Physical separation of amphiprotic-polar aprotic solvents for simultaneous extraction and clean-up of clomiphene from plasma before liquid chromatographic analyzes

Document Type : Research article

Authors

1 Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran.

2 Department of Medicinal Chemistry, School of Pharmacy-International Campus, Iran University of Medical Sciences, Tehran, Iran.

3 Department of Medicinal Chemistry, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Islamic Republic of Iran.

Abstract

An efficient and quantitative two phase freezing (TPF) method coupled with high performance liquid chromatography and UV-Vis detector was developed for the extraction, clean up and determination of clomiphene citrate (CLC) in plasma samples. The separation of two miscible solvents by TPF method permits that the CLC was efficiently removed from proteins and transferred into the relative aprotic dipolar organic phase and in consequence, gave a higher recovery. The TPF method was compared to conventional liquid-liquid extraction and it gave more clean solution with better reproducibility. Linear range, limit of detection and limit of quantification for CLC in plasma were obtained in the range of 0.06-18, 0.02 and 0.06 µg ml-1, respectively. The intraday and interday reproducibility for concentration of 1.0 µg ml-1 (%RSD) were 3.2% and 4.6%, respectively. In addition, the trueness, ruggedness, and realistic of TPF were assessment. Finally, several real plasma samples were successfully analyzed using the developed method.

Keywords

Main Subjects


Cunha GR, Taguchi O, Namikawa R, Nishizuka Y
and Robboy SJ. Teratogenic effects of clomiphene,
tamoxifen, and diethylstilbestrol on the developing

human female genital tract.
J. Human Path. (1987)
18: 1132-43.

Rostami-Hodjegan A, Lennard MS, Tucker GT and

Ledger WL. Monitoring plasma concentrations to

individualize treatment with clomiphene citrate.
Fertil.
Ster.
(2004) 81: 1187-93.
Homburg R. Clomiphene citrate—end of an era? a

mini-review.
Hum. Reprod. (2005) 20: 2043-51.
https://wada-main-prod.s3.amazonaws.com/resources/

files/wada-2016-prohibited-list-en.pdf

Ernst S, Hite G, Cantrell JS, Richardson Jr A and

Benson HD. Stereochemistry of geometric isomers

of clomiphene: A correction of the literature and a

reexamination of structure-activity relationships.
J.
Pharm. Sci.
(1976) 65:148-50 .
Amani H, Ajami M, Nasseri Maleki S, Pazoki-Toroudi

H, Daglia M, Tsetegho Sokeng AJ, Di Lorenzo A,

Nabavi SF, Devi KP and Nabavi SM. Targeting signal

transducers and activators of transcription (STAT) in

human cancer by dietary polyphenolic antioxidants.

Biochimie
(2017) 142: 63-9.
Imani B, Eijkemans MJ, de Jong FH, Payne NN,

Bouchard P, Guidice LC and Fauser BC. Free Androgen

Index and Leptin Are the Most Prominent Endocrine

Predictors of Ovarian Response during Clomiphene

Citrate Induction of Ovulation in Normogonadotropic

Oligoamenorrheic Infertility.
J. Clin. Endocrin.
Metabol.
(2000) 85:676-82 .
Imani B, Eijkemans MJ, teVelde ER, Habbema JD

and Fauser BC. Predictors of Patients Remaining

Anovulatory during Clomiphene Citrate Induction of

Ovulation in Normogonadotropic Oligoamenorrheic

Infertility.
J. Clin. Endocrin..Metabol. (1998) 83:
2361-5.

