estrogens, conjugated

7/28/2019 Estrogens, Conjugated

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R = H, HSQ3N a

Conjugated Estrogens(see Preface)

SAMPLEMatrix: bloodSample p repara t ion : 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, D uPont), he at at 37 for 1 h, cool to room tem pe rat ur e,add 15 mL diethyl ether, shake mechanically at high speed for 10 min, centrifuge at 3033g for 10 min, freeze in dry ice/acetone. Remove the organic layer and evaporate it todryne ss un der a strea m of nitrogen, rec ons titute th e residu e in 500 (xL mobile ph ase ,filter (0.45 jjim), inject a 150JJLLaliquot of the filtrate.

HPLCVARIABLESColumn: 150 X 3 5 (xm C6 (Column Engineering, Ontario CA)Mobile phase: MeCN:MeOH:50 mM pH 3.5 ammonium acetate 27:8:65Flow rate: 0.35

Inject ion volume: 150De tector: F ex 210 em 370

CHROMATOGRAMReten t ion t ime: 19.5 (17a-dihydroequilenin)In ternal s tandard: 14p-equilenin (24)Limit of quanti tat ion: 2.5 ng/mL (rat), 5 ng/mL (rabbit, monkey)

OTHER SUBSTANCES

Nonin te r fe r ing : equilenin, 17a-dihydroequilenin

Estrogens, ConjugatedMolecular formula: C18H18O2 (equilenin), C18H22O2 (17a-dihydroequilin), C18H20O2 (equilin), C18H22O2 (es-trone), 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), 53-16-7 (estrone), 338-67-5 (estrone sodium sulfate), 481-97-0 (estrone hydrogen sulfate)

Sodium Estrone Sulfate Sodium Equilin SulfateEquilenin

17a-Dihydroequilin,Sodium Sulfate Conjugate

17|3-Dihydroequilin,

Sodium Sulfate Conjugate17a-Estradiol,Sodium Sulfate Conjugate


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Flow rate: 1Injection volume: 25Detector: UV 254

CHROMATOGRAMRetention time: 5.3

Internal standard: equileninLimit of detection: 5 ng/mL

OTHER SUBSTANCESSimultaneous: betamethasone, deoxycortisol, dexamethasone, hydrocortisone, prednisone,

triamcinolone

KEYWORDSAnaLAbs. 1982, 43, 4D182; plasma=uilenin is IS

REFERENCEBouquet, S.; Brisson, A.M.; Gombert, J. Dosage du cortisol et du 11-desoxycortisol plasmatiquespar

chromatographie liquide haute performance [Cortisoland 11-desoxycortisol determinationin bloodby high performance liquid chromatography].Ann.Biol.Clin.(Paris), 1981, 39, 189-191

SAMPLEMatrix: cellsSample 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, mixthoroughly for a few s, let stand at 4 for 5 min. Remove the organic layer and evaporateit to dryness under a stream of nitrogen, reconstitute the residue in three 500 |xL portionsof acetone, evaporate to dryness under a stream of nitrogen, reconstitute with 30 |xLMeCN, inject a 20 |xL aliquot.

HPLC VARIABLESColumn: Ultrasphere ODSMobile phase: MeCN: 10 mM citric acid 40:60Column temperature: 20 1Flow rate: 1Injection volume: 20Detector: UV 280; Radioactivity

CHROMATOGRAMRetention time: 14 (17p-estradiol), 16 (equilin), 19 (estrone)

KEYWORDStritium labeled; 14C labeled

REFERENCECastagnetta, L.A.; Granata, O.M.; Lo Casto, M.; Calabro, M.; Arcuri, F.; Carruba, G. Simple approach

to measure metabolic pathwaysof steroids in living cells.J .Chromatogr., 1991, 572, 2 5 - 3 9

SAMPLEMatrix: formulationsSample preparation: Dissolve a quantity equivalent to about 25 mg of conjugated estro-

gens in 20 mL MeOH, add 20 mL water, add 4 mL concentrated HCl, add several boilingchips, 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 drynessunder nitrogen, dissolve residue in 25 mL MeOH: water 1:1, inject aliquot.


