Journal of Analytical Toxicology

Shin, Sanghee, Sarah;Borg,, Damon;Stripp,, Richard

doi: 10.1093/jat/bkz117pmid: 32115632

Abstract Antidepressant drugs are one of the most widely used medicines for treating major depressive disorders for long time periods. Oral fluid (OF) testing offers an easy and non-invasive sample collection. Detection of antidepressants in OF is important in clinical and forensic settings, such as therapeutic drug monitoring and roadside testing for driving under influence. We developed and validated a comprehensive liquid chromatography–tandem mass spectrometry method for 18 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, cyclobenzaprine, desipramine, desvenlafaxine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone, trimipramine, venlafaxine) in oral fluid collected by Quantisal® oral collection devices. One-half milliliter of Quantisal® OF (125 μL of neat OF) was submitted to solid-phase extraction. The chromatographic separation was performed employing a biphenyl column in gradient mode with a total run time of 5 min. The MS detection was achieved by multiple-reaction monitoring with two transitions per compound. The range for linearity of all analytes was from 10 to 1,000 ng/mL, with a limit of detection of 10 ng/mL. Intra and inter-day accuracy and precision (n = 15) were all within acceptable limits, ±20% error and ±15% relative standard deviation. Analyte recovery at 400 ng/mL concentration (n = 15) ranged from 91 to 129%. Matrix effect ranged from 73.7 to 157%. The internal proficiency test detected all antidepressants with accuracy ranging from 83.1 to 112.1%. The authentic patient sample showed a percentage difference compared to the previously calculated concentration of 86.3–111%. This method provides for the rapid detection of 18 antidepressants and metabolites in OF, which is readily applicable to a routine laboratory. Antidepressant, LC–MS-MS, Oral fluid Introduction Antidepressant drugs are one of the most widely used medicines to treat major depressive disorders for a long time (1). In USA, the number of adult population antidepressant users increased from 7.7 to 12.7% from 1999-2014 (2). Most of antidepressants work to regulate neurotransmitters (3,4). Tricyclic antidepressant (TCA) was the first class of antidepressant used; however, because of their narrow therapeutic window with greater risk of cardiotoxicity, central nervous system (CNS) toxicity (5), and respiratory depression (6), a new type was recommended. Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed because of their broader therapeutic range, lower risk, fewer side effects and better long-term outcomes (7). Because of their characteristic, increase in use of SSRIs, serotonin, and norepinephrine reuptake inhibitor (SNRI) were observed instead of TCA (8,9). However, since the majority of antidepressants work modifying neurotransmitter levels, cases related to reactions like serotonin syndrome are commonly observed (10), Not only but life–threatening cases and impairments from the use of serotonergic drug alone or drug–drug interactions with serotonergic drug are prevalent (11). Several method for in blood detection of antidepressant have been published previously (11, 12, 13, 14, 15), however, oral fluid (OF) detection is becoming a popular alternative method for collecting biological sample. It is known to be non-invasive and easy to collect (16). The collection devices offer the advantages of collecting a fixed amount of OF (normally 1 mL) and presence of preservative buffer that limits the instability of the drugs and metabolites in the OF such as use of Quantisal (19). OF represents recent uptake of free analyte fraction of the drugs and its metabolite (5). Compared to urine, OF is less prone to adulteration although it has a smaller window of detection. Also, OF has higher possibility of giving positive rates and sensitivity and quantification compared to urine for illicit drug (17). The tester can analyze what is in the donor’s system at the time of drug test collection. However, the disadvantages of OF include the limit of sample volume, factors affect diffusion of analyte from plasma to OF (pH, molecular size, ion trapping, degree of protein binding, drug pKa and lipophilicity) and the possibility of oral contamination (16, 18). It is important to quantify antidepressants in the OF. There are cases of serious clinical and forensic implications, such as therapeutic drug monitoring (TDM) and driving under influence (DUI). TDM of antidepressants during the treatment is key to optimizing the drug therapy experience for the individual patient (20) and for avoiding the risk of any toxicity (21). Few studies report misuse and overdoses of antidepressants which result from the combination of drugs such as monoamine