sensors
Letter Rapid Drop-Volume Electrochemical Detection of the “Date Rape” Drug Flunitrazepam in Spirits Using a Screen-Printed Sensor in a Dry-Reagent Format
Frixos Papadopoulos, Konstantinos Diamanteas, Anastasios Economou * and Christos Kokkinos Department of Chemistry, National and Kapodistrian University of Athens, 157 71 Athens, Greece; [email protected] (F.P.); [email protected] (K.D.); [email protected] (C.K.) * Correspondence: [email protected]; Tel.: +30-210-7274298 Received: 30 July 2020; Accepted: 8 September 2020; Published: 11 September 2020
Abstract: Flunitrazepam is an extremely potent benzodiazepine sedative which is associated with “drug-facilitated sexual assault” when administered within an alcoholic drink. This work describes a simple electrochemical method for on-site rapid detection of flunitrazepam in untreated spirits (whiskey, vodka and gin) using a single-use screen-printed sensor (featuring graphite working and auxiliary electrodes and an Ag/AgCl reference electrode) in a dry reagent format. Analysis was performed by placing a drop of sample on the sensor, which was previously coated with dry KCl, and recording selected reduction/oxidation peaks of the target compound in a cyclic voltammetry scan. The 1 limit of quantification of flunitrazepam was at the sub-mg L− range. The between-sensor % relative 1 standard deviation of the analytically useful reduction peak in a solution containing 11.4 mg L− flunitrazepam was 9.8% (n = 5). Quantification was performed using calibration curves constructed from pooled samples spiked with flunitrazepam with relative errors <15%. The main advantages of the methodology are that it involves no sample pretreatment (such as deoxygenation, extraction or reagent(s) addition) and requires only drop-sized volumes of the sample, thus facilitating rapid on-site screening using portable equipment.
Keywords: flunitrazepam; cyclic voltammetry; screen-printed sensor; date-rape drugs; alcoholic drinks
1. Introduction Flunitrazepam (6-(2-fluorophenyl)-2-methyl-9-nitro-2,5-diazabicyclo [5,4,0]ndeca-5,8,10,12-tetraene- 3-one) is a benzodiazepine first synthesized in 1972 by Roche as a potent sedative to treat severe insomnia [1,2]. It is legally prescribed in more than 50 countries in Europe, Africa, Latin America, Middle East, Asia, and Australia but also is illegally found in countries in which its medical use is not approved, notably in the United States [1]. Flunitrazepam is known by different trade names (Rohypnol®, Roipnol®, Fluninoc®, Silece®, Hipnosedon®, Nervocuril® etc.) and colloquially known as “roofies”, “the forget pill”, “La Rocha” and “Mexican Valium” [3]. Flunitrazepam is used as a recreational drug popular in clubs and rave parties and believed to be the most commonly used “date-rape drug” associated with “drug-facilitated sexual assault” [1,2,4,5]. In view of the increasing number of “drug-facilitated sexual assault” cases, national and international bodies have expressed their concern: the World Health Organization identifies the implicative role of “date-rape” drugs in incidents of sexual violence [6]; the Parliamentary Assembly of the Council of Europe took steps in order to raise the public’s awareness to sexual assaults linked to “date-rape” drugs [7]; and, the U.S. Congress passed the Drug-Induced Rape Prevention and Punishment Act, which imposes stiff sentences for “drug-facilitated sexual assault” crimes and for the importation and distribution of flunitrazepam [8,9]. In a typical case of “drug-facilitated sexual assault”, flunitrazepam is administered
