Development and evaluation of new aminoacids derivatizing reagents and automated sample preparation procedures with LC-MS / MS methods

In the present study, novel and automated methods were developed for the determination of pharmaceutical compounds and analytes of great biological importance in the presence of biological fluids or not, with the employment of LC-MS/MS technique. It is well known that methods developed with LC-MS/MS technique are characterized by great sensitivity and specificity. In any case, the objective beyond the spectrometric and chromatographic optimization was to find all those conditions and parameters that lead to optimizing the response of each system under consideration. In the first section, the aim was signal optimization of some basic and non-natural amino acids in pure solutions. Amino acids are poorly ionized, thus their detection with mass spectrometry often becomes problematic. Derivatization as a technique of signal boosting of these analytes is commonly used. Taking into consideration the chemical properties that must characterize a derivative detectable with LC-MS/MS, new derivatization agents C1- DAPS and C4-DAPS were synthesized. More specifically, these agents were designed so that the formed derivatives (i) to carry a permanent positive charge as a quaternary ammonium cation, (ii) to possess a significant hydrophobic part and (iii) to provide with the same characteristic daughter ion after fragmentation of precursor ions, for further selectivity. Afterwards, a chromatographic method for the derivatives determination was developed and a central composite design was applied for the optimization of derivatization reaction conditions. Finally, comparison between these novel amino acid derivatives and non derivatized ones was conducted. Comparative study was also conducted between these derivatives and amino acid butyl esters after amino acid derivatization with 1-butanol in HCl 3M. This derivatizing agent was used as a comparative measure as it is commonly used in bibliography for amino acids signal enhancement. Response evaluation was attained with column chromatography as well as with flow injection analysis. In conclusion, C1-DAPS and C4-DAPS agents enhance considerably amino acids detection signal (up to 52 times for C1-DAPS and up to 23 for C4-DAPS) and especially glycine detection signal (up to 4500 and 2800 respectively). Comparing C1-DAPS derivatives to butyl esters signal enhancement, the superiority of the first group was clear for all amino acids under examination. C4-DAPS agent exhibited either enhanced response or of the same level with that of butyl esters. As far as derivatization procedure is concerned, it can be characterized as operationally simple, and fast, since derivatives are formed instantaneously, whereas derivatization reaction by satisfactory repeatability. Referring to derivatization protocol exploitation perspectives in biological fluids, it could be applied to cases of analytes’ quantitation carrying amino