Rapid Detection of Growth Hormone-Releasing Peptides in Dried Blood Spots: a Proof of Concept Workflow for Anti-Doping Analysis

Significance of the Topic

Growth hormone releasing peptides are potent performance enhancers listed by WADA. Reliable detection in athlete samples is essential for fair competition and anti doping compliance. Dried blood spots (DBS) offer low volume sampling and improved stability.

Objectives and Study Overview

This study develops a proof of concept workflow for sensitive detection of ipamorelin in DBS using independent data acquisition on the SCIEX X500R QTOF system. The aim is to achieve reproducible quantitation at low ng per mL levels.

Methodology and Sample Preparation

• DBS card conditioning with citric acid followed by spotting 40 uL spiked whole blood and internal standard MRFA
• Ten step extraction using 50 50 water acetonitrile with 10 percent formic acid and centrifugation cycles
• UHPLC separation on Phenomenex C18 column with 7 minute gradient at 0.5 mL per minute and 5 uL injection volume
• Independent data acquisition with TOF MS scan 230 800 Da and TOF MS MS 50 720 Da
• Triplicate injections over three days for validation of linearity accuracy and precision

Instrumentation

• SCIEX X500R QTOF System operating in positive ESI and IDA mode
• SCIEX ExionLC AC UHPLC system
• Phenomenex C18 column 100 x 2.1 mm 1.7 u m
• Whatman protein saver dried blood spot cards
• Standard laboratory equipment including centrifuge shaker and pipettes

Main Results and Discussion

• Limit of detection 2.5 ng/mL and limit of quantitation 5 ng/mL for ipamorelin in DBS
• Excellent linearity over 5 to 100 ng/mL range with R2 of 0.99499
• Inter and intra day precision CV below 25 percent and accuracy bias below 20 percent except at LOQ where CV reached 32.7 percent
• Reproducible detection of precursor ion m z 356.7000 and fragment ions at m z 129.1022 223.1189 and 420.2030 with mass errors under 3 ppm

Benefits and Practical Applications

• Minimal sample volume with DBS ease of storage and transport
• High specificity and sensitivity suitable for anti doping screening
• Rapid workflow supports routine monitoring of growth hormone releasing peptides

Future Trends and Potential Applications

• Expansion to a broader panel of GHRPs on the WADA prohibited list
• Integration with high throughput platforms for large scale screening
• Adaptation to alternative microsampling devices for field testing

Conclusion

The presented workflow enables reproducible and sensitive detection of ipamorelin in dried blood spots using SCIEX X500R QTOF with independent data acquisition. This approach can support anti doping efforts and be extended to additional peptides.