Development of an LC-MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study

Accepted Manuscript Title: Development of an LC-MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study Author: Manisha Bhateria Rachumallu Ramakrishna Dora Babu Pakala Rabi Sankar Bhatta PII: DOI: Reference: S1570-0232(15)30109-4 http://dx.doi.org/doi:10.1016/j.jchromb.2015.07.042 CHROMB 19543 To appear in: Journal of Chromatography B Received date: Revised date: Accepted date: 17-5-2015 13-7-2015 20-7-2015 Please cite this article as: Manisha Bhateria, Rachumallu Ramakrishna, Dora Babu Pakala, Rabi Sankar Bhatta, Development of an LC-MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. 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Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Development of an LC-MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study Manisha Bhateria1,3#, Rachumallu Ramakrishna1,3#, Dora Babu Pakala2, Rabi Sankar Bhatta1,3* # 1 Authors contributed equally to this work Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute,Lucknow-226032, Uttar Pradesh (India) 2 Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Rae Bareli, Uttar Pradesh (India) 3 Academy of Scientific and Innovative Research, New Delhi -110001 (India) * Address for correspondence Dr. Rabi Sankar Bhatta Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow ‐ 226032 Email ID: rabi_bhatta@cdri.res.in; rabi.cdri@gmail.com Tel.: +91-522 2772974 (Ext-4853) Highlights ► ► First report on simultaneous quantification of memantine and donepezil in plasma. ► Sensitive method (0.2 ng/mL), low sample volume (50 µL) and short run time (3 min). ► Application to pharmacokinetics and drug interaction studies in rats. ► ► Abstract Recently, a fixed dose combination (FDC) of memantine (MM) and donepezil (DPZ) has been approved for the treatment of Alzheimer’s disease (AD). In the present work, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of MM and DPZ was developed and validated in rat plasma over the linearity range of 0.2-400 ng/mL using amantadine (AM) as an internal standard. Both the analytes and IS were extracted using one step liquid-liquid extraction procedure. The analytes were separated on C18 reversed phase column with mobile phase consisting of a mixture of methanol and 10 mM ammonium acetate, pH 5 (92:8 v/v) at a flow rate of 0.7 mL/min. The detection of the analytes was done on triple quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI) and quantified using multiple reaction monitoring (MRM). The method was fully validated in terms of linearity, accuracy, precision, recovery, matrix effect, dilution integrity, carry-over effect and stability. The within- and between-run precisions were < 10% and accuracy was all within ±10%. The mean recovery of MM and DPZ was found to be greater than 80%. The % RSD value at higher as well as lower concentration was well within the acceptable range (±15%) in all the stability experiments. The method was successfully applied to the oral pharmacokinetics and drug-drug interaction study of MM and DPZ in male Sprague Dawley (SD) rats. Keywords Memantine, Donepezil, Alzheimer’s disease, LC-MS/MS, Pharmacokinetics, Drug interaction 1. Introduction Alzheimer’s disease (AD) is the most frequent form of dementia, accounting 60 to 80% of the cases. In general, dementia is characterized by a decline in memory or other cognitive skills that affect a person’s capability to carry out day-to-day activities. Currently, FDA approved treatments for AD consists of three acetylcholinesterase inhibitors such as Donepezil (DPZ), rivastigmine and galantamine and the N-methyl-D-aspartate (NMDA) receptor antagonist memantine (MM) as well [1]. Recently, a fixed-dose combination (FDC) of MM hydrochloride extended release and DPZ hydrochloride has been approved by FDA under the trade name NAMZARIC™ for the treatment of moderate to severe AD in patients stabilized on MM and DPZ [2]. Clinically, adding MM to an Alzheimer Dementia Treatment Regimen which already includes DPZ showed an additive effect and proved to be the most effective therapy for AD patients who are progressing from moderate to severe dementia [36]. The FDC of MM and DPZ has been developed as a replacement indication for the patients who are often co-prescribed the individual drugs. Memantine hydrochloride, (1-amino-3, 5-dimethyladamantane hydrochloride) is a moderate affinity, voltage dependent uncompetitive N-methyl- D aspartate (NMDA) receptor antagonist which imparts a neuroprotective action and has been found to be effective in