Betamethasone

Molecular formula: C22H29FO5 Molecular weight: 392.5 CAS Registry No.: 378-44-9, 987-24-6 (acetate), 22298-29-9 (benzoate), 5593-20-4 (dipropionate), 151-73-5 (sodium phosphate), 2152-44-5 (17-valerate), 5534-05-4 (acibutate), 360-63-4 (dihydrogen phosphate)

SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 100 |xL water containing 5 jxg/mL 2,3-diaminonaphthalene and 3.5 |xg/mL 18-hydroxy-ll-deoxycorticosterone + 1 mL 250 mM NaOH + 7 mL diethyl ether, shake on a rotary shaker for 15 min, repeat extraction. Combine the organic layers and evaporate them to dryness under a stream of nitrogen at 30-40, reconstitute the residue in 70 jxL MeOH: 100 mM perchloric acid 50:50, inject a 20 |xL aliquot. HPLCVARIABLES Column: 150 X 3.9 4 |xm Nova-Pak C18 Mobile phase: Gradient. A was 58 mM NaH2PO4 containing 6 mM sodium heptanesulfonate, adjusted to pH 3.1 with concentrated phosphoric acid. B was MeCN: MeOH 85:15. A:B from 100:0 to 78:22 over 5 min, to 70:30 over 12 min, maintain at 70:30 for 4 min, to 65:35 over 9 min. Flow rate: 1 Injection volume: 20 Detector: UV 245; UV 256; UV 343 CHROMATOGRAM Retention time: 19.45 Internal standard: 2,3-diaminonaphthalene (10.71), 18-hydroxy-ll-deoxycorticosterone (15.85) Limit of detection: 1-10 ng/mL (245 nm) OTHER SUBSTANCES Extracted: chloroquine, corticosterone, cortisone, dexamethasone, fluocinolone acetonide, fluendrenolide, fhiorometholone, fluprednisolone, hydrocortisone, hydroxychloroquine, 178-hydroxyprogesterone, meprednisone, methylprednisolone, methylprednisolone acetate, paramethasone, prednisolone, prednisone, progesterone, triamcinolone Noninterfering: aspirin, ibuprofen, indomethacin, phenylbutazone, pregnenolone KEYWORDS serum REFERENCE
Volin, P. Simple and specific reversed-phase liquid chromatographic method with diode-array detection for simultaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids. J.Chromatogr.B, 1995, 666, 347-353

SAMPLE Matrix: blood Sample preparation: Prepare a Sep-Pak Plus Environmental C18 cartridge by washing with 15 mL MeOH then 15 mL water. 1 mL Serum + 200 |xL isopropanol: acetonitrile 1:1, mix, add to SPE cartridge, wash with 10 mL water, elute with 3 mL MeOH. Evap-

orate the eluate at 50 under a stream of nitrogen, reconstitute in 200 |JLL mobile phase A, inject a 20 |JLL aliquot. HPLC VARIABLES Guard column: jxBondapak C18 guard column Column: 250 X 4.6 5 |xm Hypersil ODS Mobile phase: Gradient. A was isopropanol: 50 mM pH 4.5 acetate buffer 10:90. B was isopropanol:50 mM pH 4.5 acetate buffer 30:70. A:B from 90:10 to 30:70 over 25 min, hold at 30:70 for 5 min, to 90:10 over 5 min, hold at 90:10 for 15 min before next injection. Column temperature: 40 Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 33 Internal standard: betamethasone OTHER SUBSTANCES Simultaneous: metabolites, cortisone, hydrocortisone, prednisolone, prednisone KEYWORDS serum; SPE; betamethasone is IS REFERENCE
Hirata, H.; Kasama, T.; Sawai, Y.; Fike, R.R. Simultaneous determination of deflazacort metabolites II and III, cortisol, cortisone, prednisolone and prednisone in human serum by reversed-phase highperformance liquid chromatography. J.Chromatogr.B, 1994, 658, 55-61