oxidase inhibitors (MAOI) and SSRIs (24) or intake of fluoxetine (22, 23). Patients overdosing may experience deteriorating cognition and motor function as result of antidepressant withdrawal syndrome (27, 28, 29). Much of the research was done with quantifying antidepressants in blood and urine with different methods (30, 31, 32, 33, 34, 35). There are several researches done using the OF as an alternative to plasma for TDM of different drugs and compounds (25, 26). But only a limited number of publication was done with detection of multiple antidepressant in OF (3, 5, 36). The aim of this study was to develop a fast procedure for the quantification of 18 antidepressants and metabolites in OF using SPE and LC–MS-MS analysis. Materials and Methods Reagents and chemicals All 18 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, cyclobenzaprine, desipramine, desvenlafaxine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone, trimipramine, venlafaxine) at 1 mg/mL in 1 mL methanol and some of their internal standards (IS) (amitriptyline-d3, bupropion d9, citalopram d6, desipramine d3, doxepin d3, duloxetine d3, fluoxetine d6, imipramine d3, nortriptyline d3, venlafaxine d6, paroxetine d6, sertraline d3) were purchased from Cerilliant (Round Rock, TX) at 100 μg/mL in 1 mL methanol, except for Doxepin-d3 which had a concentration of 1 mg/mL in 1 mL methanol. Solvents and chemicals, including methanol, dichloromethane, 2-propanol, formic acid, acetic acid, sodium acetate, ammonium hydroxide, isopropanol, sodium phosphate and acetone, were purchased from VWR International (Bridgeport, NJ). All solvents were of high-performance liquid chromatography grade or better. Oral fluid collection devices Quantisal devices for the collection of OF specimens were obtained from Immunalysis Corporation (Pomona, CA). The total specimen volume for the analysis is 4 mL (1 mL OF +3 mL buffer). Oral fluid specimens for method development Synthetic OF mixed with Quantisal buffer from Immunalysis Corporation (Pomona, CA) was used for the preparation of calibrators and QC. Negative authentic OF were collected from laboratory volunteers and utilized during ME, recovery and specificity experiment. Also, authentic OF from donors prescribed antidepressant medication were de-identified and provided by Cordant Health Solutions for research purposes. Working solutions A 100,000-ng/mL intermediate stock was prepared: firstly by adding nine antidepressants at 1 mg/mL in a 10-mL volumetric flask and vortexed for a few seconds. Then, the mixture was dried completely at 40°C under a stream of nitrogen gas. The same procedure was repeated after adding the rest of nine antidepressants and drying it out. Ten milliliters of methanol was added to the flask and was vortexed for 15 s. The solution was transferred to the brown bottle labeled Antidepressant Stock 100,000 ng/mL. Other intermediate stocks (10,000, 1,000 and 100 ng/mL) were made by dilution with methanol. The stock solutions were stored at a −20°C freezer. Three working quality control (QC) standards were prepared in methanol from separate stock material at concentrations of 100, 1,000 and 10,000 ng/mL. A deuterated IS working solution of 50,000 ng/mL was prepared by appropriately diluting the IS stock solution with 50% methanol in distilled water and stored at a −20°C freezer until the analysis, except for Doxepin-d3s at 500,000 ng/mL. Table I MRM Transitions of Each Compound with Constant Cell Accelerator Voltage of 7 and Positive Polarity Compound name . Precursor ion . Product ion . Ret time (min) . Fragmentor . Collision energy . Amitriptyline 278.2 105.1 2.37 45 15 278.2 232.9 2.37 45 13 Amitriptyline-d3 281.2 105.1 2.37 70 10 Bupropion 240.1 184 1.65 45 7 240.1 131.1 1.65 45 7 Bupropion-d9 249.2 185.1 1.63 80 10 Citalopram 325.2 262.2 1.96 15 18 325.2 108.9 1.96 15 18 Citalopram-d6 331.2 108.8 1.96 50 22 Clomipramine 315.2 86.1 2.3 50 22 315.2 58.1 2.3 50 22 Cyclobenzaprine 276.2 231.2 2.33 25 14 276.2 216 2.33 25 14 Desipramine 267.3 236.3 2.28 10 6 267.2 72.1 2.28 10 6 Desipramine-d3 270.3 75.1 2.27 10 6 Doxepin 280.2 235 2.07 110 16 280.2 107.4 2.07 110 20 Doxepin-d3 283.2 107.1 2.07 110 20 Duloxetine 298.2 154 2.32 80 2 298.2 44.2 2.32 80 16 Duloxetine-d3 301.1 157.2 2.32 70 2 Fluoxetine 310.1 148 2.12 70 2 310.1 44.1 2.12 70 2 Fluoxetine-d6 316.2 154.1 2.11 70 2 Imipramine 281.2 86.2 2.31 110 12 281.2 58.1 2.31 110 48 Imipramine-d3 284.3 89.1 2.31 90 14 Mirtazapine 266.2 209.1 1.42 30 16 266.2 195.1 1.42 30 16 Nortriptyline 264.2 233.3 2.34 105 16 264.2 117.2 2.34 105 24 Nortriptyline d3 267.3 117.1 2.34 30 16 O-Desmethylvenlafaxine 264.2 246.2 1.22 100 8 264.2 201.2 1.22 100 8 Paroxetine 330.2 192 2 .28 110 16 330.2 70.2 2.28 110 34 Paroxetine d6 336.2 198.5 2.28 70 10 Sertraline 307.1 275.8 2.51 85 