Veterinary Formulations Research Articles

Stability-indicating HPLC-DAD method for the simultaneous determination of fluoroquinolone in combination with a non-steroidal anti-inflammatory drug in pharmaceutical formulation

We developed and validated a stability-indicating assay method for the simultaneous determination of enrofloxacin and piroxicam in combination and in the presence of degradation products. Reversephase high-performance liquid chromatography analyses were carried out on a Vertisep C18 column and acetonitrile-water (48:52 v/v, pH 3.0) mobile phase with a 1.00 mL min−1 flow rate. The efficient chromatographic separation of these drugs and their forced degradation products was achieved in less than 5 min with a peak purity match factor higher than 950. The method used showed linearity in the concentration ranges of 0.25 to 16.0 μg mL−1 for enrofloxacin (r = 0.9997) and 0.125 to 8.0 μg mL−1 for piroxicam (r = 0.9999) as well as precision (relative standard deviation lower than 2%), accuracy (mean recovery 100 ± 2%), and robustness, according to ICH (International Conference on Harmonization) and AOAC (Association of Official Analytical Chemists) guidelines. This method can simultaneously determine the combination of these drugs in a veterinary formulation and separate the drug peaks from their forced degradation products. Additionally, its optimized chromatographic conditions can contribute to the quality control of this formulation in pharmaceutical manufacturing plants and minimize waste from the organic solvent.

Spectrophotometric determination of niclosamide Via Schiff ’s base formulations in pharmaceutical and veterinary preparations

Introduction: Niclosamide(NICS) its chemical name 5-chloro-N-(2- chloro-4-nitrophenyl)-2-hydroxybenz-amide]is the only commercially existing molluscicide optional by the WHO for large extent use in schistosomiasis be in charge of programs . NICS and its two new synthesized derivatives constructed to float on the water surface were able to kill cercariae, also obsessed promising activity in vitro nearby to an apicomplexan parasite Toxoplasma (4). Few spectrophotometric methods have been reported for the estimation of NICS as pure and in formulations, approximately these methods depend on reduction of nitro group (almost with zinc powder in acidic medium) followed by reaction with different reagents. The method based on reduction of nitro group of NICS then reaction of reduced-NICS with para- N,N dimethylaminobenzaldehyde in non-aqueous medium (methanol) to form a colored product that has been proved successfully for the estimation of NICS in pharmaceutical and veterinary formulations Material and method :All reagents used are of analytical grade and are obtained from Fluka or Aldrich , NICS wassupplied from SIGMA companies. Methanolic solution of para- N,N- dimethylanimobenzaldehyde (Fluka)3%, weighing 3 g and dissolved in 100 ml methanol in a volumetric flask. All other reagents were prepared bydissolving the propriety weight in perfect solvent. A volume in the range of 0.1 to 1.7 ml of 100 µg.ml-1RNICS solution was transferred to 10 ml calibrated flasks.2ml of PNNDMABA (3.0 %) was added, and the volume was made up to 10 ml by adding methanol. The yellow Schiff ’s base was measured at 454 nm versus a blank solution. Results and Discussion:The optimum pH for reaction of NICS with para-N, N-dimethylanimobenzaldehyde equal to 3 which resulted by mixing the components of the reaction. The absorbance increase with increasing reagent concentration (para-N,N- dimethylanimobenzaldehyde) and reached maximum on adding volume of 2.0 ml of (3%), which also gives the highest value of determination coefficient (R2).The experimental data indicated that methanol was the optimum solvent used in dilution according to high intensity of Schiff ’s base and the good stability. The formation of the yellow Schiff ’s base being complete after mixing the components of reaction and the absorbance remained constant for at least 2 hours. Conclusion: Accurate and sensitive spectrophotometric method was described for the estimation of NICS. The present method has been successfully applied for the estimation of NICS in pharmaceutical and veterinary preparations.

Optimization and validation of a new chromatographic method for the assay of veterinary formulation

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.

Impact of water hardness on oxytetracycline oral bioavailability in fed and fasted piglets.

