Analysing Opioid Peptide Demorphine in Equine Urine

Topics Covered

     Chromatography (Advance HPLC)
     Mass Spectrometry (EVOQ Elite)
Results and Discussion
About Bruker


Dermorphin, an opioid hepta-peptide, is a derivative obtained from the skin secretions of South American frogs. It is 30-40 folds more powerful as an analgesic when compared to morphine1. The presence of amino acid D-alanine makes dermorphin unique by providing resistance to protease-mediated degradation.

Although the D-amino acid peptides are preferred for their use in peptidomimetic drug design and for their low immunogenic response, its application in horse racing propels its popularity. Dermorphin’s pain-killing properties are used in improving race horse performance beyond its physical limits, thus causing injury to the animal. Due to this fact, the Association of Racing Commissioners International Model Rules have categorized dermorphin as a Class I prohibited substance.

Quantifying dermorphin in equine urine is a difficult analysis due to the associated complex matrix and the unavailability of an established labeled internal standard. This application note describes the quantitative analysis of dermorphin in equine urine using an EVOQ Elite model LC triple quadrupole mass spectrometer. The assay’s linear dynamic range was 0.05-100 ng/ mL, enabling the achievement of required low limit of quantitation (LLOQ) of 0.05 ng/ mL.


Chromatography (Advance HPLC)

  • Column: C18, 3µ, 2.1x100mm (ACE 3µ, C18, 100x2.1mm)
  • Flow rate: 0.4mL/min
  • Injection volume: 40µL
  • 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 Temperature: 350°C
  • Nebulizer gas: 70 units
  • Heated gas: 70 units
  • Cone gas: 10 units
  • Cone gas temp: 250°C
  • Active exhaust: On
  • Spray voltage: +4000 V
  • 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

Figure 1. The Bruker EVOQ

Results and Discussion

The EVOQ Elite triple quadrupole mass spectrometer was used to develop the assay of dermorphin in equine urine. The equine urine sample preparation was done with solid phase extraction. The analysis was able to achieve accurate quantification between 0.05 and 100 ng/mL easily. Figure 2 depicts a MRM chromatogram of 0.05ng/mL dermorphin in equine urine.

Figure 2. Chromatogram of demorphin at 0.05ng/mL (LOQ) and the IS deltorphin (green trace) in equine urine.

Figure 3. alibration curve for demorphin in equine urine in the range 0.05-100ng/mL

Table 1. shows the response factors of low, mid and high QC levels with a RSD% of 7.3%, illustrating a powerful ion source performance because the elimination of a divert valve that was utilized for preventing contamination of matrix onto the ion source.

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%

About Bruker

Brukeris the new name in chemical analysis. Accurate and comprehensive analysis of exogenous and discrete elements in a wide range of sample matrices are key applications for many analytical chemistry groups. To address the needs and challenges of analysts working in those areas, Bruker has expanded their product family to provide, and expertly support, a series of fully integrated solutions including:

  • Gas Chromatography-Mass Spectrometers (GC/MS and GC/MS/MS)
  • Inductively Coupled Plasma Mass Spectrometers (ICP-MS)
  • Gas Chromatography Systems (GC)

Widely used in food and consumer safety testing, forensic, industrial, environmental, and clinical laboratories, these systems are well accepted and established market leaders that universally deliver outstanding performance at a premium value.

This information has been sourced, reviewed and adapted from materials provided by Bruker.

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