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Trimethoprim 8

This document provides information for analyzing sulfamethoxazole and N-acetylsulfamethoxazole in serum samples using high-performance liquid chromatography. It describes the internal standard used, limit of detection, sample preparation methods for plasma and tissue, HPLC variables, and retention times. The document also references the original research paper describing this analytical method.

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88 views1 page

Trimethoprim 8

This document provides information for analyzing sulfamethoxazole and N-acetylsulfamethoxazole in serum samples using high-performance liquid chromatography. It describes the internal standard used, limit of detection, sample preparation methods for plasma and tissue, HPLC variables, and retention times. The document also references the original research paper describing this analytical method.

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Papaindo
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Internal standard: antipyrine (11.

0)
Limit of detection: 50 ng/mL

OTHER SUBSTANCES
Extracted: N-acetylsulfamethoxazole, sulfamethoxazole

KEYWORDS
serum

REFERENCE
Weber, A.; Opheim, K.E.; Siber, G.R.; Ericson, J.F.; Smith, A.L. High-performance liquid chromatography
quantitation of trimethoprim, sulfamethoxazole, and N4-acetylsulfamethoxazole in body fluids.
J.Chromatogr., 1983, 278, 337-345

SAMPLE
Matrix: blood, tissue
Sample preparation: Plasma. 500 jxL Plasma + 100 |xL 30 p-g/mL sulfamethazine (sul-
fadimidine) in EtOH + 150 |JLL 3% trichloroacetic acid in EtOH + 100 JJLL EtOH, vortex,
freeze at -20 for 5 min, centrifuge, freeze at -20 for 10 min, centrifuge through a Spin-
X filter tube, inject a 10 JJLL aliquot of the supernatant. Tissue. 1-3 g Tissue + 3 (muscle)
or 6 (liver) |xL 1 mg/mL sulfamethoxazole in EtOH + 2 (liver) or 3 (muscle) mL 0.7%
trichloroacetic acid in acetone, mix in Whirlmixer, sonicate for 10 min at 40, add 2 mL
10 mM pH 6 Na2HPO4, sonicate for 5 min, add 100 jxL 500 mM NaOH, add 9 (muscle)
or 10 (liver) mL dichloromethane, mix thoroughly for 1 min, centrifuge at 2240 g for 5
min. Remove 6 mL of the organic layer and evaporate it to dryness at 40under a stream
of nitrogen. Dissolve the residue in 400 (muscle) or 800 (liver) JULL MeCN: 10 mM pH 2.8
phosphate buffer 20:80, sonicate, wash with 1 mL hexane. Sonicate the aqueous phase
for 1 min, centrifuge through a Spin-X filter tube, inject a 10 JJLL aliquot of the
supernatant.

HPLCVARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-18 DB
Column: 250 X 4.6 5 ^m Supelcosil LC-18 DB
Mobile phase: MeCN: buffer 23:77 (plasma) or 20:80 (tissue) with 0.1% triethylamine
added (Buffer was 25 mM sodium phosphate and 20 mM sodium 1-hexanesulfonate, pH
adjusted to 2.8 with 5 M phosphoric acid.)
Flow rate: 0.9
Injection volume: 10
Detector: UV 270

CHROMATOGRAM
Retention time: 9 (plasma), 14 (tissue)
Internal standard: sulfamethazine (sulfadimidine) (8), sulfamethoxazole (18)
Limit of quantitation: 80 ng/g (muscle); 250 mg/mL (plasma)

OTHER SUBSTANCES
Simultaneous: sulfadiazine

KEYWORDS
plasma; fish; salmon; trout; muscle; liver

REFERENCE
Hormazabal, V.; Rogstad, A. Simultaneous determination of sulphadiazine and trimethoprim in plasma
and tissues of cultured fish for residual and pharmacokinetic studies. J.Chromatogr., 1992, 583,
201-207

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