Colloidal gold-based immunochromatographic strip assay for the rapid detection of diminazene in milk.

Diminazene (DIM) is widely used to treat trypanosomiasis in livestock, but its residues in animal products can cause food safety problems. Thus, a rapid colloidal gold-based immunochromatographic strip assay was established to analyse DIM residue in milk samples. A highly sensitive and specific monoclonal antibody (mAb) against DIM was produced with a new synthetic immunogen by the active ester method. The titre of the prepared antibody was up to 1:1.0 × 106, the affinity constant was 2.2 × 108 L mol-1 and the half-maximal inhibitory concentration (IC50) was 0.4 ng mL-1 based on enzyme-linked immunosorbent assay (ELISA). Then, the colloidal gold-labelled mAb probe was successfully prepared and used for preparing the immunochromatographic strip. The strip showed high sensitivity and specificity, the IC50 for DIM was 5.2 ng mL-1, the limit of detection was 1.2 ng mL-1 and the linear range of detection was 1.8-15.4 ng mL-1. The average recoveries ranged from 89.5% to 91.7%, and coefficients of variation were 6.6-7.2%. The immunochromatographic strip assay can be employed in the rapid detection of DIM residue in milk samples.

Development and validation of analytical method by HPLC-DAD for determination of vasodilator active in pharmaceutical ophthalmic forms

The substance 4-Aminobenzamidine dihydrochloride (4-AD) is one of the degradation products of diminazene aceturate and has demonstrated antiglaucomatous potential. Glaucoma is the second leading cause of blindness worldwide; thus, new therapeutic alternatives must be studied, for example, the molecule 4-AD vehiculated into polymeric inserts for prolonged release. The present work aims to develop and validate an analytical method to quantify 4-AD in pharmaceutical ophthalmic forms. A HPLC was used with UV-Vis detector, at 290 ƞm and ACE® C18 column (125 × 4.6 mm, 5 µm), in which the mobile phase consists of phosphate buffer (pH 7.4) and triethylamine (30 mmol/L), under an isocratic flow of 1.0 mL/min. The retention time of 3.2 minutes was observed. The method was developed and validated in accordance with ANVISA recommendations and ICH guides. The linearity range was established between the concentrations 5 and 25 µg/mL (correlation coefficient r = 0.993). The accuracy, repeatability, and intermediate precision tests obtained a relative standard deviation less than or equal to 5%. In addition, the method was considered selective, exact. and robust, with pH being its critical factor. Therefore, the HPLC analysis method is robust and can be used to quantify 4-AD in pharmaceutical forms for ocular application.(AU)

Drug quality analysis of isometamidium chloride hydrochloride and diminazene diaceturate used for the treatment of African animal trypanosomosis in West Africa.

BACKGROUND: Diminazene diaceturate (DA) and isometamidium chloride hydrochloride (ISM) are with homidium bromide, the main molecules used to treat African Animal Trypanosomosis (AAT). These drugs can be purchased from official suppliers but also from unofficial sources like local food markets or street vendors. The sub-standard quality of some of these trypanocides is jeopardizing the efficacy of treatment of sick livestock, leading thus to economic losses for the low-resource farmers and is contributing to the emergence and spread of drug resistance. The objective of this study was to assess the quality of trypanocidal drugs sold in French speaking countries of West Africa. In total, 308 drug samples including 282 of DA and 26 of ISM were purchased from official and unofficial sources in Benin, Burkina Faso, Côte d'Ivoire, Mali, Niger and Togo. All samples were analysed at LACOMEV (Dakar, Senegal), a reference laboratory of the World Organisation for Animal Health, by galenic inspection and high performance liquid chromatography. RESULTS: The results showed that 51.90% of the samples were non-compliant compared to the standards and were containing lower quantity of the active ingredient compared to the indications on the packaging. The non-compliances ranged from 63.27% in Togo to 32.65% in Burkina Faso (61.82% in Benin, 53.84% in Mali, 50% in Côte d'Ivoire, 47.36% in Niger). The rates of non-compliance were not statistically different (P = 0.572) from official or unofficial suppliers and ranged from 30 to 75% and from 0 to 65% respectively. However, the non-compliance was significantly higher for ISM compared to DA (P = 0.028). CONCLUSIONS: The high non-compliance revealed in this study compromises the efficacy of therapeutic strategies against AAT, and is likely to exacerbate chemoresistance in West Africa. Corrective actions against sub-standard trypanocides urgently need to be taken by policy makers and control authorities.

