Norethindrone

Molecular formula: G20H26O2

Molecular weight: 298.4
CAS Registry No.: 68-22-4, 51-98-9 (acetate)

SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C18 SPE cartridge with MeOH and water.
Add 500 |xL plasma to the SPE cartridge, wash with 2 mL water, wash with 2 mL
MeOH: water 20:80, elute with two 500 |JLL aliquots of MeOH. Evaporate the eluate to
dryness under a stream of nitrogen, reconstitute the residue in 50 |xL MeOH: water 20:
80, inject a 20 JULL aliquot.

HPLCVARIABLES
Column: 100 X 1 5 |xm Hypersil ODS
Mobile phase: MeCN: MeOH: water 25:25:50
Flow rate: 0.1
Injection volume: 20
Detector: UV
CHROMATOGRAM
Retention time: 20
Limit of detection: 2 ng/mL
OTHER SUBSTANCES
Extracted: androstenedione, 20a-hydroxy-4-pregnen-3-one, 17a-hydroxyprogesterone, progesterone, testosterone
KEYWORDS
microbore; rat; plasma; SPE
REFERENCE
Taylor, R.B.; Kendle, K.E.; Reid, R.G.; Hung, CT. Chromatography of progesterone and its major metabolites in rat plasma using microbore high-performance liquid chromatography columns with conventional injection and detection systems. J.Chromatogr., 1987, 385, 383392

SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 5 mL hexane: dichloromethane 1:1, shake for 10 min,
centrifuge at 2000 rpm for 10 min. Remove 4 mL of the organic phase and evaporate it at
55 under nitrogen. Reconstitute residue in 300 |xL mobile phase, inject a 150 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb RP-8
Mobile phase: MeOH: MeCN: water 20:30:50
Flow rate: 2
Injection volume: 150
Detector: UV 254
CHROMATOGRAM
Retention time: 8
Limit of quantitation: 2 ng/mL

KEYWORDS
plasma
REFERENCE
Loo, J.C.K.; Brien, R. Analysis of norethindrone in plasma by high-performance liquid chromatography.
J.Liq.Chromatogr., 1981, 4, 871-877

SAMPLE
Matrix: bulk
HPLCVARIABLES
Column: 250 X 4 10 jxin LiChrosorb RP-18
Mobile phase: MeOH: water 70:30
Flow rate: 1
Injection volume: 25
Detector: UV 240
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol, impurities, norgestrel
REFERENCE
Gdrdg, S.; Herenyi, B. Analysis of steroids. XXXVIIL The use of high-performance liquid chromatography
with diode-array UV detection for estimating impurity profiles of steroid drugs. J.Chromatogr., 1987,
400, 177-186

SAMPLE
Matrix: food
Sample preparation: Dissolve apple extracts in THF, inject a 10 |xL extract.
HPLCVARIABLES
Column: 300 X 3.9 jxBondapak C18
Mobile phase: MeOH: THF: water 11:26:63
Flow rate: 1.7
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 11.6
OTHER SUBSTANCES
Simultaneous: degradation products, impurities
KEYWORDS
apples; fruit; stability-indicating
REFERENCE
Mayberry, D.O.; Kowblansky, M.; Lane, RA.; Wray, RE. Determination of norethindrone stability on Red
Delicious apples. J.Pharm.ScL, 1990, 79, 746-749

SAMPLE
Matrix: formulations

Sample preparation: Dissolve 12 tablets in 600 mL water with stirring at 75 rpm, remove
3 mL sample, centrifuge at 3000 rpm for 15 min, inject a 250 |xL aliquot.
HPLCVARIABLES

Column: 100 X 4.6 3 jxm Phenomenex IB-SiI 3 C18

Mobile phase: MeCN: water 40:60, pH 5.6
Flow rate: 1.2
Injection volume: 250
Detector: UV 200
CHROMATOGRAM

Retention time: 7.7

OTHER SUBSTANCES
Simultaneous: ethinyl estradiol
KEYWORDS

tablets; modification of USP method

REFERENCE
Dorantes, A.; Stavchansky, S. Modification of the U.S.P. dissolution method for the analysis of norethindrone and ethinyl estradiol tablets. J.Pharm.ScL, 1994, 83, 379-381

