THE POTENTIAL OF RESTRICTED ACCESS MEDIA COLUMNS AS APPLIED IN COUPLED-COLUMN LC LC-TSP/MS/MS FOR THE HIGH-SPEED DETERMINATION OF TARGET COMPOUNDS IN SERUM - APPLICATION TO THE DIRECT TRACE ANALYSIS OF SALBUTAMOL AND CLENBUTEROL/

This study investigated the potential of restricted access media (RAM) columns used as a first column in coupled-column LC hyphenated to the rmospray tandem mass spectrometry (LC/LC-TSP/MS/MS) for the fast, sele ctive, and sensitive determination of target drugs in serum samples, B ecause of their wide range in polarity, salbutamol and clenbuterol wer e chosen as model compounds and representatives of the class of beta(2 )-agonists, Three types of RAM columns were tested: (i) Pinkerton ISRP (internal surface reversed phase, 5 mu m), (ii) SPS (semipermeable su rface, 5 mu m C18), and (iii) RP-18 ADS (alkyl-diol silica, 25 mu m). A 3-mu m C18 column (50 mm x 4.6 mm i.d.) was chosen as the second col umn, Tandem mass spectrometric detection was carried out in the select ed reaction monitoring (one parent-one daughter) mode, With regard to retention and, moreover, the peak elution volume of the analytes, the ISRP material was found to perform best: a 50-mm x 4.6-mm i.d. ISRP co lumn in combination with a 100% aqueous buffer (pH of 7.0 +/- 0.2) all owed the injection of large volumes (up to 200 mu L) of sample without additional band broadening of the analytes and provided sufficient pr eseparation between analytes and large-molecule serum constituents, Un der the selected conditions, both analytes could be determined in seru m samples up to a limit of quantification (LOQ) of 0.5 ng/mL, with a s ample throughput of 7 and 5 h(-1) for salbutamol and clenbuterol, resp ectively, Method validation was carried out by analyzing, in the cours e of several days, calf and human serum samples spiked with the analyt es. In the case of salbutamol, the overall recovery from serum samples spiked at levels between 0.5 and 50 ppb (n = 33) was 103.4%, with a r epeatability of 12.7% and reproducibility of 14.3%, The overall recove ry for clenbuterol was 99.6% (n = 15, spiked level 0.5-5 ppb), with a repeatability of 15.2% and reproducibility of 16.4%, The adopted LC/LC -TSP/MS/MS analyzer appeared to be very robust under the selected cond itions, and, after the period of analysis involving the processing of more than 100 mt of serum, neither loss of chromatographic performance nor pressure increase of columns or of the interface was observed.