Validated UPLC-MS/MS Method for the Simultaneous Quantification of Vortioxetine and Fluoxetine in Plasma: Application to Their Pharmacokinetic Interaction Study in Wistar Rats

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Introduction
Vortioxetine is 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]-piperazine (Figure 1), which is a novel antidepressant approved in the USA and EU for the treatment of major depressive disorder [1]. It administered in a dose of 5, 10, 15 and 20 mg [2]. It is a type of drug with a multimodal action specific to the serotonin neurotransmitter system. Its metabolism is mainly by cytochrome P450 (CYP450) enzymes that are responsible for oxidative metabolism of most drugs in the liver [3].
VTX has dual pharmacological modes of action, it makes inhibition of the serotonin transporter and makes immediate alteration of receptor efficiency [3]. Clinical investigations suggested that VTX has a good safety and tolerability profile [4].
The most common adverse events associated with it were nausea, headache, and dizziness [4]. VTX has been shown to be a substrate for several of the CYP450 isoforms in clinical investigations, in spite of no influence on CYP2C19 was spotted [5]. VTX had no influence on the steady-state pharmacokinetic parameters of aspirin or its metabolite salicylic acid, and had no effect on the platelet aggregation and co-administration of VTX did not alter the pharmacokinetics of warfarin and no pharmacodynamics interactions with oral contraceptives were shown [6] [7].
The clinical investigations of drug-drug interaction have shown that co-administration of bupropion (CYP2D6 inhibitor) can elevate the exposition of VTX about 2-folds [8]. Fluoxetine is considered as selective serotonin reuptake inhibitor (SSRI).
FLX is subjected to significant hepatic metabolism by cytochrome P450 enzymes (CYP2D6), thus, considered as strong CYP2D6 inhibitor [10]. The oxidative metabolism pathway of the enzyme CYP450 is implicated in drug-drug interaction mechanisms, since it is substantial for metabolism of many drugs, therefore, that interactions bring the major adverse effects with pharmacotherapy [11] [12]. Therefore, it is important to identify and quantify those interactions in vivo in order to avoid and reduce the side effects promoted from such interactions related to certain drug combination treatments. The FLX as SSRI antidepressant drug can be used in treatment-resistant depression when used in combination with other antidepressant like VTX. Hisaka el al., [13] reported that FLX is a potent CYP2D6 inhibitor and can cause some inhibition of VTX metabolism resulted in an increased VTX blood level, causing the worse VTX side effects including a significant status called the serotonin syndrome. The symptoms of this syndrome included of seizure, confusion, hallucination, elevated heart rate, quite changes in blood pressure, too much sweating, fever, blurred vision, muscle spasm,  shivering and shaking, tremor, stomach cramp, nausea, vomiting, and diarrhea and in critical situation may cause the coma and even death [14]. For these reasons, it is necessary to perform a pharmacokinetic interaction study of VTX and FLX in rats when being administered orally alone or being co-administered. An extensive literature review revealed that, VTX has been determined in biological samples by HPLC [15] [16] [17] and an LC-MS/MS technique [18] [19] [20] [21].

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Also, several analytical methods have been cited for the quantification of FLX alone or in combination with other drugs, using HPLC [22] [23] [24], GC-MS [25] [26] and LC-MS/MS [27]- [32]. However, reports describing an UPLC-S/MS-based method for simultaneous determination of VTX and FLX in plasma are not available.
In this study, a sensitive and validated UPLC-S/MS method was developed to determine the concentrations of VTX and FLX in rat plasma and pharmacoki-

