Ανάπτυξη νέων αυτοματοποιημένων μεθόδων υψηλής ταχύτητας για τον προσδιορισμό φαρμάκων σε βιολογικά υγρά με την τεχνική της δίδυμης φασματομετρίας μαζών
In the current study, three automated methods, for the quantitative determination of pharmaceutical compounds in plasma samples, which cover the whole spectrum of pre-treatment techniques, were developed and validated (Liquid – liquid extraction, Solid phase extraction either on-line or by using 96-well extraction disk plates and protein precipitation). In the case of itraconazole, an automated LLE, LC-MS/MS method was presented after conversion of the samples’ arrangement into a 96-well plate format which allowed the simultaneous determination of itraconazole and its co-active metabolite in human plasma. The application of two liquid-handling robotic workstations greatly simplified the extraction process, resulting in significant advantages over existing methods in terms of throughput and efficiency. Another advantage over these methods was the relatively small quantity of human plasma used for analysis. Moreover, the mixture of ACN / MTBE used as the extraction solvent, aside from eliminating the formation of the irregular emulsion observed when only MTBE was employed, managed to adequately adjust the polarity of the organic solvent. The proposed method was applied to a bioequivalence study of two 100 mg ITZ formulations, and it allowed the study to be completed in just 4 days. In the case of risperidone, a high-throughput ternary column (two extraction and one analytical column) on-line clean-up LC-MS/MS method was presented for the simultaneous determination of risperidone and its co-active metabolite in human plasma. This system employed two parallel columns and an analytical one allowing the equilibration of one extraction column while the analysis is taking place on the other extraction column. This resulted in a total run time of 3 min compared to run times of 4 min or more, required for other off-line and on-line methods reported for the determination of RIS and 9-OH-RIS. The use of two liquid-handling robotic workstations greatly simplified the sub-zero temperature extraction process. Furthermore, the precipitation – extraction step provided cleaner extracts with a relatively low content of proteins, especially with application of the sub-zero temperature prorocol. The latter, in combination with the use of two extraction columns instead of one allowed for longer column lifetime compared to traditional direct injection methods. The proposed method was successfully applied to a bioequivalence study of two 2mg RIS formulations and allowed its completion in just 4 days. Finally, through the use of semi-automated liquid handling systems and a 96-well plate format, including individual tubes, a rapid SPE extraction method was developed for the quantification of lisinopril in human plasma. The simultaneous SPE approach greatly simplified the 317 preparation process and decreased the time required for sample preparation till measurement versus existing methods with manual sample treatment. Therefore, hundreds of samples can be analyzed daily. Another advantage over these methods was the relatively small quantity of human plasma used for analysis. All the above developed methods were validated according to the FDA criteria for pharmaceutical compound determination in biological fluids and were found reliable with adequate accuracy and precision.
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