6 307.1 158.8 2.51 85 24 Sertraline d3 309.1 275.1 2.51 60 4 Trazodone 372.2 176.1 2 50 14 372.2 148.1 2 50 14 Trimipramine 295.2 208.1 2.42 50 14 295.2 58.1 2.42 50 14 Venlafaxine 278.2 147.2 1.77 65 18 278.2 121 1.77 65 20 Venlafaxine d6 284.3 266.2 1.76 85 8 Compound name . Precursor ion . Product ion . Ret time (min) . Fragmentor . Collision energy . Amitriptyline 278.2 105.1 2.37 45 15 278.2 232.9 2.37 45 13 Amitriptyline-d3 281.2 105.1 2.37 70 10 Bupropion 240.1 184 1.65 45 7 240.1 131.1 1.65 45 7 Bupropion-d9 249.2 185.1 1.63 80 10 Citalopram 325.2 262.2 1.96 15 18 325.2 108.9 1.96 15 18 Citalopram-d6 331.2 108.8 1.96 50 22 Clomipramine 315.2 86.1 2.3 50 22 315.2 58.1 2.3 50 22 Cyclobenzaprine 276.2 231.2 2.33 25 14 276.2 216 2.33 25 14 Desipramine 267.3 236.3 2.28 10 6 267.2 72.1 2.28 10 6 Desipramine-d3 270.3 75.1 2.27 10 6 Doxepin 280.2 235 2.07 110 16 280.2 107.4 2.07 110 20 Doxepin-d3 283.2 107.1 2.07 110 20 Duloxetine 298.2 154 2.32 80 2 298.2 44.2 2.32 80 16 Duloxetine-d3 301.1 157.2 2.32 70 2 Fluoxetine 310.1 148 2.12 70 2 310.1 44.1 2.12 70 2 Fluoxetine-d6 316.2 154.1 2.11 70 2 Imipramine 281.2 86.2 2.31 110 12 281.2 58.1 2.31 110 48 Imipramine-d3 284.3 89.1 2.31 90 14 Mirtazapine 266.2 209.1 1.42 30 16 266.2 195.1 1.42 30 16 Nortriptyline 264.2 233.3 2.34 105 16 264.2 117.2 2.34 105 24 Nortriptyline d3 267.3 117.1 2.34 30 16 O-Desmethylvenlafaxine 264.2 246.2 1.22 100 8 264.2 201.2 1.22 100 8 Paroxetine 330.2 192 2 .28 110 16 330.2 70.2 2.28 110 34 Paroxetine d6 336.2 198.5 2.28 70 10 Sertraline 307.1 275.8 2.51 85 6 307.1 158.8 2.51 85 24 Sertraline d3 309.1 275.1 2.51 60 4 Trazodone 372.2 176.1 2 50 14 372.2 148.1 2 50 14 Trimipramine 295.2 208.1 2.42 50 14 295.2 58.1 2.42 50 14 Venlafaxine 278.2 147.2 1.77 65 18 278.2 121 1.77 65 20 Venlafaxine d6 284.3 266.2 1.76 85 8 Open in new tab Table I MRM Transitions of Each Compound with Constant Cell Accelerator Voltage of 7 and Positive Polarity Compound name . Precursor ion . Product ion . Ret time (min) . Fragmentor . Collision energy . Amitriptyline 278.2 105.1 2.37 45 15 278.2 232.9 2.37 45 13 Amitriptyline-d3 281.2 105.1 2.37 70 10 Bupropion 240.1 184 1.65 45 7 240.1 131.1 1.65 45 7 Bupropion-d9 249.2 185.1 1.63 80 10 Citalopram 325.2 262.2 1.96 15 18 325.2 108.9 1.96 15 18 Citalopram-d6 331.2 108.8 1.96 50 22 Clomipramine 315.2 86.1 2.3 50 22 315.2 58.1 2.3 50 22 Cyclobenzaprine 276.2 231.2 2.33 25 14 276.2 216 2.33 25 14 Desipramine 267.3 236.3 2.28 10 6 267.2 72.1 2.28 10 6 Desipramine-d3 270.3 75.1 2.27 10 6 Doxepin 280.2 235 2.07 110 16 280.2 107.4 2.07 110 20 Doxepin-d3 283.2 107.1 2.07 110 20 Duloxetine 298.2 154 2.32 80 2 298.2 44.2 2.32 80 16 Duloxetine-d3 301.1 157.2 2.32 70 2 Fluoxetine 310.1 148 2.12 70 2 310.1 44.1 2.12 70 2 Fluoxetine-d6 316.2 154.1 2.11 70 2 Imipramine 281.2 86.2 2.31 110 12 281.2 58.1 2.31 110 48 Imipramine-d3 284.3 89.1 2.31 90 14 Mirtazapine 266.2 209.1 1.42 30 16 266.2 195.1 1.42 30 16 Nortriptyline 264.2 233.3 2.34 105 16 264.2 117.2 2.34 105 24 Nortriptyline d3 267.3 117.1 2.34 30 16 O-Desmethylvenlafaxine 264.2 246.2 1.22 100 8 264.2 201.2 1.22 100 8 Paroxetine 330.2 192 2 .28 110 16 330.2 70.2 2.28 110 34 Paroxetine d6 336.2 198.5 2.28 70 10 Sertraline 307.1 275.8 2.51 85 6 307.1 158.8 2.51 85 24 Sertraline d3 309.1 275.1 2.51 60 4 Trazodone 372.2 176.1 2 50 14 372.2 148.1 2 50 14 Trimipramine 295.2 208.1 2.42 50 14 295.2 58.1 2.42 50 14 Venlafaxine 278.2 147.2 1.77 65 18 278.2 121 1.77 65 20 Venlafaxine d6 284.3 266.2 1.76 85 8 Compound name . Precursor ion . Product ion . Ret time (min) . Fragmentor . Collision energy . Amitriptyline 278.2 105.1 2.37 45 15 278.2 232.9 2.37 45 13 Amitriptyline-d3 281.2 105.1 2.37 70 10 Bupropion 240.1 184 1.65 45 7 240.1 131.1 1.65 45 7 Bupropion-d9 249.2 185.1 1.63 80 10 Citalopram 325.2 262.2 1.96 15 18 325.2 108.9 1.96 15 18 Citalopram-d6 331.2 108.8 1.96 50 22 Clomipramine 315.2 86.1 2.3 50 22 315.2 58.1 2.3 50 22 Cyclobenzaprine 276.2 231.2 2.33 25 14 276.2 216 2.33 25 14 Desipramine 267.3 236.3 2.28 10 6 267.2 72.1 2.28 10 6 Desipramine-d3 270.3 75.1 2.27 10 6 Doxepin 280.2 235 2.07 110 16 280.2 107.4 2.07 110 20 Doxepin-d3 283.2 107.1 2.07 110 20 Duloxetine 298.2 154 2.32 80 2 298.2 44.2 2.32 80 16 Duloxetine-d3 301.1 157.2 2.32 70 2 Fluoxetine 310.1 148 2.12 70 2 310.1 44.1 2.12 70 2 Fluoxetine-d6 316.2 154.1 2.11 70 2 Imipramine 281.2 86.2 2.31 110 12 281.2 58.1 2.31 110 48 Imipramine-d3 284.3 89.1 2.31 90 14 Mirtazapine 266.2 209.1 1.42 30 16 266.2 195.1 1.42 30 16 Nortriptyline 264.2 233.3 2.34 105 16 264.2 117.2 2.34 105 24 Nortriptyline d3 267.3 117.1 2.34 30 16 O-Desmethylvenlafaxine 264.2 246.2 1.22 100 8 264.2 201.2 1.22 100 8 Paroxetine 330.2 192 2 .28 110 16 330.2 70.2 2.28 110 34 Paroxetine d6 336.2 198.5 2.28 70 10 Sertraline 307.1 275.8 2.51 85 6 307.1 158.8 2.51 85 24 