KEYWORDS

serum; SPE

REFERENCE
Laizure, S.C; Holden, CL.; Stevens, R.C. Ion-paired high-performance liquid chromatographic separa-
tion of trimethoprim, sulfamethoxazole and N4-acetylsulfamethoxazole with solid-phase extraction.
J.Chromatogr., 1990, 528, 235-242

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 JJIL 5 [LgImL sulfafurazole in MeOH: water 50:
50 + 200 |xL 1 M pH 6.8 sodium phosphate buffer + 6 mL ethyl acetate, shake for 15
min, centrifuge at 600 g for 10 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 50, reconstitute the residue in 175 |LJLL MeOH: water 20:
80, vortex for 1 min, add 25 |JLL 40% trichloroacetic acid in 100 mM hydrochloric acid,
vortex for 30 s, centrifuge at 8300 g for 5 min, inject a 100 (JLL aliquot of the supernatant.

HPLCVARIABLES
Column: 250 X 4 5 \xm LiChrosorb RP18
Mobile phase: MeCN: 150 mM pH 4.85 ammonium phosphate buffer 12.6:87.4
Flow rate: 1.5
Injection volume: 100
Detector: UV 254

CHROMATOGRAM
Retention time: 10
Internal standard: sulfafurazole (21)
Limit of detection: 160 ng/mL

OTHER SUBSTANCES
Extracted: N-acetylsulfamethoxazole, sulfamethoxazole

KEYWORDS
plasma; pharmacokinetics

REFERENCE
Van der Steuijt, K.; Sonneveld, P. Concurrent analysis of methotrexate, trimethoprim, sulfamethoxazole
and their major metabolites in plasma by high-performance liquid chromatography J.Chromatogr.,
1987, 422, 328-333

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 40 |Jig/mL timolol maleate in MeOH + 500
JULL 500 mM NaOH, vortex for 10 s, add 5 mL dichloromethane: ether 2:3, shake gently
for 5 min, centrifuge. Remove the organic layer and evaporate it to dryness at 37 under
a stream of nitrogen. Dissolve the residue in 100 |xL 100 mM HCl, add 500 jxL ether,
vortex briefly, centrifuge at 3000 rpm for 5 min, inject a 10-25 |JIL aliquot of the aqueous
phase.

HPLCVARIABLES
Column: 90 X 4.6 5 |xm Hypersil ODS
Mobile phase: MeCN: buffer 35:65 (Buffer contained 30 mM sodium lauryl sulfate and 50
mM Na2HPO4 adjusted to pH 2 with orthophosphoric acid.)
Flow rate: 2