Estrogens, Conjugated

Molecular formula: C18H18O2 (equilenin), C18H22O2 (17a-dihydroequilin), C18H20O2 (equilin), C18H22O2 (estrone), C18H24O2 (estradiol) Molecular weight: 266.3 (equilenin), 272.4 (estradiol), 268.3 (equilin), 270.4 (17a-dihydroequilin), 270.4 (estrone) CAS Registry No.: 474-86-2 (equilin), 50-28-2 (estradiol), 57-91-0 (a-estradiol), 517-09-9 (equilenin), 53-167 (estrone), 338-67-5 (estrone sodium sulfate), 481-97-0 (estrone hydrogen sulfate)

Sodium Estrone Sulfate

Sodium Equilin Sulfate Equilenin

17a-Dihydroequilin, Sodium Sulfate Conjugate

17|3-Dihydroequilin, Sodium Sulfate Conjugate

R = H, HSQ3Na

17a-Estradiol, Sodium Sulfate Conjugate

Conjugated Estrogens (see Preface)

SAMPLE Matrix: blood Sample preparation: Add 0.1 (rabbit) or 1 (rat, monkey) plasma to 1 mL 100 mM pH 5.0 acetate buffer and 50 |xL Glusulase (from Helix Pomatia, contains 10000 U/mL sulfatase and 90000 U/mL p-glucuronidase, DuPont), heat at 37 for 1 h, cool to room temperature, add 15 mL diethyl ether, shake mechanically at high speed for 10 min, centrifuge at 3033 g for 10 min, freeze in dry ice/acetone. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 500 (xL mobile phase, filter (0.45 jjim), inject a 150 JJLL aliquot of the filtrate. HPLCVARIABLES Column: 150 X 3 5 (xm C6 (Column Engineering, Ontario CA) Mobile phase: MeCN:MeOH:50 mM pH 3.5 ammonium acetate 27:8:65 Flow rate: 0.35 Injection volume: 150 Detector: F ex 210 em 370 CHROMATOGRAM Retention time: 19.5 (17a-dihydroequilenin) Internal standard: 14p-equilenin (24) Limit of quantitation: 2.5 ng/mL (rat), 5 ng/mL (rabbit, monkey) OTHER SUBSTANCES Noninterfering: equilenin, 17a-dihydroequilenin

KEYWORDS plasma; rat; rabbit; monkey; pharmacokinetics REFERENCE

Chandrasekaran, A.; Osman, M.; Adelman, S.J.; Warsheski, J.; Scatina, J.; Sisenwine, S.F. Determination of 17a-dihydroequilenin in rat, rabbit and monkey plasma by high-performance liquid chromatography with fluorimetric detection. J.Chromatogr.B, 1996, 676, 69-75

SAMPLE Matrix: blood Sample preparation: 10 |xL Plasma is injected into MeCN pumped at 0.2 mL/min, precipitated proteins are removed by 0.5 and 0.2 |xm filters in series, MeCN containing sample is switched into mobile phase allowed to pass onto analytical column. Next filter unit is switched out of circuit and back-flushed to waste with 100 mM sodium dodecyl sulfate at 2 mL/min, analytical column is eluted in normal fashion with mobile phase. Equilibrate filters with MeCN for 5 min before next injection. HPLCVARIABLES Guard column: 20 mm Brownlee C18 Column: 250 X 4.6 5 |xm Ultrasphere C18 Flow rate: 1 Injection volume: 10 Detector: UV 280 CHROMATOGRAM Retention time: 25.8 (estradiol), 36.6 (equilin), 42.6 (estrone) KEYWORDS plasma; dog REFERENCE
Asafu-Adjaye, E.B.; Su, S.Y.; Shiu, G.K. Switching-valve-filter technique for the direct injection and analysis of drugs in plasma using high-performance liquid chromatography. J.Chromatogr.B, 1994, 652, 35-42

