Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry

Accepted Manuscript Title: Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry Author: Guru R. Valicherla Priyanka Tripathi Sandeep K. Singh Anees A. Syed Mohammed Riyazuddin Athar Husain Deep Javia Kishan S. Italiya Prabhat R. Mishra Jiaur R. Gayen PII: DOI: Reference:

S1570-0232(16)30517-7 http://dx.doi.org/doi:10.1016/j.jchromb.2016.07.042 CHROMB 20180

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

5-5-2016 19-7-2016 23-7-2016

Please cite this article as: Guru R.Valicherla, Priyanka Tripathi, Sandeep K.Singh, Anees A.Syed, Mohammed Riyazuddin, Athar Husain, Deep Javia, Kishan S.Italiya, Prabhat R.Mishra, Jiaur R.Gayen, Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.07.042 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. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry Guru R. Valicherla1,4#, Priyanka Tripathi2,4#, Sandeep K. Singh1, Anees A. Syed1, Mohammed Riyazuddin1, Athar Husain1, Deep Javia3, Kishan S. Italiya3, Prabhat R. Mishra2,4, Jiaur R. Gayen1,4*. 1

Pharmacokinetics and Metabolism, 2Pharmaceutics Division, CSIR-Central Drug Research

Institute, Lucknow, India. 3

Department of Pharmaceutics, National Institute of Pharmaceutical Education and

Research, Raibarelly, India. 4

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India.

#

Equal contribution

*

Address for correspondence

Dr. Jiaur R. Gayen, Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow ‐ 226031, India Tel.: +91 522 2772450 ext 4845, E-mail : [email protected]

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Graphical abstract

Highlights  LC-MS/MS method of Miltefosine (MFS) was developed for the first time in rat plasma  The method was validated and stability studies were performed  Lower limit of quantification of MFS for the LC-MS/MS method was 1 ng/mL  Absolute bioavailability of MFS was 60.33 ± 2.32% in rats  MFS has slow absorption, long half-life and high apparent volume of distribution

Abstract Miltefosine (MFS) is the first effective oral drug for treatment of visceral, mucosal and cutaneous leishmaniasis. In this study, liquid chromatography coupled mass spectrometry (LC-MS/MS) method of MFS was validated in rat plasma and its practical utilization to pharmacokinetic studies in rats for the first time. A rapid, selective and sensitive LC-MS/MS 2

method for MFS in rat plasma was linear over the calibration range of 1-500 ng/mL. MFS and Phenacetin (internal standard) were separated on Phenomenex Luna 3µ HILIC 200A (150 x 4.6 mm) column under isocratic condition using methanol: 0.1% formic acid in triple distilled water, 90:10 (v/v) mobile phase at a flow rate of 0.8 mL/min. The total chromatographic run time was 4.0 min. The intra- and inter-day assay accuracy was observed between 99.45-102.88% and 99.92-101.58%, respectively. The intra- and inter-day assay precision was observed between 2.68-5.54% and 2.35-5.94%, respectively. The validated assay was practically applied to determine the plasma concentrations after oral and intravenous administration of MFS to rats. After oral administration, MFS showed Cmax (3200.00 ± 95.39 ng/mL) was observed at 12.00 h (tmax) and t1/2 was 102.36 ± 16.65 h. The absolute bioavailability of MFS was 60.33 ± 2.32%. Keywords: Miltefosine, Leishmaniasis, LC-MS/MS, Pharmacokinetics, Bioavailability.

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1. Introduction Leishmaniasis is a vector borne disease caused by different leishmanial species and transmitted to human subjects by phlebotomine sand flies. It contains four main clinical syndromes: cutaneous (CL), mucosal, visceral (VL, kala azar) and post kala azar dermal leishmaniasis (PKDL) [1]. Among all types of leishmaniasis, VL is systemic, most severe and fatal if untreated. VL occurs worldwide, but majority in six countries: Bangladesh, Brazil, Ethiopia, India, Nepal and Sudan [2]. According to WHO, around 0.3 million new cases and 20,000 deaths from the VL were estimated annually [3]. During the development of leishmaniasis in the human body, the amastigote forms of the parasite distributed throughout the body and multiplies within the monocytes and macrophages, resulting infiltration of the bone marrow and hepatosplenomegaly [4]. The pentavalent antimonials sodium stilbogluconate and meglumine antimoniate have been used for VL for many years, but the antimonials are toxic drugs with adverse side effects like cardiac arrhythmia and acute pancreatitis [1]. Later Amphotericin B replaced antimonials for the treatment of VL. Parenteral Amphotericin B also showed life threatening side effects like hypokalemia, nephrotoxicity and first dose anaphylaxis. Even though liposomal Amphotericin B (AmBisome) is best for the treatment of VL because of its milder toxicity, but it is an unaffordable treatment [5, 6]. Miltefosine (hexadecylphosphocholine, MFS) is the first and still only oral drug for the treatment of VL and CL. It was licensed for the treatment of VL and CL as Impavido and Miltex, respectively [7]. It showed 98 % cure for Indian VL patients with an oral dose of 2.5 mg/kg daily for 4 weeks [8]. Marketing authorization for the MFS was first granted in India in 2002, followed by Germany, Colombia; and is included in the WHO list of essential