Piloting a livestock identification and traceability system in the northern Tanzania-Narok-Nairobi trade route.

We designed and piloted a livestock identification and traceability system (LITS) along the Northern Tanzania-Narok-Nairobi beef value chain. Animals were randomly selected and identified at the primary markets using uniquely coded ear tags. Data on identification, ownership, source (village), and the site of recruitment (primary market) were collected and posted to an online database. Similar data were collected in all the markets where tagged animals passed through until they got to defined slaughterhouses. Meat samples were collected during slaughter and later analyzed for tetracycline and diminazene residues using high-performance liquid chromatography (HPLC). Follow up surveys were done to assess the pilot system. The database captured a total of 4260 records from 741 cattle. Cattle recruited in the primary markets in Narok (n = 1698) either came from farms (43.8%), local markets (37.7%), or from markets in Tanzania (18.5%). Soit Sambu market was the main source of animals entering the market from Tanzania (54%; n = 370). Most tagged cattle (72%, n = 197) were slaughtered at the Ewaso Ng'iro slaughterhouse in Narok. Lesions observed (5%; n = 192) were related to either hydatidosis or fascioliasis. The mean diminazene aceturate residue level was 320.78 ± 193.48 ppb. We used the traceability system to identify sources of animals with observable high drug residue levels in tissues. Based on the findings from this study, we discuss opportunities for LITS-as a tool for surveillance for both animal health and food safety, and outline challenges of its deployment in a local beef value chain-such as limited incentives for uptake.

Intracellular diminazene aceturate content and adenosine incorporation in diminazene aceturate-resistant Babesia gibsoni isolate in vitro.

The mechanism of the development of diminazene aceturate (DA) resistance in Babesia gibsoni is still unknown even though DA-resistant B. gibsoni isolate was previously developed in vitro. To clarify the mechanisms of DA-resistance in B. gibsoni, we initially examined the intracellular DA content in the DA-resistant isolate using high-performance liquid chromatography, and compared it with that in the wild-type. As a result, the intracellular DA content in the DA-resistant isolate was significantly lower than that in the wild-type, suggesting that the decreased DA content may contribute to DA-resistance. Additionally, the glucose consumption of the DA-resistant isolate was significantly higher than that of the wild-type, indicating that a large amount of glucose is utilized to maintain DA-resistance. It is possible that a large amount of energy is utilized to maintain the mechanisms of DA-resistance. It was reported that as the structure of DA is similar with that of adenosine, DA may be taken up by the P2 transporter, which contributes to the uptake of adenosine, in Trypanosoma brucei brucei, and that the uptake of adenosine is decreased in DA-resistant T. brucei brucei. In the present study, the adenosine incorporation in the DA-resistant B. gibsoni isolate was higher than in the wild-type. Moreover, the adenosine incorporation in the wild-type was not inhibited by the presence of DA. These results suggest that adenosine transport in B. gibsoni is not affected by DA and may not mediate DA-resistance. To clarify the mechanism of the development of DA resistance in B. gibsoni, we should investigate the cause of the decreased DA content in the DA-resistant isolate in the future.

[Simultaneous determination of diminazene aceturate and isometamidium chloride residues in cattle tissues by high performance liquid chromatography with solid-phase extraction].

