Optimization and validation of a new chromatographic method for the assay of veterinary formulation European Journal of Chemistry 10 (3) (2019) 218-223 European Journal of Chemistry View Journal Online View Article Online Optimization and validation of a new chromatographic method for the assay of veterinary formulation Hany Hunter Monir 1, Adel Magdy Michael 2, Christine Kamal Nessim 2,*, Yasmin Mohamed Fayez 1 and Nahla Salah Elshater 3 1 Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, 11562, Cairo, Egypt hany.hunter@pharma.cu.edu.eg (H.H.M.), yasminfayez1980@gmail.com (Y.M.F.) 2 Analytical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, 12566, 6th of October City, Egypt adel_magdy_m@yahoo.com (A.M.M.), christinekamal29@yahoo.com (C.K.N.) 3 Senior Researcher Poultry Disease Reference Laboratory, Veterinary Quality Control on Poultry Production, 12211, Cairo, Egypt nahlaelshater80@gmail.com (N.S.E.) * Corresponding author at: Analytical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, 12566, 6th of October City, Egypt. Tel: +20.2.38304002 Fax: +20.2.38334379 e-mail: christinekamal29@yahoo.com (C.K. Nessim). 10.5155/eurjchem.10.3.218-223.1880 Received: 14 April 2019 Received in revised form: 03 June 2019 Accepted: 14 June 2019 Published online: 30 September 2019 Printed: 30 September 2019 New, validated and accurate reversed phase HPLC method with UV detection has been established for simultaneous determination of a veterinary binary mixture of doxycycline hydrochloride (DOX) and tylosin tartrate (TYT). The stationary phase was ACE- 126-2546 AQ C-18 (250 × 4.6 mm i.d., 5 μm particle size) column at 25 °C, in an isocratic mode, using mobile phase containing a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid at the flow rate of 0.8 mL/min and UV detection was performed at 270 nm. The retention times were 4.02±0.01 and 5.62±0.01 mins for DOX and TYT, respectively. Selective determination of the cited veterinary drugs has been developed in their formulation. The method was found to be linear over 1-50 µg/mL for DOX and TYT with mean percentage recoveries 99.62±1.220 and 100.09±1.104%. The method was proven to be accurate, precise and specific. The obtained results were statistically compared with those of the official and reported methods; using Student’s t test, F test and one-way ANOVA, showing no significant difference with high accuracy. Specificity of the applied method was assessed by analysing the laboratory-prepared mixtures and their combined dosage form. The developed method was confirmed according to ICH guidelines. The validated method can be considered as alternative and basic method for the routine determination of this fixed dose combination with minimum sample preparation. UV detection Tylosin tartrate Veterinary binary mixture Doxycycline hydrochloride Validated RP-HPLC method Simultaneous determination Cite this: Eur. J. Chem. 2019, 10(3), 218-223 Journal website: www.eurjchem.com 1. Introduction Peptic ulcers are hurting lesions triggered by infection with a Helicobacter pylori or extra acid in the stomach. For microbial infection, the most current management is a mixture of two drugs (e.g. Doxycycline hydrochloride and Tylosin tartrate) [1]. Both drugs have been co-formulated and widely used for the cure of gastrointestinal infections triggered by tylosin and/or doxycycline sensitive microorganisms like Escherichia coli and Salmonella in poultry, calves and lambs. The examined drugs are formally listed in British Pharma- copeia (BP) [2]. Doxycycline hydrochloride, (4S, 4aR, 5S, 5aR, 6R,1 2aR)-4- (dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3, 12- dioxo-4a, 5, 5a, 6-tetrahydro-4H-tetracene-2-carboxamide; hydrochloride (Figure 1a) is a tetracycline derivative which is bacteriostatic with a broad spectrum of antimicrobial activity against aerobic and anaerobic Gram-positive and Gram- negative pathogenic bacteria and some protozoa [3]. It blocks binding of aminoacyl-tRNA to the mRNA-ribosome complex, thereby inhibiting protein synthesis. In addition, this agent has exhibited inhibition of collagenase activity. Tylosin tartrate, (2R,3R)-2,3-dihydroxybutanedioic acid;2- [(4R, 5S, 6S, 7R, 9R, 11E, 13E, 15R, 16R)-6-[(2R,3R,4R,5S,6R)-5- [(2S, 4R, 5S, 6S)-4, 5-dihydroxy-4, 