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medicines [5]. Several clinical studies have been reported for MFS in VL and CL patients [912]. There is little data published related to preclinical and clinical pharmacokinetics of MFS. Few bioanalytical methods are available for the quantification of MFS in liquid chromatography coupled mass spectrometry (LC-MS/MS) and high performance liquid chromatography (HPLC). MFS was quantified in fetal calf serum by using HPLC with evaporative light scattering detector [13]. There have been reports of LC-MS/MS bioanalytical method of MFS in human plasma and human peripheral blood mononuclear cells with a lower limit of quantification (LLOQ), 4 ng/mL [14, 15]. All these methods were utilized solid phase extraction (SPE) procedure for sample processing. An LC-MS/MS method has been reported for MFS quantification in dried blood spotting (DBS) of human samples with LLOQ, 10 ng/mL [16]. A recent article published for MFS quantification in human and hamster plasma using LC-MS/MS method using protein precipitation with LLOQ, 2.5 ng/mL [17]. Larger plasma volumes, costlier sample collection methods like DBS, costlier sample extraction procedures like SPE and longer analysis time are the disadvantages of the previous methods. There have been reports of MFS showing the oral pharmacokinetics study in rats by using HPTLC method with a densitometer [18]. To the best of our knowledge, there is no published report on LC-MS/MS method validation and stability studies in rat plasma. The main aim of the present study is LC-MS/MS method validation and stability studies in rat plasma and to explore in-vivo pharmacokinetic behavior and absolute oral bioavailability of MFS in Sprague Dawley (SD) rats. The present method has advantages over previous methods like smaller sample volume (50 µL), rapid analysis (4 min), sensitive (1 ng/mL), simple plasma extraction like protein precipitation. 2. Materials and Methods

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2.1. Chemicals and Reagents MFS was purchased from EMD Chemicals Inc. (San Diego, CA). Phenacetin (Internal Standard, I.S) and LC-MS/MS grade methanol were obtained from Sigma Aldrich (Mumbai, India). The chemical structures of MFS and I.S are represented in Figure.1. HPLC grade Acetonitrile (ACN), Methanol, Hexane, Tert-butyl methyl ether and Ethyl acetate were obtained from Merck Limited (Mumbai, India). Extra pure formic acid was purchased from Sisco Research Laboratories Pvt. Ltd. (SRL) (Mumbai, India). Purified triple distilled water (TDW) was obtained from MilliQ water purification system (EMD Millipore, USA). Diethyl ether was purchased from TKM Pharma (Hyderabad, India). Sodium heparin anticoagulant injection (5000 IU/mL vial) was obtained from Biological E. Ltd. (Hyderabad, India). Young male SD rats (8-10 weeks) weighing around 200-220 g were procured from the Division of Animal Laboratory, Council of Scientific and Industrial Research-Central Drug Research Institute (CSIR-CDRI) (Lucknow, India). Rats were housed in well ventilated cages at standard laboratory conditions with regular light/dark cycles for 12 h. Rats were kept for one week acclimatization prior to the experiments. Pharmacokinetic studies were conducted according to a protocol approved by the Institutional Animal Ethic Committee, CSIR-CDRI (IAEC approval no. IAEC/2012/91). 2.2. Mass spectrometric conditions All calibration standards (CS), quality control (QC) samples, stability samples, in vivo pharmacokinetic samples were analyzed on API 4000 QTRAP mass spectrometer (ABSciex, Canada) equipped with electro-spray ionization (ESI) source in a positive mode for both MFS and I.S. Quantification was performed using the multiple reaction monitoring (MRM) mode. Optimization of compound and source dependent parameters for MFS and I.S was performed by infusing the standard solutions using a Harvard infusion pump (Holliston, USA). Data