Instrumentation and Analytical Conditions
The chromatographic separation of FLX, VTX and IS had been done on the The C18 Agilent eclipses, plus analytical column (50 mm × 2.1 mm, 1.8 μm particle size) was used for chromatographic analysis was purchased from (Agilent Technologies, Palo Alto, CA, USA) and its temperature was preserved at 22˚C ± 1˚C. Data acquisition has been processed by Masslynx TM Version 4.1 (Micromass) software. The analytical separation of the analytes was carried out isocratically with a flow rate of 0.25 mL/min. The mobile phase used consisted of 60% of 10 mM ammonium formate and 40% acetonitrile at pH 4. The injection volume was 5.0 µL and the total run time was 7 min. The auto-sampler temperature was maintained at 5˚C -8˚C. Mass spectrometric detection was carried out using positive ion electrospray ionization (ESI) source. The employed MS parameters were; drying gas nitrogen flow of 11 L/min, collision nitrogen gas turned on a pressure of 50

Sample Preparation
Before analysis, rat plasma samples were defrosted at room temperature. An aliquot quantity of 50 µL of rat plasma was taken in a 1.5 mL Eppendorf tube, spiked with 50 µL of working IS solution at 500 ng/mL, and spiked with appropriate aliquots of drug standard solutions to give required concentrations. Each tube was diluted to 500 μL with deionized water and gently mixed for at least 30 s.
The mixture was treated with 500 μL of acetonitrile for deproteinization [33].
The tubes were subsequently vortexes at high speed for 1 min and centrifuged at 6000 rpm for 30 min. The supernatant (upper layer) from each tube was loaded into autosampler tray and 5 µl of it were injected (in triplicate) into the UPLC-MS/MS system. The peak area ratios of each compound to IS were processed to obtain the calibration graph of each compound. Alternatively, the corresponding regression equation was derived.

Specificity
The drug-free plasma samples were examined for the existence of any interfering peaks at the times of elusion of the tested drugs. Method specificity was esti-

Linearity
The rat plasma samples (50 µL) were spiked with ten various concentrations of the VTX and FLX in the range 2.5 -500 ng/mL, along with 50 μL of 500 ng/mL LTZ (IS) in order to construct the calibration graphs of both drugs. Following the analysis of each sample, the peak area ratios of VTX and FLX to that of IS were related to the spiked analytes concentrations to get the matrix-based calibration graph and the corresponding regression equations.

Lower Limit of Detection (LLOD) and of Quantification (LLOQ)
The LLOD and LLOQ of both VTX and FLX were established on the concentrations that make analytical responses of at least three and ten times that of the blank signals, for LLOD and LLOQ, respectively. Moreover, the analytical responses at the LLOQ should yield acceptable accuracy and precision within ±20%.

Precision and Accuracy
Intra-day accuracy and precision were computed through the analysis of QC samples at the four different concentration levels, very low LLOQ (2.5, ng/mL), low (7.5 ng/mL), medium (250 ng/mL) and high (450 ng/mL) during the same day (n = 6). While the inter-day evaluations were done on three successive days

Extraction Recovery
The rat plasma samples were spiked with previously calculated volumes of VTX and FLX along with IS to prepare the four different QC levels as in precision and the IS at the same concentration level of the assay was also calculated.

Matrix Effect
The matrix effect computed by comparing the ratio of the mean peak area of each of VTX and FLX spiked after extraction to those of standard solutions prepared at the four different QC concentration levels (2.5, 7.5, 250 and 450 ng/mL). Similarly, the matrix effect of LTZ (IS) at the same concentration level used in the analysis was evaluated.

Dilution Integrity
Dilution of highly concentrated plasma samples, with concentrations beyond the linear range of the proposed method, was evaluated for its effect on VTX and

Stability Studies
QC samples spiked at four concentration levels of VTX and FLX were analyzed (n = 6) in order to assess the drug stability in plasma.

