Objective: To establish a new method for the determination of 48 targeted small peptides in human urine using ultra-high performance liquid chromatogram coupled with Quadrupole-Orbitrap mass spectrometer (Q Exactive Plus UHPLC-HRMS). Methods: For 1.5 mL urine samples, 20 μL of the internal standard solution was spiked into the samples of which the final urinary concentration was 6.7 ng·mL-1, and 50 μL of each sample was transferred into the low-binding centrifuge tube. The remaining sample was centrifuged at 10 000 r·min-1 for 10 min, then 1 mL supernatant of each sample was applied to the OASIS WCX cartridges (1 mL, 30 mg) which was previously activated with 1 mL methanol and balanced with 1 mL deionized water. After successively washing with 1 mL 5% of ammonium hydroxide and 1 mL 20% of acetonitrile aqueous solution, the cartridges were eluted with 1 mL acetonitrile/water mixture (volume ratio, 3∶1) containing 5% of formic acid. The eluent was dried under nitrogen at 38 ℃. After re-dissolved, the extracts were mixed with the above-mentioned 50 μL urine sample, and analyzed with Q Exactive Plus UHPLC-HRMS using a Thermo Hypersil GOLD C18 column (100 mm×2.1 mm,1.9 μm). The mobile phase in the gradient elution of UHPLC was comprised of 10 mmol·L-1 ammonium formate aqueous solution with pH value of 3.5 (containing 0.05% formic acid) and methanol. The small peptides were analyzed using parallel reaction monitoring mode under positive electrospray ionization in Quadrupole-Orbitrap mass spectrometer. Results: The method was simple and reliable with good selectivity, and the limits of detection and limits of identification for 48 targeted small peptides were not higher than 0.82 ng·mL-1 and 1.30 ng·mL-1, respectively. Conclusion: The described method meets the requirements of the technical documents of World Anti-Doping Agency, and can be used in routine detection of small peptides in human urine samples.
DONG Tian-yu, MA Cong-cong, ZHANG Yu-feng, CHANG Wei, ZHANG Li-si, WANG Zhan-liang, YAN Kuan, HE Gen-ye, LIU Yun-xi
. Determination of 48 prohibited small peptides in human urine by UHPLC-HRMS method[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(12)
: 2057
-2068
.
DOI: 10.16155/j.0254-1793.2022.12.01
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