Combining restricted access material (RAM) and turbulent flow for the rapid on-line extraction of the cyclooxygenase-2 inhibitor rofecoxib in plasma samples.
Restricted access material (RAM) has been used in the packing of a solid-phase extraction (SPE) column for on-line extractions under turbulent flow conditions. The bio-compatible RAM material works by the principle of size exclusion in addition to conventional reversed-phase chromatography, thereby allowing the extraction and preconcentration of small analyte molecules from biological samples such as plasma. Using small column dimensions (0.76 mm x 50 mm) and a consequently high linear velocity, turbulent flow was achieved during online sample extractions. The improved mass-transfer rate characteristic of turbulent flow allows fast sample cleanup without decreased extraction efficiency. The novel use of the RAM column, connected upstream to a C18 monolithic column, allowed the direct injection, extraction, separation, and MS/MS detection of plasma samples spiked with rofecoxib in a span of 5 min. Calibration curves obtained using this RAM turbulent flow coupled column method showed good linearity (R2 > 0.99) and reproducibility (%RSD < or = 7%). The lower limit of quantitation of rofecoxib in plasma samples was found to be 40 ng/ml. The extraction method showed good recovery of rofecoxib from a plasma matrix with minimal signal loss and robustness after more than 200 plasma injections.
Vintiloiu
Anda
A
Pharmaceutical Research and Development, Merck Frosst Center for Therapeutic Research, 16711 Trans-Canada Highway, Kirkland, Quebec, Canada.
Mullett
Wayne M
WM
Papp
Robert
R
Lubda
Dieter
D
Kwong
Elizabeth
E
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017022:Flow Injection Analysis / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007783:Lactones / Q000097:blood* / Q000302:isolation & purification*; D009862:Online Systems; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013450:Sulfones / Q000097:blood* / Q000302:isolation & purification*
D007783:Lactones; D013450:Sulfones; C116926:rofecoxib