CHROMATOGRAM

Retention time: 3.14

Internal standard: sulfamethazine (4.19)

OTHER SUBSTANCES
Extracted: sulfamethoxazole

KEYWORDS
serum; SPE

REFERENCE
Moore, K.H.R; Brouwer, K.L.R. High-performance liquid chromatographic evaluation of the effect of
heat treatment on trimethoprim and sulfamethoxazole stability in serum. Ther.Drug Monit., 1995,
17, 356-360

SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 500 mg Sep-Pak with 10 mL MeOH and 10 mL
water at 3 mL/min (do not allow to dry). 5 mL Serum + 50 |xL 10 jxg/mL ormetoprim in
water, vortex for 15 s, allow to stand for 5 min, add 2 mL pH 11 NaH2PO4 (pH adjusted
with 5 M NaOH), vortex for 10 s, add 30 mL dichloromethane, shake for 10 min, extract
aqueous phase again with 20 mL dichloromethane. Combine organic layers and shake
them with 10 mL 150 mM sulfuric acid. Remove 8 mL of the aqueous layer 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
Column: 250 X 4 5 urn Inertsil C8
Mobile phase: MeCN: pH 3 ammonium formate-trifluoroacetic acid 20:80
Flow rate: 1
Injection volume: 50
Detector: MS, VG Trio 2 with a thermospray-plasmaspray LC interface, capillary probe tip
at 300

CHROMATOGRAM
Internal standard: ormetoprim

KEYWORDS
serum; cow; SPE; thermospray; plasmaspray

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 500 mg Sep-Pak SPE cartridge with 10 mL MeOH
and 10 mL water at 3 mL/min (do not allow to dry). 5 mL Serum + 50 |xL 10 |xg/mL
ormetoprim in water, vortex for 15 s, allow to stand for 5 min, add 2 mL pH 11 NaH2PO4
(pH adjusted with 5 M NaOH), vortex for 10 s, add 30 mL dichloromethane, shake for 10
min, extract aqueous phase again with 20 mL dichloromethane. Combine organic layers
and shake them with 10 mL 150 mM sulfuric acid. Remove 8 mL of the aqueous layer