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 1 5 mL dichloromethane, shake horizontally for 15 min, centrifuge at 1500 g for 15 min. Remove the organic layer and wash it with 100 |xL 100 mM NaOH then 1 mL water. Remove the aqueous phase and dry the organic phase over 1 g of anhydrous sodium sulfate. Evaporate the organic phase to dryness under a stream of nitrogen at not more than 37, reconstitute in 200 jxL mobile phase, inject a 175 |xL aliquot. HPLCVARIABLES Guard column: 20 X 2 30-38 ynn HC Pellosil Column: 250 X 4.6 5-6 p,m Zorbax SIL Mobile phase: Heptane: dichloromethane: glacial acetic acid: ethanol 350:600:10:35 Flow rate: 2 Injection volume: 175 Detector: UV 254 CHROMATOGRAM Retention time: 12 Internal standard: betamethasone OTHER SUBSTANCES Extracted: hydrocortisone, prednisolone, prednisone Noninterfering: cyclosporin, ethinyl estradiol, ketoconazole, levonorgestrel, rapamycin, tacrolimus, tenidap, tetrahydrocortisone

KEYWORDS plasma; normal phase; betamethasone is IS REFERENCE

Jusko, W.J.; Pyszczynski, N.A.; Bushway, M.S.; D'Ambrosio, R.; Mis, S.M. Fifteen years of operation of a high-performance liquid chromatographic assay for prednisolone, cortisol and prednisone in plasma. J.Chromatogr.B, 1994, 658, 47-54

SAMPLE Matrix: blood Sample preparation: Add 1 mL serum to a Sep Pak C18 SPE cartridge, wash with 4 mL water, elute with 4 mL MeOH, evaporate to dryness under vacuum, reconstitute in 50 \xL MeCN: water 30:70, inject whole sample. HPLCVARIABLES Column: 250 X 4.6 5 |xm Ultrasphere ODS Mobile phase: MeCN: water 30:70 Flow rate: 1 Injection volume: 50 Detector: enzyme immunoassay of fractions CHROMATOGRAM Retention time: 16 Limit of detection: 0.3 pg OTHER SUBSTANCES Extracted: dexamethasone, flumethasone, triamcinolone Noninterfering: endogenous steroids KEYWORDS serum; SPE; horse REFERENCE
Friedrich, A.; Schulz, R.; Meyer, H.H. Use of enzyme immunoassay and reverse-phase high-performance liquid chromatography to detect and confirm identity of dexamethasone in equine blood. Am.J.Vet.Res., 1992, 53, 2213-2220

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 5 mL water + 1 mL 2 |xg/mL equilenin in MeOH + 50 |xL 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 40 under a stream of nitrogen, reconstitute residue in 150 |xL mobile phase, inject 25 |JLL aliquot. HPLCVARIABLES Column: 300 X 4 10 ^m jxBondapak C18 Mobile phase: MeOH: buffer 60:40 (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: 5.5

Internal standard: equilenin (7.5) Limit of detection: 5 ng/mL

OTHER SUBSTANCES

Simultaneous: deoxycortisol, hydrocortisone, prednisolone, prednisone, triamcinolone Interfering: dexamethasone

KEYWORDS

Anal. Abs. 1982, 43, 4D182; plasma

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: formulations Sample preparation: Ointment. Add pentane:EtOH 75:25 to ointment, sonicate for 20 min, dilute an aliquot to 100 mL with MeOH, allow to settle. Centrifuge and filter an aliquot of the supernatant, inject an aliquot of the nitrate. Cream, lotion. Stir cream or lotion in EtOH: THF: water 25:25:50 at 40 for 15 min, cool in an ice bath. Centrifuge and filter an aliquot of the supernatant, inject an aliquot of the filtrate. Gel. Dissolve gel in EtOH, sonicate, filter, inject an aliquot.
HPLC VARIABLES

Guard column: present but not specified Column: 250 X 2.1 10 |xm Bondapak C18 Mobile phase: MeCN: water 48:52 containing 0.65% acetic acid, pH 3.18 (At the end of each day flush guard column only with MeOH: THF 75:25 for 30 min.) Flow rate: 1 Injection volume: 20 Detector: UV 251
CHROMATOGRAM

Retention time: 5.17 (betamethasone-17-valerate)

OTHER SUBSTANCES

Simultaneous: bamipine lactate, beclomethasone dipropionate, dexamethasone, hydrocortisone-21 -acetate

