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Automated determination of polyamines by high-performance liquid chromatography with simple sample preparation
H M van Eijk1, D R Rooyakkers, N E Deutz
1Department of Surgery, University of Limburg, Maastricht, Netherlands.
Journal of Chromatography. A
|April 12, 1996
Summary
A new Inertsil packing material enables efficient separation of polyamine derivatives, automating sample clean-up for improved reproducibility. This method accurately quantifies polyamines in urine and tissue, with potential for plasma analysis.
Area of Science:
- Analytical Chemistry
- Biochemistry
Background:
- Basic compounds, such as polyamines, present challenges in chromatographic separation.
- Ophthaldialdehyde (OPA) derivatization is a common method for detecting amino acids and polyamines.
Purpose of the Study:
- To develop and validate a new, efficient method for determining polyamine concentrations.
- To utilize a novel reversed-phase packing material, Inertsil, for improved separation of basic compounds.
Main Methods:
- Utilized Inertsil, a fully endcapped reversed-phase packing material.
- Separated OPA derivatives of amino acids from OPA derivatives of spermine (SPM), spermidine (SPD), putrescine (PUT), and cadaverine (CAD).
- Implemented an on-line sample clean-up procedure, eliminating the need for off-line extraction.
Main Results:
- Achieved complete separation of OPA derivatives, enabling automation and enhancing reproducibility.
- Demonstrated a linear response for polyamines up to 250 pmol with high R2 values.
- Established a limit of detection of 0.1 pmol, suitable for plasma concentration analysis (0.1 µmol/l).
- Reported low coefficients of variation (C.V.) for peak area (<3%) and retention times (<0.5%) in optimized conditions.
Conclusions:
- The developed method offers an easy and efficient way to measure polyamine concentrations.
- The on-line clean-up procedure significantly improves automation and reduces analytical time.
- The method is applicable to various biological samples, including urine and tissue homogenates, with potential for plasma analysis.