, 5µm) column was used. The


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instrument was equipped with pump, column oven, degasser and mass spectrometer

with turbo ion spray interface. Auto injector was used for sample injection and data

acquisition was done by using Analyst software version 1.4.2. An Afcoset electronic

balance was used for weighing the materials. Class ‘A’ Borosil glass ware was

employed for volumetric and general purpose in the method development and

validation. All the above instruments including the supporting instruments like deep

freezer, micro pipettes, centrifuge, evaporator, vortexer were appropriately calibrated

for routine usage.

11.3.2 CHEMICALS AND REAGENTS

Following are the list of chemicals and reagents used in the method.

Deferasirox

Deferasirox D4 (Internal standard)

Methanol (HPLC grade)

Ammonium formate

Formic acid

Ethyl acetate (GR grade)

Ammonia (GR grade)

Milli Q water

K2 EDTA plasma

11.3.3 PREPARATION OF SOLUTIONS

Ammonium formate buffer-1 was prepared by dissolving approximately 158 mg of

ammonium formate in 500 ml of milliQ water and the pH of the solution was adjusted

to 5.0 using formic acid. Ammonium formate buffer-2 was prepared by dissolving

approximately 315 mg of ammonium formate in 250 ml of milliQ water and the pH of

the solution was adjusted to 2.5 using formic acid. Ammoniated methanol solution

was prepared by mixing 10 ml of ammonia and 490 ml of methanol. Mobile phase

was prepared by mixing Ammonium formate buffer-1 and methanol in the ratio of

20:80. Autosampler washing solution was prepared by mixing 700 ml of methanol

and 300 ml of milliQ water.


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11.3.4 PREPARATION OF STOCK SOLUTIONS AND SPIKING SOLUTIONS

OF DRUG AND INTERNAL STANDARD

Deferasirox and deferasirox D4 stock solutions were prepared at a concentration of 2

mg/ml by dissolving in ammoniated methanol and the stock solutions were stored in

the refrigerator. Two separate stock solutions of deferasirox were prepared for

preparation of calibration curve standards and quality control samples. Spiking

solutions of deferasirox for the preparation of calibration standards and quality control

samples were prepared in ammoniated methanol solution. Internal standard dilution

was prepared at a concentration of approx. 10 µg/ml by transferring 0.25 ml of

internal standard stock solution to 50 ml volumetric flask and made up the volume

with ammoniated methanol solution.

11.3.5 PREPARATION OF CALIBRATION CURVE STANDARDS AND

QUALITY CONTROL SAMPLES

Calibration curve standards preparation:

The spiking solutions for the calibration curve were prepared from deferasirox CC

stock solution as per the table given below in the concentration range of 1261.470

µg/ml to 10.067 µg/ml using ammoniated methanol as diluent.

Stock/Spiking

Solution ID

Stock/Spiking

Solution

Conc.(µg/ml)

Volume

taken (ml)

Made up to

volume

(ml)

Concentration

(µg/ml)

Prepared SS

ID

CC stock

solution 2034.629 6.2000 10 1261.470 STD 8 SS

STD 8 SS 1261.470 8.0000 10 1009.176 STD 7 SS

STD 7 SS 1009.176 6.0000 10 605.506 STD 6 SS

STD 6 SS 605.506 5.0000 10 302.753 STD 5 SS

STD 5 SS 302.753 5.0000 10 151.376 STD 4 SS

STD 4 SS 151.376 3.5000 10 52.982 STD 3 SS

STD 3 SS 52.982 3.8000 10 20.133 STD 2 SS

STD 2 SS 20.133 5.0000 10 10.067 STD 1 SS

Above calibration curve standard spiking solutions are subsequently used for the

preparation of calibration curves standards in plasma as per the table given below in

the range of 0.201 µg/ml to 25.229 µg/ml.


