UPLC-Tandem Mass Spectrometry Method for Simultaneous Determination of Fluoxetine, Risperidone, and Its Active Metabolite 9-Hydroxyrisperidone in Plasma: Application to Pharmacokinetics Study in Rats

Risperidone (RIS) is used as an antipsychotic drug alone or with other drugs, like fluoxetine (FLX). A simple method was developed and validated for the determination of both RIS and its metabolite 9-hydroxyrisperidone (9-OH-RIS), FLX, and olanzapine (OLA) as an internal standard in rat's plasma using UPLC-MS/MS. FLX, RIS, 9-OH-RIS, and OLA were purified using acetonitrile as a protein precipitating agent. Separation was performed on an ACQUITY™ “UPLC BEH™” C18 column (50 mm × 2.1 mm i.d., 1.7 μm; Waters Corp., USA). The ranges of the calibration curves were 1.0–1000.0 ng/mL for FLX and 0.2–1000.0 ng/mL for RIS and 9-OH-RIS. Linearity, recovery, precision, and stability were within the acceptable range. This method is rapid, fast, and precise for the determination of RIS and FLX in plasma and is applicable in pharmacokinetic studies.

The drug is metabolized mainly by the liver, with less than 1% being excreted unchanged in the feces. In order of preference, the major metabolic pathways include 9-hydroxylation, Ndealkylation, and 7-hydroxylation [5]. 9-Hydroxyrisperidone (9-OH-RIS) is the principal metabolite of RIS and is the only one that has the same therapeutic effects [6].
Consequently, monitoring the level of both FLX and RIS in biological fluids is essential. However, no methods for the determination of FLX and RIS simultaneously have been published. Therefore, this study aims at developing a method for the selective determination of RIS, 9-OH-RIS, and FLX using UPLC-MS/MS.

Instrumentation.
Chromatography was performed on an ACQUITY UPLC system coupled with a triple-quadrupole tandem mass spectrometer (Waters Corp., Milford, MA, USA). Separation of the analytes was performed on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 m; Waters Corp., USA) maintained at 40 ∘ C. The mobile phase was 80 : 20 (v/v) mixture of 0.1% formic acid in acetonitrile and 0.1% formic acid in 0.25 M ammonium acetate buffer at a flow rate of 0.6 mL/min. The injection volume was 5 L in partial-loop mode, and the temperature of the autosampler was kept at 10 ∘ C. Multiple reaction monitoring (MRM) in electrospray positive ion mode was used for detection and  quantitation of all analytes. The MRM transitions selected and mass optimization parameters are summarized in Table 1. "Mass Lynx" software (Version 4.1) was used for evaluation of peak areas.

Animals.
All animal experiments were carried out under animal use regulations. Wistar rats (200−250 g) were obtained from the Laboratory Animal Center (NODCAR, Egypt). Animals were acclimated for at least five days and fasted overnight before the experiments.

Preparation of Standard
Solutions. Standard 100.0 g/mL solutions of FLX, RIS, 9-OH-RIS, and OLA were prepared in methanol. All solutions were stored at 4 ∘ C and brought to room temperature before use, and they were used for 15 days from the date of preparation.    RIS, and 9-OH-RIS, respectively, were prepared. Samples were kept at −80 ∘ C.

Sample Preparation.
After thawing the samples at room temperature, they were mixed with a vortex mixer prior to sample preparation to ensure complete mixing of the contents. A 100 L plasma sample was pipetted into a 10 mL glass test tube. Then, 10 L of IS (12.0 g/mL) was added, and the sample was mixed with vortex for 30 s. Subsequently, 300 L of acetonitrile was added for protein precipitation and the mixture was shaken by vortex and centrifuged for 10 min at 4500 rpm at 4 ∘ C. Then, the supernatant was transferred to a clean vial, and 5 L was injected into the UPLC-MS/MS apparatus for analysis.

Method
Validation. UPLC-MS/MS assay validation was performed according to the US FDA guidelines [35]. The selectivity of the method was investigated by comparing detector response at the retention times of plasma samples spiked at the lower limit of quantification (LLOQ) (1.0 ng/mL for FLX and 0.2 ng/mL for RIS and 9-OH-RIS and at 1200.0 ng/mL for the IS) with those from free-drug plasma. The linearity of the method was determined by analysis of twelve standard calibration curves with six different concentrations ranging from 1.0 to 1000.0 ng/mL for FLX and from 0.2 to 1000.0 ng/mL for RIS and 9-OH-RIS. The correlation coefficient ( 2 ) was >0.999 for all the calibration curves. The ratio of peak-area response of the analyte to IS was used for regression analysis. The concentration of the drug in rats  samples was calculated from the calibration curve ( = + ) and the regression coefficient was calculated. The LLOQ is the lowest concentration of the analyte on the calibration curve which is 1.0 ng/mL for FLX and 0.2 ng/mL for RIS and 9-OH-RIS.
Assay precision is expressed as percentage of variation (% CV) while the deviation of the concentration was found from the nominal one expressed as the accuracy.
Precision and accuracy during intraday and interday of the method were measured by injection of three QC samples (LQC, MQC, and HQC) in six replicates on the same day and on successive days, respectively. Deviation values for these parameters should be within 20% for the LLOQ and 15% for the QCs above the LLOQ.
The recovery of an analytical method is defined as a comparison between detector response for the concentration of the authentic sample and the response of the detector for the same concentration added and extracted from a biological matrix. The extraction recoveries of FLX, RIS, and 9-OH-RIS were determined at three concentration levels each. The stability of the analytes in rat plasma during sample storage as well as during processing conditions was assessed by analyzing the LQC, MQC, and HQC with six replicates. Short-term stability indicated acceptable stability behavior during the experimental conditions of the regular runs at ambient temperature for 6 h. Freeze-thaw plasma stability was checked over three freeze-thaw cycles after storage in ultradeep freezer. The long-term stability was determined after storage at −80 ∘ C for 6 weeks. Postpreparation stability was measured by reanalyzing the extracted plasma samples kept under the autosampler conditions for 24 h.

