Recombinant human erythropoietin (rhEPO) and Darbepoetin alfa (Darbe) are erythropoietic drugs that must be monitored for possible abuse in horse racing sporting events. An improved detecton method modified from literature methods was developed for confirmative and semi-quantitative analysis of these erythropoietic drugs in equine plasma samples. Anti-rhEPO antibodies coated onto magnetic beads were used for immunoaffinity separation to extract any rhEPO and/or Darbe present in equine plasma samples. The rhEPO and/or Darbe bound onto the antibody coated magnetic beads were extracted with a 0.1% polyethylene glycol (PEG) pH 2.0 buffer solution. A buffer exchange procedure was performed to eliminate PEG and to concentrate the proteins. The extracted rhEPO and/or Darbe were incubated at 80°C for 10 minutes to denature the proteins. This work found that 7 minutes incubation at 80°C was the optimal of the incubation times tested for Darbe and rhEPO proteins denaturation.
Subsequently, trypsin was added into the denatured protein sample to digest the extracted Darbe and/or rhEPO into peptide fragments generating T9 peptide from Darbe which was specific and unique to Darbe and the T5 peptide from rhEPO which was specific and unique to rhEPO. The trypsin digestion also generated T17 and T6 peptides from rhEPO and Darbe which were specific and unique to both rhEPO and Darbe. This work found that doubling the trypsin concentration in trypsin digestion method from 2 μg (24.7 μg per mL of digest) to 4 μg (49.4 μg per mL of digest) increased the detection of T17, T6, and T9 peptides from 1177, 821, 7385 integrated peak mass areas for T17, T6, and T9 peptides, respectively, to 2653, 857, and 7518 integrated peak mass areas for T17, T6, and T9 peptides, respectively.
The trypsin digested sample was incubated for 10 minutes at 80°C to deactivate trypsin in the sample digest. This work found that 5 minutes incubation at 80°C was the optimal of the incubation times tested for trypsin deactivation. PNGase F [Peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase] enzyme was added to deglycosylate the tryptic peptide fragments. This work confirmed the work of Guan et al. (2008) that deglycosylation of T9 and T5 peptides of Darbe and rhEPO, respectively, were necessary for detection in electropray ionization in positive ion mode liquid chromatography mass spectrometry (ESI+/LC-MS) of the glycosylated TT9 and T5 peptides because the glycosylation sites of the T9 and T5 peptides suppressed ionization resulting in no detecting of the compounds. This work found that the detection of T17 and T6 peptides also improved from 581 and 811 integrated peak mass areas for T17 and T6 peptides, respectively, to 1004 and 861 integrated peak mass areas for T17 and T6 peptides, respectively, after treated the tryptic digest with PNGase F. Subsequently, the sample digest was detected using ESI+/LC-MS by monitoring the molecular ions of T17, T6, T9, and T5 peptides and further confirmed by the retention times and the product ions match up with the ones generated by the peptides of the standard digest.
Darbe spiked in equine plasma from 0.05 to 2.5 ng/mL (parts per billion or ppb) and the rhEPO drug spiked in equine plasma from 0.1 to 2.5 ppb were extracted and identified by the resulting (ISU) method modified from the Guan et al. (2007 and 2008) methods. Plasma samples from a research horse before and after administration by intramuscular injection at 0.4 μg/kg of Darbe and from another research horse before and after administration by intravenous injection at 0.3 μg/kg of rhEPO were collected and analyzed by the resulting (ISU) method. The pre-injection plasma sample from both horses did not detect the presence of Darbe and rhEPO compounds. The Darbe compounds were detected for all the post-injection plasma samples from the horse administered with Darbe from 2 to 192 hours samples. The four hours post-injection plasma sample from the horse administered with rhEPO was detected for the presence of the rhEPO compounds. In summary, the resulting (ISU) method developed from the modifications of the Guan et al. (2007 and 2008) methods were capable of extracting, detecting, and confirming the presence of Darbe and/or rhEPO in spiked and administration equine plasma samples.