A method for the simultaneous determination of trypanocidal diminazene aceturate (DIM) and isometamidium chloride (ISM) that containing benzamidine groups in cattle tissues was developed by high performance liquid chromatography (HPLC) with solid-phase extraction (SPE). The tissue samples were extracted with different proportions of water-acetonitrile, then were cleaned up by Oasis WCX cartridges. DIM and ISM were separated by HPLC with a Spherisorb CN column (250 mm×4.6 mm, 5 µm). Acetonitrile-0.05 mol/L ammonium formate solution (pH 2.4) was used as mobile phases with gradient elution. The detection wavelength of UV was set at 380 nm. The limits of detection (LODs) and the limits of quantification (LOQs) of DIM and ISM in cattle tissues were 0.01 mg/kg and 0.025 mg/kg, respectively. The correlation coefficients (r) of DIM and ISM in cattle tissues were not less than 0.9993. The average recoveries of DIM and ISM at three spiked levels were 82.2%-97.6% with the intra-day relative standard derivations (RSDs) of 0.3%-5.2% (n=5) and inter-day RSDs of 1.3%-5.2% (n=15). The method was successfully applied to the analysis of DIM and ISM in cattle tissues. The method is rapid, sensitive and repeatable for the determination of diminazene aceturate and isometamidium chloride in cattle tissues.

Dual-channel optical sensing platform for detection of diminazene aceturate based on thioglycolic acid-wrapped cadmium telluride/cadmium sulfide quantum dots.

A dual-channel optical sensing platform which combines the advantages of dual-wavelength overlapping resonance Rayleigh scattering (DWO-RRS) and fluorescence has been designed for the detection of diminazene aceturate (DA). It is based on the use of thioglycolic acid-wrapped CdTe/CdS quantum dots (Q-dots). In the absence of DA, the thioglycolic acid-wrapped CdTe/CdS Q-dots exhibit the high fluorescence spectrum and low RRS spectrum, so are selected to develop an easy-to-get system. In the presence of DA, the thioglycolic acid-wrapped CdTe/CdS Q-dots and DA form a complex through electrostatic interaction, which result in the RRS intensity getting enhanced significantly with new RRS peaks appearing at 317 and 397 nm; the fluorescence is powerfully quenched. Under optimum conditions, the scattering intensities of the two peaks are proportional to the concentration of DA in the range of 0.0061-3.0 µg mL(-1). The detection limits for the two single peaks are 4.1 ng mL(-1) and 3.3 ng mL(-1), while that of the DWO-RRS method is 1.8 ng mL(-1), indicating that the DWO-RRS method has high sensitivity. Besides, the fluorescence also exhibits good linear range from 0.0354 to 10.0 µg mL(-1) with a detection limit of 10.6 ng mL(-1). In addition, the system has been applied to the detection of DA in milk samples with satisfactory results.

A validated and stability indicating HPLC method for analysis of diminazene aceturate and antipyrine combination in a ready injectable solution.

Diminazene aceturate and Antipyrine combination therapy is widely used in veterinary medicine. A simple reverse HPLC method for the analysis of samples of a ready injectable formulation containing a mixture of active ingredients and inactive excipients has been developed. The HPLC analysis was carried out using a reversed phase (RP)-C18 (250 mm×4.0 mm, 5 µm) column. The isocratic mobile phase consisted of a mixture of acetonitrile, methanol, phosphate buffer and hexane sulfonate; the flow rate was 0.6 mL/min and ultraviolet detection was at 291 nm. This method was validated in accordance with FDA and ICH guidelines and showed good linearity, accuracy, precision, selectivity and the system suitability results were within the acceptance criteria. A stability-indicating study was also carried out and indicated that this method could be used for purity and degradation evaluation of these formulations.

Validation of an HPLC method for the simultaneous determination of diminazene diaceturate and phenazone in injectable veterinary granules and bulk powders.

A validated HPLC method for the simultaneous determination of diminazene diaceturate and phenazone has been established for the analysis of the compounds in veterinary granules for injection and in bulk powders. The compounds were separated using a Symmetry RP 18 analytical column and detected by UV absorbance at 250 nm. Linearity, accuracy as well as the intra-assay precision, inter-day precision and specificity of the method were established. The limits of detection and quantification were 3.2 and 9.7 µg mL-1 for diminazene diaceturate and 9.57 and 28.99 µg mL-1 for phenazone. Method had the potential to determine these drugs simultaneously from dosage forms without any interference with each other.