KEYWORDS

ointment; creams; lotions; gels

REFERENCE
Kountourellis, J.E.; Markopoulou, C.K.; Ebete, K.O.; Stratis, J.A. Separation and determination of some corticosteroids combined with bamipine in pharmaceutical formulations by high performance liquid chromatography. J.Liq.Chromatogr., 1995, 18, 3507-3517

SAMPLE

Matrix: formulations Sample preparation: Pulverize tablets, weigh out amount equivalent to about 500 |xg betamethasone, add 10 mL water, sonicate for 15 min, extract three times with 15 mL chloroform: n-butanol 95:5. Combine extracts and filter them through 1 g anhydrous sodium sulfate moistened with chloroform: n-butanol 95:5. Collect filtrate and dilute it to 50 mL with chloroform: n-butanol 95:5. Remove a 1 mL aliquot, add 0.5 mL 40 JJLM cortisone in mobile phase, mix, inject a 5 JJLL aliquot.

HPLCVARIABLES

Guard column: 5 |xm Guard-Pak Resolve Si (dead volume 60-75 uX.) Column: 75 X 3.9 4 (xm Nova-Pak silica Mobile phase: Dichloromethane: EtOH 34:1 Flow rate: 0.7 Injection volume: 5 Detector: UV 240
CHROMATOGRAM

Retention time: 6 Internal standard: cortisone (3)

OTHER SUBSTANCES

Simultaneous: dexamethasone, hydrocortisone, 6a-methylprednisolone, prednisolone, prednisone

KEYWORDS

tablets; normal phase

REFERENCE
Liu, K.-R.; Chen, S.-H.; Wu, S.-M.; Kou, H.-S.; Wu, H.-L. High-performance liquid chromatographic determination of betamethasone and dexamethasone. J.ChromatognA, 1994, 676, 455-460

SAMPLE

Matrix: formulations Sample preparation: Triturate 1 tablet with a glass rod with 5 mL water, sonicate for 20 min, extract with 9 mL dichloromethane then three times with 5 mL dichloromethane, filter (paper), make up to 25 mL with dichloromethane. Remove a 500 |xL aliquot, add 200 |xL 1.2 mM phenacetin in dichloromethane + 100 \xL 0.5 mM 4-dimethylaminopyridine + 100 u,L 100 mM N-CBZ-Phe in dichloromethane + 100 JJLL 100 mM N, N'-dicyclohexylcarbodiimide in dichloromethane, shake mechanically at 30 for 1 h, inject a 10 IxL aliquot.
HPLCVARIABLES

Column: 75 X 3.9 4 |xm Nova-Pak silica Mobile phase: n-Hexane: dichloromethane: isopropanol 100:100:4 Flow rate: 1 Injection volume: 10 Detector: UV 240
CHROMATOGRAM

Retention time: 7 Internal standard: phenacetin (10) Limit of detection: 4.2 pmol
OTHER SUBSTANCES

Simultaneous: dexamethasone
KEYWORDS

tablets; normal phase; derivatization

REFERENCE
Chen, S.-H.; Wu, S.-M.; Wu, H.-L. Stereochemical analysis of betamethasone and dexamethasone by derivatization and high-performance liquid chromatography. J.Chromatogr., 1992, 595, 203-208

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 10 [xm Partisil 10 ODS Mobile phase: MeOH: water 75:25 Flow rate: 1.2 Detector: UV 242 CHROMATOGRAM Retention time: 5 (betamethasone 17-valerate) REFERENCE
Mithani, S.D.; Bakatselou, V.; TenHoor, CN.; Dressman, J.B. Estimation of the increase in solubility of drugs as a function of bile salt concentration. Pharm.Res., 1996, 13, 163-167

