DPC-3

Drug Profile

1. CLOMIPHENE CITRATE:

NAME Clomiphene Citrate

Official in IP, BP, USP Pharmacopeia.

Description Clomiphene Citrate is a medication used to induce

ovulation.
Structure

Chemical formulation C32H36ClNO8

Mol. Weight 598.09 g/mol

IUPAC Name 2-hydroxy-propane-1,2,3-tricarboxylic acid; {2-[4-

(2chloro-1,2-
Diphenyl ethenyl)phenoxy]ethyl}diethyl amine
Categories A triphenyl ethylene stilbene derivative which is
an estrogen agonist or antagonist depending on the
target tissue.
PHARMACOLOGY

CLASS Agonist and Antagonist

Mechnism of action Clomifene has both estrogenic and anti-

estrogenic properties, but its precise mechanism
of action has not been determined. Clomifene

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appears to stimulate the release of

gonadotropins, follicle-stimulating hormone
(FSH), and luteinizing hormone (LH), which
leads to the development and maturation of the
ovarian follicle, ovulation, and subsequent
development and function of the corpus luteum,
thus resulting in pregnancy. Gonadotropin
release may result from direct stimulation of the
hypothalamic-pituitary axis or a decreased
inhibitory influence of estrogens on the
hypothalamic-pituitary axis by competing with
the endogenous estrogens of the uterus, pituitary,
or hypothalamus. Clomifene has no apparent
progestational, androgenic, or antiandrogenic
effects and does not appear to interfere with
pituitary-adrenal or pituitary-thyroid function
Pharmacodynamics Clomifene (previously clomiphene) is an orally
administered, non-steroidal, ovulatory stimulant
that acts as a selective estrogen receptor
modulator (SERM). Clomifene can lead to
multiple ovulation, and hence increase the risk
of conceiving twins. In comparison to purified
FSH, the rate of ovarian hyperstimulation
syndrome is low. There may be an increased risk
of ovarian cancer and weight gain. Clomifene is
capable of interacting with estrogenreceptor-
containing tissues, including the hypothalamus,
pituitary, ovary, endometrium, vagina, and
cervix. It may compete with estrogen for
estrogen-receptor-binding sites and may delay
the replenishment of intracellular estrogen
receptors. Clomifene initiates a series of
endocrine events culminating in a preovulatory

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gonadotropin surge and subsequent follicular

rupture. The first endocrine event, in response to
a course of clomiphene therapy, is an increase in
the release of pituitary gonadotropins.
Properties

CAS.NO. 50-41-9

Melting point 116.5-118°C

Log p 6.08

Pka (basic) 9.31

Solubility Slightly soluble in water, Acetone, chloroform

Soluble in methanol and ethanol.
Insoluble in ether.

State white to pale yellow, crystalline powder

MARKETED FORMULATION:
Marketed Formulation of clomiphene Citrate

SR. BRAND DOSAGE MFG.

COMPOSITION
NO. NAME FROM COMPANY

1. Clomifene 50mg Cipla

Clomiphene
Citrate
2. Clopreg 25mg Kee Pharma
Clomiphene
Citrate
Genetic
Fertex-M Clomiphene 25mg
3. Pharma
Citrate

50mg Zydus – Biogen

Clomiphene
4. Folistim
Citrate

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25mg MicroNova
Clomiphene
5 Fulfyl
Citrate
50mg Torrent
Clomiphene
6 Omicite
Citrate
25mg LupinPh
Clomiphene
7 Oviphene arma
Citrate
8 25mg Win Medicare
Clomiphene
Ovipreg
Citrate
9 50mg Evacare
Clomiphene
Refert
Citrate
10 25mg Serum
Clomiphene
Siphene-M International
Citrate

2. Letrozole:

NAME

Letrozole
Official in BP, IP, USP Official Pharmacopeia.

Description Letrozole is an aromatase inhibitor used

to treat breast cancer in postmenopausal
women.
Structure

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Chemical formulation C17H11N5

Mol. Weight 285.303 g/mol

IUPAC Name 4,4'-((1H-1,2,4-triazol-1-yl) methylene)

dibenzo nitrile
Categories Letrozole is an oral non-steroidal type II
aromatase inhibitor.
PHARMACOLOGY

CLASS Nonsteroidal aromatase inhibitors.

