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
Graphical abstract
Introduction
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 depressants [1]. Suvorexant is a novel class of sedative/hypnotic drug therapeutically used to 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-promoting orexin neurons of the arousal system [2,3]. It induce both rapid eye movement (REM) 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 [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], [7], [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 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 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 GC–MS [12] and LC-Q/TOF-MS [13] with lower limit of quantification (LOQ) of ≥5 ng mL-1. In addition, detection of suvorexant was also reported in plasma samples by UPLC-MS/MS in our laboratory [14] and LC–MS/MS by Breidinger et al, 2015 [15]. Since suvorexant is mainly eliminated by metabolism and only 23% of radiolabeled dose is recovered in urine, a highly sensitive assay is required for the quantification of parent drug in urine sample.
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 as selectivity of the bioanalytical methods and complexity of biological matrices. Dispersive liquid liquid microextraction integrated with solidification of floating organic droplets ((DLLME-SFO) is an advanced technique of liquid phase microextraction (LPME) first developed by Leong and 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, 1-dodecanol) 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 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 (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], [19], [20], [21]] and urine [[21], [22], [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 most preferred techniques for bioanalytical application. However, there are no reports about the application of DLLME-SFO for HPLC-MS/MS analysis. The incompatibility of the final organic phase (extractive reagent) with the mass spectrometric-based detection system is a main drawback 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 ultrasound assisted back extraction (UABE) prior to LCMS/MS analysis for determination of non-organic aromatic amines in natural water samples [24]. By following UABE step, MS 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 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 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].
Section snippets
Chemicals and reagents
Suvorexant with percentage purity of ≥ 99% was obtained from “Beijing Mesochem 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.
Results and discussion
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 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
Conclusions
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 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 DLLME-SFO-UABE is more environment friendly since
Acknowledgement
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group project no. RGP-203.
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