Sensors 2020, 20, 5192; doi:10.3390/s20185192 www.mdpi.com/journal/sensors Sensors 2020, 20, 5192 2 of 10 to an alcoholic beverage of an unsuspecting victim; the clinical manifestations of the drug, compounded by consumption of ethanol, include drowsiness, impaired psychomotor activity, confusion, ataxia, paradoxical reaction, and anterograde amnesia [10]. Flunitrazepam is colorless, odorless, and tasteless and is easily dissolved in drinks; the recommended pharmacological dose is around 1 mg in adults, but, when used as a “date-rape” drug, it is usually spiked into alcoholic drinks at higher doses (at around 2 mg it causes mild impairment and at >5 mg it induces strong sedation and amnesia). Once ingested, its action begins after 20–30 min, peaks at 2 h while residual effects can persist up to 12 h or more [2,11]. Since the drug is rapidly metabolized and excreted from the body, its determination in body fluids becomes challenging [1,2,11]. Many analytical methods have been reviewed in literature for the determination of benzodiazepines (including flunitrazepam) in different matrices [12]. Benzodiazepines are electrochemical active (possessing a reducible azomethine group) and can be electrochemically determined by a host of dynamic electroanalytical techniques, as well as potentiometry [13]. In particular, voltammetric techniques, in combination with screen-printed sensors, enable on-site screening tests which are rapid, cost-effective, and can be realized without sample pre-treatment using portable equipment [14]. Garcia-Gutierrez and Lledo-Fernandez [15] and Smith et al. [16] have reported the voltammetric determination of flunitrazepam in drinks using graphite screen-printed electrodes; however, these methods were applicable to low alcohol content (<5% v/v) beverage samples with deoxygenation and involved immersion of the sensor in the sample. More recently, Tseliou et al. have proposed a voltammetric method for the drop-volume determination of flunitrazepam in alcoholic and non-alcoholic drinks using a graphite screen-printed sensor sparked with Fe nanoparticles (to enhance sensitivity) and modified with glucose oxidase and glucose (to alleviate the oxygen interference) [17]; however, the relative complexity of the sensor’s fabrication process and the use of an expensive enzyme limits its utility for routine analysis. In this work, we propose a method for the rapid electrochemical detection of flunitrazepam in spirits (whiskey, vodka, and gin). Spirits are a common means for administering the drug for criminal purposes since the drink is rapidly consumed in a single “shot” while the high alcohol content amplifies the symptoms. The method utilizes a plain graphite screen-printed sensor in a dry reagent format and is directly applicable to the drop-volume analysis of spirits without pre-treatment (such as deoxygenation or extraction) using portable instrumentation.
2. Materials and Methods
2.1. Chemical and Reagents All the chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA). Ketamine, flunitrazepam and gamma-hydroxy butyric acid lactone (GBL) were from Sigma-Aldrich; gamma-hydroxybutyric acid (GHB) sodium salt was prepared and characterized as described previously [18]. Doubly distilled water was used throughout. A stock 1 solution containing 240 mg L− of flunitrazepam was prepared in doubly distilled water. Screen-printed sensors (DRP 11 L featuring carbon working and counter electrodes and Ag/AgCl reference electrode) were purchased from DropSens (Oviedo, Spain). Five samples of whiskey (including 2 single-malt, 2 mixed–malt and 1 bourbon brands), as well as 5 samples of each gin and vodka from different brands, were purchased from a local store. A pooled sample of each beverage was prepared by mixing equal volumes of the 5 individual samples; diluted pooled samples were also prepared by dilution of the pooled sample 1:1 (v/v) and 1:4 (v/v) with water. Calibration plots for flunitrazepam were constructed by spiking the pooled samples and diluted pooled samples with the appropriate amounts of the flunitrazepam stock solution.
2.2. Instrumentation Electrochemical experiments were performed with a Palmsens potentiostat controlled by the PSTrace 4.2 software (Palm Sens BV, The Netherlands) installed in a laptop computer. The potentiostat was connected to the screen-printed sensor using a PalmSens SPE connector. Sensors 2020, 18, x FOR PEER REVIEW 3 of 10
2.2. Instrumentation Electrochemical experiments were performed with a Palmsens potentiostat controlled by the PSTrace 4.2 software (Palm Sens BV, The Netherlands) installed in a laptop computer. The potentiostat was connected to the screen-printed sensor using a PalmSens SPE connector.