Water hardness is a critical factor that affects oxytetracycline dissolution by chelation with cations. These interactions may lead to impaired dosing and consequently decrease absorption. Moreover, feed present in gastrointestinal tract may interact with antibiotic and alter pharmacokinetic parameters. In the present study, dissolution profiles of an oxytetracycline veterinary formulation were assessed in purified, soft and hard water. Furthermore, oxytetracycline absolute bioavailability, after oral administration of the drug dissolved in soft or hard water, was evaluated in fed and fasted piglets. A maximum dissolution of 86% and 80% was obtained in soft and hard water, respectively, while in purified water dissolution was complete. Results from in vivo study reconfirmed oxytetracycline's very low oral bioavailability. The greatest values were attained when antibiotic was dissolved in soft water and in fasted animals. Statistically significant lower absolute bioavailability was achieved when hard water was used and/or animals were fed. Moreover, Cmax attained in all treatments was lower than MIC90 of most important swine pathogens. For these reasons, the oral use of OTC formulations, that have demonstrated low oral bioavailability, should be avoided to treat systemic diseases in pigs.

Densitometric and Ratio Spectra Methods for Simultaneous Determination of Sulfaquinoxaline Sodium and Pyrimethamine in Binary Mixture

Four methods were developed for simultaneous determination of sulfaquinoxaline-Na and pyrimethamine in their combined pharmaceutical formulations. The first one was a densitometric method where chloroform-methanol (9: 1, v/v) was the developing system and the plates were scanned at 254 nm. The obtained spots appeared at Rf 0.64 and 0.35 and determined in the range of 0.5-10.0 μg/spot and 0.1-10.0 μg/spot for sulfaquinoxaline-Na and pyrimethamine, respectively. Furthermore, three spectrophotometric methods manipulating ratio spectra namely, ratio difference method, extended ratio subtraction method coupled with ratio subtraction method and mean centering method were established for the determination of the two studied drugs in the presence of propylene glycol as a solvent. Linear correlation was found over the concentration range of 2-25 μg mL-1 and 3-15 μg mL-1 for the two drugs, respectively. The proposed methods were successfully applied for analyzing the cited drugs in their veterinary pharmaceutical formulations. The obtained results were statistically analyzed and found to be in accordance with those given by a reported method. The validity of the methods was evaluated according to ICH guidelines.

Stability Indicating Assay of Florfenicol using Capillary Zone Electrophoresis in Veterinary Formulation and in Milk

A simple and fast capillary zone electrophoresis method with direct ultraviolet detection has been developed for the quantitative analysis of veterinary drug florfenicol. Migration time of 1.22 minute was achieved using 1 mM borate buffer of pH 9.3 as background electrolyte. Limit of detection and limit of quantification for this method are 3.34 μg and 19.5 μg, respectively. Stability indicating assay of florfenicol is studied by exposing the drug to various stress conditions such as acidic, basic, oxidative, thermal, and ultraviolet for inducing degradation. This method was also employed for the analysis of florfenicol in its formulation and its residue in commercially available milk.

Determination of mammalian DNA in commercial canine diets with uncommon and limited ingredients.

Over‐the‐counter (OTC) limited ingredient canine diets could be reliable alternatives to veterinary therapeutic formulations for the diagnosis and management of adverse food reaction (AFR). However, the possibility of undeclared ingredients jeopardizes the efficacious use of OTC options for medical purposes. The objective was to determine the presence of undeclared ingredients in OTC canine dry diets marketed as limited or single protein source diets. Twenty‐one OTC adult canine diets marketed as limited or single protein source diets were purchased. Multiplex PCR was used to screen for DNA of 10 mammalian species with species‐specific primers that anneal to regions of the mitochondrial cytochrome b gene. The presence of DNA from one or more species not declared on the label was identified in all 21 diets: cow (Bos taurus), pig (Sus scrofa), sheep (Ovis sp.), goat (Capra hircus) and bison (Bison bison). Twenty diets were positive for the declared protein source and one diet was negative for the declared species. Cat (Felis catus), dog (Canis sp.), horse (Equus sp.), mouse (Mus musculus) and rat (Rattus norvegicus) DNA was not identified in any samples. The presence of undeclared mammal species in OTC canine dry diets marketed as having limited or single protein source ingredients may complicate AFR diagnosis and treatment. However, PCR can detect a miniscule amount of DNA which might not be clinically significant, because the amount needed to elicit a response is unknown. Quantification of the contamination was not determined in this study, precluding discrimination of intentional adulteration from unavoidable cross‐contamination.

