Medroxyprogesterone Acetate

Molecular formula: C24H34O4

Molecular weight: 386.5
CAS Registry No.: 71-58-9 (medroxyprogesterone acetate),
520-85-4 (medroxyprogesterone)

SAMPLE

Matrix: blood
Sample preparation: Extract 0.1-1 mL plasma twice with 40 volumes of diethyl ether,
evaporate the organic solvent, dissolve the residue in 100 \xL MeOH: water 80:20, inject
a 30 \xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm Beckman RP-ODS

Mobile phase: Gradient. MeCN.water from 40:60 to 70:30 over 20 min, then at 70:30 for
5 min.
Injection volume: 30
Detector: UV 238
CHROMATOGRAM
Retention time: 20.5
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS

plasma
REFERENCE
Sturm, G.; Haberlein, H.; Bauer, T.; Plaum, T.; Stalker, D.J. Mass spectrometric and high-performance
liquid chromatographic studies of medroxyprogesterone acetate metabolites in human plasma.
J.Chromatogr., 1991, 562, 351-362

SAMPLE

Matrix: blood
Sample preparation: 2 mL Plasma + 1 mL 0.5 |xg/mL 16-methylprogesterone in 200 mM
pH 7.0 phosphate buffer, vortex for 3 s, add 7 mL hexane, mix on a rolling mixer for 30
min. Remove the hexane layer and evaporate it to dryness at 30 under nitrogen. Dissolve
residue in 200 |xL mobile phase, inject a 150 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 ^m Spherisorb 5-ODS2

Mobile phase: MeOH: 20 mM pH 4 acetate buffer 79:21
Flow rate: 1.5
Injection volume: 150
Detector: UV 240
CHROMATOGRAM

Retention time: 5.3

Internal standard: 16p-methylprogesterone (9.0)
Limit of detection: 10 ng/mL

OTHER SUBSTANCES
Simultaneous: aldosterone, androstenedione, corticosterone, cortisone, estradiol, 17a-hydroxyprogesterone, progesterone, testosterone
Noninterfering: cholesterol
Interfering: lignocaine
KEYWORDS
plasma
REFERENCE
Read, J.; Mould, G.; Stevenson, D. Simple high-performance liquid chromatographic method for the
determination of medroxyprogesterone acetate in human plasma. J.Chromatogr., 1985, 341, 437444

SAMPLE
Matrix: formulations
Sample preparation: Grind tablets to a fine powder, weigh out an amount equivalent to
about 10 mg medroxyprogesterone acetate, add 10 mL MeOH, sonicate for 5 min, dilute
to 50 mL with MeOH. Dilute 25 mL of this solution to 50 mL with MeOH, filter (0.45
jjim), inject a 20 fxL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 5 |xm Novapak C18
Mobile phase: MeOHrIO mM (NHJ2HPO4 80:20, adjust pH to 7.2 0 . 1 with 85% phosphoric acid
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 6.7
KEYWORDS
tablets; stability-indicating
REFERENCE
Fatmi, A.A.; Williams, G.V.; Hickson, E.A. Liquid chromatographic determination of medroxyprogesterone acetate in tablets. J.Assoc.Off.Anal.Chem., 1988, 71, 528-530

SAMPLE
Matrix: formulations
Sample preparation: Suspensions. Dilute 2 mL suspension to 1 L with EtOH, remove a
2.5 mL aliquot and add it to 1 mL 1 mg/mL hydrocortisone in EtOH. Dilute this mixture
to 50 mL with EtOH, inject an aliquot. Tablets. Grind tablets to a fine powder, stir with
50 mL EtOH, make up to 100 mL with EtOH, filter (Whatman No. 1 paper), reject the
first 20 mL of the filtrate. Mix 2 mL of the filtrate with 1 mL 1 mg/mL hydrocortisone in
EtOH, make this mixture up to 50 mL with EtOH, inject an aliquot.
HPLCVARIABLES
Column: 300 X 4 jxBondapak CN
Mobile phase: MeOH: 20 mM KH2PO4 30:70
Flow rate: 2
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 7.5
Internal standard: hydrocortisone (2.5)
OTHER SUBSTANCES
Simultaneous: benzyl benzoate, progesterone
Noninterfering: methylcellulose, mystristyl-gamma-picolinium chloride, polyethylene glycol 4000, thimerosal
REFERENCE
Das Gupta, V. Quantitation of hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone by reversed-phase high-pressure liquid chromatography. J.Pharm.Sci., 1982, 71, 294-297

SAMPLE
Matrix: solutions
Sample preparation: Prepare a 25 |xg/mL solution in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Partisil 10 ODS-I
Mobile phase: MeOH: water 55:45
Column temperature: 40
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: k' 6.712 (medroxyprogesterone acetate), k' 3.430 (medroxyprogesterone)
OTHER SUBSTANCES
Also analyzed: androsterone (UV 210), cortexolone (UV 240), cortisone (UV 240), estradiol
(UV 280), estrone (UV 280), ethinyl estradiol (UV 280), ethisterone (UV 240), hydrocortisone (UV 240), hydroxyprogesterone (UV 240), lynestrenol (UV 210), methandienone
(UV 240), methylandrostenediol (UV 210), methylprednisolone acetate (UV 240), methylprednisolone (UV 240), methyltestosterone (UV 240), nandrolone (UV 240), norethisterone (UV 240), prednisolone acetate (UV 240), prednisolone (UV 240), prednisone (UV
240), pregnenolone (UV 210), progesterone (UV 240), testosterone (UV 240)
REFERENCE
Sadlej-Sosnowska, N. Structure retention relationship for steroid hormones. Functional groups as structural descriptors. J.Liq.Chromatogr., 1994, 17, 2319-2330