Application to Pharmacokinetic Studies
All animal procedures employed complied with the standards set forth in the guidelines for care and use of experimental animals by the Committee for Purpose of Supervision of Experiments on Animals (CPCSEA) [34], and the National Institutes of Health (NIH) protocol [35].
All the rats could access the water freely while diet was prohibited for 12 h before drug administration. The rats were acclimatized for 7 days to laboratory conditions before conducting the experiment. Four groups of four rats each were involved in this study. Rats in Group 1 were orally administered saline by oral gavage to provide the blank rat plasma; rats in Group 2 were orally administered vortioxetine (4.0 mg/kg); rats in Group 3 were given fluoxetine (16.0 mg/kg) and rats in Group 4 were given vortioxetine (4.0 mg/kg) plus fluoxetine (16.0 mg/kg). For each group, volumes of 0.2 mL blood samples were withdrawn from the retro-orbital sinus of each rat into heparinized 1.5 mL polythene tubes. Blood samples were collected at different time intervals; 0 (prior to dosing), 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, and 24 h after oral administration, respectively. All collected blood samples were centrifuged immediately at 3000 rpm (10 min, 4˚C). The plasma obtained (100 µL) were kept frozen at −20˚C till the day of analysis.
For the fate of animals, we leave the animals for a washout period (i.e. two weeks) then we use them in other animal studies in our laboratory. PK Solver 2.0 Add-in, Excel 2010 was used to process the VTX and FLX plasma concentrations as a function of the analysis time. Data were expressed as mean ± SD. Statistically significant differences of data from two sets were compared using one-way analysis of variance. In all statistical analyses, P < 0.05 was considered to indicate a statistically significant result.

Optimization of Chromatographic Conditions
Method development was begun with the optimization of chromatographic conditions, including mobile phase composition, and flow rate. Various mobile phases consisted of mixtures of different ratios of acetonitrile-water (30% -90%), and formic acid (0.05% -0.2%). Methanol-water mixtures (30 -90%), and formic acid (0.05% -0.2%) were investigated. The ammonium formate buffer at a concentration ranged from 5 -20 mM and different pH ranges from 3 -6 was examined with different mixtures of both organic modifiers to get better separation, lower retention times and good peak shapes. Mobile phase consisted of acetonitrile-water (40%:60%) containing 10 mM ammonium formate and pH was 4.0 was shown to improve signal-to-noise ratio and thus found to be suitable for the chromatographic separation of the studied analytes. Farther investigated of selected mobile phase showed that the acetonitrile percentage of less than 40% resulted in distortion of the tested analytes peaks, more concentration of acetonitrile from 45% -80%, resulted in overlapping of the tested drugs and decreased the separation. The analysis was thus performed with an isocratic elution using a mobile phase consisted of 60% of 10 mM ammonium formate and 40% acetoni-

Optimization of Mass Spectrometric Conditions
The ± ESI ionization mode was operated to assess the best MS/MS conditions of the injected standard solutions of VTX, FLX and the IS. The positive ionization mode provided better response for FLX, VTX, and IS relative to the negative ionization mode, under different MS parameters. Therefore, the optimization was carried out in the positive ionization mode in order to monitor the precursor as well as the product ions. MRM mode was defined in this research to clear any potential interference signals and improve the sensitivity of the procedure. For the highest intensity of the protonated molecular ions, different MS/MS parameters were adjusted as follows: an ESI source temperature of 350˚C and desolvation gas flow rate of 11 L/min was found optimum in the analysis. On the other hand, the collision energy is an important parameter to get reasonable responses of the daughter fragment ions. However, increasing the collision energy resulted in an increased in the intensity of the particular fragment ion till optimum values after which a decrease in the intensity would be observed. The selected collision energies that produced maximum intensities of the selected daughter ions of all studied drugs were summarized in Table 1. Moreover, the intensity of the particular fragment ion increased gradually with increased the cone voltage till certain optimum values after which a dramatic decrease was recorded. The selected optimum values for cone voltages for all studied drugs were shown in Table 1.

Method Validation
Validation of this study was performed according to the "Guidance for Industry-Bioanalytical Method Validation" recommended by the US Food and Drug Administration [36] to evaluate the specificity, linearity, accuracy and precision, extraction recovery, matrix effects, dilution integrity and stability studies.

