1. Introduction
Depression is considered to be a serious and chronic mental illness characterized by low mood, loss of interest and desire, sleep disorders, fatigue, suicidal behavior, the ability to compromise social and occupational functions, and affecting individuals regardless of their social or economic status [
1,
2,
3,
4,
5]. The World Health Organization predicted that this disorder would affect individuals of both sexes and of all ages, being considered the second-leading cause of global disease by 2020, and leading consequently to early deaths due to physical health problems and difficulty accessing health services [
1,
6].
The most common and effective treatment for moderate-to-severe depression is the administration of antidepressants, which have been increasingly prescribed in recent decades to treat this disorder, but also for other mental health problems such as anxiety, which has led to several expert warnings [
7,
8,
9]. Currently, second-generation antidepressants are the choice of first-line treatment due to their similar efficacy to classic antidepressants and fewer side effects [
10,
11]. This medication can be prescribed along with other classes of compounds and can, consequently, lead to drug interactions that can be exacerbated by the uncertainty of the dose to be administered. In addition, antidepressants show inter-individual differences, and their therapeutic windows are narrow; as a result, therapeutic drug monitoring is of great interest and importance for patient compliance and safety [
7,
12,
13]. Monitoring allows for the optimization of treatment with these drugs, adjusting and customizing the dosages for each patient and, thus, minimizing toxicity and side effects, avoiding poisoning, lack of response, or non-adherence to treatment, saving costs through the rational use of drugs and resources, and achieving better quality of life [
13,
14,
15,
16]. For this monitoring to be possible—and also because the excessive use and abuse of these drugs is verified, culminating in clinical and forensic cases of accidental or voluntary overdose—it is extremely important that analytical methodologies are developed and made available for the identification of antidepressants and their metabolites in biological fluids [
10,
16].
One of the steps to take into account in an analytical method is the isolation and concentration of analytes of interest from the biological samples under study; the most used procedures for extracting antidepressants are liquid–liquid extraction (LLE) [
6,
17], solid-phase extraction (SPE) [
6,
18,
19,
20], and some miniaturized techniques—such as solid-phase microextraction (SPME) [
6,
21,
22], microextraction by packed sorbent (MEPS) [
6,
23,
24,
25], and dispersive liquid–liquid microextraction (DLLME) [
6,
26,
27,
28,
29,
30,
31]. There are also several methods involving gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) [
26,
30,
31,
32,
33,
34,
35,
36,
37,
38] or tandem mass spectrometry (MS/MS) [
27,
39,
40,
41,
42,
43], ultraviolet (UV) [
22,
28,
44,
45,
46,
47,
48], fluorescence [
25], diode array (DAD) or photodiode array (PDA) [
24,
37,
49,
50,
51], and flame ionization (FID) [
29,
52,
53,
54] detectors; more recently, coupling with time-of-flight mass spectrometry (TOF-MS) [
21] or quadrupole time-of-flight mass spectrometry (QTOF) [
55] has been reported. However, analyses by GC–MS and GS–MS/MS are still the methods of choice, due to their sensitivity and selectivity, which allow them to obtain low limits of quantification, possess separation power for volatile compounds such as the compounds under study, and are robust and generally available in most laboratories.
Presently, oral fluid is considered an excellent alternative in both the clinical and forensic areas for drug determination in biological samples, presenting advantages such as ease of collection, lower risk of adulteration, and a smaller drug detection window, allowing a better correlation with drug effects [
56,
57,
58]. As a way to overcome the disadvantage of classical extraction methods that apply a larger volume of biological sample, miniaturized techniques such as dried matrix spots have been explored, representing a simple and fast procedure compared to other extraction techniques. Applied to blood samples, the technique of dried blood spots (DBS) has been used to determine antidepressants [
40,
42]. Both the DBS and the dried saliva spots (DSS) techniques have been used in several areas, such as pharmacology, in clinical pharmacokinetic studies, monitoring of drugs, and in the determination of several drugs [
59,
60,
61]. Our group has extensive experience in this area of research, and has already published several papers on methods for the quantification of pharmaceutical compounds based on the DSS sampling approach. Using only 50 µL of oral fluid, Carvalho et al. [
62] have determined antiepileptic drugs, Ribeiro et al. [
63] have determined methadone and its main metabolite EDDP, and Caramelo et al. [
64] have determined antipsychotic drugs. These techniques apply a smaller volume of biological sample and have lower costs of storage and transport compared to classical sampling techniques [
60,
61], and the DSS, by applying an alternative specimen, becomes an excellent alternative in situations where the amount of sample is limited, as in the case of oral fluid [
57,
65].
