Estradiol

Molecular formula: C18H24O2 Molecular weight: 272.4 CAS Registry No.: 50-28-2, 113-38-2 (dipropionate), 979-32-8 (valerate), 57-91-0 (a-estradiol), 50-50-0 (benzoate), 313-06-4 (cypionate), 4956-37-0 (enanthate), 3571-53-7 (undecylenate)

SAMPLE Matrix: blood Sample preparation: Inject 10 jxL plasma into MeCN pumped at 0.2 mL/min so that the precipitated proteins are removed by 0.5 and 0.2 fxm filters in series. Swith the MeCN containing sample into the mobile phase and allow it to pass onto the analytical column, elute the analytical column in the usual way with mobile phase. Remove the filter unit from the circuit and back-flush it to waste with 100 mM sodium dodecyl sulfate at 2 mL/min, 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 Mobile phase: MeCN: water 33:67

Flow rate: 1 Injection volume: 10 Detector: UV 280

CHROMATOGRAM Retention time: 25.8 OTHER SUBSTANCES Simultaneous: equilin, 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: 1 mL Plasma + 500 jxL 10 M NaOH, shake on a slow rotatory mixer for 5 min, add 5 mL diethyl ether, rotomix 10 min, centrifuge at 700 g for 5 min, repeat extraction. Combine organic layers, evaporate to dryness under a stream of nitrogen at 37, dissolve in 250 |xL mobile phase, inject aliquot. HPLCVARIABLES Column: 150 X 3.9 4 jxm Novapack C18 Mobile phase: MeCN.MeOH.buffer 35:15:50 (Buffer was 50 mM KH2PO4 adjusted to pH 3.6 with phosphoric acid.)

Flow rate: 1.6

Injection volume: 50
D e t e c t o r : E , W a t e r s M o d e l 4 6 4 p u l s e d e l e c t r o c h e m i c a l d e t e c t o r , - H l Vv e r s u s A g / A g C l

CHROMATOGRAM Retention time: 2.44 Limit of detection: 50 pg/mL OTHER SUBSTANCES Simultaneous: estriol, estrone, ethinylestradiol, heparin Noninterfering: pentobarbital KEYWORDS plasma; rabbit REFERENCE
Fernandez, N.; Garcia, J.J.; Diez, M.J.; Teran, M.T.; Sierra, M. Rapid high-performance liquid chromatographic assay of ethynyloestradiol in rabbit plasma. J.Chromatogr., 1993, 619, 143 147

SAMPLE Matrix: blood Sample preparation: 100 [xL Serum + 500 |xL water + 100 jxL 10 |xg/mL 3,7-dimethoxyflavone in EtOH + 8 mL diethyl ether, shake, centrifuge at 4 at 1000 g for 5 min, freeze in acetone/dry ice. Remove the organic layer and dry it over anhydrous sodium sulfate, evaporate to dryness under a stream of nitrogen, reconstitute the residue in 100 jxL MeOH: water 40:60, inject a 50 |xL aliquot. HPLCVARIABLES Column: 250 X 4.6 3 ^m NS-GeI C18 Mobile phase: Gradient. MeOH: water from 40:60 to 55:45, maintain at 55:45 for 24 min, to 80:20 over 25 min. Column temperature: 50 Flow rate: 1 Injection volume: 50 Detector: UV 210; UV 240 CHROMATOGRAM Retention time: 30.74 Internal standard: 3,7-dimethoxyflavone (47) OTHER SUBSTANCES Extracted: aldosterone, androstenedione, dehydroepiandrosterone, deoxycorticosterone, 11-deoxycortisol, estrone, hydrocortisone, 17-hydroxyprogesterone, pregnenolone, progesterone KEYWORDS serum REFERENCE
Ueshiba, H.; Segawa, M.; Hayashi, T.; Miyachi, Y; Irie, M. Serum profiles of steroid hormones in patients with Cushing's syndrome determined by a new HPLC/RIA method. Clin.Chem., 1991, 37, 13291333

SAMPLE Matrix: blood

Sample preparation: Extract 1 mL serum twice with 5 volumes ether by vortexing for 2 min, evaporate extracts to dryness under a stream of nitrogen at 35, reconstitute in 100 JJIL MeOH.
HPLCVARIABLES

