Accepted Manuscript Title: Solid-phase dispersive extraction method for analysis of benzodiazepine drugs in serum and urine samples Author: Koichi Saito Yuu Kikuchi Rieko Saito PII: DOI: Reference:
S0731-7085(14)00353-7 http://dx.doi.org/doi:10.1016/j.jpba.2014.07.020 PBA 9656
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
10-5-2014 17-7-2014 19-7-2014
Please cite this article as: K. Saito, Y. Kikuchi, R. Saito, Solid-phase dispersive extraction method for analysis of benzodiazepine drugs in serum and urine samples, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.07.020 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.
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Solid-phase dispersive extraction method for analysis of benzodiazepine drugs in serum and
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urine samples
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Koichi Saito*, Yuu Kikuchi, Rieko Saito
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Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University,
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2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
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* Corresponding author. Tel.: +81 3 5498 5764, Fax: +81 3 5498 5764
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E-mail address:
[email protected] (Koichi Saito)
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Abstract A simple yet highly efficient pretreatment method called solid-phase dispersive extraction (SPDE) was developed and used in combination with liquid chromatography/time-of-flight
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mass spectrometry (LC/TOF-MS) for the analysis of benzodiazepines (BZPs) in serum and
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urine samples. By using a custom-made centrifugal filter, SPDE could be performed in a closed
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system, thereby minimizing exposure to infectious microbes or hazardous chemicals. The limit
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of detection and the limit of quantification of nine BZPs were 1–10 and 5–50 ng/mL,
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respectively. The average recoveries of BZPs from pooled serum samples spiked at 50 and 500
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ng/mL were 89.6–105.0% (RSD: 2.1–6.8%) and 93.6–110.4% (RSD: 2.1–4.2%), respectively,
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and those from urine samples were 88.7–105.5% (RSD: 2.9–6.4%) and 91.5–101.1% (RSD:
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3.6–5.5%), respectively. SPDE-LC/TOF-MS has potential application in forensic science and
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emergency medicine.
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Keywords: Solid-phase dispersive extraction; Benzodiazepine; LC/TOF-MS; Abused
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prescription drug; Forensic science; Emergency medicine
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1. Introduction
The abuse of prescription drugs that act on the central nervous system is one of the most
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serious social problems in the world. Among those drugs, benzodiazepines (BZPs) have largely
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replaced barbiturates as anxiolytic and hypnotic drugs [1]. As the number of people who misuse
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those drugs for recreational purposes has increased in recent years, there is an urgent need to
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develop a highly reliable analytical method that can be used in forensic science and emergency
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medicine. However, as various impurities are present in biological specimens, a pretreatment
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method that can efficiently extract, concentrate, and purify an extremely small amount of the
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target compound is critical for analyzing biological specimens. Several approaches, such as solid-phase extraction (SPE) [2,3], solid-phase microextraction,
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[4,5], stir bar sorptive extraction [6,7], and liquid-phase microextraction [8,9], have been used
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so far. Dispersive solid-phase extraction (DSPE) was used for the clean-up of crude extracts
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from different sample matrices [10]. However, the extracts still contained some matrix
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contaminants, because DSPE could not selectively purify the target compound. Furthermore,
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DSPE could not be used for sample concentration. Dispersive liquid–liquid microextraction
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(DLLME) [11] features a high preconcentration capability, a short extraction time, and low cost,
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but lacks selectivity for different analytes. In addition, a dispersion may not form during the
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analysis of biological specimens [12].
We have developed a novel extraction method called the solid-phase dispersive extraction
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(SPDE) method [13], in which microparticles (solid phase) are dispersed in a liquid sample, and
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have used it for the determination of vancomycin in serum samples [14]. After the dispersion of
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the microparticles (solid phase) in a liquid sample (liquid phase), equilibrium between the two
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phases is immediately reached. In addition, various microparticles can be used in SPDE, similar
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to SPE; thus, SPDE has high selectivity, in contrast with DLLME and DSPE.
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In this study, we evaluated the extraction efficiency of SPDE when used in combination with
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liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) for the analysis of BZPs
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in serum and urine samples. Our findings suggest the benefits of SPDE for therapeutic drug
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monitoring in emergency medicine as well as for the appraisal of abused drugs in forensic
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science.
