Methylprednisolone

Molecular formula: C22H30O5

Molecular weight: 374.5
CAS Registry No.: 83-43-2,53-36-1 (acetate), 5015-36-1
(sodium phosphate), 2375-03-3 (sodium succinate), 2921-57-5
(hemisuccinate), 90350-40-6 (suleptanate)

SAMPLE
Matrix: blood
Sample preparation: Extract 1 mL plasma containing dexamethasone with 12 mL dichloromethane. Remove the organic phase and wash it with 2 mL 100 mM NaOH, wash
with 1 mL water, dry over 1 g anhydrous sodium sulfate. Evaporate to dryness under a
stream of nitrogen, reconstitute with mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 3 |xm Spherisorb silica
Mobile phase: Hexane: dichloromethane: EtOH: glacial acetic acid 26:69:3.4:2
Flow rate: 0.75
Detector: UV 254
CHROMATOGRAM
Retention time: 7.4
Internal standard: dexamethasone (5.1)
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: hydrocortisone, prednisolone
KEYWORDS
plasma; pharmacokinetics; normal phase
REFERENCE
Mollmann, H.; Hochhaus, G.; Rohatagi, S.; Barth, J.; Derendorf, H. Pharmacokinetic/pharmacodynamic
evaluation of deflazacort in comparison to methylprednisolone and prednisolone. Pharm.Res., 1995,
12, 1096-1100

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 |xL MeOH: 100 mM perchloric acid 50:50, inject a 20 jxL
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: 18.70 (methylprednisolone), 21.36 (methylprednisolone acetate)
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: betamethasone, chloroquine, corticosterone, cortisone, dexamethasone, fluendrenolide, fluocinolone acetonide, fluorometholone, fluprednisolone, hydrocortisone, hydroxychloroquine, 178-hydroxyprogesterone, meprednisone, 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: 2 mL Plasma + 500 ng fluorometholone + 5 mL hexane, shake
horizontally at 60 cycles/min for 10 min, centrifuge at 1000 g for 5 min, discard the hexane
layer, add 8 mL dichloromethane, shake horizontally at 60 cycles/min for 10 min, centrifuge at 1000 g for 5 min, repeat extraction with 8 mL dichloromethane. Combine the
dichloromethane layers and add 300 mg anhydrous sodium sulfate, shake horizontally at
200 cycles/min for 5 min, centrifuge at 1000 g for 5 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 200
jxL mobile phase, add 2 mL hexane, vortex for 1 min, centrifuge at 1000 g for 5 min,
discard the hexane layer, inject a 100 |JLL aliquot of the mobile phase layer.
HPLCVARIABLES
Guard column: 30 X 4.6 5 jim Spheri-5 RP-18
Column: 250 X 4.6 5 |jim Ultrasphere ODS
Mobile phase: MeCN: water: glacial acetic acid 33:62:5
Flow rate: 1
Injection volume: 100
Detector: UV 254
CHROMATOGRAM
Retention time: 8.07 (methylprednisolone), 21.80 (methylprednisolone acetate)
Internal standard: fluorometholone (14.55)
Limit of detection: 1 ng/mL
Limit of quantitation: 2 ng/mL
KEYWORDS
plasma; pharmacokinetics
REFERENCE
Hopkins, N.K.; Wagner, CM.; Brisson, J.; Addison, T.E. Validation of the simultaneous determination
of methylprednisolone and methylprednisolone acetate in human plasma by high-performance liquid
chromatography. J.Chromatogr., 1992, 577, 87-93

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 ^xL 3 M sulfuric acid + 50 jxL 6 |xg/mL prednisone in MeCN: MeOH 50:50, mix, add 15 mL hexane: ethyl acetate 50:50, shake for 20
min, centrifuge, freeze at -70. Remove the organic layer and add it to 1 mL 1 M nitric
acid, shake, freeze. Remove the organic layer and dry it over anhydrous sodium sulfate,
evaporate to dryness under a stream of nitrogen at 30, reconstitute the residue in 100
|xL mobile phase, inject an aliquot.
HPLCVARIABLES
Guard column: 12.5 X 4 5 |xm Zorbax SIL
Column: three 80 X 4 5 fun Zorbax SIL Reliance 5 columns in series
Mobile phase: Dichloromethane: hexane: EtOH: glacial acetic acid 69:26:2.3:1 (Pass the
mobile phase through a 70 X 6 37-53 |jim HC-Pelloeil (Whatman) column.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 12 (methylprednisolone hemisuccinate), 19 (methylprednisolone)
Internal standard: prednisone (9)
Limit of detection: 10 ng/mL
OTHER SUBSTANCES
Extracted: hydrocortisone
KEYWORDS
plasma; pharmacokinetics; normal phase
REFERENCE
Kong, A.-N.; Slaughter, R.L.; Jusko, W.J. Simultaneous analysis of methylprednisolone hemisuccinate,
cortisol and methylprednisolone by normal-phase high-performance liquid chromatography in human
plasma. J.Chromatogr., 1988, 432, 308-314