Preparation of polyclonal antibodies to diminazene and its detection in animal blood plasma.

This work was undertaken to examine the possibility of preparing polyclonal antibodies to diminazene by using colloidal gold nanoparticles as an antigen carrier and adjuvant. The antibodies prepared by us were used for immunodot determination of diminazene in calf-blood plasma. The immunochemical results agreed with those obtained by high-performance liquid chromatographic estimation of diminazene content in animal biological fluids.

Comparative application of wavelet approaches to absorption and ratio spectra for the simultaneous determination of diminazene aceturate and phenazone in veterinary granules for injection.

A comparison of two wavelet approaches, Daubechies and reverse Biorthogonal, is described for the quantitative resolution of a binary mixture of diminazene aceturate (DIMA) and phenazone (PHE) in veterinary granules for injection without any chemical separation. These two approaches were specified as db4 (a = 180) and rbior3.7 (a = 125) respectively, after testing the signal analysis parameters for the overlapping absorption spectra and ratio spectra. In the first step db4 (a = 180) was applied to the original absorbance data vector of DIMA and PHE. In the second step rbio3.7 (a = 125) was applied to the ratio spectra data vectors of DIMA using the divisor PHE. The same approach was also subjected to the ratio spectra of PHE using the divisor DIMA. The db4 (a = 180) and rbior3.7 (a = 125) calibration graphs were constructed using the transformation values obtained in the wavelet domain. In the method validation, the wavelet calibration functions were tested using synthetic mixtures and the standard addition technique. The simultaneous quantitative analysis of DIMA and PHE in the commercial veterinary preparation was achieved by the elaborated methods. The assay results were compared with each other and good agreement was observed.

Liquid chromatographic determination of diminazene diaceturate (Berenil) in raw bovine milk.

A simple liquid chromatographic (LC) method is presented for the determination of diminazene (DZ) in raw bovine milk. DZ is extracted from raw milk by chilled aqueous centrifugation and is isolated from milk components on a cyano solid-phase extraction column. DZ is eluted by using a methanol-ion pairing reagent. A Phenomenex LUNA CN column and an acetonitrile-buffered mobile phase with a counter ion are used for gradient LC. The LC effluent is monitored at a detection wavelength of 372 nm by using a deuterium lamp. Under the parameters described, the retention time of DZ is 8-10 min with a peak area response of 6.5 mAU/ng. The method demonstrated excellent precision over all levels tested (25-400 ppb) with an overall average recovery of 90.4 +/- 14.5%. The method is applicable to the monitoring of milk for DZ residues at the 25 ppb level with a limit of quantitation of 10 ppb.

Electron paramagnetic resonance dynamic signatures of TAR RNA-small molecule complexes provide insight into RNA structure and recognition.

Electron paramagnetic resonance (EPR) spectroscopy was utilized to investigate the correlation between RNA structure and RNA internal dynamics in complexes of HIV-1 TAR RNA with small molecules. TAR RNAs containing single nitroxide spin-labels in the 2'-position of U23, U25, U38, or U40 were incubated with compounds known to inhibit TAR-Tat complex formation. The combined changes in nucleotide mobility at all four sites, as monitored by their EPR spectral width, yield a dynamic signature for each compound. The multicyclic dyes Hoechst 33258, DAPI, and berenil bind to TAR RNA in a similar manner and gave nearly identical signatures. Different signatures were obtained for the acridine derivative CGP 40336A and the aminoglycoside antibiotic neomycin, which bind to different regions of the RNA. The dynamic signature for guanidinoneomycin was remarkably similar to that obtained for argininamide and is evidence for guanidinoneomycin binding to the same site as arginine 52 of the Tat protein, rather than to the neomycin binding site. The data presented here show that the dynamic signatures provide strong insights into RNA structure and recognition and demonstrate the value of EPR spectroscopy for the investigation of small molecule binding to RNA.