Imani B, Eijkemans MJ, teVelde ER, Habbema JD

and Fauser BC. Predictors of Chances to Conceive

in Ovulatory Patients during Clomiphene Citrate

Induction of Ovulation in Normogonadotropic

Oligoamenorrheic Infertility.
J. Clin. Endocrin.
Metabol.
(1999) 84:1617-22 .
Imani B, Eijkemans MJ, teVelde ER, Habbema JD

and Fauser BC. A nomogram to predict the probability

of live birth after clomiphene citrate induction of

ovulation in normogonadotropic oligoamenorrheic

infertility
. Fertil. Ster. (2002) 77: 91-7.
Vaddempudi V, Puranik SB, Kumar GVS, Sridhar KA,

Vaddempudi A and Lakshminarayana B. Development

and validation of RP-HPLC method for estimation of

clomiphene citrate in pharmaceutical dosage form.

Inter. J. Pharm.
(2012) 4: 1953-7.
Harman PJ, Blackman GL and Phillipou G. High-

performance liquid chromatographic determination of

clomiphene using post-column on-line photolysis and

fluorescence detection.
J. Chromatogr. B (1981) 225:
131-8
.
Baustian CL and Mikkelson TJ. Analysis of

clomiphene isomers in human plasma and detection


Cunha GR, Taguchi O, Namikawa R, Nishizuka Y
(1)
of metabolites using reversed-phase chromatography
and fluorescence detection
. J. Pharm. Biomed. Anal.
(1986) 4: 237-46.

Ganchev B, Heinkele G, Kerb R, Schwab M and

Mürdter TE. Quantification of clomiphene metabolite

isomers in human plasma by rapid-resolution liquid

chromatography-electrospray ionization-tandem mass

spectrometry.
Anal. Bioanal. Chem. (2011) 400: 3429-
41.

Lu J, He G, Wang X, Yan K and He Z. Mass

spectrometric analyses of urinary clomiphene and

toremifene metabolites in doping control by liquid

chromatography quadrupole time-of-flight mass

spectrometry (LC-QTOF).
Anal. Methods (2013) 5:
6677-81.

Lu J, He G, Wang X, Bo T and Ouyang G. Mass

spectrometric identification and characterization of

new clomiphene metabolites in human urine by liquid

chromatography-quadrupole time-of-flight tandem

mass spectrometry.
J. Chromatogr. A (2012) 1243:
23-32.

Mazzarino M, Fiacco I, De La Torre X and Botrè F.

A mass spectrometric approach for the study of the

metabolism of clomiphene, tamoxifen and toremifene

by liquid chromatography time-of-flight spectroscopy.

Eur. J. Mass Spect.
(2008) 14: 171-80.
Crewe HK, Ghobadi C, Regory A, Rostami-Hodjegan

A and Lennard MS. Determination by liquid

chromatography-mass spectrometry of clomiphene

isomers in the plasma of patients undergoing treatment

for the induction of ovulation.
J. Chromatogr. B,
(2007) 847: 296-9.

Mazzarino M, Biava M, De La Torre X, Fiacco I and

Botrè F. Characterization of the biotransformation

pathways of clomiphene, tamoxifen and toremifene

as assessed by LC-MS/(MS) following in-vitro and

excretion studies.
Anal. Bioanal. Chem. (2013) 405:
5467-87.

Faridbod F, Hosseini M, Ganjali MR and Norouzi PI.

Potentiometric sensor for determination of clomiphene.

Int. J. Electrochem. Sci.
(2013) 8: 1976-85.
Hage DS and Sengupta A. Studies of Protein Binding

to Nonpolar Solutes by Using Zonal Elution and High-

Performance Affinity Chromatography: Interactions

of cis- and trans-Clomiphene with Human Serum

Albumin in the Presence of β-Cyclodextrin. Anal.

Chem. (1998) 70: 4602–09.

Clark JH and Markaverich BM. The agonistic-

antagonistic properties of clomiphene.
Pharmacol.
Ther.
(1981) 15: 467-519.
Harman PJ, Blackman GL and Phillipou G. High-

performance liquid chromatographic determination of

clomiphene using post-column on-line photolysis and

fluorescence detection.
J. Chromatogr. (1981) 225:
131-8.