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 0 mL dichloromethane, shake for 1 h, centrifuge
for 15 min. Discard the aqueous layer, wash the organic layer with 1 mL 100 mM NaOH
and 1 mL water (vortex for 30 s and centrifuge for 15 min each time). Remove the organic
layer and evaporate it to dryness under a stream of nitrogen at 40-45, reconstitute the
residue in 200 |JLL mobile phase, inject a 50 |xL aliquot onto column A in series with
column B and elute with mobile phase. After 2.1 min remove column A from the circuit
and backflush it with mobile phase at 1 mL/min for 15 min, elute column B with mobile
phase and monitor the effluent.
HPLCVARIABLES
Column: A 30 mm long Spherisorb silica; B 250 X 4.6 6 jim Zorbax silica
Mobile phase: Butyl chloride: THF: MeOH: phosphoric acid 880:100:15:0.5 (Butyl chloride
was 50% water-saturated.)
Flow rate: 2
Injection volume: 50
Detector: UV 236
CHROMATOGRAM
Retention time: 24
Internal standard: 6a-methylprednisolone

OTHER SUBSTANCES
Extracted: fluoxymesterone
KEYWORDS
serum; normal phase; column-switching; 6a-methylprednisolone is IS
REFERENCE
Capponi, V.J.; Cox, S.R.; Harrington, E.L.; Wright, CE.; Antal, E.J.; Albert, K.S. Liquid chromatographic
assay for fluoxymesterone in human serum with application to a preliminary bioavailability study.
J.Pharm.ScL, 1985, 74, 308-311

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 80 |xL 3.125 |jtg/mL dexamethasone in MeOH, mix,
add 15 mL dichloromethane, shake for 20 min, centrifuge. Remove organic phase and
wash it with 1 mL 100 mM NaOH then with 1 mL water. Remove organic phase and dry
it with 1 g anhydrous sodium sulfate. Evaporate to dryness at 45 under a stream of
nitrogen, reconstitute in 200 u,L mobile phase, inject.
HPLCVARIABLES
Guard column: 70 X 6 37-53 jxm Whatman HC-Pellocil
Column: 250 X 4.6 5-6 |xm Zorbax SIL
Mobile phase: Hexane: dichloromethane: ethanol: acetic acid 26:69:3.4:1
Flow rate: 2
Injection volume: 200
Detector: UV 254
CHROMATOGRAM
Retention time: 12
Internal standard: dexamethasone (8)
Limit of detection: 2 ng/mL
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Simultaneous: beclomethasone, betamethasone, corticosterone, cortisone, fluocinonide, hydrocortisone, methylprednisone, prednisolone, prednisone
KEYWORDS
plasma; normal phase
REFERENCE
Ebling, W.F.; Szefler, S.J.; Jusko, W.J. Analysis of cortisol, methylprednisolone, and methylprednisolone
hemisuccinate. Absence of effects of troleandomycin on ester hydrolysis. J.Chromatogr., 1984, 305,
271-280

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 2 [xg/mL dexamethasone in EtOH: water
10:90 + 100 |JLL 250 mM NaOH + 7 mL ether: dichloromethane 60:40, vortex for 30 s,
centrifuge at 2000 rpm for 5 min. Remove 6 mL of the organic layer and evaporate it to
dryness under a stream of air at 40, reconstitute the residue in 100 fxL
dichloromethane:EtOH:water 95:4:1, inject a 50 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.5 5 |xm Partisil silica

Mobile phase: Dichloromethane: EtOH: water 95:4:1

Flow rate: 1.5
Injection volume: 50
Detector: UV 239
CHROMATOGRAM
Retention time: 18
Internal standard: dexamethasone (11.5)
Limit of quantitation: 25 ng/mL
OTHER SUBSTANCES
Extracted: corticosterone, 11-deoxycortisol, hydrocortisone, 17-hydroxyprogesterone, prednisolone, prednisone, progesterone
KEYWORDS
plasma; normal phase
REFERENCE
Scott, N.R.; Chakraborty, J.; Marks, V. Determination of prednisolone, prednisone, and cortisol in human
plasma by high-performance liquid chromatography Anal.Biochem., 1980, 108, 266268