2.3. Experimental Procedure The experimental procedure is illustrated in Figure 1. Screen-printed sensors were modified in the lab by placing a 1 µL drop of a 4.0 mol L−1 KCl solution on the working electrode and left to dry at room temperature. These sensors modified with dry KCl are stable for at least a month and can be directly applied to on-site assays without further addition of reagent(s) in the sample. For the analysis, a 50 µL drop of the sample was placed on the working area of the sensor (in Sensors 2020, 20, 5192 3 of 10 order to cover the three electrodes) and, after 30 s (to allow the dry KCl to dissolve), a cyclic voltammogram (CV) was recorded in the potential range +0.50 V to −1.50 V (with respect to the Ag/AgCl2.3. Experimental quasi-reference Procedure electrode) at a scan rate of 50 mV s−1; three consecutive CV scans were recorded in each sample and all the measurements were recorded in non-deoxygenated solutions. A The experimental procedure is illustrated in Figure1. new electrode was used for each measurement.
FigureFigure 1. 1. ExperimentalExperimental procedure procedure for for the the determination determination of of flunitrazepam flunitrazepam in in alcoholic alcoholic beverages beverages using using thethe screen-printed screen-printed sensor sensor in in the the dry-reagent dry-reagent format. format.
1 3. ResultsScreen-printed and Discussion sensors were modified in the lab by placing a 1 µL drop of a 4.0 mol L− KCl solution on the working electrode and left to dry at room temperature. These sensors modified with 3.1.dry Method KCl are Development stable for at least a month and can be directly applied to on-site assays without further addition of reagent(s) in the sample. The aim of this work was to develop a simple, cost-effective, and rapid test for the target For the analysis, a 50 µL drop of the sample was placed on the working area of the sensor (in order compound in spirits with minimal sample pretreatment. to cover the three electrodes) and, after 30 s (to allow the dry KCl to dissolve), a cyclic voltammogram Regarding the electrochemical transducer element, a commercially available screen-printed (CV) was recorded in the potential range +0.50 V to 1.50 V (with respect to the Ag/AgCl quasi-reference sensor, featuring carbon working and counter electrodes− and an Ag/AgCl reference electrode, was electrode) at a scan rate of 50 mV s 1; three consecutive CV scans were recorded in each sample and chosen. The sensor can be easily connected− to portable instrumentation (Figure 1), thus enabling on- all the measurements were recorded in non-deoxygenated solutions. A new electrode was used for site analysis and is disposable minimizing the risk of carry-over effects between samples. each measurement. In previous work on the voltammetric determination of flunitrazepam, phosphate buffer (in the pH3. Results range 2.0–7.0) and Discussion has been used as supporting electrolyte [15–17]. Since it is desirable to operate in a reagentless mode, initial experiments were conducted by recording CVs by direct immersion of the sensor3.1. Method in alcoholic Development drinks without the addition of supporting electrolyte but the conductivity of these
The aim of this work was to develop a simple, cost-effective, and rapid test for the target compound in spirits with minimal sample pretreatment. Regarding the electrochemical transducer element, a commercially available screen-printed sensor, featuring carbon working and counter electrodes and an Ag/AgCl reference electrode, was chosen. The sensor can be easily connected to portable instrumentation (Figure1), thus enabling on-site analysis and is disposable minimizing the risk of carry-over effects between samples. In previous work on the voltammetric determination of flunitrazepam, phosphate buffer (in the pH range 2.0–7.0) has been used as supporting electrolyte [15–17]. Since it is desirable to operate in a reagentless mode, initial experiments were conducted by recording CVs by direct immersion of the sensor in alcoholic drinks without the addition of supporting electrolyte but the conductivity of these solutions was poor. The conductivity was restored, and satisfactory CVs were