SAMPLE Matrix: solutions Sample preparation: Condition a Bond Elut C18 SPE cartridge with 4 mL water then 3 mL MeOH. Add aqueous steroid solution to cartridge, elute with MeOH, inject a 20 \L aliquot. HPLCVARIABLES Column: 150 X 4 5 |xm Nucleosil C18 Mobile phase: MeCN: water 70:30 Flow rate: 1 Injection volume: 20 Detector: UV 239 CHROMATOGRAM Limit of detection: 120 ng/mL OTHER SUBSTANCES Also analyzed: dexamethasone, flumethasone 21-acetate KEYWORDS SPE; for betamethasone 17-valerate REFERENCE
Valenta, C; Janout, H. Corticosteroid analysis by HPLC with increased sensitivity by use of precolumn concentration. J.Liq.Chromatogr., 1994, 17, 1141-1146

SAMPLE Matrix: solutions HPLCVARIABLES Column: 500 X 1 C18 (Alltech) Mobile phase: MeOH: water 65:35 Flow rate: 0.04 Injection volume: 0.5 Detector: UV 254; MS, Hewlett Packard 5985, home-made interface (details in paper) CHROMATOGRAM Retention time: 21

OTHER SUBSTANCES Simultaneous: metabolites, hydrocortisone KEYWORDS microbore REFERENCE

Eckers, C; Skrabalak, D.S.; Henion, J. On-line direct liquid introduction interface for micro-liquid chromatography/mass spectrometry: application to drug analysis. Clin.Chem., 1982, 28, 1882-1886

SAMPLE Matrix: solutions, tissue Sample preparation: Buffer solutions. Condition a 3 mL Baker C18 SPE cartridge with 400 |xL MeOH. 100-500 |JLL 0.1 M pH 4.5 acetate buffer containing steroids + 100-1000 ng prednisone in MeOH, add to SPE cartridge, wash with 1 mL water, elute with 1'mL MeOH. Evaporate the eluate to dryness under a stream of air, reconstitute in 40-200 JULL dichloromethane: MeOH 98:2, inject a 40 |xL aliquot. Skin tissue. Crush tissue with 3 mL MeOH: 100 mM pH 4.5 acetate buffer 20:80 using a Polytron tissue homogenizer, wash homogenizer twice with the same solution, combine all solutions, add 400-4000 ng prednisone in MeOH, add 10 mL dichloromethane, vortex for 1 min, centrifuge at 3000 rpm for 5 min. Filter the organic phase through a column of Celite 545, evaporate to dryness under a stream of air, reconstitute in 40-200 fxL dichloromethane: MeOH 98:2, inject a
40 JJLL aliquot.

HPLC VARIABLES Column: 250 X 4 5 |xm LiChrosorb Si-60 Mobile phase: n-Hexane: dichloromethane:MeOH: water 63.9:30:6:0.1 Flow rate: 1.2 Injection volume: 40-200 Detector: UV 240 CHROMATOGRAM Retention time: 6.6 Internal standard: prednisone (5.3) Limit of detection: 4 ng KEYWORDS SPE; normal phase REFERENCE
Kubota, K.; Maibach, H.L In vitro percutaneous permeation of betamethasone and betamethasone 17valerate. J.Pharm.ScL, 1993, 82, 1039-1045

SAMPLE Matrix: urine Sample preparation: 3 mL Urine + 0.25 g NaCl, adjust pH to 9.0 with 0.5 g Na2HPO4, add 4 mL dichloromethane, vortex 1 min, centrifuge at 3700 g for 3 min. Remove organic phase and dry it over anhydrous sodium sulfate. Evaporate a 3 mL aliquot to dryness under vacuum, reconstitute residue with 200 \xL 5 u,g/mL IS in MeOH, inject 20 u,L aliquot.
HPLCVARIABLES

Column: 250 X 4.6 Hypersil ODS Mobile phase: MeCN: water 32:68 Column temperature: 30

Flow rate: 1 Injection volume: 20 Detector: UV 245

CHROMATOGRAM Retention time: 10

Internal standard: methylprednisolone (9)

OTHER SUBSTANCES

Extracted: corticosterone, cortisone, fluorocortisone, fluorocortisone acetate, hydrocortisone, hydroxyprogesterone, prednisolone, prednisone, triamcinolone, triamcinolone acetonide Interfering: dexamethasone
KEYWORDS

SPE also discussed

REFERENCE
Santos-Montes, A.; Gonzalo-Lumbreras, R.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Solvent and solidphase extraction of natural and synthetic corticoids in human urine. J.Chromatogr.B, 1994, 652, 83-89