Mechanism Letrozole is a non-steroidal type II

of action aromatase inhibitor. It blocks the active
site, and therefore the electron transfer
chain of CYP19A1. This competitive
inhibition prevents the conversion of
androgens to estrogen. This action leads
to a
reduction in uterine weight and
elevated luteinizing hormone. In
postmenopausal women, the action of
aromatase is responsible for the
majority of estrogen production.
With reduced availability of estrogen,
estrogendependent tumours regress.
Third generation aromatase inhibitors
do not significantly affect cortisol,
aldosterone, and thyroxine levels.
Pharmacodynamics Letrozole is an aromatase inhibitor used
in the treatment of breast cancer.
Aromatase inhibitors work by
inhibiting the action of the enzyme

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aromatase, which converts androgens

into estrogens by a process called
aromatization. As breast tissue is
stimulated by estrogens, decreasing
their production is a way of suppressing
recurrence of the breast tumour tissue.
Properties

CAS NO. 112809-51-5

Melting point 184°C-185°C.
Pka 2.17

Solubility Freely soluble in dichloromethane,

slightly soluble in ethanol, and insoluble
in water.
State white to yellowish crystalline powder

MARKETED FORMULATION:

Marketed Formulation of Letrozole:.

SR. BRAND DOSAGE MFG.

COMPOSITION
NO. NAME FROM COMPANY

1. Fempro Letrozole 2.5mg Cipla

2. Le Letrozole 2.5mg Intas

tall

Novarsis Ltd
Femara Letrozole 2.5mg
3.

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Letrozole 2.5mg Alnabiotech

4. Feofar

Letrozole 2.5mg Chandrabhagat

5. Letocor Pharma

Letrozole 2.5mg
6. Letoval Sun Pharmaceutical

Letrozole 2.5mg
7. Lets Samarth Pharma

Letrozole 2.5mg Biochem

8. Oncolet Pharmaceutical

LITERATURE REVIEW

OFFICIAL METHOD FOR CLOMIPHENE CITRATE:

SR OFFICIAL METHOD DESCRIPTION REF.NO

.
METHOD
NO
1 BP Liquid Column: 4.5mm× 25cm 14
Chromatography
Packing L26

Mobile phase:

Acetonitrile: Water: diethylamine

(40:60:0.8 v/v/v)

Wave length: 233nm

Flow rate: 1.2 ml/min

Volume of Injection: 10µl

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2 IP Liquid Column: 15
Chromatography
4.5mm× 25cm

Mobile phase:

Acetonitrile:water:diethylamine

(40:60:0.8 v/v/v)

Flow rate: 1.2 ml/min

Wave length: 233nm

Volume of injection: 10𝜇𝑙

3 USP Liquid Column: 16
Chromatography
4.6 mm 𝑥 2.5 cm L26 Mobile
phase:
Methanol: Water: Triethylamine

(55:45:0.3 v/v/v)

Wave Length: 233nm

Flow Rate: 1 ml/min

Volume of injection: 10µl

REPORTED METHOD FOR CLOMIPHENE CITRATE:

SR. DRUG NAME METHOD DESCRIPTION REF.NO

No
1 Clomiphene in UV Wavelength: 17
Methanol Spectrophotometri
c 235nm in methanol

294nm

Concentration Range:

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3-40 μg/ml at 294 nm and

3-24 μg/ml at 235 nm

LOD: 295nm-0.229µg/ml
235nm-0.129µg/ml
LOQ:295nm-0.694 µg/ml
235nm-0.382 µg/ml
Accuracy: 80-120%
2 Clomiphene RP-HPLC Column: 18
Citrate
C18 column Shimadzu

(250mm × 4.5mm × 5µm)

Mobile phase:

Methanol:acetonitrile

(90:10v/v)

Wave length: 295nm

Flow rate:

1.0ml/min

Concentration Range:

10–50µg/mL
LOD:0.1µg/ml
LOQ:0.32 µg/ml
3 Clomiphene UV- Concentration Range: 6-60 19
citrate Spectrophotometri μg/ml
c
Wave Length:
290nm
LOD: 0.954µg/ml

LOQ:0.315µg/ml

Accuracy: 90-120%

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4 Clomiphene HPTLC Plate: silica gel 60 F254 TLC 20

Citrate plate
Mobile Phase: methanol-
ethyl acetate-glacial acetic
acid
(7:2.5:0.5 v/v/v)
Wave length: 245 nm.
Concentration Range:

200–1000 ng/spot.