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acquisition and quantitation were performed using Analyst 1.6 (Applied Biosystems, MDS Sciex Toronto, Canada). 2.3. Liquid chromatographic conditions A Shimadzu UFLC system (Kyoto, Japan) equipped with a binary pump (LC-20AD), a degasser (DGU-20A3), an auto sampler (SIL-HTc) and a column oven (CTO-20AC) was used to inject the samples. MFS and I.S were separated on Phenomenex Luna 3µ HILIC 200A (150 x 4.6 mm) column with a mobile phase consists of methanol: 0.1% formic acid in TDW, 90:10 (v/v) at 0.8 mL/min flow rate. The injection volume was 10 μL and total run time was 4 min. 2.4. Preparation of CS and QC samples To achieve the desired concentration of 1 mg/mL, primary stock solutions of MFS and I.S were prepared in TDW and ACN, respectively. Working stock solutions were prepared by stepwise dilutions of stock solution in ACN. The concentrations of working stock solutions of MFS for CS were prepared as 20, 40, 100, 200, 400, 1000, 2000, 4000 and 10000 ng/mL in ACN. The concentrations of working stock solutions of MFS for QC were prepared as 20, 80, 800 and 8000 ng/mL in ACN. CS and QC samples were prepared by spiking 47.5 µL of drug free plasma with corresponding working solution 2.5 µL of MFS. In the total plasma volume the organic content (ACN) was found to be 5%. The final CS were prepared as 1, 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL in plasma matrix. The QC were prepared using a stock solution of MFS at four concentration levels, such as LLOQ (1 ng/mL), low QC (4 ng/mL), medium QC (40 ng/mL) and high QC (400 ng/mL). All the stock and working solutions were stored in the refrigerator until use for analysis. 2.5. Plasma sample extraction procedure

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A simple protein precipitation (PPT) method was used for the processing of rat plasma. To 50 µL of CS, QC and plasma samples, 250 µL of I.S (200 ng/mL) solution in mobile phase (methanol: 0.1% formic acid in TDW, 90:10 (v/v)) was added and vortexed for 10 min at 2500 rpm on Vibramax. After protein precipitation, the mixed samples were followed by centrifugation for 10 min at 10,000 rpm (Thermo scientific, USA). An aliquot of 100 µL was separated and 10 µL was injected into the LC-MS/MS system for quantification. 2.6. Assay validation procedures The LC-MS/MS bioanalytical assay for MFS was fully validated according to Guidance for Industry: Bioanalytical Method Validation of USFDA[19]. 2.6.1. Specificity and selectivity The specificity was determined by analyzing the rat control plasma samples collected from six different rats spiked with MFS and I.S to evaluate the lack of chromatographic interference at retention time from plasma matrix. 2.6.2. Calibration curve The calibration curve of MFS was developed by plotting the peak area ratio of MFS to I.S (Y-axis) against the nominal CS concentrations. The final CS concentrations were prepared as 1, 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL. The results were fitted least-squares regression analysis 1/X2 weighing factor. Correlation coefficient (r2) value should be greater than 0.995 or better. The acceptance criteria for each back calculated CS was ±15% deviation from the nominal value except for LLOQ, which was ±20%. 2.6.3. Precision and accuracy

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The intra-day assay precision and accuracy were determined by quantifying six replicates of four QC levels, i.e., 1, 4, 40 and 400 ng/mL. The inter-day assay precision and accuracy were determined by analyzing the four QC levels for three successive days. The acceptance criteria for accuracy was within ±15% standard deviation from the nominal values and precision of within ±15% relative standard deviation (RSD), except for LLOQ, where it should not exceed ±20% for accuracy and precision [20]. 2.6.4. Recovery The percentage recovery of MFS through PPT was determined by comparing the peak areas of MFS extracted (pre-spiked) plasma sample at three QC levels (4, 40 and 400 ng/mL) from post extracted samples spiked at the equivalent concentrations. The percentage recovery of I.S was estimated at 200 ng/mL concentration. 2.6.5. Matrix effect Matrix effect of MFS and I.S were estimated by using the post extraction spike method. The matrix effect was evaluated at three different QC levels (n=6) such as LQC, MQC and HQC whereas the matrix effect of I.S was determined at 200 ng/mL concentration. The post extracted QC plasma samples were compared with drug free control plasma spiked with equivalent QC solution after extraction [21, 22]. The matrix effect of MFS and I.S were determined using the equation (1). A value of matrix factor (MF)>1 indicates ionization enhancement and a value of MF<1 indicates ionization suppression.