SAMPLE

Matrix: urine Sample preparation: Dilute, if necessary, 100 u,L-l mL urine to 1 mL with water, add to a Chem Elut high surface-area diatomaceous earth extraction column, after 5 min elute with two 6 mL portions of ethyl acetate, combine the eluates and wash them twice with 1 mL 200 mM NaOH. Dry the organic layer over 1 g anhydrous sodium sulfate, evaporate to dryness at 30 under a stream of nitrogen, reconstitute the residue in 250 |xL mobile phase, inject a 50 |xL aliquot.
HPLCVARIABLES

Guard column: 70 X 6 37-53 \xm HC-Pellocil Column: 250 X 4.6 5-6 |xm Zorbax SIL Mobile phase: Dichloromethane:glacial acetic acid:MeOH 91.3:7.5:1.2 Flow rate: 2 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Retention time: 8.5 Internal standard: betamethasone

OTHER SUBSTANCES

Extracted: metabolites, hydrocortisone, 6p-hydroxycortisol, 20-hydroxyprednisone, 6p-hydroxyprednisolone, 20a-hydroxyprednisolone, 20p-hydroxyprednisolone, prednisolone, prednisone
KEYWORDS

betamethasone is IS; normal phase

REFERENCE
Garg, V.; Jusko, W.J. Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performanee liquid chromatography. J.Chromatogr., 1991, 567, 39-47

SAMPLE

Matrix: urine Sample preparation: 3 mL Urine + 100 mg K2HPO4 + 500 mg anhydrous sodium sulfate + 5 mL diethyl ether, shake mechanically for 10 min, centrifuge at 2500 g for 5 min. Remove the organic layer and evaporate it to dryness under vacuum, reconstitute the residue in 200 |xL MeOH, filter (0.45 |xm), inject a 15 JJLL aliquot.
HPLCVARIABLES

Column: 60 X 4.6 3 |xm Hypersil ODS Mobile phase: Gradient. MeOH: 150 mM ammonium acetate from 40:60 to 50:50 over 6 min, maintain at 50:50 for 1 min, to 60:40 over 3 min, maintain at 60:40 for 5 min Flow rate: 0.8 Injection volume: 15 Detector: MS, Hewlett-Packard HP 5988A, vaporizer probe 92 decreased to 89 over 6 min, decreased to 86 over 3 min, maintain at 86 for 5 min, ion source 276, emission current 150 |JLA, electron energy 955 eV, positive ion mode, filament on
CHROMATOGRAM

Retention time: 7 Internal standard: betamethasone

OTHER SUBSTANCES

Extracted: corticosterone, cortisone, deoxycorticosterone, hydrocortisone, lla-hydroxyprogesterone, prednisolone, prednisone, triamcinolone, triamcinolone acetonide
KEYWORDS

betamethasone is IS
REFERENCE
Park, S.-J.; Kim, Y-J.; Pyo, H.-S.; Park, J. Analysis of corticosteroids in urine by HPLC and thermospray LC/MS. J.Anal.Toxicol., 1990, 14, 102-108