6-dimethyloxan-2-yl]oxy-4- (dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-16-ethyl- 4-hydroxy-15-[[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6- methyloxan-2-yl]oxymethyl]-5, 9, 13-trimethyl-2, 10-dioxo-1- oxacyclohexadeca-11, 13-dien-7-yl]acetaldehyde (Figure 1b) belongs to the group of broad-spectrum macrolide antibiotics and is approved for the control of mycoplasmosis in poultry. It is used widely in veterinary medicine as anticoccidial feed additives in poultry and livestock, as growth promoters, for improved feed efficiency in ruminants and due to their antibiotic activities against gram-positive microorganisms also acts as inhibitor of bacterial protein synthesis. Due to its anti- inflammatory properties, it is prescribed for pets in inflame- matory issues of the bowel [4,5]. ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2019 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.10.3.218-223.1880 http://dx.doi.org/10.5155/eurjchem.10.3.218-223.1880 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.3.218-223.1880&domain=pdf&date_stamp=2019-09-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.10.3.218-223.1880 mailto:hany.hunter@pharma.cu.edu.eg mailto:yasminfayez1980@gmail.com mailto:adel_magdy_m@yahoo.com mailto:christinekamal29@yahoo.com mailto:nahlaelshater80@gmail.com mailto:christinekamal29@yahoo.com http://www.eurjchem.com/ https://pubchem.ncbi.nlm.nih.gov/compound/Doxycycline https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.3.218-223.1880&domain=pdf&date_stamp=2019-09-30� Monir et al. / European Journal of Chemistry 10 (3) (2019) 218-223 219 O NH2 O O OH OHOH OH OH N HCl HH (a) O O O O O O O O OH OH O O OH N O O OH HO (b) Figure 1. Chemical structure of doxycycline HCl (a) and tylosin tartrate (b). Various methods have been reported for the quantitative determination of DOX both in pharmaceutical preparations and biological samples based on UV-Visible spectrophoto- metry [6-10], spectrofluorimetry [11-13], chromatographic methods [14-22] and electrochemical methods [23,24]. Several analytical procedures have been reported for the quantitative determination of TYT including spectrophoto- metric method [25] and chromatographic methods [26-30]. To the best of our knowledge, the survey of literature shows that no RP-HPLC method has been published for the assay of DOX and TYT in their veterinary formulation, so there is a need for method that permits simple, accurate and precise simultaneous quantification of the proposed drugs in their solid dosage form. The aim of this work was to develop and validate new RP-HPLC method for resolving this binary mixture. Furthermore, the separation and quantification of the proposed method HPLC was simple, rapid, selective, accurate and less time-consuming (only about 6 min for a single run). The new proposed method is simple and allows rapid analysis of drugs in bulk and dosage form for quality control labora- tories. 2. Experimental 2.1. Apparatus and software Analysis was performed on a chromatographic system Agilent1200 equipped with multi-wave detector (G1365B), Quat pump (G1311A) and 1260 HiP degasser (G1322A). A chromatographic separation was achieved by ACE-126-2546 AQ C-18 (250×4.6 mm i.d., 5 μm particle size) analytical column. Data acquisitions were made with ChromNAV software C. 2.2. Materials 2.2.1. Samples DOX was kindly supplied by Pharma Swede Veterinary Company, Egypt. Its purity was found to be 99.72±0.92 according to BP 2013 [2]. TYT was kindly supplied by Pharma Swede Veterinary Company, Egypt. Its purity was found to be 99.17±0.55 according to BP 2013 [2]. 2.2.2. Veterinary dosage form Tydovet® powder was manufactured by Pharma Swede Veterinary Company, the industrial zone in the 10th of Ramadan city, Egypt, One gram of Tydovet® powder contains 133 mg doxycycline hydrochloride and 110 mg tylosin tartrate. 2.2.3. Chemicals Water and methanol (99.9%) (HPLC grade) were purchased by E. Merck, Darmstadt, Germany. Acetonitrile (99.9%) (HPLC grade) was supplied from Lab Scan Limited, Dublin, Ireland. Trichloroacetic acid was supplied from (Adwic-El Nasr Pharmaceutical Chemicals Co. Egypt). 