Previous page

Azathioprine
Molecular formula: C9H7N7O2S Molecular weight: 277.3 CAS Registry No.: 446-86-6

SAMPLE Matrix: blood Sample preparation: 500 fxL Serum + 25 ng 9-methylazathioprine + 4.5 mL ethyl acetate, vortex for 1 min, centrifuge for 1 min, repeat extraction. Combine the organic layers and evaporate them to dryness under reduced pressure at 35, reconstitute the residue in 250 IxL mobile phase, vortex for 10 s, sonicate for 10 min, inject a 200 |xL aliquot. HPLCVARIABLES Guard column: 4 X 4.6 5 |xm LiChrospher 100 RP 18 Column: 250 X 4.6 5 jxm LiChrospher 60 Rp-select B Mobile phase: MeCN: 10 mM pH 2.3 potassium phosphate buffer 12:88 (Flush with MeCN: buffer 50:50 for 2 min after each run.) Column temperature: 22 Flow rate: 1 Injection volume: 200 Detector: UV 285 CHROMATOGRAM Retention time: 16 Internal standard: 9-methylazathioprine (Add 400 mg anhydrous potassium carbonate and 200 \xL methyl iodide to a solution of 220 mg azathioprine in 7 mL DMF at 0-5, stir under nitrogen for 24 h, add 14 mL water, neutralize with 1 M HCl and sodium bicarbonate solution, filter, wash the solid with water, dry under vacuum to give 9-methylazathioprine (mp 174-5). Purify by precipitating from DMF solution with water.) (29) Limit of quantitation: 2.5 ng/mL OTHER SUBSTANCES Noninterfering: 6-mercaptopurine KEYWORDS serum; pharmacokinetics REFERENCE
Binscheck, T.; Meyer, H.; Wellhomer, H.H. High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples. J.Chromatogr.B, 1996, 675, 287-294

SAMPLE Matrix: blood Sample preparation: Condition a 100 mg Isolute C8 SPE cartridge (International Sorbent Technology) with 2.5 mL MeOH and 3.5 mL 10 mM pH 7.0 phosphate buffer. 1 mL Plasma + 50 |xL 1.5 |xg/mL guaneran + 2 mL 10 mM pH 7.0 phosphate buffer, mix, add to the SPE cartridge at 2.5 mL/min, wash with 3 mL MeCN :pH 7.0 phosphate buffer 1:99, air dry for 2 min, elute with 2 mL MeOH: ethyl acetate 5:95. Evaporate the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 50 |xL mobile phase, inject a 20 |xL aliquot.

HPLCVARIABLES

Column: 80 X 4.6 3 [Lm HSpecosphere 3CR C8 (Perkin-Elmer) Mobile phase: MeCN: 10 mM pH 6.2 sodium phosphate buffer 9:91 Flow rate: 1.2 Injection volume: 20 Detector: UV 280
CHROMATOGRAM Retention time: 8.9

Internal standard: guaneran (6-[(l-methyl-4-nitro-5-imidazolyl)thio]-2-aminopurine, Wellcome Foundation) (7.7) Limit of detection: 0.5 ng/mL OTHER SUBSTANCES Simultaneous: caffeine Noninterfering: aspirin, chloroquine, cyclosporin, diltiazem, nifedipine, prednisolone
KEYWORDS

plasma; pharmacokinetics; SPE

REFERENCE
Albertioni, R; Pettersson, B.; Ohlman, S.; Peterson, C. Analysis of azathioprine and 6-mercaptopurine in plasma in renal transplant recipients after administration with oral azathioprine. J.Liq. Chromatogr., 1995, 18, 3991-4005

SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL Sep-Pak silica SPE cartridge with 3 mL ethyl acetate and vacuum dry for 1 min. 1 mL Plasma + 2.8 [xg antipyrine, add to SPE cartridge, wash with 5 mL benzene (Caution! Benzene is a carcinogen!) or hexane over 1 min, vacuum dry for 1 min, elute with 5 mL ethyl acetate. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 200 |JLL mobile phase, sonicate, inject all of the sample.
HPLCVARIABLES