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2. Experimental
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2.1. Materials and Reagents
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Bromazepam, nitrazepam, lorazepam, alprazolam, triazolam, flunitrazepam, etizolam, and
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diazepam standards were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
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Erimin® tablet (5 mg), which was used as the nimetazepam standard, was obtained from
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Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). Desalkylflurazepam, which was used as
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the surrogate for TOF-MS measurements, was purchased from Cerilliant Corporation (Round
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Rock, TX, USA). Escherichia coli β-glucuronidase for deconjugation was purchased from
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Sigma-Aldrich Corp. (St. Louis, MO, USA). Reversed-phase (RP) polymeric Oasis HLB®
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solid-phase gel (30 μm O.D.) was obtained by removing the gel from the corresponding Oasis
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HLB® series SPE cartridges (Waters Corporation, Milford, MA, USA). Before use, the
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solid-phase gel was first conditioned with methanol and purified water. Then, a turbid aqueous
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solution of the solid-phase gel was prepared at the concentration of 5 mg/50 μL. The centrifugal
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filter prototype was custom-made by Frontier Science Co. (Hokkaido, Japan). The filter paper
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used was No. 5B of Japanese Industrial Standards P3801.
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2.2. Serum and Urine Samples
The lyophilized serum product, Consera® (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan),
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was used as a blank for the serum tests. Urine sample from a healthy non-dosed volunteer was
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used as a blank for the urine tests. Blood and urine samples were collected from a patient
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receiving clinical doses of etizolam. This study was performed in accordance with the ethical
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standards of the Declaration of Helsinki. The sample donors were recruited on a voluntary basis
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and informed about the study objectives. The samples were kept at −20 °C until analysis.
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2.3. Basic SPDE Method
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An aqueous suspension (50 μL) of Oasis® HLB gel was introduced into a 2 mL micro test tube. Then, 500 µL of BZP standard solution (500 ng/mL) and 500 µL of 10 mM aqueous
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ammonium formate were added sequentially. Subsequently, a centrifugal filter and a new micro
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test tube for receiving the filtrate were attached (Figure 1). After the solid phase was
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homogeneously dispersed by light vortexing for a few seconds, the whole device was inverted
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and then centrifuged at 3000 x g for 10 s (Figure 1). The micro test tube containing the filtrate
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was removed, and a new micro test tube containing a washing solution (1 mL of 5% aqueous
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methanol solution) was attached. The whole centrifugal filter device was again inverted (i.e.,
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returned to the original position), and then centrifuged at 3000 x g for 10 s to introduce the
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washing solution into the opposite micro test tube containing the Oasis® HLB gel. The
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solid-phase gel was then dispersed and centrifuged as described above. Again, the micro test
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tube containing the filtrate was removed, and a new micro test tube containing an eluting
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solution (500 µL of methanol) was attached to introduce the eluting solution into the opposite
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micro test tube containing the solid-phase gel. The solid-phase gel was dispersed by light
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vortexing followed by ultrasonication for 10 s, and the solution was centrifuged as described
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above. The filtrate was analyzed by LC/TOF-MS.
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2.4. Pretreatment of Serum and Urine Samples
Five hundred microliters of serum was spiked with 10 μL of desalkylflurazepam (10 μg/mL)
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as the surrogate, and mixed well with 500 μL of acetonitrile to precipitate proteins. Then, the
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mixture was filtered through the centrifugal filter (3000 x g, 10 min). The filtrate was purged
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under nitrogen at 40 °C to near dryness. Next, 500 μL of 1 M ammonium acetate aqueous
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solution and 10 μL of β-glucuronidase (10000 units/mL) were added to the residue, and the
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mixture was incubated at 37 °C for 1 h to achieve deconjugation. The deconjugation was
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performed in accordance with the information provided by the reagent manufacturer. Then, the
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whole solution was subjected to SPDE. As for the urine sample, 10 μL of desalkylflurazepam
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(10 μg/mL), 100 μL of 1 M ammonium acetate aqueous solution, and 10 μL of β-glucuronidase
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(10000 units/mL) were added to 1 mL of urine sample, and the whole mixture was incubated at
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37 °C for 1 h to achieve deconjugation. Then, the whole solution was subjected to SPDE.