Matrix Factor  MF  

Peak area of MFS in post extracted plasma Peak area of MFS in mobile phase

1

2.6.6. Carry over and dilution integrity

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Carry over effect was assessed by injecting the highest CS (500 ng/mL) followed by blank sample. Dilution integrity was performed to evaluate the ability for extending the 500 ng/mL concentration. Dilution integrity was determined by diluting 20 times the plasma (n=6) containing 10000 ng/mL of MFS with control drug free plasma to obtain 500 ng/mL of MFS. The accuracy and precision should be within ±15%. 2.7. Stability experiments All stability studies were conducted in six replicates at each concentration of three different QC levels, such as LQC, MQC and HQC. Replicate injections of extracted plasma samples were analyzed after 12 h to estimate autosampler stability at 4±2 °C. Bench top stability for 2 and 12 h (ambient temperature, 25±2 °C), long term temperature for 30 days (-20 °C and -80 °C) were performed in rat plasma with three QC levels. Freeze thaw stability was performed after freezing (-80±10 °C for 12 h) and thawing for three cycles. All QC samples were extracted and quantified against fresh calibration curves and fresh QC samples. The acceptance criteria of accuracy and precision for all stability samples should be within ±15%. 2.8. Application to intravenous and oral pharmacokinetic study in rats The intravenous and oral pharmacokinetic (PK) studies were performed on young male SD (8-10 weeks) weighing around 200-220 g rats. For the oral PK study, rats were kept overnight fasting with water only. MFS was dissolved in 0.9% saline (0.5 mg/mL) and administered intravenously at the dose of 0.5 mg/kg with a maximum dose volume of 250 µL to each rat without fasting (n=6). MFS was dissolved in TDW (5 mg/mL) and orally administered at the dose of 5 mg/kg with a maximum dose volume of 250 µL to each fasted rat (n=6). After i.v dosing, blood samples (200 µL) were collected in microcentrifuge tubes for each time point at 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 48, 72, 96, 120, 144, 192, 240 and 264 h. After oral dosing, blood samples (200 µL) were collected in microcentrifuge tubes for each time point 10

at 0.25, 0.5, 1, 2, 6, 12, 24, 48, 72, 96, 120, 144 and 264 h. Under mild anaesthesia of rats, blood samples were collected at predetermined time intervals from the retro-orbital plexus into heparinized microcentrifuge tubes. Blood samples were centrifuged at 5000 rpm for 10 min and kept at -80±10 °C until analyzed. The pharmacokinetic profile and parameters were evaluated by non-compartmental model approach using Pheonix 6.3 WinNonlin (Pharsight corporation, USA) including t1/2, elimination half-life; Cmax, maximum drug concentration in plasma; tmax, time to reach Cmax; AUC0-t, area under curve from zero to the last time point; AUC0-∞, area under curve from zero to infinity; MRT, mean residence time; CL, clearance and Vd, apparent volume of distribution. The percentage absolute oral bioavailability (F%) of MFS was calculated by using the equation (2).

F

AUC  p.o   Dose  i.v  AUC  i.v   Dose  p.o 

100

 2

3. Results and Discussion 3.1. Mass spectrometric conditions An API 4000 Q-trap mass spectrometer equipped with an ESI source was utilized for the quantification of MFS. For optimization of source and compound dependent parameters of MFS and I.S, continuous direct infusion of neat solution was performed using an infusion pump. Ionization was performed both in positive and negative polarity mode and stable and higher intensity was observed in positive mode for both MFS and I.S. The source and compound parameters were optimized in positive polarity mode to obtain reproducible results for both MFS and I.S which are tabulated in Table 1. In Q1 scan mode, the predominant precursor ions [M+H]+ were observed as 408.20 m/z for MFS and 180.20 m/z for I.S. In MRM mode, the predominant transitions of parent ion 408.20 m/z to selected product ion

