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.
Vienneau
D S
DS
Procter and Gamble Co., Health Care Research Center, Mason, OH 45040-8006, USA.
Kindberg
C G
CG
D002021:Buffers; D002122:Calcium Chloride; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004318:Doxycycline / Q000032:analysis; D004492:Edetic Acid; D005883:Gingival Crevicular Fluid / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010510:Periodontal Diseases / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013752:Tetracycline / Q000032:analysis*
D002021:Buffers; D004492:Edetic Acid; D013752:Tetracycline; D002122:Calcium Chloride; D004318:Doxycycline