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2.5. Apparatus
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An Alliance HT 2795 system equipped with an LCT Premier XE TOF-MS (Waters Corporation, Milford, MA, USA) was used. LC separation was performed with a Poroshell 120
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EC-C18 column (100 mm × 4.6 mm I.D., 2.7 µm; Agilent Technologies, Inc., Santa Clara, CA,
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USA). Column temperature was maintained at 40 °C. The mobile phase was a mixture of
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ammonium formate (pH 3.0, 10 mM) – acetonitrile (70:30, v/v), and was delivered at the flow
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rate of 0.5 mL/min. A 5 µL aliquot of the sample was injected into the system.
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ionization positive; capillary voltage, 2000 V; and cone voltage, 50 V. The proton adduct of each
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compound was used as the monitoring ion (Figure 2). Mass accuracy was maintained using
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Lock Spray with leucine-enkephalin [M+H]+ ion, m/z = 556.2771 as lock mass. Resolution was
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at least 10,000 as calculated by using the full width at half maximum method.
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3. Results and Discussion
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3.1. Optimization of SPDE Method
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The centrifugal filter was composed of a double lap filter paper and two types of
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polypropylene units, such that the filter paper was sandwiched between the polypropylene units
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(Figure 1). The optimum solid-phase gel volume for the recovery of BZP by SPDE was investigated.
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Because relatively high recoveries of over 80% were obtained when 5 mg of solid-phase gel
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was used, that amount was considered optimum for mixing with a sample.
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We also analyzed the filtrate and the washing solution after one SPDE. As no BZP was
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detected, we assumed that almost all the BZPs in the liquid phase were extracted by the
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solid-phase gel after one SPDE. The extraction yield of each BZP was determined by
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calculating the recovery of the drug in the standard solution, and was in the range of 81.3 to
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89.2% (RSD: 1.8–6.9%). This result indicates that adsorption to and desorption from the
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solid-phase gel occurred almost instantaneously. As microparticles in a suspension can move
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around freely and have a large surface area, they have a greater chance of coming into contact
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with the target compound in the liquid solution, and this was thought to result in the enhanced
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extraction. The enrichment factors of BZPs in the serum and urine samples were 100 and 200,
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respectively.
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As the SPDE method is performed in a closed system with high sealing property compared to
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a previously reported system [14], exposure to infectious microbes or hazardous chemicals is
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minimized.
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3.2 Method Validation of SPDE-LC/TOF-MS for Serum Analysis
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The limit of detection (S/N = 3) and the limit of quantification (LOQ; S/N > 10) of nine BZPs
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were in the range of 1–10 and 5–50 ng/mL, respectively. Each calibration curve showed good
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linearity (r > 0.998) over the range of LOQ (5–20 ng/mL) to 100 times those concentrations
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(500–2000 ng/mL). Figure 2 shows the typical chromatograms for 200 ng/mL BZPs in serum,
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as measured by SPDE-LC/TOF-MS. For serum samples, the average recoveries of BZPs spiked at 50 ng/mL and 500 ng/mL were
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89.6%–105.0% (RSD: 2.1%–6.8%) and 93.6%–110.4% (RSD: 2.1%–4.2%), respectively (Table
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1). For urine samples, the average recoveries of BZPs spiked at the same concentrations were
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88.7%–105.5% (RSD: 2.9%–6.4%) and 91.5%–101.1% (RSD: 3.6%–5.5%), respectively. Table
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2 shows intra-day and inter-day assay data for BZPs in serum and urine samples spiked at 100
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ng/mL. Statistical analyses were performed using one-way analysis of variance. The
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repeatability values ranged from 1.6 % to 7.0% (serum), and 2.8 % to 7.4% (urine), and the
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intermediate precision values were from 2.4% to 8.0% (serum), and 3.4 % to 7.0% (urine).
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These data suggested that SPDE-LC/TOF-MS has sufficient sensitivity and precision for
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monitoring residual BZPs in biological samples.