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Spiking

Solution ID

Spiking

Solution

Conc.(µg/ml)

Volume

taken (ml)

Made up

to volume

(ml)

Final STD

concentration

(µg/ml)

CC STD

ID

STD 8 SS 1261.470 0.2 10 25.229 STD 8

STD 7 SS 1009.176 0.2 10 20.184 STD 7

STD 6 SS 605.506 0.2 10 12.110 STD 6

STD 5 SS 302.753 0.2 10 6.055 STD 5

STD 4 SS 151.376 0.2 10 3.028 STD 4

STD 3 SS 52.982 0.2 10 1.060 STD 3

STD 2 SS 20.133 0.2 10 0.403 STD 2

STD 1 SS 10.067 0.2 10 0.201 STD 1

Quality Control samples preparation:

The spiking solutions for the quality control samples were prepared from deferasirox

QC stock solution as per the table given below in the concentration range of 966.668

µg/ml to 10.104 µg/ml using ammoniated methanol as diluent.

Stock/Spiking

Solution ID

Stock/Spiking

Solution

Conc.(µg/ml)

Volume

taken

(ml)

Made up

to volume

(ml)

Concentration

(µg/ml)

Prepared SS

ID

QC stock

solution 2056.740 4.7000 10 966.668 HQC SS

HQC SS 966.668 6.0000 10 580.001 MQC SS

MQC SS 580.001 0.5200 10 30.160 LQC SS

LQC SS 30.160 3.3500 10 10.104 LLOQQC SS

Above quality control sample spiking solutions are subsequently used for the

preparation of quality control samples in plasma as per the table given below in the

range of 0.202 µg/ml to 19.333 µg/ml.


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Spiking

Solution ID

Spiking

Solution

Conc.(µg/ml)

Volume

taken

(ml)

Made up

to volume

(ml)

Final QC

concentration

(µg/ml)

QC STD

ID

HQC SS 966.668 0.2 10 19.333 HQC

MQC SS 580.001 0.2 10 11.600 MQC

LQC SS 30.160 0.2 10 0.603 LQC

LLOQQC SS 10.104 0.2 10 0.202 LLOQQC

The prepared calibration curve standards and quality control samples are aliquoted

(0.3 ml) into individual polypropylene tubes and stored in the deep freezer.

11.3.6 LINEARITY RANGE VERIFICATION

The acceptability of the linearity range of the calibration curve was verified by the Q

test at 95% percent confidence limits for both lower and higher concentration of the

range.

Calibration Standards concentrations

STD 1 0.201

STD 2 0.403

STD 3 1.060

STD 4 3.028

STD 5 6.055

STD 6 12.110

STD 7 20.184

STD 8 25.229

Q Test at lowest concentration:

Range (STD 8-STD 1) 25.028

Module D1 (STD 2-STD 1) 0.201

Q1 Ratio (D1/Range) 0.008

Theoretical value of Q 95%

for 8 standards 0.526

As the Q1 ratio is less than the theoretical value, the lowest concentration of the

calibration curve range is acceptable.


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Q Test at highest concentration:

Range (STD 8-STD 1) 25.028

Module D2 (STD 8-STD 7) 5.046

Q2 Ratio (D2/Range) 0.202

Theoretical value of Q 95%

for 8 standards 0.526

As the Q2 ratio is less than the theoretical value, the highest concentration of the

calibration curve range is acceptable.

11.4 OPTIMIZATION OF THE CHROMATOGRAPHIC CONDITIONS AND

METHOD DEVELOPMENT

As the method development involves both liquid chromatographic separation and

mass spectrometric detection, parameters effecting both the phases were considered

during method development and optimized for consistent response.

Selection of internal standard

During liquid chromatographic and mass spectrometric detection, selection of internal

standard was very important for reliable method. Deferasirox D4 was selected as

internal standard as it was the deuterated form deferasirox. Labeled internal standards

behave similar to the analyte and matrix effects shall be controlled. As the molecular

weight of deferasirox D4 (377.4) was different to deferasirox (373.4), the ions are

easily differentiated during mass spectrometric detection.

