Journal xyz 2017; 1 (2): 122–135 Open Chem., 2018; 16: 614–620

The First Decade (1964-1972) Research Article Open Access Research Article Haitham Alrabiah, Mohammed Abunassif, Sabry Attia, Gamal Abdel-Hafiz Mostafa* Max Musterman, Paul Placeholder What Is So Different About A new selective, and sensitive method for the Neuroenhancement? Was ist so anders am Neuroenhancement? determination of lixivaptan, a 2 (V2)- receptor antagonist, in mouseJournal xyz 2017; plasma 1 (2): 122–135 and its Pharmacological and Mental Self-transformation in Ethic Comparison application in a pharmacokinetic study Pharmakologische und mentale Selbstveränderung im The First Decade (1964-1972) ethischen Vergleich https://doi.org/10.1515/chem-2018-0063Research Article received September 19, 2017; accepted April 9, 2018. mouse plasma. A mean maximum plasma concentration https://doi.org/10.1515/xyz-2017-0010 of lixivaptan of 113.82 ng mL-1 was achieved in 0.5 h after received February 9, 2013; accepted March 25, 2013; published online July 12, 2014 Max Musterman, Paul Placeholder Abstract: A new, selective and sensitive HPLC method oral administration of a 10 mg kg-1 dose in mouse as Abstract: In the concept of the aesthetic formation of knowledge and its as soon for Whatthe determination Is So Differentof lixivaptan, anAbout oral selective determined using the developed method. as possible and success-oriented application, insights and profits without the vasopressin 2 (V2)-receptor antagonist, was investigated reference to the arguments developed around 1900. The main investigation also andNeuroenhancement? validated. A Waters symmetry C18 column was used Keywords: Lixivaptan; HPLC/UV; mouse plasma; includes the period between the entry into force and the presentation in its current as a stationary phase in isocratic elution mode using a pharmacokinetic. version. Their function as part of the literary portrayal and narrative technique. Was ist so anders am Neuroenhancement? mobile phase composed of KH2PO4 (100 mM)-acetonitrile Keywords: Function, transmission, investigation, principal, period (40: 60, v/v) at a flow rate of 1.5 mL min-1. Diclofenac was Pharmacological and Mental Self-transformation in Ethic used as the internal standard (IS). Lixivaptan and the IS 1 Introduction Dedicated to Paul Placeholder wereComparison extracted from plasma by protein precipitation and werePharmakologische detected at 260 nm. Lixivaptan und mentale and diclofenac Selbstveränderung were Lixivaptan im (LIX) is an orally-administered elutedethischen at 3.6 and Vergleich 6.2 min, respectively. The developed pharmacological compound used as an antagonist of 1 Studies and Investigations method showed good linearity over the calibration range vasopressin, a hormone that is associated with heart of https://doi.org/10.1515/xyz-2017-001050 -1000 ng mL-1 with a lower limit of detection of 16.5 failure [1, 2] and has a role in maintaining water balance The main investigation also includes the period between the entry into force and received-1 February 9, 2013; accepted March 25, 2013; published online July 12, 2014 the presentation in its current version. Their function as part of the literary por- ng mL . The extraction percentage of lixivaptan in the in the body. Lixivaptan functions by blocking the trayal and narrative technique. mouse plasma was in the range of 88.88 - 114.43%, which vasopressin 2 (V2) receptor and can potentially be used Abstract: In the concept of the aesthetic formation of knowledge and its