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3.3. Analysis of Serum and Urine Samples from Patient Receiving Clinical Doses of Etizolam
Serum and urine samples from a patient receiving clinical doses of etizolam (Depas®) were
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analyzed. The major metabolites of etizolam are 8-hydroxy etizolam and α-hydroxy etizolam,
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and their glucuronide conjugates, all of which are excreted in urine [15]. The serum and urine
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samples were subjected to deconjugation with β-glucuronidase. Blood and urine of the patient
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receiving clinical doses of Depas® (2 mg) were periodically sampled (3, 6, and 9 h after oral
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administration) and subjected to SPDE-LC/TOF-MS analysis. The typical chromatograms of the
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serum and urine samples obtained 3 h after oral administration exhibited an etizolam peak
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(Figure 3(A), (B)). The accurate mass (359.0733 ± 20 mDa) of the proton adducts of expected
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etizolam metabolites in the total ion chromatogram of urine sample was determined and drawn
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as a mass chromatogram. Peaks (a) and (b) were observed (Figure 3(C)) at the retention times of
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~4 min and ~10 min, respectively. The chemical formula C17H15ClN4SO for both etizolam
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metabolites is consistent with the molecular ion peaks from mass spectrometry, and isotopic
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peaks indicate the presence of chlorine (Figure 3(D)), as expected. Thus, we attributed peaks (a)
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and (b) to the etizolam metabolites. However, because 8-hydroxy etizolam and α-hydroxy
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etizolam are structural isomers, we used the retention times from LC to differentiate them. In
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RPLC, the lipophilicity (log Pow) of a substance is used for the prediction of elution order. Then,
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using the software program “Chem Draw Pro,” the log Pow values for 8-hydroxy etizolam,
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α-hydroxy etizolam, and etizolam were calculated as 4.15, 4.58, and 5.17, respectively. As
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8-hydroxy etizolam has lower lipophilicity than α-hydroxy etizolam, peak (a) was speculated to
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be 8-hydroxy etizolam, and peak (b), α-hydroxy etizolam.
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3.4. Drug Monitoring in Blood and Urine
Figure 4 shows time course of the concentrations of etizolam and its two metabolites in serum
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and urine samples following oral administration. For the urine sample, the drug concentration
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was corrected by creatinine values. The maximum concentration of etizolam in blood was
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reached 3 h after oral administration, consistent with previously reported data [16]. Accordingly,
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the metabolites and the parent compound should be monitored when urine is analyzed to
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identify the causative agent when drug poisoning or abuse is suspected.
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In conclusion, SPDE-LC/TOF-MS is useful for therapeutic drug monitoring in emergency
medicine and forensic science.
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Acknowledgement This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No. 20590043).
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References
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[1] S.J. Marin, R. Coles, M. Merrell, G. McMillin, G., Quantitation of benzodiazepines in urine,
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serum, plasma, and meconium by LC-MS-MS, J. Anal. Toxicol., 32 (2008) 491–498.
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[2] M. Nakamura, T. Ohmori, Y. Itoh, Simultaneous determination of benzodiazepines and their
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metabolites in human serum by liquid chromatography-tandem mass spectrometry using a
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high-resolution octadecyl silica column compatible with aqueous compounds, Biomed.
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Chromatogr., 23 (2009) 357–364.
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[3] B.A. Bidlingmeyer, Guidelines for proper usage of solid phase extraction devices, LC GC, 2
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(1984) 578–580.
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[4] C.L. Arthur, J. Pawliszyn, Solid phase microextraction with thermal desorption using fused
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silica optical fibers, Anal.Chem., 62 (1990) 2145–2148.
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[5] K.J. Reubsaet, H. Ragnar Norli, P. Hemmersbach, K.E. Rasmussen, Determination of
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benzodiazepines in human urine and plasma with solvent modified solid phase micro extraction
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and gas chromatography; rationalization of method development using experimental design
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strategies, J.Pharm. Biomed. Anal., 18 (1998) 667–680.
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[6] E. Baltussen, P. Sandra, F. David, C. Cramers, Stir bar sorptive extraction(SBSE), a novel
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extraction technique for aqueous samples, J.Microcol.Sep., 11 (1999) 737–739.
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[7] B. Tienpont, F. David, T. Benijts, P. Sandra, Stir bar sorptive extraction-thermal
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desorption-capillary GC-MS for profiling and target component analysis of pharmaceutical
22
drugs in urine, J.Pharm. Biomed. Anal., 32 (2003) 569–579.
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[8] Y. He, H.K. Lee, Liquid phase microextraction in a single drop of organic solvent by using a
24
conventional microsyringe, Anal. Chem., 69 (1997) 4634–4640.