Optimization of chromatographic conditions

Chromatographic conditions were optimized for analyte and internal standard using

isocratic flow rate. Initially mobile phase was optimized by using organic solvents

like methanol and acetonitrile and buffers like formic acid and acetic acid. Usage of

phosphate buffers was avoided due to non evaporative nature. Mobile phase

containing mixture of organic solvents and milliQ water does not produced optimum

response due to low ionization. Addition of ammonium buffers like ammonium

formate and ammonium acetate to the organic solvents produced increased and

consistent response due to increased ionization. Mobile phase consisting of 5 mM

ammonium formate buffer and methanol in the ratio of 20:80 was proved to be the


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most suitable of all the combinations. Separation of analyte and internal standard was

achieved on non polar C18 column of Discovery C18 (50x4.6mm, 5µm) column with

the flow rate of 0.9 ml/min.

Optimization of mass spectrometric conditions

Deferasirox and deferasirox D4 (internal standard) were tuned for the identification of

parent ion and the product ions (daughter ion) by infusing around 500 ng/ml of the

pure aqueous solutions individually. Tuning was initially checked in both positive and

negative modes from 100 to 400 amu and better sensitivity was observed in negative

mode due to the ability to donate protons. Parent ions were selected and confirmed

based on the molecular weight and parent ions of deferasirox and deferasirox D4 were

observed at 372.5 and 376.4. Product ions were selected based on reproducibility by

varying collision energy which provides fragmentation. 328.2 and 332.3 were selected

as product ions of deferasirox and deferasirox respectively. Other source and gas

parameters are optimized to get consistent response of both analyte and internal

standard. Mass spectra of analyte and internal standard are shown in figure 11.3 and

11.4.

Figure 11.3 Product ion spectra of deferasirox


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Figure 11.4 Product ion spectra of deferasirox D4

Optimization of sample extraction procedure

As deferasirox was having high partition coefficient, liquid-liquid extraction

technique was evaluated for suitability. Even though liquid-liquid extraction

technique are prone to cause matrix effects during mass spectrometer detection, due to

the usage of labeled internal standard (deferasirox D4), the matrix effects shall be

compensated. Protein precipitation with methanol or acetonitrile yielded very low

recoveries and hence liquid-liquid extraction was used. During liquid-liquid

extraction, ethyl acetate provided optimal response and chromatography. During

extraction, before addition of organic solvent, the plasma sample was added with

ammonium formate buffer-2 solution. Plasma volume of 0.2 ml was used to achieve

lower limit of quantification.

Extraction procedure involves the following steps.

Aliquot of 0.2 ml (200µl) was added to the polypropylene tube containing

50µl of internal standard dilution and vortex the tubes.

To the above tubes add 200 µl of ethyl acetate and vortex the tubes.

To the above tubes add 2 ml of ethyl acetate and vortex the tubes for about 10

to 15 minutes.

Centrifuge the tubes at 3000 rpm for about 5 to 10 minutes to separate the

supernatant.


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Transfer the supernatant into another polypropylene tube and evaporate to

dryness under the stream of nitrogen gas at 50oC.

After evaporation, reconstitute with 0.4 ml of mobile phase and after vortexing

transfer to autosampler vials for injection.

After evaluation of various chromatographic and mass spectrometric conditions, the

following optimized conditions mentioned in table 11.1 were finalized for the method

validation.

Table 11.1 Optimized chromatographic and mass spectrometric conditions

Parameter Value

Chromatographic conditions:

Column Discovery C18 (50x4.6mm, 5µm)

Mobile phase Ammonium formate buffer methanol in the

ratio of 20:80

Flow rate 0.9 ml/min

Column temperature 40˚C

Autosampler temperature 4˚C

Injection volume 5 μl

Retention time Deferasirox: 1.0 minute

Deferasirox D4: 1.0 minute

Run time 2.0 min

Mass spectrometric conditions:

Ionization mode Turbospray negative mode

Deferasirox m/z ratio 372.4/328.2

Deferasirox D4 m/z ratio 376.4/332.3

Dwell time 200 msec

Temperature 450˚C


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Ion Source Gas1 45

Ion Source Gas 2: 45

Curtain gas 35

CAD gas 7

Ion spray voltage -4500

Data acquisition and processing

The chromatographic data was processed by peak area ratio of deferasirox to internal

standard using analyst software. The concentration of the calibration standards and

quality control samples were back calculated using the following equation derived for

each calibration curve. The preparation of calibration curve was prepared using

1/Conc2 as weighting factor.

