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Pravastatin

Molecular formula: C23H36O7


Molecular weight: 423.5
CAS Registry No.: 81131-70-6 (pravastatin sodium),
81093-37-0 (pravastatin)

SAMPLE
Matrix: blood
Sample preparation: Condition an immobilized antibody column (preparation details in
paper) with 4 mL water, 4 mL MeOH: water 80:20, and 4 mL PBS. Add 1 mL plasma to
the column, let stand for 15 min, wash with two 4 mL portions of water, wash with 4 mL
PBS, wash with two 4 mL portions of water, elute with 4 mL MeOH. Add 100 (xL 100
ng/mL IS in water to the eluate, evaporate to dryness, reconstitute with 100 (xL DMF,
add 100 |xL 47.6 jxg/mL triethylamine in dioxane (Caution! Dioxane is a carcinogen!), add
100 |xL 51.8 |xL/mL diethyl phosphorocyanidate in dioxane, add 100 |xL 100 jxg/mL Ndansylethylenediamine (Molecular Probes, Inc., Eugene OR) in dioxane, stir for 10 s, let
stand at room temperature for 10 min, inject a 10 |xL aliquot onto column A and elute to
waste with mobile phase A. After 7.5 min elute the contents of column A onto column B
with mobile phase B. After 2.5 min remove column A from the circuit. Elute column B
with mobile phase B and monitor the effluent from column B.
HPLCVARIABLES
Column: A 150 X 4.6 Ultron 300-C4 (Shinwa Chemical Industries); B 150 X 6 Cosmosil
5C18-AR (Nacalai Tesque)
Mobile phase: A MeCN: 10 mM pH 2.4 citric acid 30:70; B MeCN: 5 mM pH 2.6 citric acid
50:50

Flow rate: 1
Injection volume: 10
Detector: F ex 325 (40 mW He-Cd laser); F ex 350 em 530; UV 239
CHROMATOGRAM
Retention time: 17.3
Internal standard: R-416 (Sankyo) (19)
Limit of quantitation: 0.1 ng/mL (laser fluorescence)
KEYWORDS
column-switching; dog; rat; laser; plasma; SPE; derivatization; pharmacokinetics
REFERENCE
Dumousseaux, C; Muramatsu, S.; Takasaki, W.; Takahagi, H. Highly sensitive and specific determination of pravastatin sodium in plasma by high-performance liquid chromatography with laser-induced fluorescence detection after immobilized antibody extraction. J.Pharm.Sci., 1994, 83, 16301636

SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL C2 Bond-Elut SPE cartridge with 2 mL water, 2
mL MeOH, and 2 mL water. 1 mL Plasma + 1 mL 100 mM pH 7.2 KH2PO4, add to the
SPE cartridge, wash with 2 mL water, elute with 500 JJLL MeCN-.water 75:25. Evaporate
the eluate to dryness under reduced pressure, reconstitute with 80 JULL IS solution, cen-

trifuge, inject a 50 |xL aliquot. (Prepare the IS solution by mixing 2 mL 100 |xg/mL simvastatin p-hydroxyacid in MeOH, 1 mL MeOH, and 1 mL 100 mM pH 5 KH2PO4.)
HPLC VARIABLES
Guard column: 20 mm long Supelguard LC-18
Column: 50 X 4.6 3 jim Supelcosil LC-18
Mobile phase: MeCN: 50 mM pH 3.5 ammonium phosphate 26:74
Column temperature: 50
Flow rate: 1.6
Injection volume: 50
Detector: UV 238
CHROMATOGRAM
Retention time: 3.0
Internal standard: simvastatin p-hydroxyacid (4.8)
Limit of detection: 2 ng/mL
KEYWORDS
pharmacokinetics; plasma; SPE
REFERENCE
Iacona, L; Regazzi, M.B.; Buggia, I.; Villani, P.; Fiorito, V.; Molinaro, M.; Guarnone, E. High-performance
liquid chromatography determination of pravastatin in plasma. Ther.Drug Monit., 1994, 16, 191
195

SAMPLE
Matrix: blood, feces, urine
Sample preparation: Plasma. 2 mL Plasma + 4 mL MeCN, centrifuge at 700 g for 10
min, remove the supernatant and wash the precipitate twice with 2 mL MeCN: water 2:
1. Combine the supernatants and evaporate them to dryness under vacuum, reconstitute
the residue in 1 mL MeCN: water 2:1, centrifuge at 10000 g, remove the supernatant and
add it to 500 |xL water, centrifuge at 10000 g, inject a 1 mL aliquot. Urine. Centrifuge at
10000 g, inject an aliquot. Feces. 1 g Homogenized feces + 2 mL MeCN, sonicate for 5
min, shake in a wrist action shaker for 20 min, centrifuge at 700 g for 10 min. Remover
the supernatant and wash the precipitate twice with 1 mL MeCN: water 2:1, combine
the supernatants, inject a 500 |xL aliquot.
HPLC VARIABLES
Guard column: present but not specified
Column: 500 X 9.4 Partisil 10 ODS-3 C18
Mobile phase: Gradient. MeCN: 10 mM pH 7.2 potassium phosphate buffer containing 5
mM tetrabutylammonium hydrogen sulphate at 25:75 for 20 min, then to 50:50 over 45
min, hold at 50:50 for 10 min.
Flow rate: 4
Injection volume: 500-1000
Detector: Collect fractions and measure radioactivity; UV 245
CHROMATOGRAM
Retention time: 33
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
plasma; semi-preparative; radiolabeled starting material

REFERENCE
Everett, D.W.; Chando, T.J.; Didonato, G.C.; Singhvi, S.M.; Pan, H.Y.; Weinstein, S.H. Biotransformation
of pravastatin sodium in humans. Drug Metab.Dispos., 1991, 19, 740-748

SAMPLE

Matrix: solutions
Sample preparation: Inject an aliquot of an aqueous solution.
HPLCVARIABLES

Column: Cosmosil 5C18-AR (Nacalai Tesque)


Mobile phase: A MeCN: 10 mM pH 2.4 citric acid 30:70
Flow rate: 1
Detector: UV 235
REFERENCE
Dumousseaux, C; Muramatsu, S.; Takasaki, W.; Takahagi, H. Highly sensitive and specific determination of pravastatin sodium in plasma by high-performance liquid chromatography with laser-induced fluorescence detection after immobilized antibody extraction. J.Pharm.ScL, 1994, 83, 16301636

SAMPLE

Matrix: solutions
Sample preparation: Centrifuge at 2000 rpm, inject an aliquot.
HPLCVARIABLES

Column: 300 X 3.9 jxBondapak


Mobile phase: MeOH: water: triethylamine: glacial acetic acid 500:500:1:1
Column temperature: 30
Flow rate: 1.3
Detector: UV 238
CHROMATOGRAM

Retention time: 17.5 (10.1 hydroxy acid form)


Limit of detection: 10 ng/mL
Limit of quantitation: 25 ng/mL
REFERENCE
Serajuddin, A.T.; Ranadive, S.A.; Mahoney, E.M. Relative lipophilicities, solubilities, and structurepharmacological considerations of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors pravastatin, lovastatin, mevastatin, and simvastatin. J.Pharm.Sci., 1991, 80, 830-834