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125.10 m/z for MFS (Figure 2) and parent ion 180.20 m/z to selected product ion 110.20 m/z for I.S were selected for quantitative analysis. 3.2. Liquid chromatographic conditions After mass spectrometric parameter optimization, liquid chromatographic conditions were optimized such as choice of composition of the mobile phase, the pH of the buffer, column selection and flow rate. After extensive optimization to achieve better peak shape and better sensitivity, methanol and 0.1% formic acid in TDW mobile phase showed good peak shape and good intensity. Different columns were tried to obtain better resolution, selectivity and rapid analysis. The Phenomenex Luna 3μ HILIC 200A (150 x 4.6 mm) column with isocratic mobile phase ratio methanol: 0.1% formic acid in TDW, 90:10 (v/v) at 0.8 mL/min flow rate was suitable for MFS and I.S. The overall analysis run time was 4 min. Phenacetin was selected as internal standard after several compounds were tried based on the similarities of chromatographic retention, ionization efficiency and extraction behavior with MFS. 3.3. Extraction optimization and recovery Different extraction procedures such as PPT and liquid-liquid extraction were performed initially, but acceptable recovery and reduced matrix effect were resulted with PPT. The simple PPT procedure was preferred because able to give good recovery, cleaner samples and it was not showing the matrix effect. Several organic solvents like ACN, methanol, isopropanol, ethanol, 0.1% formic acid in ACN, 0.1% acetic acid in ACN, 0.1% formic acid in methanol, mobile phase of the method, methanol with 0.1% acetic acid, combination of ACN and methanol and combination of ACN and methanol with 0.1% formic acid were investigated as precipitation solvents for PPT. Methanol and 0.1% formic acid in TDW (90:10) was chosen for PPT based on higher extraction recovery and reduced matrix effect for both MFS and I.S. The absolute mean recovery for MFS was found to be more than 95% 12

of three QC samples. The absolute recovery of MFS for LQC, MQC and HQC was found to be 102.47 ± 5.14%, 96.54 ± 3.52% and 104.23 ± 4.76%, respectively. The absolute mean recovery for I.S was more than 98.75 ± 1.48%. 3.4. Bioanalytical method validation 3.4.1. Selectivity and specificity After optimization of LC-MS/MS conditions, the retention times of MFS and I.S were observed at 2.41 and 2.45 min, respectively. The sample preparation was specific and selective as there was no significant interference from plasma endogenous components was observed at the retention time of MFS and I.S. Figure 3 depicts the chromatograms of blank plasma (MFS and I.S free), zero plasma (MFS free and spiked with I.S), MFS (LLOQ, 1 ng/mL) and I.S spiked rat plasma sample, plasma sample showing MFS and I.S peak obtained following oral administration at 2 h and plasma sample showing MFS and I.S peak obtained following i.v administration at 2 h. The LLOQ for MFS was achieved at 1 ng/mL, where signal to noise ratio (S/N) was equal to 29 and LOD was 0.5 ng/mL, where S/N was equal to 10. 3.4.2. Calibration curve The calibration curve of MFS in rat plasma was linear and reproducible over the calibration range of 1-500 ng/mL. It was determined by using linear regression equation y = mx+c with 1/x2 weighing factor. The average correlation coefficient (r2) (n=6) was found to be ≥0.998 (0.9982, 0.9998, 0.9999, 0.9985, 0.9995 and 0.9999). The accuracy and precision observed from the mean of back calculated CS were within the acceptance criteria as listed in Table 2. 3.4.3. Accuracy, precision and carry over effect

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The intra- and inter-day accuracy and precision of MFS in rat plasma at four QC levels are listed in Table 3. The results of accuracy and precision were found within acceptable limits. No carry over effect was observed, there was no peak of MFS and I.S in blank and zero samples processed with PPT were injected after ULOQ. 3.4.4. Dilution integrity and matrix effect Dilution integrity was performed by diluting the MFS spiked plasma (10000 ng/mL) with blank plasma to obtain 500 ng/mL (HQC). The accuracy after 20 times dilution was found 99.10 ± 3.40%. The dilution integrity was found within acceptable limits for accuracy and precision. Matrix effect of MFS and I.S were determined by using the post extraction spike method. The average matrix effect of MFS was found 1.03, 0.92 and 1.11 for LQC, MQC and HQC, respectively. The average matrix effect of I.S was found 1.04. 3.5. Stability studies The stability of MFS was performed in rat plasma according to the procedure as described in the methods section. The autosampler, bench top, freeze thaw, long term stability results indicated that MFS was stable in rat plasma under different storage conditions that may be encountered during the routine study sample analysis (Table 4). 3.6. Application to pharmacokinetics and bioavailability studies The developed and validated LC-MS/MS method of MFS was practically applied to the pharmacokinetic studies and bioavailability assessment in rats. The average MFS plasma concentration-time profiles following i.v and oral administration in male SD rats are shown in Figure 4. The pharmacokinetic parameters of MFS after i.v and oral administration to rats are listed in Table 5. After oral administration (5 mg/kg), the plasma concentration of MFS was slowly reached to Cmax (3200.00 ± 95.39 ng/mL) at 12.00 h (tmax) and slowly declined with 14

