Simple and Sensitive Molecularly Imprinted Polymer â Mn-Doped ZnS

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A simple and sensitive molecularly imprinted polymer – Mn-doped ZnS quantum dots based fluorescence probe for cocaine and metabolites determination in urine Maria Pilar Chantada-Vázquez, Juan Sánchez-González, Elena Peña-Vazquez, Maria Jesús Tabernero, Ana María Bermejo, Pilar Bermejo-Barrera, and Antonio Moreda-Piñeiro Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04250 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Analytical Chemistry

A simple and sensitive molecularly imprinted polymer – Mn-doped ZnS quantum dots based fluorescence probe for cocaine and metabolites determination in urine María Pilar Chantada–Vázquez1, Juan Sánchez–González1, Elena Peña–Vázquez1, María Jesús Tabernero2, Ana María Bermejo2, Pilar Bermejo–Barrera1, Antonio Moreda–Piñeiro1* (1) Department of Analytical Chemistry, Nutrition and Bromatology. Faculty of Chemistry. University of Santiago de Compostela. Avenida das Ciencias, s/n. 15782 – Santiago de Compostela. Spain. (2) Department of Pathologic Anatomy and Forensic Sciences. Faculty of Medicine. University of Santiago de Compostela. Rúa de San Francisco, s/n. 15782 – Santiago de Compostela. Spain.

*

Corresponding author: E–mail address: [email protected]

ACS Paragon Plus Environment

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Abstract A new molecularly imprinted polymer (MIP)-based fluorescent artificial receptor has been prepared by anchoring a selective MIP for cocaine (COC) on the surface of polyethylene glycol (PEG) modified Mn-doped ZnS quantum dots (QDs). The prepared material combines the high selectivity attributed to MIPs, and the sensitive fluorescent property of the Mndoped ZnS QDs. Simple and low cost methods have therefore been optimized for assessing cocaine abuse in urine by monitoring the fluorescence quenching when the template (COC) and also metabolites from COC [benzoylecgonine (BZE), and ecgonine methyl ester (EME)] are present. Fluorescence quenching was not observed when performing experiments with other drugs of abuse (and their metabolites), or when using non-imprinted polymer (NIP)coated QDs. Under optimized operating conditions (1.5 mL of 200 mg L-1 MIP-coated QDs solution, pH 5.5, and 15 min before fluorescence scanning) two analytical methods were developed/validated. One of the procedures (direct method) consisted of urine sample 1:20 dilution before fluorescence measurements. The method has been found to be fast, precise and accurate, but the standard addition technique for performing the analysis was required because of the existence of matrix effect. The second procedure performed a solid phase extraction (SPE) first, avoiding matrix effect and allowing external calibration. The limit of detection of the methods were 0.076 mg L-1 (direct method), and 0.0042 mg L-1 (SPE based method), which are lower than the cut-off values for confirmative conclusions regarding cocaine abuse.


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Introduction Despite the decrease in cocaine use between the 2007-2009 and 2010-2012 periods in Western and Central Europe as reported by the United Nations Office on Drugs and Crime (UNODC), cocaine is still one of the most widely used illicit substances in European countries.1 Cocaine abuse and cocaine overdose are diagnosed mainly through laboratory tests of various biological fluids. The tests must be sensitive and specific enough to ensure a correct assessment. Urinary cocaine and its metabolites are routinely used to confirm the abuse of this substance, and immunoassay methods have been widely used for screening purposes.2 Screening methods must guarantee the avoidance of false-negative results, be sensitive and reliable, and require little or no sample pre-treatment. However, some immunoassays are expensive, and low-cost and rapid methodologies for assessing cocaine abuse are therefore needed for screening and quantification. Semiconductive nanocrystal QDs have emerged as excellent nanostructures for developing sensor probes mainly due to their luminescent properties, which allow a simple and inexpensive fluorescent and room temperature phosphorescent measurement.3 QD-based sensors are therefore appealing methodologies for performing sensitive screening and quantification methods. Although CdSe QDs were first proposed for analytical applications, toxicity of cadmium led to the development of less toxic ZnSe and ZnS QDs.3-5 More recently, Mn- and Cu-doped ZnS, in which transition metals are used as doping substances have been found to diminish self-quenching and to provide greater strength to thermal, chemical and photochemical disturbances.6-8 However, QDs based probes have been reported to offer low selectivity, the quenching phenomena occurring in the presence of certain substances (analytes), and also in the presence of sample’s constituents. Improvements in selectivity of QDs for a certain analyte can be successfully achieved by coating the QD core with a film layer of an MIP, resulting in the so-called molecularly imprinted optosensing


