My laboratory does pharmacological studies to understand the best medicines to use in many different animal species.
Dr Benn Bryant who is a veterinarian at Western Plains Zoo, at Dubbo, a regional town in Australia in conjunction with my laboratory, is doing a study for the International Rhino Foundation to investigate how well non-steroidal anti-inflammatories (NSAIDs) work for the black rhinoceros! The NSAIDs we are looking at are meloxicam, phenylbutazone and flunixin.
Benn and his teams medicate the rhinos with the NSAID and the animals have been trained to provide saliva samples and to allow blood to be taken at set-time points after the NSAID has been administered. My job is to measure the NSAID’s concentration in the blood and the saliva and work out what may be the best dose for these animals. This is the first time that a study has been done looking at the efficacy of any medicine for the rhinoceros.
The following show our chromatograms to detect the various NSAIDs in rhino blood:
Assay to detect meloxicam, phenylbutazone and flunixin in plasma of the black rhinoceros
Our lab previously developed and validated HPLC condition for measuring meloxicam plasma concentrations in the koala [1] and sheep [2] for pharmacokinetic studies.
The assay conditions are the following:
The HPLC system comprises of a Shimadzu CBM-20A module (Kyoto, Japan) equipped with a LC-20AT delivery unit with DGU-20As degassing solvent delivery unit and SIL-20AC auto injector. A reversed phase C18 column (Synergi 4μm MAX-RP 80A, 150 x 4.6mm, Phenomenex, Lane Cove, NSW) was used for separation. The isocratic mobile phase comprised of 50 mM potassium phosphate buffer (pH 2.15) and acetonitrile (55:45 v/v). The mobile phase was run at a flow rate of 1 mL min-1 with an oven temperature of 30°C. Meloxicam and peroxicam as the internal standard (IS) were detected via an SPD-20A UV detector (Kyoto, Japan) at a wavelength of 355 nm. Shimadzu class VP data system (software version 7.4) (Kyoto, Japan) calculated the area under the drug peaks.
Several standards of known meloxicam concentration (that ranged from 0.048 to 25 ug/mL) were made with blank rhinoceros plasma to construct a ‘standard meloxicam concentration curve. QC samples (0.01, 0.2 and 1 µg/mL) were spiked not only with known concentrations of meloxicam but also with the internal standard (IS) piroxicam (Sigma‐Aldrich, St. Louis, MO, USA). The standards and quality control samples were cleaned with solid phase extraction prior to HPLC analysis.
The lowest concentration of meloxicam [known as the lowest level of quantification (LLOQ)] that can be accurately and reliably measured by this assay is 0.01 ug/mL.
Precision and accuracy of the LOQ (0.01 μg/mL) were <15% [coefficient of variation (CV)] and within 20% of nominal concentration, respectively. For quality control (QC) samples (0.01, 0.2 and 1 μg/mL), the intra‐ (n = 3) and inter‐day (5 days) precision ranged from 1.60 to 9.37% and 0.43 to 8.16% (CV), respectively. Intra‐day and inter‐day accuracy of QC samples expressed as a percentage of the bias ranged from 0.42 to 4% and 0.19 to 5.60%, respectively. The absolute recovery of meloxicam in QC samples (n = 5) ranged from 89 – 93%. The retention times of meloxicam and the internal standard were approximately at 8.03 and 5.56 min, respectively.
Variations of the assay for meloxicam (wavelength of 355 nm) could also detect phenylbutazone (at a wavelength of 285 nm) and flunixin (wavelength 290 nm) in blank rhinocerous plasma.
Assay chromatograms
Meloxicam
Figure 1: Blank Rhinoceros plasma with UV detector set at 355 nm
Figure 2: Blank rhinoceros plasma spiked with meloxicam (blue circle) and IS (piroxicam) (red circle)
Phenylbutazone
Phenylbutazone plasma concentrations in rhinoceros plasma were measured via HPLC-UV. The HPLC system comprised of a Shimadzu CBM-20A module (Kyoto, Japan) equipped with a LC-20AT delivery unit with DGU-20As degassing solvent delivery unit and SIL-20AC auto injector. A reversed phase C18 column (Synergi 4μm MAX-RP 80A, 150 x 4.6mm, Phenomenex, Lane Cove, NSW) was used for separation. The isocratic mobile phase comprised of 50 mM potassium phosphate buffer (pH 2.15) and acetonitrile (60:40 v/v). The mobile phase was run at a flow rate of 1 mL min-1 with ambient temperature. Phenylbutazone and the internal standard (IS) detection were detected via an SPD-20A UV detector (Kyoto, Japan) at a wavelength of 265 nm and Shimadzu class VP data system (software version 7.4) (Kyoto, Japan) calculated the area under the drug peaks. Prior to HPLC analysis, protein precipitation was used for sample cleaning (plasma: acetonitrile 1:2, v/v) and the injection volume was 20 uL. Total run time is 15min and retention times for phenylbutazone and IS are 13 min and 11 min, respectively (Figure 3 and 4). No endogenous peak as detected in both retention times of phenylbutazone or IS.
Figure 3 Blank rhinoceros plasma with UV detector set at a wavelength of 265 nm
Figure 4 Blank rhinoceros plasma spiked with phenylbutazone (blue circle) and IS (meloxicam – red circle)
Flunixin
Flunixin plasma concentrations in rhinoceros were measured via HPLC-UV. The HPLC system comprised of a Shimadzu CBM-20A module (Kyoto, Japan) equipped with a LC-20AT delivery unit with DGU-20As degassing solvent delivery unit and SIL-20AC auto injector. A reversed phase C18 column (Synergi 4μm MAX-RP 80A, 150 x 4.6mm, Phenomenex, Lane Cove, NSW) was used for separation. The isocratic mobile phase comprised of 50 mM potassium phosphate buffer (pH 2.15) and acetonitrile (55:45 v/v). The mobile phase was run at a flow rate of 1 mL min-1 with ambient temperature. Flunixin and the internal standard (IS) detection were detected via an SPD-20A UV detector (Kyoto, Japan) at a wavelength of 290 nm and Shimadzu class VP data system (software version 7.4) (Kyoto, Japan) calculated the area under the drug peaks. Prior to HPLC analysis, protein precipitation was used for sample cleaning (plasma: acetonitrile, 1:2, v/v) and the injection volume was 20uL. Total run time is 11min and retention times for phenylbutazone and IS are 3.68 min and 9.02 min, respectively (Figure 5). No endogenous peak as detected in both retention times of flunixin or IS.
Figure 5 Blank rhinoceros plasma spiked with flunixin (blue circle) and IS (meloxicam-red circle )
More information to come on this study over the next few months
- Kimble B, Li KM, Govendir M. Quantitation of meloxicam in the plasma of koalas (Phascolarctos cinereus) by improved high performance liquid chromatography Journal of Veterinary Science. 2013;14:7-14.
- Woodland AN, Van der Saag D, Kimble B, White PJ, Govendir M, Lomax S. Plasma pharmacokinetic profile and efficacy of meloxicam administered subcutaneously and intramuscularly to sheep. PloS one. 2019;14(4)
Koala Medicines 