recorded, after spiking 1 the samples with KCl (200 µL of a 4.0 mol L− KCl solution in 10 mL of sample). KCl was additionally selected as the supporting electrode in order to provide a constant Cl- concentration to stabilize the Sensors 2020, 18, x FOR PEER REVIEW 4 of 10 solutions was poor. The conductivity was restored, and satisfactory CVs were recorded, after spiking the samples with KCl (200 µL of a 4.0 mol L−1 KCl solution in 10 mL of sample). KCl was additionally selected as the supporting electrode in order to provide a constant Cl- concentration to stabilize the potential of the Ag/AgCl quasi-reference electrode. A CV of an aqueous solution containing 15.7 mg L−1 flunitrazepam in 0.08 mol L−1 KCl is illustrated in Figure 2b. The redox mechanism of flunitrazepam has been studied earlier [15–17]: the reduction peak R2 arises from reduction of the 7- nitro group to hydroxylamine, the reduction peak R3 is due to reduction of the hydroxylamine group to the respective amine, the oxidation peak O1 is likely to result from the oxidation of the hydroxylamine to the nitroso analogue which is reduced back to hydroxylamine (peak R1). It was observed that the reduction peak R1 was not reproducible while the reduction peak R3 was hardly observed as it coincided with the hydrogen evolution wave. For analytical purposes, the reduction peak R2 and the oxidation peak O1 could be used successfully. In order to simplify the analytical protocol and convert it into a dry reagent drop-volume format, the sensor was modified with dry KCl (by placing a 1 µL drop of 4.0 mol L−1 KCl solution on the working electrode of the screen-printed sensor and leaving to dry). Then a 50 µL drop of the sampleSensors was2020 placed, 20, 5192 on the working area of the sensor and the CV was recorded. A CV taken4 of 10using the dry reagent drop-volume format using a 50 µL drop of an aqueous solution containing 15.7 mg L−1 flunitrazepam on the sensor modified with dry KCl is illustrated in Figure 2c. It is clear1 that potential of the Ag/AgCl quasi-reference electrode. A CV of an aqueous solution containing 15.7 mg L− well-defined peaks were obtained1 using the dry-reagent format only exhibiting a potential shift flunitrazepam in 0.08 mol L− KCl is illustrated in Figure2b. The redox mechanism of flunitrazepam withhas respect been studiedto the earlier solution-pha [15–17]: these reductionformat in peak Figure R2 arises 2b from(which reduction is attributed of the 7-nitro to groupincomplete to dissolutionhydroxylamine, of the dry the reductionKCl in the peak time-scale R3 is due toof reductionthe experiment). of the hydroxylamine In this way, group several to the sensors respective can be easilyamine, prepared the oxidation with the peak dry O1 isreagent, likely to alleviating result from the the oxidation need for of thereagent hydroxylamine addition to in the the nitroso sample solution,analogue hence which simplifying is reduced the back analytical to hydroxylamine procedure. (peak R1). It was observed that the reduction peak RIt1 iswas a further not reproducible critical aspect while of the the reduction procedure peak that, R3 was under hardly theobserved conditions as itof coincided the analysis, with oxygen the did nothydrogen interfere evolution with the wave. detection For analytical of flunitrazepam purposes, the and reduction no prior peak deoxygenation R2 and the oxidation step of peak the sample O1 could be used successfully. was required.
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O1 20
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FigureFigure 2. Cyclic 2. Cyclic voltammograms voltammograms in in the the range range 0.50 0.50 V to −1.50 V:V: ((aa)) immersing immersing the the sensor sensor in an in aqueousan aqueous −1 1 − 1−1 solutionsolution of 0.08 of 0.08 mol mol L LKCl;− KCl; (b) ( bas) as(a ()a after) after addition addition of 15.715.7 mgmg L L− flunitrazepam;flunitrazepam; (c) ( atc) aat 50 a µ50L dropµL drop −11 of anof aqueous an aqueous solution solution containing containing 15.7 15.7 mgmg LL− flunitrazepamflunitrazepam at theat the sensor sensor modified modified with drywith KCl dry (1 µKClL (1 1 µL dropdrop of of 4.0 4.0 mol LL−−1 KCl).KCl).