5 Clomiphene UV- Concentration Range: 21

Citrate spectrophotometric
4-20µg/mL

Wave length:

234 & 290 nm

LOD: 0.19 µg/ml
LOQ: 0.56 µg/ml
Accuracy: 86.5-100.24%

6 Clomiphene HPLC-UV Solvent: 22

Citrate
Octanol

Concertation range:

5-1000 ng/ml.
LOD: 1.5 ng/ml
LOQ: 5 ng/ml

7 Clomiphene RP-HPLC Column: C18 23

Citrate (250mm x 4.6mm, 5µm)
Mobile Phase:
Potassium dihydrogen
Phosphate: Acetonitrile
(50:50v/v)

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Flow rate: 1.0ml/min

Wave length: 290nm

8 Clomiphene RP-UPLC Column: 24

Citate + N
acetyl C18 (50mm x 4.6 mm x
cysteinein
3µm)

Mobile Phase: Potassium

phosphate buffer: methanol:
acetonitrile 40:52:8 v/v/v.
Flow rate: 1.0 ml/min
Wave length: 233nm
Run time: 5 min

9 Clomiphene LC-MS/MS Column: 25

Citrate (Human Plasma) ZORBAX Eclipse plus

C18 (1.8 μm)

Mobile Phase:

Formic Acid: Acetonitrile

(1:10 v/v)

Wave Length: 254 nm

Dosage From : 0.06 ng/mL
for clomiphene-N-oxides to
0.3 ng/mL for (E)-
Ndesethylclomiphene

Summary of Clomiphene citrate:

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SR DRUG MATRIX HPL LC-MS GC-MS LC-MSMS

No. C

1 Clomiphene Human - - - 1

Citrate plasma

OFFICIAL METHOD FOR LETROZOLE:

SR OFFICIA METHOD DESCRIPATION REF.N

. L O
NO IN
1 IP Liquid Column: 26
chromatography
30cm𝑥4mm

Mobile Phase:

Triethylamine: ethanol: hexane

(1:20:80v/v/v)

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Flow rate: 2ml/min

Wave length: 302 nm

Injection volume: 50𝜇𝑙

2 BP Liquid Colum: 27
chromatography
4.6mm×0.125mm

Mobile Phase: water:

Acetonitrile (70:30v/v)

Flow rate: 1.0 ml/min

Wave length: 230nm

Injection volume: 20𝜇𝑙

3 USP Liquid Colum: 28
Chromatography
4.6 mm 𝑥 12.5 cm 𝑥 5 µm Mobile
Phase:
Acetonitrile: water (48:52 v/v)

Flow Rate: 1 ml/min

Wave Length: 230nm

Injection Volume: 20µl

REPORTED METHOD FOR LETROZOLE:

SR. DRUG METHOD DESCRIPTION REF.

NO NO

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1 Letrozole RP-HPLC Column: 29

C18 column (250 mm.6 mm;

5μ)
Mobile Phase:
acetonitrile (ACN): acetate
buffer (pH 4.5) mixture
(50:50% v/v)

wavelength: 240nm
Flow rate: 0.8 ml/min

2 Letrozole LC-MS/MS Column: 30

C18 column (2.0 × 100 mm, 3

µm)

Flow rate: 0.3 ml/min

Accuracy: 97.43-105.17%

3 Letrozole Liquid Colum: 31

Chromatography
C-18 (250 mm×4.6 mm𝑥5 μm)
Mobile Phase:
Methanol: Tetra butyl
ammonium hydrogen sulphate
(80:20V/V)

Flow rate: 1.0 ml/min

Wave length: 240nm

LOD: 0.012 µg/ml

LOQ: 0.043 µg/ml

Accuracy: 80-120%(99.51%)

4 Letrozole RP-HPLC Column: 32

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+Palbociclib C8 (4.6 mm × 250 mm particle

size 5 μm)

Mobile phase:

Sodium dihydrogen
phosphate buffer (pH 5.5):
Acetonitrile: Methanol
(80:10:10 v/v/v)
Flow rate: 1.0ml/min
Concentration range: 5–