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materials (MIOM).9 MIOM combines the high selectivity of MIPs and the substantial (quenchable) fluorescence of QDs. The high selectivity of MIPs is a consequence of the recognition cavities generated after analyte (template molecule) reaction with an adequate monomer, polymerization, and template removal stages. Examples of successful MIP-QDs (Cd based QDs) approaches include sensors for tocopherol assessment in rice;10,11 for bovine hemoglobin and serum albumin detection;12,13 for zearalenone determination in cereal samples;14 and for nitroaromatic explosives,15 clenbuterol and melamine,16 ractopamine,17 and cystein18 sensing. Regarding Mn-doped ZnS QDs, fluorescent MIP-QDs probes have been reported for sensing proteins,19-21 organophosphate6 and pyrethroid22 insecticides, pesticides,23 and tetrabromobisphenol A.24 In addition, room temperature phosphorescent sensing has also been proposed for chlorophenols,25,26 domoic acid,27 proteins,28 and mercury.29 Recently, a fluorescent probe based on MIP-Mn-doped ZnS QDs for COC and its metabolites (BZE and EME) sensing was prepared and fully characterized.30 The aim of the current work has been to explore the possibilities of using this fluorescent MIP-QD based probe for developing simple and low cost analytical methods for cocaine abuse assessment when analyzing complex samples such as urine. To date, immunochemical techniques, mainly enzyme-multiplied immunoassay technique (EMIT), and radioimmunoassay (RIA), have commonly been used for cocaine screening purposes in urine;31 whereas, high performance liquid chromatography (HPLC) and gas chromatography (GC) with mass spectrometer detectors have been proposed as confirmatory techniques.31 These methodologies are quite expensive, and reliable and low cost methodologies in the forensic laboratory are required. In addition, false-negative results can be obtained by using immunochemical assays when BZE (main metabolite from cocaine in urine) occurs at low concentrations.31 This is common when analyzing urine samples after 1 or 2 days since last use of cocaine (the time frame


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within which cocaine metabolites can be detected in urine).32 Therefore, direct urine analysis and SPE sample pre-treatment before the MIP-QD based fluorescent measurement have been developed in a novel way in the current work. The practical utility of the proposed application lies in the use of inexpensive (world-wide) analytical instrumentation for assessing cocaine abuse by analyzing very complex samples such as urine. It could be therefore an appealing alternative to the use of more sophisticated and expensive instrumentation such as that based on inmunoanalysis and chromatography.

Experimental section Instrumentation A Hitachi F-2500 fluorescence spectrometer (Schaumburg, IL, USA) equipped with a xenon lamp and 10 mm quartz cells was used for fluorescence measurements. A 3200 Q TRAP LC/MS/MS system (ABSciex, Concord, Canada), equipped with a Flexar FX-15 UHPLC binary chromatographic pump (Perkin Elmer, Waltham, MA, USA), and a Flexar UHPLC autosampler (Perkin Elmer) was used for confirming template (COC) removal from MIPQDs, and also for determining COC, BZE, and EME (comparative purposes). Separations were performed with a Kinetex 5µ C18 100 Å reverse phase column (100 mm length × 2.10 mm i.d., 5.0 µm particle diameter) from Phenomenex (Torrance, CA, USA) connected to a Phenomenex C8 guard column (4 mm length × 3.0 mm i.d). A Raypa Model UCI-150 ultrasonic cleaner bath (power of 325 W and frequency of 35 kHz) from R. Espinar S.L. (Barcelona, Spain) was used for synthesizing MIP-QD composites. Other laboratory devices were: Miniplus 3 peristaltic pump (Gilson, Middleton, WI, USA) with Tygon 2-stop tubing (2.06 mm i.d.) from Perkin Elmer, Basic20 pH–meter with a glass–calomel electrode (Crison, Barcelona, Spain), Reax 2000 mechanical stirrer (Heidolph, Kelheim, Germany), Centromix centrifuge (Selecta, Barcelona, Spain), vacuum pump (Millipore Co., Bedford, MA, USA),


