Haemolysis Effect Estimation of Doxycycline Hyclate 150 mgDelay Release Tablet in Bio-analysis (Human Plasma) by LiquidChromatography-Tandem Mass Spectrometry
A fast, simple and particular method for estimation of Doxycycline in healthy human plasma was validated using Minocycline as IS. The analyte and IS were extracted from plasma using SPE. The compound was estranged on a RP column with an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (12:88, v/v), and detected by tandem mass spectrometry in positive ion mode. The ion transition recorded in several reaction monitoring mode were m/z 294.1→225.1 for Doxycycline and m/z 286.1→217.1 for IS. Linearity in plasma was observed over the concentration range 0.3-30 ng/mL for Doxycycline. The mean recovery for Doxycycline was 83.7%, with a lower limit of quantification of 0.3 ng/mL. The coefficient of variation of the assay was less than 6.8%, and accuracy of 96.1% to 102.2%. The validated method was applied to bioequivalence study of 150 mg Doxycycline Hyclate tablet in healthy human volunteers. The validated method was used to expose study samples of bioequivalence study of 150 mg Doxycycline Hyclate delay release tablet in 36 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed, which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range. Bioequivalence was prove for test and reference using validated method to experimental samples (Figure 1).
Development and validation of a liquid chromatographic/ tandem mass spectrometric method for determination of chlortetracycline, oxytetracycline, tetracycline, and doxycycline in animal feeds.
A selective and accurate LC/MS/MS method for the simultaneous determination of chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC), and doxycycline (DC) in animal feeds was developed. Samples were extracted with Na2EDTA-McIlvaine buffer and further purified with Oasis HLB SPE columns. The purified extract was separated on an Xbridge C18 column and detected by LC/MS/MS with positive electrospray ionization in the multiple reaction monitoring mode. This method provided average recoveries of 80.9 to 119.5%, with CVs of 1.7 to 9.8% in the range of 0.5 to 50 mg/kg CTC, OTC, TC, and DC in feeds, except the average recovery of CTC was 76.0%, with a CV of 14.6% in pig feed spiked with 0.5 mg/kg CTC. The linear ranges for the four TCs determined by LC/MS/MS ranged from 0.005 to 2.5 microg/mL with a linear correlation coefficient (R2) >0.99. The LOD and LOQ for CTC, OTC, TC, and DC in pig and poultry feeds ranged from 0.003 to 0.02 and 0.01 to 0.05 microg/g, respectively. The method was successfully applied for the analysis of 30 real feed samples, and no illegal use was detected.
Systemic detection of doxycycline after local administration
Objectives. Controlled release delivery (CRD) systems are used to extend the half-life of topical antibiotics in gingival crevicular fluid (GCF), while avoiding systemic contamination with antibiotics. When multiple periodontitis sites are treated by subgingival application of a one-component copolymer gel containing 14% doxycycline, it is likely that low levels of the antibiotic can be detected in blood by high performance liquid chromatography (HPLC). Methods. Twelve patients with severe periodontitis and one single defect per patient were treated with one single subgingival application of a new one-component doxycycline gel (14%) in each defect (the UNISITE group). Furthermore, 12 patients with between 3 and 9 periodontal defects were treated with a single application of the same doxycycline gel in each defect, resulting in 3–9 applications per patient (the MULTISITE group). Doxycycline was separated and quantitatively measured with HPLC using a UV detector. Results. In saliva, the maximum doxycycline concentration of the MULTISITE patients was nearly 10-fold higher than in the UNISITE group. In GCF specimens, maximum doxycycline concentrations were the same magnitude in both the MULTISITE and UNISITE groups. Only one UNISITE patient showed detectable levels of doxycycline in blood serum (maximum application: 0.18 µg/ml). Six MULTISITE patients exhibited measurable concentrations of doxycycline in their serum samples (maximum values: 0.12–0.76 µg/ml). The mean systemic concentration following application of the doxycycline-containing gel to multiple sites was as high as 160 ng/ml within minutes following application. Within approximately 1 h, this fell to levels below the limit of detection by HPLC (<50 ng/ml). Conclusions. Systemic contamination with doxycycline after topical administration may occur even after unisite application if no periodontal dressing is used. Locally administered doxycycline can be identified in the systemic circulation at levels far below those expected to have antibacterial effects. Systemic concentration following application to a single site was always below levels capable of detection by HPLC.