Quantitation of surfactant protein B by HPLC in bronchoalveolar lavage fluid.
A sensitive reversed phase HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of the hydrophobic surfactant protein B (SP-B) in human bronchoalveolar lavage fluid. Samples were extracted two times with CHCl(3):MeOH:HCl (2:3:0.005N) solution in a ratio of 1:2 by volume. The extract of the lower phase was separated on a C4 butyl silica gel column with an isocratic elution using a mobile phase, consisting of 97% methanol, 2.75% chloroform and 0.25% 0.1 M trifluoroacetic acid (by volume), at a flow rate of 1 ml/min. SP-B was detected by ELSD and quantified by comparison to an external standard. The duration of a run was 7 min, the quantification limit 30 ng and the limit of detection was at about 15 ng of SP-B. This method is suitable for the rapid routine quantification of SP-B in human bronchoalveolar lavage fluid samples.
Paschen
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Department of Pediatrics, Dr. von Haunersches Kinderspital, University of Munich, Lindwurmstrasse 4, D-80337 Munich, Germany.
Griese
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D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D037701:Pulmonary Surfactant-Associated Protein B / Q000032:analysis*; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity
D037701:Pulmonary Surfactant-Associated Protein B