50 μg mL−1

LOD:PB-0.098 µg/ml

LT-0.082 µg/ml

LOQ: PB- 0.381µg/ml

LT- 0.315µg/ml

5 Letrozole RP-HPLC Column: 33

C18 (250 mm × 4.6 mm, 5

μm)
Mobile phase: methanol–
water (70:30v/v)

Flow rate: 1.0ml/min

wave length: 239nm

Accuracy:Intra-day:
-11.52%

Inter-day: -2.26%

Linearity range: 0.15-

100µg/ml

6 Letrozole UV- Concentration range: 34

spectrophotometric

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1 -10 µg /ml

Wave length: 240nm

Assay: 99.66%

7 Letrozole HPLC Column: 35

C18 (250mm×4.6mm,5µm)

Mobile phase :

Acetate: Acetonitrile(60:40
v/v)

Flow rate: 1.0 ml/min

Wave length: 240nm

Accuracy: 97.44-102.70%

LLOQ: 75 ng/ml

Precision: 2.61-7.48%

8 Letrozole UV- Concentration Range: 36

Spectrophotometric
5 to 20 µg·mL-1

Wave Length: 240nm

LOD: 0.99 µg/ml

LOQ: 3.29 µg/ml

9 Letrozole RP-LC Column: 37

C18 column(100mmx4.6mm,

3.5µm)

Mobile phase:

potassium
phosphate:methanol(70:30)

Flow rate: 1.0ml/min

Wave length: 230nm

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10 Letrozole Rp-HPLC(Rat COLUMN: 38`

Serum)
C18 (250 mm × 4.6 mm, 5
μm) Mobile Phase:
Methanol: Water (70:30 v/v)
Concentration Range:
0.15–100 μg mL–
1
wave length: 239nm
Flow rate:0.15–100 μg/ mL

11 Letrozole + LC-MS/MS (Human Concentration range : 39

Palbociclib Plasma)
+ Palbociclib: 0.3 – 250 ng/mL
Ribociclib
Ribociclib :10-10000 ng/mL

Letrozole :0.5-500 ng/mL

12 Letrozole HPLC (Rat plasma) Column: 40

(250 mm × 4.6 mm, 5 μm)

Mobile phase:

Acetate: acetonitrile
(60:40v/v)

Flow rate: 1.0ml/min

Wave length: 240 nm

13 Letrozole LC-ESI-MS/MS Column:C18 41

(Human Plasma)
(50mm𝑥3mm𝑥 5𝜇𝑔)
Extraction teq:SPF
Mobile Phase:
Formic acid: water
(60:40 v/v)

Flow rate: 0.6 ml/min

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Run time: 4.0 min

LLOD: 0.43 ng/ml

LLOQ:1.56 ng/ml

Accuracy: 99.63-102.0%

14 Letrozole + LC-MS/MS Extraction teq: SPF 42

Metformin (Human Plasma) Column:
(50mm×4.6mm, 5µm)
Mobile phase:
Ammonium:Acetonitrile
(20:80 v/v)
Run time: 2.5 min
LLOQ: MF- 5 ng/ml
LT- 0.5 ng/ml
Accuracy:
MF- 97.44-101.62%
LT- 92.50-104.35%
15 Letrozole UPLC-MS/ Extraction teq: SPF 43
MS(Human Plasma) Column:

C18(50mm×1.7mm,5µm)
Mobile phase:
Formic acid: water (85:15 v/v)
Run time: 2min
Flow rate: 0.3 ml/min
LQC: 0.10 ng/ml
HQC: 80.0 ng/ml
16 Letrozole LC/MS-MS Column: 44
(Human Plasma) C18 MG column (100 mm ×
4.6 mm, 5 µm)
Mobile phase :
Methanol : ammonium acetate
(65:35, v/v)
Flow Rate: 0.6 ml/min

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Run time: 4.0 min

Summary of letrozole:

SR DRUG MATRIX HPLC LC- GC- LC-

No. MS MS MSMS

1 Letrozole Human - - - 4
plasma

2 Letrozole Rat plasma 1 - - -

3 Letrozole + Human - - - 1
Palbociclib + plasma
Ribociclib
4 Letrozole Rat serum 1 - - -

5 Letrozole Human - - - 1
+Metformin
Plasma

Material and Method

• REAGENTS, MATERIALS, INSTRUMENT USED:

• MATERIALS:
1. Clomiphene citrate API (CHEMLAND IND ,VAPI)
2. Clomiphene citrate tablet (Cipla)
 REAGENTS:
1. Methanol

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2. Mili Q water
3. Triethylamine
4. Acetonitrile
5. Ammonium Acetate
6. Water

• Instrument use in method development:

1. HPLC Model: Shimadzu 2030
2. Column: Shim-pack NT-ODS
3. Software: Cromalion 7.2
4. Analytical balance: Shimadzu
5. Flask and pipettes: Class A-Calibrated
6. pH meter: Toshcon Industries.
7. LC-MS: Sciex QTRAP 4500
8. Software: Analyst

 APPARATUSUSED

1. Beakers
2. Volumetricflasks
3. Riavials
4. Tarsontubes
5. Glassautosamplervials

Experimental Work

Solution Preparation:
Optimization of chromatography in HPLC Method:
 Mobile Phase:
Methanol: Milli Q Water: Triethylamine (55:45:0.3 v/v/v)Adjustment of
pH with 0.1% TFA pH to 2.5 (+-0.05).
 Standard Stock Solution:

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Weigh accurately about 50 mg of Clomiphene Citrate standard API and

transfer into a100 mL volumetric flask add about 70 mL of Mobile phase,
and sonicate to dissolve. Make up to the mark with the Mobile phase and
mix well.
 Clomiphene Related Compound A Stock Solution:
Weigh accurately about 1.197 mg of Clomiphene Related Compound A
and transfer into a 20 mL volumetric flask add about 14 mL of
Acetonitrile, and sonicate to dissolve. Make up to the mark with
Acetonitrile, and mix well.
• System Suitability Solution:
Dilute 1.7 mL of Clomiphene Related Compound A Stock Solution and
5.0 mL of Standard Stock Solution to 50 mL with Mobile phase and mix
well.
Standard solution: Dilute 5.0 mL of Standard Stock Solution to 50 mL
with Mobile phase and mix well.
 Sample stock solution:
Weigh accurately about 50 mg of Clomiphene Citrate sample and transfer
into a 100 mL volumetric flask add about 70 mL of Mobile phase, and
sonicate to dissolve. Make up to the mark with the Mobile phase and mix
well.
 Sample solution:
Dilute 5.0 mL of Sample stock solution diluted to 50 mL with Mobile
phase and mix well.

Optimization of chromatography condition:

Sr No. Chromatography parameter Condition

1 Mobile phase Methanol: Mili Q

Water: Triethylamine(55:45:0.3 v/v/v)
2 Column 4.6mm×25cm

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3 Flow Rate 1 mL/min

4 Column Temperature 25°C

5 Run time 30 min

6 Injection Volume 50ml

7 L26 detector 233nm

Trials for Optimization Chromatography:

SR. RETANTION COLUMN MOBILE REMARK
NO TIME PHASE
(ml/min)
1 2.3 min C18 MG column Methanol : No peak
(100 mm × 4.6 ammonium observed.
mm, 5 μm) acetate (65:35,
v/v)
2 1.5 min C18(50mm×4.6m Formic acid : Peak was
m, 5μm) Acetonitrile observed but
(10:90,v/v) separation not
good.
3 12min C18(50mm×4.6m Ammonium No peak
m, 5μm) Acetate : observed.
Acetonitrile
(20:80 v/v)
4 4.95 min C18(4.6 mm ×2.5 Methanol: Milli Proper peak
mm ) Q Water: was
Triethylamine observed.
(55:45:0.3 v/v/v)

Optimization of LC-MS method:

Solution Preparation:
Mobile Phase A: 126gm Ammonium Formate in 2000mL of Purified
Water with the addition of 2.0mL of Formic Acid and Mixed well.
Sonicate it for 10 Min.
Mobile Phase B: Dissolve 1.0 mL of Formic Acid in 1000mL of
Acetonitrile and Mixed well. Sonicate it of 10 mL Min.

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Diluent:Purified Water and Methanol in the ratio of (20:80)%v/v.

