Plasma, urine and tissue concentrations of Flunixin and Meloxicam in Pigs.

BACKGROUND: The objective of this study was to determine the renal clearance of flunixin and meloxicam in pigs and compare plasma and urine concentrations and tissue residues. Urine clearance is important for livestock show animals where urine is routinely tested for these drugs. Fourteen Yorkshire/Landrace cross pigs were housed in individual metabolism cages to facilitate urine collection. This is a unique feature of this study compared to other reports. Animals received either 2.2 mg/kg flunixin or 0.4 mg/kg meloxicam via intramuscular injection and samples analyzed by mass spectrometry. Pigs were euthanized when drugs were no longer detected in urine and liver and kidneys were collected to quantify residues. RESULTS: Drug levels in urine reached peak concentrations between 4 and 8 h post-dose for both flunixin and meloxicam. Flunixin urine concentrations were higher than maximum levels in plasma. Urine concentrations for flunixin and meloxicam were last detected above the limit of quantification at 120 h and 48 h, respectively. The renal clearance of flunixin and meloxicam was 4.72 ± 2.98 mL/h/kg and 0.16 ± 0.04 mL/h/kg, respectively. Mean apparent elimination half-life in plasma was 5.00 ± 1.89 h and 3.22 ± 1.52 h for flunixin and meloxicam, respectively. Six of seven pigs had detectable liver concentrations of flunixin (range 0.0001-0.0012 µg/g) following negative urine samples at 96 and 168 h, however all samples at 168 h were below the FDA tolerance level (0.03 µg/g). Meloxicam was detected in a single liver sample (0.0054 µg/g) at 72 h but was below the EU MRL (0.065 µg/g). CONCLUSIONS: These data suggest that pigs given a single intramuscular dose of meloxicam at 0.4 mg/kg or flunixin at 2.2 mg/kg are likely to have detectable levels of the parent drug in urine up to 2 days and 5 days, respectively, after the first dose, but unlikely to have tissue residues above the US FDA tolerance or EU MRL following negative urine testing. This information will assist veterinarians in the therapeutic use of these drugs prior to livestock shows and also inform livestock show authorities involved in testing for these substances.

A study to assess the correlation between plasma, oral fluid and urine concentrations of flunixin meglumine with the tissue residue depletion profile in finishing-age swine.

BACKGROUND: Flunixin meglumine (FM) was investigated for the effectiveness of plasma, oral fluid, and urine concentrations to predict tissue residue depletion profiles in finishing-age swine, along with the potential for untreated pigs to acquire tissue residues following commingled housing with FM-treated pigs. Twenty pigs were housed in groups of three treated and one untreated control. Treated pigs received one 2.2 mg/kg dose of FM intramuscularly. Before treatment and at 1, 3, 6, 12, 24, 36, and 48 h (h) after treatment, plasma samples were taken. At 1, 4, 8, 12 and 16 days (d) post-treatment, necropsy and collection of plasma, urine, oral fluid, muscle, liver, kidney, and injection site samples took place. Analysis of flunixin concentrations using liquid chromatography/tandem mass spectrometry was done. A published physiologically based pharmacokinetic (PBPK) model for flunixin in cattle was extrapolated to swine to simulate the measured data. RESULTS: Plasma concentrations of flunixin were the highest at 1 h post-treatment, ranging from 1534 to 7040 ng/mL, and were less than limit of quantification (LOQ) of 5 ng/mL in all samples on Day 4. Flunixin was detected in the liver and kidney only on Day 1, but was not found 4-16 d post-treatment. Flunixin was either not seen or found less than LOQ in the muscle, with the exception of one sample on Day 16 at a level close to LOQ. Flunixin was found in the urine of untreated pigs after commingled housing with FM-treated pigs. The PBPK model adequately correlated plasma, oral fluid and urine concentrations of flunixin with residue depletion profiles in liver, kidney, and muscle of finishing-age pigs, especially within 24 h after dosing. CONCLUSIONS: Results indicate untreated pigs can be exposed to flunixin by shared housing with FM-treated pigs due to environmental contamination. Plasma and urine samples may serve as less invasive and more easily accessible biological matrices to predict tissue residue statuses of flunixin in pigs at earlier time points (≤24 h) by using a PBPK model.

Plasma and urine pharmacokinetics of intravenously administered flunixin in greyhound dogs.