Gold nanoparticles coupled with graphene quantum dots in organized medium to quantify aminoglycoside anti-biotics in yellow fever vaccine after solid phase extraction using a selective imprinted polymer

The determination of kanamycin sulfate was made indirectly by measuring its effect on photoluminescent amino functionalized graphene quantum dots (GQDs-amino) associated with gold nanoparticles (AuNPs) that were produced by the reduction of AuCl4 with NaBH4 in an aqueous dispersion of GQDs-amino (obtained by the pyrolysis of citric acid and glutathione) also containing the cationic surfactant CTAB. The AuNPs-GQDs-amino-CTAB system presents a suppressed photoluminescence that is amplified in the presence of kanamycin. Under optimized experimental conditions, the photoluminescence amplification of the nanomaterial system showed a linear response as a function of kanamycin concentration, covering three orders of magnitude (10−7 to 10−5 mol L−1). The use of solid phase extraction with a cartridge packed with aminoglycoside selective molecularly imprinted polymer ensured selectivity in determinations made on yellow-fever vaccine and veterinary pharmaceutical formulations. The analytical results were statistically similar to those obtained with an HPLC-based fluorescence method (after chemical derivatization). The proposed method is a simple, sensitive and selective approach that does not involve the use of toxic reagents employed for chemical derivatization of aminoglycoside antibiotics.

Smartphone-based colorimetric determination of sulfadiazine and sulfasalazine in pharmaceutical and veterinary formulations

ABSTRACTA novel, simple, accurate, and low-cost colorimetric device based on an Android smartphone was developed for sulfonamide determination. A software program was developed to be used for analyzing the samples. Various parameters for digital colorimetric detection were investigated and optimized, such as the volume of the sample drop, the type of sample holder, the distance from the mobile phone camera to the sample holder, and the effect of ambient light. From the optimized conditions, a calibration curve was created by the intensity of blue channel for sulfonamides for the concentration range of 0.5–2.5 µg mL−1 with good linearity and a regression coefficient (R2) of 0.996. The results obtained by the smartphone method were compared with a spectrophotometric procedure at the 95% confidence level (n = 3). Both methods correlated well with a regression coefficient (R2) of 0.997. The limit of detection of both methods was equal to 0.11 µg mL−1. The developed smartphone system was successfully used for the determination of sulfonamides in pharmaceutical and veterinary formulations with recoveries of 102 and 98.7%, respectively. The developed method provides good accuracy (relative error <5%) and precision (relative standard deviation <7%) and offers simple, convenient, rapid, and inexpensive determination of sulfonamides.

Preparation and characterization of a carbon nanotube-based ceramic electrode and its potential application at detecting sulfonamide drugs

In this work, we report the construction, characterization, and the potential application of carbon-ceramic electrodes prepared with functionalized multi-walled carbon nanotubes (CCE/f-MWCNT) to detect two sulfonamide drugs: sodium sulfacetamide (SFC) and sulfadiazine (SFD). Prior to the construction of CCE/f-MWCNT, MWCNT samples were functionalized by using an acid treatment (HNO3/HClO4), and then characterized by FTIR spectroscopy, XRD, FESEM, and Raman spectroscopy. These techniques were also employed to characterize the prepared carbon-ceramic electrodes, and the obtained results demonstrated that the MWCNT were properly dispersed in the silica matrix. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy experiments showed that the f-MWCNT could enhance the electrochemical performance of the electrodes, as a consequence of its high electrical conductivity and large surface area. The antibacterial drugs were determined with CCE/f-MWCNT by employing CV and differential pulse voltammetry (DPV). A linear relationship between DPV peak currents and SFC and SFD concentrations was verified separately in the range from 9.9 to 177.0 µmol L− 1 in 0.04 mol L− 1 BR buffer solution pH 6.0. Detection and quantification limits were found to be 1.06 and 3.54 µmol L− 1 for SFC and 2.31 and 7.71 µmol L− 1 for SFD, respectively. Besides the good sensitivity, the CCE/f-MWCNT showed reproducibility and specificity, since its response was not significantly affected by the presence of electroactive interfering compounds. The promising analytical performance of the CCE/f-MWCNT was confirmed by determining SFC and SFD in commercial veterinary formulations, with percent recoveries ranging from 99.9 to 101.1% for SFC and from 99.8 to 102.2% for SFD.

