SLOVENIAN VETERINARY RESEARCH

84Z. Bajc, D. Z. Doganoc, K. Šinigoj Gačniktermined as the corresponding concentration at they-intercept of the calibration curve plus 2.33-timesthe standard deviation of the within-laboratoryreproducibility at 2 µg/kg. The detection capability(CCβ) was the corresponding concentration at theCCα plus 1.64 times the standard deviation of thewithin-laboratory reproducibility at 2 µg/kg.Results and DiscussionMethod developmentMost methods published so far are based on thepost-column oxidation of LMG to MG and detectionof MG using visible light absorption. As mentionedbefore, oxidation has been commonly performed usingPbO 2(7-11). While the methods that used PbO 2reactor provide adequate sensitivity and recovery,the manually prepared lead-oxide reactor can beplagued by problems, including rapid depletionand peak broadening, which lead to a decrease inmethod sensitivity (12). For this reason we avoidedthe oxidation of LMG to MG and thus both analyteswere detected as such. MG and LMG were detectedsimultaneously, MG with an absorbance detectorand LMG with a fluorescence detector. To optimizethe analytical procedure the absorption spectrumof the MG solution and fluorescence spectra of theLMG solution was measured. According to the absorbancespectrum, the absorbance detector wasset at 618 nm. That wavelength is usually used fordetection of MG (7, 8, 11). With regard to previouslypublished methods (13, 18) and to the apex in therecorded fluorescence spectrum, the wavelength ofemission was raised from 360 to 370 nm, while thewavelength of excitation was left the same (256 nm).This change improved the sensitivity of the method.Three different analytical columns were tested foreffective resolution of MG, CV, LMG and LCV. CV isa triphenylmethane dye with very similar structureto MG and anti-parasitic and anti-microbial properties(13). On all tested columns MG and CV wereseparated with baseline resolution, but the separationof LMG and LCV was not easy to achieve. Thebest resolution of LMG and LCV was achieved on aPhenyl-Hexyl column at 27°C with a mobile phase of55% acetonitrile and 45% 0.01 M acetate buffer (pH= 3.6) and a flow-rate at 1 ml/min. At this condition,the resolution (R) between LMG and LCV was higherthan 2.5, but the retention time was relatively long(t rfor LMG was 22 min). On a SynChropak SCD-100column at 27°C with 62% acetonitrile and 38% 0.01M acetate buffer (pH = 4.1) as mobile phase and aflow-rate at 1 ml/min, near-baseline separation betweenLMG and LCV was achieved (R = 1.4) in lessthan 11 min (Figure 1). But on a PerkinElmer-HS5 C-18 column using a different mixture of ammoniumacetate buffer (0.01 M) and acetonitrile asthe mobile phase, the resolution between LMG andLCV was always less than 1. Because the aim of ourstudy was to develop a fast and economical method,we chose the SynChropak SCD-100 column, whichwas also used by Rushing and Hansen (13).Figure 1: Typical chromatogram of mixed solution (50ng/mL)We tested two procedures of sample extractionand sample clean-up. First we followed the procedureof Rushing and Hansen (13). With this procedure,extraction of MG and LMG was performedusing a mixture of ammonium acetate buffer andacetonitrile; liquid-liquid extraction into dichloromethaneand solid-phase extraction followed.With regard to the original method we decreased theweight of the sample and the volumes of chemicalsused for the extraction by a factor of two. Althoughthe method was performed more economically, theconsumption of organic solvents was still very highand the time used for sample preparation was long,especially because separatory funnels were used forliquid-liquid extraction. For these reasons we performedliquid-liquid extraction according to the proceduredescribed by Halme and co-workers (4). Thesample extraction in this method is performed withsmaller volumes of organic solvents. Instead of separatoryfunnels for liquid-liquid extraction, solventswere separated by centrifugation and the lower layerwas transferred using the pipette. All these changesreduced the time used for sample preparation anddecreased the recovery of LMG from 90% to 78%,which was still acceptable for the determination ofveterinary drug residues in food. The recovery of MGremained the same (around 50–60%).