ABSTRACT
In-tube solid phase microextraction (IT-SPME) coupled on-line to capillary liquid chromatography with diode array detection provides a simple and fast analytical methodology for the simultaneous quantitation of caffeine and its three primary metabolites (theobromine, paraxanthine and theophylline) in micro samples of serum, saliva and urine matrices. The sample amount required for one analysis was only 2.5⯵L of saliva, 6.25⯵L of serum or 40⯵L of urine, a requirement for its implementation in a hospital laboratory for preterm newborns, where sample availability is a major problem. In standard conditions, 25⯵L of diluted saliva or serum (or 100⯵L of urine) were processed by IT-SPME in 30â¯cm of commercially available capillary GC column coated with ZB-FFAP (100% nitroterephthalic modified polyethylene glycol). The retained compounds were desorbed from the IT-SPME capillary by the mobile phase (a gradient mixture of water and methanol) and the separation was carried out in a C18 column (150â¯mmâ¯×â¯0.5â¯mm i.d., 5⯵m particle size). Analytes eluted before 14â¯min, at a flow rate of 15⯵Lâ¯min-1, and were detected by absorbance at 275â¯nm. The calibration graphs presented good linearity (R2â¯>â¯0.99), without the presence of matrix effect, and recoveries between 84 and 112% were obtained. Limits of detection (S/Nâ¯=â¯3) were 0.1⯵g·mL-1 in serum and 0.5⯵g·mL-1 in saliva and urine samples, for all compounds, and the intra- and inter-day variation coefficients (nâ¯=â¯3) were between 3 and 17%. Analytical figures of merit were similar to those proposed by other methodologies, but using lower sample volume and a faster and simpler sample treatment and analysis. Paired samples of serum and saliva from preterm newborns treated with caffeine at the pediatric intensive care unit were analyzed by the method, with statistically equivalent results for caffeine concentrations.