Analysis of An Opioid Peptide Dermorphin in Equine Urine using EVOQ™ Elite LC-MS/MS System

Application Note #704423 Analysis of An Opioid Peptide Dermorphin in Equine Urine using EVOQ™ Elite LC-MS/MS System B ru ke r D al to ni cs is c on tin ua lly im pr ov in g its p ro du ct s an d re se rv es t he r ig ht to c ha ng e sp ec ifi ca tio ns w ith ou t no tic e. © B ru ke r D al to ni cs 0 9 -2 01 2, # 70 4 42 3 Abstract Dermorphin, a naturally occurring hepta-peptide, is a potent analgesic and a Class I prohibited substance in horse racing. In this work, dermorphin was quantified in equine urine on an EVOQ Elite model LC triple quadrupole mass spectrom- eter. The linear dynamic range of the assay was 0.05-100ng/ mL and required low limit of quantitation (LLOQ) of 0.05 ng/ mL was easily achieved. Introduction Dermorphin is an opioid hepta-peptide derived from the skin secretions of South American frogs (Phyllomedusa sauvagei). Dermophine is 30-40 times more potent as an analgesic than morphine1 and is unique because it contains D-alanine, an amino acid that makes the peptide highly resistant to protease-mediated degradation. While the D-amino acid peptides are sought after for their low immu- nogenic response and in peptidomimetic drug design, it was dermorphin’s use in horse racing that catapulted this opioid peptide from obscurity into the headlines. The pain-killing properties of dermorphin were used to drive race horse performance beyond its physical limits, often injuring the animal. As a result, dermorphin is now a Class I prohibited sub- stance as decreed by the Association of Racing Commis- sioners International (RCI) Model Rules. The analysis of dermorphin in equine urine is a challenging assay because of the associated complex matrix and lack of a stable labeled internal standard. Results and Discussion The assay of dermorphin in equine urine was developed on an EVOQ Elite triple quadrupole mass spectrometer. The equine urine sample was prepared by solid phase extrac- tion. Accurate quantification from 0.05 to 100ng/mL was easily achieved. Figure 1 shows an example MRM chro- matogram of 0.05ng/mL dermorphin in equine urine, and Figure 2 the calibration curve. The response factors of three QC levels (low, mid, and high) in Table 1 have a RSD% of 7.3%, demonstrating a robust ion source performance, as no divert valve was used to prevent contamination of matrix onto the ion source. Chromatogram of Demorphin Calibration Curve for Demorphin B ru ke r D al to ni cs is c on tin ua lly im pr ov in g its p ro du ct s an d re se rv es t he r ig ht to c ha ng e sp ec ifi ca tio ns w ith ou t no tic e. © B ru ke r D al to ni cs 0 9 -2 01 2, # 70 4 42 3 Bruker Daltonik GmbH Bremen · Germany Phone +49 (0)421-2205-0 Fax +49 (0)421-2205-103 sales@bdal.de Bruker Daltonics Inc. Billerica, MA · USA Phone +1 (978) 663-3660 Fax +1 (978) 667-5993 ms-sales@bdal.com Bruker Daltonics Inc. Fremont, CA · USA Phone +1 (510) 683-4300 Fax +1 (510) 490-6586 ms-sales@bdal.com www.bruker.com/ms For research use only. Not for use in diagnostic procedures. Experimental Chromatography (Advance™ HPLC) • Column: C18, 3µ, 2.1x100mm (ACE 3µ, C18, 100x2.1mm) • Injection volume: 40µL • Flow rate: 0.4mL/min • Mobile phase A: Water with 0.2% Formic acid • Mobile phase B: Acetonitrile with 0.2% Formic acid • Gradient conditions: • 0.00min 5% B • 0.20min 5% B • 8.00min 95% B • 8.50min 95% B • 8.51min 5% B • 12.50min 5% B Mass Spectrometry (EVOQ™ Elite) • VIP Heated-ESI Temp: 350°C • Heated gas: 70 units • Nebulizer gas: 70 units • Cone gas temp: 250°C • Cone gas: 10 units • Spray voltage: +4000v • Active exhaust: On • MRM for Dermorphin and Deltorphin II (as Internal Standard): • m/z 803.4>602 (CE=20v), Quantifier ion • m/z 803.4>202 (CE=32v), Qualifier ion • m/z 783>277 (CE=40 v), Internal Standard Chromatogram of Demorphin Figure 1: Chromatogram of demorphin at 0.05ng/mL (LOQ) and the IS deltorphin (green trace) in equine urine. Table 1: Response factor %RSD for the QC QC Concentration (ppb) Area Ratio Response Factor Low QC 0.3 0.01000 0.03335 Low QC 0.3 0.01031 0.03438 Mid QC 6.0 0.22314 0.03719 Mid QC 6.0 0.17834 0.02972 High QC 50.0 1.67945 0.03359 High QC 50.0 1.62102 0.03242 Response Factor RSD 7.3% Figure 2: Calibration curve for demorphin in equine urine in the range 0.05-100ng/mL Calibration Curve for Demorphin Author Rohan Thakur Acknowledgements Professor Scott Stanley of University of California of Davis for providing the Dermophin standard and the prepared equine urine samples.