Determination of diminazene aceturate in pharmaceutical formulations by HPLC and identification of related substances by LC/MS.

A validated, reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous assay of diminazene aceturate, antipyrine (excipient) and diminazene impurities in pharmaceutical formulations is described. The chromatographic system consisted of a Lichrospher-60 RP-select B column with a mobile phase composition of acetonitrile-methanol-ammonium formate (pH 4.0, 20 mM) (10:10: 80 v/v/v) and UV detection at 254 nm. The method is specific, precise and accurate for the determination of diminazene in the presence of its manufacturing and degradation impurities with a limit of detection and quantification of 50 ng/ml and 10 microgram/ml (RSD<3.0%), respectively. The major manufacturing impurity [1-(4 amidino phenyl)3-(4 carbamoyl phenyl)-triazene] and a degradant (p-aminobenzamidine) of diminazene aceturate have been resolved and identified by liquid chromatography/electrospray ionization-mass spectrometry operated in a positive ion mode.

Determination by capillary zone electrophoresis of berenil, phenamidine, diampron and dibromopropamidine in serum and urine.

A quick, simple and reliable analysis method has been developed in order to determine berenil, phenamidine, diampron and dibromopropamidine by capillary zone electrophoresis in samples of serum and urine. In order to define the operation parameters in CZE, we have carried out a study on how the apparent electrophoretic mobility (mu(app)) varies when pH, buffer concentration, voltage and temperature are modified. Ohm's law plot has been studied, too. With the data obtained from this study we have determined the optimum work conditions, which are: citrate buffer 25 mM, pH=3.70, 14 kV, 30 degrees C, wavelength of the UV detector: 200 nm, capillary tube: 570 mm x 75 microm. Under these conditions, all the products appear in times between: 7.6 min phenamidine and 8.8 min dibromopropamidine, limits of detection being: berenil: 0.50, phenamidine: 0.25, diampron: 0.40 and dibromopropamidine: 0.80 microg ml(-1). We have carried out a recovery study with three kinds of extraction cartridges: Sep-pak C-18 plus, Sep-pak C-8 plus and Oasis HBL for each one of the products in blood and urine.

Pharmacokinetics of diminazene in plasma and lymph of goats.

OBJECTIVE: To characterize the pharmacokinetics of diminazene in plasma and pseudo-afferent lymph of East Africa X Galla goats. DESIGN: The efferent prescapular lymphatic duct of 3 goats was cannulated 8 weeks after surgical removal of the lymph node. Thereafter, 3.5 mg of diminazene base/ kg of body weight was administered to these goats and to 3 noncannulated goats. PROCEDURE: Using high-performance liquid chromatography, concentration of diminazene was determined in plasma and lymph collected up to 96 hours after treatment. RESULTS: Maximal concentrations of diminazene in plasma of noncannulated goats (median [range], 4.30 [4.28 to 5.01] micrograms/ml), plasma of cannulated goats (3.94 [2.94 to 4.06] micrograms/ml), and lymph (1.06¿0.73 to 1.86] micrograms/ml) were significantly different (P < 0.05); values in lymph were considerably lower than those in plasma from noncannulated and cannulated animals. Time to reach maximal concentration did not differ significantly between lymph and plasma of noncannulated and cannulated goats. Over the first 24 hours after drug administration, concentration of diminazene in plasma of noncannulated goats was generally higher than that in lymph, but thereafter was similar. Apparent volume of distribution of diminazene in the plasma of noncannulated (2.57 [1.93 to 2.60] L/kg) and cannulated (2.30 [1.04 to 2.40] L/kg goats did not differ significantly. Penetration ratio of diminazene into lymph, compared with plasma, of cannulated goats was 1.69:1. CONCLUSIONS: Disposition of diminazene in goats is characterized by higher concentration in plasma than in lymph. However, the drug persists longer in lymph than in plasma. CLINICAL RELEVANCE: The longer persistence of diminazene in lymph than in plasma may account for the enhanced therapeutic efficacy of diminazene in the early stage, compared with later stages, of a tsetse fly-transmitted trypanosome infection.