Validation of a dried blood spot method for therapeutic drug monitoring of citalopram, mirtazapine and risperidone and its active metabolite 9-hydroxyrisperidone using HPLC-MS.

Citalopram, mirtazapine and risperidone are frequently prescribed for psychiatric illnesses such as depression and psychosis or for aggressive behavior in elderly patients with dementia. The plasma concentrations vary greatly between patients, especially in elderly patients. Thus, therapeutic drug monitoring (TDM) increases the safety of antipsychotic treatment and a more rapid response to treatment may be achieved. To facilitate TDM, the objectives of this study were to develop and validate a reliable dried blood spot method to simultaneously quantify citalopram, mirtazapine and risperidone including its active metabolite 9-hydroxyrisperidone. The blood punches were extracted by methanol using an ultrasonic bath, purified by liquid-liquid extraction and analyzed by liquid chromatography/mass spectrometry (LC-MS). All acceptance criteria of the EMA and FDA guidelines for method validation were fulfilled. Linearity was shown over the range of 2.5-300µg/L for all substances. The analytes were stable for at least one month at all investigated storage conditions, including storing at room temperature exposed to light. Retrieving capillary blood by finger-pricking the assay was successfully applied in elderly patients. Venous serum samples were drawn simultaneously to compare capillary blood with serum concentrations. Given the validated results and the calculated capillary blood:serum ratio, the studied dried blood spot method offers an excellent application in TDM and can be applied in ambulatory care.

Interactions of carboxypeptidase G2 with 6S-leucovorin and 6R-leucovorin in vitro: implications for the application in case of methotrexate intoxications.

Carboxypeptidase G2 (CPG2) is used when unexpected toxicity or renal failure occurs during high-dose methotrexate therapy. Leucovorin is also administered to antagonise the effects of methotrexate on purine anabolism. To investigate the effects of CPG2 on leucovorin rescue, we incubated the enzyme with both stereoisomers and analysed the degradation. A method for separating the stereoisomers of leucovorin, the internal standard aminopterin and the degradation products by capillary electrophoresis with 2.6-dimethyl-beta-cyclodextrin as a chiral selector has been developed. The active 6S-leucovorin is degraded much faster than the inactive 6R-isomer. The maximum observed degradation velocity was 31 microM/min for 6S-leucovorin and 20 microM/min for 6R-leucovorin, respectively, with an initial concentration of each stereoisomer of 250 microM. Similar results were obtained at lower concentrations of leucovorin isomers. Thus, the selectivity of CPG2 for methotrexate in comparison to leucovorin is not as high as anticipated in the literature as only the active 6S-leucovorin and not the mixture of the diastereomers should be taken into account. We conclude that the protective effects of leucovorin are antagonized by CPG2. Therefore, CPG2 should be administered to patients with caution.