Specificity
The specificity of the method was assessed by comparing the chromatograms obtained from six batches of blank and plasma samples with those spiked with low

Linearity
The

Lower Limit of Detection (LLOD) and Lower Limit of Quantification (LLOQ)
The lower limit of quantification (LLOQ) was established as 2.5 ng/mL for both FLX and VTX, while the LLOD for FLX was 1.00 ng/mL and for VTX and FLX.

R E T R A C T E D
R. Al-Shalabi et al.  The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were calculated according to the FDA guidelines [36]. The MRM chromatograms of plasma samples spiked with FLX and VTX at their LLOQ were approached in Figure 3(b). The low values of LLOQ performed the succeeded implementation of the developed method in the trace analysis of the two drugs in clinical investigations.

Precision and Accuracy
The developed method was approved to be reproducible according to the resulted values of precision and accuracy of the intra-and inter-day assessment process of FLX and VTX QC samples. The data for intra-day and inter-day precision and accuracy were expressed in (not more than ± 15.0%), this specified that this study was with a high degree of accuracy and precision and was dependable and reproducible for the simultaneous quantitative analysis of vortioxetine and fluoxetine in rat plasma samples.

Extraction Recovery
Analyzing of the rat plasma samples with the tested analytes at four different concentration levels; very low (2.5 ng/mL), low ( 99.54% ± 4.55%. In addition the, the mean recovery of the IS was 98.56% ± 3.46%. Recovery results were summarized in Table 5. Table 4. Intra-day and iner-day precision and accuracy results of fluoxetine (FLX) and vortioxetine (VTX) in rat plasma (Mean ± SD, n = 6).

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R. Al-Shalabi et al.

Matrix Effect
The matrix effect assessment is very valuable in the analytical method, this due to the significant effect of the biological samples on the ionization of the tested drugs either by inhibition or elevating of the ionization. This issue was carried out by the same process as per recovery assessment, but the processed samples quently, the % matrix factors (% relative error) at the four selected concentration levels were found not more than −2.78% for FLX and −0.91% for VTX, the results were presented in Table 6. The % matrix factor for the IS at the actual concentration applied in the assay was found −2.51%. These results replied matrix effect had negligible influence on the ionization of the tested compounds.

Stability Studies
Stability studies were assessed using plasma samples spiked at two different FLX and VTX concentrations, namely 7.5 and 450 ng/mL. The results were presented in Table 8, Table 9 explained that all the resulted values of recovery did not excessed the permitted limits (± 15), where the recoveries values of FLX were ranged between 97.19% -99.92% and for VTX were 97.56% -99.67%. The RSD % values of the results did not exceed the accepted limits, 4.32% for FLX and 4.75% for VTX. Negligible loss of the tested compounds during sample storage under different conditions and during sample handling of the QC samples at the analysis conditions indicating a high degree of sample stability.

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R. Al-Shalabi et al.  Furthermore, shorten analysis run time (7 min) which approved the convenience of this method in high throughput bioanalysis.

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R. Al-Shalabi et al.