as soon indicates acceptable extraction. The aforementioned for the treatment of [3-5]. Hyponatremia as possible and success-oriented application, insights and profits without the method was validated according to guidelines of the is the condition in which the concentration of sodium in reference to the arguments developed around 1900. The main investigation also *Max Musterman: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning International Council on Harmonization (ICH). The the blood is lower than normal. Lixivaptan can eliminate Road Keelung 20224, Taiwan (R.O.C), e-mail: [email protected] includes the period between the entry into force and the presentation in its current intra- and inter-day coefficients of variation did not excess fluids from the body and keep blood sodium Paul Placeholder: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning version. Their function as part of the literary portrayal and narrative technique. Road Keelung 20224, Taiwan (R.O.C), e-mail: [email protected] exceed 5.5%. This method was presented to be simple, levels within the normal range. It is a selective non- sensitive, and Function, accurate transmission,and was successfully investigation, adapted principal, in peptide period V2 antagonist [3, 4] and a chemical derivative Open Access. © 2017 Mustermann and Placeholder, published by De Gruyter. This work is Keywords: licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. a pharmacokinetic study of the profile of lixivaptan in of benzodiazepine, with the chemical name 5-fluoro- Dedicated to Paul Placeholder 2-methyl-N-[4-(5H-pyrrolo[2,1-c]-[1,4]benzodiazepin- 10(11H)-ylcarbonyl)-3 chlorophenyl]benzamide (Figure1) *Corresponding author: Gamal Abdel-Hafiz Mostafa, Department [6]. Lixivaptan strongly binds to V2 receptors in the of Pharmaceutical Chemistry, College of Pharmacy, King Saud kidneys, preventing the insertion of aquaporin channels University,1 Studies P.O.Box 2457, and Riyadh Investigations 11451, Saudi Arabia; Micro- into the apical membrane layer, thus resulting in an analytical Lab., Applied Organic Chemistry Department, National increase in solute-free water excretion and sodium ResearchThe main Center, investigation Dokki, Cairo, Egypt, also includes E-mail: [email protected] the period between the entry into force and retention [7]. Haithamthe presentation Alrabiah: Department in its current of Pharmaceutical version. Chemistry,Their function College as part of the literary por- of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Several studies were directed toward assessing the trayal and narrative technique. Saudi Arabia pharmacodynamics and pharmacokinetics of lixivaptan Mohammed Abunassif: Department of Pharmaceutical Chemistry, [8-11]. Lixivaptan is absorbed quickly with a mean College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh estimated T of < 1 h. A crosswise analysis showed 11451,*Max Saudi Musterman: Arabia Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ningmax that the rate of clearance, volume of distribution, and SabryRoad Attia: Keelung Department 20224, ofTaiwan Pharmacology (R.O.C), e-mail: and Toxicology, [email protected] College of Pharmacy,Paul Placeholder: King Saud University,Institute of P.O.BoxMarine Biology,2457, Riyadh National 11451, Taiwan Saudi Ocean half-life University, of 2 Pei-Ning lixivaptan were in a correlation between the ArabiaRoad Keelung 20224, Taiwan (R.O.C), e-mail: [email protected] LIX concentration with free water clearance [10]. The