Sonicate it for 10 Min.
Standard Stock Preparation: Diluted 3mg of STD API into to 50mL
volumetric flask with Methanol and mixed it well.
Standard Final Preparation: Diluted 5.0 mL of the above standard
stock solution into100.0 mL of volumetric flask with diluent and mixed
it well. Further,dilute 5.0 mL of this solution to up to 100.0 mL with
Blood Plasma Solution and mix well, centrifuge it at 5000 RPM for 10
Minutes. After that Filter the above supernatant with 0.45µ PTFE MDI
filter and collect the filtrate by discarding not less than 5mL of filtrate
through it. Then again further diluted 5.0 mL of this solution to up to
100.0 mL with diluent and mixed well.
Sample Preparation:Weigh accurately 10 tablets and calculate the
average weight. Crush it into fine powder and take sample powder
equivalent to about 500mg of Clomiphene Citrate into 25mL of
volumetric flask. Add about 15mL of diluent and Sonicate it for 30
minutes with shaking of every 5 minutes for a period of 30 seconds.
Make it up to mark with diluent and mix well. Centrifuge it for 5000
RPM for 10 minutes.
Now diluted 1 mL above solution to 10 mL into Blood Plasma and
mixed it well. Centrifuge it for 5000 RPM for 10 minutes. Then again
diluted above 5 mL of sample solution to up to 10 mL with Diluent.
Filter the above supernatant with 0.45µ PTFE MDI filter and collect the
filtrate by discarding not less then 5mL of filtrate through it.

Optimization of chromatography condition:

SR. Chromatography Condition
N NO Parameter

1 Column Biphenyl
Kinetic(150mm×4.6m
m,5.0µm)

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2 Mode MRM mode

3 Flow rate 0.5 mL/min

4 Injection Volume 10µL

5 Column 35°C
Temperature
6 Auto Sampler 1500µL
Rinsing Volume
7 Auto Sampler 5µL/sec
sampling speed
8 Autosampler 10°C
Temperature
9 Auto Sampler 35µL/sec
Rinsing Volume
10 Auto Sampler 48mm
needle Stock
11 Run Time 20 min

BIOANALYTICALMETHODVALIDATION:
Accuracy:
Accuracy The accuracy of an analytical method describes the closeness
of mean test results obtained by the method to the nominal value
(concentration) of the analyte. Accuracy is determined by replicate
analysis of samples containing known amounts of analyte. Accuracy
should be measured using a minimum of 3 concentrations and 5
determinations per concentration. The mean value should be within 15%
of the nominal value except at lower limit of quantification (LLOQ, see

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below), where it should not deviate by more than 20%. The deviation of
the mean from the nominal value (relative error) serves as the measure
of accuracy.
Recovery:
Recovery is a measure of efficiency at which an analytical method
recovers the analyte through the sample processing step. Recovery
should be performed by comparing analytical results for extracted
samples at three concentrations (low,
medium and high) and three replicates with un-extracted standards that
represents 100% recovery. Recovery of analyte need not to be 100%,
but extent of recovery of an analyte and of ISTD should be consistent,
precise, and reproducible.
Precision:
The precision of an analytical method describes the closeness of
individual measures of an analyte when the procedure is applied
repeatedly to multiple aliquots of a single homogeneous volume of
biological matrix. Precision should be measured using a minimum of 3
concentrations and 5 determinations per concentration. The imprecision
determined as coefficient of variation (CV) at each concentration level
should not exceed 15% except for the LLOQ (see below), where it
should not exceed 20%. Precision is further subdivided into – Within-
day precision, which assesses precision during a single analytical run,
and – Between-day precision, which measures precision with time and
may involve different analysts, equipment, reagents, a

Result and Analysis

Gradient Program:

SR Time Modul %AMobile Phase % B Mobile Phase

NO (min e

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)
1 0.01 Pumps 95 5
2 4.00 Pumps 85 15
3 9.00 Pumps 60 40
4 12.00 Pumps 15 85
5 15.50 Pumps 95 5
6 20.00 Pumps 95 5
7 20.01 Pumps 95 5

Graph 1:

100
90
80
70
60
50 Sum of %A Mobile Phase
40 Sum of % B Mobile Phase
30
20
10
0
0.01 4 9 12 15.5 20 20.01 (min)

Figure 1. Typical gradient graph chart

Calculation for linearity:

Linearity Calculation Sheet
Sr. No Concentration ng/mL Peak Area

1 18.75 12723
2 37.5 23746
3 60 37496
4 75 48237
5 90 58397

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6 112.5 71701

80000

70000 f(x) == 637.621930870084

f(x) 637.621930870084 x + x206.060786650771
+ 206.060786650771
R² = 0.999109831745275
0.999109831745275
60000

50000
Peak Area

40000

30000

20000

10000

0
0 20 40 60 80 100 120
Concentration

Figure 2. Linearity graph of clomiphene Citrate by LC-MS

Trials of Optimization of Chromatography Condition in HPLC:

Trial 1:

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Figure3. Typical HPLC chromatogram of Mobile Phase- Methanol:

ammonium acetate (65:35, v/v)

Trial 2:

Figure4, Typical HPLC chromatogram of Mobile Phase -Formic

acid: Acetonitrile (10:90,v/v)

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Trial 3:

Figure 5, Typical HPLC Chromatogram of Mobile phase -

Ammonium Acetate: Acetonitrile (20:80 v/v)
Trial 4:

Figure 6, Typical HPLC Chromatogram Mobile Phase - Methanol:

Milli Q Water: Triethylamine (55:45:0.3 v/v/v)

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Figure 7, Typical LC-MS Chromatogram of Blank blood

plasma Sample

Figure 8, Typical LC-MS Chromatogram of

Recovery at25%.

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Figure 9, Typical LC-MS Chromatogram of

Recovery at 100% .

Figure 10, Typical LC-MS Chromatogram of

Recovery at 150%.

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Trial 1.

Figure 11, Typical LC-MS Chromatogram of Clomiphene

Citrate Standard Solution in blood plasma
Trial 1.1

Figure 11.1, Typical LC-MS Chromatogram of Clomiphene

Citrate Sample Solution in Blood plasma

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Trail 2. STD and sample in blood Plasma:

Figure 12, Typical LC-MS Chromatogram of

Clomiphene Citrate Standard and sample solution in
Blood Plasma
Calculation for Recoveryat DifferentLevels:

Recovery at Different Levels

Recovery at Recovery at Recovery at
Sr.
25% 100% 150%
No
(18.75ng/ml) (75 ng/ml) (112.5ng/ml)
1 12654 47894 71510
2 12761 48121 71721
3 12802 48283 71956
4 12781 48106 71723
5 12799 48321 71821
6 12697 47954 71696
Means 12749.00 48113.17 71737.83
SD 60.34 170.60 147.43
% RSD 0.47 0.35 0.21
Mean Accuracy% 99.53% 99.63% 99.79%

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Table of Clomiphene Citrate API:

Clomiphene Citrate API
Sr.No Precision Intermediate Precision
1 47894 46984
2 48121 45892
3 48283 47952
4 48106 45951
5 48321 46111
6 47954 47346
Means 48113.17 46706.00
SD 170.60 851.59
% RSD 0.35 1.82

Clomiphene Citrate
Sr.No Standard SST Standard CAL Clomiphene Citrate Tab (100mg)
1 48213 48962
Sr.No Tablet Area
2 48564 48771
3 48612 - 1 47635
4 48372 2 47765
5 48466
6 47321 Means 47700.00
Means 48258.00 48866.50 SD 91.92
SD 480.67 135.06
% RSD 0.19
% RSD 1.00 0.28

Result of Analysis of Drug Product and Recovery Study of Clomiphene Citrate

by LC-MS

Amount Present
Label Claim % Label % Recovery ±
Sample (mg/Tablet) ±
(mg) Claim %RSD
%RSD
Sample-
100 97.48 ± 0.19 99.98 ± 0.23 97.49 ± 0.82
1

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SUMMARY OUTPUT

Regression Statistics

Multiple R 0.999555

R Square 0.99911

Adjusted R 0.998887
Square
Standard Error 1.145785

Observations 6

LOQ 2.31 ng/ml

LOD 0.312 ng/ml

Future Work of Plan

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• Procurement of API (letrozole).

• Identification study of API
• LC-MS Method development of proceeding API.

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DPC-II Compliance

DPC COMMENT JUSTIFICATION

2 Do interstices study of FDA guideline of bioanalytical method
bioanalytical method had been studied.

Correct formatting Formatting had need done.

Check Spelling mistake Spellings had been corrected.

Submit review copy DPC-II DPC II revised copy had been

submitted.

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