In order to simplify the analytical protocol and convert it into a dry reagent drop-volume format, 3.2. Analytical Features 1 the sensor was modified with dry KCl (by placing a 1 µL drop of 4.0 mol L− KCl solution on the workingCalibration electrode of flunitrazepam of the screen-printed in the sensor concentration and leaving range to dry). 0–25.7 Then mg a 50 Lµ−L1 was drop performed of the sample using was the pooledplaced samples on the and working the diluted area of pool the sensor samples and spiked the CV waswith recorded. flunitrazepam. A CV taken The using calibration the dry features reagent are µ 1 summarizeddrop-volume in Table format 1. using Representative a 50 L drop CVs of an showing aqueous solutionthe reduction containing peak 15.7 R2 mg and L− theflunitrazepam oxidation peak on the sensor modified with dry KCl is illustrated in Figure2c. It is clear that well-defined peaks were O1, together with the respective calibration plots for a gin sample diluted 1:1 (v/v), are illustrated in obtained using the dry-reagent format only exhibiting a potential shift with respect to the solution-phase format in Figure2b (which is attributed to incomplete dissolution of the dry KCl in the time-scale of the experiment). In this way, several sensors can be easily prepared with the dry reagent, alleviating the need for reagent addition in the sample solution, hence simplifying the analytical procedure. It is a further critical aspect of the procedure that, under the conditions of the analysis, oxygen did not interfere with the detection of flunitrazepam and no prior deoxygenation step of the sample was required.
3.2. Analytical Features
1 Calibration of flunitrazepam in the concentration range 0–25.7 mg L− was performed using the pooled samples and the diluted pool samples spiked with flunitrazepam. The calibration features are summarized in Table1. Representative CVs showing the reduction peak R 2 and the oxidation peak Sensors 2020, 20, 5192 5 of 10
O1, together with the respective calibration plots for a gin sample diluted 1:1 (v/v), are illustrated in Figure3; the signal-to-noise ratio (S /N) of the reduction peak R1 is shown to be markedly better than the S/N ratio of the oxidation peak O1.
Table 1. Calibration features in pooled spirit samples using the reduction peak R2 and the oxidation peak O1.
1 1 1 2 Sample Slope (µA mg− L) R LOQ (mg L− )
Reduction peak R2 No dilution 0.288 0.023 0.993 0.93 (0.93) − ± Whiskey Diluted 1:1 0.365 0.011 0.994 1.0 (2.0) − ± Diluted 1:4 0.955 0.024 0.997 0.63 (3.2) − ± No dilution 0.136 0.003 0.996 0.81 (0.81) − ± Gin Diluted 1:1 0.456 0.009 0.997 0.57 (1.1) − ± Diluted 1:4 0.688 0.022 0.993 0.71 (3.6) − ± No dilution 0.255 0.007 0.996 0.76 (0.76) − ± Vodka Diluted 1:1 0.271 0.009 0.993 1.1 (2.2) − ± Diluted 1:4 0.473 0.017 0.990 0.75 (3.8) − ± Oxidation peak O1 No dilution 0.087 0.021 0.989 0.96 (0.96) ± Whiskey Diluted 1:1 0.143 0.033 0.997 0.67 (1.2) ± Diluted 1:4 0.342 0.009 0.996 0.79 (4.0) ± No dilution 0.075 0.0032 0.986 1.7 (1.7) ± Gin Diluted 1:1 0.169 0.006 0.993 1.0 (2.0) ± Diluted 1:4 0.368 0.013 0.992 1.1 (4.4) ± No dilution 0.124 0.003 0.995 1.0 (1.0) ± Vodka Diluted 1:1 0.134 0.005 0.989 1.5 (3.0) ± Diluted 1:4 0.379 0.014 0.993 0.91 (4.6) ± 1 Coefficient of determination. 2 Limit of quantification (in parenthesis the LOQ values in the original sample before Sensorsdilution). 2020, 18, x FOR PEER REVIEW 6 of 10
Figure 3. Cont.