the symptomatic treatment of patients with moderate to severe AD [7]. It also blocks the neurotoxicity of glutamate without interfering with its physiological actions required for learning and memory [8]. Donepezil hydrochloride, [(±)-2-[(1-benzyl-piperidine-4-yl)ethyl]5,6-dimethoxyindan-1-one hydrochloride] is a piperidine-based reversible and selective acetylcholinesterase inhibitor, the predominant cholinesterase in the brain [9, 10]. The chemical structures of MM and DPZ are presented in Fig.1a and 1b, respectively. The aim of the present work was to develop a bioanalytical method with single extraction step for the simultaneous determination of MM and DPZ in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To date, numerous reports have been published to determine the concentration of MM and DPZ individually in several biomatrices by using instruments such as high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing various techniques such as fluorimetric derivatization, solid-phase extraction, automated liquid– liquid extraction (LLE) and so on [11-20]. However, to the best of our knowledge, reports determining their simultaneous estimation have not been published yet. Although, simultaneous determination of all the four FDA approved antidementia drugs (DPZ, galantamine, MM, rivastigmine and its metabolite NAP226-90) has been reported using ultra performance liquid chromatography (UPLC-MS/MS) in human plasma [21]. On the contrary, the proposed method is the first report on the simultaneous determination of MM and DPZ in rat plasma with high specificity, sensitivity (LLOQ ~0.2 ng/mL for both MM and DPZ), low sample processing volume (50 µL), shorter analysis run time (3 min) which becomes fairly essential when large number of samples are to be analyzed in a short span of time. The availability of such bioanalytical assay in rodent plasma would facilitate the ease of adaptability in human plasma. Additionally, the method would prove to be of great assistance in investigating the ADME of the combination for which no reports are present till date. The plasma concentration-time profile of MM and DPZ would assist in correlating the pharmacodynamic activity with the exposure of drug in AD and other comorbid disorders affecting geriatric population. Thus, we aim to develop a simple and sensitive LC-MS/MS the state-of-the-art technique for the simultaneous estimation of MM and DPZ which was validated based on US-FDA guidelines. Further, the developed method was applied to determine MM and DPZ concentration in rat plasma samples and to study pharmacokinetic interaction between MM and DPZ in a group of healthy male Sprague-Dawley (SD) rats following single oral administration. 2. Experimental 2.1 Chemicals and reagents MM (C12H21N.HCl, purity ≥98%), DPZ (C24H29NO3.HCl, purity ≥98%) and amantadine hydrochloride (C10H17N.HCl, purity ≥99%) were obtained from Sigma-Aldrich (St. Louis, MO). HPLC-grade ammonium acetate and tert-butyl methyl ether (TBME) was purchased from Sigma-Aldrich (St. Louis, MO). Chromatographic grade methanol was obtained from Merck Chemicals (Darmstadt, Germany). Heparin was purchased from Gland Pharma Ltd (Hyderabad, India). Ultrapure water was obtained in-house using a Milli-Q PLUS PF water purifying system (Millipore, Bedford, MA). All other reagents and solvents were of analytical grade and purchased from standard chemical suppliers. Drug-free rat plasma containing heparin as anticoagulant was collected from adult healthy male SD rats. 2.2 Instrumentation The liquid chromatography (LC) system consisted of a WATERS HPLC system equipped with WATERS binary 515 HPLC pump and WATERS 2707 auto sampler. The LC system was coupled to API 3200 triple quadrupole mass spectrometer (ABI SCIEX, ON, Canada). Chromatography was performed isocratically on Thermo Syncronis-C18 column (100x4.6 mm i.d., 5 µm) preceded with a C18 guard column (Security Guard, Phenomenex, USA) maintained at ambient temperature. Mobile phase consisting of a mixture of methanol and 10 mM ammonium acetate, pH 5 (adjusted using acetic acid) (92:8 v/v) was pumped at a flow rate of 0.7mL/min. The injection volume was 10 µL. Methanol-water (50:50 v/v) was used as the rinsing solvent. The total analysis run time was set to 3 min. Neat solutions of MM, DPZ and internal standard (IS) was infused separately for compound and source dependent parameters optimization using Harvard infusion pump (Holliston, MA, USA). Nitrogen was used as curtain gas, collision gas and source gas. Tuning of the instrument was done in the positive ionization mode to set various compound and source dependent mass parameters. Multiple reaction monitoring (MRM) was operated at unit resolution for both Q1 and Q3 quadrupoles. Dwell time was set to 200 msec per MRM channel. Analytical data was acquired and integrated by Analyst software 1.6 version (AB SCIEX, ON, Canada). 