2.3. Solutions 2.3.1. Standard stock solutions Accurately weighed 50 mg of each pure drug was dissolved in methanol in 100 mL volumetric flasks, and then the volume was accomplished to the mark with the same solvent (each, 500 μg/mL). The final prepared solutions were put in storage in a fridge at 4 °C. 2.3.2. Standard working solutions Aliquots of the prepared stock solutions were further diluted with methanol to a final volume of 100 mL. The obtained diluted solutions were used as the working solutions for DOX and TYT (each, 100 µg/mL). All solutions were put in storage in a fridge at 4 °C. 2.4. Procedures 2.4.1. Spectral characteristics DOX and TYT were scanned separately using methanol as blank in a wavelength range of 200-400 nm and maxima for each component were measured in zero-order absorption spectra. It was found that 270 nm is best wavelength for UV detection. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.218-223.1880 220 Monir et al. / European Journal of Chemistry 10 (3) (2019) 218-223 Table 1. Statistical analysis of parameters required for system suitability of RP-HPLC chromatographic method. Parameter RP- HPLC method Reference value [31] DOX TYT t R (Retention time) 4.02±0.01 5.62±0.01 - N (Column efficiency) (Theoretical plate count) 8989 17651 N > 2000 Increases with efficiency of the separation HETP (Height equivalent to theoretical plates) 0.0028 0.0014 The smaller the value, the higher the column efficiency T (tailing factor) 1.33 1.5 T = 1 for symmetric peak Rs (Experimental Resolution) 9.45 Rs > 1.5 2.4.2. Chromatographic conditions RP-HPLC was carried out at ambient temperature on ACE AQ-C18 column. The mobile phase consisted of a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid in an isocratic mode. The mobile phase was filtered using 0.45 μm Millipore membrane filter (Billerica, MA) and delivered at a flow rate of 0.8 mL/min. The injection volume was 50 μL and the detection was done at 270 nm. 2.4.3. System suitability Fifty microliters of the working solutions were injected and applied to the chromatographic conditions. The system suitability parameters including tailing factor (T), theoretical plate count (N), height equivalent to theoretical plates (HETP), and resolution (Rs) were calculated according to USP guidelines [31]. 2.4.4. Construction of calibration graphs Aliquots equivalent to 1-50 μg of DOX and 1-50 μg of TYT were accurately transferred from their working solutions (each, 100 μg/mL) into separate series of 10 mL volumetric flasks, and then the volume was completed to the mark with methanol. The corresponding chromatographic conditions were applied for these solutions and the chromatograms were recorded. The calibration graphs of DOX and TYT were constructed by plotting the relative peak area (the recorded peak area of each concentration of DOX and TYT to that of an external standard 10 µg/mL of DOX or 10 µg/mL of TYT, respectively) against the corresponding concentration at 270 nm and the regression equations were computed. The validation parameters as accuracy, precision and specificity were assessed according to ICH guidelines. 2.4.5. Analysis of laboratory-prepared mixtures For the preparation of laboratory mixtures, aliquots equi- valent to 1-50 µg of DOX and 1-50 µg of TYT were accurately transferred from their working solutions (each, 100 µg/mL) with different ratios of the two drugs into a series of 10 mL volumetric flasks, and the volume was completed to the mark with methanol and mixed well to prepare different mixtures of DOX and TYT. The chromatographic conditions of the developed method were adopted for each laboratory-prepared mixture and the concentrations of each drug were calculated from the corresponding regression equation. Each concent- ration was conducted from the average of three experiments. 2.4.6. Application to veterinary formulation Ten grams of Tydovet® powder pack were mixed thoroughly, and then one gram of it equivalent to 133 mg DOX and 110 mg TYT was accurately weighed. The weighed amount was quantitatively transferred into 100 mL beaker. The powder was extracted with 30 mL methanol for 20 min by vortex shaker then the solution was filtered using through Whatman® filter paper No. 41 into a 100 mL volumetric flask. The volume was completed with methanol. Further dilution was applied from the filtrate to obtain a final concentration claimed to be 13.3 µg/mL of DOX and 11 µg/mL of TYT. The proposed method was used for the analysis of the cited drugs in the previously prepared solution in three replicates. The concentrations of the studied drugs were calculated from the corresponding regression equations. The corresponding chromatographic conditions of the applied method were adopted for each working solution and the average of three replicates was calculated for each concentration. The concentrations of each studied drug were calculated via substitution in the corresponding regression equation. 