SAMPLE

Matrix: formulations
Sample preparation: Dissolve 6 tablets in 600 mL water: isopropanol 97:3, remove 5 mL
samples, centrifuge at 1500 rpm for 10 min, inject a 50-200 |xL aliquot.
HPLCVARIABLES

Column: 300 X 3.9 10 jxm Bondapak C18

Mobile phase: MeCN: water 55:45
Flow rate: 1
Injection volume: 50-200
Detector: UV 200
OTHER SUBSTANCES
Simultaneous: mestranol
KEYWORDS

tablets; modified USP method

REFERENCE
Nguyen, H.T.; Shiu, G.K.; Worsley, W.N.; Skelly, J.R Dissolution testing of norethindrone: ethinyl estradiol, norethindrone: mestranol, and norethindrone acetate: ethinyl estradiol combination tablets.
J.Pharm.ScL, 1990, 79, 163-167

SAMPLE

Matrix: formulations
Sample preparation: Dissolve 6 tablets in 600 mL 100 mM HCl containing 0.02% sodium
lauryl sulfate, remove 5 mL samples, centrifuge at 1500 rpm for 10 min, inject a 50-200
IxL aliquot.
HPLCVARIABLES

Column: 220 X 4.6 5 |jim Spheri-5 C18

Mobile phase: MeCN: 20 mM pH 6.0 phosphate buffer 35:65

Flow rate: 1.5

Injection volume: 50-200
Detector: UV 200
CHROMATOGRAM
Retention time: 20.26
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol
KEYWORDS
tablets; modified USP method
REFERENCE
Nguyen, H.T.; Shiu, G.K.; Worsley, W.N.; Skelly, J.R Dissolution testing of norethindrone: ethinyl estradiol, norethindrone: mestranol, and norethindrone acetate: ethinyl estradiol combination tablets.
J.Pharm.ScL, 1990, 79, 163-167

SAMPLE
Matrix: formulations
Sample preparation: Dissolve 6 tablets in 600 mL 100 mM HCl containing 0.02% sodium
lauryl sulfate, remove 5 mL samples, centrifuge at 1500 rpm for 10 min, inject a 50-200
jxL aliquot.
HPLCVARIABLES
Column: 220 X 4.6 5 |xm Spheri-5 C18
Mobile phase: MeCN: 20 mM pH 6.0 phosphate buffer 60:40
Flow rate: 1
Injection volume: 50-200
Detector: UV 240
CHROMATOGRAM
Retention time: 12.73 (norethindrone acetate)
KEYWORDS
tablets; modified USP procedure
REFERENCE
Nguyen, H.T.; Shiu, G.K.; Worsley, W.N.; Skelly, J.R Dissolution testing of norethindrone: ethinyl estradiol, norethindrone: mestranol, and norethindrone acetate: ethinyl estradiol combination tablets.
J.Pharm.ScL, 1990, 79, 163-167

SAMPLE
Matrix: formulations
Sample preparation: Oils. 1 mL Sample + 25 mL MeOH: water 90:10, shake vigorously
for 5 min, centrifuge, inject a 10 jiL aliquot of the supernatant. Tablets. Grind a tablet
to a fine powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |xm), discard first
5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aqueous).
Make up 5 mL to 50 mL with MeOH, filter (0.45 |xm), discard first 5 mL of filtrate, inject
a 10 |xL aliquot of the remaining filtrate.
HPLCVARIABLES

Column: 250 X 4.6 5 ^m Zorbax ODS

Mobile phase: MeOH: water 75:25
Flow rate: 1.5

Injection volume: 10
Detector: UV 240
CHROMATOGRAM
Retention time: 4.6 (norethindrone), 8.6 (norethindrone acetone)
Limit of detection: 5 jig/mL
OTHER SUBSTANCES
Simultaneous: aspirin, benzyl alcohol, benzyl benzoate, caffeine, calusterone, cortisone, dehydroepiandrosterone (UV 210), formebolone, mesterolone (UV 210), methandriol (UV
210), methandrostenolone, methenolone acetate, methyltestosterone, mibolerone, nandrolone, nandrolone acetate, nandrolone propionate, norethandrolone, norgestrel, oxymetholone, stanozolol, testolactone, testosterone, testosterone acetate, testosterone propionate,
trenbolone acetate
Interfering: boldenone, ethisterone, fluoxymesterone, oxandrolone (UV 210)
KEYWORDS
oils; tablets; suspensions
REFERENCE
Walters, M.J.; Ayers, R.J.; Brown, D.J. Analysis of illegally distributed anabolic steroid products by
liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry J.Assoc.Off.Anal.Chem., 1990, 73, 904-926