2.3 Preparation of calibration standards and quality control samples Stock solutions of MM and DPZ for calibration standards (CS) and quality controls (QCs) were prepared individually in methanol-water (50:50, v/v) at a concentration of 1 mg/mL. Mixed working stocks of MM and DPZ were prepared by serial dilution in methanol to give a series of standard solutions with different concentration levels. Stock solution of amantadine (internal standard, IS) (1 mg/mL) was diluted with methanol to obtain a concentration of 100 ng/mL. Mixed working stock solutions were spiked (2% of total plasma volume) to blank plasma in order to obtain calibration standards of 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 400 ng/mL. Four levels of QCs fixed at 0.2, 0.6, 15 and 320 ng/mL as lower limit of quantification (LLOQ), low QC (LQC), mid QC (MQC) and high QC (HQC), respectively, were prepared. All stock solutions were stored at refrigerated conditions (2-8 °C) and were withdrawn prior to use on the day of analysis. 2.4 Sample preparation MM, DPZ and IS were extracted using one step liquid-liquid extraction (LLE) method. To an aliquot of 50 µL of drug spiked plasma, 10 µL of IS (equivalent to 10 ng/mL) and 50 µL of 0.1% v/v, ammonia were added sequentially and vortex mixed. Subsequently, 2.5 mL of extraction solvent (TBME) was added and vortex-mixed (Vibrax VXR basic, staufen, Germany) for 10 minutes to extract the analytes of interest. Further, centrifugation was done at 5000 ×g for 15 min at 10 °C (Eppendorf, Hamburg, Germany). Then, the supernatant organic phase was evaporated to dryness using an evaporator (Turbovap®, MA, United States) at 40 °C, 20 psi under a stream of nitrogen gas. The dried extract was reconstituted in 50 µL of mobile phase, vortexed briefly, and transferred to pre-labeled autosampler vials. From these, 10 µL was injected in to the LC-MS/MS system for analysis. 2.5 Method validation The validation of the optimized method was based on the current guidelines and acceptance criteria recommended by FDA [22]. Validation was performed in terms of selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect, carry-over effect, dilution integrity and stability studies under different storage conditions. Selectivity of the method was evaluated by analyzing the plasma extracts from six different lots of blank heparinized rat plasma to confirm the absence of interfering peaks from endogenous matrix components at the retention times (RT) of the analytes and IS. Additionally, the extent of matrix interferences with the analytes and IS was determined by comparing the chromatograms obtained from blank plasma samples and spiked plasma samples at the LLOQ concentration of the analytes. Lowest standard for the calibration curves of MM and DPZ that can be measured with precision and accuracy was considered to be LLOQ. Signal-to noise ratio (S/N) at LLOQ should be greater than 10 with an acceptable precision (±20%) and accuracy (80-120%) [22]. The calibration curve contained blank sample (plasma matrix processed without analyte and IS), zero sample (plasma matrix processed with IS) and 11 non-zero standards (plasma matrix processed with analytes and IS) over the concentration range of 0.2 to 400 ng/mL, a range that was appropriate for the pharmacokinetic study after the oral administration of MM and DPZ. Calibration curves were developed by plotting the peak area ratio of the analyte to the IS (Y-axis) against the nominal concentration of the analytes (X-axis). Six calibration curves were obtained by weighted (1/x2) linear regression analysis and assessed for linearity. Further, suitability of the calibration model was confirmed by back-calculating the concentrations of the calibration standards which should be within ± 15% deviation of the nominal value, except for LLOQ for which it should be within ± 20% deviation. At least 67% of the non-zero standards should meet the above acceptance criteria, including LLOQ and upper limit of quantification (ULOQ). Within-run accuracy and precision was determined by analyzing a set of QC samples (n=6) at each of the four concentration levels (LLOQ-QC, LQC, MQC and HQC) on a single day. Similarly, between-run accuracy and precision was determined by analyzing a set