Determination of caffeine using high-performance liquid chromatography
Gilar, M.; Perlík, F.; Patzelová, V.
Ceska a Slovenska Farmacie Casopis Ceske Farmaceuticke Spolecnosti a Slovenske Farmaceuticke Spolecnosti 43(2): 78-80
1994
ISSN/ISBN: 1210-7816 PMID: 8019829 Document Number: 439158
The present paper reports a method of caffeine assay in human saliva using high-performance liquid chromatography (HPLC). Samples of saliva were extracted into a mixture of chloroformisopropanol, the extract was evaporated at 40 degrees C and reconstituted with 100 microliters of methanol. The internal standard was 8-chlorotheophylline, the mobile phase consisted of 30 parts of methanol and 70 parts of 0.01 KH2PO4 buffer, the acidity of which was adjusted to pH = 4 conc.H3PO4. The column NUCLEOSIL C18 5 microns, 250 x 4 mm, was employ for separation, UV detection, 254 nm. The chromatograms are shown in Fig. 1. The introduced method was verified when determining caffeine concentrations in patients with liver cirrhosis and in control persons. The results are shown in Table 2. The calibration curve is linear in the range of the concentrations determined from 0.5 to 10 mg/l with the regression coefficient r1,2 = 0.990. Good reproducibility of the method is characterized by the low values of the relative standard deviations (Table 1). The reported manner of caffeine determination in saliva can be utilized to evaluate liver metabolic capacities.