Mobile phase: MeOH-.

water 55:45
Column temperature: 40
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: k' 1.509 (methylprednisolone), k' 2.190 (methylprednisolone acetate)
OTHER SUBSTANCES
Also analyzed: androsterone (UV 210), cortexolone (UV 240), cortisone (UV 240), estradiol
(UV 280), estrone (UV 280), ethinyl estradiol (UV 280), ethisterone (UV 240), hydrocortisone (UV 240), hydroxyprogesterone (UV 240), lynestrenol (UV 210), medroxyprogesterone acetate (UV 240), medroxyprogesterone (UV 240), methandienone (UV 240), methylandrostenediol (UV 210), methyltestosterone (UV 240), nandrolone (UV 240),
norethisterone (UV 240), prednisolone acetate (UV 240), prednisolone (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 20 |mL aliquot of a MeOH solution.
HPLCVARIABLES
Column: 250 X 4.6 5 u,m Hypersil 5-ODS
Mobile phase: THF: water 23:77
Column temperature: 30
Flow rate: 1
Injection volume: 20
Detector: UV 245
CHROMATOGRAM
Retention time: k' 11.36
OTHER SUBSTANCES
Simultaneous: metabolites, betamethasone, corticosterone, cortisone, deflazacort, deoxycorticosterone, dexamethasone, fludrocortisone, fludrocortisone acetate, fluorocortisone,
fluorocortisone acetate, hydrocortisone, 21-hydroxydeflazacort, lla-hydroxyprogesterone,
prednisolone, prednisone, triamcinolone, triamcinolone acetonide
REFERENCE
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

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 |jim SI-100 (Brownlee)
Mobile phase: Butyl chloride:THF:MeOH-.glacial acetic acid 95:7:3.5:3 (Butyl chloride
was 50% water saturated.)
Injection volume: 20
Detector: UV 254

KEYWORDS

normal phase
REFERENCE
Kane, M.P.; Tsuji, K. Radiolytic degradation scheme for
1983, 72, 30-35

60

Co-irradiated corticosteroids. J.Pharm.Sci.,

SAMPLE

Matrix: synovial fluid

Sample preparation: 100 fxL Synovial fluid + 10 |xL 10 |xg/mL flumethasone in MeOH +
1 mL 100 mM NaOH -I- 10 mL dichloromethane, shake for 10 min, centrifuge at 8400 g
for 10 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 40, reconstitute the residue in 100 |xL mobile phase, vortex, inject the whole
amount.
HPLCVARIABLES

Column: 100 X 8 10 |xm Radial Pak B silica (Waters)

Mobile phase: Dichloromethane: MeOH: glacial acetic acid 96.8:2.4:0.8
Flow rate: 1.4
Injection volume: 100
Detector: UV 254
CHROMATOGRAM

Retention time: 8 (methylprednisolone), 4 (methylprednisolone acetate)

Internal standard: flumethasone (6)
Limit of detection: 200 ng/mL
KEYWORDS

normal phase; cow; pharmacokinetics

REFERENCE
Alvinerie, M.; Toutain, RL. Determination of methylprednisolone and methylprednisolone acetate in
synovial fluid using high-performance liquid chromatography. J.Chromatogr., 1984, 309, 385-390

SAMPLE

Matrix: tissue
Sample preparation: Homogenize rat brain in 10 mL n-hexane: isopropanol 60:40 in a
glass homogenizing tube with a PTFE pestle at 4 using 15 passes, transfer to an acidwashed glass tube, wash out homogenizing tube with three 2.5 mL aliquots of n-hexane:
isopropanol 60:40, centrifuge with a table-top centrifuge, remove the supernatant, reextract the pellet with 8 mL n-hexane: isopropanol 60:40. Combine the supernatants and
filter (0.2 |xm) them, evaporate to dryness under a stream of nitrogen, reconstitute the
residue in n-hexane: isopropanol 60:40, inject an aliquot into the gradient elution system.
Collect the methylprednisolone peak and rechromatograph isocratically.
HPLCVARIABLES

Column: 250 X 4.6 5-6 |xm Zorbax silica

Mobile phase: Gradient. A was n-hexane: isopropanol 60:40. B was n-hexane: isopropanol:
water 56.7:37.8:5.5. A:B from 55:45 to 24:76 over 7 min, to 0:100 over 5 min, maintain
at 0:100 for 22 min. (Methylprednisolone peak can be collected and rechromatographed
isocratically at A:B 90:10 at 0.8 mL/min, retention time 7.17 min.)
Column temperature: 34
Flow rate: 1.8
Detector: UV 254