of four QC levels (n=6) on three different days. Analysis of all the QC’s was done against the calibration curve developed on the same day and the obtained concentrations were compared with the nominal values. Precision and accuracy of the assay was expressed in terms of percent relative standard deviation (% RSD) and percent nominal (% nominal), respectively. The acceptable limit for intra- and inter-run precision is 15% and for accuracy is ± 15% (±20% for LLOQ-QC) of the nominal value. Extraction recovery of MM, DPZ and IS from the rat plasma were determined at three QC levels (LQC, MQC and HQC) in six replicate by comparing the peak area ratio of the analyte to the IS in extracted plasma sample against post-extracted samples spiked with analytes at the corresponding concentration. %RSD of ≤ 15% was considered acceptable at all the QC levels [23, 24]. Matrix effect was determined both qualitatively and quantitatively. Primarily, qualitative matrix effect was examined by post-column infusion of the analytes (MM and DPZ) into the MS/MS detector during chromatographic analysis of blank plasma extract (n=3). The standard solution of analytes or IS was infused separately at with a flow rate of 10 µL/min with simultaneous injection of blank plasma extracts in to the MS/MS system. Any suppression or enhancement of the signal at the RT of the analytes or IS was observed. Quantitatively, matrix effect was determined by comparing the post extracted plasma samples spiked with analytes and IS at two concentration levels (LQC and HQC) in six replicates, to the samples from neat solutions at the same concentrations. Matrix factor (MF) of MM and DPZ and subsequently IS-normalized MF was determined. To meet the acceptable criteria, inter-subject variability and MF at all the concentration levels should not be greater than ±15% [25]. MF and IS-normalized MF was calculated by the following formula: MF = (1) IS-normalized MF = (2) To evaluate the carry-over effect blank plasma extract in triplicate was injected after ULOQ. Carry-over should not be greater than 20% of the LLOQ and 5% for the IS in the blank sample. Dilution integrity experiment was performed with six replicates of blank plasma spiked with about two times of the highest calibration standard (ULOQ) which was further diluted to tenand twenty-fold with blank plasma. The samples were processed as per the optimized extraction procedure. Accuracy and precision should be within ±15%. Analytes stability was determined at two QC concentrations levels (LQC and HQC) in six replicates as per the specified conditions and time period. To determine stability of analytes in stock solution and plasma at ambient temperature (22 °C) for 24 h and refrigerated conditions (2-8 °C) for 30 days peak area ratio of analyte to the IS in stability samples was compared to the peak area ratio of the freshly prepared QC samples. Similarly, stability studies such as freeze-thaw stability (–80 °C for three cycles), bench-top stability (ambient temperature for 24 h), long-term stability (-80ºC and -20 ºC for 2 months), autosampler stability (6±2 ºC for 24 hours) and dry extract stability (ambient temperature for 12 h) were determined. All the stability samples were processed and quantified from fresh calibration curve and compared with the peak area ratio of the freshly prepared QC samples. The samples were considered stable if the deviation was within ±15% from the nominal concentration. 2.6 Application of the LC-MS/MS method The developed LC-MS/MS method was successfully applied to determine the plasma concentration of MM and DPZ and to assess pharmacokinetic interaction between MM and DPZ following oral administration to healthy male SD rats (n=6). Animals were housed in temperature- and humidity-controlled rooms (23-25οC and 50-70%, respectively) with 12/12 h light/dark cycle. Animals were kept on standard chow diet with free access to water. All the animal experiments were carried out as per guidelines of Institutional Animal Ethical Committee (IAEC approval no: IAEC/2014/155) at CSIR-Central Drug Research Institute. Prior to the start of the experiment rats were kept on overnight fasting. MM (2 mg/kg) and DPZ (1 mg/kg) were orally administered as a suspension in 0.5% sodium carboxy methylcellulose to facilitate their oral (gavage) administration. Rats were divided in to three groups (six rats in each group): group I, MM only (2 mg/kg body weight); group II, DPZ (1mg/kg body weight) only and group III, MM in combination with DPZ (2/1 mg/kg body weight). The rat dose for MM and DPZ was