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C. J. Giasson, R. Ratchkova, L. Timmer, L. Michaud; Comparison Between 2 Methods of Processing Extracts From Worn Contact Lenses Prior to Their Analysis for Lysozyme by HPLC. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4840. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To compare 2 methods of sample processing of extracts from contact lenses worn by patients prior to a gradient elution reversed-phase HPLC method in order to analyze their lysozyme contents.
A total of 37 worn contact lenses incubated in a 50:50 solution of 0.2% trifluoroacetic acid (TFA): acetonitrile (ACN) for a minimum of 17 hours in order to extract protein. Extracts from each contact lenses were separated into 2 aliquots. Aliquots no 1 were injected without any modification into a Varian HPLC according to the method of Keith et al (50 µL, half-loop mode) (Method 1). Aliquots no 2 were evaporated under vacuum, after which the remaining solids were dissolved into a 0.1% TFA solution of 15% ACN:85% water (initial mobile phase) to produce an enrichment factor of 8, and then injected (10 µL, µL pick-up mode) (Method 2) into the system. Proteins from the extracts (M1) and enriched extracts (M2) were separated by gradient elution with 0.1% TFA in ACN (eluant A) and 0.1% TFA in water (eluant B) under similar conditions. The initial gradient of 15% of eluant A ramped to reach 65% at 6 minutes on a C18 TSK NPR (4.6 x 35 mm) column of a HPLC system equipped with a UV-visible diode array detector set at 220 nm. Lysozyme contents of contact lens extracts were calibrated with the linear regression equation of lysozyme peak surface area as a function of known lysozyme content of the injected external standards.
Seven extracts from lenses had measurable amounts of lysozyme. Lysozyme eluted at 4.73 ± 0.10 minutes on average in both methods. After correction for the different volumes injected and enrichment factor, lysozyme levels averaged for the 7 lenses 20.5 ± 18.2 µg per injection (range: 0.14-39.0 µg) in the enriched extract (M2) and 15.0 ± 13.3 µg per injection (range: 0.04-29.1 µg) in the unmodified extract (M1). This difference was statistically significant as tested with paired t tests. On average, lysozyme levels were 1.37 times higher in the enriched extracts compared to the unmodified extract.
A step of enrichment of the protein extract coupled with solubilization into the initial mobile phase can be useful to improve sensitivity of this HPLC method compared to a method that injects the unmodified extract in the HPLC. Furthermore, because proteins in the enriched extracts are solubilized into a mixture identical to the initial mobile phase of the chromatogram, presumably there will be less unpredictable chemical interactions than those which occur in the unmodified extract that contains a proportion of solvents different than those found in the initial mobile phase.
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