This article reports a methodology for the identification of some of the most frequently prescribed antidepressants, such as fluoxetine (FLX), norfluoxetine (NFLX), citalopram (CIT), sertraline (SRT) and paroxetine (PXT)—which are selective serotonin reuptake inhibitors—and venlafaxine (VLX) and O-desmethylvenlafaxine (DVLX) as an antidepressant and metabolite-selective serotonin–norepinephrine reuptake inhibitor, respectively, within the limits of their therapeutic range in only 100 µL of oral fluid samples, using DSS as an extraction procedure and GC–MS/MS analysis. To the best of our knowledge, this is the first application of DSS as an extraction technique to identify these drugs in oral fluid samples, which can be considered as an alternative to the classical techniques normally used in routine laboratory analysis.
3. Materials and Methods
3.1. Reagents and Standards
Standard solutions of fluoxetine hydrochloride (FLX), venlafaxine hydrochloride (VLX), norfluoxetine (NFLX), citalopram (CIT), and paroxetine (PXT) were provided by Sigma-Aldrich, (St. Louis, MO, USA).
O-desmethylvenlafaxine (DVLX) was acquired from LGC-Standards (Teddington, London), and sertraline hydrochloride (SRT) was kindly offered by Pfizer (Groton, MA, USA), and their molecular structures and molecular weights are shown in
Figure S2 (
Supplementary Materials). The internal standard (IS) protriptyline (PTP) was acquired from Sigma-Aldrich (Lisbon, Portugal). Methanol (Merck Co, Darmstadt, Germany) and acetonitrile (Carlo Erba Reagents, Val-de-Reuil, France) were both of analytical grade. Ethyl acetate, 2-propanol, hexane, and dichloromethane were acquired from Fisher Scientific (Loughborough, UK). N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and trimethyl chlorosilane (TMCS) were acquired from Macherey-Nagel (Düren, Germany). Whatman™ 903 protein saver cards were acquired from Sigma-Aldrich (Sintra, Portugal).
All standards were acquired at 1 mg/mL. Working standard solutions were prepared by properly diluting the starting solutions with methanol to the final concentrations for the two compound mixtures. Mixture 1 contained FLX, VLX, DVLX, and NFLX at 10 µg/mL, CIT at 4 µg/mL, SRT at 5 µg/mL, and PXT at 2 µg/mL, while mixture 2 contained FLX and VLX at 5 µg/mL, DVLX and NFLX at 2.5 µg/mL, CIT at 1 µg/mL, SRT at 2 µg/mL, and PXT at 0.5 µg/mL. A working solution of the IS was prepared in methanol at a concentration of 1 µg/mL. All of the above solutions were stored in the absence of light at 4 °C.
3.2. Biological Specimens
Blank oral fluid samples used in all experiments for the present work were obtained by laboratory staff. Authentic oral fluid samples were analyzed routinely and were obtained from patients under treatment at the Centro Hospitalar Cova da Beira. These samples were sent to our laboratory (Laboratório de Fármaco-Toxicologia, UBIMedical, Covilhã, Portugal) for analysis. All oral fluid specimens were collected by spitting, and without the use of specific collection devices. These samples were stored refrigerated at −20 °C until analysis.
3.3. Sample Preparation
The final extraction procedure for the antidepressants was as follows: After homogenization in the vortex mixer, 100 μL of oral fluid was applied to Whatman® 903 protein saver cards and dried for 1 h at room temperature. Then, the spots of each sample were cut with scissors, placed in tubes, and 1 mL of methanol and 20 μL of IS (1 μg/mL) were added, followed by the extraction process—performed with a roller mixer for 5 min at room temperature. The samples were centrifuged for 5 min at 3500 rpm, and the spots were removed from the tubes. The extract was evaporated to dryness under a gentle nitrogen stream, and was subsequently derivatized with 50 μL of MSTFA with 5% TMCS for 2 min in a microwave oven at 800 W. Finally, a 2 μL aliquot of the derivatized extract was injected into the GC–MS/MS system.