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[9] H.G. Ugland, M. Krogh, K.E. Rasmussen, Liquid-phase microextraction as a sample
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preparation technique prior to capillary gas chromatographic-determination of benzodiazepines
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in biological matrices, J. Chromatogr. B., 749 (2000) 85–92.
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[10] M. Anastassiades, S. J. Lehotay, D. Štajnbaher, F.J. Schenck, Fast and easy multiresidue
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method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction”
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for the determination of pesticide residues in produce, J. AOAC Int. 86 , (2003) 412–431.
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[11] M. Rezaee, Y. Assadi, M.R. Millani Hosseini, E. Aqhaee, F. Ahmadi, S. Berijani,
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Determination of organic compounds in water using dispersive liquid-liquid microextraction, J.
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Chromatogr. A., 1116 (2006) 1–9.
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[12] M.I. Leoung, S.D. Huang, Dispersive liquid-liquid microextraction method based on
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solidification of floating organic drop combined with gas chromatography with electron-capture
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[13] R. Saito, Y. Kikuchi, Y.Iwasaki, R. Ito, K. Saito, Analysis of benzodiazepine drugs in serum
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by LC/TOF-MS coupled with solid-phase dispersive extraction method, in Proceedings of The
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54th Symposium of the Pharmaceutical Society of Japan Kanto Branch, 2010, Tokyo, 107.
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Table 1 Recoveries of BZPs in spiked serum and urine samples as measured by SPDE-LC/TOF-MS Spiked amount Sample
50 ng/mL Serum Mean (%) (RSD, %)
500 ng/mL Serum Mean (%) (RSD, %)
(5.2)
93.6
(4.1)
101.8
(5.8)
Nitrazepam
89.6
(2.1)
98.4
(3.9)
88.7
(3.5)
Lorazepam
95.9
(6.8)
98.7
(4.2)
104.6
(6.4)
Alprazolam
104.8
(3.6)
104.7
(3.1)
99.1
(3.5)
Triazolam
105.0
(2.9)
106.7
(3.8)
100.6
(3.8)
100.7
(4.5)
Flunitrazepam
96.4
(3.0)
98.0
(2.5)
98.4
(3.7)
94.5
(4.4)
Nimetazepam
94.8
(4.5)
97.0
(2.1)
Etizolam
100.8
(3.8)
110.4
(4.0)
Diazepam
93.7
(3.0)
98.7
(4.2)
10 11 12
99.8
(3.6)
100.1
(4.2)
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(4.3)
(2.9)
93.9
(3.8)
105.5
(4.8)
98.8
(3.6)
99.4
(3.4)
91.5
(4.1)
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94.6
(n = 7)
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93.7
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4
8
(5.5)
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101.1
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93.1
2
6
500 ng/mL Urine Mean (%) (RSD, %)
Bromazepam
1
5
50 ng/mL Urine Mean (%) (RSD, %)
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Table 2 Intra-day and inter-day precisions of BZPs in spiked serum and urine samples as measured by SPDE-LC/TOF-MS
5 6 7 8 9
RSD (%) Urine
Bromazepam
103.4
96.5
4.1
7.4
Nitrazepam
89.6
95.5
1.6
4.2
Lorazepam
95.9
95.9
7.0
4.5
Alprazolam
104.8
98.5
5.3
Triazolam
105.0
98.7
6.4
Flunitrazepam
96.4
97.1
Nimetazepam
94.8
99.0
100.8
99.9
93.7
102.4
Urine 7.0
3.0
5.1
6.6
5.1
4.5
6.8
4.5
3.4
8.0
4.4
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3.8
2.8
5.9
4.6
2.1
3.2
2.4
3.4
3.5
4.1
4.8
4.5
3.6
2.8
5.5
3.6
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3.5
(n = 2 × 5 test)
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Spiked amount: 100 ng/mL
Serum
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Serum
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Intermediate precision
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RSD (%)
Urine
Diazepam
2
Repeatability
Mean (%) Serum
Etizolam
1
Accuracy
10 11 12 13
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Figure captions
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Figure 1 Schematic of the centrifugation filter device used for solid-phase dispersive extraction
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(SPDE).
(A) Cross section of the SPDE filter device, (B) cross section of the centrifugal filter
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unit, (C) photograph of centrifugal filter and its separated parts, and (D) overview of
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the SPDE operation.