3. Results The literature review shows that no published analytical method has been established for simultaneous determination of DOX and TYT in their bulk powders and/or their veterinary formulation. The main goal of this work is to establish new, simple, sensitive and accurate analytical method for simultaneous analysis of DOX and TYT in their bulk powders, laboratory-prepared mixtures and veterinary dosage form with satisfactory accuracy and specificity. A simple isocratic RP-HPLC method was developed for the determination of DOX and TYT in their mixtures. The best resolution was obtained by using mobile phase containing a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid as shown in Figure 2. To optimize the UV maxima, various HPLC experi- ments were performed at various wavelengths starting from 250 to 320 nm. The best response has achieved with UV detection at 270 nm. This HPLC method can represent a good analytical tool for the simultaneous analysis of DOX and TYT, especially there is no any technique has been published in the literature review. The newly proposed RP-HPLC applied a simple isocratic mobile phase in which, less time-consuming (only about 6 min for a single run). System suitability parameters of the proposed method was calculated and listed in Table 1. 3.1. Optimization of the developed method The chromatographic conditions were optimized to afford robust performance so to optimize the RP-HPLC method, it was necessary to test the effect of different variables since different developing systems with different ratios were tried especially there is no published paper on this mixture. 3.2. Solvent effect Different types of solvents were tried including distilled water, methanol and acetonitrile. The developed system was the best mobile phase and showed satisfactory results for DOX and TYT regarding selectivity as well as it gave good linearity with high correlation coefficient. Higher methanol and acetonitrile concentrations (>85%) in the mobile phase caused DOX and TYT peaks to be superimposed with inadequate separation. At lower methanol and acetonitrile concentrations (<75%), the retention time of the drugs increased. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.218-223.1880 Monir et al. / European Journal of Chemistry 10 (3) (2019) 218-223 221 Table 2. Assay parameters and validation sheet obtained by applying RP-HPLC chromatographic method. Parameters RP-HPLC method Literature DOX TYT DOX [7] TYT [29] Linearity a Slope 0.1025 0.0985 0.0387 1652.1 Intercept -0.017 0.0201 -0.0253 30311 Correlation coefficient (r) 0.9998 0.9999 0.9997 0.9996 Range (µg/mL) 1-50 1-50 6-21 10-250 Mean±SD a 99.62±1.22 100.09±1.11 100.23±1.43 102.36±1.56 RSD 1.220 1.104 1.432 1.561 Accuracy b 100.02±0.998 99.76±0.736 99.02±0.240 100.23±0.456 Repeatability c 100.42±1.099 99.91±0.962 100.21±1.211 99.45±1.244 Intermediate precision c 100.49±1.131 99.82±1.553 101.22±1.321 100.45±1.456 Specificity d (laboratory-prepared mixtures) mean±SD 98.99±1.34 99.59±1.29 100.32±1.342 - Tydovet® mean±SD 98.78±0.55 100.21±0.95 110.68±0.26 98.36±0.45 a Six calibration points, average of three experiments. b Mean±RSD. c Relative standard deviation (RSD) % of three different concentrations. d For five laboratory-prepared mixtures. Figure 2. RP-HPLC chromatogram of 20 μg/mL of DOX at 4.02 min (a) and 40 μg/mL of TYT at 5.62 min (b) using a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid at 270 nm. Figure 3. The effect of aqueous percent on the resolution of the separated drugs. Different mobile phase systems were investigated for appropriate chromatographic separation. The mobile phase selection was based on peak shape parameters, ease of preparation and run time. Acetonitrile and methanol were tried as the organic part and gave better separation and high resolution efficiency. Water containing trichloroacetic acid of different ratio was tried as the aqueous part. By varying the concentration of aqueous part in the mobile phase, it was observed that 20% of it provided the optimum resolution and gave better results as shown in Figure 3. The mobile phase was composed of a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid at the flow rate 0.8 mL/min and UV detection was performed at 270 nm and the separation was completed in 6 minutes. 