CHROMATOGRAM
Retention time: 3.27
Limit of detection: 4.9 ng
Limit of quantitation: 9.8 ng
KEYWORDS
rat; brain; normal phase
REFERENCE
Murphy, E.J.; Slivka, A.R; Rosenberger, T.A.; Horrocks, L.A. High-performance liquid chromatography
separation and quantitation of methylprednisolone from rat brain. Anal.Biochem., 1993, 209, 339
342

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 jxL MeOH, inject 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Hypersil 5-ODS
Mobile phase: MeCN: water 30:70
Column temperature: 30
Flow rate: 1
Injection volume: 20
Detector: UV 245
CHROMATOGRAM
Retention time: 14
Internal standard: methylprednisolone
OTHER SUBSTANCES
Extracted: cortisone, hydrocortisone
Simultaneous: fluorocortisone
Noninterfering: corticosterone, deflazacort, deoxycorticosterone, fluorocortisone acetate,
21-hydroxydeflazacort, lla-hydroxyprogesterone, prednisolone, prednisone, triamcinolone
acetonide
KEYWORDS
methylprednisolone is IS
REFERENCE
Santos-Montes, A.; Gonzalo-Lumbreras, R.; Izquierdo-Hornillos, R. Simultaneous determination of cortisol and cortisone in urine by reversed-phase high-performance liquid chromatography. Clinical and
doping control applications. J.Chromatogr.B, 1995, 673, 27-33

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 [xg/mL IS in MeOH, inject 20 |xL
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: 9
Internal standard: prednisone (6.3)
OTHER SUBSTANCES

Simultaneous: betamethasone, corticosterone, cortisone, dexamethasone, fluorocortisone,

fluorocortisone acetate, hydrocortisone, hydroxyprogesterone, prednisolone, triamcinolone, triamcinolone acetonide
KEYWORDS

SPE also discussed

REFERENCE
Santos-Montes, A.; Gonzalo-Lumbreras, R.; Gasco-Lopez, A.L; 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: 1 mL Urine + 1 mL 1.5 M HCl + 500 jxL water + 10 mL dichloromethane, shake 20 min, centrifuge at 250 g for 3 min. Remove the organic phase and
evaporate it to dryness under a stream of nitrogen at 40. Reconstitute with 200 |JLL
MeOH, inject 10-30 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.5 Partisil 10 ODS-3

Mobile phase: Gradient. A was MeCN: water 28:72 containing 0.05% phosphoric acid and
0.05% acetone. B was MeCN:50 mM KH2PO4 50:50. A:B 100:0 for 8 min then to 0:100
over 6 min
Flow rate: 2
Injection volume: 10-30
Detector: UV 220
CHROMATOGRAM

Retention time: 13.6

Internal standard: methylprednisolone
OTHER SUBSTANCES

Extracted: ibuprofen, ibuprofen metabolites

KEYWORDS

methylprednisolone is IS
REFERENCE
Lockwood, G.F.; Wagner, J.G. High-performance liquid chromatographic determination of ibuprofen and
its major metabolites in biological fluids. J.Chromatogr., 1982, 232, 335-343

Next page

ANNOTATED BIBLIOGRAPHY
Herman, B.D.; Sinclair, B.D.; Milton, N.; Nail, S.L. The effect of bulking agent on the solid-state stability
of freeze-dried methylprednisolone sodium succinate. Pharm.Res., 1994, 11, 1467-1473
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 [gradient; reverse phase; normal phase; for 6a-methylprednisolone, 6a-methylprednisolone 21-acetate, 6a-methylprednisolone 21-sodium succinate; also
beclomethasone, beclomethasone 17,21-dipropionate, betamethasone, betamethasone 21-acetate, betamethasone 17,21-dipropionate, betamethasone 21-disodium phosphate, betamethasone 17-valerate,
cortisone, cortisone 21-acetate, 11-deoxycorticosterone 21-acetate, dexamethasone, dexamethasone
21-acetate, dexamethasone 21-disodium phosphate, fluocinolone, fluocinolone acetonide, 9afluorohydrocortisone 21-acetate, 9a-fluorohydrocortisone, 9a-fluoroprednisolone, 9a-fluoroprednisolone 21-acetate, hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 21-hemisuccinate, 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 betamethasone, corticosterone, cortisone, deoxycorticosterone, dexamethasone, fluorocortisone, hydrocortisone, hydroxyprogesterone, prednisolone, prednisone,
triamcinolone]
McGinley, RA.; Braughler, J.M.; Hall, E.D. Determination of methylprednisolone in central nervous
tissue and plasma using normal-phase high-performance liquid chromatography. J.Chromatogr.,
1982, 230, 29-35