equivalent to the clinical dose and calculated according to FDA guidelines for human to animal dose extrapolation [26]. Blood samples were withdrawn via oculi chorioideae vein and collected into the heparinized polypropylene tubes at 0.083 to 72 h post dose under light ether anesthesia. After each blood sampling, plasma was separated by centrifuging the blood at 4000 rpm for 10 min and kept frozen at - 80 °C until analysis. Plasma concentration-time profile and pharmacokinetic parameters of MM and DPZ were calculated by applying non-compartmental statistics model using Phoenix WinNonlin version 6.3 (Pharsight Corporation, Mountain view, USA). The pharmacokinetic parameters includes maximum plasma concentration (Cmax); the time to reach maximum plasma concentration (Tmax); the area under the plasma concentration-time curve from zero to infinity (AUC0-∞); mean residence time (MRT); the terminal elimination half-life (t½); and clearance (Cl/F). Statistical analysis was evaluated by Student’s t-test with p < 0.05 being considered significant. 3. Results and Discussion 3.1 Method development 3.1.1 Optimization of the ESI-MS/MS conditions The optimization of mass spectrometry (MS) parameters was done by infusing the standard solutions of analytes and IS directly into the ESI source of the mass spectrometer using a Harvard infusion pump. Positive ESI mode was selected as the signal intensity was higher and stable for the analytes and IS. Other compound and MS specific optimized critical parameters which ascertain reproducible responses are tabulated in Table 1. In Q1 scan, singly charged protonated precursor ion (M + H) + for MM, DPZ and IS were obtained in abundance at m/z 180.2, 380.2 and 152.2, respectively. The Q1 for MM, DPZ and IS were used as the precursor ion to obtain product ion spectra. In Q3 scan mode, the characteristic product ion for MM, DPZ and IS was observed at m/z163.2, 91.1 and 135.0, respectively and was used for quantification. Hence, the most sensitive MRM transition used for MM, DPZ and IS were 180.2/163.2, 380.2/91.1 and 152.2/135.0, respectively. The product ion mass spectra of MM, DPZ and IS, along with their high abundance fragment ions, are shown in Fig. 2. 3.1.2 Optimization of chromatographic conditions Chromatographic conditions mainly mobile phase composition, flow-rate, column were optimized through several trials to achieve symmetrical peak shape, good resolution and short run time. Different varieties of reversed-phase columns such as Waters Symmetry C18 (100 x 4.6 mm, 5 µm), Phenomenex Luna C18 (100 x 4.6 mm, 5 µm), Thermo Syncronis C18 (100 x 4.6 mm, 5 µm) and Supelco Discovery C18 (100 x 4.6 mm, 5 µm) were tried. In case of Waters and Phenomenex columns, RT of the analytes and IS was more than 3 minutes, whereas, in case of Supelco, peaks with poor shapes were obtained. However, it was found that Thermo Syncronis C18 (100 x4.6 mm, 5 µm) served the purpose and was selected for the analytes separation. Likewise, various mobile phase compositions were tried which included water, 0.05 to 0.5% formic acid or acetic acid, 5-20 mM ammonium formate or ammonium acetate as an aqueous component and acetonitrile and methanol as an organic component. Changing the aqueous component from water to ammonium acetate (10 mM) showed a marked enhancement in the sensitivity and peak shape of the analytes which was well associated with the increase in the ionization of the analytes and IS in positive MRM mode. Furthermore, adjusting the 10 mM ammonium acetate mobile phase component to a pH of 5 improved peak shapes of the analytes with high S/N ratio at LLOQ. Methanol and acetonitrile were tested as the organic part of the mobile phase. In comparison to acetonitrile, methanol gave sharper and symmetric peaks. Hence, methanol and ammonium acetate buffer, pH 5 (adjusted with acetic acid) in the ratio 92:8 (v/v) was considered as the mobile phase composition of choice as it gave the best chromatographic results. Flow rate of 0.7 mL/min was found to be optimal as it gave best chromatographic results within a short run-time of 3 min. 3.1.3 Optimization of sample extraction procedure Among various sample extraction techniques, LLE was used for the extraction of the analytes and IS from rat plasma. From general laboratory experience, it has been demonstrated that LLE typically provides cleaner extracts with less matrix effects, minimum ion suppression and low propensity of building backpressure in the chromatographic column due to large number of sample injection [27]. When protein precipitation was used