Investigation of absolute and relative response for three different liquid chromatography/tandem mass spectrometry systems; the impact of ionization and detection saturation.

RATIONALE: The investigations in this article were triggered by two observations in the laboratory; for some liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems it was possible to obtain linear calibration curves for extreme concentration ranges and for some systems seemingly linear calibration curves gave good accuracy at low concentrations only when using a quadratic regression function. METHODS: The absolute and relative responses were tested for three different LC/MS/MS systems by injecting solutions of a model compound and a stable isotope labeled internal standard. The analyte concentration range for the solutions was 0.00391 to 500 µM (128,000×), giving overload of the chromatographic column at the highest concentrations. The stable isotope labeled internal standard concentration was 0.667 µM in all samples. RESULTS: The absolute response per concentration unit decreased rapidly as higher concentrations were injected. The relative response, the ratio for the analyte peak area to the internal standard peak area, per concentration unit was calculated. For system 1, the ionization process was found to limit the response and the relative response per concentration unit was constant. For systems 2 and 3, the ion detection process was the limiting factor resulting in decreasing relative response at increasing concentrations. CONCLUSIONS: For systems behaving like system 1, simple linear regression can be used for any concentration range while, for systems behaving like systems 2 and 3, non-linear regression is recommended for all concentration ranges. Another consequence is that the ionization capacity limited systems will be insensitive to matrix ion suppression when an ideal internal standard is used while the detection capacity limited systems are at risk of giving erroneous results at high concentrations if the matrix ion suppression varies for different samples in a run.

Development and validation of a liquid chromatography and tandem mass spectrometry method for determination of roscovitine in plasma and urine samples utilizing on-line sample preparation.

Roscovitine, a purine analogue that selectively inhibits cyclin-dependent kinases, has been considered as a potential anti-tumor drug. The determination of roscovitine in plasma and urine was performed using microextraction in packed syringe as on-line sample preparation method with liquid chromatography and tandem mass spectrometry. The sampling sorbent utilized was polystyrene polymer. 2H3-lidocaine was used as internal standard. The limit of detection for roscovitine was as low as 0.5 ng/mL and the lower limit of quantification was 1.0 ng/mL. The accuracy and precision values of quality control samples were between +/-15% and < or =11%, respectively. The calibration curve was obtained within the concentration range 0.5-2000 ng/mL in both plasma and urine. The regression correlation coefficients for plasma and urine samples were > or =0.999 for all runs. The present method is miniaturized and fully automated and can be used for pharmacokinetic and pharmacodynamic studies.

Stability, pKa and plasma protein binding of roscovitine.

In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 degrees C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 degrees C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to alpha1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, alpha1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 degrees C compared to 37 degrees C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.