Background and aims: The analysis of blood collected and dried on a paper substrate (i.e., "dried blood spot" or DBS) represents an alternative blood sample collection that nowadays is routinely used in neonatal screening. The goals of this study were: 1) to develop a LC-MS/MS method for the determination of 5 antidepressants and 2 antipsychotics in DBSs; 2) to establish the diagnostic reliability of DBSs with the routine blood analyses of these substances; 3) to prove the stability of these analytes on DBS within a 3-month period of storage. Method: 85 µL of blood were put on a card for DBS analysis and allowed to dry overnight in the dark, at room temperature. The whole blood stain was cut and put in a plastic tube, containing 1 mL phosphate buffer at pH 6 and deuterated internal standards, extracted using SPE cartridges and injected in the LC-MS/MS system. The analytes were separated through a reverse phase chromatography on a C18 column and detected on a triple quad operating in Multiple Reaction Monitoring mode and positive polarization. A calibration curve was prepared in the range LOQ-500 ng/mL. The procedure was applied to 60 authentic postmortem cases. For each case, 15 DBSs were collected at T0. 4 DBSs were analyzed in triplicates within 4 consecutive weeks. The last samples were analyzed after three months. Results and discussion: The analytical procedure is simple, sensitive and specific. LODs were detected in a range of 0.1 to 4.1 ng/mL(g) for antidepressants, and 0.1 to 2.7 ng/mL for antipsychotics. LOQs vary from 0.3 to 13.6 ng/ml for antidepressants, and 0.3 to 9.1 ng/mL for antipsychotics. Validation parameters fulfilled all the acceptance criteria. 4 cases were positive for citalopram and quetiapine (84-2309 ng/mL), 3 for desvenlafaxine (479-686 ng/mL) and venlafaxine (25-362 ng/mL), 2 for paliperidone (17-56 ng/mL) and trazodone (91-162 ng/mL). The concentrations on DBSs, stored at room temperature, were in good agreement with the ones obtained on blood samples analyzed with routine methods (<30% CV, both at T0 and after 3 months). The degradation percentage for most of the substances was lower than 20% within the three-month period. Citalopram and paliperidone were stable for the first month, while a degradation higher than 50% was observed after three-month storage. Quetiapine was found to be stable into 2 samples, while a significant degradation was noticed in other two cases.
Background e obiettivi: L’analisi del campione ematico raccolto e lasciato ad asciugare su un supporto cartaceo (“dried blood spot” o DBS) rappresenta un’alternativa ai metodi attuali di raccolta del sangue ed è ad oggi usata, in particolare, nello screening neonatale di routine. Gli obiettivi di questo studio sono: 1) di sviluppare un metodo LC-MS/MS per la determinazione di 5 antidepressivi e 2 antipsicotici nei DBS; 2) di stabilire l’affidabilità diagnostica dei DBS nell’analisi routinaria di queste sostanze nel sangue; 3) di provare la stabilità di questi analiti nei DBS durante un periodo di conservazione di 3 mesi. Metodo: 85 µL di sangue sono stati depositati sul supporto cartaceo per l’analisi dei DBS e lasciati asciugare durante la notte, al buio e a temperatura ambiente. L’intera macchia di sangue è stata inizialmente tagliata e, successivamente, posta in una provetta contenente 1 mL di tampone fostato a pH 6 e gli standard interni deuterati. L’estrazione è stata condotta mediante l’utilizzo di colonne SPE; il campione è stato quindi iniettato nella LC-MS/MS. Gli analiti sono stati separati attraverso cromatografia a fase inversa su colonna C18 e rilevati tramite triplo quadrupolo, operando in modalità Multiple Reaction Monitoring a polarità positiva. È stata preparata una curva di calibrazione nel range compreso tra 10 e 500 ng/mL. La procedura è stata applicata a 60 casi post mortem reali. Per ogni caso sono stati raccolti 15 DBS al T0. 