Sertraline d3 309.1 275.1 2.51 60 4 Trazodone 372.2 176.1 2 50 14 372.2 148.1 2 50 14 Trimipramine 295.2 208.1 2.42 50 14 295.2 58.1 2.42 50 14 Venlafaxine 278.2 147.2 1.77 65 18 278.2 121 1.77 65 20 Venlafaxine d6 284.3 266.2 1.76 85 8 Open in new tab Calibrators and QCs Six calibrators at concentrations of 10, 20, 40, 200, 400 and 1,000 ng/mL and three QC samples at concentrations of 40, 200 and 800 ng/mL were prepared by spiking the corresponding calibrators’ and controls’ methanolic working solutions in 0.5 mL of drug-free synthetic OF-buffer mixture (0.125 mL OF +0.375 mL Quantisal buffer) from Immunalysis Corporation (Pomona, CA). The IS at a concentration of 500 ng/mL were used throughout the experiment. Extraction procedure An automated liquid dispenser Cerex® 48, SPEware (Baldwin Park, CA) and mixed mode Cerex® Trace-B cartridges 3 mL 35 mg, SPEware (Baldwin Park, CA), were used in the extraction procedure. Each cartridge was conditioned with 500 μL of methanol and 500 μL of distilled water. Then, 1 mL of 0.1 M sodium phosphate buffer pH 6 was added. Five hundred microliters of the Quantisal sample and 30 μL of IS at 500 ng/mL were mixed and loaded on to the cartridge. Washing was performed with 3 mL of distilled water, 3 mL of 0.1 M of acetic acid and 3 mL of 25% methanol in distilled water. Cartridges were dried with nitrogen heated to 40°C (14 min). The drugs were eluted with 750 μL of freshly prepared dichloromethane: isopropyl alcohol: ammonium hydroxide (70:26:4). The eluent was evaporated at 40°C under a stream of nitrogen gas. The dried extract was reconstituted in 150 μL of 0.1% formic acid in distilled water. The reconstituted samples were transferred to auto-sampler vials, and 10 μL was injected onto the LC–MS-MS. Instrumental analysis (LC–MS-MS) Agilent Technologies 6460 LC equipped with triple-quadrupole liquid chromatography tandem mass spectrometer (QQQ LC–MS) was used for chromatographic separation. We employed mobile phase combination of 0.1% formic acid in distilled water (A) and methanol (B) at a flow rate of 0.7 mL/min and column temperature of 50°C and using a Kinetex Biphenyl column (2.1 mm × 50 mm × 2.6 μm). The following gradient mode was applied: 20% until 0 min; then, percentage was gradually increased to 65% until 1.8 min; another increase to 75% at 2.2 min, with a total run time of 5 min and injection volume of 10 μL. For detection, a direct injection of 1 μL of each analyte (1,000 ng/mL) to QQQ LC–MS with a Jet Stream electrospray source was done. The molecular weight and the electrospray ion source were in positive mode, the precursor ion was obtained and product ion scans at different collision cell energies were performed to obtain a list of fragment ions. All tandem mass spectrometer parameters were optimized to produce the greatest analyte response: 350°C gas temperature, 10 L/min nebulizer gas flow, 50 psi nebulizer gas pressure, 4000 V capillary voltage, 400°C sheath gas temperature, 11 L/min sheath gas flow and 1500 V of nozzle voltage. The mass spectrometer operated in electrospray positive mode with a cell accelerator voltage of 7. Scheduled multiple reaction monitoring (MRM) mode was used for compound detection with a detection window set of 1 min around the expected retention time (Table I). Method validation The proposed method was validated by evaluating the linearity, accuracy, precision, limit of quantification (LOQ), matrix effect (ME), selectivity, interference, carryover, process efficiency, stability and specificity based on the Scientific Working Group for Forensic Toxicology (37). Determination of linearity for the calibration curve was investigated over at least five different days using five non-zero calibrators using 500 mL of synthetic OF (125 μL of neat OF). The calibration curve was evaluated using a least-square residual model incorporating different weighting factors (none, 1/x, 1/x2) to produce an accurate model (linear and quadratic). Linearity was considered acceptable if the individual residual was within a 20% range of the expected concentration, and the coefficient of determinations (r2) was greater than 0.985. The lower limit of quantitation (LLOQ) was chosen to be at the same concentration as the lowest non-zero calibrator and was examined in 10 different donor samples. The accuracy and precision of the LLOQ samples were required to be within 20% of the expected concentration in order to pass. Six calibration curves, negative samples, and three QC were extracted on the same day and analyzed by LC–MS-MS on five separate days and expressed by relative mean error (RME%), standard deviation (SD%) and coefficient variation (CV%) which were expressed for intra-day and inter-day precision and accuracy. The precision of this method was determined by triplicate analysis of