Figure 3. CVs for the determination of 0–25.7 µg L−1 of flunitrazepam in a pooled gin sample diluted
1:1 (v/v) using the drop-volume detection format using: (A) the reduction peak R2, and; (B) the
oxidation peak O1.
3.3. Interference Study No peaks interfering with the analytical peaks of flunitrazepam were observed in the CVs of the whiskey, vodka and gin samples analyzed, therefore no endogenous redox species in the samples interfered electrochemically with the detection of the target compound. The interference study included the two other main date-rape drugs [2], gamma-hydroxybutyric acid (GHB) and ketamine, as well as scopolamine which is also well known to be used for predatory purposes [19]. None of these compounds produced peaks in the CV scan or caused any change in the flunitrazepam peak heights (Figure S2, Supplementary Materials). Indeed, GHB is known to be oxidized only under very specific conditions [18] while both ketamine [20] and scopolamine [19] are oxidized at more positive potentials.
Sensors 2020, 18, x FOR PEER REVIEW 6 of 10
Sensors 2020, 20, 5192 6 of 10
Figure 3. CVs for the determination of 0–25.7 µg L1−1 of flunitrazepam in a pooled gin sample diluted Figure 3. CVs for the determination of 0–25.7 µg L− of flunitrazepam in a pooled gin sample diluted 1:1 1:1 (v/v) using the drop-volume detection format using: (A) the reduction peak R2, and; (B) the (v/v) using the drop-volume detection format using: (A) the reduction peak R2, and; (B) the oxidation oxidation peak O1. peak O1.
3.3. Interference Study 1 The limits of detection obtained with the proposed sensor were in the range 0.57–4.6 mg L− , dependingNo peaks on the interfering type of sample with the and analytical the dilution peaks level. of flunitrazepam To put these were data observed into perspective, in the CVs to of achieve the thewhiskey, 1 mg pharmacological vodka and gin dose, samples a pill analyzed, containing therefor 1 mge of no flunitrazepam endogenous shouldredox species be added in the in a samples portion of 1 a spiritinterfered (around electrochemically 40 mL) and the with resulting the detection concentration of the intarget the drinkcompound. would be 25 mg L− . This is actually the minimumThe interference concentration study included expected the in two a typical other scenariomain date-rape of “drug-facilitated drugs [2], gamma-hydroxybutyric sexual assault” since flunitrazepamacid (GHB) and is usually ketamine, spiked as well into as alcoholicscopolamine drinks which at higheris also well doses known than theto be pharmacological used for predatory dose. Therefore,purposes the [19]. limits None of of quantitation these comp (LOQs)ounds produced of the proposed peaks in methodology the CV scan or are caused sufficient any change for quantifying in the theflunitrazepam minimum concentration peak heights of(Figure the drug S2, evenSupplemen at 1:4tary (v/v )Materials). dilution of Indeed, the sample GHB (correspondingis known to be to oxidized1 only under very specific conditions [18] while both ketamine [20] and scopolamine [19] are 5 mg L− of flunitrazepam in the solution to be analyzed). The LOQs using the reduction peak R2 were oxidized at more positive potentials. generally lower than using the oxidation peak O1. From inspection of the slopes of the calibration plots, it is clear that a significant matrix effect occurred since the slope increased with increasing sample dilution. Statistically significant differences in sensitivity between the pooled whiskey, gin, and vodka samples were also observed. In view of the different slopes in different types of samples and dilutions, quantification issues are further discussed in paragraph 3.4. The between-sensor reproducibility was estimated by performing measurements of a standard 1 solution containing 11.4 mg L− of flunitrazepam at five different sensors prepared on the same day; the % relative standard deviations were 9.8% for the reduction peak R2 and 11.1% for the oxidation peak O1. Considering the LOQs, the S/N ratios and repeatability/reproducibility, the reduction peak R2 was selected for quantification in subsequent experiments. The shelf-life of the KCl-modified sensors (i.e., the stability of the KCl-modified sensors under storage in ambient conditions) was assessed by preparing 21 different sensors on the same day and performing 3 consecutive measurements of a 1 standard solution containing 11.4 mg L− of flunitrazepam every 4 days. As demonstrated in Figure S1 (Supplementary Materials), the response remained statistically stable within the 25 days of the study.