Column: 240 X 4.5 Bio-Rad ODS-5S Mobile phase: Gradient. MeOH:MeCN.water at 20:60:20 for 3 min then to 5:85:10 over 26 min. Flow rate: 1 Injection volume: 50 Detector: UV 230
OTHER SUBSTANCES

Simultaneous: androstenedione, progesterone, testosterone

KEYWORDS

serum
REFERENCE
Yu, F.H.; Yun, Y.W.; Yuen, B.H.; Moon, YS. Effects of hydroxyflutamide on rats treated with a superovulatory dose of pregnant mare serum gonadotropin. Can.J.Physiol.Pharmacol., 1991, 69, 185-190

SAMPLE

Matrix: blood Sample preparation: 0.5-1 mL Plasma + 1 mL 500 mM pH 7 phosphate buffer + 12 mL hexane: ethyl acetate 70:30, extract. Remove a 10 mL aliquot of the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 100 J U L L mobile phase, inject a 20-50 |JLL aliquot. (Hydrolyze 500 |xL plasma by adding 500 |JLL 200 mM pH 5 acetate buffer and 100 pX beef liver p-glucuronidase (Sigma) or 10 J U L L pglucuronidase/sulfatase (Glusulase), heat at 37 overnight, add 1 mL 500 mM pH 7 phosphate buffer + 12 mL hexane:ethyl acetate 70:30, extract. Remove a 10 mL aliquot of the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 100 ]xL mobile phase, inject a 20-50 JJLL aliquot.)
HPLCVARIABLES

Column: 250 X 4.6 5 p,m Partisil 5/25 silica gel Mobile phase: Hexane:EtOH 92.5:7.5 Flow rate: 1.5 Injection volume: 20-50 Detector: F ex 195 em 250 (cut-off filter)
CHROMATOGRAM

Retention time: 8.9 Limit of detection: 3 ng/mL

OTHER SUBSTANCES

Extracted: metabolites, estramustine, estromustine, estrone

KEYWORDS

plasma; rat; dog; human; pharmacokinetics; normal phase

REFERENCE
Dixon, R.; Brooks, M.; Gill, G. Estramustine phosphate: Plasma concentrations of its metabolites following oral administration to man, rat and dog. Res.Commun.Chem.Pathol.PharmacoL, 1980, 27, 17-29

SAMPLE

Matrix: blood, tissue Sample preparation: 1 mL Blood or brain + 1 mL 50 mM ammonium acetate buffer, homogenize (Polytron PT-1200C), add 4 mL MeCN, vortex, add 1 mL concentrated brine, allow to stand at -5 for 1 h. Remove the organic phase and centrifuge it at 3000 g, filter, inject an aliquot.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm Spherisorb C8 Mobile phase: MeCN: 50 mM pH 6.8 ammonium acetate 52:48 containing 10 mM tetraethylammonium perchlorate Flow rate: 1 Detector: UV 280
CHROMATOGRAM

Retention time: 3.8 Limit of detection: 133 ng/g

KEYWORDS

whole blood; rat; brain

REFERENCE
Brewster, M.E.; Druzgala, P.J.; Anderson, W.R.; Huang, M.-J.; Bodor, N.; Pop, E. Efficacy of a 3-substituted versus 17-substituted chemical delivery system for estradiol brain targeting. J.Pharm.Sci., 1995, 84, 38-43

SAMPLE

Matrix: culture medium Sample preparation: Extract culture medium twice with 2 volumes of ether, combine the extracts and evaporate them to dryness, reconstitute with MeOH, inject an aliquot.
HPLCVARIABLES

Column: 300 X 3.9 Techopak 10 C18 (HPLC Technology) Mobile phase: MeOH: 0.5% pH 3.0 (NH4)H2PO4 62:39 Flow rate: 0.7 Detector: UV 280; Radioactivity
CHROMATOGRAM

Retention time: 20 OTHER SUBSTANCES Extracted: estrone

KEYWORDS

tritium labeled
REFERENCE
Wild, M.J.; Rudland, P.S.; Back, D.J. Metabolism of the oral contraceptive steroids ethynylestradiol and norgestimate by normal (Huma 7) and malignant (MCF-7 and ZR-75-1) human breast cells in culture. J.Steroid Biochem.MoLBioL, 1991, 39, 535-543

