DPC-3 Word

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
1
Enrolment No: 2108071322005 BMCAR, BMU
DPC-3
Mechanism of Clomifene has both estrogenic and

action anti-estrogenic properties, but its
precise mechanism of action has not
been determined. Clomifene 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
Pharmacodynam Clomifene (previously clomiphene)
ics 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
2
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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 estrogen receptor-
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 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
3
DPC-3
MARKETED FORMULATION:
Marketed Formulation of clomiphene Citrate
SR. BRAND DOSAGE MFG.

COMPOSITION
NO. NAME FROM COMPANY
1. Clomifene 50mg Cipla

Clomiphene
Citrate
2. 25mg Kee Pharma
Clopreg Clomiphene
Citrate
Genetic
Fertex-M Clomiphene 25mg
3. Pharma
Citrate
50mg Zydus – Biogen

Clomiphene
4. Folistim
Citrate
25mg MicroNova
Clomiphene
5 Fulfyl
Citrate
50mg Torrent
Clomiphene
6 Omicite
Citrate
25mg Lupin
Clomiphene
7 Oviphene Pharma
Citrate
8 25mg Win Medicare
Clomiphene
Ovipreg
Citrate
9 50mg Evacare
Clomiphene
Refert
Citrate
10 25mg Serum
Clomiphene
Siphene-M International
Citrate
4
DPC-3
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
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
5
DPC-3
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,
estrogen dependent 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
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:
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Marketed Formulation of Letrozole: .
SR. BRAND DOSAGE MFG.

COMPOSITION
NO. NAME FROM COMPANY
1 Fempro Letrozole 2.5mg Cipla

.
2 Letrozole 2.5mg Intas

. Letall
Novarsis Ltd
Femara Letrozole 2.5mg
3
.
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 OFFICIA METHOD DESCRIPTION REF.NO

. L
NO METHOD
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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

2 IP Liquid Column: 15
Chromatography
4.5mm× 25cm
Mobile phase:
Acetonitrile:water:diethylamine
(40:60:0.8 v/v/v)
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
8
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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:
3-40 μg/ml at 294 nm and
3-24 μg/ml at 235 nm

LOD: 295nm-0.229µg/ml
235nm-0.129µg/ml
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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:
1.0 ml/min
10–50µg/mL
LOD: 0.1 µg/ml
LOQ:0.32 µg/ml
3 Clomiphene UV- Concentration Range: 6-60 19
citrate Spectrophotometri μg/ml
c
Wave Length:
290nm
LOD: 0.954 µg/ml
LOQ: 0.315 µg/ml
Accuracy: 90-120%
4 Clomiphene HPTLC Plate: silica gel 60 F254 TLC 20

Citrate plate
Mobile Phase: methanol-
ethyl acetate-glacial acetic
acid
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(7:2.5:0.5 v/v/v)
Wave length: 245 nm.
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)
Flow rate: 1.0ml/min
Wave length: 290nm
8 Clomiphene RP-UPLC Column: 24

Citate + N
acetyl C18 (50mm x 4.6 mm x
11
DPC-3
cysteinein 3µm)
Mobile Phase: Potassium

phosphate buffer: methanol:
acetonitrile 40:52:8 v/v/v.
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:
SR DRUG MATRIX HPL LC-MS GC-MS LC-MSMS

No. C
1 Clomiphene Human - - - 1
Citrate plasma
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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)
Flow rate: 2ml/min
Wave length: 302 nm
Injection volume: 50𝜇𝑙
13
DPC-3
2 BP Liquid Colum: 27
chromatography
4.6mm×0.125mm
Mobile Phase: water:
Acetonitrile (70:30v/v)
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
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
14
DPC-3
2 Letrozole LC-MS/MS Column: 30
C18 column (2.0 × 100 mm, 3

µm)
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)
Wave length: 240nm
LOD: 0.012 µg/ml
LOQ: 0.043 µg/ml
Accuracy: 80-120% (99.51%)