The amount of the derivatization agent used is a common parameter in the development of analytical procedures, but also an internal factor optimized by the research group, whereby a compromise is required between the amount used and the chromatographic behavior and signal of the analytes of interest. The derivatizing agent is always added in excess, so as to not be the limiting reagent of the reaction.
3.4. Gas Chromatographic and Mass Spectrometric Conditions
Chromatographic analysis was performed using an HP 7890A gas chromatography system equipped with a model 7000B triple-quadrupole mass spectrometer, both from Agilent Technologies (Waldbronn, Germany), along with an MPS2 autosampler and a PTV injector from Gerstel (Mülheim an der Ruhr, Germany). Separation of the antidepressants was achieved using a capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness) with 5% phenylmethylsiloxane (HP-5MS), provided by J&W Scientific (Folsom, CA, USA).
The initial oven temperature was maintained at 150 °C for 1 min, and then increased to 280 °C at 5 °C/min and held for 4 min, giving a total runtime of 31 min. The injection inlet temperature was set at 250 °C, and the detector temperature was set at 280 °C. The 2 µL of derivatized sample was introduced into the gas chromatograph via splitless injection mode, and the helium was used as a carrier gas with a constant flow rate of 0.8 mL/min. The mass spectrometry was conducted with a filament current of 35 μA and electron energy of 70 eV in the positive electron ionization mode, and nitrogen was utilized as a collision gas at a flow rate of 2.5 mL/min. Data were acquired in the MRM mode using the MassHunter WorkStation Acquisition Software rev. B.02.01 (Agilent Technologies).
The retention time and mass-to-charge ratio (
m/z) spectra were initially obtained by individually injecting each of the standard antidepressant solutions at a comfortable concentration (100 µg/mL), and then used to identify the different compounds under study. Then, two transitions were chosen for each of the compounds, of which the most abundant transition was used for compound quantitation and the second transition for confirmation purposes. This choice of these transitions was made in order to obtain better selectivity and sensitivity for the analytes and less matrix interference, and the choice of ions for these same transitions was based on the highest masses and most abundant mass peaks (including more specific masses for each compound) in order to maximize the signal-to-noise ratio in the matrix extracts.
Table 6 shows the detection criteria—such as retention time, quantifier transition, qualifier transition, and collision energy—selected for each analyte.
4. Conclusions
A fully optimized and validated analytical method, which has been shown to be accurate, sensitive, and selective, is described for the simultaneous detection and quantification of five selective serotonin reuptake inhibitor antidepressants (fluoxetine, citalopram, sertraline, and paroxetine) and a selective serotonin–norepinephrine reuptake inhibitor and metabolite (venlafaxine and O-desmethylvenlafaxine) in oral fluid samples using DSS and GC–MS/MS. This method was linear within the range of 10–100 ng/mL for all analytes under study, with adequate accuracy and precision, and using only 100 µL of biological sample. The combination of DSS extraction and GC–MS/MS chromatographic analysis proved to be adequate for the determination of these drugs in oral fluid samples. Acceptable recovery values were obtained (13–46%), and good limits of quantification were achieved considering the therapeutic concentration ranges of the studied antidepressants. The low volume of specimen applied and the good sensitivity verified provide significant advantages, especially when there is little specimen availability, which is a problem in the case of the oral fluid, which allows multiple exams to be performed on the same sample. As the first report on the use of DSS as a sampling approach for these compounds, our findings can be considered to provide an alternative to the classical techniques normally implemented, which will result in lower consumption of sample, solvents, and analysis time. Furthermore, the ease of operation allows the routine use of this method in the identification of antidepressants in clinical and forensic toxicology analysis, and its application in authentic biological samples has proven its usefulness in drug monitoring.