SAMPLE

Matrix: microsomal incubations Sample preparation: 1 mL Microsomal incubation + 5 mL ethyl acetate, vortex, centrifuge at 2000 g for 8 min, remove organic phase, repeat extraction. Combine the organic layers

and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeOH.water 50:50, inject a 50 JULL aliquot. HPLCVARIABLES Column: 150 X 4.6 5 \xm Ultracarb 30 ODS (Phenomenex) Mobile phase: Gradient. MeCN: 0.1% acetic acid in MeOH: 0.1% acetic acid in water 16: 12:72 for 3 min, to 20:21:59 over 25 min (Waters no. 3 convex gradient), to 24:23:53 over 10 min (linear), to 55:24:21 over 10 min (linear), to 92:5:3 over 1 min, maintain at 92:5:3 for 7 min, return to initial conditions over 15 min. Flow rate: 1.2 Injection volume: 50 Detector: UV 280 CHROMATOGRAM Retention time: 46 OTHER SUBSTANCES Extracted: metabolites, estrone KEYWORDS

rat
REFERENCE
Suchar, L.A.; Chang, R.L.; Rosen, R.T.; Lech, J.; Conney, A.H. High-performance liquid chromatography separation of hydroxylated estradiol metabolites: Formation of estradiol metabolites by liver microsomes from male and female rats. J.Pharmacol.Exp.Ther., 1995, 272, 197-206

SAMPLE Matrix: microsomal incubations Sample preparation: 1 mL Human placental microsome suspension + 1 mL dichloromethane, extract, centrifuge, remove organic layer and evaporate it under vacuum, dissolve residue in 30 fxL MeCN: water 50:50, centrifuge for 3 min, inject supernatant. After each run wash column with MeCN for 1 min, re-equilibrate for 1 min. HPLCVARIABLES Column: 50 X 4.6 3 jjim Spherisorb ODS-2 Mobile phase: MeCN: water 50:50 Column temperature: 60 Flow rate: 2 Injection volume: 30 Detector: UV 200 CHROMATOGRAM Retention time: 0.6 Limit of detection: <0.1 nmol/mL OTHER SUBSTANCES Simultaneous: androstenedione, estrone, testosterone KEYWORDS human; placenta REFERENCE
Taniguchi, H.; Feldmann, H.R.; Kaufmann, M.; Pyerin, W. Fast liquid chromatographic assay of androgen aromatase activity. Anal.Biochem., 1989, 181, 167-171

SAMPLE Matrix: solutions

HPLCVARIABLES

Column: 150 X 4.6 5 (xm Ultrasphere Mobile phase: MeCN: EtOH: water 54:1:45 Flow rate: 1.5 Detector: UV 270
CHROMATOGRAM

Retention time: 2.4 (17p-estradiol)

REFERENCE
Fridriksdottir, H.; Loftsson, T.; Gudmundsson, J.A.; Bjarnason, G.J.; Kjeld, M.; Thorsteinsson, T. Design and in vivo testing of 17(3-estradiol-HPpCD sublingual tablets. Pharmazie, 1996, 51, 39-42

SAMPLE Matrix: solutions

HPLCVARIABLES

Column: 250 X 4.6 5 |xm Nucleosil phenyl Mobile phase: Gradient. Carbon dioxide: MeOH from 98:2 to 78:22 over 40 min. Column temperature: 50 Flow rate: 2 Detector: UV
CHROMATOGRAM

Retention time: 10.3

OTHER SUBSTANCES

Simultaneous: other steroids, estriol, hydrocortisone, hydroxyprogesterone, norethisterone, testosterone

KEYWORDS

SFC; 200 bar

REFERENCE
Hanson, M. Aspects of retention behaviour of steroids in packed column supercritical fluid chromatography. Chromatographia, 1995, 40, 58-68

SAMPLE

Matrix: solutions Sample preparation: Prepare an aqueous solution, inject a 20 (xL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 3.5 |xm Zorbax SB C18 Mobile phase: MeCN: MeOH: buffer 15:45:40 (Buffer was 10 mM KH2PO4 and 50 mM tetrabutylammonium chloride, pH adjusted to 3.0 with 1 M HCl.) Flow rate: 0.9 Injection volume: 20 Detector: UV 220
CHROMATOGRAM

Retention time: 9.6 (17p-estradiol), 6.9 (17p-estradiol-3-phosphate)

OTHER SUBSTANCES Simultaneous: estriol, estrone, estrone-3-phosphate KEYWORDS stability-indicating (for 17(3-estradiol-3-phosphate) REFERENCE
Miller, R.B.; Chen, C. A stability-indicating HPLC method for the determination of 17p-estradiol-3phosphate in an ophthalmic solution. Chromatographia, 1995, 40, 204-206