Guard column: C18 Guard-Pak (Waters) Column: 100 X 8 4 |xm 8 NV C18 Radial-Pak (Waters) Mobile phase: MeOH .10 mM pH 4.5 sodium phosphate 13:87 Flow rate: 3 Injection volume: 200 Detector: UV 280
CHROMATOGRAM Retention time: 7

Internal standard: antipyrine (17) Limit of quantitation: 5 ng/mL

OTHER SUBSTANCES

Simultaneous: acetaminophen, aspirin, carmustine, chlorambucil, cytarabine, dacarbazine, diclofenac, etoposide, 5-fluorouracil, ifosfamide, indomethacin, lomustine, methotrexate, naproxen, salicylic acid, tegafur, teniposide, thioguanine Noninterfering: betamethasone, carboplatin, cyclophosphamide, cyclosporine A, ibuprofen, thiotepa
KEYWORDS

plasma; rabbit; SPE; human; pharmacokinetics

REFERENCE
el-Yazigi, A.; Abdel Wahab, F. Expedient liquid chromatographic analysis of azathioprine in plasma by use of silica solid phase extraction. Ther.Drug Monit, 1992, 14, 312-316

SAMPLE

Matrix: blood Sample preparation: 200 u,L Serum + 5 uJL 50 |xg/mL 2-ethyl-4-oxoquinazoline in EtOH + 100 \xL reagent, let stand at room temperature for 1 h, add 1.8 mL ethyl acetate, mix, centrifuge at 1800 g for 5 min, remove 1.5 mL of the supernatant, repeat the extraction. Combine the organic layers and evaporate them to dryness under reduced pressure below 30, reconstitute the residue in 100 |xL initial mobile phase, inject a 90 |xL aliquot. (Reagent was 30 mg N-ethylmaleimide in 2 mL 50 mM pH 7.0 phosphate buffer, prepare fresh daily.)
HPLCVARIABLES

Column: 10 |xm ixBondapak C18 Mobile phase: Gradient. MeCN: 10 mM KH2PO4 9:91 for 26 min, then 50:50 for 1 min (step gradient). Flow rate: 1.5 Injection volume: 90 Detector: UV 280
CHROMATOGRAM

Retention time: 13.4 Internal standard: 2-ethyl-4-oxoquinazoline (28) Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES

Extracted: 6-mercaptopurine
KEYWORDS

serum
REFERENCE
Tsutsumi, K.; Otsuki, Y; Kinoshita, T. Simultaneous determination of azathioprine and 6-mercaptopurine in serum by reversed-phase high-performance liquid chromatography. J.Chromatogr., 1982,231, 393-399

SAMPLE

Matrix: formulations Sample preparation: Dissolve crushed tablets or the freeze-dried compound for injection in 20 mM NaOH. Add a 10 mL aliquot of this solution (or a saline injection) to 10 mL 3 mg/mL theophylline in 20 mM NaOH, inject a 1.5 |xL aliquot.
HPLCVARIABLES

Column: 100 X 5 5 |xm ODS-Hypersil Mobile phase: MeOH: 25 mM KH2PO4: glacial acetic acid 20:79:5 adjusted to pH 4.50 (Flush column with MeOH: water 60:40 at the end of each day.) Flow rate: 1.5 Injection volume: 1.5 Detector: UV 240
CHROMATOGRAM

Retention time: 4.2 Internal standard: theophylline (3.5)

OTHER SUBSTANCES Simultaneous: impurities, degradation products, 6-mercaptopurine KEYWORDS stability-indicating; injections; tablets REFERENCE Fell, A.F.; Plag, S.M.; Neil, J.M. Stability-indicating assay for azathioprine and 6-mercaptopurine by reversed-phase high-performance liquid chromatography. J.Chromatogr., 1979, 186, 691-704

ANNOTATED BIBLIOGRAPHY Ding, T.L.; Benet, L.Z. Determination of 6-mercaptopurine and azathioprine in plasma by high-performance liquid chromatography. J.Chromatogr., 1979, 163, 281-288