Medication control in greyhound racing requires information from administration studies that measure drug levels in the urine as well as plasma, with time points that extend into the terminal phase of excretion. To characterize the plasma and the urinary pharmacokinetics of flunixin and enable regulatory advice for greyhound racing in respect of both medication and residue control limits, flunixin meglumine was administered intravenously on one occasion to six different greyhounds at the label dose of 1 mg/kg and the levels of flunixin were measured in plasma for up to 96 hr and in urine for up to 120 hr. Using the standard methodology for medication control, the irrelevant plasma concentration was determined as 1 ng/ml and the irrelevant urine concentration was determined as 30 ng/ml. This information can be used by regulators to determine a screening limit, detection time and a residue limit. The greyhounds with the highest average urine pH had far greater flunixin exposure compared with the greyhounds that had the lowest. This is entirely consistent with the extent of ionization predicted by the Henderson-Hasselbalch equation. This variability in the urine pharmacokinetics reduces with time, and at 72 hr postadministration, in the terminal phase, the variability in urine and plasma flunixin concentrations are similar and should not affect medication control.

Possible active transport mechanism in pharmacokinetics of flunixin-meglumin in rabbits.

The plasma and urine kinetics of flunixin-meglumin (FNX, 2 mg/kg, i.v.) in rabbits were examined. Unusual pharmacokinetic profiles were obtained, including high binding percentage with plasma protein (> 99%), a short elimination half-life (< 4 hr) and a relatively large Vd-area (0.5 L/kg). These profiles indicate that some active transport mechanisms are involved in FNX disposition. The recovery of FNX from urine was approximately 9 % of the dose within 24 hr following the injection. The estimated renal clearance of the unbound drug nearly corresponded to the renal blood flow rates, indicating that active tubular secretion in the renal re-absorptive tract may be involved in the disposition. The effect of a concomitant administration of pravastatin (PV) on FNX disposition was also examined. PV is a representative substrate of a transporter in human liver cells (OATP-2). After the PV administrations, the Vd-area of FNX and total body clearance markedly decreased, indicating that FNX is actively taken up and metabolized in liver cells by an OATP-2 like transporter. In conclusion, there are at least 2 active transport pathways for FNX pharmacokinetics in rabbits, one is renal tubular secretion and the other is in the sinusoidal section of the liver.

Determination of clonixin in plasma and urine by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method that enables the determination of clonixin in human plasma and urine samples is described. Recovery of the drug was over 87.6 and 80.7% for plasma and urine, respectively. The limit of quantitation of the method was established as 10 ng/ml in plasma and 20 ng/ml in urine samples, with RSDs of less than 11.1%. The applicability of the method was further assessed by determining the plasma concentrations time course of clonixin in six healthy volunteers after single oral dose administration of 150 and 300 mg of clonixin and Clonix.

Detection and identification of flunixin after multiple intravenous and intramuscular doses to horses.

The objectives of the study were to compare various methods to determine flunixin in test samples collected periodically from horses after intramuscular (IM) and intravenous (IV) dosing at the maximum recommended dosage and to document detection times for this drug in test samples. Flunixin, a nonsteroidal anti-inflammatory drug approved for use in horses, was administered to eight mares in five consecutive daily doses of 1.1 mg per kilogram of body weight by the IM or IV route. Flunixin was detected in urine samples collected at various times after drug administration by flunixin enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatographic-mass spectrometric (GC-MS) methods. Detection time was defined as the time period over which flunixin was detected and was dependent on the method used. The shortest detection times were 24 to 48 h and were observed when the TLC method was used. On the other hand, detection times were as long as 15 days when HPLC, GC-MS, and flunixin ELISA methods were used. The use of these more sensitive tests to monitor official samples collected from racehorses could result in positive tests for flunixin when it is exerting no detectable clinical effects because it produces clinical effects lasting only 24-36 h in horses.

Identification of a flunixin metabolite in camel by gas chromatography-mass spectrometry.

A flunixin metabolite, a hydroxylated product, has been identified in camel urine and plasma samples using gas chromatography-mass spectrometry (GC-MS) and GC-MS-MS in the electron impact and chemical ionization modes. Its major fragmentation pattern has been verified by GC-MS-MS in daughter ion and parent ion scan modes. The method could detect flunixin and its metabolite in camel urine after a single intravenous dose of 2.2 mg of flunixin/kg body weight for 96 and 48 h, respectively, which increases the reliability of antidoping control analysis.

Pharmacokinetics, metabolism and urinary detection time of flunixin after intravenous administration in camels.

The pharmacokinetics of flunixin were determined after an intravenous dose of 1.1 mg/kg body weight in six camels and 2.2 mg/kg body weight in four camels. The data obtained (mean +/- SEM) for the low and high dose, respectively, were as follows: The elimination half-lives (t1/2 beta) were 3.76 +/- 0.24 and 4.08 +/- 0.49 h, the steady state volumes of distribution (Vdss) were 320.61 +/- 38.53 and 348.84 +/- 35.36 mL/kg body weight, total body clearances (ClT) were 88.96 +/- 6.63 and 84.86 +/- 4.95 mL/h/kg body weight and renal clearances (Clr) were 0.52 +/- 0.09 and 0.62 +/- 0.18 mL/h/kg body weight. A hydroxylated metabolite of flunixin was identified by gas chromatography/mass spectrometry (GC/MS) under electron and chemical ionization and its major fragmentation pattern was verified by tandem mass spectrometry (GC/MS/MS) using neutral loss, daughter and parent scan modes. The detection times for flunixin and its hydroxylated metabolite in urine after an intravenous (i.v.) dose of 2.2 mg/kg body weight were 96 and 48 h, respectively.

