RP-HPLC assay of doxycycline in human saliva and gingival crevicular fluid in patients with chronic periodontal disease

[Display omitted] ► RP-HPLC-FLD method for determination of doxycycline was developed and validated. ► Tetracycline was used as internal standard. ► The simple extraction with ACN without the sample concentration was described. ► The proposed method was successfully applied to the analysis of DOXY in human saliva and GCF. ► Low values for LODs and LOQs allowed precise identification and quantification. A reversed-phased HPLC method with fluorescence detection was optimized and validated for determination of DOXY in human saliva and gingival crevicular fluid (GCF) with tetracycline as internal standard. Single step extraction with acetonitrile for both types of samples was performed. The separation was achieved at Zorbax Extend-C18 analytical column at 30°C. Mobile phase was consisted of an aqueous phase containing magnesium acetate, ammonium acetate, Na2EDTA, triethyl-ammonium acetate buffered to pH 7.5 with ammonium hydroxide solution and acetonitrile. The volume ratio of the buffered water mixture of salts and acetonitrile was 86:14. Fluorescence detector was set at λex=380nm and λem=520nm. Under the optimized experimental conditions, good linearity was found in the range of 5.0–250.0ng/mL for GCF with LOD of 1.63ng/mL and LOQ of 4.93ng/mL and 20.0–500.0ng/mL for saliva with LOD of 6.36ng/mL and LOQ of 19.28ng/mL. This method was successfully applied for determination of DOXY in saliva and GCF obtained from patients with chronic periodontal disease.