Y = mX + C

Where, Y = Analyte area / Internal standard area (Area ratio)

X = Analyte concentration

m = Slope of the calibration curve

C = Intercept value

11.5 METHOD VALIDATION

The method was validated in compliance with international regulatory guidelines. The

parameters evaluated during the method validation are selectivity, accuracy, precision,

linearity, robustness, dilution integrity, recovery, carryover and stability during

various stress conditions including bench top stability, freeze thaw stability, auto

sampler stability, stability of stock solutions etc. During method validation,

calibration curve was analysed at the starting of the batch.

11.5.1 SELECTIVITY

Present method utilizing liquid chromatographic separation and detection by mass

spectrometer shown to be selective for the analyte and internal standard. Analyte and

internal standard are selectively extracted from the biological matrix and retention


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time of both the analyte and internal standard was 1.0 minutes. Mass spectrometric

detection of deferasirox and deferasirox D4 was selective as the parent and product

ion ratio was unique and possibly no other component have the similar ratio. No

interfering peaks were observed with the same retention time of the analyte when

plasma samples from different volunteers were analysed. Figures 11.5, 11.6 and 11.7

represent the chromatogram of blank plasma sample and lowest calibration standard

and highest calibration standards respectively.

Figure 11.5 Representative chromatogram of blank plasma sample

Figure 11.6 Representative chromatogram of lowest calibration standard


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Figure 11.7 Representative chromatogram of highest calibration standard

11.5.2 LINEARITY

Linearity of the method was demonstrated using three precision and accuracy batches

by least square regression analysis of the standard plots associated with eight

calibration standards which are distributed throughout the range. The calibration plot

was linear in the range of 0.201-25.229 µg/ml as shown in figure 11.7. The average

correlation coefficient was found to be 0.9986 with goodness of fit. Table 11.2 show

data from calibration curves analysed for the evaluation of precision and accuracy

during different days.

Figure 11.7 Calibration curve of deferasirox


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Table 11.2 Precision and accuracy of calibration standards

Batch

STD 1 STD 2 STD 3 STD 4 STD 5 STD 6 STD 7 STD 8 Regression

Coefficient

(r value)

Concentration (µg/ml)

0.201 0.403 1.060 3.028 6.055 12.110 20.184 25.229

Precision and

Accuracy Batch-1 0.206 0.366 0.910 3.379 6.491 12.061 21.447 26.207 0.9986

Precision and

Accuracy Batch-2 0.194 0.404 0.930 3.358 6.569 11.884 21.815 26.218 0.9981

Precision and

Accuracy Batch-3 0.202 0.402 1.001 3.462 6.610 11.567 21.069 25.915 0.9992

Average 0.2007 0.3907 0.9470 3.3997 6.5567 11.8373 21.4437 26.1133

0.9986 Standard

Deviation 0.00611 0.02139 0.04782 0.05499 0.06045 0.25028 0.37301 0.17185

%CV 3.0 5.5 5.0 1.6 0.9 2.1 1.7 0.7

11.5.3 PRECISION AND ACCURACY

Precision and accuracy was evaluated by analyzing three precision and accuracy

batches. Accuracy was calculated by computing the ratio of the calculated mean

values of the QC samples to their nominal concentrations, expressed as percentage

nominal. Each precision and accuracy batch consists of calibration curve standards,

blank sample, blank sample with internal standard and six replicates of LOQQC,

LQC, MQC and HQC. Precision and accuracy was evaluated both inter and intra

batches. The intraday and inter day precision and accuracy of the method for each

deferasirox concentration level are presented in Table 11.3. The mean accuracy for

each concentration level ranged from 96.3 to 102.9 and the mean precision for each

concentration level ranged from 3.4 to 6.7.