SAMPLE
Matrix: formulations
Sample preparation: Inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb CN
Mobile phase: MeCNrIOO mM pH 4.5 NaH2PO4 15:85
Injection volume: 20
Detector: UV 300
CHROMATOGRAM
Retention time: 4.2
Internal standard: propylparaben (3.3)
OTHER SUBSTANCES
Simultaneous: dexamethasone, granisetron
KEYWORDS
injections; 5% dextrose; saline; water
REFERENCE
Pinguet, R; Rouanet, P.; Martel, P.; Fabbro, M.; Salabert, D.; Astre, C. Compatibility and stability of
granisetron, dexamethasone, and methylprednisolone in injectable solutions. J.Pharm.Sci., 1995,84,
267-268

SAMPLE
Matrix: formulations
Sample preparation: Prepare a solution in chloroform: n-butanol 95:5, inject a 5 |xL
aliquot.
HPLCVARIABLES
Guard column: 5 |jim Guard-Pak Resolve Si (dead volume 60-75 |xL)
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: 10.5 (6a-methylprednisolone)

OTHER SUBSTANCES

Simultaneous: betamethasone, cortisone, dexamethasone, hydrocortisone, prednisolone

Interfering: 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.Chromatogr.A, 1994, 676, 455-460

SAMPLE

Matrix: formulations
Sample preparation: Dilute 20-fold with mobile phase, inject a 10 |JLL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 pjn jxBondapak C18

Mobile phase: MeCN: 50 mM pH 5.7 sodium acetate 33:67
Flow rate: 1
Injection volume: 10
Detector: UV 248
CHROMATOGRAM

Retention time: 6.4 (methylprednisolone sodium succinate)

KEYWORDS

water; injections; stability-indicating

REFERENCE
Nahata, M.C; Morosco, R.S.; Hippie, T.F. Stability of diluted methylprednisolone sodium succinate injection at two temperatures. Am.J.Hosp.Pharm., 1994, 51, 2157-2159

SAMPLE

Matrix: perfusate
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL MeOH and 10
mL water. Add 3 mL Perfusate to the SPE cartridge, wash three times with 10 mL aliquots of water, elute with 5 mL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen at 35, reconstitute the residue in 100 |xL mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 15 X 3.2 Newguard RP-18

Column: two 250 X 4.6 Spheri-5 RP-18 columns in series
Mobile phase: MeOH: water 53:47
Column temperature: 40
Flow rate: 1.1
Injection volume: 50
Detector: UV 242

CHROMATOGRAM

Retention time: 30
Internal standard: 6a-methylprednisolone
OTHER SUBSTANCES

Extracted: metabolites, cortisone, dihydrocortisol, dihydrocortisone, hydrocortisone

Simultaneous: prednisolone
Noninterfering: acetaminophen, albuterol, betamethasone, bupivacaine, carbamazepine,
cholesterol, clonazepam, dehydroepiandrosterone, dexamethasone, diazepam, estradiol,
estriol, hydroxyprogesterone, methimazole, phenobarbital, prednisone, progesterone, ritodrine, scopolamine, testosterone
KEYWORDS

SPE; methylprednisolone is IS
REFERENCE
Dodds, H.M.; Maguire, D.J.; Mortimer, R.H.; Addison, R.S.; Cannell, G.R. High performance liquid chromatographic separation of cortisol, cortisone, and their 20-reduced metabolites in perfusion media.
J.Liq.Chromatogr., 1995, 18, 1809-1820

SAMPLE

Matrix: solutions
Sample preparation: Inject an aliquot of a 1 |JIM solution in MeOH.
HPLC VARIABLES

Column: 470 X 4.6 5 |xm Spheri-5 RP-18

Mobile phase: MeOH: water 56:44
Flow rate: 0.5
Injection volume: 10
Detector: UV 240
CHROMATOGRAM

Retention time: 55
OTHER SUBSTANCES

Simultaneous: cortisone, dehydrocorticosterone, hydrocortisone, prednisolone, prednisone,

tetrahydrocortisol, tetrahydrocortisone
REFERENCE
Lukulay, RH.; McGuffin, V.L. Comparison of solvent modulation with premixed mobile phases for the
separation of corticosteroids by liquid chromatography J.Liq.Chromatogr., 1995, 18, 4039-4062

SAMPLE

Matrix: solutions
HPLC VARIABLES

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, chlorphenesin, 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, estradiol, 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, Iidocaine, 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, 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, triprolidine, 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 |xg/mL solution in mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Partisil 10 ODS-I