Column temperature: 50

Flow rate: 1.5

Detector: UV 280 for 5 min then UV 254

CHROMATOGRAM
Retention time: 6.75
Internal standard: heptanophenone (19.2)

OTHER SUBSTANCES
Extracted: acetazolamide, ampicillin, bromazepam, caffeine, carbamazepine, chloramphen-
icol, chlorothiazide, diazepam, droperidol, ethionamide, furosemide, isoniazid, methadone,
penicillin G, phenobarbital, phenytoin, prazepam, propoxyphene, pyrazinamide, rifampin,
trimeprazine

KEYWORDS
serum; plasma; column-switching

REFERENCE
Seifart, H.I.; Kruger, RB.; Parkin, D.P.; van Jaarsveld, P.P.; Donald, P.R. Therapeutic monitoring of
antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatography sys-
tem. J.Chromatogr., 1993, 619, 285-290

SAMPLE
Matrix: blood, CSF, gastric fluid, urine
Sample preparation: 200 |JLL Serum, urine, CSF, or gastric fluid + 300 |xL reagent. Flush
column A to waste with 500 JJLL 500 mM ammonium sulfate, inject sample onto column
A, flush column A to waste with 500 |JLL 500 mM ammonium sulfate, backflush the con-
tents of column A onto column B with mobile phase, monitor the effluent from column B.
(Reagent was 8.05 M guanidine hydrochloride and 1.02 M ammonium sulfate in water.)

HPLCVARIABLES
Column: A 40 |xm preparative grade C18 (Analytichem); B 75 X 2.1 pellicular C18 (What-
man) + 250 X 4.6 5 iJim C8 end-capped (Whatman)
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCN: isopropanol 80:20.
A:B 90:10 for 1 min, to 30:70 over 20 min.
Column temperature: 50
Flow rate: 1.5
Detector: UV 220

CHROMATOGRAM
Retention time: 6.5
Internal standard: heptanophenone (19)

OTHER SUBSTANCES
Extracted: acetaminophen, allobarbital, azinphos, barbital, brallobarbitone, bromazepam,
butethal, caffeine, carbamazepine, carbaryl, cephaloridine, chloramphenicol, chlordiaze-
poxide, chlorothiazide, chlorvinphos, clothiapine, cocaine, coomassie blue, desipramine,
diazepam, diphenhydramine, dipipanone, ethylbromphos, flufenamic acid, formothion,
griseofulvin, indomethacin, lidocaine, lorazepam, malathion, medazepam, midazolam, ox-
azepam, paraoxon, penicillin G, pentobarbital, prazepam, propoxyphene, prothiophos, qui-
nine, salicylic acid, secobarbital, strychnine, sulfamethoxazole, theophylline, thiopental,
thioridazine

KEYWORDS
serum; column-switching