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and VLM EC1 metal block thermostat and N2 sample concentrator from VLM (Leopoldshöhe-Greste, Germany). Reagents Ultrapure water, 18 MOhm cm-1 of resistivity, obtained from a Milli-Q purification device (Millipore). Drug stock standard solutions were prepared from COC, and EME (1000 mg L-1 dissolved in acetonitrile), and BZE (1000 mg L-1 dissolved in methanol) purchased from Cerilliant (Round Rock, TX, USA). Other drug stock standard solutions were codeine (COD),

morphine

(MOR),

∆9-tetrahydrocannabinol

(∆9-THC),

11-hydroxy-∆9-

tetrahydrocannabinol (∆9-THC-OH), and cannabinol (CBN), 1000 mg L-1 dissolved in methanol; and 6-monoacetylmorphine (6-MAM), 1000 mg L-1 dissolved in acetonitrile (Cellirant). Cannabidiol (CBN), 2000 mg L-1, was prepared by dissolving 10 mg of CBD (National Measurement Institute Australian Government, Sidney, Australia) in 5 mL of methanol. Mn-doped ZnS QDs were synthesized by using heptahydrate zinc sulfate (Panreac, Barcelona, Spain), sodium sulfide (Fluka, Buchs, Switzerland), and manganese dichloride (Merck, Darmstadt, Germany). Polyethylene glycol (PEG 6000), dimethyl sulfoxide (DMSO), 2-propanol, toluene, ammonium hydroxide, dipotassium hydrogen phosphate, chloroform, hydrochloric acid (35%), and silica gel (5-6 mm) were from Panreac. MIP was synthesized by using divinylbenzene (DVB) from Sigma-Aldrich (Steinheim, Germany), and ethylene glycol dimethacrylate (EGDMA) and 2,2´-azobisisobutyronitrile (AIBN) from Fluka. Acetonitrile and methanol (supragradient HPLC grade), ammonium acetate, neutral alumina, and sodium hydroxide were from Merck. Potassium dihydrogen phosphate was from BDH (Poole, UK). Other used consumables were: Durapore 0.20 µm membrane filters (Millipore), 0.20 µm cellulose acetate syringe filters (LLG, Meckenheim, Germany), ACCUREL® PP membrane (Membrana, Wuppertal, Germany), and Bond Elut Certify cartridges (130 mg, 3 mL) from Agilent Technologies (Santa Clara, CA, USA).


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Urine samples Urine samples were from polydrug abusers under control in an addiction research center in Santiago de Compostela, Spain. Drug-free urine samples (used for method validation) were obtained from laboratory staff volunteers. Urine samples were collected in clean sealed polyethylene vials and kept at -20°C when necessary. Synthesis of MIP-coated QDs Mn-doped ZnS QDs synthesis was performed as described elsewhere.30 Surface QDs modification was performed by adding 10 mL of an aqueous solution containing 3.33 g of PEG and subjecting the mixture to ultrasounds (325 W, 37 kHz, 4 h). The mixture was centrifuged (3000 rpm, 20 min). After rinsing (5 mL of methanol, three times), nanoparticles were isolated by centrifugation (3000 rpm, 20 min) and dried at room temperature inside a desiccator for 24 h. For MIP synthesis onto the modified PEG-QDs, COC (0.20 g) and EGDMA (126 µL) were dissolved in 4 mL of DMSO, sparged with argon, and kept at room temperature in the dark for 12 h (template-monomer self-assembly). PEG-QDs (0.500 g) were then dispersed in 25 mL of ultrapure water, and mixed with the pre-polymerization mixture (COC-EGDMA in DMSO). The beakers were sealed after adding the cross-linker (1.25 mL of DVB) and the initiator (0.10 g of AIBN), and the mixture was subjected to ultrasounds (325 W, 37 kHz, room temperature, 4 h). The prepared composite was washed with methanol, centrifuged (3000 rpm, 20 min), and dried at room temperature inside a desiccator for 24 h. Non imprinted polymers (NIP-QDs) were also prepared as above but without using the template. Template (COC) was removed from MIP-QDs by enclosing 200 mg inside a rectangular (3.0 × 2.0 cm) PP membrane, and subjecting the device to ultrasound (325 W, 37 kHz, 30 min) with 10 mL of 70:20:10 hexane/2-propanol/ammonium hydroxide (ten 30-min sonication cycles with a fresh solution in each cycle)30. Negligible COC concentrations were found in