Isolation, purification, and structural characterization of flunixin glucuronide in the urine of greyhound dogs.

A urinary metabolite of flunixin in greyhound dogs was isolated and purified by a gradient-elution solid-phase extraction technique. The purified metabolite was shown to be hydrolyzed to free flunixin by strong base and by beta-glucuronidase, suggesting the presence of a C1-beta-glucuronide ester of flunixin. The metabolite was further characterized by positive-ion, tandem MS with electrospray ionization. Mass spectral data showed the presence of a protonated molecular ion (M+1) at m/z 473, which was consistent with the molecular weight of protonated flunixin glucuronide, and a product ion at m/z 297, which was consistent with the molecular weight of protonated flunixin. Collisionally induced dissociation of the m/z 297 product ion showed a fragmentation pattern consistent with that of standard flunixin. These data support the contention that this metabolite of flunixin in greyhound urine is the C1-beta-glucuronide of flunixin. Acyl glucuronide metabolites of some organic acid drugs have been shown to bind covalently to tissue proteins in vitro, in vivo, and ex vivo. The presence of this metabolite may, therefore, have pharmacokinetic and pharmacodynamic implications for flunixin in greyhound dogs, as well as in other animal species in which the acyl glucuronide of flunixin is a metabolite.

Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay.

A two-step kinetic enzyme-linked immunosorbent assay was developed to detect the presence of flunixin in the urine of greyhound dogs. The assay system was developed using polyclonal antiflunixin antisera, a rabbit albumin-flunixin conjugate adsorbed onto polystyrene microtiter strips, and flunixin reference standards for calibration. The assay parameters were optimized and the performance characteristics were determined. The quantitative intra- and inter-run precisions (%CV) of the analysis of replicate (n = 10) flunixin-spiked urine samples were 9.9-12.5% and 10.2-13.6%, respectively. The linear dynamic range was 1-100 ng/mL, and the quantitative accuracy, as determined by calculation of percent error of measured flunixin in flunixin-spiked drug-free greyhound urine, was -16% to +14% over this range. The I50 of the ELISA was 17.3 ng/mL. The limit of detection was 25 ng/mL in greyhound urine. The reactivity in the assay system relative to flunixin (100%) was 147% for flunixin glucuronide, 25% for clonixin, and 5% for niflumic acid. The ELISA was capable of detecting total flunixin for up to 72 h in dogs administered flunixin at 0.55 mg/kg orally and up to 96 h in a dog that was administered flunixin at 1.0 mg/kg orally.

Determination of flunixin in equine urine and serum by capillary electrophoresis.

A capillary electrophoresis (CE) and a solid-phase extraction method was developed for the determination of flunixin in equine urine and serum. The suitable CE run conditions were described. The factors affecting flunixin recovery rates were investigated and optimum solid-phase extraction conditions for flunixin in equine urine and serum were established. Limits of detection and quantitation were 3.4 and 5.6 ng/ml for serum and 16.9 and 33.1 ng/ml for urine, respectively. The recoveries exceeded 96% for urine and 79% for serum. Urine samples from race horses and urine and serum samples from a mare administrated with flunixin were analyzed with this procedure.

Detection of flunixin in equine urine using high-performance liquid chromatography with particle beam and atmospheric pressure ionization mass spectrometry after solid-phase extraction.

A normal-phase HPLC method combined with particle-beam mass spectrometry (PB-MS) was developed for the analysis of non-steroidal anti-inflammatory drugs (NSAIDs). The forty one NSAIDs analysed responded in one or more (electron impact, positive and negative chemical ionisation) modes and highly characteristic spectra were produced. A mixed-mode solid-phase extraction (SPE) method for isolating acidic NSAIDs was developed using the Bond Elut Certify II cartridge. The average recovery was 88.5%. Flunixin, extracted by SPE from urine of a mare to which the meglumine salt had been administered was positively identified by HPLC-PB-MS and HPLC-atmospheric pressure ionization (API) MS methods.

