Minimizing Carry-over for High Throughput Analysis
In this study a strategy to minimize carry-over for HT-analysis was developed by a research team from the School of Life Sciences FHNW and CTC Analytics.
Carry-over is the appearance of an analyte signal in a blank after the analysis of samples with higher analyte concentrations. It is compound and method dependent. Minimal carry-over is an important quality criterion of modern auto sampler technology and critical in HT-Analysis. In this study a strategy to minimize carry-over for HT-analysis was developed. The sources and relative contributions of carry-over were evaluated.
A total cycle time of <2 min is achieved by overlapped wash and cleaning steps. Wash efficiency/low carry-over and cycle time compete against each other.
Dead volumes caused by bad connections between tubing and fittings
Scratches on rotor/stator of valves
Generally badly flushed volumes (cavities)
ESI/APCI source components (needle, spray shield)
Sorptive carry-over:
Chemical adsorption of molecules to surfaces of tubings, loops, injection needles, or valves
Sample adsorption to the column’s stationary phase or inner surfaces
Solvent contaminants concentrated on and released from the column during a gradient run
Carry-over, if no wash step is applied (injection of 220 ng chlorhexidin):
HPLC-Method development Evaluate MS-Parameters (Spray, parent-mass, fragments, MS/MS), and HPLC separation method.
Optimize HTS Minimize the time for flushing and the time to restore and maintain starting conditions.
The regeneration time is evaluated by comparing 3 consecutive injections. The regeneration is too short as soon as the signals don’t overlap. This is buffer, column and flow dependent. (Example chlorhexidin)
Matrix is injected and the gradient is run in cycles. Several runs are carried out with shorter flushing times. As soon as significant effects are observed in the 2nd and 3rd gradient, the method is considered as too short. (Example cyclosporine in MeCN crashed blood)
Optimize sampler wash steps
The wash steps (see schedule above) are optimized to reduce wash time, solvent consumption and to maintain reproducible retention times.
Solvent 1 equals eluent A to avoid any effect on the retention (e.g. shorter retention-time). Solvent 2 must solve the analyte and matrix perfectly (Example chlorhexidin).
Always check your carry-over with an non-selective method (TIC-Scan)
Always check the solubility of your analyte in the wash solutions
Reduce dwell volume and check for cavities (bad connectors)
Reduce matrix by good sample preparation and dilution if possible
[1] Dolan, JW; LCGC 19, Feb 2001, 164-68 [2] Dolan, JW; LCGC 19, Oct 2001, 1050-54 [3] Determination of carry-over and contamination for MS – Based chrom sssays: Hughes, NC et al. AAPS Journal 2007; 9 (3), Article 42 [4] EMA Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009 ) [5] Carryover, and how to minimize it http://www.palsystem.com/ index.php?id=280
Research by: Christian Berchtold[1], Reto Bolliger[2], Guenter Boehm[2], Götz Schlotterbeck[1] [1] University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Institute of Chemistry and Bioanalytics, 4132 Muttenz, Switzerland [2] CTC Analytics AG; Industriestrasse 20, 4222 Zwingen, Switzerland
