part-[b] simultaneous reverse phase high performance...

PART-[B]

Simultaneous reverse phase

high performance liquid

chromatographic [RP-HPLC]

determination of salicylamide,

salicylic acid and deferasirox in

the bulk API dosage forms

SECTION-[III]

HPLC method development and

validation of deferasirox

Section- [III] Deferasirox

PART-B Page 95

1.0 Introduction of deferasirox

Deferasirox is chemically 4-(3, 5-bis (2-hydroxyphenyl) benzoic acid. Its

molecular formula is C21H15N3O4 and molecular is weight 373.36 gm/mole.

Deferasirox is an oral iron chelator. 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 anemia [1-5].

Deferasirox is an oral iron chelator.

Deferasirox is a white to slightly yellow powder. Inactive Ingredients:

Lactose monohydrate, crospovidone, povidone (K3O), sodium lauryl sulphate,

microcrystalline cellulose, silicon dioxide, and magnesium stearate. 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 [6-8]. It is the first oral medication approved in the USA for this

purpose. It was approved by the United States Food and Drug Administration

(FDA) in November 2005 [9]. According to FDA (May 2007), renal failure and

cytopenias have been reported in patients receiving deferasirox oral suspension

tablets. It is approved in the European Union by the European Medicines

Agency (EMA) for children 6 years and older for chronic iron overload from

repeated blood transfusions [10-12]. Its low molecular weight and high

lipophilicity allows the drug to be taken orally unlike deferoxamine which has to

be administered by IV route (intravenous infusion). Together with deferiprone,

deferasirox seems to be capable of removing iron from cells (cardiac myocytes

and hepatocytes) as well as removing iron from the blood.

[2.0] Chemical structure, IUPAC and common name of deferasirox

Common name

20kg, Exjade, ICL-670, CGP 72670, Deferasirox, Deferasirox (CGP-72670),

Deferasirox for research, 4-[3,5-bis(2-hydroxyphenyl),4-[3,5-bis(2-


PART-B Page 96

hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid 4-[3,5-bis(2-hydroxyphenyl)-

1,2,4-triazol-1-yl]benzoic acid.

3.0 Brief overview of synthetic pathway of deferasirox

Deferasirox can be prepared from simple commercially available starting

materials (salicylic acid, salicylamide and 4-hydrazinobenzoic acid) in the

following two-step synthetic sequence.

4.0 Description

[4.1] Pharmacodynamics

Deferasirox is an orally active chelator that is selective for iron (as Fe3+).

It is a tridentate ligand that binds iron with high affinity in a 2 : 1 ratio.

Although deferasirox has very low affinity for zinc and copper there are variable

decreases in the serum concentration of these trace metals after the

administration of deferasirox. The clinical significance of these decreases is

uncertain.


PART-B Page 97

[4.2] Mechanism of action

Two molecules of deferasirox are capable of binding to 1 atom of iron.

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

which it has a strong affinity), forming a stable complex which is eliminated via

the kidneys.

[4.3] Pharmacokinetics

Pharmacokinetic data

Bioavailability 70 %

Protein binding 99 %

Metabolism Hepatic glucuronidation

Half-life 8 to 16 hours

Metabolism

Hepatic. 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. Indication for the

treatment of chronic iron overload due to blood transfusions (transfusional

hemosiderosis) in patients 2 years of age and older.

Table 1: Physical and chemical properties

Physical and chemical properties

Property Value

Molecular Weight 373.3615 g/mol

Molecular Formula C21H15N3O4

Molecular mass 373.362 g/mol

Exact Mass 373.106256 g/mol

Density 1.5 g/cm3

Solubility Soluble in water (< 1 mg/mL at 25°C), water

solubility (0.038 mg/mL at 37°C), soluble in

acetonitrile and methanol

CAS number 201530-41-8

Physical state White to Off-White Solid

Melting point 116-117°C

Boiling point 260-2620°C

Stability Less stable up to 45°C

Categories oral iron chelator


PART-B Page 98

[5.0] Survey of analytical method/Literature reviews

The literature reviews regarding deferasirox suggest that various

analytical methods were reported for its determination in pharmaceutical

formulation and in various biological fluids. As per discussion in the literature

reviews UV, LC-MS, HPLC methods for the determination of deferasirox in

pharmaceutical dosage forms are reported. Most of the reported methods do not

include stress degradation studies or are not completely optimized, validated

and they are cumbersome, time-consuming and expensive. The literature

reviews for analysis of deferasirox are as under:

[1] VS. Rao Somisetty, D. Dhachinamoorthi, SA. Rahaman, CH MM Prasada

Rao et al, have developed a simple, sensitive, specific UV-spectroscopic and

RP-HPLC methods are developed for the estimation of deferasirox in bulk and

pharmaceutical formulation. The first method was based on UV–spectroscopic

determination of the drug. It involves absorbance measurements at 319 nm in

0.1 M sodium hydroxide. Calibration curve was linear with the correlation

coefficient was 0.9997 % over a concentration range of 5 to 30 μg/mL for the

drug. The second method was based on HPLC separation of the drug in reverse

phase mode using C18 column (150 mm × 4.6 mm i.d. 5μ). The mobile phase

constituted of acetonitrile: water pH 3.5 adjusted with orthophosphoric acid (70

: 30 v/v) and flow rate 1.0 mL/min. Detection was performed at 248nm.

Separation completed within 5 minutes. Calibration curve was linear with the

correlation coefficient was 0.9996 % over a concentration range of 1 to 6 μg/mL

for the drug. The relative standard deviation (R.S.D) was found < 2.0 % for UV–

spectroscopic and RP-HPLC methods [13].

[2] PK. Tiwari, Padmakar Sathe, Navin Devadiga et al, have developed HPLC

method using Zorbax SB C18 column (250 4.6 mm i.d.), 5 μm, with UV

Detector was used. The proposed method was cost effective, specific, linear,

accurate, rugged and precise. The calibration curves showed good linearity over

the concentration range of 0.5 μg/mL-1.5 μg/mL with respect to the sample.

The correlation coefficient was 0.999 %. Excellent recoveries of 102 % were

obtained at the level 0.5 μg/mL [14].

[3] S. Saravanan, R. Swetha et al, have developed and validated a simple and

isocratic reverse phase high performance liquid chromatography (RP-HPLC)

method for determination of deferasirox and its impurities. The method was


PART-B Page 99

validated as per International Conference on Harmonization (ICHQ2A (R1))

guidelines for system suitability, precision, linearity, specificity, solution

stability, robustness and ruggedness, limit of detection and limit of

quantitation. Deferasirox was analyzed by using Inertsil ODS-3V (150 mm X 4.6

mm, 5 μm) at 35°C column temperature, with isocratic elution. The analysis

was performed at a wavelength of 245 nm using dual λ absorbance detector.

Efficient UV detection at 245 nm enabled determination of deferasirox without

any interference of impurities salicylic acid & salicylamide. The retention time

(RT) for deferasirox was around 8.7 min [15].

[4] S. Vattikuti, RS. Phani, R. Seetharaman, KS. Lakshmi et al, have

developed a simple rapid and precise reversed phase high performance liquid

chromatography method for the analysis of deferasirox. Chromatography

separation of deferasirox was performed by using a kromosil C18 column (250 x

4.6 mm, 5 μm) as stationary phase with a mobile phase comprising of

Methanol: O-phosphoric acid : 0.01 % Tetrahydrofuran [90 : 7 : 3 (v/v)] at a

flow rate of 1.5 mL/min and UV detection at 245 nm. The linearity of

deferasirox in the range of 0.01 mg/mL to 0.09 mg/mL. The limit of detection

for deferasirox was found to be 40 nanograms. The recovery was calculated by

standard addition method [16].

[5] M. Khan, S. Sinha, M. Todkar, V. Parashar, KS. Reddy, U. Kulkarni et

al, have developed a reverse phase high performance liquid chromatography

(RP-HPLC) method was developed and validated for the assay and process and

degradation related impurities of deferasirox. The method involves quaternary

gradient pumps and variable wavelength UV detector. Chromatographic

conditions include a 20 μL injection volume Xterra RP-18 (150 X 4.6 mm, 5 μm)

column (column temperature: 25°C), flow rate of 1.0 mL/min and detection at

250 nm. The separation of drug from its impurities was achieved by a gradient

elution method, using the mobile phase composed of solution A (water adjusted

to pH 2.5 with perchloric acid) and solution B (methanol). The retention times of

deferasirox, impurity A and impurity B are 37.7 min, 43.7 min and 49.3 min,

respectively. The method has been validated both for assay and impurity

profiling as per ICH guideline [17].