Pharmacokinetic Interaction between Vortioxetine and Fluoxetine Study
This study was the firstly reported method utilized the UPLC-MS/MS technique for simultaneous determination of of VTX and FLX in rat plasma and its application to a pharmacokinetic interaction study. According to the studies of the VTX metabolism bath way, it's extensively metabolized primarily through oxidation via multiple cytochrome P450 (CYP) isozymes (predominantly CYP2D6) and subsequent glucuronic acid conjugation [37]. In pharmacokinetic interactions, the majority of clinically relevant pharmacokinetic interactions with antidepressants arise as a consequence of drug induced changes in hepatic metabolism, through inhibition or induction of CYP isoenzymes [38]. In line with the previous observations, only the co-administration of a CYP2D6 inhibitor was able to increase the area under the plasma concentration curve (AUC), and the maximum plasma vortioxetine concentration in healthy adults, also increased the incidence of adverse effects when co-administered with vortioxetine [39]. Since the fluoxetine is an antidepressant drug with strong CYP2D6 inhibition effect due to its metabolism at the CYP2D6 isoenzyme [40] [41], and since VTX drug metabolized by the same mechanism, therefore, FLX co-administration with VTX expected to cause synergistic increase in the VTX concentration resulting in worse SSRI side effects [42] [43]. For this reason the presented UPLC-MS/MS method developed in this work was utilized to explore the probability of PK interaction between VTX and FLX. The present method was successfully applied to pharmacokinetic study of vortioxetine with/without fluoxetine in rats and this assay was designed for the purpose of comparison between the rats groups. The groups II and III were given oral doses of only VTX (4 mg/kg) and FLX (16 mg/kg), respectively, while the group IV were administered with a combination of VTX and FLX in a dose of 4 mg/kg for VTX and 16 mg/kg for FLX. The mean plasma concentration-time profiles of vortioxetine with/without fluoxetine were shown in Figure 5. The main relevant pharmacokinetic parameters from non-cpmpartment model analysis were listed in Table 10. The typical MRM chromatograms gained from rat plasma 1 h after VTX oral administration alone/with FLX were shown in Figure 6, while MRM chromatograms gained from rat plasma 4 h after administration of FLX alone/with VTX were presented in Figure 7. After oral administration of vortioxetine with/without fluoxetine, the standard pharmacokinetic va-      It was found that the results were in close agreement to that represented in our study. The PK parameters calculated for VTX and FLX given in combinations for rats in group IV were compared with those obtained following single administration of either of the two drugs in group II and III. Table 10 revealed that about 240% and 226% increase in C max and AUC (AUC 0-∞ and AUC 0-t ) of VTX respectively were recorded with the co-administration of FLX together with VTX in studied rats group IV. Half-life of VTX was slightly longer, for T max , it has been observed that there was no disparity between the obtained values of the rat groups treated with VTX and FLX combination in group IV, and those obtained following single administration of either of the two drugs in group II and III.

R E T R A C T E D
This study concluded that when co-administration of FLX and VTX, the FLX could promote higher concentration of VTX in blood due to the inhibition of FLX to the liver enzyme responsible for VTX metabolism leading to increase in the plasma concentration the VTX. Therefore, reduce VTX dose by two thirds when the FLX is co-administered could be considered. The aim of antidepressant therapy is to induce remission and prevent relapses of major depressive disorder with minimum adverse effects during the treatment [51].

Conclusion
This work was the first analytically scanned the influence of FLX on VTX in rat plasma. A sensitive and simple UPLC-MS/MS method for simultaneous quantification of VTX and FLX in rat plasma has been developed and validated as per FDA guidelines. This method showed a linear range between 2.5 -500 ng/mL for both studied drugs FLX and VTX with LLOD of 1.00 ng/mL. The developed simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for quantification of the tested drugs. It is noticeable remind that the high sensitivity of the method permitted the accurate assessments of the PK parameters and the high capacity for the precise measurements and estimations for the very low doses of the tested medications that could be applied in any further clinical investigations. The short run time (7.0 min), and simple preparation process of the developed method was instituted to be accurate, precise and specific, and was succeeded to be used in the pharmacokinetic interaction study of vortioxetine and fluoxetine in rats. Results indicate that co-administration of vortioxetine and fluoxetine might bring a considerable change in vortioxetine plasma level. According to the product labeling, adminis-

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C T E D American Journal of Analytical Chemistry tration of vortioxetine with the potent CYP450 2D6 inhibitor such as FLX resulted in greater than 2 fold increases in vortioxetine peak plasma concentration (C max ) and systemic exposure (AUC) compared to administration of vortioxetine alone. Accordingly, we recommended that the dosage of vortioxetine should be reduced by two thirds when used in combination with potent CYP450 2D6 inhibitors such as fluoxetine. Further investigations required to study that pharmacokinetic interaction on human in order to adjust the dose regimen of VTX when combined treatment with FLX in some cases of depression.