Table 11.3 Between and within batch precision and accuracy of QC samples

LOQQC LQC MQC HQC

Nominal

Concentration 0.202 0.603 11.600 19.333

Precision and

Accuracy Batch-1

0.198 0.591 12.030 20.287

0.212 0.571 11.852 21.249

0.201 0.593 10.868 20.190

0.221 0.590 11.846 21.020

0.215 0.614 11.512 18.738

0.204 0.590 11.469 19.144


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Average 0.2085 0.5915 11.5962 20.1047

Standard Deviation 0.00892 0.01366 0.41703 0.99785

%CV 4.3 2.3 3.6 5.0

% Nominal 103.2 98.1 100.0 104.0

Precision and

Accuracy Batch-2

0.204 0.593 11.331 21.125

0.206 0.598 11.760 20.216

0.216 0.567 11.295 20.324

0.212 0.579 11.200 20.946

0.201 0.570 11.043 19.611

0.215 0.609 11.015 20.098

Average 0.2090 0.5860 11.2740 20.3867


%CV 3.0 2.8 2.4 2.8

% Nominal 103.5 97.2 97.2 105.5

Precision and

Accuracy Batch-3

0.209 0.598 11.141 19.903

0.214 0.557 10.603 19.431

0.162 0.578 10.758 19.156

0.192 0.536 10.613 19.055

0.190 0.554 10.479 18.822

0.194 0.592 10.268 18.737

Average 0.1935 0.5692 10.6437 19.1840


%CV 9.4 4.2 2.8 2.2

% Nominal 95.8 94.4 91.8 99.2

Global Precision and Accuracy

Average 0.2037 0.5822 11.1713 19.8918


%CV 6.7 3.4 4.6 4.3

% Nominal 100.8 96.6 96.3 102.9


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11.5.4 RECOVERY

Recovery was evaluated by comparing response of extracted and unextracted samples.

Extracted samples include six replicates of extracted LQC, MQC and HQC samples.

Unextracted samples included the aqueous solutions equivalent to extracted samples.

Internal standard recovery was evaluated in the same manner at MQC level. The

average recovery for deferasirox in plasma was ranged from 75.9 to 88.1% for the

low, medium and high quality control samples respectively with an average of 81.8%.

The recovery of the internal standard was 86.6%.

Table 11.4 Recovery of deferasirox

S.No. LQC level MQC level HQC level

Extracted

samples

Un extracted

samples

Extracted

samples

Un extracted

samples

Extracted

samples

Un extracted

samples

1 53918 66372 1022805 1369538 2544024 2818791

2 54936 67098 999132 1349185 2516300 2775170

3 51766 65408 1028040 1324149 2465148 2743798

4 54543 65893 1007641 1243902 2428421 2746830

5 54423 65236 977932 1318166 2381047 2789387

6 54180 67206 1004054 1354192 2345453 2789185

Mean 53961.0 66202.2 1006600.7 1326522.0 2446732.2 2777193.5

% Recovery 81.5 75.9 88.1

Average %

Recovery 81.8

Table 11.5 Recover of deferasirox D4

S.No. MQC level

Extracted samples Un extracted samples

1 1884575 2169517

2 1857398 2176413

3 1876463 2172132

4 1860366 2033945

5 1855704 2208685

6 1886984 2197147

Mean 1870248.3 2159639.8

% Recovery 86.6


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11.5.5 MATRIX EFFECT

Matrix effect was evaluated by processing two samples of blank plasma of six

different lots till the extraction and the cartridges were extracted by equivalent

aqueous solution at low (LQC) and high (HQC) quality control levels along with

internal standard. These samples were considered as post extracted samples and are

compared against aqueous solutions. Matrix factor for analyte and internal standard

was calculated by comparing the peak response in presence of matrix ions to that of

peak response in absence of matrix ions. After calculation of analyte and internal

standard matrix factors separately, internal standard normalized matrix factor was

calculated by the ratio of analyte/internal standard matrix factor. The mean internal

standard normalized factor for deferasirox at low (LQC) and high (HQC) quality

control samples ranged from 0.98 to 1.06.