SAMPLE Matrix: solutions Sample preparation: Prepare a solution in n-propanol: water 80:20 or DMF: water 80:20, inject an aliquot. HPLCVARIABLES Column: 250 X 4 5 jxm LiChrospher 100 Diol Mobile phase: Gradient. A was hexane. B was ethyl acetate. C was 0.1% formic acid in MeCN. D was 0.1% formic acid in water. A:B:C:D 100:0:0:0 for 5 min, to 0:100:0:0 over 15 min, maintain at 0:100:0:0 for 5 min, to 0:0:100:0 over 5 min, maintain at 0: 0:100:0 for 5 min; to 0:0:0:100 over 25 min, maintain at 0:0:0:100 for 5 min. Flow rate: 0.9 Detector: ELSD (Sedex 55, Sedere) CHROMATOGRAM Retention time: 18.22 OTHER SUBSTANCES Simultaneous: acetylcholine, cholesterol, choline, cortisone, dextrose, glycine, phenylalanine, testosterone REFERENCE
Treiber, L.R. Normal-phase high-performance liquid chromatography with relay gradient elution. I. Description of the method. J.Chromatogr.A, 1995, 696, 193-199

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210 OTHER SUBSTANCES Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphe-

nesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine REFERENCE
Hill, D. W.; Kind, A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J.Anal.ToxicoL, 1994, 18, 233-242

SAMPLE Matrix: solutions Sample preparation: Prepare a 25 jxg/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 280 CHROMATOGRAM Retention time: k' 3.462

OTHER SUBSTANCES Also analyzed: androsterone (UV 210), cortexolone (UV 240), cortisone (UV 240), estrone (UV 280), ethinyl estradiol (UV 280), ethisterone (UV 240), hydrocortisone (UV 240), hydroxyprogesterone (UV 240), lynestrenol (UV 210), medroxyprogesterone (UV 240), medroxyprogesterone acetate (UV 240), methandienone (UV 240), methylandrostenediol (UV 210), methylprednisolone (UV 240), methylprednisolone acetate (UV 240), methyltestosterone (UV 240), nandrolone (UV 240), norethisterone (UV 240), prednisolone (UV 240), prednisolone acetate (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 Sample preparation: Inject an aliquot of a solution in MeOH. HPLCVARIABLES Column: Radial-PAK jxBondapak C18 Mobile phase: MeCN: water 50:50 Flow rate: 2 Injection volume: 100 Detector: UV 254; UV 214 CHROMATOGRAM Retention time: 5.6 OTHER SUBSTANCES Simultaneous: estriol, estrone, progesterone Interfering: estradiol, testosterone REFERENCE
Erkoc, RU.; Ozsar, S.; Giiven, B.; Kalkandelen, G.; Ugrar, E. High-performance liquid chromatographic analysis of steroid hormones. J.Chromatogr.Sci., 1989, 27, 8690

SAMPLE Matrix: solutions Sample preparation: Dissolve in MeOH: water 1:1 at a concentration of 50 |xg/mL, inject a 10 fxL aliquot. HPLCVARIABLES Column: 300 X 3.9 10 |xm piBondapak C18 Mobile phase: MeOH: acetic acid: triethylamine: water 80:1.5:0.5:18 Flow rate: 1.5 Injection volume: 10 Detector: UV CHROMATOGRAM Retention time: k' 0.41 (estradiol), k' 3.53 (estradiol benzoate), k' 7.45 (estradiol cypionate), k' 3.49 (estradiol valerate) 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: solutions Sample preparation: Prepare a solution in EtOH, inject an aliquot.

HPLCVARIABLES

Column: 150 X 4.6 5 ^m 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: 13 (17p-estradiol-3-sulfate), 23 (17p-estradiol)

OTHER SUBSTANCES

Simultaneous: estriol, estriol-3-sulfate, estrone, estrone-3-sulfate

REFERENCE
Simonian, M.H.; Capp, M.W. Reversed-phase high-performance liquid chromatography of steroid 3-sulfates and the corresponding unconjugated steroids. J.Chromatogr., 1984, 287, 97104

SAMPLE

Matrix: tissue Sample preparation: Incubate endometrial tissue with buffer, remove tissue, extract medium twice with 2 volumes of diethyl ether, evaporate to dryness, reconstitute in a small volume of MeOH, inject an aliquot.
HPLCVARIABLES

