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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 bicar-
bonate solution, filter, wash the solid with water, dry under vacuum to give 9-methyla-
zathioprine (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 mea-
surement 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, Well-
come 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 car-
tridge, 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, dacarba-
zine, diclofenac, etoposide, 5-fluorouracil, ifosfamide, indomethacin, lomustine, metho-
trexate, 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. (Re-
agent 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-mercaptopu-
rine 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-perfor-
mance liquid chromatography. J.Chromatogr., 1979, 163, 281-288
Azithromycin
Molecular formula: C38H72N2O12
Molecular weight: 749.0
CAS Registry No.: 83905-01 -5

SAMPLE
Matrix: blood
Sample preparation: 50 |xL Serum + 50 jxL 60 mM potassium carbonate + 50 |xL 50
ng/mL IS in MeCN water 50:50, mix, add 200 |JLL water, vortex for several s, add 3 mL
MTBE, vortex for 20 s, centrifuge at 3300 rpm for 3 min. Remove the organic layer and
evaporate it to dryness in a vortex evaporator at 40°, reconstitute the residue in 100 jxL
mobile phase, sonicate, vortex, inject a 50 |xL aliquot.

HPLCVARIABLES
Column: 30 X 4.6 3 |xm Hypersil ODS
Mobile phase: MeCN: MeOH: THF: 50 mM ammonium acetate 44:19:3:34
Flow rate: 1
Injection volume: 50
Detector: MS, SCIEX API III, atmospheric pressure ionization, nebulizer probe 450°, 2.5
|xA Corona discharge needle, quadrupole mass filter, 0.002 inch pinhole aperture, SIM
m/z 749 and 752

CHROMATOGRAM
Retention time: 1.9
Internal standard: trideuteroazithromycin
Limit of quantitation: 10 ng/mL

KEYWORDS
serum

REFERENCE
Fouda, H.G.; Schneider, R.P. Quantitative determination of the antibiotic azithromycin in human serum
by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization mass
spectrometry: Correlation with a standard HPLC-electrochemical method. Ther.Drug Monit., 1995,
17, 179-183

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 25 JULL 10 |xg/mL IS in MeCN + 1 mL 30 mM potas-
sium carbonate, vortex, add 5 mL MTBE, vortex for 30 s, centrifuge at 2000 g for 10 min.
Remove the organic layer and evaporate it to dryness under vacuum at 37°, reconstitute
the residue in 300 |xL mobile phase, vortex for 30 s, add 1 volume hexane, vortex, cen-
trifuge, remove the aqueous layer, inject a 50 jxL aliquot of the aqueous layer (J. Chro-
matogr. 1991, 565, 321).

HPLCVARIABLES
Guard column: 20 X 4.6 5 \xm Nucleosil C18
Column: 125 X 4.6 5 |xm Nucleosil C18
Mobile phase: MeCN: 40 mM Na2HPO4:5 mM tetrabutylammonium phosphate 33:50:50,
adjusted to pH 7.0 with 25% phosphoric acid
Flow rate: 1
Injection volume: 50
Detector: E, ESA 5100A Coulochem, guard cell +1 V, dual electrode analytical cell +0.7
and +0.8 V

CHROMATOGRAM
Retention time: 5.8
Internal standard: n-propyl analog of azithromycin (7.8)
Limit of quantitation: 8 ng/mL

OTHER SUBSTANCES
Extracted: metabolites

KEYWORDS
serum; pharmacokinetics

REFERENCE
Riedel, K.-D.; Wildfeuer, A.; Laufen, H.; Zimmermann, T. Equivalence of a high-performance liquid
chromatographic assay and a bioassay of azithromycin in human serum samples. J.Chromatogr.,
1992, 576, 358-362

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum -h 25 |ULL 10 jutg/mL IS in MeCN + 1 mL 30 mM potas-
sium carbonate, vortex, add 5 mL MTBE, vortex for 30 s, centrifuge at 2000 g for 10 min.
Remove the organic layer and evaporate it to dryness under vacuum at 37°, reconstitute
the residue in 300 |xL mobile phase, vortex for 30 s, add 1 volume hexane, vortex, cen-
trifuge, remove the aqueous layer, inject a 100 JJLL aliquot of the aqueous layer.