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HPLCVARIABLESColumn: 250 X 4.6 3 ^m Nucleosil C18Mobile phase: MeOH: water: isopropanol: dichloromethane 45:42.5 :7.5:5Flow rate: 0.7Injection volume: 20Detector: UV 280; E, Laboratorni Pristroje ADLC 2 detector, carbon fiber working elec-

trode, stainless steel counter electrode, 1.1 V vs Ag/AgCl reference electrode, 0.6%Na2HPO4.12H2O added to mobile phase which was adjusted to pH6.0-6.05 with aceticacid

CHROMATOGRAMRetention 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

KEYWORDStablets; capsules

REFERENCENovakovic, 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 -3 63

SAMPLEMatrix: formulationsSample preparation: Dissolve in mobile phase, inject an aliquot.HPLC VARIABLESColumn: 150 X 3.9 4 |xm NovaPak C18Mobile phase: MeOH: 25 mM KH2PO4 40:60Flow rate: 0.8Injection volume: 50Detector: UV 200

CHROMATOGRAM

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

KEYWORDSinjections; tablets

REFERENCEFlan n, B.; Lodge, B. Analysis of estrogen s ulp hat e m ixture s in pharm aceu tical form ulations by reversed-

phase chromatography.J.Chromatogr., 1987, 402, 273-282

SAMPLEMatrix: formulationsSample preparation: Dissolve in mobile phase, inject an aliquot.

HPLCVARIABLESColumn: 250 X 4.6 5 jxm Ultrasphere OctylMobile ph ase: MeOH: water: trichloroethanol 23:75:2, all 0.1 M in silver nitra teColumn temperature: 45

Flow rate: 1.0


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In jec t ion vo lum e: 10Detector : UV 270

CHROMATOGRAMReten t ion t ime: 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-es-

tradiol sulfate), 30.37 (equilenin sulfate), 23.28 (17a-dihydroequilenin sulfate)

KEYWORDStablets; injections

REFERENCEFlan n, B.; Lodge, B. Analysis of estrogen su lpha te m ixture s in pharm aceu tical form ulations by reversed-

phase chromatography.J.Chromatogr., 1987, 402, 273 -282

SAMPLEMatrix: formulationsSample p repara t ion : Dissolve in mobile phase, inject an aliquot.

HPLCVARIABLESColumn: 250 X 4.6 5 |xm Ultrasphere OctylMobile phase: M eCN : M eOH : buffer 32 :18 :50 (Buffer w as 1.7 mM cetyltrimethy lamm on-

ium phosphate and 25 mM KH2PO4.)Flow rate: 0 .9In jec t ion vo lume: 50Detector : UV 200

CHROMATOGRAMReten t ion t ime: 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-es-tradiol sulfate), 48.41 (equilenin sulfate), 37.91 (17a-dihydroequilenin sulfate), 17.50 (es-trone), 15.75 (equilin), 14.00 (17a-dihydroequilin), 11.08 (17p-dihydroequilin), 15.75 (17a-estradiol), 13.42 (17-estradiol), 14.58 (equilenin sulfate), 9.92 (17p-dihydroequilenin)

KEYWORDS

tablets; injectionsREFERENCEFlan n, B.; Lodge, B. Analysis of estrogen su lph ate m ixtu res in pharm aceu tical form ulations by reversed-

phase chromatography.J.Chromatogr., 1987, 402, 273-282

SAMPLEMatrix: formulationsSample p repara t ion : Powder tablets, weigh out amount corresponding to 6.9 mg conju-

gated estrogens, add 6 g Celite 545, add 4 mL water, mix, add to a mixture of 2 g Celiteand 1 mL water in a 150 X 25 tube, dry rinse container with 1 g Celite and add this tothe tube, elute with 100 mL water-saturated ether, collect this eluate (A), elute with 5mL 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 ona steam bath, reconstitute with 20 mL MeOH, add 6 mL 5% HCl, reflux for 12 min, coolin an ice bath, add 5 mL 400 |xg/mL ethinyl estradiol in MeOH, add 70 mL water, add50 mL benzene (Caution! Benzen e is a carcinogen!), sha ke for 1 min. Remove the organiclayer and wash it with 10 mL water, three 15 mL portions of 2% sodium carbonate so-lution, and two 10 mL portions of water. Pass the organic layer through 30 g anhydroussodium sulfate in a column to give C. Evaporate a 2 mL aliquot of the solution (or an

aliquot of elua te A) to dryn ess u nd er a str eam of air, reco nstitu te w ith 10 mL 200 |xg/mL


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dansyl chloride in acetone, add 15 mL buffer, let stand in the dark for 30 min, add 50mL water, add 50 mL ether, shake for several min, extract the aqueous layer with 25 mLether. Combine the ether layers and wash them with two 25 mL portions of water, passthe 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 drynessunder 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 bereduced 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! Flammablehydrogen gas is evolved!), let stand for 45 min, add 70 mL water, add 50 mL benzene,sha ke for 1 min. Remove the organic layer and w ash 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 mLaliquot to dryness and proceed with the derivatization as described above. (Prepare thebuffer by dissolving 366.7 mg anhydrous sodium carbonate in 300 mL water and adding150 mL acetone. Note that the initial elution with ether (A) gives free estrogens and theelution with chloroform (B) gives 3-sulfate derivatives which are then hydrolyzed.)