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1: a 2 mL test tube without a locked cap; 2: a 1.5 mL test tube without a locked cap; 3: centrifugal filter; 4: filter paper (No. 5B); 5: upper part of centrifugal filter; 6: lower part
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of centrifugal filter
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Figure 2 Chromatograms of BZPs in serum spiked with 200 ng/mL BZP standards.
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Figure 3 Mass chromatograms of etizolam in (A) serum and (B) urine three hours after oral
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administration of etizolam (2 mg). (C) Mass chromatogram of etizolam metabolites in
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urine. (D) Mass spectra of peaks (a) and (b) in chromatogram (C).
Figure 4 Time course of the concentrations of etizolam and its metabolites in (A) serum and (B) urine samples
* Peak area ratio = analyte peak area/IS (desalkylflurazepam) peak area
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(A)
2
(B)
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5
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7 8
an
9
66
10 11
M
(C)
12
d
13
te
14
17 18 19 20 21
Ac ce p
15 16
ip t
3
(D) solid-phase gel
3000 xg 3000 10 10 ssec centrifuge centrifuge
22
solvent
23 24 25
Figure 1
15
Page 15 of 19
1
m/z 316.0083
0
Intensity
4 5
20
m/z 282.0874
40 0 10
20
Intensity
Time (min)
0
10
20
Time (min)
300 200 100 0 0
10
20
200 100 0 0
10
Flunitrazepam 150 100 50 0 0
15
10
Nimetazepam Nimetazapam Niometazepam 200
Ac ce p
Intensity
20
m/z 343.0509
30
40
m/z 314.0904
d
14
40
Time (min)
te
Intensity
13
30
Time (min)
M
Intensity
12
40
m/z 309.0913
Triazolam
11
30
an
Intensity
10
40
m/z 321.0218
20
Alprazolam
9
30
40
0
8
40
Time (min)
Lorazepam
7
30
80
0
6
16
10
Nitrazepam
ip t
3
60 40 20 0
cr
2
us
Intensity
Bromazepam
20
30
40
Time (min)
m/z 296.1017
100 0
17
0
10
19
Intensity
Etizolam
18
22
m/z 343.0756
0
10
30
40
m/z 285.0798
200 100 0
0
10
20
30
40
Time (min)
Intensity
400
24 25
20
Time (min)
Desalkylflurazepam
23
40
200
Diazepam Intensity
21
30
400
0
20
20
Time (min)
m/z 289.0535
200 0 0
Figure 2
10
20
30
40
Time (min)
16
Page 16 of 19
300 200 100 0 0
5
10
15
20
25
us
Intensity
250 200 150 100
an
50 0 10
Time (min)
20
30
M
0
(C)
Peak (b)
2000
(9.8 9.8min)
1500
Peak (a) (4.24.2 min)
1000
te
500
d
Intensity Intensity
30
cr
Time (min)
(B)
ip t
Intensity
(A)
0
5
Ac ce p
0
(D)
Peak (a)
10
15
20
25
30
Time (min)
Peak (b)
[M+H]+
[M+H]+ (359.0733)
Intensity
Intensity
(359.0733)
m/z
m/z
1 2 3
Figure 3
17
Page 17 of 19
1 2 3
6 7 8 9
1.2 0.9 etizolam
us
Peak area ratio*
5
ip t
(A)
1.5
0.6
8-hydroxy etizolam
▲ -hydroxy etizolam
0 3
20 21
d 0.06
Ac ce p
15
te
Peak area ratio* / creatine
14
19
(B)
0.09
13
18
9
Time (hr) (h)
12
17
6
M
0
11
16
an
0.3
10
cr
4
▲ -hydroxy etizolam
8-hydroxy etizolam
0.03
etizolam
0
0
3
6 Time (hr) (h)
9
22 23 24 25
Figure 4
26 18
Page 18 of 19
1
Benzodiazepine drugs were quantified in human serum and urine by LC/TOF-MS.
2 3
Sample preparation was performed by solid-phase dispersive extraction (SPDE).
5
ip t
4
Solid-phase gels immediately reached equilibrium, resulting in rapid extraction.
SPDE is simple, and superior to conventional solid-phase extraction for cleanup.
us
7
cr
6
8
This SPDE-LC/TOF-MS method is useful in forensic science and emergency medicine.
an
9 10
Ac ce p
te
d
M
11
19
Page 19 of 19