+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)
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DPC-3
Concentration range: 5–
50 μg mL−1
LOD: PB-0.098 µg/ml
LT-0.082 µg/ml
LOQ: PB- 0.381 µg/ml
LT- 0.315 µg/ml
C18 (250 mm × 4.6 mm, 5

μm)
Mobile phase: methanol–
water (70:30v/v)
wave length: 239nm
Accuracy: Intra-day:
-11.52%
Inter-day: -2.26%
Linearity range: 0.15-100

µg/ml
6 Letrozole UV- Concentration range: 34

spectrophotometric
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
16
DPC-3
v/v)
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)
Wave length: 230nm
10 Letrozole Rp- HPLC (Rat COLUMN: 38`

Serum)
C18 (250 mm × 4.6 mm, 5
μm) Mobile Phase:
Methanol: Water (70:30 v/v)
0.15–100 μg mL–1
wave length: 239nm
Flow rate: 0.15–100 μg/ mL
17
DPC-3
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)
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)
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)
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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
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
Run time: 4.0 min
Summary of letrozole:
SR DRUG MATRIX HPLC LC- GC- LC-

No. MS MS MSMS
19
DPC-3
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
2. Mili Q water
3. Triethylamine
4. Acetonitrile
5. Ammonium Acetate
6. Water
• Instrument use in method development:

1. HPLC Model: Shimadzu 2030
20
DPC-3
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
 APPARATUS USED
1. Beakers
2. Volumetric flasks
3. Ria vials
4. Tarson tubes
5. Glass autosampler vials
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:
Weigh accurately about 50 mg of Clomiphene Citrate standard API 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.
 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
21
DPC-3
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
3 Flow Rate 1 mL/min
4 Column Temperature 25°C
5 Run time 30 min
6 Injection Volume 50ml
7 L26 detector 233nm
22
DPC-3
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.
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 into 100.0 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
23
DPC-3
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)
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
24
DPC-3
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
BIOANALYTICAL METHOD VALIDATION:

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
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,
25
DPC-3
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 %A Mobile Phase % B Mobile Phase

NO (min) e
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
26
DPC-3
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
6 112.5 71701
27
DPC-3
80000
70000 f(x) = 637.621930870084 x + 206.060786650771

R² = 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:
28
DPC-3
Figure 3. Typical HPLC chromatogram of Mobile Phase- Methanol:

ammonium acetate (65:35, v/v)
Trial 2:
Figure 4, Typical HPLC chromatogram of Mobile Phase -Formic

acid: Acetonitrile (10:90, v/v)
Trial 3:
Figure 5, Typical HPLC Chromatogram of Mobile phase -

Ammonium Acetate: Acetonitrile (20:80 v/v)
29
DPC-3
Trial 4:
Figure 6, Typical HPLC Chromatogram Mobile Phase - Methanol:

Milli Q Water: Triethylamine (55:45:0.3 v/v/v)
30
DPC-3
Figure 7, Typical LC-MS Chromatogram of Blank blood

plasma Sample
Figure 8, Typical LC-MS Chromatogram of

Recovery at 25%.
31
DPC-3

Recovery at 100% .

Recovery at 150%.
Trial 1.
32
DPC-3
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
33
DPC-3
Trail 2. STD and sample in blood Plasma:

Clomiphene Citrate Standard and sample solution in
Blood Plasma
Calculation for Recovery at Different Levels:
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%
Table of Clomiphene Citrate API:

Clomiphene Citrate API
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DPC-3
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
SUMMARY OUTPUT
Regression Statistics
Multiple R 0.999555
35
DPC-3
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
• Procurement of API (letrozole).

• Identification study of API
• LC-MS Method development of proceeding API.
36
DPC-3
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Academic Press, 2007, pp. 485-489.
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Issue - 7, Year – 2021.
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And Validation Of The UV Spectrophotometric Method Of Clomiphene Citrate In
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Scientific & Technology Research Volume 8, Issue 10, October 2019.
20. Sowndaravel. P., Kokilambigai. K. S., Seetharaman.R., Lakshmi. K. S “Analytical
Method Development And Validation Of Clomiphene Citrate By Hptlc Method In
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21. Venkatakiran Vaddempudi1, S.B. Puranik2 , G.V.S. Kumar3 , K. A Sridhar4
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2020, 16, 000-000.
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Validation of RP-HPLC method for estimation of Clomiphene Citrate in
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Simultaneous Assessment Of Clomiphene Citrate And N-Acetyl Cysteinein
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110369005
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E. Murdter “Quantification of clomiphene metabolite isomers in human plasma by

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Reddy, Meka S.; Mutalik, Srinivas “ Full Factorial Experimental Design for
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187-194 | Received 20 May 2017, Accepted 17 Sep 2017, Accepted author version
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33. Sasmita Kumari ACHARJYA , Subrat Kumar BHATTAMISRA , Bhanoji Rao E.

MUDDANA 3, Ravikumar V. V. BERA 1, Pinakini PANDA 1, Bibhu
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Liquid Chromatographic Method for Determination of Letrozole in Wistar Rat
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Srivatsava1 , T.Sivakumar and Swastika Ganguly “A Validated Uv

Spectrophotometric Method For The Determination Of Letrozole In Bulk And Solid
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36. Aura Rusu, Maria-Alexandra Sbanca, Nicoleta Todoran, Camil-Eugen Vari
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42
DPC-3
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Official in IP, BP, USP Pharmacopeia.

Description Clomiphene Citrate is a medication

Chemical formulation C32H36ClNO8

Mol. Weight 598.09 g/mol

IUPAC Name 2-hydroxy-propane-1,2,3-tricarboxylic

CLASS Agonist and Antagonist

Mechanism of Clomifene has both estrogenic and

rate of ovarian hyperstimulation

Melting point 116.5-118°C

Pka (basic) 9.31

Solubility Slightly soluble in water, Acetone,

SR. BRAND DOSAGE MFG.

1. Clomifene 50mg Cipla

50mg Zydus – Biogen

Description Letrozole is an aromatase inhibitor used

Chemical formulation C17H11N5

Mol. Weight 285.303 g/mol

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

CLASS Nonsteroidal aromatase inhibitors.

Mechanism Letrozole is a non-steroidal type II

androgens to estrogen. This action leads

CAS NO. 112809-51-5

Solubility Freely soluble in dichloromethane,

Marketed Formulation of Letrozole: .

SR. BRAND DOSAGE MFG.

1 Fempro Letrozole 2.5mg Cipla

2 Letrozole 2.5mg Intas

Letrozole 2.5mg Alnabiotech

Letrozole 2.5mg Biochem

OFFICIAL METHOD FOR CLOMIPHENE CITRATE:

SR OFFICIA METHOD DESCRIPTION REF.NO

1 BP Liquid Column: 4.5mm× 25cm 14

Acetonitrile: Water: diethylamine

Wave length: 233nm

Flow rate: 1.2 ml/min

Volume of Injection: 10µl

Flow rate: 1.2 ml/min

Wave length: 233nm

Volume of injection: 10𝜇𝑙

Wave Length: 233nm

Flow Rate: 1 ml/min

Volume of injection: 10µl

REPORTED METHOD FOR CLOMIPHENE CITRATE:

SR. DRUG NAME METHOD DESCRIPTION REF.NO

3-40 μg/ml at 294 nm and

3-24 μg/ml at 235 nm

LOQ: 295nm-0.694 µg/ml

(250mm × 4.5mm × 5µm)

Wave length: 295nm

LOQ: 0.315 µg/ml

4 Clomiphene HPTLC Plate: silica gel 60 F254 TLC 20

5 Clomiphene UV- Concentration Range: 21

234 & 290 nm

6 Clomiphene HPLC-UV Solvent: 22

7 Clomiphene RP-HPLC Column: C18 23

8 Clomiphene RP-UPLC Column: 24

Mobile Phase: Potassium

9 Clomiphene LC-MS/MS Column: 25

Formic Acid: Acetonitrile

Wave Length: 254 nm

Summary of Clomiphene citrate:

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