as the extraction procedure, clean extracts were not obtained which resulted in severe signal suppression. On the contrary, when LLE was used as the extraction procedure clean extracts with satisfactory recoveries of the analytes were obtained. Different extraction solvents were tried such as ethyl acetate, chloroform, n-hexane, TBME and diethyl ether. In case of n-hexane and chloroform, although DPZ showed a recovery of ~50% but MM and IS showed negligible recovery. When diethyl ether and ethyl acetate were used the recovery of the analytes and IS was low (~20-30%). TBME proved to be the best extraction solvent as it yielded highest recovery (>80%) for both the analytes and IS. Moreover, it gave clean chromatograms with no significant interferences in the MRM of the analytes and IS at the relevant retention times. However, on trying several possible combinations of TBME with chloroform and n-hexane, desirable recovery of the analytes and IS was not obtained. Consequently, TBME was used as the extraction solvent of choice. Extraction recovery of the analytes and IS in the presence of basifying agent such as ammonia and sodium hydroxide at different concentrations (0.05, 0.5 and 1%) was also assessed. Ammonia at the concentration of 0.1% showed an increase in the recovery of the analytes and IS (>80%). When different volume ratio (1:1, 1:2, 1:3, v/v) of plasma: basifying agent (0.1%) was tested, no improvement in the recovery of the analyte and IS was observed above volume ratio of 1:1, v/v. Hence, addition of 50 µL of 0.1% ammonia followed by extraction with TBME was chosen as the sample clean-up procedure as it yielded high extraction recoveries and negligible matrix effect for both the analytes and IS. The mean percent recoveries of MM and DPZ obtained with different extraction solvents were compared and are shown in Supplementary Fig. 1. 3.1.4 Selection of internal standard Addition of IS to the biological samplesis important in the determination of analytes using LC-MS/MS methods, as it compensates for inescapable assay variance in extraction efficiency, ionization effects and transfer losses. The chosen IS should resemble the analyte of interest during the sample preparation and analysis [28]. It should perfectly track the analyte and improves both accuracy and precision of the assay. AM was the IS of choice as it behaved similar to MM and DPZ during extraction and chromatographic separation. It also showed excellent ionization in positive mode and good extraction recovery which is comparable to both the analytes. 3.2 Method validation The analysis of MM and DPZ using MRM mode was highly selective with no interfering peaks at the RT of the analytes and IS. Fig. 3 shows the MRM chromatograms of blank-, zero- and analyte spiked- rat plasma samples. These chromatograms indicated that the adopted LLE procedure gave a clean sample extract free from endogenous substances. The variability in the RT of both analytes and IS was well within the acceptable limit (RSD ± 5%). The LLOQ for the determination of MM and DPZ in rat plasma was 0.2 ng/mL with acceptable accuracy and precision (within ± 20%). The signal to noise (S/N) ratio for the analyte peak at LLOQ was ~10 times higher than the drug-free blank plasma. Linearity was found over the concentration range of 0.2-400 ng/mL for both MM and DPZ in rat plasma. The correlation coefficient (r2) was found to be greater than 0.996 for all the calibration curves of both the analytes. Deviation of the back calculated calibration standard concentrations from the nominal values were within the acceptable range. All the mean calibration curve parameters for MM and DPZ are represented in Table 2. Within- and between-run accuracy and precision of this method were determined at four different QC samples and the corresponding assay values were well within the acceptable criteria (both % nominal and % CV ≤10%) and are illustrated in the Table 3. These results indicated that the developed bioanalytical method was reliable and reproducible within its analytical range. The extraction recoveries for MM and DPZ from rat plasma was determined by comparing the analyte peak area at three QC levels as well as IS prior to extraction with those from the plasma samples spiked with the analytes at the same concentrations post-extraction. The results are shown in Table 4. The data suggested that the optimized LLE procedure was efficient and reproducible in extracting the analytes and IS from rat plasma with % RSD < 10%. Qualitative matrix effect study using post-column infusion experiment