11.5.6 DILUTION INTEGRITY

Dilution integrity at 50% (1/2) and 25%(1/4) was evaluated by preparing a quality

control pool (containing 38.231 µg/ml of deferasirox) at a concentration of

approximately 2 times of HQC concentration to assess the dilution integrity. The

precision and accuracy for dilution integrity at 50% dilution and 25% dilution of the

QC pool sample with screened blank human plasma were determined by using fresh

calibration curve standards. The precision for dilution integrity of deferasirox was

5.6% at 50 percent dilution and 4.2% at 25 percent dilution. The accuracy for dilution

integrity of deferasirox was 94.1% for 50 percent dilution and 102.6% for 25 percent

dilution. Results were shown in the following table 11.6.


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Table 11.6 Dilution integrity of deferasirox

Nominal

Concentration 38.231 µg/ml

Dilution factor 50% dilution 25% dilution

S.No. Calculated


Calculated


1 39.694 39.741

2 36.459 39.386

3 35.596 36.813

4 35.231 39.047

5 34.897 41.861

6 33.926 38.581

Mean 35.9672 39.2382

Standard Deviation 2.00612 1.64340

% CV 5.6 4.2

% Nominal 94.1 102.6

11.5.7 STABILITY STUDIES

Stability evaluations of deferasirox in both aqueous solutions and in biological matrix

were evaluated after subjecting to different stress conditions and temperatures that

could encounter during regular analysis. All the stability assessments were made at

LQC and HQC level (n=6) by comparing the stability samples against freshly

prepared samples (comparison samples). Stability of internal standard stock solution

and dilution were also evaluated.

Stability of deferasirox in plasma was evaluated in terms of bench-top stability,

freeze-thaw stability, long-term stability and auto sampler stability. Bench-top

stability was assessed at room temperature for a period of 19 hours and freeze thaw

stability was evaluated for three cycles. During the freeze thaw cycles, freezing was

performed at -70°C and thawing was performed at room temperature. Long term

stability was evaluated at -70°C for 87 days. Stability of deferasirox during

autoinjector storage was evaluated at 4°C for 43 hours. Reinjection stability was

evaluated by injecting the previously injected samples and the reinjection


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reproducibility met the specifications. Stock solutions of deferasirox and deferasirox

D4 were evaluated by comparing against freshly prepared solutions and were stable

for 6 days in refrigerator including the short term storage at room temperature for 8

hours. Stock dilutions of deferasirox and deferasirox D4 were stable for 19 hours.

Stability evaluations of deferasirox and deferasirox met the acceptance criteria for %

stability demonstrating insignificant degradation at the specified storage conditions

and durations. The stability results are presented in table 11.7.

Table 11.7 Stability results of deferasirox

Stability QC

level

Mean

concentration of

stability samples

(µg/ml)

Mean concentration

of comparison

samples (µg/ml)

% stability

Bench top stability

(19 hours at 25°C)

LQC 0.6268 0.5936 105.6

HQC 20.4353 19.6894 103.8

Freeze thaw stability

(3 cycles)

LQC 0.6568 0.6023 109.0

HQC 20.4353 20.8458 98.0

Long term stability

(87 days at -70°C)

LQC 0.6053 0.6521 92.8

HQC 20.3549 19.4354 104.7

Autoinjector stability

(43 hours at 4°C)

LQC 0.6253 0.5938 105.3

HQC 20.3549 19.0733 106.7

11.6 SUMMARY OF THE METHOD

The present bioanalytical method involves the development of a selective, precise and

accurate method for the quantification of deferasirox in human plasma using LC-

MS/MS detection and liquid-liquid extraction technique, which is suitable for

application to a pharmacokinetic, bioequivalence and drug interaction studies for the

estimation of deferasirox from plasma. Summary of method validation results are

presented in table 11.8. Based on the results obtained during the method validation, it

is concluded that the present method can be successfully applied for the determination

of deferasirox in human plasma over the concentration range of 0.201 to 25.229

µg/ml. The method established a lower limit of quantification of 0.201 µg/ml and uses

low plasma volume of 0.2 ml with simple and cost effective liquid-liquid extraction


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technique ensuring reproducible recovery. The method met the acceptance criteria

with respect to selectivity, linearity, precision, accuracy, recovery, dilution integrity

and the stability evaluations performed in both plasma and stock solutions.

