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A fast, sensitive, and specific liquid chromatographic/tandem mass spectrometric method was developed and validated for determination of tetracycline in human plasma. Tetracycline and oxytetracycline [internal standard (IS)] were extracted from the plasma by protein precipitation. The mobile phase consisted of acetonitrile-formic acid 0.1% (48 52, v/v), run at a flow rate of 1 mL/min (split 1:5). Detection was performed by positive electrospray ionization in multiple reaction monitoring mode, monitoring the transitions 444.8 > 410.0 and 461.0 > 426.0 for tetracycline and IS, respectively. The analysis was performed in 3.5 min and the method was linear in the plasma concentration range of 50-6000 ng/mL. The mean extraction recoveries for tetracycline and IS from plasma were 92.14 and 94.04%, respectively. Method validation investigated parameters such as the linearity, precision, accuracy, specificity, and stability, giving results within the acceptable range. The proposed method was successfully applied for determination of tetracycline in human plasma samples to support bioequivalence studies.

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The group of tetracyclines, especially tetracycline, oxytetracycline, and chrlortetracycline, are widely used to treat human illness, in veterinary practice, and as feed additives. Because of the increased awareness among consumers, there is growing demand for faster and more sensitive methods for determination of tetracycline, especially in biological matrixes (Figure 1).
Due to the widespread use of tetracyclines, the literature on the analysis of these compounds is fairly extensive and includes methods for the extraction and analysis in various matrixes, e.g., animal tissues, urine, plasma, feces, water, and soil. In recent years, several methods for the determination of tetracyclines in human plasma have been based on liquid chromatography (LC). The methods have all used reversed-phase separation with UV and fluorescence detection. A few methods based on LC/mass spectrometric (MS) determination of tetracycline in human plasma have been published (1-4).
Bioanalytical methods used for the quantitative determination of drugs and their metabolites in biological samples are the key determinants in generating reproducible and reliable data, which, in turn, are used in the evaluation and interpretation of bioavailability, bioequivalence, and pharmacokinetic findings. It is essential to use well-characterized and fully validated analytical methods to yield reliable results which can be interpreted satisfactorily. Bioanalytical method validation includes all the experimental procedures and documentation which demonstrate that a particular method used for quantitative measurement of analytes is reliable and suitable for the intended analytical applications. Fundamental parameters that require determination are linearity, accuracy, precision, specificity, and stability (5, 6).
LC coupled with MS detection has been used for both identification and quantitation of drugs at low concentrations in raw materials, various pharmaceutical formulations, and biological matrixes, due to the improved sensitivity and specificity of this technique. Moreover, as a consequence of mass selectivity, it was expected that the time for method development and sample turnover could be significantly reduced. However, matrix ion suppression requires that most of the biological matrix constituents be removed before LC/MS/MS analysis, making sample preparation a time-consuming step in the development of an LC/MS/MS procedure (7-12).
Despite the advances in the development of highly efficient analytic instrumentation for the end point determination of analytes in biological matrixes, sample pretreatment is still necessary to extract the analyte of interest. This can be performed using solid-phase extraction (SPE), liquid-liquid extraction (LLE), or protein precipitation methods. SPE is used to increase the reproducibility and decrease the volume of organic solvents needed in comparison to LLE. However, traditional offline SPE needs either relatively large volumes of plasma or organic solvents for the elution of the analyte from the solid phase, so that concentration of the extract before injection in the LC/MS/MS system is usually necessary. In contrast, the protein precipitation method requires smaller amounts of plasma, does not need concentration of extracts before injection, and requires a shorter sample preparation time.
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The aim of the present work was to develop and validate a sensitive and fast LC/MS/MS method for determination of tetracycline in human plasma, improving the current published procedures, in order to support human clinical studies, and to demonstrate the applicability of the method for the quantitation of tetracycline in bioequivalence studies.
Experimental
Chemicals and Reagents
Tetracycline chloridrate reference standard was purchased from Brazilian Pharmacopeia (Santa Maria, Brazil) and oxytetracycline was purchased from Sigma (St. Louis, MO). Tablets containing 500 mg tetracycline were obtained from Prati, Donaduzzi & Cia Ltda (Toledo, Brazil) and Parenzyme[R] from Medley (Sao Paulo, Brazil) within their shelf-life period. HPLC grade acetonitrile, methanol, formic, and acetic acid were purchased from Tedia (Fairfield, OH). Ammonium acetate and trichloroacetic acid were purchased from Merck (Darmstadt, Germany). All chemicals used were of pharmaceutical or special analytical grade. For all the analyses, ultrapure water (Gehaka, Sao Paulo, Brazil) filtered through a 0.22 [micro]m membrane filter was used.