Urmos I, Benko SM and Klebovich I. Simple and

rapid determination of clomiphene cis and trans

isomers in human plasma by high-performance

liquid chromatography using on-line post-column

photochemical derivatization and fluorescence

detection.
J. Chromatogr. (1993) 617: 168-72.
Steven L, Young MD, Michael S, Opsahl MD and

Fritz MA. Serum concentrations of enclomiphene and

zuclomiphene across consecutive cycles of clomiphene

citrate therapy in anovulatory infertile women.
Fertil.
Ster.
(1999) 71: 639-44.
Ahmadi F, Asgharloo H, Sadeghi S, Gharehbagh-

Aghababa V andAdibi H. Post-derivatization procedure

for determination of hippuric acid after extraction by

an automated micro solid phase extraction system and

monitoring by gas chromatography.
J. Chromatogr. B.
(2009) 877: 2945–51.

Bempong DK and Honigberg IL. Multivariate analysis

of capillary electrophoresis separation conditions for

Z-E isomers of clomiphene.
J. Pharm. Biomed. Anal.
(1996) 15: 233-9.

Rahimi-Nasrabadi M, Pourmohamadian S,

Pourmortazavi SM, Ahmadi F, Ganjali MR and

Moghimi A. NMR study of the stoichiometry and

stability of complexation reaction between Mg
2+,
Ca
2+, Sr 2+ and Ba 2+ ions and 60-crown-20 in binary
acetonitrile solvent mixtures.
J. Inc. Phenom. Macro.
Chem.
(2012) 73: 263–7.
Joshaghani M, Gholivand MB and Ahmadi F.

Spectrophotometric and conductometric study of

complexation of salophen and some transition metal

ions in nonaqueous polar solvents.
Spectrochim. Acta
A
(2008) 70:1073–78.
Ahmadi F, Rezaei H and Tahvilian R. Computational-

aided design of molecularly imprinted polymer for

selective extraction of methadone from plasma and

saliva and determination by gas chromatography.
J.
Chromatogr. A
(2012) 1270: 9–19.
Schenck F, Callery P, Gannett P, Daft J and Lehotay

S. Comparison of Magnesium Sulfate and Sodium

Sulfate for Removal of Water from Pesticide Extracts

of Foods.
J. AOAC (2002) 85: 1177-80.
Kaufmann A, Butcher P, Maden K, Walker S and

Widmer M. Multi-residue quantification of veterinary

drugs in milk with a novel extraction and clean

up technique: Salting out supported liquid extraction

(SOSLE).
Anal. Chim. Acta (2014) 820: 56–68.
Kaufmann A, Butcher P, Maden K and Widmer M.

Quantitative multiresidue method for about 100

veterinary drugs in different meat matrices by sub 2-μm

particulate high-performance liquid chromatography

coupled to time of flight mass spectrometry.
J.
Chromatogr. A
(2008) 1194: 66-79.
Farajzadeh A, Feriduni B and Afshar Mogaddam

MR. Development of counter current salting-out

homogenous liquid–liquid extraction for isolation and

preconcentration of some pesticides from aqueous

samples.
Anal. Chim. Acta (2015) 885: 122–31.
Ahmadi F, Shahbazi Y and Karami N. Determination

of tetracyclines in meat using two phases freezing

extraction method and HPLC-DAD.
Food Anal. Meth.
(2015) 8: 1883–91.

Ahmadi F, Mahmoudi-Yamchi T and Azizian H. Super

paramagnetic core–shells anchored onto silica grafted
with C
8 /NH2 nano-particles for ultrasound-assisted
magnetic solid phase extraction of imipramine and

desipramine from plasma.
J. Chromatogr. B (2018)
1077-1078: 52-9

(37)
Kincses L, Falkay G, Sas M and Morvay J. Therapeutic
blood level
of clomiphen citrate. Magyar Nöorvosok
Lapja
(1978) 41: 29-31.