A New Automated Approach for the Determination of Peptides in Human Plasma Using On-line SPE–LC–MS–MS

October 2, 2011

The Application Notebook

The Application Notebook, The Application Notebook-10-02-2011, Volume 0, Issue 0

Spark Application Note


A new approach for peptide analysis has been developed and evaluated — the integrated clean up and analysis of peptides using an on-line SPE procedure with a mixed mode strong anion exchange cartridge. Sample preparation has been simplified to just one operator step before the automated analysis of the samples.


A Spark Holland Symbiosis Pico system with an Applied Biosystems API 4000 ESI-MS system were used for this application.


Three peptides where received from Novo Nordisk (Malov, Denmark) for whom the method was developed (1). This application note shows the results for the 5900 AMU peptide but similar results were achieved for the 2100 and 6500 AMU peptide. Samples were prepared by spiking pooled human plasma with a 5900 amu peptide drug. Prior to extraction, all plasma samples were diluted 1:4 with water. 100 µL of diluted sample was then placed directly into the Symbiosis autosampler to undergo fully automated analysis. On-line SPE is performed on a disposable cartridge; each sample uses a fresh SPE cartridge. After extraction the peptides were eluted from the cartridge onto a reverse phase analytical column, using the HPD focusing mode. The mobile phases consist of formic acid (0.1%), water and methanol (95%) with separation on a Phenomenex Jupiter 4µm Proteo 50 × 2 mm LC column.


The sample clean-up was optimized by increasing the percentage of organic modifier in the wash solvent. The mixed mode anion exchange cartridge allows for a high organic wash without reducing the analyte recovery. For optimal clean-up a first wash step with 20% ACN was programmed to remove interfering proteins (no denaturing on SPE cartridge), followed by a second high organic wash at 80% ACN to remove non-polar matrix compounds.

Figure 1: 5900 AMU peptide. Spiked plasma sample extracted on Oasis MAX (10 × 1 mm).

A clean and reproducible chromatogram was obtained (Figure 1). Subsequently, a calibration curve was determined by injecting a full set of calibration standards ranging from 0.5 to 500 ng/mL spiked human plasma standards. The calibration curve is calculated with a 1/X weighting. The curve for the peptide is displayed in Figure 2 (r > 0.996).

Figure 2: 5900 AMU peptide. Spiked plasma calibration curve from 0.5 to 500 ng/mL.


A simple sample preparation method has been developed for the determination of peptides in plasma. Automated on-line SPE–LC–MS–MS delivers clean and reproducible results. Good linearity was obtained in human plasma over the 0.5–500 ng/mL range (r > 0.996). The developed XLC–MS–MS method has an absolute recovery of more than 90%. The accuracy ranges from 89–117% and the precision 2.1–7.9%CV.

The Symbiosis Pico method is currently used routinely in several pharmaceutical labs for analysing different size of peptides (<10K amu) with similar results.


Spark Holland poster ASMS 2010: A new automated approach for the determination of peptides in human plasma using online SPE-LC-MS/MS

For more information please contact applications@sparkholland.com

Spark Holland BV

P. de Keyserstraat 8, PO Box 388, 7800 AJ Emmen, The Netherlands

E-mail: applications@sparkholland.com

Website: www.sparkholland.com