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and add it to 10 mL phosphate buffer (adjusted to pH 6 with 1 M NaOH), add this to the

SPE cartridge, wash with 10 mL water, draw air through the cartridge for 1 min, elute
with 1 mL MeOH, evaporate the eluate at 50 under a stream of nitrogen, reconstitute
the residue in 1 mL mobile phase, filter (0.45 |xm), inject a 50 |xL aliquot.

HPLCVARIABLES
Guard column: C18 pre-column filter
Column: 250 X 4.6 5 |xm Spherisorb ODS(2)
Mobile phase: MeCN: MeOH: buffer 150:50:800 (Buffer was 2.5 mL triethylamine in 900
mL water, add 5 mL glacial acetic acid, make up to 1 L with water.)
Flow rate: 1.5
Injection volume: 50
Detector: UV 225

CHROMATOGRAM
Retention time: 7.5
Internal standard: ormetoprim (10)
Limit of detection: 5 ng/mL

KEYWORDS
serum; cow; SPE

REFERENCE
Nachilobe, P.; Boison, J.O.; Cassidy, R.M.; Fesser, A.C.E. Determination of trimethoprim in bovine serum
by high-performance liquid chromatography with confirmation by thermospray liquid chromatogra-
phy-mass spectrometry. J.Chromatogr., 1993, 616, 243-252

SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL C18 Bond Elut SPE cartridge with 500 |xL MeOH
then 1 mL wash solution. 500 jxL Serum + 500 |xL wash solution, vortex for 3 s, add 25
IxL 240 |xg/mL p-nitrophenol in MeOH, vortex for 3 s, add to the SPE cartridge, wash
with 1 mL wash solution, dry with vacuum applied for 30 s, elute with two 500 |xL aliquots
of MeOH: triethylamine 10:1. Combine the eluates and evaporate them to dryness under
a stream of nitrogen, reconstitute in 200 |xL mobile phase, inject a 50-100 jxL aliquot.
(Wash solution was 7 mL 3 M HCl + 1.6 g 1-octanesulfonic acid + 150 mL 100 mM
disodium citrate made up to 800 mL with water, pH adjusted to 3.00 with 3 M HCl.)

HPLCVARIABLES
Guard column: 20 X 2 40 fxm Upchurch pellicular C18
Column: 300 X 3.9 10 \xm jxBondapak C18
Mobile phase: MeCN: buffer 24:76 with 0.8 g/L 1-octanesulfonic acid (Buffer was 7 mL 3
M HCl plus 150 mL 100 mM disodium citrate made up to 760 mL with water, pH adjusted
to 3.00 with 3 M HCl.)
Flow rate: 1.5
Injection volume: 50-100
Detector: UV 230

CHROMATOGRAM
Retention time: 5.6
Internal standard: p-nitrophenol (9.3)
Limit of quantitation: 25 ng

OTHER SUBSTANCES
Simultaneous: N4-acetylsulfamethoxazole, sulfamethoxazole