HPLCVARIABLES
Guard column: 21 X 3 40 |xm glass bead column (Waters)
Column: 50 X 4.6 5 |xm Chromegabond alkylphenyl (ES Industries)
Mobile phase: MeCN:MeOH:20 mM ammonium acetate:20 mM sodium perchlorate 45:
10:22:23, adjust apparent pH to 6.8-7.2 with glacial acetic acid
Flow rate: 1
Injection volume: 100
Detector: E, ESA 5100A Coulochem, ESA 5020 guard cell +1V, ESA 5010 dual electrode
analytical cell, screen electrode +0.7 V, detector electrode +0.8 V, porous carbon electrodes

CHROMATOGRAM
Retention time: 9
Internal standard: 9a-N-propyl analog of azithromycin (13)
Limit of quantitation: 10 ng/mL

OTHER SUBSTANCES
Extracted: metabolites

KEYWORDS
serum; human; mouse; rat; dog; rabbit

REFERENCE
Shepard, R.M.; Duthu, G.S.; Ferraina, RA.; Mullins, M.A. High-performance liquid chromatographic
assay with electrochemical detection for azithromycin in serum and tissues. J.Chromatogr., 1991,
565, 321-337
SAMPLE
Matrix: blood, tissue
Sample preparation: Serum. 500 JXL Serum + 25 |xL 2 [xg/mL IS in MeCN + 500 |JLL 60
mM potassium carbonate, vortex, add 5 mL MTBE, vortex for 30 s, centrifuge at 2000 g
for 10 min. Remove the organic layer and add it to 500 JJLL 15 mM pH 3.1 citric acid,
vortex for 30 s, centrifuge at 2000 g for 5 min. Remove the aqueous layer and add it to
1 mL 60 mM potassium carbonate, vortex for 1 min, add 5 mL MTBE, vortex for 30 s,
centrifuge at 2000 g for 10 min. Remove the organic layer and evaporate it to dryness
under vacuum at 37°, reconstitute the residue in 300 fxL MeCN: water 50:50, vortex for
30 s, add 1 volume hexane, vortex, centrifuge, remove the aqueous layer, inject a 60 JULL
aliquot of the aqueous layer. (For high azithromycin concentrations extraction into citric
acid and back extraction is not necessary.) Tissue. 1 g Tissue + 9 volumes MeCN + 50
IxL 20 fxg/mL IS in MeOH, homogenize (Polytron PT 10/35) for 10 s, centrifuge at 2000 g
for 10 min. Remove a 500 u,L aliquot of the supernatant and evaporate it to dryness
under vacuum at 50°, reconstitute the residue 500 |JLL 60 mM potassium carbonate, add
5 mL MTBE, vortex for 30 s, centrifuge at 2000 g for 10 min. Remove the organic layer
and evaporate it to dryness under vacuum at 37°, reconstitute the residue in 300 |xL
MeCN: water 50:50, vortex for 30 s, add 1 volume hexane, vortex, centrifuge, remove the
aqueous layer, inject a 20-60 |JLL aliquot of the aqueous layer.

HPLCVARIABLES
Guard column: 21 X 3 40 fim glass bead column (Waters)
Column: 150 X 4.6 5 jjim Chromegabond 7-RP-l alumina (ES Industries)
Mobile phase: MeCN: 50 mM potassium phosphate 30:70, adjusted to an apparent pH of
11.0 with 1 M KOH
Flow rate: 1
Injection volume: 60
Detector: E, BAS LC-4B (Bioanalytical Systems), glassy carbon electrode +0.8 V, Ag/AgCl
reference electrode

CHROMATOGRAM
Retention time: 8
Internal standard: 9a-N-propargyl analog of azithromycin (10)
Limit of detection: 100 ng/g (tissue)
Limit of quantitation: 10 ng/mL (serum)

OTHER SUBSTANCES
Extracted: metabolites

KEYWORDS
serum; human; mouse; rat; dog; rabbit; brain; muscle; liver; kidney

REFERENCE
Shepard, R.M.; Duthu, G.S.; Ferraina, R.A.; Mullins, M.A. High-performance liquid chromatographic
assay with electrochemical detection for azithromycin in serum and tissues. J.Chromatogr., 1991,
565, 321-337