SAMPLE Matrix: blood Sample preparation: 100 (JLL Plasma + 10 /JLL IS in water, extract twice by shaking for 1 min with 1.2 mL dichloromethane, evaporate organic layer below 40 under reduced pressure, dissolve residue in 100 |JLL MeCN. Add 10 |xL reagent 1, add 10 JULL reagent 2, heat at 50 for 15 min, cool to room temperature, add 100 fxL water, add 200 |JLL MeOH: water 1:1, add to Sep-Pak C18 cartridge, wash vial with 2 mL MeOH: water 1:1 and add washings to cartridge, wash cartridge with 40 mL MeOH-.water 1:1, elute with 5 mL MeOH. Concentrate eluent to 500 |JLL by evaporation at 40 under reduced pressure, inject 20 \xh aliquot. (Reagent 1 was 30 mg 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole in 3 mL pyridine, add 700 mg 4-piperidinopyridine, dilute to 10 mL with MeCN. Reagent 2 was 700 mg l-isopropyl-3-(3-dimethylaminopropyl)carbodiimide perchlorate in 10 mL MeCN.) HPLCVARIABLES Guard column: 50 X 4 5 jxm Wakosil 5C18 Column: 300 X 4 5 |xm Wakosil 5C18 Mobile phase: MeOH: water 90:10 Flow rate: 0.7 Injection volume: 20 Detector: F ex 336 em 440

CHROMATOGRAM Retention time: 10.5 (estriol), 15.4 (ethynylestradiol), 16.5 (equilin), 16.5 (equilenin), 17.2 (estrone), 18.2 (estradiol), 19.2 (estetrol), 28.1 (4-hydroxyestradiol), 35.5 (2hydroxyestradiol) Internal standard: sec-butyl p-hydroxybenzoate (14.3) Limit of detection: 1-2 pg/mL KEYWORDS plasma; equilin and equilenin not resolved REFERENCE
Katayama, M.; Taniguchi, H. Determination of estrogens in plasma by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole. J.Chromatogr., 1993, 616, 317-322

SAMPLE Matrix: blood Sample preparation: 0.5 mL Serum + 0.5 mL MeCN: water 1:1, vortex 15 s, add 3 mL MeCN, shake 1 min, centrifuge at 1800 rpm for 10 min. Remove supernatant and dry it under a stream of nitrogen at 55, add 2 mL MeCN: MeOH 1:1, vortex 15 s, centrifuge at 1800 rpm for 10 min. Remove supernatant and dry it under a stream of nitrogen at 55. Reconstitute in 200 \xL MeCN: water 1:1. HPLCVARIABLES Column: 250 X 4.6 5 |xm Beckman ODS Mobile phase: A 2% tetrabutylammonium hydroxide adjusted to pH 3 with phosphoric acid. B MeCN:water 33:67 A:B was 6.5:93.5 Flow rate: 0.8 Injection volume: 20 Detector: UV 210; F ex 280 em 312 CHROMATOGRAM Retention time: 53 (estrone), 48 (equilin), 41 (estrone sulfate), 38 (equilin sulfate), 32 (estradiol), 26 (17-a-dihydroequilin sulfate) Limit of detection: 10-100 ng/mL KEYWORDS serum REFERENCE
Su, S.Y.; Shiu, G.K.; Simmons, J.; Viswanathan, C.T.; Skelly, J.P. High performance liquid chromatographic analysis of six conjugated and unconjugated estrogens in serum. Biomed.Chromatogr., 1992, 6, 265-268

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma H - 5 mL water + 1 mL 2 |xg/mL equilenin in MeOH + 50 fxL 0.1 M NaOH to adjust pH to 10, vortex briefly after each addition, shake with 10 mL dichloromethane for 10 min, centrifuge at 2000 g for 10 min. Wash organic layer twice with 2 mL water, centrifuge 5 min, evaporate 8 mL of organic phase to dryness at 40under a stream of nitrogen, reconstitute residue in 150 jxL mobile phase, inject 25 fxL aliquot. HPLCVARIABLES Column: 300 X 4 10 |xm jxBondapak C18 Mobile phase: MeOH-.buffer 65:35 (Buffer was 10 mL 200 mM acetic acid + 15 mL 200 mM sodium acetate made up to 1 L, pH 4.8.)