the half-life (t1/2) of 102.36 ± 16.65 h. The AUC0-∞ after oral administration was found to be 258831.57 ± 9965.36 h*ng/mL. MFS has a slow elimination as it has a high t1/2. The apparent volume of distribution (Vd) was found of 2.86 ± 0.52 L/kg. Vd was found to be much larger than the total body water of rat, suggests that high tissue distribution [23]. MFS showed slow absorption after oral dosing and it could provide chronic onset of action as the detectable levels of MFS were observed after 11 days. After i.v administration, Cmax and AUC0-∞ was found to be 2253.33 ± 460.58 ng/mL and 42900.11 ± 3835.49 h*ng/mL, respectively. The absolute bioavailability of MFS was 60.33 ± 2.32% estimated using AUC0-∞ values of oral and i.v administration. The high bioavailability of MFS in rats was probably due to slow and complete absorption from the intestinal tract [18, 24, 25]. The permeability of MFS across the intestinal epithelium involves the non-specific passive pathway and promotes its own transport throughout the intercellular spaces by opening the tight junctions [26, 27]. In preclinical in vitro metabolism, no oxidative metabolism by any reconstituted cytochrome (CYP) isoenzymes and metabolized by phospholipases [28]. The reason for higher oral bioavailability of MFS is probably due to slow and complete absorption from the gastrointestinal tract and no oxidative metabolism. 4. Conclusion A sensitive, rapid and reproducible LC-MS/MS bioanalytical assay was validated for the determination of MFS in rat plasma for the first time. The stability studies were also reported for the first time in rat plasma. The present assay contributes good linearity and reproducibility over the concentration range of 1-500 ng/mL. The LC-MS/MS method was sufficient to generate the pharmacokinetics and absolute bioavailability was calculated from AUC0-∞ values of oral and i.v administration. The advantages of the method are single step protein precipitation, no significant matrix effect, low sample volume (50 µL) and short run time (4 min). The present LC-MS/MS method can be useful for understanding the drug-drug 15

interactions and PK-PD modeling studies. This method could be used for the preclinical and clinical pharmacokinetic studies even for the pediatric population. Acknowledgements Authors acknowledge CSIR for providing research fellowship to GRV, PT and MR. JRG is thankful to Department of Science and Technology (DST), Department of Biotechnology (DBT) and CSIR for financial support. Author KSI, DJ are thankful to Ministry of Chemicals and Fertilizers for financial assistance. The authors are thankful to the Director, CSIR-CDRI for constant encouragement and support. Declaration of interest The authors report no conflict of interest.

Author Contributions Statement GRV, PT, SKS, AAS, MR, AH, DJ and KSI, performed the experimentations. GRV, PT, PRM, and JRG wrote the manuscript. GRV, PT, SKS and MR prepared the figures and tables; PRM and JRG supervised the work. All authors reviewed the manuscript.

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[12] T.P. Dorlo, A.D. Huitema, J.H. Beijnen, P.J. de Vries, Optimal dosing of miltefosine in children and adults with visceral leishmaniasis, Antimicrob. Agents Chemother, 56 (2012) 3864-3872. [13] A. Lemke, O. Kayser, HPLC detection of miltefosine using an evaporative light scattering detector, Pharmazie, 61 (2006) 406-408. [14] T.P. Dorlo, M.J. Hillebrand, H. Rosing, T.A. Eggelte, P.J. de Vries, J.H. Beijnen, Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 865 (2008) 55-62. [15] A.E. Kip, H. Rosing, M.J. Hillebrand, M.M. Castro, M.A. Gomez, J.H. Schellens, J.H. Beijnen, T.P. Dorlo, Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 998999 (2015) 57-62. [16] A.E. Kip, H. Rosing, M.J. Hillebrand, S. Blesson, B. Mengesha, E. Diro, A. Hailu, J.H. Schellens, J.H. Beijnen, T.P. Dorlo, Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection, Antimicrob. Agents Chemother, 60 (2016) 2081-2089. [17] S. Jaiswal, A. Sharma, M. Shukla, J. Lal, LC-coupled ESI MS for quantification of miltefosine in human and hamster plasma, Bioanalysis, 8 (2016) 533-545. [18] P. Kaufmann-Kolle, J. Drevs, M.R. Berger, J. Kotting, N. Marschner, C. Unger, H. Eibl, Pharmacokinetic behavior and antineoplastic activity of liposomal hexadecylphosphocholine, Cancer Chemother. Pharmacol, 34 (1994) 393-398. [19] FDA guidance for industry: bioanalytical method validation US Department of Health and Human Services, Food and Drug Administration, CDER, Rockville, 2013. [20] D. Mandloi, P. Tripathi, P. Mohanraj, N.S. Chauhan, J.R. Patel, Development and validation of a stability-indicating hplc method for analysis of zidovudine (zdv) in bulk drug and in vitro release studies of tablets, J Liq Chrom Relat Tech, 34 (2011) 601-612. [21] A. Van Eeckhaut, K. Lanckmans, S. Sarre, I. Smolders, Y. Michotte, Validation of bioanalytical LC-MS/MS assays: evaluation of matrix effects, J. Chromatogr. B, 877 (2009) 2198-2207. [22] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal. Chem, 75 (2003) 3019-3030.

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[23] B. Davies, T. Morris, Physiological parameters in laboratory animals and humans, Pharm. Res, 10 (1993) 1093-1095. [24] A. Breiser, D.J. Kim, E.A. Fleer, W. Damenz, A. Drube, M. Berger, G.A. Nagel, H. Eibl, C. Unger, Distribution and metabolism of hexadecylphosphocholine in mice, Lipids, 22 (1987) 925-926. [25] N. Marschner, J. Kotting, H. Eibl, C. Unger, Distribution of hexadecylphosphocholine and octadecyl-methyl-glycero-3-phosphocholine in rat tissues during steady-state treatment, Cancer Chemother. Pharmacol, 31 (1992) 18-22. [26] C. Menez, M. Buyse, H. Chacun, R. Farinotti, G. Barratt, Modulation of intestinal barrier properties by miltefosine, Biochem. Pharmacol, 71 (2006) 486-496. [27] C. Menez, M. Buyse, C. Dugave, R. Farinotti, G. Barratt, Intestinal absorption of miltefosine: contribution of passive paracellular transport, Pharm. Res, 24 (2007) 546-554. [28] H. Sindermann, J. Engel, Development of miltefosine as an oral treatment for leishmaniasis, Trans. R. Soc. Trop. Med. Hyg, 100 Suppl 1 (2006) S17-20.

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Figure legends Figure 1. Representation of chemical structures of A) Miltefosine (MFS) and B) Phenacetin (I.S).

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Figure 2. MS/MS spectra of MFS showing prominent parent ion and selected product ion transitions.

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Figure 3. Typical MRM chromatograms of MFS and I.S (200 ng/mL) in A) blank rat plasma (MFS and I.S free), B) zero sample (MFS free and spiked with I.S), C) plasma spiked with MFS (LLOQ, 1 ng/mL) and I.S, D) plasma sample showing MFS and I.S peak obtained following oral administration at 2 h and E) plasma sample showing MFS and I.S peak obtained following intravenous administration at 2 h.

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Figure 4. Plasma concentration vs time profile of MFS following A) oral administration at dose of 5 mg/kg to rats; B) intravenous administration at dose of 0.5 mg/kg to rats. The data was analyzed in n=6 with mean±SD.

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Table 1. Source and compound dependent parameters of tandem mass spectrometer for Miltefosine (MFS) and Phenacetin (I.S). Source parameters Value Ion source temperature (°C) 550 Curtain gas (psi) 30 Collison gas (CAD) Medium Nebulizer gas (GS1, psi) 40 Turbo ion gas (GS2, psi) 60 Ion source voltage (eV) 5500 Polarity Positive Compound parameters Miltefosine Phenacetin (I.S) Parent ion ([M+H]+, m/z) 408.20 180.20 Product ion (m/z) 125.10 110.20 Dwell time (ms) 150 150 Declustering potential (eV) 121 71 Entrance potential (eV) 10 10 Collison energy (eV) 39 30 Collison exit potential (eV) 22 10

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Table 2. Precision and accuracy data of back calculated concentrations of calibration samples of MFS in rat plasma (n=6).