proved that the signals at the retention times of both the analytes and IS were not appreciably altered when drug-free plasma extract was injected into the LC-MS/MS system suggesting the absence of ion suppression/enhancement using the proposed method (Fig. 4). The findings from postcolumn infusion experiment were further validated by quantitative assessment wherein, ISnormalized MF was calculated at two QC levels (at LQC and HQC). The mean value of ISnormalized MF at LQC and HQC was 1.02 and 0.922, respectively for MM and 1.01 and 0.98, respectively for DPZ. The %RSD of IS-normalized MF was <3.85% and <4.09% for all QC replicates of MM and DPZ, respectively, which indicated the reproducibility of the peak areas for the six extracted aliquots from the blank plasma spiked with the analytes and IS. It also showed that the extracts obtained were cleaner without any “unseen” co-eluting components that could interfere with the ionization of the analytes. When blank plasma was injected after concentration corresponding to ULOQ, no significant carry-over from both the analytes and IS was observed. Dilution integrity was confirmed by ten- and twenty-fold dilution of QC samples at concentrations exceeding two-fold the highest calibration level (ULOQ). The accuracy and precision of the diluted samples were within the acceptance criteria of ±15%. Therefore, the plasma samples exceeding the ULOQ can be adequately diluted with blank plasma by using any of the tested dilution factors before analysis. The results of the stock solution, bench-top, long-term, freeze-thaw, autosampler and dry extract stability studies revealed that all the QC samples were found to be stable under the different storages conditions expected during the routine analysis of the samples (Table 5). No significant loss of the analytes and IS was observed under any of these conditions and deviations at all QC levels were within 10% from the nominal values. 3.3 Application of the LC-MS-MS method The proposed method was successfully applied to the simultaneous determination of MM and DPZ in rat plasma after oral administration of MM and DPZ alone as well as in combination. Typical mean plasma concentration-time profiles of MM and DPZ are shown in the Fig. 5 and the corresponding values of the pharmacokinetic parameters based on noncompartmental method (Tmax, Cmax, AUC0−∞, MRT, t1/2 and Cl/F) are shown in the Table 6. As shown in the Fig. 5, the Cmax for MM (26.02±1.25) and DPZ (18.42±6.9) was achieved within 1.5±0.5 h and 1.6±1.15 h with an elimination half-life of 0.27±0.04 h and 0.06±0.01 h, respectively. No significant difference (P<0.05) in the pharmacokinetic parameters of MM and DPZ were observed when given in combination substantiating the absence of pharmacokinetic interaction between them. 4. Conclusion This report is the first demonstration of a sensitive, reliable and reproducible LC-MS/MS assay for the simultaneous analysis of MM and DPZ in rat plasma. The present method provides excellent specificity and linearity over the concentration range of 0.2–400 ng/mL which was sufficient to generate the pharmacokinetics and interaction profiles for MM and in rats after oral administration. The other major advantages of the method are sensitivity (0.2 ng/mL), one step LLE sample clean-up process, low plasma processing volume (50 µL) and short run time (3 min). Thus, the proposed method could be considered to be a valuable tool for pharmacokinetic interaction studies of MM and DPZ with other drugs. In addition, the present methodology could be used for supporting human clinical studies involving co-dosing with MM and DPZ. Conflict of interest There is no potential conflict of interest. Acknowledgements We are thankful to the Director, CSIR-CDRI, for providing facilities and infrastructure for the study. We also acknowledge Council of Scientific and Industrial Research (CSIR) for providing fellowship. CSIR-CDRI communication number for this manuscript is: -----. References [1] National Institute on Aging, Alzheimer’s disease fact sheet, 2011, http://www.nia.nih.gov/alzheimers/publication/alzheimers-disease-fact-sheet. [2] I.N. Spray, FDA Approves New Drug to Treat Dementia, Journal of Gerontological Nursing, 41 (2015). [3] K.C. Riordan, C.R. Hoffman Snyder, K.E. Wellik, R.J. Caselli, D.M. Wingerchuk, B.M. 