Flow rate: 1 Injection volume: 25 Detector: UV 254 CHROMATOGRAM Retention time: 5.3 Internal standard: equilenin Limit of detection: 5 ng/mL OTHER SUBSTANCES Simultaneous: betamethasone, deoxycortisol, dexamethasone, hydrocortisone, prednisone, triamcinolone KEYWORDS AnaLAbs. 1982, 43, 4D182; plasma=uilenin is IS REFERENCE
Bouquet, S.; Brisson, A.M.; Gombert, J. Dosage du cortisol et du 11-desoxycortisol plasmatiques par chromatographie liquide haute performance [Cortisol and 11-desoxycortisol determination in blood by high performance liquid chromatography]. Ann.Biol.Clin.(Paris), 1981, 39, 189-191

SAMPLE Matrix: cells Sample preparation: Homogenize (glass-glass homogenizer) cells in medium, centrifuge at 1000 g for 5 min. Add 3 mL homogenate to 10 mL diethyl ether: acetone 90:10, mix thoroughly for a few s, let stand at 4 for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in three 500 |xL portions of acetone, evaporate to dryness under a stream of nitrogen, reconstitute with 30 |xL MeCN, inject a 20 |xL aliquot. HPLC VARIABLES Column: Ultrasphere ODS Mobile phase: MeCN: 10 mM citric acid 40:60 Column temperature: 20 1 Flow rate: 1 Injection volume: 20 Detector: UV 280; Radioactivity CHROMATOGRAM Retention time: 14 (17p-estradiol), 16 (equilin), 19 (estrone) KEYWORDS tritium labeled; 14C labeled REFERENCE
Castagnetta, L.A.; Granata, O.M.; Lo Casto, M.; Calabro, M.; Arcuri, F.; Carruba, G. Simple approach to measure metabolic pathways of steroids in living cells. J .Chromatogr., 1991, 572, 25-39

SAMPLE Matrix: formulations Sample preparation: Dissolve a quantity equivalent to about 25 mg of conjugated estrogens in 20 mL MeOH, add 20 mL water, add 4 mL concentrated HCl, add several boiling chips, boil for 5 min, cool to room temperature, add 2.5 mL 0.5 mg/mL estriol in MeOH, extract twice with 10 mL and once with 5 mL portions of chloroform, combine extracts, wash with 5 mL water, pass through 1 g anhydrous sodium sulfate, evaporate to dryness under nitrogen, dissolve residue in 25 mL MeOH: water 1:1, inject aliquot.

HPLCVARIABLES

Column: 250 X 4.6 3 ^m Nucleosil C18 Mobile phase: MeOH: water: isopropanol: dichloromethane 45:42.5:7.5:5 Flow rate: 0.7 Injection volume: 20 Detector: UV 280; E, Laboratorni Pristroje ADLC 2 detector, carbon fiber working electrode, stainless steel counter electrode, 1.1 V vs Ag/AgCl reference electrode, 0.6% Na2HPO4.12H2O added to mobile phase which was adjusted to pH 6.0-6.05 with acetic acid
CHROMATOGRAM

Retention time: 23.18 (estrone), 20.59 (equilin), 18.59 (equilenin), 15.14 (17a-estradiol), 13.36 (17a-dihydroequilin), 11.85 (17a-dihydroequilenin) Internal standard: estriol (6.44) Limit of detection: 2-4 |xg/mL
KEYWORDS

tablets; capsules
REFERENCE
Novakovic, J.; Tvrzicka, E.; Pacakova, V. High-performance liquid chromatographic determination of equine estrogens with ultraviolet absorbance and electrochemical detection. J.Chromatogr.A, 1994, 678, 359-363

SAMPLE

Matrix: formulations Sample preparation: Dissolve in mobile phase, inject an aliquot.