Journal xyz 2017; 1 (2): 122–135 Open Open Access. Access. © © 2018 2017 HaithamMustermann Alrabiah and Placeholder, et al., published published by by De De Gruyter. Gruyter. ThisThis work work is is licensed under the Creative Commons Attribution-NonCommercial-NoDerivativeslicensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. 4.0 License. The First Decade (1964-1972) Research Article

Max Musterman, Paul Placeholder What Is So Different About Neuroenhancement? Was ist so anders am Neuroenhancement?

Pharmacological and Mental Self-transformation in Ethic Comparison Pharmakologische und mentale Selbstveränderung im ethischen Vergleich https://doi.org/10.1515/xyz-2017-0010 received February 9, 2013; accepted March 25, 2013; published online July 12, 2014

Abstract: In the concept of the aesthetic formation of knowledge and its as soon as possible and success-oriented application, insights and profits without the reference to the arguments developed around 1900. The main investigation also includes the period between the entry into force and the presentation in its current version. Their function as part of the literary portrayal and narrative technique.

Keywords: Function, transmission, investigation, principal, period

Dedicated to Paul Placeholder

1 Studies and Investigations

The main investigation also includes the period between the entry into force and the presentation in its current version. Their function as part of the literary por- trayal and narrative technique.

*Max Musterman: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning Road Keelung 20224, Taiwan (R.O.C), e-mail: [email protected] Paul Placeholder: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning Road Keelung 20224, Taiwan (R.O.C), e-mail: [email protected]

Open Access. © 2017 Mustermann and Placeholder, published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. A new selective, and sensitive method for the determination of lixivaptan, a vasopressin 2 (V2)-receptor... 615

Figure 1: Chemical structure of: (A) lixivaptan and (B) the internal standard (IS) diclofenac (DC). pharmacokinetic study need to highly sensitive technique 2 Materials and Methods to determine LIX in lower concentration as well as their detection in plasma metrics without interferences. 2.1 Reagents and materials Therefor this study aimed to develop an HPLC for determination of LIX in mice plasma and its application to Lixivaptan and diclofenac sodium (DC) (Figure 1) of pharmacokinetic study. purity > 99% (Sigma-Aldrich, Steinheim, Germany) were Validation of any developed method is essential used as references in this study. HPLC-grade acetonitrile in order to prove that the method is acceptable for the and methanol were purchased from Zeus Quimica proposed use. The proposed method should also satisfy S.A (Barcelona, Spain). Analytical grade “potassium criteria related to estimating the drug in biological fluids dihydrogen phosphate, phosphoric acid, perchloric acid or various matrices (biological samples) as well as in and trifluoroacetic acid were acquired from AVONCHEM clinical studies [12-14]. We therefore developed a new (Macclesfield, Cheshire, England). Double distilled HPLC method to which international standards, such as water was produced using a cartridge system (Waters the guidelines of US Food and Drug Administration (FDA) Millipore, Milford, USA)”. Naltrexone hydrochloride, [15] and the International Council for Harmonization (ICH) cyclobenzaprine hydrochloride, clonidine hydrochloride, [16], were accordingly applied for validation. tizanidine hydrochloride, pravastatin sodium, fenofibrate, The rationale for choosing HPLC is based on the fenofibric acid, phenobarbital sodium, and 7-Methyl- advantages of this technique, including its simplicity, 6,7,8,9,14,15-hexahydro-5H-benz[d]indolo[2,3-g]azecine sensitivity and accuracy, which have made it commonly (LE 300) were purchased from Sigma (St. Louis, Mo, USA). used for drug analysis in many laboratories [17-19]. The proposed HPLC method involves detection via ultraviolet spectroscopy, and it was optimized according to the 2.2 Apparatus and chromatographic selected validation criteria [15, 16]. This work represents conditions a complementary study to our initial work, which was based on HPLC-MS/MS analysis [20]. “The HPLC analysis was carried out using a Waters HPLC In the present investigation, a sensitive, selective, system (Milford, USA) equipped with 1500 series HPLC and accurate HPLC-UV method is proposed for the assay pump”, operated at a flow rate of 1.5 mL min-1. A dual- of lixivaptan in mouse plasma. A protein precipitation wavelength “ultraviolet detector (2489) and an auto- procedure as an extraction method was used for extracting sampler (717plus)” were used. Data were collected with lixivaptan from mouse plasma. The validation parameters an “Empower pro Chromatography Manager for data of lixivaptan determination as a bioanalytical method acquisition and analysis”. were optimized according to ICH guidelines. The suggested Chromatographic separations were completed using method was successfully applied in a pharmacokinetic an analytical Waters Symmetry “C18 analytical column study of lixivaptan in mouse plasma. (125 mm ´ 4.6 mm i.d. ´ 3 µm particle size (Waters, USA) coupled with a Symmetry C18 sentry guard column (20 mm)”. All solutions were degassed by “ultra-sonication and filtered through a 0.45 µm filter (Millipore)”. 616 Haitham Alrabiah et al.