HPLCVARIABLESColumn: 250 X 4.6 5 |jim Zorbax-SilMobile phase: n-H eptan e: chloroform: EtOH 50:49 .5:0.5Flow rate : 2In jec t ion vo lum e: 10Detec to r : F ex 240-420 (filter) em 440 (cutoff filter)

CHROMATOGRAMReten t ion t ime: 4 (estrone), 4 (equilin), 4 (equilenin), 12.5 (a-estradiol), 15 (a-dihydroe-

quilin), 16 (a-dihydroequilenin), 18 (p-estradiol), 19.5 (p-dihydroequilin), 22 O-dihydroe-quilenin)In ternal s tandard: ethinyl estradiol (9)

KEYWORDSnormal pha se; tablets; d erivatization

REFERENCERoos, R.W.; Lau-Cam, CA. Liquid chromatographic analysis of conjugated and esterified estrogens in

tablets. J.Pharm.ScL, 1985, 74, 201-204

SAMPLEMatrix: formulationsSample p repara t ion : Finely powder tablets. Weigh out an amount equivalent to 3 mg

piperaz ine es trone su lfate, add 10 mL mobile pha se con taining 100 |xg/mL biphenyl, s ha ke30 min, inject 10 |xL aliquot.

HPLCVARIABLESColumn: 250 X 4.8 Brownlee RP-18Mobile phase: M eCN: 20 mM pH 5.0 ph osp hate buffer 55 :45 co ntaining 3 mM cetyltri-

methylammonium bromideFlow rate : 2In ject ion volume: 10Detector : UV 225

CHROMATOGRAMReten t ion t ime: k' 8.06 (estrone sulfate), k' 7.63 (equilin sulfate), k' 6.45 (a-estradiol sul-

fate), k' 5.30 (p-estradiol sulfate), k' 2.78 (estrone), k' 2.16 (a-estradiol), k' 1.95 (|S-estradiol)

In ternal s tandard: biphenyl (k' 11.04)

Limit of detect ion: 1 |xg/mL


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OTHER SUBSTANCESSimul taneous : methylparaben, propylparaben

KEYWORDStablets

REFERENCECarig nan , G.; Lodge, B.A.; Skak um , W. Analy sis of pipera zine estrone sulfate in ta ble ts by ion-pair high-

performance liquid chromatography.J.Chromatogr., 1982, 234, 240-243

SAMPLEMatrix: formulationsSample p repara t ion : 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 th e re st of th e filtrate (A). Ta ke a 26 mL

aliquot, add 1 mL HCl, add boiling chips, heat on a steam bath for 5 min, cool, add 70mL water, extract with 75 mL benzene (Caution! Benzene is a carcinogen!). Wash thebenzene layer with 15 mL water, four times with 15 mL 2% sodium carbonate in water,and twice with 10 mL water. Pas s the benzene throug h a tube containing 30 g anhy droussodium sulfate, wash the tube with 25 mL benzene, evaporate to dryness. Add 10 mL 200|xg/mL dansyl ch loride in acetone, sw irl to dissolve, add 15 mL ba se solution , mix, stopper,allow to stand in the dark for 30 min. Extract twice with 50 mL ether, wash each extracttwice with 25 mL water, pass the ether through a 150 X 25 mm tube containing 50 ganhydrous sodium sulfate, wash the column with 25 mL ether, evaporate the ether layersto dryness, dissolve residue in 5 mL chloroform, inject a 10JULL aliquot. (Prepare basesolution by dissolving 366.7 mg anh ydro us sodium carb onate in 300 mL wat er an d add ing150 mL acetone. Estro ne, equilin, a nd equilenin co-elute. They can be reduced to p-estra-diol, p-dihydroequilin, and p-dihydroequilenin, respectively as follows. Add 150 mg so-dium borohydride to 25 mL filtrate (A) (Caution! Flammable hydrogen gas is evolved!),let stan d for 45 min, add 1 mL HC l, let stand for 15 min, add 25 mL water, add 25 mLbenzene, shake, wash the organic layer with four 20 mL portions of water, pass the or-ganic layer through 25 g anhydrous sodium sulfate in a 150 X 25 tube. Evaporate a 1mL aliquot to dryness under a stream of air and proceed with the derivatization as de-scribed above.)

