-
- Posts: 292
- Joined: Wed Jan 19, 2005 2:20 pm
Hi there,
We have experienced issues recently with an accuracy spiking method validation exercise, spiking drug into a liquid nebulizer solution that contains 10 mM of a citrate buffer. The concentration of drug is prepared accurately at about 5 mg/mL. The drug itself is a zwitterionic .HCL salt. The sample preparation for LC injection involves a 50 fold dilution with 25/75 AcN/water to 0.1 mg/mL concentration. So the final sample diluent contains 2% v/v of 10 mM citrate buffer.
The assay standards are prepared slightly differently, by dissolving directly into a 25/75 AcN/water v/v diluent. So there is no citrate in these standard solutions.
Assaying the accuracy (spiked) validation samples against the standards we consistently got between 93-94% recoveries (outside of our validation SOP criteria). We then investigated adding different levels of citrate to the samples and obtained the following results.
Citrate Addition % v/v Drug Recovery %
0 99.9
1 97.1
2 95.6
4 94.7
6 94
8 94.2
10 94.1
So there appears to be some kind of leveling off effect with the citrate concentration in the sample solution.What could be the cause of this missing 6-7% when we run mismatched samples and standards? Is it some kind of ion-pairing/chelating effect with citrate that is causing a decrease in our assay recovery by LC? It is the first time I have come across something like this of this big a significance.
Consequently when we added 2% v/v citrate to our standard diluent preparation we achieve ca. 100% recoveries for our samples. I also compared UV stand-alone spectra of standards (no citrate) with standards with 2 and 10% citrate (diluted from the same stock standard so all at the same concetration) and there was perfectly overlay - i.e. no shifts in response or wavelength.
We have experienced issues recently with an accuracy spiking method validation exercise, spiking drug into a liquid nebulizer solution that contains 10 mM of a citrate buffer. The concentration of drug is prepared accurately at about 5 mg/mL. The drug itself is a zwitterionic .HCL salt. The sample preparation for LC injection involves a 50 fold dilution with 25/75 AcN/water to 0.1 mg/mL concentration. So the final sample diluent contains 2% v/v of 10 mM citrate buffer.
The assay standards are prepared slightly differently, by dissolving directly into a 25/75 AcN/water v/v diluent. So there is no citrate in these standard solutions.
Assaying the accuracy (spiked) validation samples against the standards we consistently got between 93-94% recoveries (outside of our validation SOP criteria). We then investigated adding different levels of citrate to the samples and obtained the following results.
Citrate Addition % v/v Drug Recovery %
0 99.9
1 97.1
2 95.6
4 94.7
6 94
8 94.2
10 94.1
So there appears to be some kind of leveling off effect with the citrate concentration in the sample solution.What could be the cause of this missing 6-7% when we run mismatched samples and standards? Is it some kind of ion-pairing/chelating effect with citrate that is causing a decrease in our assay recovery by LC? It is the first time I have come across something like this of this big a significance.
Consequently when we added 2% v/v citrate to our standard diluent preparation we achieve ca. 100% recoveries for our samples. I also compared UV stand-alone spectra of standards (no citrate) with standards with 2 and 10% citrate (diluted from the same stock standard so all at the same concetration) and there was perfectly overlay - i.e. no shifts in response or wavelength.