The mobile phase consisted of KH2PO4 (100 mM) and calculated using the following formula: ([peak area ratio acetonitrile (40: 60, v/v). All separations were achieved of extract/mean peak area ratio of un-extracted drug] in isocratic mode at a flow rate of 1.5 mL min-1 at 25°C. The ´100). injection volume was 50µL and the absorbance of eluents The intra-day and inter-day precisions of the was recorded at 260 nm. lixivaptan assay were calculated by the repeatability of the analysis of three quality control samples (75, 300 and 700 ng mL-1) within the same day or in three different days, 2.3 Preparation of solutions and plasma respectively. The accuracy and precision of the established samples method for lixivaptan were determined according to ICH guidelines for bio-analytical method validation [16]. Stock solutions of lixivaptan and DC were prepared The stability of lixivaptan in mouse plasma by dissolving a quantity of the drug and standard was evaluated using three replicate samples of QC in acetonitrile and methanol, respectively, to yield a concentrations (75, 300, and 750 ng mL-1). The stability concentration of 1 mg mL.-1 Three working solutions of conditions were: at 25°C (room temperature) for 8 h, at LIX (100 and 10 and 1 µg mL-1 ) and two working solution - 4°C for one week, and in the auto-sampler tray for 12 of DC (100 and 10 µg mL-1) were prepared by suitable h. Calculation of accuracy and precision of the quality dilution. All solutions were stored at 4°C. control samples was carried out using a calibration curve Mouse plasma (100 µL) was spiked with a suitable based on fresh mouse plasma. amount of lixivaptan ( 10 and 1 µg mL-1) and DC (10 µg mL-1) to give a final concentration of 50, 100, 200, 400, 800, and 1000 ng mL-1 lixivaptan and 2 µg mL-1 DC, with each sample 2.5 Pharmacokinetic Application being prepared in a 2.0 mL disposable polypropylene micro-centrifuge tube. Then, 500 µL acetonitrile was 2.5.1 Animal maintenance added to induce protein precipitation, and each tube was vortexed for about 1 min. The samples were then Adult male white” Swiss albino mice” weighing 25-30 g centrifuged at 10,000 rpm at room temperature (25°C) for (10 - 12 weeks old) were obtained from the “Experimental 9 min. The upper layer solution was filtered through a Animal Care Center, King Saud University”. The animals simple pure filter (0.22 µm) and the clarified sample was were maintained in an “air-conditioned animal house at injected into the HPLC system. Analysis of each lixivaptan a temperature of 25-28 , relative humidity of 50%, and concentration was repeated six times. The average peak 12:12h light and dark photo-cycle”. The animals were ℃ area ratio for each sample was estimated and plotted provided with standard diet pellets and water ad libitum. against LIX concentration. Blank mouse plasma samples The experiments were approved by the “Ethics Committee were prepared in the same way using the diluting solvent of the Experimental Animal Care Society, College of instead of lixivaptan and DC. Pharmacy, King Saud University, Riyadh, Saudi Arabia.”

2.4 Bioanalytical method of validation 2.5.2 Pharmacokinetic study

The linearity of the method was evaluated according to After two days of housing, the mice were randomly ICH guidelines [16]. A calibration graph was obtained by divided into eight groups (six mice each), then seven of plotting the area ratios of lixivaptan to DC (IS) against the groups were orally administered with 10 mg kg-1 of the initial lixivaptan concentration. The equation of the lixivaptan, and the remaining group was administered calibration curve was obtained using the fitting of the “dimethyl sulfoxide in saline to serve as blank mouse plot. Calibration plots for lixivaptan were prepared using plasma” [21]. The injection volume of the drug was 0.01 six concentration points (50, 100, 200, 400, 800, and 1000 mL g-1 body weight. Lixivaptan was administered at 0.5, ng mL-1). Each concentration point was repeated six times, 1, 1.5, 2, 3, 4, and 8h before blood sampling. Each time and the average value was calculated and used to plot the point was repeated six times with different mice. The calibration graph. blood samples were withdrawn from the heart (1.0 mL). The accuracy of the determination of lixivaptan was The plasma samples were separated from the serum by assessed explicitly by spiking known concentrations centrifugation at 4000 rpm for 5 min, and then preserved of lixivaptan into mouse plasma. The % recovery was at -20 until analysis.

℃ A new selective, and sensitive method for the determination of lixivaptan, a vasopressin 2 (V2)-receptor... 617

Table 1: Extraction % recovery of lixivaptan from spiked mouse 3 Results and discussion plasma using solvent deproteinization procedure.

This study describes an HPLC-UV method for the assay of Conc., Peak area ratio Peak area ratio of Recovery, % lixivaptan. To select an internal standard (IS), we tested ng mL-1 of standard spiked plasma different drugs with similar chemical structures or pKa 50 0.048 0.044 91.66 values to the drug, including naltrexone, cyclobenzaprine, clonidine, tizanidine, pravastatin, fenofibrate, fenofibric 100 0.090 0.080 88.88 acid, LE 300, and phenobarbital sodium. Most of the tested 200 0.180 0.170 94.44 drugs failed because they had short elution times (less 400 0.370 0.420 113.51 than 1 min), because they interfered with lixivaptan, or 800 0.840 0.760 90. 47 because an excessively large separation was obtained. DC was selected as the IS for the quantification of lixivaptan 1000 0.970 1.110 114.43 because it was separated at a suitable elution time of n=6 approximately 3.6 min under optimal chromatographic separation conditions. Suitable chromatographic conditions were studied resulted in a good recovery (see Table 1). The extraction and optimized through multiple trials to achieve good recovery of lixivaptan from mouse plasma was in the range resolution and a symmetric peak shape for lixivaptan and of 88.88-114.43%, which is consistent with published DC, as well as a suitable elution time. For the optimization standards [12]. of the mobile phase composition, different mobile phase components, such as methanol, water and acetonitrile in different ratios, were tested. Acetonitrile: KH2PO4 showed 3.2 Specificity better separation than methanol: KH2PO4; however, the resolution was still not complete, and approximately To investigate the specificity of the new method, a variety of 1.2 (about 90%) resolution was obtained. Another trial blank mouse plasma samples were examined individually involved the use of KH2PO4 of different ionic strengths (25, to detect any potential interference. Chromatograms of 50, and 100 mM), and 100 mM appeared to be the most mouse blank plasma and mouse plasma spiked with suitable ionic strength to obtain satisfactory resolution. 2 μg mL-1 DC and 200 ng mL-1 lixivaptan are presented in The absorbance spectrum of lixivaptan showed two Figure 2. The peaks of lixivaptan and DC were well resolved maxima, at 210 and 260 nm. The peak at 260 nm resulted at the correct retention times of 6.2 and 3.6 min, respectively. in acceptable selectivity when compared with that at 210 In mouse plasma, no peaks similar to either lixivaptan or nm. In addition, at 210 nm, the blank showed a higher DC were observed over the elution time of both the drug absorbance intensity compared with that at 260 nm. and the IS. The analysis was completed within 10 min with Therefore, the optimal conditions of chromatographic complete separation. The protein precipitation procedure separation were found to be a mobile phase consisting of of the plasma samples was appropriate for the separation acetonitrile:100 mM KH2PO4, 60:40 (v/v) and detection at and extraction of the drug from the plasma in the absence wavelength 260 nm. Under these conditions, lixivaptan of any interference from similar peaks (Figure 3). The analysis demonstrated a good capacity factor, separation system suitability parameters for lixivaptan are listed in factor, resolution, and peak symmetry. Table 2 based on isocratic elution of the drug at a flow rate of 1.5 mL min-1 and a detection wavelength of 260 nm.

3.1 Extraction procedure 3.3 Validation of the Method A clean-up procedure is often essential to remove plasma proteins prior to analysis. The extraction step 3.3.1 Linearity and sensitivity in general involves time-consuming and laborious sample pretreatments, which often include the use of The investigated method showed good linearity for liquid or solid phase extraction. A solvent-based protein lixivaptan over the calibration range of 50-1000 ng mL-1 precipitation method was developed using different lixivaptan. The regression correlation coefficient was solvents such as perchloric acid, trifluoroacetic acid and r2 = 0.9989. The calibration curve equation was y = 0.001x- acetonitrile. The optimal solvent was acetonitrile, which 0.0127. The good linearity of the calibration graph is 618 Haitham Alrabiah et al.

Table 2: Chromatographic system suitability parameters.

Parameters RT K Selectivity Resolution Symmetry factor Number of theoretical

“a” “RS” plates “N” IS 3.6 127.16 1.5 - 1.06 2452

Lixivaptan 6.2 210.34 2.47 6.98 1.0 3956

RT = Retention time K = Capacity factor for drug and IS

α = Separation factor, calculated as K2/K1, where k = the capacity factor for drug and IS

Rs = 2 (t2 – t1) / (w1 + w2), where t2 and t1 are the retention times of the drug and IS and w2 and w1 are the half-peak width values for the drug and IS, respectively N = number of theoretical plates = (RT/w)2

Table 3: Analytical parameters of the investigated HPLC method.

Parameter Value aa

Slope 0.001

Standard error of slope 3.2×10-5

Intercept 0.0127

Standard error of intercept -0.005

STE YX 0.005

Correlation coefficient, (r2) 0.9989 Figure 2: HPLC chromatogram of the analysis of lixivaptan in drug-free plasma: (A) blank plasma, (B) mouse plasma spiked with 200 ng mL-1 Calibration range ( ng mL-1) 50.0 - 1000.0 and 2 μg mL-1 of lixivaptan and DC, respectively. LOD (ng mL-1) 16.50

LOQ (ng mL-1) 50.0

Retention time of lixivaptan (min) 3.6

Retention time of IS(min) 6.2 a Values are presented the mean of three determinations.

3.3.2 Accuracy and precision

The accuracy and precision of the studied method were evaluated by assaying different concentrations plotted in the calibration curve, encompassing a broad range of Figure 3: HPLC chromatograms of mouse plasma (A) at zero time -1 (Blank) and (B) 0.5 h after injection. concentrations (75, 300 and 700 ng mL ) of lixivaptan. The precision of the method was estimated in terms of injection repeatability, analysis repeatability during a single indicated by the high value of the correlation coefficient day, and intermediate precision over multiple days. The and the standard deviation parameters of the obtained accuracy and precision of the investigated method were calibration curve [22]. Table 3 gives an outline of the within an adequate range as defined by ICH guidelines general analytical characterization of the investigated [16]. The intra-day accuracy and precision were within the method. The lower limit of quantification and lower limit range 95.0-97.77% and 0.94 - 4.63%, respectively. The inter- of detection of lixivaptan in mouse plasma was estimated day accuracy ranges were 91.0-92.3% and 0.97 - 4.66%, at 50.0 and 16.5 ng mL,-1 respectively (signal-to-noise ratio respectively (Table 4). of 10 or 3) A new selective, and sensitive method for the determination of lixivaptan, a vasopressin 2 (V2)-receptor... 619

Table 4: Intra- and inter-day accuracy and precision of lixivaptan quality control samples determined for the investigated HPLC method.

Conc. Intra-day Inter-day -1 (ng mL ) Mean SD RSD% Accuracy % Mean SD RSD% Accuracy %

75.0 71.2 3.3 4.63 95.0 69.22 3.23 4.66 92.3

300 294.5 5.3 1.79 98.18 273.00 5.23 1.91 91.0

700.0 684.4 6.46 0.94 97.77 642.53 6.25 0.97 91.79

Table 5: Stability of lixivaptan in mouse plasma based on the proposed HPLC method.

Concentration Mean Recovery, % RSD, % RE, % (ng mL-1)

Room Temp. for 8h

75 71.25 95 5.0 -5%

300 288 96 4.5 -4%

750 720 96 4.0 -4%

Storage at -4o C

75 70.5 94 6 -6%

300 285 95 4.5 -5% Figure 4: Concentration-time curve of lixivaptan in mouse plasma -1 750 720 96 4.0 -4% after oral administration of 10 mg kg­ lixivaptan. Each point represents mean value ± SD. Injector stability for 12h

75 71.25 95 5.0 -5%

300 288 96 4.9 -4% individually. A plasma concentration time curve (AUC) of lixivaptan was plotted (Figure 4) using the lixivaptan 750 720 94 4.5 6.0% concentration determined at each time point. Lixivaptan pharmacokinetic parameters were estimated from the 3.3.3 Stability concentration time curve. After oral administration of 10 mg Kg-1 lixivaptan, the mean values of the pharmacokinetic

Drug stability was assessed under standard conditions. parameters Tmax and Cmax were determined to be 0.5 h and The concentrations calculated following the trials 113.82±28.1 ng mL-1, respectively. Determination of these varied only within ±10%. During optimization, sample parameters represents a demonstration of the utility of the processing, or bench study, no degradation of lixivaptan developed method in pharmacokinetic research. was observed (Table 5). In addition, no loss of lixivaptan concentration was recorded for a relatively short period of time or during refrigeration. The newly developed 4 Conclusion procedure for the assay of lixivaptan can be performed under ordinary research facility conditions with a high In the current study, we investigated, for the earliest degree of reproducibility without any recorded loss. time, a simple, sensitive, and selective method for the determination of lixivaptan in mouse plasma samples using HPLC separation and UV detection. Acetonitrile 3.4 Pharmacokinetic Study was used as protein precipitation solvent for extracting lixivaptan from mouse plasma, and the method was The investigated method was used to perform a validated using ICH guidelines. The method proved to be preliminary pharmacokinetic study of lixivaptan in mouse of acceptable accuracy, precision, recovery, selectivity, plasma. The concentrations of lixivaptan in plasma and sensitivity. The assay was further applied in a samples at different time points after dosing were assessed pharmacokinetic study of lixivaptan in mouse plasma, 620 Haitham Alrabiah et al.

which shows the applicability of the method for use in [11] Swan S., Lambvrecht L., Orczyk G., E. Ellis-Grosse E, clinical studies. Interaction between VPA-985, an ADH (V2) antagonist, and furosemide, J. Am. Soc. Nephrol., 1999, 10, 124A. [12] US Food and Drug Administration. Guidance for Industry: The authors declare no conflict of Conflict of interest: Bioanalytical Method Validation. US Department of Health interest. and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Acknowledgments: The authors extend their Medicine: Rockville, MD, 2013. appreciation to the Deanship of Scientific Research [13] González O., Blanco M.E., Iriarte G., Bartolomé L., Maguregui M.I., Alonso R.M., Bioanalytical chromatographic method at King Saud University for funding the work through validation according to current regulations, with a special focus research group project no. RGP-1436-024. on the non-well defined parameters limit of quantification, robustness and matrix effect, Journal of Chromatography A, 2014, 1353, 10-27. [14] Shabir G., Validation of HPLC methods for pharmaceutical References analysis: Understanding the differences and similarities between validation requirements of the US Food and Drug [1] Ku E., Nobakht N., Campese V.M., Lixivaptan: a novel Administration, the US Pharmacopoeia and the International antagonist, Expert opinion on Conference on Harmonization, J. Chromatogr. A, 2003, 987, investigational drugs, 2009, 18, 657-662. 57-66. [2] Zmily H.D, Alani A., Ghali J.K, Evaluation of lixivaptan in [15] Health U.D.O., Services H., Food and Drug Administration euvolemic and hypervolemic hyponatremia and Center for Drug Evaluation and Research Center for Veterinary treatment, Expert opinion on drug metabolism & toxicology, Medicine, Guidance for Industry, Bioanalytical Method 2013, 9, 645-655. Validation, 2001. [3] Liamis G., Filippatos T.D., Elisaf S., Treatment of hyponatremia: [16] ICH I., In Harmonised Tripartite Guideline, Validation of the role of lixivaptan, Expert review of clinical pharmacology, Analytical Procedures: Test and Methodology, International 2014, 7, 431-441. conference on harmonisation of technical requirements for [4] Ring T., Lixivaptan and hyponatremia, Kidney international, registration of pharmaceuticals for human use, 1996. 2013, 83, 1205- 1206. [17] Ermer J., Miller J. H. M., Method validation in pharmaceutical [5] Bowman B.T., Rosner M.H., Lixivaptan–an evidence-based analysis: A guide to best practice; John Wiley & Sons, 2006. review of its clinical potential in the treatment of hyponatremia, [18] Snyder L.R., Kirkland J.J., Glajch J.L., Practical HPLC method Core evidence, 2013, 8, 47. development, John Wiley & Sons, 2012. [6] O’Neil M.J., The Merck index: an encyclopedia of chemicals, [19] Snyder, L. R., Kirkland, J. J., Dolan., J.W.; Introduction to drugs, and biologicals, RSC Publishing, 2013. modern liquid chromatography; John Wiley & Sons: 2011. [7] Rusinaru D., Tribouilloy C., Berry C., Richards A.M., Whalley [20] Kadi A.A., Alrabiah H., Attwa M.W., Attia S., Mostafa G.A.E., G.A., Earle N., Poppe K.K., Guazzi M., Macin S.M., Komajda Development and validation of HPLC-MS/MS method for M., Relationship of serum sodium concentration to mortality the determination of lixivaptan in mouse plasma and in a wide spectrum of heart failure patients with preserved its application in a pharmacokinetic study, Biomedical and with reduced ejection fraction: an individual patient data Chromatography, 2017. meta‐analysis, European journal of heart failure, 2012, 10, [21] Bhat M.A., Al-Omar M.A., Ansari M.A., Zoheir K.M., Imam 1139-1146. F., Attia S.M., Bakheet S.A., Nadeem A., Korashy H.M., [8] Muralidharan G., Meng X., DeCleene S., evallos W.C, Fruncillo Voronkov A., Berishvili V., Ahmad S.F., Design and Synthesis Hicks D., Orczyk, G., Pharmacokinetics and Pharmacodynamics of N-Arylphthalimides as Inhibitors of Glucocorticoid-Induced of A Novel Vasopressin Receptor Antagonist, VPA‐985, in TNF Receptor-Related Protein, Proinflammatory Mediators, and Healthy Subjects, Clinical Pharmacology & Therapeutics, 1999, Cytokines in Carrageenan-Induced Lung Inflammation, J. Med 65, 189-198. Chem., 2015, 58 , 8850-67. [9] Ellis-Grosse E., Meng X., Orczyk G., Single dose [22] Miller J.N., Miller J.C., Statistics and chemometrics for pharmacokinetic (PK)-pharmacodynamic (PD) profile of analytical chemistry, Pearson Education, 2005. VPA-985, a novel, V2 receptor antagonist, in patients with congestive heart failure (CHF), AAPS Pharm. Sci., 1999, 1, S1. [10] Guyader D., Patat A., Ellis‐Grosse E.J., Orczyk G., Pharmacodynamic effects of a nonpeptide antidiuretic hormone V2 antagonist in cirrhotic patients with ascites Hepatology, 2002, 36, 1197-1205.