Development and validation of a sensitive method for tetracycline in gingival crevicular fluid by HPLC using fluorescence detection.

Increased interest in the clinical use of antibiotics for periodontal therapy required the development of a sensitive assay for the quantitation of tetracycline in gingival crevicular fluid (GCF). An HPLC method was developed and validated for tetracycline which separates and identifies the degradation component epi-tetracycline. The HPLC assay employs a C18 reversed-phase Hypersil column with a mobile phase composed of methanol and sodium acetate buffer containing CaCl2 and EDTA disodium salt. The chromatographic separation was monitored by a fluorescent detector with an excitation wavelength of 375 nm and an emission wavelength of 512 nm. Tetracycline was extracted from GCF collected on Periopapers by addition of a methanol solution containing the internal standard, doxycycline, and the mobile phase buffer (25:75, v/v). The mean percent recovery for the extraction method was 107.8% with all the % R.S.D. below 7.5%. The mean inter- and intra-batch accuracy was 104.1 and 105.3%, respectively with a coefficient of variation of less than 9.5%. The lower limit of detection was 2.5 ng on the Periopapers. The typical GCF volumes collected were 0.1-1 microliter. The method was validated for the linear concentration range 2.5-1000 ng of tetracycline on the Periopaper. This assay for tetracycline was shown to be an accurate, precise and rugged method.