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Hypersil ODS
Mobile phase: MeOH: water 60:40
Injection volume: 250
Detector: UV
CHROMATOGRAM
Retention time: 10 (for medroxyprogesterone)
OTHER SUBSTANCES
Simultaneous: dienestrol, diethylstilbestrol, hexestrol, 17ct-methyltestosterone, nandrolone, trenbolone, zeranol

REFERENCE
Jansen, E.H.J.M.; Both-Miedema, R.; van den Berg, R.H. Application of optimization procedures for the
separation of anabolic compounds by high-performance liquid chromatography. J.Chromatogr., 1989,
489, 57-64

SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH: water 1:1 at a concentration of 50 |xg/mL, inject
a 10 |JLL aliquot.
HPLCVARIABLES
Column: 300 X 3.9 10 imm iuiBondapak C18
Mobile phase: MeOH: acetic acid: triethylamine: water 70:1.5:0.5:28
Flow rate: 1.5
Injection volume: 10
Detector: UV
CHROMATOGRAM
Retention time: k' 3.12
REFERENCE
Roos, R.W.; Lau-Cam, CA. General reversed-phase high-performance liquid chromatographic method
for the separation of drugs using triethylamine as a competing base. J. Chromatogr., 1986, 370,
403-418

SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Waring blender) tissue at full speed for 2 min, lyophilize, grind. Extract with supercritical carbon dioxide at 60 at 400 atmospheres with
a 20 cm X 21 jxm restrictor for 1 h, collect the extract in 1 mL MeOH cooled to 5.
Evaporate the MeOH to dryness under a stream of nitrogen, reconstitute the residue in
100 |xL MeCN: MeOH: 20 mM ammonium formate 15:15:70, inject an aliquot. Alternatively, vortex 5 g ground tissue with 10 mL 40 mM sodium acetate, adjust pH to 4.2-4.7
with glacial acetic acid, add 100 uX p-glucuronidase (Sigma), heat at 37 for 8 h, add 20
mL MeCN, vortex for 30 s, centrifuge at 5000 rpm for 20 min. Remove a 30 mL aliquot
of the supernatant and add it to 8 mL hexane and 2 mL dichloromethane, rotate for 3
min, centrifuge at 2000 rpm for 2 min. Remove a 15 mL aliquot of the middle layer and
evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 1 mL
dichloromethane, inject an aliquot.
HPLCVARIABLES
Column: 50 X 4.6 5 |xm Supelcosil
Mobile phase: Gradient. MeCN: MeOH: 20 mM ammonium formate from 2.5:2.5:95 to
47.5:47.5:5 over 19 min.
Flow rate: 1
Injection volume: 20
Detector: UV 245; MS, Sciex TAGA 6000E tandem triple quadrupole, APCI
CHROMATOGRAM
Retention time: 13.7
Limit of detection: 100 ppb
OTHER SUBSTANCES
Extracted: dexamethasone, diethylstilbestrol, melengestrol acetate, trenbolone, triamcinolone acetonide, zeranol

KEYWORDS

cow; muscle; liver; SFE

REFERENCE
Huopalahti, R.P.; Henion, J.D. Application of supercritical fluid extraction and high performance liquid
chromatography/mass spectrometry for the determination of some anabolic agents directly from bovine tissue samples. J.Liq.Chrom.Rel.TechnoL, 1996, 19, 69-87

SAMPLE

Matrix: urine
Sample preparation: 10 mL Urine + glucuronidase/sulfatase (Helix pomatia), incubate at
37 for 1 h, extract twice with 5 mL diethyl ether, add 225 |xL water and evaporate ether
under nitrogen, add 400 |xL MeOH, inject a 250 |xL aliquot of this mixture.
HPLC VARIABLES

Guard column: 75 X 2.1 Corasil C18

Column: 150 X 4.6 5 |xm Hypersil ODS
Mobile phase: MeOH: water 60:40
Flow rate: 2
Injection volume: 250
Detector: UV 240
CHROMATOGRAM

Retention time: 14 (for medroxyprogesterone)

Limit of detection: about 6 ng/mL
OTHER SUBSTANCES

Simultaneous: trans-diethylstilbestrol, 17a-methyltestosterone, nandrolone, 17p-trenbolone, zeranol

KEYWORDS
cow

REFERENCE
Jansen, E.H.; Both-Miedema, R.; van Blitterswijk, H.; Stephany, R.W. Separation and purification of
several anabolics present in bovine urine by isocratic high-performance liquid chromatography.
J.Chromatogr., 1984, 299, 450-455

ANNOTATED BIBLIOGRAPHY
Mould, G.R; Read, J.; Edwards, D.; Bye, A. A comparison of the high-performance liquid chromatography
and RIA measurement of medroxyprogesterone acetate. J.Pharm.Biomed.AnaL, 1989, 7, 119-122
Rees, H.D.; Bonsall, R.W.; Michael, R.R Pre-optic and hypothalamic neurons accumulate
[3H]medroxyprogesterone acetate in male cynomolgus monkeys. Life Sci., 1986, 39, 1353-1359
[brain; tissue; UV detection]
Milano, G.; Carle, G.; Renee, N.; Boublil, J.L.; Namer, M. Determination of medroxyprogesterone acetate
in plasma by high-performance liquid chromatography. J.Chromatogr., 1982, 232, 413-417