ANNOTATED BIBLIOGRAPHY

Jonsson, G.; Astrom, A.; Andersson, P. Budesonide is metabolized by cytochrome P450 3A (CYP3A) enzymes in human liver. Drug Metab.Dispos., 1995, 23, 137-142 [human; liver; microsomal incubations; extracted budesonide; betamethasone is IS; SPE; gradient] Garg, V.; Jusko, W. J. Effects of indomethacin on the pharmacokinetics and pharmacodynamics of prednisolone in rats. J.Pharm.ScL, 1994, 83, 747-750 [rat; plasma; extracted corticosterone, prednisolone, prednisone; betamethasone is IS] Santos-Montes, A.; Gonzalo-Lumbreras, R.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Extraction and high-performance liquid chromatographic separation of deflazacort and its metabolite 21-hydroxydeflazacort. Application to urine samples. J.Chromatogr.B, 1994, 657, 248-253 [interfering triamcinolone acetonide; simultaneous corticosterone, cortisone, deflazacort, deoxycorticosterone, dexamethasone, fludrocortisone, fludrocortisone acetate, hydrocortisone, 21-hydroxydeflazacort, l l a hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcinolone] Santos-Montes, A.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Simultaneous determination of dexamethasone and betamethasone in pharmaceuticals by reversed-phase HPLC. Chromatographia, 1994, 39, 539-542 [simultaneous dexamethasone; methylprednisolone (IS); tablets; column temp 30; LOD 6 ng/mL; non-interfering corticosterone, cortisone, deflazacort, fludrocortisone, fludrocortisone acetate, hydrocortisone, hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcinolone; interfering triamcinolone acetonide] Valvo, L.; Paris, A.; Savella, A.L.; Gallinella, B.; Ciranni Signoretti, E. General high-performance liquid chromatographic procedures for the rapid screening of natural and synthetic corticosteroids. J.Pharm.Biomed.AnaL, 1994, 12, 805-810 [for betamethasone, betamethasone 21-acetate, betamethasone 17,21-dipropionate, betamethasone 21-disodium phosphate, betamethasone 17-valerate; gra-

dient; reverse phase; normal phase; also beclomethasone, beclomethasone 17,21-dipropionate, cortisone, cortisone 21-acetate, 11-deoxycorticosterone 21-acetate, dexamethasone, dexamethasone 21-acetate, dexamethasone 21-disodium phosphate, fluocinolone, fluocinolone acetonide, 9a-fluorohydrocortisone 21-acetate, 9a-fluorohydrocortisone, 9a-fluoroprednisolone, 9a-fluoroprednisolone 21-acetate, hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 21-hemisuccinate, 6a-methylprednisolone, 6a-methylprednisolone 21-acetate, 6a-methylprednisolone 21-sodium succinate, prednisolone, prednisolone 21-acetate, prednisolone 21-disodium phosphate, prednisolone 21-pivalate, prednisolone 21-sodium succinate, prednisone, triamcinolone, triamcinolone acetonide] Santos-Montes, A.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Optimization of the high-performance liquid chromatographic separation of a mixture of natural and synthetic corticosteroids. J.Chromatogr., 1993, 620, 15-23 [simultaneous corticosterone, cortisone, deoxycorticosterone, dexamethasone, fluorocortisone, hydrocortisone, hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcinolone] Smith, E.W.; Haigh, J.M. In vitro diffusion cell design and validation. I. A stability-indicating highperformance liquid chromatographic assay for betamethasone 17-valerate in purified isopropyl myristate receptor phase. Pharm.Res., 1989, 6, 431-435 Maron, N.; Cristi, E.A.; Ramos, A.A. Determination of betamethasone 17-benzoate in lipophylic vehicles by reversed-phase high-performance liquid chromatography. J.Pharm.Sci., 1988, 77, 638-639 Skrabalak, D.S.; Cuddy, K.K.; Henion, J.D. Quantitative determination of betamethasone and its major metabolite in equine urine by micro-liquid chromatography-mass spectrometry. J.Chromatogr., 1985, 341, 261-269 Tokunaga, H.; Kimura, T.; Kawamura, J. Determination of glucocorticoids by liquid chromatography. III. Application to ointments and a cream containing cortisone acetate, dexamethasone acetate, fluorometholone, and betamethasone valerate. Chem.Pharm.BulL, 1984, 32, 4012-4016 Cairns, T.; Siegmund, E.G.; Stamp, J.J.; Skelly, J.P. Liquid chromatography mass spectrometry of dexamethasone and betamethasone. Biomed.Mass.Spectrom., 1983, 10, 203-208 Okumura, T. Application of thin-layer chromatography to high-performance liquid chromatographic separation of steroidal hormones and cephalosporin antibiotics. J.Liq.Chromatogr., 1981, 4, 10351064 [normal phase; also cephalexin, cephaloglycine, cephaloridine, cephalothin, cortisone, dexamethasone, hydrocortisone] Petersen, M. C; Nation, R.L.; Ashley, J. J. Simultaneous determination of betamethasone, betamethasone acetate and hydrocortisone in biological fluids using high-performance liquid chromatography. J.Chromatogr., 1980, 183, 131-139