SAMPLE
Matrix: formulations
Sample preparation: Dissolve 5 g cream containing 0.00925% fluocinonide, 0.00365% procinonide, and 0.0021% ciprocinonide in 2.5 mL THF, add norethindrone, dilute to 25 mL
with MeOH, centrifuge, inject a 25 JULL aliquot onto column A with mobile phase A and
allow components to elute from column A to column B for 7 min. After 7 min remove
column A from circuit, monitor effluent from column B. Back-flush column A with mobile
phase B for 5 min, equilibrate column A with mobile phase A for 5 min before next
injection.
HPLCVARIABLES
Column: A 30 X 4.6 5 >jim Spheri-5 ODS (Brownlee); B 70 X 2.1 Whatman Co:Pell ODS +
250 X 4.6 5 |jim Ultrasphere C18
Mobile phase: A MeCN:THF:water 43:4:53; B MeOH:THF 75:25
Flow rate: A 1.5; B 1
Injection volume: 25
Detector: UV 260 for 22 min then UV 236
CHROMATOGRAM
Retention time: 12
Internal standard: norethindrone
OTHER SUBSTANCES
Simultaneous: ciprocinonide, fluocinolone acetonide, fluocinonide, procinonide
KEYWORDS
creams; column-switching; norethindrone is IS
REFERENCE
Conley, D.L.; Benjamin, E.J. Automated high-performance liquid chromatographic column switching
technique for the on-line clean-up and analysis of drugs in topical cream formulations. J.Chromatogr.,
1983, 257, 337-344

SAMPLE
Matrix: formulations
Sample preparation: 1 Tablet + 4 mL 50 mM KH2PO4, rotate 15 min, add 2 mL 1 ixg/mL
o-phenylphenol in mobile phase, add 4 mL MeOH, rotate 15 min, centrifuge. Remove
supernatant, extract residue twice with 5 mL mobile phase (10 min rotation), combine
supernatants, inject 50 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 (xm LiChrosorb RP8
Mobile phase: MeOH:50 mM KH2PO4 3:2
Flow rate: 2
Injection volume: 50
Detector: UV 220
CHROMATOGRAM
Retention time: 9 (norethindrone), 19.3 (norethindrone acetate)
OTHER SUBSTANCES
Simultaneous: methyltestosterone, norgestrel
Interfering: ethinyl estradiol
KEYWORDS
tablets; stability-indicating
REFERENCE
Strusiak, S.H.; Hoogerheide, J.G.; Gardner, M.S. Determination of ethinyl estradiol in solid dosage forms
by high-performance liquid chromatography. J.Pharm.Sci., 1982, 71, 636-640

SAMPLE
Matrix: reaction mixtures
Sample preparation: If necessary, remove oxidizing power of solution by adding sodium
metabisulfite, inject a 20 JJLL aliquot.
HPLC VARIABLES
Guard column: 15 X 4.6 5 |xm Microsorb C8
Column: 250 X 4.6 5 |xm Microsorb C8
Mobile phase: MeCN: 100 mM KH2PO4 adjusted to pH 7 with 1 M KOH 50:50
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 8.7
Limit of detection: 100 ng/mL
REFERENCE
Lunn, G.; Rhodes, S.W.; Sansone, E.B.; Schmuff, N.R. Photolytic destruction and polymeric resin decontamination of aqueous solutions of pharmaceuticals. J.Pharm.Sci., 1994, 83, 1289-1293

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 50 X 4.6 5 Jim Supelcosil LC-18
Mobile phase: MeOH: THF: water 10:20:70

Flow rate: 2
Injection volume: 20
Detector: UV 220
CHROMATOGRAM
Retention time: 3 (norethindrone), 7.4 (norethindrone acetate)
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol, norethynodrel acetate, norgestrel
REFERENCE
Supelco Catalog, Supelco, Inc, Bellefonte PA, 1996, p. A130