4 DBS sono stati analizzati in triplicato durante 4 settimane consecutive. L’ultimo campione è stato analizzato dopo 3 mesi. Risultati e discussione: la procedura analitica è risultata semplice, sensibile e specifica. I valori dei LOD sono compresi tra 0.1 e 4.1 ng/mL per gli antidepressivi, e tra 0.1 e 2.7 ng/mL per gli antipsicotici. I LOQ variano da 0.3 a 13.6 ng/ml per gli antidepressivi, e da 0.3 a 9.1 ng/mL per gli antipsicotici. I parametri di validazione rientrano all’interno delle soglie di accettabilità secondo quanto riportato nelle linee guida internazionali di riferimento. 4 casi sono stati trovati positivi per Citalopram e Quetiapina (84-2309 ng/mL), 3 per Desvenlafaxina (479-686 ng/mL) e Venlafaxina (25-362 ng/mL), 2 per Paliperidone (17-56 ng/mL) e Trazodone (91-162 ng/mL). Le concentrazioni nei DBS, conservati a temperatura ambiente, concordavano con quelle ottenute nei campioni di sangue analizzati con i metodi di routine (<30% CV, entrambi a T0 e dopo 3 mesi). La degradazione in percentuale per la maggior parte delle sostanze era minore del 20% durante i 3 mesi. Citalopram e Paliperidone sono rimasti stabili per il primo mese, mentre una degradazione maggiore del 50% è stata osservata dopo 3 mesi di conservazione. La Quetiapina è rimasta stabile in 2 campioni, mentre una degradazione significativa è stata osservata in altri due casi.
Campionamento “Dried Blood Spot” ed analisi quali-quantitativa LC-MS/MS di farmaci psicoattivi in sangue cadaverico
VALENTINI, BEATRICE
2017/2018
Abstract
Background and aims: The analysis of blood collected and dried on a paper substrate (i.e., "dried blood spot" or DBS) represents an alternative blood sample collection that nowadays is routinely used in neonatal screening. The goals of this study were: 1) to develop a LC-MS/MS method for the determination of 5 antidepressants and 2 antipsychotics in DBSs; 2) to establish the diagnostic reliability of DBSs with the routine blood analyses of these substances; 3) to prove the stability of these analytes on DBS within a 3-month period of storage. Method: 85 µL of blood were put on a card for DBS analysis and allowed to dry overnight in the dark, at room temperature. The whole blood stain was cut and put in a plastic tube, containing 1 mL phosphate buffer at pH 6 and deuterated internal standards, extracted using SPE cartridges and injected in the LC-MS/MS system. The analytes were separated through a reverse phase chromatography on a C18 column and detected on a triple quad operating in Multiple Reaction Monitoring mode and positive polarization. A calibration curve was prepared in the range LOQ-500 ng/mL. The procedure was applied to 60 authentic postmortem cases. For each case, 15 DBSs were collected at T0. 4 DBSs were analyzed in triplicates within 4 consecutive weeks. The last samples were analyzed after three months. Results and discussion: The analytical procedure is simple, sensitive and specific. LODs were detected in a range of 0.1 to 4.1 ng/mL(g) for antidepressants, and 0.1 to 2.7 ng/mL for antipsychotics. LOQs vary from 0.3 to 13.6 ng/ml for antidepressants, and 0.3 to 9.1 ng/mL for antipsychotics. Validation parameters fulfilled all the acceptance criteria. 4 cases were positive for citalopram and quetiapine (84-2309 ng/mL), 3 for desvenlafaxine (479-686 ng/mL) and venlafaxine (25-362 ng/mL), 2 for paliperidone (17-56 ng/mL) and trazodone (91-162 ng/mL). The concentrations on DBSs, stored at room temperature, were in good agreement with the ones obtained on blood samples analyzed with routine methods (<30% CV, both at T0 and after 3 months). The degradation percentage for most of the substances was lower than 20% within the three-month period. Citalopram and paliperidone were stable for the first month, while a degradation higher than 50% was observed after three-month storage. Quetiapine was found to be stable into 2 samples, while a significant degradation was noticed in other two cases.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/18530