HPLC VARIABLES

Column: 150 X 3.9 4 |xm NovaPak C18 Mobile phase: MeOH: 25 mM KH2PO4 40:60 Flow rate: 0.8 Injection volume: 50 Detector: UV 200
CHROMATOGRAM

Retention time: 19.01 (estrone sulfate), 16.54 (equilin sulfate), 17.87 (17a-dihydroequilin sulfate), 16.16 (17p-dihydroequilin sulfate), 25.28 (17a-estradiol sulfate), 19.01 (17p-estradiol sulfate), 13.88 (equilenin sulfate), 14.26 (17a-dihydroequilenin sulfate)
KEYWORDS

injections; tablets
REFERENCE
Flann, B.; Lodge, B. Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversedphase chromatography. J.Chromatogr., 1987, 402, 273-282

SAMPLE

Matrix: formulations Sample preparation: Dissolve in mobile phase, inject an aliquot.

HPLCVARIABLES

Column: 250 X 4.6 5 jxm Ultrasphere Octyl Mobile phase: MeOH: water: trichloroethanol 23:75:2, all 0.1 M in silver nitrate Column temperature: 45 Flow rate: 1.0

Injection volume: 10 Detector: UV 270 CHROMATOGRAM Retention time: 50.61 (estrone sulfate), 33.40 (equilin sulfate), 19.74 (17a-dihydroequilin sulfate), 14.17 (17p-dihydroequilin sulfate), 47.07 (17a-estradiol sulfate), 33.43 (17p-estradiol sulfate), 30.37 (equilenin sulfate), 23.28 (17a-dihydroequilenin sulfate) KEYWORDS tablets; injections REFERENCE
Flann, B.; Lodge, B. Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversedphase chromatography. J.Chromatogr., 1987, 402, 273-282

SAMPLE Matrix: formulations Sample preparation: Dissolve in mobile phase, inject an aliquot. HPLCVARIABLES Column: 250 X 4.6 5 |xm Ultrasphere Octyl Mobile phase: MeCN: MeOH: buffer 32:18:50 (Buffer was 1.7 mM cetyltrimethylammonium phosphate and 25 mM KH2PO4.) Flow rate: 0.9 Injection volume: 50 Detector: UV 200 CHROMATOGRAM Retention time: 58.33 (estrone sulfate), 53.08 (equilin sulfate), 45.50 (17a-dihydroequilin sulfate), 34.41 (17p-dihydroequilin sulfate), 50.75 (17a-estradiol sulfate), 39.66 (17p-estradiol sulfate), 48.41 (equilenin sulfate), 37.91 (17a-dihydroequilenin sulfate), 17.50 (estrone), 15.75 (equilin), 14.00 (17a-dihydroequilin), 11.08 (17p-dihydroequilin), 15.75 (17aestradiol), 13.42 (17-estradiol), 14.58 (equilenin sulfate), 9.92 (17p-dihydroequilenin) KEYWORDS tablets; injections REFERENCE
Flann, B.; Lodge, B. Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversedphase chromatography. J.Chromatogr., 1987, 402, 273-282

SAMPLE Matrix: formulations Sample preparation: Powder tablets, weigh out amount corresponding to 6.9 mg conjugated estrogens, add 6 g Celite 545, add 4 mL water, mix, add to a mixture of 2 g Celite and 1 mL water in a 150 X 25 tube, dry rinse container with 1 g Celite and add this to the tube, elute with 100 mL water-saturated ether, collect this eluate (A), elute with 5 mL 20 mg/mL dicyclohexylamine acetate in chloroform, elute with 145 mL chloroform (B). Combine the chloroform eluates and evaporate them to dryness under a stream of air on a steam bath, reconstitute with 20 mL MeOH, add 6 mL 5% HCl, reflux for 12 min, cool in an ice bath, add 5 mL 400 |xg/mL ethinyl estradiol in MeOH, add 70 mL water, add 50 mL benzene (Caution! Benzene is a carcinogen!), shake for 1 min. Remove the organic layer and wash it with 10 mL water, three 15 mL portions of 2% sodium carbonate solution, and two 10 mL portions of water. Pass the organic layer through 30 g anhydrous sodium sulfate in a column to give C. Evaporate a 2 mL aliquot of the solution (or an aliquot of eluate A) to dryness under a stream of air, reconstitute with 10 mL 200 |xg/mL