Simultaneous analysis of flunixin, naproxen, ethacrynic acid, indomethacin, phenylbutazone, mefenamic acid and thiosalicylic acid in plasma and urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

Simple and reproducible high-performance liquid chromatographic (HPLC) and gas chromatographic-mass spectrometric (GC-MS) methods have been developed for the simultaneous analysis of several acidic drugs in horse plasma and urine. Although the capillary GC-MS column provided better separation of the drugs than the reversed-phase C8 (3 microns, 75 mm) HPLC column, the total analysis time with HPLC was shorter than the total analysis time with GC-MS. The HPLC system equipped with a diode-array detector provided simultaneous screening (limit of detection 100-500 ng/ml) and confirmation (limit 1.0 micrograms/ml) of the drugs. The HPLC system equipped with fixed-wavelength ultraviolet and fluorescence detectors provided a relatively sensitive screening [limit of detection 50-150 ng/ml for ultraviolet and 10 ng/ml for fluorescence (naproxen only) detectors] of the drugs. However, the positive samples had to be confirmed by using either the diode-array detector or the GC-MS system. The GC-MS system provided simultaneous screening and confirmation of the drugs at very low concentrations (20-50 ng/ml).

Influence of furosemide on the detection of flunixin meglumine in horse urine samples.

The possibility of false negative results from TLC when a diuretic is administered concomitantly with flunixin was studied. Samples were subjected to solvent extraction from acidic aqueous solutions; duplicate samples were also subjected to alkaline hydrolysis at pH 12.5. The internal standard was flufenamic acid. The quantification of flunixin was performed by HPLC and the results confirmed by GC/MS. The data show that furosemide influences the urinary concentration of flunixin.

Disposition and excretion of flunixin meglumine in horses.

The disposition of flunixin meglumine administered IV at a dosage of 1.1 mg/kg was described by a 2-compartment model; the alpha and beta half-lives (t1/2) were 0.61 and 1.5 hours, respectively. When administered IV at a rate of 2.2 mg/kg, the disposition was best described by a 3-compartment model, and the alpha, beta, and lambda t1/2 were 0.16, 1.52, and 6.00 hours, respectively. The zero-time plasma concentrations after flunixin meglumine was administered at 1.1 and 2.2 mg/kg were 9.3 +/- 0.76 and 21.5 +/- 7.4 mg/L, respectively. The bioavailability after oral administration of 1.1 mg/kg was 85.8%. The absorption t1/2 was 0.57 hours, with a peak concentration of 2.50 +/- 1.25 mg/L. The cumulative urinary recoveries for IV and oral administrations were 61.0% and 63.3%, respectively, of the dose for the 12-hour collection period. The final asymptotic points of urine excretion after IV and oral administrations were 406.4 +/- 65.5 and 357.7 +/- 53.5 mg, respectively, which represented 75.5 and 77.5% of the drug accounted for between 30 and 35 hours after administration. Flunixin meglumine was rapidly excreted in urine over a 2- to 4-hour period after drug administration and was highly bound to protein in plasma.

Gas chromatographic analysis of flunixin in equine urine after extractive methylation.

A quantitative method for the analysis of flunixin, 2-(2-methyl-3-trifluoromethylanilino) nicotinic acid, in equine urine by gas chromatography with nitrogen-phosphorus detection has been developed. Flunixin and the internal standard, mefenamic acid, N-(2,3-xylyl) anthranilic acid, were analysed after extractive methylation of the carboxylic acid group using methyl iodide. The extraction and alkylation conditions of flunixin and mefenamic acid have been studied. The detection limit of the method was 0.25 mumol/l flunixin in urine (74 ng/ml). Flunixin was found to be conjugated to 96.5% in equine urine, and the conjugate was spontaneously hydrolysed to free flunixin. This approach can also be used to confirm the presence of flunixin or mefenamic acid in horse urine in the doping control of racehorses.

Identification of a flunixin metabolite in the horse by gas chromatography-mass spectrometry.

The main metabolite of flunixin, a hydroxylated product, has been identified by gas chromatography-mass spectrometry and 1H NMR spectroscopy in equine urine and plasma. The method also permits the qualitative monitoring of the urinary elimination of the drug and its metabolite. The two products are detected up to 175 and 54 h, respectively, after a single intravenous administration at the dose of 1 mg/kg. Simultaneous detection of the two compounds increases the reliability of anti-doping control analysis.

Evaluation of the potential for interference by dimethyl sulfoxide (DMSO) in drug detection in racing animals.

Dimethyl sulfoxide (DMSO) had been postulated to be a 'masking agent' when used concurrently with therapeutic or prohibited drugs in racing animals. Eight drugs (flunixin, furosemide, caffeine, apomorphine, phenylbutazone, lidocaine, cocaine, and acepromazine maleate) were administered to six horses singly and with concurrent intravenous DMSO. Urine samples were analyzed for the presence of the drugs and/or their metabolites by thin layer chromatography. Direct comparison of thin layer chromatograms of extracts of positive urine samples with and without DMSO verified that DMSO did not interfere with the detection of these drugs.