Evaluation of physicochemical properties as supporting information on quality control of raw materials and veterinary pharmaceutical formulations

This study aimed to show that the physicochemical proprieties obtained by Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TG), and scanning electronic microscopy (SEM) can be useful tools for evaluating the quality of active pharmaceutical ingredients (APIs) and pharmaceutical products. In addition, a simple, sensitive, and efficient method employing HPLC-DAD was developed for simultaneous determination of lidocaine (LID), ciprofloxacin (CFX) and enrofloxacin (EFX) in raw materials and in veterinary pharmaceutical formulations. Compounds were separated using a Gemini C18 (250 mm × 4.6 mm, 5 µm) Phenomenex® column, at a temperature of 25 °C, with a mobile phase containing 10 mM of phosphoric acid (pH 3.29): acetonitrile (85.7:14.3, v/v) and a flow rate of 1.5 mL/min. Physicochemical characterization by TG, FTIR, and SEM of raw materials of LID, CFX, and EFX provided information useful for the evaluation, differentiation, and qualification of raw materials. Finally, the HPLC method was proved to be useful for evaluation of raw material and finished products, besides satisfying the need for an analytical method that allows simultaneous determination of EFX, CFX, and LID, which can also be extended to other matrices and applications.

Tissue depletion study of enrofloxacin and its metabolite ciprofloxacin in broiler chickens after oral administration of a new veterinary pharmaceutical formulation containing enrofloxacin

• A new veterinary drug formulation containing enrofloxacina was studied in broiler chickens.

Analysis of lomustine drug content in FDA-approved and compounded lomustine capsules.

OBJECTIVE To determine the lomustine content (potency) in compounded and FDA-approved lomustine capsules. DESIGN Evaluation study. SAMPLE 2 formulations of lomustine capsules (low dose [7 to 11 mg] and high dose [40 to 48 mg]; 5 capsules/dose/source) from 3 compounders and from 1 manufacturer of FDA-approved capsules. PROCEDURES Lomustine content was measured by use of a validated high-pressure liquid chromatography method. An a priori acceptable range of 90% to 110% of the stated lomustine content was selected on the basis of US Pharmacopeia guidelines. RESULTS The measured amount of lomustine in all compounded capsules was less than the stated content (range, 59% to 95%) and was frequently outside the acceptable range (failure rate, 2/5 to 5/5). Coefficients of variation for lomustine content ranged from 4.1% to 16.7% for compounded low-dose capsules and from 1.1% to 10.8% for compounded high-dose capsules. The measured amount of lomustine in all FDA-approved capsules was slightly above the stated content (range, 104% to 110%) and consistently within the acceptable range. Coefficients of variation for lomustine content were 0.5% for low-dose and 2.3% for high-dose FDA-approved capsules. CONCLUSIONS AND CLINICAL RELEVANCE Compounded lomustine frequently did not contain the stated content of active drug and had a wider range of lomustine content variability than did the FDA-approved product. The sample size was small, and larger studies are needed to confirm these findings; however, we recommend that compounded veterinary formulations of lomustine not be used when appropriate doses can be achieved with FDA-approved capsules or combinations of FDA-approved capsules.

