Oxytetracycline (OTC) has been used for years in medical field. Chemically, it is 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro3,5,6,10,12,12a-hexahydroxy-6-methyl-
1,11-dioxo-2-naphthacenacarboxamide. OTC is bacteriostatic agent with broad-spectrum of antimicrobial activity. OTC is used in the treatment of respiratory, urinary and alimentary tract infections, because of its activity, low toxicity and good penetration into tissues [2,3].
This paper presents a simple, highly sensitive, rapid and economical HPLC method for determination of OTC in plasma.
The chromatographic system used was a Varian liquid chromatograph (Varian, Walnut Creek, CA, USA). It consisted of a solvent delivery pump (STAR 9002), a 10 µL volume manual injector, a variable wavelength UV-VIS detector (all Varian Analytical Instruments, USA). Chromatographic separations were performed using a Varian ChromSep HPLC OmniSpher 5 C18 (250 × 4.6 mm) column. A centrifuge (MPW 210), an analytical balance (Sartorius BP 61S), cartridges (Shimadzu C18, 500 mg), a vacuum pump (AGA Labor, Warsaw, Poland), Vortex (WL-1, Bio-mix, Warsaw, Poland), and extraction chamber SPE (Varian, USA, 16 × 75 mm) were also used.
Sample preparation:1 mL of plasma was mixed with 1 mL of methanol. After centrifugation (15 min. at 5500 rpm) the upper supernatant layer was discharged. The solution was diluted to 30 ml using the buffor 0.01 M EDTA-McIlvaine. Next following reagents were applied to a cartridge (in reported sequence): methanol, deionized water, buffor, an analyte and buffor. After this analyte was eluted using ACN-buffor 0.01M EDTA-McIlvaine solution. Sample was evaporated and redissolved in mobile phase, and the analysis was preformed by HPLC. A mobile phase consisted of ACN-MeOH-(HCOO)2 (17.5/17.5/65, v/v/v) (pH=2) was pumped at a flow rate of 1.4 mL/min. The variable wavelength UV detector was set at 280 nm [1,4]. The method was validated by the determination of the following parameters: linearity (r=0.999) precision and accuracy (n=4), limit of detection (LOD=3.58 ng/mL) and limit of quantification (LOQ=11.93 ng/mL). The equation for the curve was
y = 55.035 x + 28.875. The calculated standard deviation (SD) was 1.40 ng/mL and relative standard deviation (RSD) was 1.39 %. The calculated of OTC recovery was 92.50%.
Described HPLC method for the measurement of OTC in plasma was fully validated and showed good sensitivity, reproducibility, linearity and selectivity. This makes it valuable and adequate in many applications, particularly in veterinary medicine studies. Other authors determined residues of tetracyclines (including oxytetracycline) in animal tissues, milk and cheese using HPLC method. According to our best knowledge the recovery has never reached the level of 90%. As was already mentioned the recovery was 92.50%.
It can be concluded that the developed method in the present study can be successfully applied for analysis of OTC in plasma.
References:
1. Sun Y. et al., J Contr. Rel., 2002, 85, 125-134.
2. El Korchi G. et al., J. Vet. Pharmacol. Therap., 2001, 24, 274-250.
3. Dowling P.M., Russell A.M., J. Vet. Pharmacol. Therap., 2000, 23, 107-110.
4. Oka, H., Ito Y., Matsumoto H., J.Chromatogr. A, 2000, 882, 109-133. |