3.3. Method validation The proposed method was applied for the determination of pure drugs since the statistical parameters of the regression equations, the concentration ranges and the linear equations were summarized and the satisfactory results were obtained. ICH parameters [32] are represented in Table 2. 3.3.1. Linearity The linearity of the method was evaluated by analyzing the concentrations of DOX and TYT. Each concentration was repeated three times. The concentrations of DOX and TYT range from 1-50 µg/mL. The assay was performed according to the experimental conditions previously mentioned. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.218-223.1880 222 Monir et al. / European Journal of Chemistry 10 (3) (2019) 218-223 Table 3. Statistical comparison between the results obtained by the proposed and the official methods for the determination of DOX and TYT, respectively in pure powder form. Parameters RP-HPLC method Literature Official methods b DOX TYT DOX TYT DOX TYT Mean±SD 99.62±1.22 100.09±1.11 100.23±1.43 102.36±1.56 99.72±0.92 99.17±0.55 No. of experiments 6 6 6 6 6 6 Student’s t test (2.571) a 0.9035 0.0861 1.9800 0.1254 F test (5.050) a 1.7633 3.9846 2.6700 1.2654 a Figures between parentheses represent the corresponding tabulated values of t and F at p = 0.05. b The official methods for DOX & TYT are HPLC methods. British Pharmacopoeia, Her Majesty’s Stationary Office, London, vol. I & II (pp. 552) and vol. V (pp. 6) (2013) [2]. Table 4. One way ANOVA testing for the different proposed and the official methods used for the determination of DOX and TYT in pure powdered form at the 0.05 level, the population means are not significantly different. Source DF Sum of squares Mean square F value F critical DOX Between exp. 1 0.028 0.028 0.024 4.965 Within exp. 10 11.570 1.157 TYT Between exp. 1 2.553 2.553 3.340 4.965 Within exp. 10 7.643 0.764 Table 5. One way ANOVA testing for the different proposed and the reported method used for the determination of DOX and TYT in their dosage form *. F critical F value Mean square Sum of squares DF Source 7.709 0.029 0.005 0.005 1 Between exp. DOX 0.167 0.669 4 Within exp. 7.709 4.736 9.282 9.282 1 Between exp. TYT 1.959 7.839 4 Within exp. * At the 0.05 level, the population means are not significantly different. 3.3.2. Range The calibration range was established through considera- tions of the practical range necessary according to adherence to Beer’s law and the concentration of DOX and TYT present in the veterinary preparations to give accurate precise and linear results. 3.3.3. Accuracy Accuracy of the obtained results was examined by using the developed method for analysis of another concentrations of more different blind samples of DOX and TYT in between those of regression equation to clarify that the point in between are fitted on the line of regression equation with satisfactory recovery percentage thus good mean and low standard deviation. The concentrations were obtained from the corresponding regression equations, from which the percentage recoveries suggested good accuracy of the proposed method. 3.3.4. Precision Repeatability and intermediate precision express the precision under the same operating conditions over a short interval of time. They were determined using three concent- rations of each of DOX and TYT, which were analyzed three times intra-daily and inter-daily on three different days using the proposed method. The relative standard deviations were calculated. 3.3.5. Specificity Specificity of the proposed method was successfully evaluated by the analysis of different laboratory-prepared mixtures of DOX and TYT within the linearity range. Satis- factory results were shown in Table 2. 