FOURIER TRANSFORM INFRARED SPECTROSCOPY, THERMOGRAVIMETRIC ANALYSIS, SCANNING ELECTRON MICROSCOPY AS SUPPORTING TOOLS IN QUALITY CONTROL OF ANTIPARASITICS

This study shows that Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) can be used as supporting tools for the evaluation of the quality of antiparasitics. In addition, an analytical methodology was developed and validated to quantify simultaneously thiabendazole (TB), febantel (FB), toltrazuril (TZ), and fluazuron (FZ) in bulk and in their veterinary pharmaceutical formulations using reverse phase high performance liquid chromatography (RP-HPLC). In order to investigate stability, pharmaceuticals were submitted to degradation processes under different conditions, such as recommended by the International Conference on Harmonization. The chromatographic conditions were optimized and the validation parameters, such as selectivity, linearity, detection limit, quantification limit, precision, accuracy, and robustness showed results within acceptable standards. All analytes were stable in the stability assays in acid and basic media and thermal conditions, except in the oxidation process, which presented two degradation peaks. Physicochemical characterization by TGA, FTIR, and SEM of raw materials of TB, FB, TZ, and FZ provided information about the authenticity of the analytes, proving the wide applicability of the instrumental techniques. The RP-HPLC proposed method was found to be accurate, precise, and reproducible and can in addition be used for routine quality control analysis.

Functional characterization of recombinant major envelope protein (rB2L) of orf virus.

Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa 1M-D334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (~60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as co-administration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations.

Graphene-doped Bi2S3 nanorods as visible-light photoelectrochemical aptasensing platform for sulfadimethoxine detection

Bismuth sulphide (Bi2S3) nanorods doped with graphene (G) were synthesized and explored as photoactive materials for constructing a photoelectrochemical (PEC) aptasensor for sulfadimethoxine (SDM) detection. The formation of Bi2S3 nanorods and G nanosheets was observed by scanning electron microscopy (SEM) and further characterized by X-ray diffraction (XRD) spectroscopy. The PEC measurements indicated that the photocurrent response of Bi2S3 was obviously improved by doping suitable amount of G. The G-Bi2S3 composite coated electrode was utilized for fabricating a PEC aptasensor by covalently immobilizing a 5′-amino-terminated SDM aptamer on the electrode surface. Based on the specific interaction between SDM and the aptamer, a PEC sensor responsive to SDM was obtained. Under optimal conditions, the proposed sensor showed a linear photocurrent response to SDM in the concentration range of 1.0–100nM, with a low detection limit (3S/N) of 0.55nM. Moreover, the sensor showed high sensitivity, stability and reproducibility. The potential applicability of the PEC aptasensor was confirmed by detecting SDM in veterinary drug formulation and milk.

Spectrofluorimetric analysis of menbutone in veterinary formulations: Application to residue determination in bovine meat and milk

A simple, rapid, and sensitive spectroflourimetric method was developed for the determination of menbutone. The method is based on measuring the native fluorescence of the alkaline aqueous methanolic solution of the cited drug at its optimum excitation and emission wavelengths at λ em 378 nm, upon excitation at λ ex 300 nm. The method showed good linearity over the range of 0.2-2.0 µg/mL with a detection and quantitation limits of 7.25 and 24.15 ng/mL, respectively. The suggested method was successfully applied to the analysis of menbutone in its commercial veterinary formulations and the results obtained were in good agreement with those given with the manufacturer method. The method was further extended to the determination of menbutone residues in bovine meat and milk, and the results were satisfactory. The recoveries obtained were in the 98.50-102.25% range. No organic solvents were used in the extraction procedures, therefore, the proposed method can be considered as a type of 'green' chemical process.

Metabolism of aceclofenac in cattle to vulture-killing diclofenac.

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac is highly toxic to Gyps vultures, and its recent widespread use in South Asia caused catastrophic declines in at least 3 scavenging raptors. The manufacture of veterinary formulations of diclofenac has since been banned across the region with mixed success. However, at least 12 other NSAIDs are available for veterinary use in South Asia. Aceclofenac is one of these compounds, and it is known to metabolize into diclofenac in some mammal species. The metabolic pathway of aceclofenac in cattle, the primary food of vultures in South Asia, is unknown. We gave 6 cattle the recommended dose of aceclofenac (2 mg/kg), collected blood thereafter at intervals for up to 12 h, and used liquid chromatography with mass spectrometry in a pharmacokinetic analysis of aceclofenac and diclofenac in the plasma. Nearly all the aceclofenac administered to the cattle was very rapidly metabolized into diclofenac. At 2 h, half the aceclofenac had been converted into diclofenac, and at 12 h four-fifths of the aceclofenac had been converted into diclofenac. Therefore, administering aceclofenac to livestock poses the same risk to vultures as administering diclofenac to livestock. This, coupled with the risk that aceclofenac may replace diclofenac in the veterinary market, points to the need for an immediate ban on all aceclofenac formulations that can be used to treat livestock. Without such a ban, the recovery of vultures across South Asia will not be successful.