Application of the Method in a Clinical Pharmacokinetic
Study. The present method was fruitfully applied for determinations of FLX, RIS, and 9-OH-RIS levels in rat plasma samples. A pharmacokinetic study was conducted using six male Wistar rats (200−250 g). After overnight fasting, the rats received simultaneous oral doses of FLX (10 mg/kg) and RIS (0.3 mg/kg). Blood samples (0.5 ml) were collected at different time intervals. Plasma samples were centrifuged at 4000 rpm and the separated plasma samples were stored in an ultradeep freezer until analysis. Different pharmacokinetic parameters were estimated for each rat.

Results and Discussion
In biological matrices, quantification of drugs by LC-MS/MS is widespread due to the high sensitivity and selectivity of this technique. Such sensitivity is fundamental to establish a method capable of quantifying FLX, RIS, and 9-OH-RIS at a level down to 1.0 for FLX and 0.2 ng/mL for RIS and 9-OH-RIS. The ingrained selectivity of MS/MS detection was expected to be helpful in developing a selective and sensitive method. Furthermore, this method would be suitable for efficient analysis of a large number of plasma samples for pharmacokinetic, bioavailability, and bioequivalence studies of FLX and RIS.
There is no reported method for the determination of FLX and RIS and 9-OH-RIS in plasma simultaneously; therefore, the aim of this study was to develop and validate a simple, fast, and specific UPLC-MS/MS assay method for simultaneous extraction, separation, and quantification of the cited drugs. To achieve this goal, different selections were estimated during the development of the method to optimize detection parameters, chromatographic separation, and sample extraction.
LC-multiple reaction monitoring (MRM) is a great technique as it provides the sensitivity and selectivity required for accurate analysis. Thus, the MRM technique was chosen for our method. Electrospray ionization (ESI) was employed in order to obtain a better response from the analytes. The best signals were achieved using ESI-positive ion mode. The product ion mass spectra for FLX, RIS, 9-OH-RIS, and OLA present a high abundance of fragment ions of m/z 44. 18, 191.12, 206.97, and 256.03, respectively (Figure 2).

Method Development.
The constituents of the mobile phase were changed several times to achieve a chromatogram with symmetric peak and good resolution for the analytes and IS. A mixture of 0.1% formic acid in acetonitrile and 0.1% formic acid in 0.25 M ammonium acetate buffer (80 : 20, v/v) with a flow rate of 0.6 mL/min achieves this purpose   and permits a run time of 2.0 min. Endogenous substances in the plasma may affect the column, MS system, and analytes and the IS, which leads to ion suppression. The advantage of protein precipitation is that it helps in preparing a clean sample and consequently avoids this suppression effect in UPLC-MS/MS analysis.

Method Performance and Validation.
A representative chromatogram obtained from blank plasma is shown in Figure 3. The MRM chromatograms obtained from spiked plasma samples are shown in Figure 4. No endogenous compounds appear at the retention times of FLX, RIS, 9-OH-RIS, or the IS to interfere with their peaks. Moreover, the base line is relatively free from drift.
The linearity of the method was determined using coefficient of variation of the standard. Calibration curves were obtained by plotting the peak-area ratio (drug/IS) against the concentration of the analyte in the plasma. The linearity  of the calibration curves ( = 12) was verified from 1.0 to 1000.0 ng/mL for FLX and from 0.2 to 1000.0 ng/mL for RIS and 9-OH-RIS ( Figure 5). The LLOQ is defined as the lowest concentration of an analyte that can be measured accurately under the mentioned experimental condition and meet the acceptable criteria (precision < 20% and an accuracy between 80% and 120%). The LLOQ is 1.0 ng/mL for FLX and 0.2 ng/mL for RIS and 9-OH-RIS. Results of precisions (% CV) and accuracy for the intra-and interday analysis of FLX, RIS, and 9-OH-RIS in plasma are presented in Table 2.
The extraction recovery determined for FLX, RIS, and 9-OH-RIS is shown to be consistent, accurate, and reproducible. The average recovery was 90.54%, 96.41%, and 84.34% for FLX, RIS, and 9-OH-RIS, respectively, which is acceptable for the routine measurement of these analytes (Table 3). Table 4 summarizes stability data for FLX, RIS, and 9-OH-RIS during analysis. All the results indicate reliable stability behavior during these tests. Therefore, there is no stability-related problem during the routine analysis of samples for the bioavailability study.
Six male rats received a single oral dose of 10 mg/kg of FLX and 0.3 mg/kg of RIS concurrently and plasma drug levels were determined. The chromatogram of a plasma sample extracted from a rat at 1 h is shown in Figure 6. The concentration-time profiles of FLX, RIS, and 9-OH-RIS are shown in Figure 7. The pharmacokinetic parameters are listed in Table 5.

Conclusion
In this study, a consistent, selective, and specific and fully validated UHPLC-MS/MS method was developed for the determination of FLX, RIS, and 9-OH-RIS in rat plasma. This method was successfully applied in pharmacokinetic

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.