Affiliations: Institute of Microbiology and Biomedical Sciences,
Faculty of Medicine, Università Politecnica delle Marche, Ancona,
Italy | Institute of Biochemistry, Faculty of Medicine,
Università Politecnica delle Marche, Ancona, Italy | Institute of Legal Medicine, Faculty of Medicine,
Università Politecnica delle Marche, Ancona, Italy
Note: [] Address for correspondence: Dr. Maurizio Battino, PhD, DSc, MD
Hon, Inst. of Biochemistry, Faculty of Medicine, Università Politecnica
delle Marche, Via Ranieri, 65, 60100 Ancona, Italy. Tel.: +39 071 2204646; Fax:
+39 071 2204398; E-mail: [email protected]
Abstract: A novel isocratic modified column-switching HPLC method for
automated quantitative analysis of Vitamin E and Coenzyme Q, in the reduced and
oxidized form, is described. Many column-switching HPLC methods are found in
the literature, also for determining antioxidant substances, but we developed a
different system of column-switching. An empty column, 5 cm long, was connected
to the switching valve, before the sample loop and the extraction column. The
sample loop was connected directly after the empty column. The inserted column,
containing about 1.4 ml of the extraction eluent simulated a gradient elution,
enhancing sensitivity and resolution. When switching the columns, the empty
column is placed right before the extraction column and acts as a static mixer
for the extraction phase and the incoming analytical phase. Samples were
cleaned from interfering compounds by transfer onto a extraction-column, using
a C-8 silica. Separation was performed onto an analytical column C18 3
μm, 150 mm × 4.6 mm at a flow
rate of 1.0 ml/min with 20 mmol/l lithium perchlorate/perchloric acid, pH3.0 in
Ethanol as analytical eluent. Detection was performed with a ESA Coulochem 5100
A model. The method was found to be suitable for automated analysis of Coenzyme
Q, reduced and oxidized form, and Vitamin E in serum.