HPLCVARIABLESColumn: 250 X 3.2 5 \xm LiChrosorb Si-60Mobile phase: n-H ep tan e: chloroform 50:50Flow rate : 0.98In ject ion volum e: 10Detec to r : F ex 240-420 em 440 (cut-off)

CHROMATOGRAMReten t ion t ime: 5 (estrone), 5 (equilin), 5 (equilenin), 14 (a-estradiol), 16 (a-dihydroe-

quilin), 18 (a-dihydroequilenin), 20 (p-estradiol), 21 (p-dihydroequilin), 24 (p-dihydroe-quilenin)

KEYWORDStablets; normal phase; derivatization

REFERENCERoos, R. W.; Medwick, T. Application of dansy l derivatization to the high pre ssure liquid ch roma tographic

identification of equine estrogens.J.Chromatogr.ScL, 1980, 18, 626-630

SAMPLE

Matrix: solutions


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

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

REFERENCELamparczyk, 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, 5 4 3 - 5 4 9

SAMPLEMatrix: solutionsSample preparation: Inject a 2 JULLaliquot of a 1 mg/mL solution in MeOH.

HPLCVARIABLESColumn: 150 X 4.6 5 \xmZorbax ODSMobile phase: MeOH: 50 mM KH2PO4 45:55 containing 5 mg/mL heptakis(2,6-di-O-

methyl)-p-cyclodextrinInjection volume: 2Detector: UV 200

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

OTHER SUBSTANCESSimultaneous: 2-hydroxyestrone, 4-hydroxyestrone, 16a-hydroxyestrone

REFERENCE

Spencer, B.J .; Purdy, W.C. High-performance liquid chrom atographic separa tion of equilin, estrone, andestrone derivatives with cyclodextrins as mobile phase additives.J.Liq.Chromatogr., 1995, 18,4063-4080

SAMPLEMatrix: solutionsSample preparation: Inject an aliquot of a solution in EtOH.

HPLC VARIABLES

Column: 150 X 4.6 5 |xm Spherisorb S5-0DSMobile phase: Gradient. MeOH: 20 mM ammonium sulfate from 30:70 to 100:0 over 35min.

Column temperature: 45Flow rate: 1Injection volume: 50Detector: F ex 214 em 340 (cut-off); UV 280

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

(estriol), 22 (estrone), 23 (17p-estradiol)


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REFERENCEWei, J .Q.; Wei, J .L.; Zhou, X.T. Op timizatio n of an isocratic reve rsed p ha se liquid chrom atograp hic

system for the separation of fourteen steroids using factorial design and computer simulation.Bio-med.Chromatogr., 1990, 4, 3 4 - 3 8

SAMPLEMatrix: solutionsSample preparation: Inject an aliquot of a solution in mobile phase.

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

CHROMATOGRAMRetention time: 32 (estrone), 28.5 (equilin), 25.5 (equilenin), 23 (17p-estradiol), 18.5 (17a-

dihydroequilin), 16 (17a-dihydroequilenin)

KEYWORDSalso details of normal phase procedure

REFERENCELin, J.-T.; Heftmann, E. High-performance liquid chrom atography of natu rally occurring estrogens.

J.Chromatogr., 1981, 212, 239-244

SAMPLEMatrix: urineSample 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 + 100JJIL 1.5 M pH 3 acetatebuffer, add to the SPE cartridge, wash with 10 mL 150 mM pH 3 acetate buffer, elutewith 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-nitro-

benzo-2-oxa-l,3-diazole (NBD-Cl) in MeCN containing 25 nM 18-crown-6 and 15 mM po-tassium carbonate, heat at 80 for 30 min, filter, inject a 10-15 |xL aliquot.

HPLCVARIABLESColumn: 5 \xmHypersil ODSMobile phase: MeOH: water 75:25Flow rate: 1Injection volume: 10-15Detector: UV 380

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

KEYWORDSderivatization; SPE; derivatives are not fluorescent

REFERENCETirendi, S.; Lan cetta, T.; Bousq uet, E. Estrogens determ ination in urine by RP-HPLC with UV detection.

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