Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography.
A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred microl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively.
Chiap
P
P
Department of Analytical Pharmaceutical Chemistry, Institute of Pharmacy, University of Liège, CHU, B36, B-4000 Liège 1, Belgium.
Piette
M
M
Evrard
B
B
Frankenne
F
F
Christiaens
B
B
Piel
G
G
Cataldo
D
D
Foidart
J-M
JM
Delattre
L
L
Crommen
J
J
Hubert
Ph
P
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors / Q000097:blood*; D012680:Sensitivity and Specificity; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet
D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors