Cytotoxicity of cobalt chloride in brain cell lines - a comparison between astrocytoma and neuroblastoma cells.

High levels of circulating cobalt ions in blood have been reported to induce systemic reactions in patients with metal-on-metal (MoM) hip implants. We still lack information regarding these adverse effects, which may specifically impact on patients showing adverse neurological symptoms. To investigate this, we used a battery of in vitro viability and proliferation assays to identify toxic cobalt chloride (CoCl2) concentrations in two different brain cell types: SH-SY5Y neuroblastoma and U-373 astrocytoma cells. Cobalt cytotoxicity was characterised by MTT and Neutral Red (NR) assays at concentrations ranging from 0 to 500 µM after 24, 48, and 72 h exposure. MTT and NR showed a dose- and time-dependent toxicity with cobalt decreasing cell viability at high concentrations. IC50s for MTT at 72 h (astrocytes: 333.15 ±â€¯22.88; neurons: 100.01 ±â€¯5.91 µM) and for BrdU proliferation assays (astrocytes: 212.89 ±â€¯9.84; neurons: 88.86 ±â€¯19.03 µM) demonstrate that SH-SY5Y neurons are significantly more vulnerable to cobalt than astrocytes. Increased BrdU and MTT assay sensitivity suggested that DNA synthesis and metabolism disruption were involved in Co toxicity. Intracellular cobalt level measured by ICP-MS was significant after 100 µM treatment. Astrocytes displayed improved resistance to cobalt toxicity and higher uptake, which may reflect their neuroprotective nature. In summary, exposure to high concentrations of extracellular cobalt has deleterious effects in neurons and astrocytes, with neurons showing particular sensitivity.

Development and mechanical characterisation of self-compressed collagen gels.

Collagen gels are considered a promising biomaterial for the manufacturing of tissue engineering scaffolds, however, their mechanical properties often need to be improved to enable them to provide enough mechanical support during the course of tissue regeneration process. In this paper, we present a simple self-compression technique for the improvement of the mechanical properties of collagen gels, identified by the fitting of bespoke biphasic finite element models. Radially-confined highly hydrated gels were allowed to self-compress for 18h, expelling fluid, and which were subsequently subjected to unconfined ramp-hold compression. Gels, initially of 0.2%, 0.3% and 0.4% (w/v) collagen and 13mm thickness, transformed to 2.9±0.2%, 3.2±0.3% and 3.6±0.1% (w/w) collagen and 0.45±0.06mm, 0.69±0.04mm and 0.99±0.07mm thickness. Young's moduli of the compressed gels did not increase with increasing collagen fibril density, whilst zero-strain hydraulic permeability significantly decreased from 51 to 21mm4/Ns. The work demonstrates that biphasic theory, applied to unconfined compression, is a highly appropriate paradigm to mechanically characterise concentrated collagen gels and that confined compression of highly hydrated gels should be further investigated to enhance gel mechanical performance.

Inactivation of micro-organisms isolated from infected lower limb arthroplasties using high-intensity narrow-spectrum (HINS) light.

High-intensity narrow-spectrum (HINS) light is a novel violet-blue light inactivation technology which kills bacteria through a photodynamic process, and has been shown to have bactericidal activity against a wide range of species. Specimens from patients with infected hip and knee arthroplasties were collected over a one-year period (1 May 2009 to 30 April 2010). A range of these microbial isolates were tested for sensitivity to HINS-light. During testing, suspensions of the pathogens were exposed to increasing doses of HINS-light (of 123mW/cm(2) irradiance). Non-light exposed control samples were also used. The samples were then plated onto agar plates and incubated at 37°C for 24 hours before enumeration. Complete inactivation (greater than 4-log10 reduction) was achieved for all of the isolates. The typical inactivation curve showed a slow initial reaction followed by a rapid period of inactivation. The doses of HINS-light required ranged between 118 and 2214 J/cm(2). Gram-positive bacteria were generally found to be more susceptible than Gram-negative. As HINS-light uses visible wavelengths, it can be safely used in the presence of patients and staff. This unique feature could lead to its possible use in the prevention of infection during surgery and post-operative dressing changes. Cite this article: Bone Joint J 2015;97-B:283-8.

Toxicity of cobalt-chromium nanoparticles released from a resurfacing hip implant and cobalt ions on primary human lymphocytes in vitro.

Adverse tissue responses to prostheses wear particles and released ions are important contributors to hip implant failure. In implant-related adverse reactions T-lymphocytes play a prominent role in sustaining the chronic inflammatory response. To further understand the involvement of lymphocytes in metal-on-metal (MoM) implant failure, primary human lymphocytes were isolated and treated with cobalt-chromium (Co-Cr) wear debris and Co ions, individually, and in combination, for 24, 48 and 120 h. There was a significant increase in cell number where debris was present, as measured by the Neutral Red assay. Interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) secretion levels significantly decreased in the presence of metal particles, as measured by ELISA. Interleukin-2 (IL-2) secretion levels were significantly decreased by both debris and Co ions. Flow cytometry analysis showed that the metal nanoparticles induced a significant increase in apoptosis after 48-h exposure. This investigation showed that prolonged exposure (120 h) to metal debris induces lymphocyte proliferation, suggesting that activation of resting lymphocytes may have occurred. Although cytokine production was affected mainly by metal debris, cobalt toxicity may also modulate IL-2 secretion, and even Co ion concentrations below the MHRA guideline levels (7 ppb) may contribute to the impairment of immune regulation in vivo in patients with MoM implants.

Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR(-)) rats.

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km=3.2±0.8µM (SD), 9.0±3.1µM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km=192.1±291.5µM (SD), 69.2±36.2µM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h)=2.0±0.5) and vesicles (h=1.6±0.2) expressing Mrp2 and from SD (h=1.6±0.4) and Wistar (h=4.0±0.6) hepatocytes suggests transport involves more than one binding site. In TR(-) hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km=100.7±36.0µM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.

405 nm Light exposure of osteoblasts and inactivation of bacterial isolates from arthroplasty patients: potential for new disinfection applications?

Infection rates after arthroplasty surgery are between 1-4 %, rising significantly after revision procedures. To reduce the associated costs of treating these infections, and the patients' post-operative discomfort and trauma, a new preventative method is required. High intensity narrow spectrum (HINS) 405 nm light has bactericidal effects on a wide range of medically important bacteria, and it reduced bacterial bioburden when used as an environmental disinfection method in a Medical Burns Unit. To prove its safety for use for environmental disinfection in orthopaedic theatres during surgery, cultured osteoblasts were exposed to HINS-light of intensities up to 15 mW/cm2 for 1 h (54 J/cm2). Intensities of up to 5 mW/cm2 for 1 h had no effect on cell morphology, activity of alkaline phosphatase, synthesis of collagen or osteocalcin expression, demonstrating that under these conditions this dose is the maximum safe exposure for osteoblasts; after exposure to 15 mW/cm2 all parameters of osteoblast function were significantly decreased. Viability (measured by protein content and Crystal Violet staining) of the osteoblasts was not influenced by exposure to 5 mW/cm2 for at least 2 h. At 5 mW/cm2 HINS-light is an effective bactericide. It killed 98.1 % of Staphylococcus aureus and 83.2 % Staphylococcus epidermis populations seeded on agar surfaces, and is active against both laboratory strains and clinical isolates from infected hip and knee arthroplasties. HINS-light could have potential for development as a method of disinfection to reduce transmission of bacteria during arthroplasty, with wider applications in diverse surgical procedures involving implantation of a medical device.

The influence of the choice of digestion enzyme used to prepare rat hepatocytes on xenobiotic uptake and efflux.

Isolated rat hepatocytes are widely used to assess the metabolism and toxicity of xenobiotics. The choice of digestion enzyme used to prepare the cells has been shown previously to influence their metabolic capability. This study investigates the effect of the digestion enzyme (collagenase II, collagenase A/trypsin inhibitor, or collagenase plus dispase) on the uptake of xenobiotics into, and efflux from, hepatocytes. The choice of digestion enzymes used in this study does not affect uptake of either pravastatin (an organic anion probe substrate for Oatp transporter) or metformin (an organic cation probe substrate for Oct transporter). With regard to efflux transporters, hepatocyte differentiation was better maintained when cells were isolated using collagenase II alone.

Effects of feeding high-linoleate safflower seeds on postpartum reproduction in beef cows.

Two experiments were conducted to evaluate reproductive responses to supplemental high-linoleate safflower seeds in postpartum beef cows. In Exp. 1, 18 primiparous, crossbred beef cows (411 +/- 24.3 kg of BW) were fed Foxtail millet hay starting 1 d postpartum at 1.68% of BW (DM basis) and a low-fat control (control: 63.7% cracked corn, 33.4% safflower seed meal, and 2.9% liquid molasses; DM basis) at 0.35% of BW (n = 9) or a supplement (linoleate) containing 95.3% cracked high-linoleate (79% 18:2n-6) safflower seeds and 4.7% liquid molasses (DM basis) at 0.23% of BW (n = 9). Beginning 1 d postpartum, blood was collected every 3 d for sera. Cows were slaughtered at 37 +/- 3 d postpartum for collection of hypothalami, anterior pituitary glands, liver, ovarian follicles, and uterine tissue. By 37 +/- 3 d postpartum, dietary treatment did not influence ovarian follicular development (P >or= 0.17), hypophyseal concentrations of LH (P = 0.14), or concentrations of IGF-I in liver (P = 0.15). In contrast, anterior pituitary glands from linoleate cows contained more FSH (P = 0.02) than control cows and linoleate cows had less IGF-I in the medial basal hypothalamus (P = 0.05), preoptic area (P = 0.06), and in follicular fluid (P

The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes.

The preparation of hepatocytes using the two-stage perfusion technique usually involves the use of collagenase (CII) alone or in combination with dispase (C/D) or trypsin inhibitor (CA/TI) as digestion enzymes. The effect of CII, C/D and CA/TI on cell viability, yield, cytochrome P450 mediated oxidation of testosterone, glucuronidation and sulfation of 7-hydroxycoumarin, glutathione content, glutathione-S-transferase activity and glutathione-conjugation capacity of hepatocytes has been assessed. Cytochrome P450 mediated oxidation of testosterone was significantly (p < 0.05) decreased with CII isolated hepatocytes (81.7 +/- 3.3 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (96.6 +/- 1.9 nmol/10(6) cells) or C/D (95.1 +/- 2.1 nmol/10(6) cells). In contrast, glutathione conjugation of the non-specific substrate 1-chloro-2,4-dinitrobenzene was significantly (p < 0.05) increased with CII isolated hepatocytes (56.9 +/- 5.9 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (36.0 +/- 3.7 nmol/10(6) cells) or C/D (31.6 +/- 3.7 nmol/10(6) cells). These findings have significant implications for the interpretation of metabolism data derived from hepatocytes in suspension, particularly in terms of glutathione conjugation of potentially toxic reactive intermediates of xenobiotic metabolism. Indeed, data presented show that the presence of trypsin inhibitor in the preparation of isolated rat hepatocytes significantly affects the formation of glutathione conjugates of reactive intermediate products of troglitazone metabolism.

Release of chromium from orthopaedic arthroplasties.

Many orthopaedic implants are composed of alloys containing chromium. Of particular relevance is the increasing number of Cobalt Chromium bearing arthroplasies being inserted into young patients with osteoarthritis. Such implants will release chromium ions. These patients will be exposed to the released chromium for over 50 years in some cases. The subsequent chromium ion metabolism and redistribution in fluid and tissue compartments is complex. In addition, the potential biological effects of chromium are also controversial, including DNA and chromosomal damage, reduction in CD8 lymphocyte levels and possible hypersensitivity reactions (ALVAL). The establishment of these issues and the measurement of chromium in biological fluids is the subject of this review.

The interaction of chromium (VI) with macrophages: depletion of glutathione and inhibition of glutathione reductase.

There are reports of alterations in the number and functions of the cells of the immune system in patients with metal-on-metal (MOM) orthopaedic implants. These effects have been correlated with elevated chromium levels in the patients' blood. We have investigated the interactions of clinically relevant concentrations of Cr VI with macrophages in vitro, and the mechanisms responsible for its toxicity. Cr VI causes a concentration dependent decrease in macrophage viability above 1 microM as measured by the MTT and Neutral Red assays. This falls well within the range of circulating chromium serum concentrations measured in patients with MOM. Intracellular reduced glutathione (GSH) levels fall as a result, and most of the loss (86%) is accounted for by oxidation to the dimer, GSSG. Prior depletion of GSH does not sensitise the cells to Cr VI toxicity, implying that it is not involved in protecting the cells against the effects of Cr VI. During the metabolism of Cr VI, glutathione reductase activity is inhibited. In contrast, the activities of catalase and superoxide dismutase are not significantly altered. Prior inhibition of glutathione reductase activity protects against the toxicity of Cr VI to a significant extent, suggesting that it reduces Cr VI to a toxic metabolite.

Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated rats.

Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold.

Metabolism of troglitazone in hepatocytes isolated from experimentally induced diabetic rats.

Troglitazone (TGZ), the prototype 2,4-thiazolidinedione antidiabetic agent, is associated with hepatotoxicity in patients with Type 2 diabetes. Although the mechanism of toxicity has not been established, alterations in the clearance of TGZ from in-vitro hepatocyte cultures through metabolic conjugation reactions are believed to modulate the toxicity of the compound. In this study, the metabolism of TGZ in freshly isolated hepatocytes from the fat-fed streptozotocin-treated rat model of Type 2 diabetes is described. Biochemical parameters such as cellular reduced glutathione content, content of cytochromes P450 and b5, and the expression of glutathione-S-transferase alpha (subunits Ya and Yc2) were not affected by the induced diabetes. TGZ was metabolized primarily to a sulfonate, a quinone and a glucuronide in both control and experimentally diabetic animals. However, metabolism after induction of diabetes was characterized by a moderate increase in sulfation, a decrease in the elimination half-life of TGZ and the absence of the minor metabolites of TGZ, notably the glutathione adduct of the putative reactive intermediate (m/z = 747 (M + H)+; m/z = 745 (M - H)-).

The mechanical properties of calcium phospate ceramics modified by collagen coating and populated by osteoblasts.

Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP) or composites which combine the best properties of both materials are among the principal candidates for bone replacement grafts. In this study we have investigated the mechanical strength of HA, TCP and composites of the two in the ratios 75;25 (H75), 50:50 (H50) and 25:75 (H25). The strength of each material was investigated in the presence and absence of collagen coating, and the influence of osteoblast culture for up to 28 days on strength was determined. TCP, H25 and H75 were significantly weakened by collagen coating, the strengths of the other materials were either not affected (HA) or increased (H50). Culture with osteoblasts significantly increased the strength of uncoated HA and H50, but this effect was not observed when the materials were coated with collagen. Our results indicate that ceramic composition affects the interactions between collagen coating, culture with osteoblasts and mechanical strength of the material. Although collagen coating has been found to increase the proliferation of osteoblasts into these ceramic materials, it may be necessary to stabilise and optimise the coating process to minimise effects on mechanical strength.

Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen.

Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.

Bacterial adhesion to bisphosphonate coated hydroxyapatite.

Staphylococcus aureus (S. aureus) is commonly associated with microbial infection of orthopaedic implants. Such infections often lead to osteomyelitis, which may result in failure of the implant due to localised bone destruction. Bacterial adhesion and subsequent colonisation of the device may occur as a consequence of contamination during surgery, or by seeding from a distant site through the blood circulation. Coating of the hydroxyapatite (HA) ceramic component of artificial hip joints with the bisphosphonates clodronate (C) and pamidronate (P) has been proposed as a means to minimise osteolysis and thereby prevent loosening of the implant. However, the effect of the bisphosphonate coating on bacterial adhesion to the HA materials must be determined before this approach can be implemented. In this study coated HA materials were incubated with the S. aureus and the number of adherent bacteria determined using the Modified Vortex Device (MVD) method. The number of bacteria adherent to the P coated HA material was significantly greater than that adherent to uncoated HA (60-fold increase) or to the C coated HA (90-fold increase). Therefore, even though earlier studies suggested that P bound to HA may improve osseointegration, the results presented would suggest that the use of this coating may be limited by the potential increased susceptibility of the coated device to infection.

Metabolism of the dihydropyridine calcium channel blockers mebudipine and dibudipine by isolated rat hepatocytes.

The prototype 1,4-dihydropyridine (1,4-DHP) nifedipine, indicated for the management of hypertension and angina pectoris, has drawbacks of rapid onset of vasodilating action and a short half-life. Several newer analogues have been designed to offset these problems and these include mebudipine and dibudipine. These analogues contain t-butyl substituents that have been selected to alter the fast metabolism without altering pharmacological activity. In this study, the metabolism of mebudipine and dibudipine by isolated rat hepatocytes has been investigated. These compounds were extensively metabolized in 2 h by oxidative pathways, analogous to those known for nifedipine, and by O-glucuronidation after hydroxylation of the t-butyl substituents. The in-vitro half-lives of mebudipine (22 +/- 7.1 min) and dibudipine (40 +/- 9.8 min) were significantly longer than that of nifedipine (5.5 +/- 1.1 min), which was investigated in parallel in this study. These newer 1,4-DHPs address the problem of the short half-life of nifedipine and have potential for further development in view of their comparable potency to nifedipine.

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation.

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at -78 degrees C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7+/-6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7+/-14.2% protein concentration, 55.4+/-4.2% carboxyfluorescein diacetate de-acetylation, 27.2+/-7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3+/-7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.

The effect of inducing agents on the metabolism of trypanocidal diamidines by isolated rat hepatocytes.

This study has investigated the effect of phenobarbitone (PB), 3-methylcholanthrene (3-MC), and deltamethrin (DM) on the metabolism of two trypanocidal diamidines; pentamidine isethionate and diminazene aceturate in freshly isolated Sprague-Dawley rat hepatocytes. There were significant increases in the total cytochrome p450 content of hepatocytes obtained from rats pre-treated with PB and 3-MC, whereas pre-treatment with DM did not produce any significant induction of cytochrome p450. However, pre-treatment of rats with each of the three agents led to inhibition of pentamidine metabolism following a 3h incubation of pentamidine (100 microM) with freshly isolated rat hepatocytes (5 x 10(6) cells ml(-1)). Pre-treatment with 3-MC caused the highest inhibitory effect on pentamidine metabolism (8-fold inhibition), compared with PB (4.8-fold) and DM (2.2-fold). Six previously reported phase I metabolites of pentamidine were identified in cells from all the pre-treated animals as well as controls. When compared to the control group, there were significant differences between the profiles of the three major metabolites of pentamidine, 1,5-di(4'-amidinophenoxy)-2-pentanol, 1,5-di(4'-amidinophenoxy)-3-pentanol and 5-(4'-amidinophenoxy) pentanoic acid, in hepatocytes from the DM and 3-MC pre-treated rats, whereas no significant differences were observed in the cells from the PB pre-treated group. In contrast, diminazene was not metabolised with the same experimental conditions. Differences in the metabolic profiles of pentamidine and its metabolites as a result of concomitant exposure to environmental xenobiotics could have important toxicological and pharmacological implications for patients that receive the drug.

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.