3.3. Interference Study No peaks interfering with the analytical peaks of flunitrazepam were observed in the CVs of the whiskey, vodka and gin samples analyzed, therefore no endogenous redox species in the samples interfered electrochemically with the detection of the target compound. Sensors 2020, 18, x FOR PEER REVIEW 7 of 10
3.4. Application
Sensors 2020While, 20, 5192 the primary goal is screening for the target compound in spirits, quantification is also7 of 10 possible using the proposed drop-volume methodology. For this purpose, calibration curves were constructed from pooled samples and diluted pooled samples spiked with flunitrazepam and were Thestored interference in PSTrace study4.2 operating included in thethe twoanalytical other mode. main date-rapeTo test the drugs accuracy [2], gamma-hydroxybutyricof the methodology acid (GHB)developed and in ketamine, this work, asthree well individual as scopolamine samples which each of is gin, also whiskey well known and vodka to be (undiluted used for predatory and diluted 1:1 (v/v) and 1:4 (v/v)) were spiked with flunitrazepam and were analyzed. The concentration purposes [19]. None of these compounds produced peaks in the CV scan or caused any change in was calculated using the respective stored calibration plot constructed using pooled samples or the flunitrazepam peak heights (Figure S2, Supplementary Materials). Indeed, GHB is known to be diluted pooled samples using electrodes randomly selected from three batches purchased at different oxidizedtimes. only The undervalues veryof the specific relative conditionserrors for all [ 18the] samples while both analyzed ketamine were [<15%20] and and scopolamine the method was [19 ] are oxidizeddeemed at more accurate positive for the potentials. intended purpose (Table 2). Quantification using stored calibration curves of pooled samples is faster and more convenient than applying the method of standard additions 3.4. Applicationsince only a single measurement per sample is required; the total analysis time (from sample addition Whileto reporting the primary of the result) goal is is screening less than for90 thes. Vo targetltammograms compound for inthe spirits, detection quantification and quantification is also possible of usingflunitrazepam the proposed in drop-volume individual samples methodology. of spirits are For illustrated this purpose, in Figure calibration 4. curves were constructed from pooledTable samples 2. Accuracy and for diluted the determination pooled samples of flunitrazepa spiked withm in individual flunitrazepam spirits andusing were the reduction stored in PSTrace
4.2 operatingpeak inR2. the analytical mode. To test the accuracy of the methodology developed in this work, three individual samples each of gin, whiskey and vodka (undiluted and diluted 1:1 (v/v) and 1:4 Sample Dilution (v/v) Added (mg L−1) Found (mg L−1) Er (%) 1 (v/v)) were spiked with flunitrazepam and were analyzed. The concentration was calculated using the 1 2 No 13.0 14 respective stored calibration plot constructed using pooled samples or diluted pooled samples using Whiskey 2 3 1:1 12.6 10 electrodes randomly selected3 4 from three1:4 batches purchased at different12.2 times. The6.8 values of the relative errors for all the samples analyzed1 wereNo <15% and the method was 10.4 deemed accurate−8.8 for the intended purpose (Table2). QuantificationGin 2 using1:1 stored calibration11.4 curves of pooled 12.1 samples 6.2 is faster and more convenient than applying the3 method1.4 of standard additions since only 10.8 a single measurement−5.2 per sample is required; the total analysis1 time (fromNo sample addition to reporting 12.8 of the result) 12 is less than 90 s. VoltammogramsVodka for the detection2 and1:1 quantification of flunitrazepam 12.6 in individual 10 samples of spirits are illustrated in Figure4. 3 1:4 11.2 −11 1 % error; 2 single-malt; 3 mixed-malt; 4 bourbon.