group, and particularlyΣτην παρούσα εργασία, αναπτύχθηκαν νέες και αυτοματοποιημένες μέθοδοι για τον προσδιορισμό φαρμάκων και ενώσεων βιολογικής σημασίας παρουσία βιολογικών υγρών ή μη με την τεχνική LC-MS/MS. Ως γνωστόν, η ανάπτυξη μεθόδων με την τεχνική LC-MS/MS χαρακτηρίζεται από μεγάλη ευαισθησία και ειδικότητα. Στόχος, σε κάθε περίπτωση, πέρα από τη φασματομετρική και χρωματογραφική βελτιστοποίηση, ήταν η εύρεση όλων εκείνων των συνθηκών και παραμέτρων που οδηγούσαν στη βελτιστοποίηση της απόκρισης του εκάστοτε υπό εξέταση σύστηματος. Στο πρώτο μέρος, στόχος ήταν η βελτιστοποίηση του σήματος ορισμένων βασικών και συνθετικών αμινοξέων σε καθαρά διαλύματα. Τα αμινοξέα, ως ελεύθεροι αναλύτες, δεν έχουν μεγάλη ικανότητα ιοντισμού με αποτέλεσμα η ανίχνευσή τους με τη φασματομετρία μαζών να είναι συχνά προβληματική. Η παραγωγοποίηση ως τεχνική ενίσχυσης του σήματος των συγκεκριμένων αναλυτών είναι συνήθης και ευρέως διαδεδομένη πρακτική. Μελετώντας τις χημικές ιδιότητες που πρέπει να διαθέτει ένα παράγωγο ως προuπόθεση για τον προσδιορισμό του με την τεχνική LC-MS/MS, συνετέθησαν νέοι παράγοντες παραγωγοποίησης C1-DAPS και C4-DAPS οι οποίοι θα προσέδιδαν στα παραγωγοποιημένα αμινοξέα τα επιθυμητά χαρακτηριστικά. Συγκεκριμένα, οι παράγοντες αυτοί σχεδιάστηκαν κατά τέτοιο τρόπο ώστε τα παράγωγα (i) να φέρουν σταθερό θετικό φορτίο υπό τη μορφή του άλατος του τεταρτοταγούς αμμωνίου, (ii) να διαθέτουν σημαντικό υδρόφοβο τμήμα και (iii) να δίνουν όλα το ίδιο θυγατρικό ιόν έπειτα από θραυσματοποίηση των μητρικών τους ιόντων, για μεγαλύτερη εκλεκτικότητα. Ακολούθως, αναπτύχθηκε χρωματογραφική μέθοδος προσδιορισμού των παραγώγων, ενώ βελτιστοποιήθηκαν μέσω πειραματικού σχεδιασμού, και συγκεκριμένα με κεντρικό σύνθετο σχεδιασμό, οι παράμετροι εκείνες οι οποίες επηρεάζουν την αντίδραση παραγωγοποίησης. Επόμενο στάδιο ήταν η σύγκριση των αποκρίσεων των παραγώγων των αμινοξέων με τους νέους παράγοντες C1-DAPS και C4-DAPS με αυτές των μη παραγωγοποιημένων αμινοξέων. Επίσης, έγινε σύγκριση μεταξύ των νέων αυτών παραγώγων και των βουτυλεστέρων των αμινοξέων που προκύπτουν έπειτα από παραγωγοποίηση αυτών με 1-βουτανόλη σε υδροχλωρικό οξύ 3Μ. Ο παράγοντας αυτός παραγωγοποίησης χρησιμοποιήθηκε ως μέτρο σύγκρισης, καθώς είναι βιβλιογραφικά αποδεδειγμένο πως ενισχύει το σήμα ανίχνευσης των αμινοξέων συνδεόμενος με αυτά. Η αξιολόγηση των αποκρίσεων πραγματοποιήθηκε τόσο με χρήση χρωματογραφικής στήλης όσο με απευθείας έγχυση δείγματος. Συμπερασματικά, οι παράγοντες C1-DAPS και C4-DAPS ενισχύουν σημαντικά το σήμα ανίχνευσης των αμινοξέων (έως και 52 φορές για τον παράγοντα C1-DAPS και έως 23 για τον C4-DAPS) και ιδιαίτερα εκείνο της γλυκίνης (περί τις 4500 και 2800 φορές αντίστοιχα). Όσον αφορά, δε, στην αύξηση σήματος που επιτυγχάνεται έναντι των βουτυλεστέρων, η υπεροχή των C1-DAPS παραγώγων ήταν ξεκάθαρη με την ένταση του σήματος σαφώς σημαντικά ενισχυμένη για όλα τα αμινοξέα. Ο παράγοντας C4-DAPS εμφάνισε στην πλειοψηφία των αμινοξέων βελτιωμένη ή παραπλήσια ένταση σήματος με αυτή των βουτυλεστέρων..

Dried plasma spots as an alternative sample collection technique for the quantitative LC-MS/MS determination of gabapentin

Dried plasma spots were employed as an alternative sample collection technique for the quantitative determination of gabapentin in human plasma, using an automated liquid chromatography tandem mass spectrometry method. After the methanolic extraction of single plasma, 1/8-in. disks which contained only 1.98 μL plasma volume, were placed in 96-well format plates, then gabapentin and its internal standard, 4-aminocyclohexanecarboxylic acid, were derivatized with n-butanol/HCl (3 M) and detected as butyl esters. The assay exhibited excellent linearity over the concentration range of 40.0-10.0 × 10 3 ng/mL, which is suitable for the determination of gabapentin after per os administration of a single tablet. Variations in intra- and inter-assay accuracy and precision were within internationally accepted criteria. Homogeneity of plasma spots was proven, whilst butyl ester stability for both analytes was estimated and found very satisfactory. The quantitative analysis of Gabapentin with dried plasma spot specimens seems to be a prominent and advantageous technique, especially when applied to pharmacokinetic studies, where plasma sampling procedure becomes rapid and required plasma volumes negligible. © 2010 Springer-Verlag

Advantages of automation in plasma sample preparation prior to HPLC/MS/MS quantification: Application to the determination of cilazapril and cilazaprilat in a bioequivalence study

An HPLC/MS/MS method characterized by complete automation and high throughput was developed for the determination of cilazapril and its active metabolite cilazaprilat in human plasma. All sample preparation and analysis steps were performed by using 2.2 mL 96 deep-well plates, while robotic liquid handling workstations were utilized for all liquid transfer steps, including liquid-liquid extraction. The whole procedure was very fast compared to a manual procedure with vials and no automation. The method also had a very short chromatographic run time of 1.5 min. Sample analysis was performed by RP-HPLC/MS/MS with positive electrospray ionization using multiple reaction monitoring. The calibration curve was linear in the range of 0.500-300 and 0.250-150 ng/mL for cilazapril and cilazaprilat, respectively. The proposed method was fully validated and proved to be selective, accurate, precise, reproducible, and suitable for the determination of cilazapril and cilazaprilat in human plasma. Therefore, it was applied to a bioequivalence study after per os administration of 2.5 mg tablet formulations of cilazapril

Validation of a novel, fully automated high throughput high-performance liquid chromatographic/tandem mass spectrometric method for quantification of pantoprazole in human plasma

An automated high-throughput HPLC/MS/MS method was developed for the quantitative determination of pantoprazole in human plasma. Only 100 mL plasma was placed in 2.2 mL 96 deep-well plates, and both pantoprazole and omeprazole (IS) were extracted from human plasma by liquid-liquid extraction, using diethyl ether-dichloromethane (70:30, v/v) as the organic solvent. Robotic liquid-handling workstations were used for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation, and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. Sample analysis was performed by utilizing the combination of RP-HPLC/MS/MS, with positive-ion electrospray ionization and multiple reaction monitoring detection. The chromatographic run time was set at 1.8 min with a flow rate of 0.6 mL/min on a Nucleosil octylsilyl (C8) analytical column. The method was proven to be sensitive, specific, accurate, and precise for the determination of pantoprazole in human plasma. The method was applied to a bioequivalence study after per os administration of a 40 mg pantoprazole gastric retentive tablet

An improved and fully validated LC-MS/MS method for the simultaneous quantification of simvastatin and simvastatin acid in human plasma

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of simvastatin (SV) and simvastatin acid (SVA) in human plasma. Plasma samples were treated by acetonitrile (ACN) addition for protein precipitation (PP) and subsequent two-step liquid-liquid extraction (LLE) in 96-deepwell plates, using methyl t-butyl ether (MTBE) as the organic solvent. ACN addition step was proven to enhance method sensitivity, as well as producing cleaner samples for injection. Lovastatin (LV) and lovastatin acid (LVA) were used as internal standards (IS) for SV and SVA quantification respectively. A relatively small plasma volume (300 μL) was employed and all procedure liquid transfer steps were performed automatically, by the use of robotic liquid handling workstations. Both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources were applied and compared for LC-MS/MS sample analysis, with ESI proven to be more sensitive for the specific analytes. Polarity switch (from negative to positive ionization mode) was performed during the same analytical run, so as for the simultaneous SV and SVA determination to be possible. The method had a short sample preparation time, as well as a chromatographic run time of just 1.9 min, the shortest so far reported for SV determination. It was validated and fulfilled all preset criteria for sensitivity, specificity, linearity (0.100-40.0 ng/mL), inter- and intra-accuracy and precision for both molecules. The proposed method was applied to the rapid and reliable simultaneous determination of SV and SVA in a bioequivalence study, after per os administration of a SV tablet (80 mg). © 2008 Elsevier B.V. All rights reserved