dansyl chloride in acetone, add 15 mL buffer, let stand in the dark for 30 min, add 50 mL water, add 50 mL ether, shake for several min, extract the aqueous layer with 25 mL ether. Combine the ether layers and wash them with two 25 mL portions of water, pass the organic layer through a 150 X 25 column containing 50 g anhydrous sodium sulfate, wash the column with 25 mL ether. Combine the eluates and evaporate them to dryness under a stream of air on a steam bath, reconstitute with 10 mL chloroform, inject an aliquot. (Under these conditions estrone, equilin, and equilenin co-elute. They can be reduced to p-estradiol, p-dihydroequilin, and p-dihydroequilenin, respectively, as follows. Evaporate a 10 mL aliquot of eluate (A) or solution (C) to dryness under a stream of air, reconstitute with 20 mL MeOH, add 150 mg sodium borohydride (Caution! Flammable hydrogen gas is evolved!), let stand for 45 min, add 70 mL water, add 50 mL benzene, shake for 1 min. Remove the organic layer and wash it with four 20 mL portions of water, pass through 30 g of anhydrous sodium sulfate in a 150 X 25 tube, evaporate a 10 mL aliquot to dryness and proceed with the derivatization as described above. (Prepare the buffer by dissolving 366.7 mg anhydrous sodium carbonate in 300 mL water and adding 150 mL acetone. Note that the initial elution with ether (A) gives free estrogens and the elution with chloroform (B) gives 3-sulfate derivatives which are then hydrolyzed.) HPLCVARIABLES Column: 250 X 4.6 5 |jim Zorbax-Sil Mobile phase: n-Heptane: chloroform: EtOH 50:49.5:0.5 Flow rate: 2 Injection volume: 10 Detector: F ex 240-420 (filter) em 440 (cutoff filter) CHROMATOGRAM Retention time: 4 (estrone), 4 (equilin), 4 (equilenin), 12.5 (a-estradiol), 15 (a-dihydroequilin), 16 (a-dihydroequilenin), 18 (p-estradiol), 19.5 (p-dihydroequilin), 22 O-dihydroequilenin) Internal standard: ethinyl estradiol (9) KEYWORDS normal phase; tablets; derivatization REFERENCE
Roos, R.W.; Lau-Cam, CA. Liquid chromatographic analysis of conjugated and esterified estrogens in tablets. J.Pharm.ScL, 1985, 74, 201-204

SAMPLE Matrix: formulations Sample preparation: Finely powder tablets. Weigh out an amount equivalent to 3 mg piperazine estrone sulfate, add 10 mL mobile phase containing 100 |xg/mL biphenyl, shake 30 min, inject 10 |xL aliquot. HPLCVARIABLES Column: 250 X 4.8 Brownlee RP-18 Mobile phase: MeCN: 20 mM pH 5.0 phosphate buffer 55:45 containing 3 mM cetyltrimethylammonium bromide Flow rate: 2 Injection volume: 10 Detector: UV 225 CHROMATOGRAM Retention time: k' 8.06 (estrone sulfate), k' 7.63 (equilin sulfate), k' 6.45 (a-estradiol sulfate), k' 5.30 (p-estradiol sulfate), k' 2.78 (estrone), k' 2.16 (a-estradiol), k' 1.95 (|Sestradiol) Internal standard: biphenyl (k' 11.04) Limit of detection: 1 |xg/mL