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Figure 4. Cont. Sensors 2020, 20, 5192 8 of 10
Sensors 2020, 18, x FOR PEER REVIEW 8 of 10
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Figure 4. CVs for the determination of flunitrazepam using the reduction peak R2 in: (Α) gin, ((B) Figure 4. CVs for the determination of flunitrazepam using the reduction peak R2 in: (A) gin, ((B) vodka) and, (C) whiskey samples diluted 1:1 (v/v), (a) before and (b) after spiking with 11.4 mg L−1 of vodka) and, (C) whiskey samples diluted 1:1 (v/v), (a) before and (b) after spiking with 11.4 mg L 1 flunitrazepam. − of flunitrazepam. 4. Conclusions Table 2. Accuracy for the determination of flunitrazepam in individual spirits using the reduction peak R2. In this work, an electrochemical method is developed for on-site rapid detection of the date-rape Sample Dilution (v/v) Added (mg L 1) Found (mg L 1) E (%) 1 drug flunitrazepam in spirits (whiskey, vodka, and gin) using− a single-use screen-printed− r sensor in a 1 2 No 13.0 14 dry reagent format. The main advantages of the methodology are that it is extremely simple and fast, Whiskey 2 3 1:1 12.6 10 involves neither sample pretreatment3 4 1:4(such as deoxygenation, extraction 12.2 or reagent(s) 6.8addition) nor electrode modification, requires1 only Nodrop-sized volumes of the sample 10.4 (thus facilitating8.8 rapid on- 11.4 − site screeningGin using portable2 equipment) 1:1 while a single measurement 12.1is sufficient for 6.2 quantitative 3 1.4 10.8 5.2 analysis. The method proved fit-for-purpose and is suitable both for screening purposes− and for quantification of the target1 compound No in alcoholic drinks. 12.8 12 Vodka 2 1:1 12.6 10 3 1:4 11.2 11 − 1 % error; 2 single-malt; 3 mixed-malt; 4 bourbon. Sensors 2020, 20, 5192 9 of 10
4. Conclusions In this work, an electrochemical method is developed for on-site rapid detection of the date-rape drug flunitrazepam in spirits (whiskey, vodka, and gin) using a single-use screen-printed sensor in a dry reagent format. The main advantages of the methodology are that it is extremely simple and fast, involves neither sample pretreatment (such as deoxygenation, extraction or reagent(s) addition) nor electrode modification, requires only drop-sized volumes of the sample (thus facilitating rapid on-site screening using portable equipment) while a single measurement is sufficient for quantitative analysis. The method proved fit-for-purpose and is suitable both for screening purposes and for quantification of the target compound in alcoholic drinks.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/20/18/5192/s1, Figure S1: Shelf-life test of the sensors, Figure S2: Selectivity of the sensors towards ketamine, GHB and scopolamine. Author Contributions: Conceptualization, A.E. and C.K.; Methodology, A.E. and C.K; Validation, K.D., A.E. and F.P.; Investigation, K.D., A.E. and F.P.; Data Curation, K.D., C.K. and F.P.; Writing—Original Draft Preparation, A.E.; Writing—Review & Editing, A.E. and C.K.; Supervision, A.E. and C.K.; Project Administration, A.E. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.
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