An isocratic high-performance liquid chromatography (HPLC) procedure was developed for the quantitative determination of bromadiolone (hydroxycoumarins) in Ratitox F product – rodenticide. HPLC separation was carried out by reversed phase chromatography ODS 2 Hypersil C18 (250 mm x 4.6 mm i.e.; 5 ìm particle size), held in thermostat at 25°C. The mobile phase consisted of methanol/0.1% aqueous solution phosphoric acid (90/10v/v), with a flow rate of 1 ml/min and with UV detection at 265 nm. In order to validate the method, the following parameters have been investigated- linearity (r2 = 0.9999), range, precision, accuracy, specificity, limit of detection and limit of quantification. The described method can be successfully applied for the analysis of Ratitox F – rodenticide

Elena Gabriela Oltean

Nica, A.

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Elena G. Oltean                                    Medicamentul Veterinar / Veterinary Drug                                                           Vol. 5 (2) 2011, December 62    DEVELOPMENT AND VALIDATION OF A RP- HPLC METHOD FOR THE QUANTITATION STUDIES OF BROMADIOLONE IN RATITOX F   DEZVOLTAREA ùI VALIDAREA METODEI RP- HPLC DE DETERMINARE CANTITATIVĂ A BROMADIOLONE DIN PRODUSUL RATITOX F  Elena Gabriela Oltean, A. Nica Romvac Company SA  Summary  An isocratic high-performance liquid chromatography (HPLC) procedure was developed for the quantitative determination of bromadiolone (hydroxycoumarins) in Ratitox F product – rodenticide. HPLC separation was carried out by reversed phase chromatography ODS 2 Hypersil C18 (250 mm x 4.6 mm i.e.; 5 µm particle size), held in thermostat at 25°C. The mobile phase consisted of methanol/ 0.1% aqueous solution phosphoric acid (90/ 10 v/ v), with a flow rate of 1 ml/ min and with UV detection at 265 nm. In order to validate the method, the following parameters have been investigated- linearity (r2=0.9999), range, precision, accuracy, specificity, limit of detection and limit of quantification. The described method can be successfully applied for the analysis of Ratitox F – rodenticide.  Key words: bromadiolone, Ratitox F, reversed phase high performance liquid chromatography RP-HPLC UV - VIS, validation  Rezumat  A fost dezvoltată úi validată o metodă isocratică de lichid cromatografie de performanĠă înaltă pentru determinarea cantitativă a bromadiolone din produsul Ratitox F - raticid. Separarea HPLC a fost realizată prin cromatografie în fază inversă pe o coloana ODS 2 Hypersil C18 (mărimea particulelor 5 µm; 250 x 4.6 mm diametrul intern), termostatată la 25°C. Faza mobilă a fost metanol/ acid fosforic 0.1% solutie apoasa (90/ 10 v/ v), cu debit de 1 ml/ min. úi detecĠie UV la 265 nm. Pentru validarea metodei au fost urmăriĠi următorii parametri - linearitatea (r2=0,9999), intervalul, precizia, acurateĠea, specificitatea, cantitatea minimă decelabila LOD, cantitatea minimă masurabilă LOQ. Metoda descrisă poate fi utilizată cu succes pentru analiza compusului activ din produsul Ratitox F  Cuvinte cheie: bromadiolone, Ratitox F, UV-VIS cromatografie de lichide de performanĠă ridicată de fază inversă, validare  This paper aimed to develop and validate an HPLC sensitive applicable method to determine the quantity of bromadiolone in Ratitox F, contributing to the quality and safety  control  of  these  types  of pharmaceutical preparations.  Materials and methods  Reagents  The standard reference bromadiolone has been provided by SIGMA (Germany). Methanol, dimethylformamide and phosphoric acid have been provided by MERCK (Germany).  Ratitox F has been provided by Romvac Company and used during shelf-life.  All the chemical substances used had pharmaceutical or analytical degree. Double distilled water, filtered on 0.45 µm membrane was used.  System and chromatographic conditions  HPLC method was carried out on a LC SURVEYOR (Thermo  Electron  Corporation,  USA) provided with quaternary pump, auto sampler, 25 µl loop and UV-VIS detector – diode array (Thermo Electron Corporation, USA). The integration of chromatographic peaks has been carried out with the ChromQuest soft (Thermo Electron). The analyses have been performed by using an ODS 2 Hypersil C18 (5 µm particle size; 250 x 4.6 mm inner diameter).  The samples have been isocratically eluted in methanol and phosphoric acid aqueous solution 0.1% (90/ 10 v/ v), with flow of 1 ml/min. Each sample has been filtered before injection with PVDF 0.45 µm filter (Thermo Electron).  The injection volume of the sample was 5 µl, and detection was carried out at 265 nm, at 25°C.   Elena G. Oltean                                    Medicamentul Veterinar / Veterinary Drug                                                           Vol. 5 (2) 2011, December 63 Preparing the standard reference solutions  Bromadiolone standard working solution had a final concentration of 0.025 mg/ ml, prepared in methanol.  The standard solutions for linearity fell within the area of 0.005 – 0.05 mg/ ml starting from a stock solution of bromadiolone of 1 mg/ ml prepared in dimethylformamide. All samples have been triplicated. The stock solution of bromadiolone is kept at +4°C for one week.  Preparing the test solutions  Weigh 30 g of product and add over 5 ml dimethylformamide and keep at ultrasound for 15 minutes; add 10 ml methanol and keep at ultrasound for 15 minutes. Filter through filter paper. Before injection filter the solutions through a 0.45 µm PVDF filter.  Chromatographic method validation  After establishing the chromatographic conditions, the method has been validated by observing the following parameters: linearity, working range, precision, accuracy, limit of detection, limit of quantification, specificity and system compliance, using ICH guide.  Linearity and working range    The analytical curve has been obtained with 5 different concentrations of bromadiolone placed between 0.005–0.05 mg/ml, prepared in triplicate.  The linearity was evaluated by linear regression analysis. The system has been balanced for minimum 30 minutes. 3 replicates have been injected from each concentration of standard bromadiolone at a volume of 5 µl, in order to verify the reproducibility of the detector response at each level of concentration.   Precision The  method  precision  has  been determined through repeatability (same day) and intermediate precision (different days). The repeatability has been determined through 12 repeated analyses of the same test sample of Ratitox F, on the same day, in the same experimental conditions. The intermediate precision of the method has been determined through the analysis during 2 days (same day), and by other analyst within  the  same  laboratory  (different analysts).  Accuracy In order to certify the accuracy of the recommended method, 9 samples have been analyzed using 3 levels of concentration which cover the working range.  System compliance  In order to ensure the validity of the analytical method, the test of system compliance has been carried out. 6 samples with 0.025 mg/ ml bromadiolone have been injected on this purpose at a volume of 5 µl.  The evaluation of the system compliance has been carried out with the ChromQuest soft, by analyzing the parameters – area, retention time and asymmetry.   The analysis of bromadiolone in the product The analysis of the content in bromadiolone in Ratitox F has been carried out  under  the  developed  method recommended for validation using  the reference standard.   Results and discussions  In order to determine the quantity of bromadiolone in RATITOX F a HPLC method of reversed phase has been suggested, choosing  the  optimum  conditions  of chromatographic separation.  The analysis of the chromatograms reveals that there are no interferences between the compound of interest and the rest of the matrix constituents, the retention time being 4.292 min. The asymmetry of the peak was good, equal to 1.0. The calibration curves for bromadiolone have been formed by representing the peak area towards concentration.  The linearity has been observed in the selected reference field.  The concentration range was 20 – 200% towards the working concentration.  By applying the linear regression for the calibration  curve,  a  coefficient  of determination r2 = 0.99929 has been established. The method precision represents the degree of compliance between the results of Elena G. Oltean                                    Medicamentul Veterinar / Veterinary Drug                                                           Vol. 5 (2) 2011, December 64 the individual  tests,  through repeated application of the method on multiple samples of a homologue batch.  Repeatability has been studied by calculating the relative standard deviation (RSD) of 12 samples with a concentration of 0.1 mg/ ml bromadiolone, carried out on the same day and experimental conditions.  The intermediate precision involves the estimation of the variability of analysis when the method is used in different laboratories, on different days, by different analysts or with different equipment.  The results are detailed in Table 1.   Table1 Concentration, precision and intermediate precision in HPLC method for bromadiolone  Parameter  Value Concentration  0.025  mg/  ml RSD%  (same  day)  0.489% RSD%  (different  day)  0.458%  The accuracy of method is the degree of similarity between the results practically obtained with the method, compared to the theoretical value.  The accuracy has been determined by analyzing 9 samples with bromadiolone in concentration of 80, 100, 120% towards the suggested  working  concentration  (0.02, 0.025, 0.03 mg/ ml).  Table 2 Recovery of bromadiolone from samples analyzed through RP-HPLC  Theoretical amount mg/ ml % Recovery % Accuracy 0.02  106.666% 121.594%  0.025  126.673% 0.03  116.516%  a mean of three replicates  The analysis of data presented in Table 2 reveals that the method is accurate within the recommended range, the average recovery rate being 121.594% for the compound of interest - bromadiolone.  In order to evaluate the resolution and reproducibility of the recommended system of analysis, compliance tests have been carried out.  The results presented in Table 3 prove that the parameters are within the limits of compliance.   Table 3 Results of the system compliance test for bromadiolone  Parameter  Minimum  Maximum  RSD (%)  Status Asymmetry  0.91104  0.91993  0.384  complies Retention time  4.282  4.292  0.105  complies Area  2013881  2032493  0.377  complies  The limits of detection and quantification have been calculated, reaching the following values: Table 4 Limit of detection and limit of quantification for bromadiolone  Component  LOD  LOQ Bromadiolone  0.000384  mg/ml  0.001280  mg/ml  Conclusions  1.  The results presented for the validation of RP–HPLC method prove its accuracy, linearity and precision and show the limits of detection and quantification.  2.  The method can be successfully used for the quantification of bromadiolone as active substance.   3.  The recommended method provides the advantage  of  using  a  comfortable analytical method, which requires a simple preparation of samples. Therefore, the method can be used for the routine analysis.   Bibliography  1.  http://www.ich.org 2.  http://www.waters.com  3.  http://www.ncbi.nlm.gov.com 4. Jin, M.C., Chen, X. H., (2006).  Rapid determination  of  three  anticoagulant rodenticides in whole blood by liquid chromatography  coupled  with  electrospray ionization  mass  spectrometry.  Rapid Communications in Mass Spectrometry. 20(18), 2741-2746. 5.  http://www.thermoscientific.com 6.  Jin, M., Ren, Y., Xu, X., Chen, X.,  (2006). Determination of bromadiolone in whole blood by high-performance liquid chromatography coupled with electrospray ionization tandem mass  spectrometry.  Forensic  Science International.,171(1), 52-56. 7.  http://onlinelibrary.wiley.com 8.  http://findarticles.com 9.  http://medind.nic.in 10.  http://www.inchem.org 

Development and validation of a RP- HPLC method for the quantitation studies of bromadiolone in Ratitox F

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