Evaluation of baseline cortisol concentration to monitor efficacy of twice-daily administration of trilostane to dogs with pituitary-dependent hyperadrenocorticism: 22 cases (2008-2012).

To evaluate use of cortisol concentration prior to ACTH stimulation (baseline) to monitor efficacy of twice-daily administration of trilostane to dogs with pituitary-dependent hyperadrenocorticism (PDH). Retrospective case series. 22 dogs with PDH. The database of a veterinary hospital was searched to identify dogs with PDH that were treated with the FDA-approved veterinary formulation of trilostane twice daily between January 1, 2008, and December 31, 2012. For each dog, signalment and details regarding each hospital visit including comorbidities, electrolyte concentrations, and clinical signs were extracted from the record. For each ACTH stimulation test performed, the respective correlations between baseline cortisol concentration and the cortisol concentration after ACTH stimulation (ACTH-stimulated cortisol concentration) and resultant decision regarding trilostane dose adjustment were determined. Excessive suppression of cortisol production was defined as an ACTH-stimulated cortisol concentration < 2.0 μg/dL. The ability of various baseline cortisol concentrations to predict whether a dog had excessive suppression of cortisol production was determined. 109 ACTH stimulation tests were performed for the 22 dogs. A baseline cortisol concentration > 3.2 μg/dL predicted that ACTH-stimulated cortisol concentration would be ≥ 2.0 μg/dL with 100% certainty; however, 14 of 64 tests with a baseline cortisol concentration > 3.2 μg/dL had an ACTH-stimulated cortisol concentration ≤ 3.2 μg/dL, which was suggestive of inadequate adrenocortical cortisol reserves. Results indicated that baseline cortisol concentration should not be used as the sole monitoring tool for management of dogs with PDH treated with trilostane twice daily.

A Validated HPLC Method for the Determination of Tiludronate Disodium on a Novel Brominated Stationary Phase

An analytical method for analysis of Tiludronate; [4-chlorothiophenyl) methylene] bisphosphonate known as Tildren® is developed without pre or post derivatization on a newly introduced halogenated stationary phase namely pentabromobenzyl column (PBr column) for the first time. The optimum HPLC conditions used are: A mobile phase composed of acetonitrile: Water: triethylamine: Acetic acid (50: 50: 0.05: 0.05; v: v: v: v), at a flow rate 0.5 mL/min, at temperature 35°C. Two wavelengths were selected from the UV-spectrum namely 197 and 267 nm. The method is linear in the range of 0.06-0.6 mg/mL with square of the regression coefficient (r2) 0.9998 at 197 nm. The limit of quantification (LOQ) is 0.040 mg/mL and the limit of detection (LOD) is 0.013 mg/mL. The method is precise with %RSD 0.41-1.36 (intra-day) and %RSD 0.38-1.91 (inter-day). The method is found to be robust at temperature 35°C ± 3°C, wavelength 197 nm ± 1 nm, pH of the aqueous phase ± 0.03 and %ACN 50% ± 1%. At 267 nm, the method is linear in the range of 0.08-0.8 mg/mL with r2 0.9997, LOQ is 0.050 mg/mL and LOD is 0.016 mg/mL. The %RSD for intra-day precision ranges from 0.96-1.74, while for inter-day precision ranges from 1.09-2.32. The method is proved robust in temperature 35°C ± 3°C, wavelength 297 nm ± 3 nm, pH of the aqueous phase ± 0.03 and %ACN 50% ± 1%. The method is accurate with % recovery ranged from 98.82-99.42% with %RSD ranging from 0.210-1.023. This method is suitable for the assay of tiludronate in both bulk and in its veterinary pharmaceutical formulations.