3.3.6. Stability DOX and TYT working solutions in methanol showed no spectrophotometric changes up to 4 weeks when stored at 4 °C. 3.4. Application of the methods in assay of Tydovet® powder The proposed UV methods were applied for the deter- mination of DOX and TYT in their combined formulation Tydovet® powder and the results were shown in Table 2. The good percentage recoveries confirm the suitability of the proposed methods for the routine determination of these components in their dosage form. 3.5. Statistical analysis Statistical comparison between the results obtained by the proposed method and those obtained by the official methods [2] showed no significant difference as given in Table 3. In order to compare the ability of the proposed methods for the determination of DOX and TYT, the results obtained by applying the proposed method were subjected to statistical analysis using one way ANOVA test, there was no significant difference among the proposed method and those obtained by the official methods [2] as shown in Table 4 and the reported method [7,29] as shown in Table 5. 4. Conclusion This work represents the first trial to analyze the veterinary binary mixture of DOX and TYT by simple, rapid and accurate RP-HPLC in their pure powdered form and laboratory-prepared mixtures with good accuracy and precision. A simple optimized study was done to determine the run time, the resolution and the validation of the studied method. RP-HPLC method provided rapid analysis and better separation efficiency and resolution. The linearity ranges were the same. The described method was applied successfully to determine the studied drugs in the veterinary formulation. It can be considered as an alternative tool for the routine analysis of this fixed dose combination with minimum sample preparation so it could be easily applied in quality control laboratories for the simultaneous determination of DOX and TYT without any tedious separation steps. Overall, the adopted methodology is readily transferable to quality control laboratories with advantage of rapid analysis showing high robustness using simple isocratic mobile system with no buffer solution, unlike the complicated spectrophoto- metric methods [33,34] or tedious chromatographic method 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.218-223.1880 Monir et al. / European Journal of Chemistry 10 (3) (2019) 218-223 223 [35]. The final separation conditions yielded separations that were complete in 6 min for a single run, thus resulting in minimal analytical dead time and save the reset of the system computer necessary to reset the next injection. Acknowledgements The authors would like to appreciate Pharma Swede Veterinary Company for its support in supplying the raw materials. Disclosure statement Conflict of interests: The authors have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Hany Hunter Monir http://orcid.org/0000-0001-5992-8848 Adel Magdy Michael http://orcid.org/0000-0002-9875-6003 Christine Kamal Nessim http://orcid.org/0000-0001-8857-8218 Yasmin Mohamed Fayez http://orcid.org/0000-0003-4446-0853 Nahla Salah Elshater http://orcid.org/0000-0002-1426-5312 References [1]. 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Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.218-223.1880 http://orcid.org/0000-0001-5992-8848 http://orcid.org/0000-0002-9875-6003 http://orcid.org/0000-0001-8857-8218 http://orcid.org/0000-0003-4446-0853 http://orcid.org/0000-0002-1426-5312 http://www.med.umich.edu/1info/FHP/%20practiceguides/newpud/pud.pdf http://www.med.umich.edu/1info/FHP/%20practiceguides/newpud/pud.pdf http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Apparatus and software 2.2. Materials 2.2.1. Samples 2.2.2. Veterinary dosage form 2.2.3. Chemicals 2.3. Solutions 2.3.1. Standard stock solutions 2.3.2. Standard working solutions 2.4. Procedures 2.4.1. Spectral characteristics 2.4.2. Chromatographic conditions 2.4.3. System suitability 2.4.4. Construction of calibration graphs 2.4.5. Analysis of laboratory-prepared mixtures 2.4.6. Application to veterinary formulation 3. Results 3.1. Optimization of the developed method 3.2. Solvent effect 3.3. Method validation 3.3.1. Linearity 3.3.2. Range 3.3.3. Accuracy 3.3.4. Precision 3.3.5. Specificity 3.3.6. Stability 3.4. Application of the methods in assay of Tydovet® powder 3.5. Statistical analysis 4. Conclusion Acknowledgements Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: