MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets

Accepted Manuscript Title: UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets Authors: Muzaffar Iqbal, Essam Ezzeldin, Nasr Y. Khalil, Prawez Alam, Khalid A. Al-Rashood PII: DOI: Reference:

S0731-7085(18)31375-X https://doi.org/10.1016/j.jpba.2018.10.005 PBA 12254

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

10-6-2018 29-9-2018 1-10-2018

Please cite this article as: Iqbal M, Ezzeldin E, Khalil NY, Alam P, Al-Rashood KA, UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquidliquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.10.005 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.

UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets Muzaffar Iqbal

a,b*,

Essam Ezzeldin

a,b,

Nasr Y. Khalila, Prawez Alamc, Khalid A. Al-

Rashooda Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh,

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a

11451, Saudi Arabia b

Bioavailability Laboratory, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi

Arabia

Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University,

Al-Kharj 11942, Kingdom of Saudi Arabia

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*Author to whom correspondence should be addressed

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c

Dr. Muzaffar Iqbal,

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Associate Professor, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, KSA. 11451, PO BOX No. 2457

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Fax: +966-14676220

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Tel: +966-14697565, Mobile: +966535667290 E-Mail: [email protected]; [email protected]

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Graphical Abstract

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HIGHLIGHTS

A highly sensitive UPLC-MS/MS assay was developed for analysis of suvorexant in urine sample A simplified DLLME-SFO-UABE procedure was used for sample preparation SWGTOX guideline was followed for assay validation

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First report of DLLME-SFO-UABE application in biological fluid

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Abstract

Suvorexant is a novel sedative/hypnotic drug approved for treatment of insomnia. It has significant

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forensic importance due to its hypnotic and depressant effects on central nervous system. In this study, a highly sensitive UPLC-MS/MS assay was developed and validated for the determination

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of suvorexant in urine sample. A simplified dispersive liquid-liquid microextraction followed by ultrasound assisted back extraction from solidified floating organic droplets was employed for

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sample preparation. The 20 µL of 1-undecanol and 200 µL of acetonitrile were used as extraction solvent and dispersive solvent, respectively. An ultrasound assisted back extraction step was employed to enable the cleanup procedure compatible with mass spectrometric detection. Acquity CSHTM C18 column with mobile phase composition of 15 mM ammonium acetate: acetonitrile:

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formic acid (15:85:0.1%; v/v/v) were used for chromatographic separation. The multiple reaction monitoring transition of 451.12 →104.01 and 451.12→186.04 were used for identification and quantification of suvorexant, respectively, whereas 237.06→194.1 was used for IS in mode. The

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assay demonstrated good linearity in the range of 0.27-1000 ng mL-1 with limit of detection (LOD) and quantification (LOQ) of 0.10 and 0.27 ng mL-1, respectively. Assay validation was performed by following SWGTOX guidelines and all validation results were found to be within acceptable limits. This is the first report of dispersive liquid-liquid microextraction based on solidification of floating organic droplets employed to UPLC-MS/MS for application in biological fluids.

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Keywords: Suvorexant; UPLC-MS/MS; DLLME-SFO; UABE; SWGTOX; Urine

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1. Introduction

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Sedative/hypnotic drugs are of forensic importance and potential for misuse due to their widespread use and ability to produce additive effects with other central nervous system

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depressants [1]. Suvorexant is a novel class of sedative/hypnotic drug therapeutically used to

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induce sleep in patients with insomnia. It is highly selective and potent dual orexin receptor (OX1R and OX2R) antagonist which produces rapid onset of sleep by inhibiting the wakefulness-

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promoting orexin neurons of the arousal system [2,3]. It induce both rapid eye movement (REM)

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and non-REM sleep and also preserve the cognitive performance plus ability to arouse to salient stimuli [4]. Suvorexant is sparingly soluble in water (0.117 mg mL-1) and has LogP value of 4.04

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[5]. Suvorexant is well absorbed with maximum plasma concentration usually achieved within 2 h after oral administration. It has high oral bioavailability (82 %), extensively bind to plasma protein (99 %), and has high volume of distribution (Vd) of 49 Lkg-1 [6-8]. It was safe and well tolerated after single and multiple dose administration which support the once-nightly dosing regimen [9]. Like other sedative/hypnotics, suvorexant has abuse potential and was listed in 3

Schedule IV of the Federal Controlled Substances Act shortly after approval [10]. Recently, Suvorexant was successfully detected and quantified in the real postmortem specimens of three separate autopsy cases suggesting that this compound will be encountered more often by the

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forensic toxicology community [11]. Due to its forensic importance, illegal use of suvorexant is expected and therefore a sensitive assay is required for their detection and quantification in biological samples. Among the biological matrices, urine is the most preferred specimen for forensic analysis due to its simple and non-invasive procedure of collection [12]. Previously, suvorexant has been quantified in urine specimen, but without any application in real samples by

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GC-MS [12] and LC-Q/TOF-MS [13] with lower limit of quantification (LOQ) of ≥5 ng mL-1. In

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addition, detection of suvorexant was also reported in plasma samples by UPLC-MS/MS in our

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laboratory [14] and LC-MS/MS by Breidinger et al, 2015 [15]. Since suvorexant is mainly

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eliminated by metabolism and only 23 % of radiolabeled dose is recovered in urine, a highly

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sensitive assay is required for the quantification of parent drug in urine sample.

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Owing to the presence of trace level of suvorexant in urine and complexity of biological matrices, sample extraction and pre-concentration steps are crucial to improving sensitivity as well

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as selectivity of the bioanalytical methods and complexity of biological matrices. Dispersive liquid liquid microextraction integrated with solidification of floating organic droplets ((DLLME-SFO)

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is an advanced technique of liquid phase microextraction (LPME) first developed by Leong and

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co-workers in 2008 [16]. Compared to DLLME procedure, DLLME-SFO is more environment friendly approach in which trace amount of less toxic extraction solvents (e.g. 1-undecalol, 1dodecanol) is dispersed (with the aid of small amount of disperser solvent) into the sample. After dispersion, the samples become cloudy and the analyte is transferred to the organic solvents, which on centrifugation floats at the top of the extraction tube. The organic droplets use to solidify in ice

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bath and is immediately transfer into a suitable vial and become melted at room temperature; then it is finally injected for analysis. Despite its successful combination with many chromatographic techniques, DLLME-SFO procedure is widely applied for trace analysis of multiclass pollutants

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(pesticides, plasticizers, pharmaceuticals and personal care products) in environmental water samples [17] and rarely used for the analysis of drugs in biological matrixes e.g. plasma [18-21] and urine [21-23]. Limited application of DLLME-SFO procedure in biological fluids might be due to its restricted analysis by HPLC only by using ultraviolet (UV), photodiode array (PDA) and Florescence (FL) detectors. Due to high sensitivity and selectivity, HPLC-MS/MS is one of the

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most preferred techniques for bioanalytical application. However, there are no reports about the

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application of DLLME-SFO for HPLC-MS/MS analysis. The incompatibility of the final organic

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phase (extractive reagent) with the mass spectrometric-based detection system is a main drawback

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for its limited application in HPLC-MS/MS analysis. In order to overcome this issue, recently Canales et al, proposed a novel alternative procedure in which DLLME-SFO is followed by

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ultrasound assisted back extraction (UABE) prior to LC- MS/MS analysis for determination of

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non-organic aromatic amines in natural water samples [24]. By following UABE step, MS

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compatible organic solvents e.g. acetonitrile, methanol or mobile phase can be used to facilitate the UPLC-MS/MS analysis. In this study, a highly sensitive UPLC-MS/MS assay coupled with

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DLLME-SFO-UABE procedure was developed for the determination of suvorexant in human urine sample. To the best of our knowledge, DLLME-SFO-UABE has not yet been applied in

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biological fluids and this is first report about the bioanalytical application of DLLME-SFO procedure in UPLC-MS/MS analysis. Since the assay was developed for application in forensic toxicology, the validation was performed by following the “Scientific Working Group for Toxicology” (SWGTOX) guidelines [25].

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2. Experiments 2.1. Chemicals and reagents Suvorexant with percentage purity of ≥ 99% was obtained from “Beijing Mesochem

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Technology Co. Ltd.” Beijing, China. Carbamazepine (purity, 99%) used as internal standard (IS), was obtained as gratis sample from “Tabuk Pharmaceutical” Tabuk, Saudi Arabia. HPLC grade dimethyl sulphoxide and acetonitrile were obtained from “VWR International Ltd.” Poole, England. AR grade ammonium acetate and formic acid were obtained from Qualikems Fine Chemical Private. Ltd. (Vadodara, India) and Loba Private. Ltd. (Mumbai, India), respectively.

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Acetate buffer of pH 4.6 and 1-undecanol (99%) were purchased from Sigma Aldrich (St. Louis,

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MO, USA). 1-dodecanol (98 %) was from Acros Organic (New Jersey, USA). Milli-QR grade

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ultrapure water (pore size of 0.22µm) was used for aqueous samples preparation. Drug-free blank

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human urine sample was obtained from healthy volunteer and was stored in refrigerator whereas

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the blank rat urine was collected from healthy rats after housing them in separate metabolic cages

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2.2. Preparation of stock solution, calibration standard (CS) and quality control (QC) samples The stock solution of suvorexant (100 µg mL-1) was prepared in dimethyl sulfoxide (DMSO)

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using an accurately weighed amount of a reference standard of the drug. It was further diluted by 50% acetonitrile in water to achieve working standards of CSs ranging between 5.44- 20000 ng

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mL-1. The working standard solution was further fortified into a drug free urine sample to achieve

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CS in urine in the range of 0.27- 1000 ng mL-1. The same procedure was followed to prepare the QC samples of 0.90, 45 and 900 ng mL-1 concentration by using separate stock solution of suvorexant and were treated as LQC, MQC and HQC, respectively. Stock solution of IS (160 µg mL-1) was also prepared in methanol and was diluted with 50% of acetonitrile to achieve working solution of 400 ng mL-1. All working solutions were stored in pharmaceutical refrigerator 6

maintained at 4 ±2 ° C, whereas fortified urine CS and QC were stored in Deep Freezer maintained at ─ 80 ±5 ° C. 2.3. Mass spectrometry instrumentation and chromatographic conditions

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The mass spectrometry analysis was performed on UPLC-MS/MS instrument comprising of Acquity H-Class UPLC system coupled to Acquity TQD detector “Waters Corp., Milford, MA, USA.” Sample ionization was performed by electrospray ionization (ESI) which was operated in positive mode. Detection and quantification was performed in multiple reaction monitoring (MRM) mode. Separate MRM transitions were used for qualification and quantification of the

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analyte. The MRM transition of 451.12 →104.01 and 451.12 →186.04 were used for identification

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and quantification of suvorexant, respectively, whereas 237.06→ 194.1 was used for IS. The ratio

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of quantifiers to qualifiers ions were limited to be within 20 % in QC samples. The optimized

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MS/MS parameters were: capillary voltage, 3.10 kV; source temperature, 150 ° C; desolvation temperature, 350° C; desolvation gas (nitrogen) flow 600 L h-1, collision gas flow, 0.15 mL min-1.

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The compound specific parameters like cone voltage for suvorexant and IS were 34 and 32 V,

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respectively.

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respectively whereas collision energy was 68 eV (qualifier); 20 eV (quantifier); 24 (IS),

An Acquity UPLC In-Line Filter (contains 0.2 μm stainless steel filter disc) coupled to

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UPLCCSHTM (100 x 2.1 mm) C18 column was used for chromatographic separation by using mobile phase composition of 15 mM ammonium acetate: acetonitrile: formic acid (15:85:0.1%;

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v/v/v) eluted at 0.225 mL min-1 flow rate in isocratic mode. The optimized column heater temperature was 40° C whereas sample manager temperature was maintained at 10 ° C. MassLynx software (Version 4.1) was used for operating MS/MS system and collected data was processed by the Target Lynx TM program.

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2.4.

Sample extraction by DLLME-SFO-UABE procedure For extraction of the analyte from urine, an aliquot of 250 µL of CS and QC samples were

transferred into 2 mL capacity Eppendorf tubes. Then 25 µL of IS was added to all tubes except

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the blank. Samples were vortexed for 30 sec and 1 mL of cold acetate buffer (pH 4.6) was added and again vortex-mixed. Then 200 µL of acetonitrile (dispersive solvent) containing 20 µL of 1undecanol (extraction solvent) was added which resulted in formation of a cloudy mixture due to dispersion of fine droplets of 1-undecanol. Consequently, the mixtures were again vortex mixed which resulted in quantitative extraction of the analyte and IS into fine droplets of 1-undecanol.

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This cloudy mixture was cold centrifuged at 10,000 rpm for 8 min maintained at 3 °C which results

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in a floating solidified droplet on the surface in form of disc shape pellets (Fig.1). These pellets

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were carefully transferred into 1.5 mL of Eppendorf tubes by the help of curved spatula. All pellets

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were instantaneously melted and 150 µL of acetonitrile was added into each tube. The samples were vortex-mixed and placed in an ultrasonic bath for 30 min maintained at 40 °C for back

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extraction. The final extract was centrifuged at 4500 rpm at 8 °C for 4 min, which resulted in

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solidification of 1-undecanol at the bottom of the tube this time. Then 100 µL of supernatant was

2.5.

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transferred into UPLC insert vial and 5 µL was injected for UPLC-MS/MS analysis. Evaluation of sample extraction efficiency

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The enrichment factor (EF) and extraction efficiency (ER) were determined to evaluate the extraction efficiency of the proposed sample extraction procedure. The EF was defined as the ratio

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of the analyte concentration in the floated phase (Cf) to the analyte concentration in the aqueous samples (C0): EF = Cf / C0

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The percentage extraction recovery (ER%) was expressed as the percentage amount of the total analyte extracted into the floated phase: ER% = EF X (Vf / V0) X 100

2.6.

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Where Vf and V0 are the volume of the floating phase and the sample solution, respectively. Method Validation

The method validation was accomplished following SWGTOX guidelines for forensic laboratory in human urine [21]. A partial validation in term of selectivity, linearity, accuracy precision and matrix effects were also evaluated in rat urine to enable the application in real sample

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analysis in rats. The limit of detection (LOD) and LOQ were identified by replicate measurements

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of five blank human urine samples by spiking at decreasing concentration of the analyte. LOD and

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LOQ were defined as the concentration of the analyte which signal to noise (S/N) ratio was ≥3 and

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≥10, respectively. Moreover, for LOQ the variation in precision and bias must be limited to within 20 %. The calibration curves (CCs) at nine different concentration (0.27, 0.91, 3.02, 10.08, 33.6,

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112, 280, 700 and 1000 ng/mL) in human and rat urine were prepared by plotting the peak area

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ratio of the analyte to that of the IS versus nominal concentration of the analyte. Assay linearity

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was evaluated by duplicate processing of the CCs using weighted least squares regression method. Curves were best fitted using y = mx+ b, where y is the peak area ratio, m is slope, b is the y-axis

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intercept of the CCs and x is the analyte concentration. Five different urine CCs were processed and analyzed and concentration of each calibrator were back-calculated from these curves to

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determine the concentration of suvorexant and the resulting calculated parameters were used to determine concentrations of analyte in quality control samples. The coefficient of determination (R2) was limited to 0.99 for all the CCs. Three weighing factors none, 1/x and 1/x2 were used and the accuracy (% nominal) at each calibrator of the CCs was back calculated. Ten different blank

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urine samples were used to determine the assay selectivity in human and rat urine. The samples showing responses below the LOD was considered as selective and absence of any interference form endogenous substance from urine. The selectivity of the assay towards some commonly

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prescribed medicines like atorvastatin, metformin, valsartan, risperidone and celecoxib were also analysed after sample extraction by fortifying at their 1 µg/mL concentration in human urine. Carry-over effect was also evaluated by injecting the processed blank urine sample just after the highest calibrator of CC in triplicate. Intra- and inter-day precision and bias were determined in human and rat urine by using LOQ and all the three QC samples in four replicates. The coefficient

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of variation (%, CV) was used to determine the precision whereas percentage deviation from

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nominal concentration was the bias of the assay.

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The matrix effects (ME), which is one of the main drawbacks of MS/MS detection was

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determined in human and rat urine by post extraction addition approach. The low and high QC samples of neat standard solution were injected six times and their average responses were

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compared with the average responses of 10 urine samples which obtained from different individual

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and rats and were fortified with low and high QC samples. 𝐴𝑀 −𝐴𝑆 𝐴𝑆

× 100

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% ME =

Where AM is the average peak area response of the analyte in extracted urine and AS is the

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average peak response in neat standards.

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The stability of suvorexant in urine at different anticipated storage condition were evaluated

by using LQC and HQC samples in three replicates. The bench top, freeze thaw, autosampler and long-term stability were evaluated and analyzed against freshly prepared CCs.

3. Results and discussion

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The extraction efficiency of suvorexant from the proposed DLLME-SFO-UABE procedure is conditioned by its mass transfer from matrix to extraction followed by BE steps and depend upon the type and volume of extraction solvent and dispersing agent, time and rate of centrifugation and

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vortexing, type and volume of BE solvents, ultrasonic time and temperature. Therefore, the experimental conditions of these variables were carefully evaluated to achieve maximum recovery/enrichment of the analyte in a short time frame. 3.1. Optimization of DLLME-SFO conditions 3.1.1. Effect of type and volume of extraction solvent

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The selection of an appropriate extraction solvent is a crucial step in DLLME-SFO procedure.

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An ideal solvent must have low volatility, lower density than water, low solubility in water and

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can easily solidify at low temperature. Considering the aforementioned requirements, two

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extraction solvent (1-undecanol, 1-dodecanol) were investigated in this study. The experiment was performed by using 20 µL of each extractant containing 200 µL of acetonitrile in triplicates (n=3).

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According to the results, 1-undecanol produced maximum % ER and was selected as the extraction

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solvent for further steps. Moreover, the droplet shape produced by 1-undecanol was more suitable

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from solidification point of view than 1-dodecanol. To study the effect of extractant volume on the extraction efficiency, five different volumes

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of 1-undecanol (10, 15, 20, 25, 30 µL) containing fixed volumes of acetonitrile (200 µL) were evaluated to get suitable volume for further experiments in triplicates (n=3). It was observed that

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the % ER was decreased with increasing the volume of extractant due to dilution effects [19], suggesting low volume for better recovery (Fig. 2). However, it was difficult to handle the sediment volume less than 20 µL. Therefore, 20 µL of 1-undecanol was selected as an optimal extraction solvent volume.

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3.1.2. Effect of type and volume of dispersive solvent Like conventional DLLME, the type and volume of dispersive solvent affect the efficiency of the extraction procedure. An ideal dispersive solvent must have property to be miscible in both

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aqueous samples and extraction solvent. In this study, acetone, acetonitrile and methanol were tested using 300 µL of each solvent containing 20 µL of 1-undecanol in triplicate (n=3). Among them, acetonitrile was considered as the most suitable dispersive solvent because it produced very fine droplets in cloudy state with high recovery.

Further experiment was performed to optimize the different volume of acetonitrile (100, 200,

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200, and 400 L) containing 20 µL of 1-undecanol in triplicates (n=3). It was observed that the

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variation in volume also resulted in change in the volume of the floated phase which also affected

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the extraction efficiency. As illustrated in Fig. 3, by increasing the volume of acetonitrile the

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solubility of the analyte in the aqueous phase increased, which resulted in decrease in % ER. Moreover, the cloudy state was not formed satisfactory with 100 µL, thereby 200 µL of acetonitrile

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was selected as the dispersive solvent for further study.

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3.2. Optimization of UABE conditions

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To avoid direct injection of highly viscous melted organic droplets in UPLC-MS/MS, initially sample drying followed by reconstitution step was followed. But it was taken too much time in

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drying due to highly viscous nature of organic droplets. Then UABE step (direct transfer of analyte form enriched floating organic droplet to solvent compatible with MS/MS detection) was used.

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This strategy made feasible the coupling of DLLME-SFO with UPLC-MS/MS detection. During BE experimental condition optimization, type of BE solvent and use of organic modifiers, effects of temperature and time of ultrasonic bath were evaluated. 3.2.1. Effect of type and volume of BE solvent

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An ideal BE solvent should be compatible with chromatographic separation and good extraction capability for the compound of interest. Considering this, pure acetonitrile, 0.1% formic acid in acetonitrile and the mobile phase were evaluated. Among them, the BE efficiency was

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highest with pure acetonitrile and was chosen as BE solvent. The low BE efficiency with 0.1 % formic acid in water or mobile phase might be due to its ion suppressing effects. Further different volumes of acetonitrile (100, 150, 200, 250 and 300 µL) were evaluated. It was observed that the BE efficiency was gradually decreased after 150 µL volume. As illustrated in Fig. 4, 150 µL of the acetonitrile was selected for UABE procedure.

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3.2.2. Time and temperature of ultrasonic bath for BE

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In order to provide optimum ultrasonic assisted quantitative contact mixing between melted

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SFO and BE solvent, exposure of different ultrasonication time (10, 20, 30 and 40 min) on BE

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efficiency were evaluated. The time of ultrasonication was fixed at 30 min, as poor recovery was observed once ultrasonication time was decreased. The temperature of ultrasonic bath was fixed

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at 40 °C to avoid evaporation of BE solvent.

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3.3. Optimization of mass spectrometry and chromatographic conditions

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Mass spectrometry parameters were optimized by infusing of 400 ng mL-1 solution of suvorexant and IS at a flow rate of 20 μLmin-1. Initial tuning was performed by direct infusion followed by

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combine flow with mobile phase solution. The first two dominant precursors to product ion transitions of suvorexant were found at m/z of 451.12 →186.04 and 451.12 →104.01 which is

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same as our previous report [14]. The optimized precursor to product ion transitions of IS was m/z of 237.06→ 194.1. In previous study, we used revaroxaban as IS, but due to its poor recovery with DLLME-SFOUABE procedure, carbamazepine was considered for IS in this study. Addition of 0.1 % formic

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acid in previous mobile phase condition produced better signal intensity and ionization efficiency for both analyte and IS and therefore considered for chromatographic separation. Similarly, UPLCCSHTM column produced better symmetrical peaks compared to Acquity BEH column which was

3.4.

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used in previous study. Method validation

All CCs which were prepared by fortifying the suvorexant in blank human and rat urine were found to be linear with coefficient of determination ranged between 0.992-0.997. The weighing factor by using 1/x2 was weighted to obtain the lowest total bias to correlate the analyte

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concentrations with detector response, and was used for method validation. The mean regression

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parameters ±RSD were represented by equation y=0.0471(±0.00716) x + 0.0171± (0.0047) in

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human urine and y=0.0418±(0.00216) x + 0.0401(±0.010) in rat urine. Furthermore, the back

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calculated concentration for each calibrator was found to be within the acceptable limit of accuracy (±15 %) and precision (≤ 15 %, RSD), except for lowest calibrator (LOQ), which was within ±20

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% and ≤ 20 %, %RSD. Suvorexant was detected and quantified down to 0.1 ng mL-1 and 0.27 ng

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mL-1, which were considered as LOD and LOQ of the assay, respectively. The precision and bias

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data of human and rat plasma were obtained from analysis of PA batches of LOQ and three QC samples (LQC, MQC and HQC) on the same day and five consecutive days are presented in Table

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1. According to results, the intra and inter-day precision didnot exceed 12.08 % and 11.0 % (RSD, %) in human and rat urine, respectively. The percentage bias was ranged from ‒7.52 to 14.43 %

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and ‒8.15 to 12.0 % in human and rat urine, respectively. As per SWGTOX guidelines’ these data are within the acceptable limits, therefore the method is accurate and precise. Moreover, no significant interfering peaks were obtained at the retention time of analyte and IS, which confirmed that the assay is selective and specific and free from interference from endogenous substances and

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matrices. Similarly, no significant peak was detected in chromatograms of analyte and IS samples fortified with five commonly prescribed drugs. The retention time of suvorexant and IS were 1.46 ± 0.02 and 1.11± 0.02 min, respectively. Representative MRM chromatogram of suvorexant and

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IS in blank urine and urine fortified with LOQ concentration are shown in Fig. 5. During carryover effects evaluation, no significant interferences were observed in blank human urine samples which were injected just after the highest calibrator. Therefore, no carry-over effects would be expected during routine analysis by proposed assay. The matrix effects in form of ions suppression effects were observed for suvorexant and IS in both human and rat urine sample, however it was

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within the acceptable range of ±25 % with ±15 % RSD. This result confirmed that the matrix

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effects is negligible and it did not affect the analytical performance by applying DLLME-SFO-

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UABE extraction procedure.

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The stability of suvorexant in urine sample evaluated at different storage conditions were also found to be within the acceptable limit of ±15 % as per SWGTOX guideline. The results confirmed

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that suvorexant is stable up to 24 h at room temperature; remained intact up to 1 month at ─ 80 ±

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freeze-thaw cycles.

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5° C in deep freezer, upto 24 h in sample manager and no degradation was observed after three

3.5. Comparison of the proposed method of suvorexant with previously reported methods

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The results of the proposed method with other reported extraction methods with reference to sensitivity, amount of organic solvent consumed, calibration range, and run-time are presented in

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Table 2. As can be seen, all previously reported assays were based on liquid-liquid extraction (LLE) method which consumed high amount (≥1.2 mL) of toxic organic solvent (like TBME, diethyl ether and toluene). However, the proposed DLLME-SFO-UABE need only small amount of organic solvent (350 µL acetonitrile and 20 µL 1-undecanol) without sample evaporation step.

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Therefore, the proposed sample extraction procedure is more environment friendly than previously reported assays. Moreover, the LOD and LOQ (sensitivity) of the proposed assay is lower with large calibration range (0.27-1000) and shorter run-time than all the previous reported assays in

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urine/plasma. 3.6. Comparison of the proposed DLLE-SFO-UABE method with previously reported DLLE--SFO Due to small sample volume, all extraction steps were performed in disposable eppendorf tubes. Moreover, no further step for solidification in ice bath was required due to sue of cooling centrifuge. Therefore, this DLLE-SFO procedure is simpler than previously reported DLLE-SFO

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assays.

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3.7. Application in real samples

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Since suvorexant is not clinically approved in Saudi Arabia and also due to ethical issue

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pertaining to use of drug of abuse in human, method application in human subjects could not be taken up. However, this newly developed assay was applied for quantification of suvorexant in

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experimental rats. Wistar Albino male rats (n = 3) weighing between 190-210 gm were housed in

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separate metabolic cages after receiving a single oral dose of suvorexant at 1 mg/kg suspension

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[14]. All animals were kept overnight fasting before the drug administration. All rats were accessed both food and water while they were housed in metabolic cages. The study was carried out

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according to “guidelines of the animal care and use committee at King Saud university.” The urine samples were collected at different time intervals (0–2, 2–6, 6–12 and 12–24 h) post dosing.

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Suvorexant was successfully detected and quantified up to 24 h in all experimental rats. Representative MRM chromatogram of suvorexant and IS between 6-12 h after administration of 1 mg/kg suspension are shown in Fig. 5

4. Conclusions 16

In this study, a highly sensitive DLLME-SFO-UABE coupled with UPLC-MS/MS method was developed for determination of suvorexant in urine samples. Due to the forensic importance of suvorexant, the validation was accomplished by following SWGTOX guidelines. In spite of

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using low matrix volume, this assay offers high sensitivity with LOD and LOQ of 0.1 ng mL-1 and 0.27 ng mL-1, respectively. Comparing to previously reported assays of suvorexant, this DLLMESFO-UABE is more environment friendly since only small amounts of organic solvents were used without sample evaporation step. The DLLME-SFO procedure was simplified by performing all extraction steps in disposable eppendorf tubes and by using cooling centrifuge, no further step for

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solidification in ice bath was required. Addition of UABE step in DLLME-SFO enables this assay

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feasible to UPLC-MS/MS analysis. This is the first report of DLLME-SFO procedure combined

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with UPLC-MS/MS for application in biological fluids.

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Acknowledgements

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The authors extend their appreciation to the Deanship of Scientific Research at King Saud

References

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University for funding the work through the research group project no. RGP-203.

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Figure legends

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Toxicol., 2013, 37, 452–474.

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Fig.1 Solidified disc shape extraction solvent droplets floating on the surface after cold centrifugation Fig.2 Effect of volume of 1-undecanol (extraction solvent) on the extraction recovery of suvorexant by DLLME-SFO procedure Fig.3 Effect of volume of acetonitrile (dispersive solvent) on the extraction recovery of suvorexant by DLLME-SFO procedure Fig.4 Effect of volume of acetonitrile (BE solvent) on the extraction recovery of suvorexant by DLLME-SFO-UABE procedure Fig.5 Representative MRM chromatogram of suvorexant (qualifier and quantifier) and IS in (a) blank human urine (b) urine fortified with LOQ concentration and (c) between 6-12 h after administration of 1 mg/kg suspension of suvorexant in rats

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Table 1 Precision and accuracy (bias) data of suvorexant fortified in urine sample Human urine Nominal Conc. (ng/mL ± SD)

Bias (%)

Measured conc. ( ng mL-1 ±SD)

Precision (% RSD)

Bias (%)

Intra-day variation ( n=4) 0.27 0.31 ± 0.03 0.90 0.95 ± 0.07 45 47.12 ± 2.26 900 884.8 ± 56.82

9.93 7.36 4.79 6.42

14.43 5.62 4.70 ─1.69

0.30 ±0.02 0.97±0.06 44.34 ±4.67 826.67±70.09

8.26 5.85 10.53 8.48

12.0 7.90 ─1.46 ─8.15

Inter-day variation (n=20) 0.27 0.28 ± 0.04 0.90 0.98 ± 0.07 45 45.61 ± 3.73 900 832.3 ± 65.55

12.08 7.17 8.19 7.88

7.41 8.35 1.34 ─7.52

0.29± 0.03 0.98±0.06 43.87 ±4.82 847.78 ±75.72

10.27 6.52 11.0 8.93

7.47 9.07 ─2.51 ─5.81

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Measured conc. (ng mL-1 ±SD)

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Table 2 Comparison of the proposed method with previously reported methods Extraction Instrumentation Matrix Organic LOD LOQ Calibration Method solvent (ngmL- (ngmL- range(ng/mL 1 1 (Solvent) (mL) ) ) ) LLE ( GC/MS Urine 5 10 10 10-1000 ether/toluene) LLE (diehtyl UPLC-MS/MS Plasma 2 0.1 0.33 0.33-200 ether) LLE (ter butyl HPLC-MS/MS Plasma 1.2 1 1-1000 methyl ether) LLE ( LC-Q/TOF-MS Urine 2.5 0.5 5 5-250 ether/toluene) DLLME-SFO- UPLC-MS/MS Urine 0.37* 0.1 0.27 0.27-1000 UABE (1undecanol)

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*0.35ml acetonitrile and 0.02 ml 1-undecanol

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Run Time ( min) 14

Reference

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[12]

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[13]

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[10]

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This work