[6] VK. Chakravarthy, DG. Sankar et al, have developed an isocratic reverse

phase liquid chromatography (RP-HPLC) method has been developed and


PART-B Page 100

subsequently validated for the determination of deferasirox in pharmaceutical

formulation. Separation was achieved with a Develosil ODS HG-5 (150 mm

4.6 mm I.D; particle size 5 μm) and Sodium dihydrogen phosphate

monohydrate Buffer (pH adjusted to 3.0 with dilute orthophosphoric acid):

acetonitrile (55 : 45) as eluent at flow rate 2.0 mL/min. UV detection was

performed at 245 nm. The method is simple, rapid, and selective. The described

method of Deferasirox is linear over a range of 11.999 μg/mL was 35.997

μg/mL. The method precision for the determination of assay was below 2.0 %

RSD. The percentage recoveries of active pharmaceutical ingredient (API) from

dosage forms ranged from 100.5 to 101.0 [18].

[7] N. Padmaja, N. Ramathilangam et al, have developed a simple, accurate,

precise and linear isocratic RP-HPLC has been developed and subsequently

simultaneous for determination of deferasirox in pharmaceutical formulations.

Develosil ODS HG-S (150 X 4.6 mm) 5 μ with flow rate of 2 mL/min. By using

HPLC water PU-2695 pump and photodiode array detector-2996 at 245 nm.

The separation was carried out using a mobile phase consisting of mixture of

sodium dihydrogen phosphate monohydrate buffer (pH = 3.0) and acetonitrile in

the ratio of 55 : 45. The retention time of Deferasirox was found to be 6.31 min.

The mean percentage recovery was found to be 99.55 %. The correlation

coefficient was found to be 0.998 %. The percentage estimation of the drug was

found near to 100 % representing the accuracy of the method [19].

[8] JR. Kothawade, KM. Agrawal, VS. Lad et al, have a simple stability

indicating RP-HPLC assay method has been developed and validated for the

determination of deferasirox from tablet dosage form. Drugs were determined on

a Hypresil BDS 150 × 4.6 mm column packed with 5μm particles. The

optimized mobile phase was a 50 : 50 (v/v) Buffer (1 mL OPA in 2 lit. Milli-Q

Water) and Acetonitrile, pumped at a flow rate of 1 mL/min. UV detection was

performed at 245 nm. The method was validated in the concentration ranges of

50 ppm to 150 ppm where it demonstrated good linearity with R2 = 0.998 %.

The method was found to be robust, resisting to small deliberate changes in

flow rate, column temperature and composition of the mobile phase [20].


PART-B Page 101

[6.0] Aim and scope of present work

The primary objective of the present work was thus to develop and

validate a RP-HPLC method for the assay of deferasirox from API dosage forms.

Hence, the method is useful for routine quality control analysis and also for

determination of stability. Purpose of the present study was to develop and

validate a RP-HPLC Method for determination of deferasirox in API

pharmaceutical dosage forms. The aim and scopes of the proposed work are as

under:

1. To select suitable mobile phase (solvent buffer ratio)

2. To optimize RP-HPLC conditions

3. To develop suitable HPLC method for deferasirox

4. Perform the validation for the developed method

[7.0] Experimental

[7.1] Materials

Deferasirox obtained received from Cadila pharma dholka Ahmedabad.

Orthophosphoric acid was obtained from s d Fine Chemical Limited. Acetonitrile

(fisher Qualigens, HPLC grade) were obtained from Thermo Fisher Scientific

India Pvt. Ltd. and potassium dihydrogen phosphate was obtained from (Merck

Specialties Private Limited).