QC samples at the following concentrations: 40, 200 and 800 ng/mL. Samples were analyzed as part of one batch to determine intra-day precision of the assay (n = 3). Additionally, for inter-day precision and accuracy, three replicates of QC at three different concentrations were prepared and analyzed in five different days (n = 15). For carryover, samples were extracted at the upper limit of linearity, ULOL (1,000 ng/mL) and 10 times the concentration of ULOL (10,000 ng/mL). Two blank samples were placed after ULOL, and three blank samples were placed after 10 × ULOL. The detection of the analytes above the limit of detection in blank samples would indicate that carryover exists in this method. Figure 1 Open in new tabDownload slide Representative chromatograms showing separation of all analytes at the LLOQ (10 ng/mL). Doxepin’s unique peak shape is due to the mixture of cis- and trans-isomers (in a 15:85 ratio in prescribed medication as well as the Cerilliant standards). Figure 1 Open in new tabDownload slide Representative chromatograms showing separation of all analytes at the LLOQ (10 ng/mL). Doxepin’s unique peak shape is due to the mixture of cis- and trans-isomers (in a 15:85 ratio in prescribed medication as well as the Cerilliant standards). ME was evaluated comparing the neat, pre-extraction and post-extraction response (38). Negative OF specimens from 15 donors were selected and analyzed to determine if any significant LC–MS-MS response. Three neat bottles are prepared by adding 1,000 ng/mL of a concentration drug panel directly into a vial to achieve 400 ng/mL. Two sets of 15 real matrices (500 μL) were prepared by spiking with a stock solution to achieve 400 ng/mL concentration: pre-extraction and post-extraction. All samples were evaporated and followed with reconstitution of 150 μL of 0.1% formic acid in distilled water. ME was calculated comparing pre-extraction and neat samples. Analyte recovery was calculated comparing post-extraction and pre-extraction samples. Process efficiency was calculated comparing post-extraction and neat samples. An ME value less than 100% indicates ion suppression, and values greater than 100% indicate ion enhancement. ULOL (1,000 ng/mL) was diluted 1:2, 1:5 and 1:10 with negative OF (n = 4). Results were expected to be within 20% of the target value. The limit of quantification (LOQ) was defined as the lowest concentration, where the analyte could be quantified with a mean relative error less than 20%. Ten samples were spiked at LOQ (level l = 10 ng/mL) in the synthetic OF. To evaluate the potential interference from the OF matrix, negative OF specimen from 15 donors were selected and analyzed to determine if any significant LC–MS-MS response representing antidepressant could be detected. The presence of an LC–MS-MS peak could indicate possible IS impurity. Each drug response and IS response were compared to see the significance of each. The presence of any peak that was not expected would indicate possible impurity. For endogenous factors, 15 authentic donor samples, without any presence of analytes of interest or IS. Interference of endogenous factors was absent if the response at the point of interest was below one half of the response of LOD. To evaluate the interference of exogenous factors, 10 samples with an antidepressant panel spiked at 10 ng/mL and a multiple-drug panel (aripiprazole, asenapine, brexpiprazole, chlorpromazine, clozapine, fluphenazine, haloperidol, lurasidone, olanzapine, paliperidone, perphenazine, quetiapine, risperidone, ziprasidone, 6-acetylmorphine, benzoylecgonine, cocaine, codeine, EDDP, hydrocodone, hydromorphone, MDA, MDEA, MDMA, methadone, methamphetamine, morphine, norhydorcodone, noroxycodone, oxycodone, oxymorphone, phencyclidine, fentanyl) spiked at 1,000 ng/mL with IS in OF samples. The panel includes drugs that are often prescribed together. Interference of exogenous factors was absent if the concentration of the antidepressant drugs was within ±20% of 10 ng/mL. To determine if the addition of IS could contribute to the analyte response, oral fluid specimen from 15 volunteers were spiked with IS and analyzed to determine if any significant LC–MS-MS response representing any of the antidepressants. Auto-sampler stability was evaluated in extracted samples. Three levels of QC samples were left out at room temperature for 72 h. They were compared with newly extracted QCs. Any concentration accuracy of over 20% should be considered unstability of sample and taken in consideration. Authentic and internal control oral fluid specimens The method was applied to seven authentic OF samples previously analyzed by Cordant Health Solution Laboratory, by SPE and LC–MS-MS. The samples were collected with Quantisal® l and stored at room temperature until analysis from