OTHER SUBSTANCES Simultaneous: methylparaben, propylparaben KEYWORDS tablets REFERENCE

Carignan, G.; Lodge, B.A.; Skakum, W. Analysis of piperazine estrone sulfate in tablets by ion-pair highperformance liquid chromatography. J.Chromatogr., 1982, 234, 240-243

SAMPLE Matrix: formulations Sample preparation: Powder tablets (60 mesh), take powder equivalent to about 3.2 mg conjugated estrogens, add 50 mL MeOH, shake 30 min, dilute to 100 mL with MeOH, mix, filter, discard first 20 mL filtrate, collect the rest of the filtrate (A). Take a 26 mL aliquot, add 1 mL HCl, add boiling chips, heat on a steam bath for 5 min, cool, add 70 mL water, extract with 75 mL benzene (Caution! Benzene is a carcinogen!). Wash the benzene layer with 15 mL water, four times with 15 mL 2% sodium carbonate in water, and twice with 10 mL water. Pass the benzene through a tube containing 30 g anhydrous sodium sulfate, wash the tube with 25 mL benzene, evaporate to dryness. Add 10 mL 200 |xg/mL dansyl chloride in acetone, swirl to dissolve, add 15 mL base solution, mix, stopper, allow to stand in the dark for 30 min. Extract twice with 50 mL ether, wash each extract twice with 25 mL water, pass the ether through a 150 X 25 mm tube containing 50 g anhydrous sodium sulfate, wash the column with 25 mL ether, evaporate the ether layers to dryness, dissolve residue in 5 mL chloroform, inject a 10 JULL aliquot. (Prepare base solution by dissolving 366.7 mg anhydrous sodium carbonate in 300 mL water and adding 150 mL acetone. Estrone, equilin, and equilenin co-elute. They can be reduced to p-estradiol, p-dihydroequilin, and p-dihydroequilenin, respectively as follows. Add 150 mg sodium borohydride to 25 mL filtrate (A) (Caution! Flammable hydrogen gas is evolved!), let stand for 45 min, add 1 mL HCl, let stand for 15 min, add 25 mL water, add 25 mL benzene, shake, wash the organic layer with four 20 mL portions of water, pass the organic layer through 25 g anhydrous sodium sulfate in a 150 X 25 tube. Evaporate a 1 mL aliquot to dryness under a stream of air and proceed with the derivatization as described above.) HPLCVARIABLES Column: 250 X 3.2 5 \xm LiChrosorb Si-60 Mobile phase: n-Heptane: chloroform 50:50 Flow rate: 0.98 Injection volume: 10 Detector: F ex 240-420 em 440 (cut-off) CHROMATOGRAM Retention time: 5 (estrone), 5 (equilin), 5 (equilenin), 14 (a-estradiol), 16 (a-dihydroequilin), 18 (a-dihydroequilenin), 20 (p-estradiol), 21 (p-dihydroequilin), 24 (p-dihydroequilenin) KEYWORDS tablets; normal phase; derivatization REFERENCE
Roos, R. W.; Medwick, T. Application of dansyl derivatization to the high pressure liquid chromatographic identification of equine estrogens. J.Chromatogr.ScL, 1980, 18, 626-630

SAMPLE Matrix: solutions

HPLCVARIABLES

Column: 120 X 4 5 |xm ODS-2 (Knauer) Mobile phase: MeCN: water 30:70 containing 16 mM p-cyclodextrin Column temperature: 40 Flow rate: 1 Injection volume: 20 Detector: UV 280
CHROMATOGRAM

Retention time: 3 (17p-estradiol), 5 (17a-estradiol), 6 (equilin)

REFERENCE
Lamparczyk, H.; Zarzycki, RK. Effect of temperature on separation of estradiol stereoisomers and equilin by liquid chromatography using mobile phases modified with p-cyclodextrin. J.Pharm. Biomed.AnaL, 1995, 13, 543-549

SAMPLE

Matrix: solutions Sample preparation: Inject a 2 J U L L aliquot of a 1 mg/mL solution in MeOH.