Table 10.8 Summary of method and validation results

Analyte Deferasirox

Internal Standard Deferasirox D4

Method Description Liquid-liquid extraction

Regression Model Linear regression with 1/Conc2 weighing

Analysis method Peak area ratios

Limit of Quantification 0.201 µg/ml

Selectivity No interference from the endogenous matrix

components

Recovery of analyte 81.8%

Recovery of internal

standard

86.6%

Linearity Range 0.201 to 25.229 µg/ml

Quality Controls

concentrations

LLOQQC: 0.202 µg/ml

LQC: 0.603 µg/ml

MQC: 11.600 µg/ml

HQC: 19.333 µg/ml

QC interday accuracy range 96.3 to 102.9%

QC interday precision range 3.4 to 6.7

Bench Top stability 19 hours at room temperature

Freeze-Thaw stability 3 Cycles

Auto injector stability 43 hours

Long term stability 87 days at -70oC

Stock solution stability 6 days

Stock dilution stability 19 hours

Dilution integrity 1/2 and 1/4

Re-injection

Reproducibility

One time


Page 254

11.7 REFERENCES

1. Chauzit E, Bouchet S, Micheau M, Mahon FX, Moore N, Titier K and

Molimard M; A method to measure deferasirox in plasma using HPLC coupled

with MS/MS detection and its potential application; The Drug Monitoring,

2010 Aug;32(4):476-81.

2. Choudhry VP and Rahul Naithani; Current status of iron overload and

chelation with Deferasirox; Indian Journal of Pediatrics, 2007, 74 (8), 759-64.

3. Felix Waldmeier, Gerard J Bruin, Ulrike Glaenzel, Katharine Hazell, Romain

Sechaud, Steve Warrington and John B Porter; Pharmacokinetics, Metabolism,

and Disposition of Deferasirox in β-Thalassemic Patients with Transfusion-

Dependent Iron Overload Who Are at Pharmacokinetic Steady State; Drug

Metabolism and Disposition, 2010, 38(5), 808-816.

4. Kalyana Chakravarthy V and Gowri Sankar D; LC Determination of

Deferasirox in Pharmaceutical Formulation; Journal of Global Trends in

Pharmaceutical Sciences, 2010, 1(1), 42-52.

5. Mubeen Ahmad Khan, Sukumar Sinha, Mangesh Todkar, Vikas Parashar,

Swamy Reddy K and Umesh Kulkarni; Validated HPLC-UV method for

simultaneous estimation of Deferasirox and its impurities; International Journal

of Pharmaceutical and Biomedical Research, 2011, 2(2), 128-134.

6. Padmaja N and Ramathilagam N; Method development and validation of RP-

HPLC method for the detremination of Deferasirox in tablets; International

Journal of Pharmacy and Biological Sciences, 2012, 2(4), 338-343.

7. Pligoropoulou H, Vonaparti A, and Panderi I; Hydrophilic interaction liquid

chromatography/positive ion electrospray mass spectrometry for the

quantification of deferasirox, an oral iron chelator, in human plasma; Journal of

Chromatography B, 2012, 893-894, 114-20.


Page 255

8. Ramakrishna Nirogi, Devender Reddy Ajjala, Vishwottam Kandikere,

Raghupathi Aleti, SuryaRao Srikakolapu and Himabindu Vurimindi; Dried

blood spot analysis of an iron chelator Deferasirox and its potential application

to therapeutic drug monitoring; Journal of Chromatography B, 907, 2012, 65-

73.

9. Sambasivarao Vattikuti, Phani RSCh, Seetharaman R, Dr. Lakshmi KS; RP-

HPLC Method Development for Analysis of Deferasirox in Formulation; IJPI’s

Journal of Analytical Chemistry, 2011, 1(4), 30-35.

10. Sampath Saravanan and Rama Swetha; Method development and validation for

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Silvia Racca, Francesco Di Carlo and Antonio Piga; A new HPLC UV

validated method for therapeutic monitoring of deferasirox in thalassaemic

patients; Journal of chromatography. B, 2012, 893-894, 127-33.

12. Yang LP, Keam SJ, Keating GM; Deferasirox: a review of its use in the

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