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Figure legends Figure 1.Structural representation of (a) MM, (b) DPZ and (c) IS. Figure 2.Full-scan product ion mass spectra in positive mode of (a) MM, (b) DPZ and (c) IS. Figure 3.Typical MRM chromatograms of MM, DPZ and IS (left to right) in rat plasma resulting from analysis of (a) blank (analyte and the IS free) plasma, (b) zero sample (analyte-free spiked with the IS) plasma, (c) plasma spiked with analytes at LLOQ (0.2 ng/mL) and IS (80 ng/mL) and (d) 3 h post dose plasma sample after oral administration of MM (2 mg/kg) and DPZ (1 mg/kg) to SD rats. Retention time of MM, DPZ and IS were 2.08, 2.37 and 1.93 min, respectively. Figure 4.Post column infusion chromatograms for (a) MM and (b) DPZ. Figure 5.Mean plasma concentration-time profiles of (a) MM (2 mg/kg) and (b) DPZ (1 mg/kg) after oral administration of MM and DPZ alone and in combination to SD rats. Each point represents mean and standard deviation bars of six observations. Table 1. Optimized Source and compound dependent tandem mass spectrometer parameters Source parameters Value Source temperature, οC 400 Nebulizer gas (Gas1), psi 40 Turbo Ion gas (Gas2), psi 50 Curtain gas, psi 15 Collision gas (CAD), psi Medium Ion spray voltage (eV) 5500 Dwell time (msec) 200 Entrance potential (eV) 10 a Interface heater On Polarity Positive Quadrupoles resolution unit Compound parameters MM DPZ IS Precursor ion [M+H]+, m/z 180.2 380.2 152.2 Product ion, m/z 163.2 91.1 135.0 DP (eV)a 46 61 48.8 CE (eV)b 18.3 70 25.41 CXP (eV)c 3 3 3 Declustering potential; b Collision energy; c Collision exit potential Table 2. Calibration curve parameters for MM and DPZ in rat plasma (n=6) Parameters MM DPZ Mean slope (SD) 0.064 0.306 Mean intercept (SD) (0.0034) (0.025) 0.0146 0.0139 (0.0012) (0.0013) 0.9972 0.9967 (0.0002) (0.0015) Mean r2 (SD) Table 3. Precision and accuracy for determination of MM and DPZ in rat plasma Nominal conc., (ng/mL) MM LLOQ LQC DPZ MQC HQC LLOQ LQC MQC HQC (15) (320) (0.2) (0.6) (15) (320) 0.203 0.576 14.536 286 (0.014) (0.021) (1.03) (18.64) (0.2) (0.6) 0.205 (0.009) 0.566 (0.04) 14.26 (0.77) 309.4 (7.64) Precision (% RSD)a 4.434 7.023 5.400 2.468 7.020 3.606 7.084 6.518 Accuracy (% nominal)b 102.30 94.33 95.07 103.13 99.00 91.50 101.33 100.67 0.202 (0.018) 0.595 (0.034) 14.86 (0.873) 313.2 (3.83) Precision (% RSD)a 8.751 5.784 5.878 1.224 4.530 7.921 4.873 5.987 Accuracy (% nominal)b 101.00 99.20 99.07 104.40 99.50 91.50 101.33 100.67 Within-run (n=6) Mean calculated conc., ng/mL (SD) Between-run (n=6) 0.209 0.604 15.26 302.4 (0.009) (0.048) (0.744) (18.10) Mean calculated conc., ng/mL (SD) a % RSD = Relative standard deviation, [SD/mean ×100] b % nominal = [measured concentration/spiked concentration × 100] Table 4.Extraction recovery for MM and DPZ at three QC levels (n=6). MM Nominal conc (ng/mL) DPZ IS LQC MQC HQC LQC MQC HQC 0.6 15 320 0.6 15 320 10 % Mean recovery (SD) %RSDa a 82.6 (6.08) 7.36 84.47 (4.99) 5.92 89.49 (2.92) 3.26 87.22 (8.25) 9.98 80.39 (3.17) 3.75 80.69 (5.26) 5.88 % RSD = Relative standard deviation, [SD/mean ×100] Table 5. Stability of MM and DPZ in rat plasma at two QC levels (n=6) Storage conditions Drug LQCa Nominal (%) HQCb Nominal (%) Bench-top stability MM 0.62 (0.05) 103.38 314.5 (6.24) 98.28 DPZ 0.55 (0.04) 91.33 316.25 (13.15) 98.83 MM 0.62 (0.03) 102.92 324.25 (12.82) 101.33 DPZ 0.57 (0.06) 95.0 314.25 (9.95) 98.2 MM 0.59 (0.03) 97.92 313.25 (11.18) 97.89 DPZ 0.61 (0.06) 101.25 305.0 (18.28) 95.31 MM 0.64 (0.04) 105.83 307.5 (12.23) 96.09 DPZ 0.58 (0.04) 96.67 320.25 (15.95) 100.08 MM 0.6 (0.05) 99.17 329.25 (10.37) 102.89 DPZ 0.59 (0.05) 97.50 310.25 (25.64) 96.95 (22 οC, 24 h) Long-term stability (-80 οC, 2 months) Long-term stability (-20 οC, 2 months) Three freeze-thaw cycles stability Auto sampler stability (6 ± 2 οC, 24 h) 78.93 (5.01) 6.34 Dry extract stability (22 οC, 12 h) MM 0.61 (0.05) 101.25 303.75 (17.04) 94.92 DPZ 0.60 (0.03) 100.0 331.13 (14.06) 103.48 All values are expressed as mean (SD). a LQC: 0.6 ng/mL; b HQC: 320 ng/mL Table 6. Pharmacokinetic parameters of MM and DPZ(alone and in combination) following oral administration of MM and DPZ to rats (n=6) Pharmacokinetic parameters MM Alone DPZ In combination p-valuea Alone In combination p-valuea Cmax (ng/mL) 26.02 (1.25) 28.2 (6.51) 0.438 18.42 (6.9) 18.2 (6.77) Tmax 1.5 (0.5) 2.16 (0.76) 0.105 1.66 (1.5) 2.66 (1.52) AUC0-∞ (h.ng/mL) 153.69 (6.05) 161.80 (7.66) 0.393 t ½ (h) 2.61 (0.48) 1.87 (0.8) 0.807 10.32 (1.82) 9.75 (1.57) 0.574 MRT0-∞ (h) 5.42 (0.32) 5.07 (0.36) 0.105 11.27 (2.68) 10.87 (2.32) 0.787 Cl/F (L/h/Kg) 13.02 (5.09) 13.05 (1.02) 0.988 8.11 (3.80) 8.32 (4.09) 0.928 Each value represents the mean (±SD) 1 145.19 (72.82) 143.81 (75.44) 0.956 0.227 0.974 a Student’s t‐test with p < 0.05 being considered significant.