few days to weeks. The method was also applied to three samples fortified at the three levels (48, 280, 780 ng/mL), as part of an internal proficiency test in the laboratory. Results Calibration curves were linear with a correlation coefficient >0.985 over the range from 10 to 1,000 ng/mL of OF. The ratio of the intensity of the qualifying transitions to the intensity of the quantifying transitions was acceptable. The 15 different OF samples spiked at LLOQ levels range from 94 to 117%. A clear chromatogram of each analyte at the lowest level, LLOQ, was achieved (Figure 1). For accuracy, inter-day analyses were between 0 and 11.6% and intra-day analyses were between 0.7 and 9.9%. For precision, inter-day analyses were between 3.8 and 11.3% and intra-day analyses were between 0 and 7.3%. All the accuracy was within 20% and precision was within 15% as shown in Table II. Table II Intra- (n = 3) and Inter-day (n = 15) Precision and Accuracy Data (Q1: 40 ng/mL, Q2: 200 ng/mL, Q3: 800 ng/mL) . Precision (CV%) . Accuracy (% error) . . Intra-day . Inter-day . Intra-day . Inter-day . . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Amitriptyline 1.4 0.8 4.1 6.7 5.5 8.9 4.1 3.8 3.7 0.6 0.8 2.9 Bupropion 0.5 0.2 0.7 4.5 5 7.2 5.2 6.2 5.8 0.5 1 4.4 Citalopram 2.5 0.9 0.7 5.6 4.7 7.1 2.5 5.6 5.8 1.1 0.2 3.9 Clomipramine 2.5 1.2 1 6.2 6.7 7.2 1.9 4.9 6.2 1.6 0.1 3.5 Cyclobenzaprine 4.3 2.4 2.6 7.7 8.5 7.3 8.5 9.3 5 0.2 0 1.8 Desipramine 1.1 1.6 0.4 6.7 5.8 7.5 2.6 6.2 5.3 1.5 0 4.9 Doxepin 2.9 2.4 1.2 5.2 7 7.6 5.3 6.9 9.2 1.2 3.2 2.1 Duloxetine 4.2 2.2 3 8.1 12 5.3 3.3 1.8 5.3 1.9 4.7 5.2 Fluoxetine 5.6 3.8 0.6 7.6 9.9 8.1 5.3 4.1 5.2 2.5 1.4 5.9 Imipramine 0.7 1 0 6.9 7.5 7.5 6.1 7.1 7.1 1.5 1.3 3.6 Mirtazapine 1.5 1.5 0.1 6.7 7 8.2 0.7 5 4.4 1.8 1.7 5.4 Nortriptyline 3.2 2 2 6.6 5.9 7.7 3.1 4.1 5.6 0.1 0.9 4 O-Desmethylvenlafaxine 0.5 1.1 1 6 6.4 9.8 4.1 9.1 7.5 4.9 5.4 2.1 Paroxetine 6.2 5.4 7.3 5.9 6.4 11.3 9.9 9.1 8.3 11.6 7.9 2.6 Sertraline 6 0.9 1.4 8 6.8 9.7 4 0.8 7.6 1.7 4 3.9 Trazodone 1.2 1.1 0.2 3.8 5.6 6.6 1.1 3.9 6 3 0.6 4.5 Trimipramine 2.6 0.9 4.6 7.3 6.5 4.8 2 5.1 3.3 1.3 0.2 5 Venlafaxine 2.1 0.3 1 5 4.6 7.6 2.6 6.6 7.7 0.6 2.3 2.7 . Precision (CV%) . Accuracy (% error) . . Intra-day . Inter-day . Intra-day . Inter-day . . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Amitriptyline 1.4 0.8 4.1 6.7 5.5 8.9 4.1 3.8 3.7 0.6 0.8 2.9 Bupropion 0.5 0.2 0.7 4.5 5 7.2 5.2 6.2 5.8 0.5 1 4.4 Citalopram 2.5 0.9 0.7 5.6 4.7 7.1 2.5 5.6 5.8 1.1 0.2 3.9 Clomipramine 2.5 1.2 1 6.2 6.7 7.2 1.9 4.9 6.2 1.6 0.1 3.5 Cyclobenzaprine 4.3 2.4 2.6 7.7 8.5 7.3 8.5 9.3 5 0.2 0 1.8 Desipramine 1.1 1.6 0.4 6.7 5.8 7.5 2.6 6.2 5.3 1.5 0 4.9 Doxepin 2.9 2.4 1.2 5.2 7 7.6 5.3 6.9 9.2 1.2 3.2 2.1 Duloxetine 4.2 2.2 3 8.1 12 5.3 3.3 1.8 5.3 1.9 4.7 5.2 Fluoxetine 5.6 3.8 0.6 7.6 9.9 8.1 5.3 4.1 5.2 2.5 1.4 5.9 Imipramine 0.7 1 0 6.9 7.5 7.5 6.1 7.1 7.1 1.5 1.3 3.6 Mirtazapine 1.5 1.5 0.1 6.7 7 8.2 0.7 5 4.4 1.8 1.7 5.4 Nortriptyline 3.2 2 2 6.6 5.9 7.7 3.1 4.1 5.6 0.1 0.9 4 O-Desmethylvenlafaxine 0.5 1.1 1 6 6.4 9.8 4.1 9.1 7.5 4.9 5.4 2.1 Paroxetine 6.2 5.4 7.3 5.9 6.4 11.3 9.9 9.1 8.3 11.6 7.9 2.6 Sertraline 6 0.9 1.4 8 6.8 9.7 4 0.8 7.6 1.7 4 3.9 Trazodone 1.2 1.1 0.2 3.8 5.6 6.6 1.1 3.9 6 3 0.6 4.5 Trimipramine 2.6 0.9 4.6 7.3 6.5 4.8 2 5.1 3.3 1.3 0.2 5 Venlafaxine 2.1 0.3 1 5 4.6 7.6 2.6 6.6 7.7 0.6 2.3 2.7 Open in new tab Table II Intra- (n = 3) and Inter-day (n = 15) Precision and Accuracy Data (Q1: 40 ng/mL, Q2: 200 ng/mL, Q3: 800 ng/mL) . Precision (CV%) . Accuracy (% error) . . Intra-day . Inter-day . Intra-day . Inter-day . . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Amitriptyline 1.4 0.8 4.1 6.7 5.5 8.9 4.1 3.8 3.7 0.6 0.8 2.9 Bupropion 0.5 0.2 0.7 4.5 5 7.2 5.2 6.2 5.8 0.5 1 4.4 Citalopram 2.5 0.9 0.7 5.6 4.7 7.1 2.5 5.6 5.8 1.1 0.2 3.9 Clomipramine 2.5 1.2 1 6.2 6.7 7.2 1.9 4.9 6.2 1.6 0.1 3.5 Cyclobenzaprine 4.3 2.4 2.6 7.7 8.5 7.3 8.5 9.3 5 0.2 0 1.8 Desipramine 1.1 1.6 0.4 6.7 5.8 7.5 2.6 6.2 5.3 1.5 0 4.9 Doxepin 2.9 2.4 1.2 5.2 7 7.6 5.3 6.9 9.2 1.2 3.2 2.1 Duloxetine 4.2 2.2 3 8.1 12 5.3 3.3 1.8 5.3 1.9 4.7 5.2 Fluoxetine 5.6 3.8 0.6 7.6 9.9 8.1 5.3 4.1 5.2 2.5 1.4 5.9 Imipramine 0.7 1 0 6.9 7.5 7.5 6.1 7.1 7.1 1.5 1.3 3.6 Mirtazapine 1.5 1.5 0.1 6.7 7 8.2 0.7 5 4.4 1.8 1.7 5.4 Nortriptyline 3.2 2 2 6.6 5.9 7.7 3.1 4.1 5.6 0.1 0.9 4 O-Desmethylvenlafaxine 0.5 1.1 1 6 6.4 9.8 4.1 9.1 7.5 4.9 5.4 2.1 Paroxetine 6.2 5.4 7.3 5.9 6.4 11.3 9.9 9.1 8.3 11.6 7.9 2.6 Sertraline 6 0.9 1.4 8 6.8 9.7 4 0.8 7.6 1.7 4 3.9 Trazodone 1.2 1.1 0.2 3.8 5.6 6.6 1.1 3.9 6 3 0.6 4.5 Trimipramine 2.6 0.9 4.6 7.3 6.5 4.8 2 5.1 3.3 1.3 0.2 5 Venlafaxine 2.1 0.3 1 5 4.6 7.6 2.6 6.6 7.7 0.6 2.3 2.7 . Precision (CV%) . Accuracy (% error) . . Intra-day . Inter-day . Intra-day . Inter-day . . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Q1 . Q2 . Q3 . Amitriptyline 1.4 0.8 4.1 6.7 5.5 8.9 4.1 3.8 3.7 0.6 0.8 2.9 Bupropion 0.5 0.2 0.7 4.5 5 7.2 5.2 6.2 5.8 0.5 1 4.4 Citalopram 2.5 0.9 0.7 5.6 4.7 7.1 2.5 5.6 5.8 1.1 0.2 3.9 Clomipramine 2.5 1.2 1 6.2 6.7 7.2 1.9 4.9 6.2 1.6 0.1 3.5 Cyclobenzaprine 4.3 2.4 2.6 7.7 8.5 7.3 8.5 9.3 5 0.2 0 1.8 Desipramine 1.1 1.6 0.4 6.7 5.8 7.5 2.6 6.2 5.3 1.5 0 4.9 Doxepin 2.9 2.4 1.2 5.2 7 7.6 5.3 6.9 9.2 1.2 3.2 2.1 Duloxetine 4.2 2.2 3 8.1 12 5.3 3.3 1.8 5.3 1.9 4.7 5.2 Fluoxetine 5.6 3.8 0.6 7.6 9.9 8.1 5.3 4.1 5.2 2.5 1.4 5.9 Imipramine 0.7 1 0 6.9 7.5 7.5 6.1 7.1 7.1 1.5 1.3 3.6 Mirtazapine 1.5 1.5 0.1 6.7 7 8.2 0.7 5 4.4 1.8 1.7 5.4 Nortriptyline 3.2 2 2 6.6 5.9 7.7 3.1 4.1 5.6 0.1 0.9 4 O-Desmethylvenlafaxine 0.5 1.1 1 6 6.4 9.8 4.1 9.1 7.5 4.9 5.4 2.1 Paroxetine 6.2 5.4 7.3 5.9 6.4 11.3 9.9 9.1 8.3 11.6 7.9 2.6 Sertraline 6 0.9 1.4 8 6.8 9.7 4 0.8 7.6 1.7 4 3.9 Trazodone 1.2 1.1 0.2 3.8 5.6 6.6 1.1 3.9 6 3 0.6 4.5 Trimipramine 2.6 0.9 4.6 7.3 6.5 4.8 2 5.1 3.3 1.3 0.2 5 Venlafaxine 2.1 0.3 1 5 4.6 7.6 2.6 6.6 7.7 0.6 2.3 2.7 Open in new tab Table III ME and Recovery Data (n = 15) . ME (%) . %CV of ME . AR (%) . PE (%) . Amitriptyline 111.4 7 94.6 105.4 Bupropion 157.6 4.5 91.5 144.1 Citalopram 95.1 9.8 103.1 98 Clomipramine 86 6.4 104.5 89.9 Cyclobenzaprine 97.2 3.2 101.3 98.5 Desipramine 76 8.9 110.9 84.3 Doxepin 91.1 2.3 100.2 91.3 Duloxetine 73.7 8.5 129 95 Fluoxetine 78 4.9 104.4 81.5 Imipramine 110.5 1.8 100.1 110.6 Mirtazapine 124.1 3.1 99.1 123 Nortriptyline 89.5 7.7 105.8 94.7 O-Desmethylvenlafaxine 114.4 8.7 98.3 112.5 Paroxetine 82.3 3.6 113.9 93.7 Sertraline 77.7 8 107.1 83.2 Trazodone 97.9 9.2 99.6 97.5 Trimipramine 103.7 13.5 99.4 103 Venlafaxine 122.5 3.9 97.7 119.7 . ME (%) . %CV of ME . AR (%) . PE (%) . Amitriptyline 111.4 7 94.6 105.4 Bupropion 157.6 4.5 91.5 144.1 Citalopram 95.1 9.8 103.1 98 Clomipramine 86 6.4 104.5 89.9 Cyclobenzaprine 97.2 3.2 101.3 98.5 Desipramine 76 8.9 110.9 84.3 Doxepin 91.1 2.3 100.2 91.3 Duloxetine 73.7 8.5 129 95 Fluoxetine 78 4.9 104.4 81.5 Imipramine 110.5 1.8 100.1 110.6 Mirtazapine 124.1 3.1 99.1 123 Nortriptyline 89.5 7.7 105.8 94.7 O-Desmethylvenlafaxine 114.4 8.7 98.3 112.5 Paroxetine 82.3 3.6 113.9 93.7 Sertraline 77.7 8 107.1 83.2 Trazodone 97.9 9.2 99.6 97.5 Trimipramine 103.7 13.5 99.4 103 Venlafaxine 122.5 3.9 97.7 119.7 Open in new tab Table III ME and Recovery Data (n = 15) . ME (%) . %CV of ME . AR (%) . PE (%) . Amitriptyline 111.4 7 94.6 105.4 Bupropion 157.6 4.5 91.5 144.1 Citalopram 95.1 9.8 103.1 98 Clomipramine 86 6.4 104.5 89.9 Cyclobenzaprine 97.2 3.2 101.3 98.5 Desipramine 76 8.9 110.9 84.3 Doxepin 91.1 2.3 100.2 91.3 Duloxetine 73.7 8.5 129 95 Fluoxetine 78 4.9 104.4 81.5 Imipramine 110.5 1.8 100.1 110.6 Mirtazapine 124.1 3.1 99.1 123 Nortriptyline 89.5 7.7 105.8 94.7 O-Desmethylvenlafaxine 114.4 8.7 98.3 112.5 Paroxetine 82.3 3.6 113.9 93.7 Sertraline 77.7 8 107.1 83.2 Trazodone 97.9 9.2 99.6 97.5 Trimipramine 103.7 13.5 99.4 103 Venlafaxine 122.5 3.9 97.7 119.7 . ME (%) . %CV of ME . AR (%) . PE (%) . Amitriptyline 111.4 7 94.6 105.4 Bupropion 157.6 4.5 91.5 144.1 Citalopram 95.1 9.8 103.1 98 Clomipramine 86 6.4 104.5 89.9 Cyclobenzaprine 97.2 3.2 101.3 98.5 Desipramine 76 8.9 110.9 84.3 Doxepin 91.1 2.3 100.2 91.3 Duloxetine 73.7 8.5 129 95 Fluoxetine 78 4.9 104.4 81.5 Imipramine 110.5 1.8 100.1 110.6 Mirtazapine 124.1 3.1 99.1 123 Nortriptyline 89.5 7.7 105.8 94.7 O-Desmethylvenlafaxine 114.4 8.7 98.3 112.5 Paroxetine 82.3 3.6 113.9 93.7 Sertraline 77.7 8 107.1 83.2 Trazodone 97.9 9.2 99.6 97.5 Trimipramine 103.7 13.5 99.4 103 Venlafaxine 122.5 3.9 97.7 119.7 Open in new tab ME ranged 82.3–114.4%, within ±20% respect to neat, which showed no significant effect, except for bupropion, desipramine, duloxetine, fluoxetine, mirtazapine, sertraline and venlafaxine (Table III). The enhancement was observed for bupropion (157%), venlafaxine (122%) and mirtazapine (124%). Suppression was observed for duloxetine (73.7%), fluoxetine (78%), sertraline (77.7%), and desipramine (76%). CV% of ME were between 1.8 and 13.5%, which is within 15%. All analytical recoveries (AR) were ranging from 94.6 to 129%, indicating that the elution solvent (dichloromethane: isopropyl alcohol: ammonium hydroxide; 70:26:4) extraction effectively recovers analyte from oral fluid samples. All process efficiencies (PE) were ranging from 81.5 to 144.1%. The results of carryover showed no significant carryover, as calculated results for the reinjected blank were below 50% of the LOQ response. These data indicated that there will be no or little contamination from the subsequent sample. All diluted samples were quantitated within 20% of the expected concentration for all drugs. The quantitative values of all analyses in samples 1:2, 1:5 and 1:10 quantitated within 20% were not influenced by the