[7.2] Equipment

Equipment Apparatus

HPLC System Dionex ultimate 3000 (Germany) High performance liquid chromatographic system equipped with ultimate 3000 Pump, Auto Sampler, Column Compartment and RS Diode Array Detector

Software Dionex Chromeleon ® 7 (Version 7.1, Simply Intelligent)

Column oven Ambient

Column Waters symmetry C18 (4.6 x 250 mm, 5 µm, 110 Å)

[7.3] Preparation of stock and sample solutions

[7.3.1] Preparation of buffer

Dissolved 1.36 g of potassium dihydrogen phosphate in 1000 mL high

purity demonized Milli-Q water [Millipore, Milli-Q, Bedford, MA, USA,

purification system] and pH was adjusted 3.2 with ortho-phosphoric acid

and filtered through 0.22 μ size nylon filter under vacuum.


PART-B Page 102

[7.3.2] Preparation of mobile phase

The mobile phase was prepared by mixing 400 mL phosphate buffer pH

3.2 and 600 mL of acetonitrile [HPLC Grade]. The mixture was sonicated

in Expo-Hi Tech sonicator for 5 minutes.

[7.3.3] Preparation of diluent

Diluent for deferasirox was 99.5 mL acetonitrile and 0.5 mL water

[7.3.4] Preparation of standard and sample solution

The Standard stock solutions were prepared by accurately weighing 100

mg of deferasirox in 100 mL volumetric flask (1000 µg/mL) in

acetonitrile. Sample solutions were prepared by appropriate dilution of

the standard solutions with the diluent.

[8] Method development and optimization of chromatographic conditions

(UV graph/chromatograms)

To develop a precise, accurate and suitable RP-HPLC method for the

simultaneous estimation of salicylamide different mobile phases, solvent-buffer

ratios and pH were tried to proposed final chromatographic conditions.

Deferasirox is soluble in methanol and acetonitrile : water mixture. The peak

shape, resolution and symmetry of deferasirox were good with above gradient

elution at a 1.0 mL/min flow rate. The method developed was unique in

determining the impurities even at low levels than that of specifications. The

developed method was successfully applied to estimate the amount of

deferasirox.

Optimized chromatographic conditions

Parameter Optimized condition

Flow rate 1.0 mL/min

Mobile phase 40 : 60 v/v (Buffer : ACN)

Buffer pH Potassium phosphate buffer pH 3.2 adjusted by OPA

Wavelength 245 nm

Injection volume 10 µL

Run time 15 min

Column and column oven temperature

30ºC


PART-B 3

Figure 1: Chromatogram and UV calibration curve for deferasirox standard

(Mobile phase: ACN : buffer, 60 : 40 v/v, pH 3.2 with OPA).

Figure 2: Chromatogram and UV calibration curve for deferasirox sample



PART-B Page 104




PART-B Page 105



[9] Analytical method development/validation and its result and

discussion

The optimized RP-HPLC assay method was validated for specificity, linearity,

accuracy, precision (repeatability and intermediate precision), recovery and

system suitability according to International Conference on Harmonization

(ICH) guidelines for the validation of bioanalytical method [21] and the US Food

and Drug Administration (FDA) [22].

[9.1] System suitability

System suitability was performed by using 100 µg/mL of salicylamide by

making six replicate injections. Chromatographic parameters calculated from

experimental data, such as Number of theoretical plates, % RSD of peak area

and resolution factors (Rs) are given in table-2. The system was deemed to be


PART-B Page 106

suitable for use if the capacity factors were in the range of 2-20 (2 < K’ < 20),

lower than 2 for tailing factor, more than 2 for resolution (Rs), greater than

6988 number of theoretical plates (N), resolution between salicylamide of at

least two and less than 2 % relative standard deviation (% RSD) for peak area.

Table 2: System suitability parameters

System suitability parameters

Sr. No. Parameters Salicylic acid

1 Linearity range (µg/mL) 10.0-100.0 µg/mL

2 Retention time (Min.) 7.030

3 Theoretical plates (N) 6988

4 Peak Asymmetry (T) 2.46

5 Resolution (Rs) 2.4

6 Accuracy 99.99 %

7 Precision 99.70 %

8 % RSD (For peak area) 0.089 %

[9.2] Precision

The precision of the assay was studied with respect to both intra-day

(Repeatability) and Inter-day (Intermediated) precisions. Repeatability was

calculated from five replicate injections of three different concentrations of

deferasirox in the same equipment on the same day. Inter day precision was

checked with the same concentrations as intra-day assay and the

determination of each compound was repeated day by day during three days.

