Simultaneous Quantification of Antipsychotic and Antiepileptic Drugs and Their Metabolites in Human Saliva Using UHPLC-DAD

Abstract

Neuroleptics and antiepileptics are excreted in saliva, which can, therefore, be very useful in determining their concentration in the body. This study presents a method developed to simultaneously identify five neuroleptics—olanzapine, quetiapine, risperidone, aripiprazole, and clozapine—and the antiepileptic carbamazepine together with their metabolites: N-demethyl olanzapine, norquetiapine, 9-OH-risperidone, dehydroaripiprazole, N-desmethylclozapine, and carbamazepine-10,11 epoxide. Chlordiazepoxide was used as the internal standard. Strata-X-C columns were used for isolation of the compounds. Chromatographic analysis was carried out using UHPLC with a diode array detector (DAD). A mixture of acetonitrile and water with the addition of formic acid and 0.1% triethylamine was used as the mobile phase. The developed method was validated by determining the linearity for all analytes in the range 10–1000 ng/mL and the value of R2 > 0.99. Intra- and inter-day precision were also determined, and the relative standard deviation (RSD) value in both cases did not exceed 15%. To determine the usefulness of the developed method, saliva samples were collected from 40 people of both sexes treated with the tested active substances both in monotherapy and in polypragmasy. In all cases, the active substances tested were identified.

Deproteinization as a Rapid Method of Saliva Purification for the Determination of Carbamazepine and Carbamazepine-10,11 Epoxide

“…The methods for the determination of anti-epileptic drugs in blood have often been modified to determine carbamazepine in saliva [2,3,6,8,9,17,18]. Most often, saliva samples were 1 mL [14,18,22,23,25] or more [3,6,8], and were rarely less than 1 mL [15,21,24,27]. The method developed in this study allows for the determination of carbamazepine and its active metabolite, in as little as 200 µL of saliva.…”

“…Applied to the analysis of neuroleptics and carbamazepine, it allowed for good saliva purification and an analysis of patient samples. However, when the saliva contained the active metabolite carbamazepine, the extraction process was inefficient, as evidenced by the relatively low recovery [23]. Therefore, it was decided to use Phree columns, which do not require activation, and the purification process is simplified by adding to the analyzed sample a 1% formic acid solution in methanol or acetonitrile as a deproteinizing agent.…”

“…Saliva sampling for the analysis of the carbamazepine content is most often taken following stimulating its secretion by chewing Parafilm [3,9,14] or eating citric acid [15][16][17][18]. Saliva samples can also be collected in a tube without prior stimulation [8][9][10][11][12][13][14][15][16][17][18][19][20] or on cotton swabs specially designed for this purpose, which are placed in the mouth to soak up the saliva [21][22][23]. After removing the swab from the mouth, it is placed in a tube and is centrifuged.…”

“…The most common method of carbamazepine isolation from saliva samples is liquid-liquid extraction [8,14,16,[24][25][26]. Saliva samples are also purified using a modified liquid-liquid extraction, cloude-point extraction [26], solid phase extraction [22,23], and saliva deproteinization [15,20].…”

“…Carbamazepine in saliva can be quantified by an enzymatic method (FPIA-fluorescence polarization immunoassay) [7,19], as well as by separation techniques, gas chromatography [2,16,27], and liquid chromatography coupled with UV detection [3,8,9,14,15,17,18,[20][21][22][23][24][25][26].…”