dilution. The stability results showed that 18 antidepressants were stable at room temperature for 72 h, with accuracy between 88.7 and 109.8%, within ±20%. The result of exogenous interference, from 10 different sources of blank, showed average accuracy of 18 antidepressant concentrations between 92.5 and 118.2%, except for duloxetine (124%), mirtazapine (123%), paroxetine (129%) and trimipramine (124.6%). The results of endogenous interferences from 10 different sources of blank OF showed no interfering peaks at either analyte or the IS retention time. The result of IS interference from 10 different sources of blank OF with IS showed no interfering peaks at analyte. Authentic OF samples The authentic OF samples were already quantified in the Cordant Health Solution lab. Ten different antidepressant drugs were detected in these seven authentic patient samples. Among the positive samples, the percentage difference compares to previously calculated concentration ranged from 86.3 to 111% (Table IV). For the internal proficiency test, quantitative sample analysis accuracy was within ±20% which ranged from 83.1 to 112.1%. Table IV Positivity Results of Patient Samples Compared to Previously Quantified Concentration Patient . Administered AD . Calculated concentration (ng/mL) . Accuracy (%) . 1 Amitriptyline 12.3 86.3 Nortriptyline 16.8 105.1 2 Citalopram 160.1 105.5 3 Cyclobenzaprine 12.5 111 Venlafaxine 741.2 104.9 4 Desipramine 89.5 103.1 Imipramine 45.4 100.8 5 Fluoxetine 46.3 103.5 6 Paroxetine 10.8 107.6 7 Sertraline 217.7 108.6 Patient . Administered AD . Calculated concentration (ng/mL) . Accuracy (%) . 1 Amitriptyline 12.3 86.3 Nortriptyline 16.8 105.1 2 Citalopram 160.1 105.5 3 Cyclobenzaprine 12.5 111 Venlafaxine 741.2 104.9 4 Desipramine 89.5 103.1 Imipramine 45.4 100.8 5 Fluoxetine 46.3 103.5 6 Paroxetine 10.8 107.6 7 Sertraline 217.7 108.6 Open in new tab Table IV Positivity Results of Patient Samples Compared to Previously Quantified Concentration Patient . Administered AD . Calculated concentration (ng/mL) . Accuracy (%) . 1 Amitriptyline 12.3 86.3 Nortriptyline 16.8 105.1 2 Citalopram 160.1 105.5 3 Cyclobenzaprine 12.5 111 Venlafaxine 741.2 104.9 4 Desipramine 89.5 103.1 Imipramine 45.4 100.8 5 Fluoxetine 46.3 103.5 6 Paroxetine 10.8 107.6 7 Sertraline 217.7 108.6 Patient . Administered AD . Calculated concentration (ng/mL) . Accuracy (%) . 1 Amitriptyline 12.3 86.3 Nortriptyline 16.8 105.1 2 Citalopram 160.1 105.5 3 Cyclobenzaprine 12.5 111 Venlafaxine 741.2 104.9 4 Desipramine 89.5 103.1 Imipramine 45.4 100.8 5 Fluoxetine 46.3 103.5 6 Paroxetine 10.8 107.6 7 Sertraline 217.7 108.6 Open in new tab Discussion This study employs 500 μL Quantisal (125 μL of neat oral fluid) compared to Coulter et al. who used 1 mL from the Quantisal (250 μL of neat oral fluid), Knihnicki et al. (2014) who used 1 mL of oral fluid from the Salivette swabs and Castro et al. (2008) who used 0.2 mL of sample that was spat into polypropylene tubes directly from volunteers. Methodology employing a lower amount of sample is beneficial if there is a limited amount of sample, as it happens in oral fluid. Also, no additional steps were taken before the extraction compared to Knihnicki et al. who pretreated the oral fluid by adding 0.5 mL of phosphate buffer (pH 7.4), which was sonicated for 30 min and centrifuged twice. Coulter et al. added potassium phosphate buffer (0.1 M, pH 6) to oral fluid before extraction. Castro et al. added sodium acetate buffer pH 3.6 to oral fluid before the extraction. This method helped the lab to save the material and the time to handle when preparing the oral fluid sample before the extraction. Similar to previous studies (3, 5, 36), an extraction column of mixed mode cation exchange and reversed phase was used. This method has a broader calibration range compared to the previous studies (3, 5, 31). Our LLOQ (10 ng/mL) is higher than most of the previous methods because some of the drugs did not fulfill the criterion for accuracy, making it not suitable for the determination of analyte at a lower level. The use of the biphenyl column made the method faster compared to use of the C18 column from previous studies (3, 5, 31). For exogenous interference, the accuracy of duloxetine, mirtazapine, paroxetine and trimipramine was out of range so this should be taken into consideration when analyzing the concentration. For selectivity and especially for exogenous interference, this study was analyzed by adding 34 different drugs of antipsychotic and other commonly abused drugs. The accuracy of duloxetine, mirtazapine, paroxetine and trimipramine was out of range so this should be taken into consideration, since their concentration could be higher than expected for future analysis. 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