SAMPLE
Matrix: solutions
Sample preparation: Inject a 5 JJLL aliquot of a 10 jxg/mL solution in MeOH.
HPLCVARIABLES
Column: 75 X 4.6 3 fxm Ultrasphere ODS
Mobile phase: MeCN: 10 mM ammonium acetate buffer 45:55
Flow rate: 0.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 5.941
OTHER SUBSTANCES
Simultaneous: boldenone, epimethandienone, epitestosterone, fluoxymesterone, 6p-hydroxymethandienone, methandienone, oxymetholone (UV 280), trenbolone
REFERENCE
Barron, D.; Pascual, J.A.; Segura, J.; Barbosa, J. Prediction of LC retention of steroids using solvatochromic parameters. Chromatographia, 1995, 41, 573-580

SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 \iL aliquot of a solution in MeOH: water 50:50.
HPLCVARIABLES
Column: 250 X 4 7 |jim LichroCART RP-8 (Merck)
Mobile phase: MeCN: MeOH: water 32:37:31
Flow rate: 1
Injection volume: 20
Detector: UV 230
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: fluoxymesterone, medrogestone, mestranol, progesterone, testosterone
propionate

REFERENCE
Gau, YS.; Sun, S.W.; Chem, R.R.-L. Optimization of high-performance liquid chromatographic separation for progestogenic, estrogenic, and androgenic steroids using factorial design. J.Liq.Chromatogr.,
1995, 18, 2373-2382

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 10 ^m Nucleosil C18
Mobile phase: MeCN:THF-.water 12.9:22.4:64.7
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 8.5 (norethindrone), 15.5 (norethindrone acetate)
OTHER SUBSTANCES
Simultaneous: estrone, ethinyl estradiol, mestranol, norgestrel
REFERENCE
Gazdag, M.; Szepesi, G.; Szeleczki, E. Selection of high-performance liquid chromatographic methods in
pharmaceutical analysis. I. Optimization for selectivity in reversed-phase chromatography.
J.Chromatogr., 1988, 454, 83-94

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 ^m LiChrosorb Si 60
Mobile phase: Hexane: dioxane: isopropanol 95:3:2
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 20 (norethindrone), 10 (norethindrone acetate)
OTHER SUBSTANCES
Simultaneous: estrone, ethinyl estradiol, mestranol, norgestrel
KEYWORDS
normal phase
REFERENCE
Gazdag, M.; Szepesi, G.; Fabian-Varga, K. Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. II. Optimization for selectivity in normal-phase systems.
J.Chromatogr., 1988, 454, 95-107

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 |xm (xBondapak C18

Next page
Mobile phase: MeOH: acetic acid: triethylamine: water 60:1.5:0.5:38
FIo5W rate: 1.5
Injection volume: 10
Detector: UV 240
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: hydrocortisone acetate, methyltestosterone, prednisolone, prednisolone succinate, prednisone, progesterone
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

ANNOTATED BIBLIOGRAPHY
Tang, G.P.; Chen, Q.Q. [Column switching HPLC method for determination of in vivo release of norethindrone-alpha, beta-poly (3-hydroxypropyl)-DL-asparamide conjugate]. Yao Hsueh Hsueh Pao, 1994,
29, 301-305
Lee, G.J.-L.; Oyang, M.-H.; Bautista, J.; Kushinsky, S. Determination of ethinylestradiol and norethindrone in a single specimen of plasma by automated high-performance liquid chromatography and
subsequent radioimmunoassay. J.Liq.Chromatogr., 1987, 10, 2305-2318 [LOD 20-50 pg/mL]
Papas, A.N.; Marchese, S.M.; Delaney, M.F. Rapid determination of norethindrone and ethinylestradiol
in oral contraceptive tablets by reversed-phase liquid chromatography. LC Mag., 1985, 3, 354-358
[tablets; column temp 25; simultaneous ethinylestradiol; Chem.Abs., 102, 226114]
Swynnerton, N.R; Fischer, J.B. Determination of ethynylestradiol and norethindrone in synthetic intestinal fluid and in timed-release oral formulations. J.Liq.Chromatogr., 1980, 3, 1195-1204 [formulations; also ethinylestradiol]