The method was found to be precise with RSD values within for intra-day and

inter day assay. Evaluation of the intra-day and inter-day precision for the

determination of deferasirox by the proposed HPLC method according to ICH

guidelines.

Intra Day (Repeatability) precision

Repeatability can be defined as the precision of the procedure when repeated by

same analyst under the same operating conditions over a short interval of time

or same day. It is normally expected that at least six replicates be carried out

and individual result provided from mean, standard deviation and coefficient of

variation should be calculated for set of n value. The RSD values are important

for showing degree of variation expected when the analytical procedure is

repeated several time in a standard situation (RSD below 2 % for assays in

finished product).


PART-B Page 107

Inter day (Intermediate) precision

Repeatability can be defined as the precision of the procedure when repeated by

same analyst under the same operating conditions and the determination of

each compound was repeated day by day during three days or study repeat

three days over a long interval of time.

Table 4: Intra-day and Inter-day precision data for deferasirox standard

Standard con. (µg/mL)

Intra day Inter day

Area (mAU*min) Area (mAU*min)

1000 596.335 596.337

596.252 596.389

596.399 596.299

596.288 596.249

596.372 596.399

Average 596.3292 596.3346

% SD 0.059989 0.062664

% RSD 0.01006 0.010508

Standard potency 99.77 %

Table 3: Intra-day and Inter-day precision data for deferasirox sample Sample

Con.

(µg/mL)

Intra day Inter day

Area

(mAU*m

in)

Mean

area

% SD %

RSD

Area

(mAU*m

in)

Mean

area

% SD %

RSD

100 60.359 60.323 0.064 0.106 60.358 60.342 0.053 0.089

60.288 60.252

60.345 60.375

60.232 60.339

60.395 60.389

500 291.159 291.301 0.139 0.048 291.157 291.201 0.056 0.019

291.219 291.198

291.374 291.217

291.245 291.148

291.508 291.289

1000 596.335 596.362 0.064 0.010 596.338 596.381 0.065 0.010

596.399 596.399

596.427 596.332

596.388 596.489

596.265 596.351

[1] % Assay =

P

AT = Average area of obtained in sample preparation

AS = Average area of obtained in standard preparation

W1 = Weight taken of reference standard (mg)


PART-B Page 108

W2 = Weight taken of test sample (mg)

P = Potency of reference standard (%)

[2]

[1] Intra day

1. % Assay =

99.77

= 1.00 0.9993 99.77 = 99.70 %

2.

=

= 0.010 %

[2] Inter day

1. % Assay =

99.77

= 1.00 0.9995 99.77 = 99.72 %

2.

=

= 0.010 %

[9.3] Limit of detection (LOD) and Limit of Quantification (LOQ)

The limit of detection (LOD) is defined as the lowest concentration of an analyte

that can reliably be differentiated from background levels. The standard

solutions of the compounds for LOD were prepared by diluting them

sequentially. Limit of quantification (LOQ) of an individual analytical procedure

is the lowest amount of analyte that can be quantitatively determined with

suitable precision and accuracy (ICH Guideline Q2B, 2005). LOD and LOQ were

determined calculating the signal-to-noise ratio of each compound by injecting

a series of solution until the S/N ratio 3 for LOD and 10 for LOQ. where S is the

standard deviation of y-intercepts of regression.

[9.4] Specificity

Specificity of method can be absence of any interference at retention

times of samples. The specificity of the method was demonstrated by injection

of standard solution of deferasirox at concentration of 100 µg/mL. The elution

peaks of salicylamide, salicylic acid and deferasirox presented in representative

chromatograms. The representative chromatogram for simultaneous

determination of the studied drugs in API pharmaceutical dosages forms.


PART-B Page 109

Table 5: Specificity study of deferasirox

Con. (µg/mL) Sample Standard

Area (mAU*min) Area (mAU*min)

100 59.978 59.99

59.992 59.98

59.957 59.97

59.983 59.99

59.978 59.95

Average 59.9776 59.976

% SD 0.012856905 0.016733201

% RSD 0.021436177 0.027899827

Standard potency 99.77 %

[1] % Assay =

99.66

= 1.00 0.9998 99.66 = 99.64 %

[2]

=

= 0.01018 %

[9.5] Linearity

The linearity of deferasirox was studied by preparing standard solution at

five different concentrations ranging from 10.0-100.0 µg/mL. Each

concentration was injected in a five replicates and mean value of peak area was

taken for calibration curve.