Nominal conc. (ng/mL) 1 2 5 10 20 50 100 200 500

Observed conc. (ng/mL, mean ± SD) 1.02 ± 0.03 2.13 ± 0.14 5.02 ± 0.28 10.29 ± 0.37 19.85 ± 0.96 50.75 ± 1.99 100.89 ± 1.83 200.00 ± 3.08 513.00 ± 6.16

% CV

% Accuracy

3.40 6.31 5.76 3.56 4.93 3.85 1.81 1.29 1.22

102.00 106.25 100.42 102.94 99.35 101.35 100.89 99.92 102.75

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Table 3. Precision and accuracy data of MFS quality controls in rat plasma.

Level

Nominal conc. (ng/mL)

LLOQ

1

LQC

4

MQC

40

HQC

400

Intra-day (n=6) Observed conc. Precision Accuracy (ng/mL, (%) (%) mean ± SD) 1.02 ± 3.69 101.70 0.04 4.12 ± 5.94 102.88 0.25 39.75 ± 1.50 403.83 ± 10.13

3.88

99.45

2.35

100.80

Inter-day (n=18) Observed conc. Precision Accuracy (ng/mL, (%) (%) mean ± SD) 1.00 ± 5.54 100.30 0.06 4.05 ± 4.37 101.18 0.18 40.20 ± 1.03 400.50 ± 12.77

2.68

100.57

3.06

99.92

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Table 4. Stability of MFS quality controls in rat plasma at three QC levels (n=6). Stability

0h (for all)

Autosampler (4 °C, 12h)

Bench-top (2h)

Bench-top (12h)

Three freeze-thaw cycles

Long term (-20°C, 30 days)

Long term (-80°C, 30 days)

Nominal conc. (ng/mL) 4

Observed mean conc. (ng/mL, mean ± SD) 4.00 ± 0.03

Precision (%) 0.85

Accuracy (%) 100.10

40

40.20 ± 2.26

5.62

100.50

400

387.00 ± 11.36

2.95

96.73

4

4.41 ± 0.02

0.52

110.33

40

40.23 ± 1.10

2.84

100.70

400

405.00 ± 12.12

3.16

101.30

4

4.53 ± 0.12

2.34

113.00

40

40.47 ± 0.84

2.22

101.23

400

403.33 ± 8.96

2.06

100.80

4

4.17 ± 0.24

5.78

104.33

40

41.57 ± 1.37

3.39

103.97

400

432.67 ± 11.15

2.45

108.00

4

3.93 ± 0.17

4.38

98.37

40

40.63 ± 0.81

2.04

101.80

400

396.67 ± 6.35

1.57

99.20

4

3.88 ± 0.13

3.27

97.10

40

39.93 ± 0.25

0.36

99.70

400

404.67 ± 3.21

0.57

101.00

4

3.98 ± 0.05

1.41

99.67

40

40.63 ± 0.57

1.56

101.63

400

427.67 ± 4.04

0.93

107.00

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Table 5. Pharmacokinetic parameters of MFS following intravenous and oral administration to rats (n=6). Pharmacokinetic

Intravenous

Oral

parameters

(Mean ± SD)

(Mean ± SD)

Dose (mg/kg)

0.5

5

t1/2 (h)

64.13 ± 19.99

102.36 ± 16.65

Tmax (h)

-

12.00

Cmax (ng/mL)

MRT0-∞ (h)

2253.33 ± 460.58 39881.06 ± 4393.34 42900.11 ± 3835.49 97.01 ± 9.38

3200.00 ± 95.39 250780.01 ± 12258.79 258831.57 ± 9965.36 83.78 ± 4.88

CL (mL/h/kg)

11.72 ± 1.08

19.34 ± 0.73

Vd (L/kg)

1.09 ± 0.36

2.86 ± 0.52

Bioavailability (%)

-

60.33 ± 2.32

AUC0-t (h*ng/mL) AUC0-∞ (h*ng/mL)

28