Column: 300 X 3.9 Technopak 10 C18 Mobile phase: MeOH: 0.5% pH 3.0 (NH4)H2PO4 62:38 Flow rate: 0.7 Detector: UV 280
CHROMATOGRAM

Retention time: 25
OTHER SUBSTANCES

Simultaneous: estrone
KEYWORDS

endometrial tissue
REFERENCE
Wild, M.J.; Rudland, RS.; Back, D.J. Metabolism of the oral contraceptive steroids ethynylestradiol, norgestimate and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-IA) and endometrial tissue in vitro. J.Steroid Biochem.Mol.BioL, 1993, 45, 407-420

SAMPLE

Matrix: tissue Sample preparation: Dry pack 60 X 8 mm glass columns with 250 mg Carbopack B (200400 mesh) and 60 X 4 mm glass columns with 50 mg Amberlite CG-400 I (100-200 mesh). Wash Carbopack column with 5 mL MeOH, 15 mL dichloromethane: MeOH 70:30, and MeOH: water 85:15. Wash Amberlite column with 3 mL 0.5 M NaOH, 8 mL

dichloromethane: MeOH 70:30, 1 mL water, and 3 mL 1 M HCl. Repeat this cycle 4 times. Finally pass through 20 mL 50 mM NaOH then 1 mL water. Keep column in water. (Process converts Amberlite to OH form.) Homogenize 1 g of tissue in 5 mL MeOH, sonicate 5 min, centrifuge at 6000 rpm for 10 min. Add another 5 mL MeOH to pellet and repeat. Combine supernatants, make up to 6.8 mL with MeOH, add 1.2 mL water. Pass through Carbopack column, wash column with 2 mL MeOH: water 85:15 then 2 mL MeOH, elute column with 8 mL dichloromethane: MeOH 70:30. Pass eluate onto Amberlite column, wash with 1 mL MeOH, 1 mL 1 M HCl, elute with 2 mL 30 mM HCl in MeCN: MeOH 20:80. Evaporate eluate to dryness with nitrogen at 40, take up in 100 JJLL MeCN:MeOH:THF: 10 mM KH2PO4 adjusted to pH 3.0 with phosphoric acid 22:8: 13:57, inject 40 JXL aliquot HPLC VARIABLES Guard column: 20 X 4.6 5 ^m Supelguard LC-18 Column: 250 X 4.6 5 \xm Supelco C18 Mobile phase: MeCN: 10 mM KH2PO4 adjusted to pH 3.0 with phosphoric acid 46:54 Flow rate: 1.2 Injection volume: 40 Detector: F ex 280 em 308 CHROMATOGRAM Retention time: 7 Limit of detection: 1 ng/g KEYWORDS muscle; liver; chicken; ox; cow REFERENCE
Lagana, A.; Marino, A. General and selective isolation procedure for high-performance liquid chromatographic determination of anabolic steroids in tissues. J.Chromatogr., 1991, 588, 89-98

SAMPLE Matrix: urine Sample preparation: 50 mL Urine + 7 mL concentrated HCl, heat at 90 for 1 h, add 10 jxL 1 mg/mL 4-phenylphenol in MeOH, extract 3 times with 10 mL diethyl ether, combine organic phases, wash twice with 20 mL portions of pH 10.5 NaHCO3/NaOH buffer, wash with 20 mL water, dry over 5 g anhydrous sodium sulfate. Filter, evaporate under reduced pressure almost to dryness, take up residue in 1 mL mobile phase, inject 20 |xL aliquot. HPLCVARIABLES Column: 250 X 4.5 Beckman ODS Mobile phase: MeCN: water 25:75 containing 14 mM p-cyclodextrin Column temperature: 40 Flow rate: 1 Injection volume: 20 Detector: UV 280; F ex 280 em 312 CHROMATOGRAM Retention time: 8.1 Internal standard: 4-phenylphenol Limit of detection: 1-3 ng/mL OTHER SUBSTANCES Simultaneous: estriol, estrone

REFERENCE
Lamparczyk, H.; Zarzycki, RK.; Nowakowska, J.; Ochocka, R.J. Application of (3-cyclodextrin for the analysis of estrogenic steroids in human urine by high-performance liquid chromatography. Chromatographia, 1994, 38, 168-172

ANNOTATED BIBLIOGRAPHY

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