Construction of the calibration curves

Working solutions containing (10.0-100.00) μg/mL were prepared by serial

dilution of standard solution with the acetonitrile. In all cases, 10 µL aliquots

were injected (triplicate) and eluted with the mobile phase under the following

chromatographic conditions. The average peak area ratio of each drug and the

internal standard were plotted versus the final concentration of the drug in

μg/mL to get the calibration graph. Alternatively, the corresponding regression

equation was derived.

Sr. No. Concentration (µg/mL) Area (mAU*min)

1 1 0.968

2 10 6.51

3 20 10.12

4 30 14.29

5 40 21.277

6 50 28.353

7 100 59.977


PART-B Page 110

Figure 5: Linearity curve

Table 6: Summary of linearity data

Summary of linearity data

Sr. No. Parameters Deferasirox

1 Linearity range (µg/mL) 1.0-100.0 µg/mL

2 Slope ± Standard error 1.2427

3 Intercept ± Standard error 0.5984x

4 Linearity equation 0.5984x - 1.2427

5 r2 0.99 %

[9.6] Accuracy

Accuracy of the assay method was calculated for deferasirox by recovery studies

at three concentrations of 50 %, 100 %, 150 % and 200 % levels by standard

addition method. The mean % recovery for deferasirox was found is given in the

follow table 7.


PART-B Page 111

Table 7: Accuracy data for deferasirox

Sr. No.

Deferasirox Added (µg/mL)

Found (µg/mL)

% Recovery

% Mean Recovery

SD % RSD

1 50% 4.754 4.754 99.99 99.99 0.026 0.026

4.755 4.754 100.02

4.752 4.754 99.96

4.755 4.755 100.00

4.753 4.754 99.96

2 100% 10.057 10.057 99.99 99.99 0.028 0.028

10.059 10.056 100.03

10.054 10.057 99.96

10.057 10.058 99.98

10.052 10.056 99.96

3 150% 14.664 14.664 99.99 99.98 0.088 0.088

14.671 14.664 100.04

14.647 14.665 99.88

14.679 14.665 100.09

14.652 14.664 99.91

4 200% 20.388 20.388 100.00 100.01 0.081 0.081

20.378 20.388 99.95

20.371 20.387 99.91

20.413 20.388 100.12

20.399 20.387 100.05

Table 8: Sample and standard area for deferasirox

Sample Area (mAU*min)

Standard

Area (mAU*min)

50% 100% 150% 200% 1000%

28.353 59.977 87.45 121.58 596.335

28.359 59.989 87.49 121.52 596.252

28.343 59.959 87.35 121.48 596.399

28.358 59.973 87.54 121.73 596.288

28.345 59.948 87.38 121.65 596.372

Average 28.351 59.969 87.442 121.592 596.329

% SD 0.0073 0.0159 0.0779 0.0897 0.0599

% RSD 0.0258 0.0266 0.0890 0.0738 0.0100

Standard potency

99.77 %

[1] Amount added (µg/mL) =

=

= 4.754 µg/mL

[2] Amount found (µg/mL) =

=

= 4.754 µg/mL


PART-B Page 112

[3] % Recovery =

= 99.99 %

[10.0] Summary and Conclusion

A new RP-HPLC-DAD method has been developed for the estimation of

deferasirox in bulk API dosages forms. The developed method was

validated as per ICH guidelines and was found to be simple, precise,

accurate, and sensitive. The proposed method is rapid, where the total

analytical run time for deferasirox (Rt = 7.030 min.) and the internal standard is

less than 10 min and can be used for routine analysis of deferasirox in

bulk API dosages forms.

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[5] V. Sambasivarao, RS. Phani, R. Seetharaman, KS. Lakshmi. IJPI’s

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[9] FDA Approves First Oral Drug for Chronic Iron Overload (Press release).

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[11] http://www.lancet.com/journals/lancet/article/PIIS01406736%2813%

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