HPLCVARIABLES

Column: 150 X 4.6 5 \xm Zorbax ODS Mobile phase: MeOH: 50 mM KH2PO4 45:55 containing 5 mg/mL heptakis(2,6-di-Omethyl)- p-cyclodextrin Injection volume: 2 Detector: UV 200
CHROMATOGRAM

Retention time: 14 (equilin), 17 (estrone)

OTHER SUBSTANCES

Simultaneous: 2-hydroxyestrone, 4-hydroxyestrone, 16a-hydroxyestrone

REFERENCE
Spencer, B. J.; Purdy, W.C. High-performance liquid chromatographic separation of equilin, estrone, and estrone derivatives with cyclodextrins as mobile phase additives. J.Liq.Chromatogr., 1995, 18, 4063-4080

SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a solution in EtOH.

HPLC VARIABLES

Column: 150 X 4.6 5 |xm Spherisorb S5-0DS Mobile phase: Gradient. MeOH: 20 mM ammonium sulfate from 30:70 to 100:0 over 35 min. Column temperature: 45 Flow rate: 1 Injection volume: 50 Detector: F ex 214 em 340 (cut-off); UV 280
CHROMATOGRAM

Retention time: 5 (estriol-3-sulfate), 12 (estrone-3-sulfate), 13 (17p-estradiol-3-sulfate), 16 (estriol), 22 (estrone), 23 (17p-estradiol)

Next page

REFERENCE
Wei, J.Q.; Wei, J.L.; Zhou, X.T. Optimization of an isocratic reversed phase liquid chromatographic system for the separation of fourteen steroids using factorial design and computer simulation. Biomed.Chromatogr., 1990, 4, 34-38

SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a solution in mobile phase.

HPLCVARIABLES

Column: 250 X 4.6 7-8 |xm Zorbax BP-ODS Mobile phase: MeCN: water 35:65 Flow rate: 2 Injection volume: 50 Detector: UV 280
CHROMATOGRAM

Retention time: 32 (estrone), 28.5 (equilin), 25.5 (equilenin), 23 (17p-estradiol), 18.5 (17adihydroequilin), 16 (17a-dihydroequilenin)
KEYWORDS

also details of normal phase procedure

REFERENCE
Lin, J.-T.; Heftmann, E. High-performance liquid chromatography of naturally occurring estrogens. J.Chromatogr., 1981, 212, 239-244

SAMPLE

Matrix: urine Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL water, 5 mL MeOH, and 10 mL water. 1 mL Urine + 2 nmoles equilin + 100 JJIL 1.5 M pH 3 acetate buffer, add to the SPE cartridge, wash with 10 mL 150 mM pH 3 acetate buffer, elute with 3 mL MeOH. Add HCl to the eluate so that the concentration of HCl is 500 mM, heat at 100 for 1.5 h, neutralize with sodium bicarbonate, extract with 2 mL chloroform. Evaporate the organic layer to dryness, reconstitute with 1 mL 5 [xM 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole (NBD-Cl) in MeCN containing 25 nM 18-crown-6 and 15 mM potassium carbonate, heat at 80 for 30 min, filter, inject a 10-15 |xL aliquot.
HPLCVARIABLES

Column: 5 \xm Hypersil ODS Mobile phase: MeOH: water 75:25 Flow rate: 1 Injection volume: 10-15 Detector: UV 380
CHROMATOGRAM

Retention time: 6.50 (estrone), 8.39 (estradiol), 3.23 (estriol) Internal standard: equilin (4.95) Limit of detection: 30-50 nM
KEYWORDS

derivatization; SPE; derivatives are not fluorescent

REFERENCE
Tirendi, S.; Lancetta, T.; Bousquet, E. Estrogens determination in urine by RP-HPLC with UV detection. Farmaco, 1994, 49, 427-430