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the tenth washing solution after HPLC-MS/MS analysis33. Template-free MIP-QD was rinsed twice with methanol and ultrapure water, and dried at room temperature inside a desiccator for 24 h. The dried synthesized material was kept at 4°C in the dark. Under optimum conditions, fluorescence measurements were performed using NIP/MIP-coated QDs (100 mg) re-dispersed in 250 mL of 0.1M/0.1M KH2PO4-NaOH pH 5.5 (NIP/MIP coated QD concentration of 200 mg L-1). These solutions were also stored at 4°C in the dark. Solid phase extraction procedure Cartridges were conditioned by pumping successively 2 mL of methanol and 2 mL of 0.1M phosphate buffer (pH 6) before urine sample (1 mL) loading (flow rate of 1.67 mL min-1).34 After sample loading, cartridges were rinsed with 3 mL of ultrapure water, 3 mL of 0.1 N hydrochloric acid, 9 mL of methanol, and 3 mL of 0.3 M ammonium hydroxide, and finally vacuum-dried for 5 min. Analytes were eluted with 3 mL of 4:1 chloroform–isopropanol. After evaporation to dryness (N2 stream, 60°C), the residue was re-dissolved with 100 µL of 0.1M/0.1M KH2PO4-NaOH (pH 5.5) for fluorescence analysis (section 2.7.1.). Fluorescence measurements Measurements were performed using an excitation wavelength of 297 nm (2.5 nm slit width), and recording a 400–800 nm emission range (2.5 nm emission slit, maximum fluorescence emission wavelength at 590 nm, photomultiplier tube voltage at 700 V). External calibration External calibration was performed by mixing 1.5 mL of MIP-QDs solution (200 mg L-1), volumes within the 0-0.5 mL range of COC previously prepared in 0.1M/0.1M KH2PO4NaOH (pH 5.5), and volumes within the 0.5-0 mL range of the 0.1M/0.1M KH2PO4-NaOH buffer pH 5.5 (fixed volume of 2 mL). The mixture s were mixed thoroughly and kept at 4°C for at least 15 min before fluorescence scanning. Three replicates were performed for each COC concentration tested (from 0 to 1.0 mg L-1). When analyzing eluates from urine after


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SPE (section 2.6), the eluate (100 µL) was mixed with 1.5 mL of MIP-QDs solution (200 mg L-1) and 0.4 mL of 0.1M/0.1M KH2PO4-NaOH (pH 5.5) buffer, and fluorescence was measured after 15 min. Standard addition calibration Standard addition calibration was performed by mixing 1.5 mL of MIP-QDs solution (200 mg L-1) with a fixed volume of urine of 100 µL, volumes within the 0-0.4 mL range of COC previously prepared in 0.1M/0.1M KH2PO4-NaOH (pH 5.5), and volumes within the 0.4-0 mL range of the 0.1M/0.1M KH2PO4-NaOH buffer pH 5.5 (fixed volume of 2 mL, urine dilution 1:20). Measurements (three independent replicates for each concentration level within the 0 to 1.0 mg L-1 range) were performed after 15 min (mixtures stored at 4°C).

Results and discussion Optimization of operating conditions Optimal fluorescence quenching response of MIP-QDs was achieved by testing the effect of the pH, the concentration of MIP-QDs, and the interaction time between MIP-QDs and analytes. COC and metabolites (BZE and EME) are slightly alkaline in an aqueous medium and the MIP-QDs were suspended in aqueous (0.1M/0.1M KH2PO4/NaOH) buffer solutions at fixed acid pHs to allow an efficient interaction with the composite nanoparticles. Experiments for studying the pH used several 1.5 mL aliquots of MIP-QDs solutions (200 mg L-1) prepared in KH2PO4/NaOH buffer solutions at pHs of 5.0, 5.5, 6.0, and 6.5. A final volume of 2.0 mL was completed by adding volumes of COC (template) and BZE (major metabolite in urine) solutions (10 mg L-1 each one) within the 0 – 0.20 mL range (prepared in KH2PO4/NaOH buffer at the tested pH), and volumes of the KH2PO4/NaOH buffer at the selected pH (from 0.50 to 0.30 mL). After a delay time of 10 min to allow COC/BZE and MIP-QD interaction,


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the fluorescence was recorded. Figure 1 (three measurements for each COC/BZE concentration and pH tested) shows that the highest slopes (the highest fluorescence quenching) were obtained for experiments at the lowest pHs tested (5.0 and 5.5). A pH of 5.5 was finally selected. The effect of the MIP-QDs concentration was studied within the 100-400 mg L-1 range. For all cases, MIP-QDs were prepared in KH2PO4/NaOH buffer (pH 5.5), and a fixed volume of 1.5 mL was used. As in preceding experiments, volumes ranging from 0 to 0.20 mL of solutions containing 10 mg L-1 COC/BZE (prepared in KH2PO4/NaOH buffer, pH 5.5), and volumes of KH2PO4/NaOH buffer pH 5.5 (from 0.50 to 0.30 mL) were added (COC/BZE concentrations within the 0.0 – 1.0 mg L-1 range), and the fluorescence emission was recorded after 10 min. Results in triplicate (plotted in Figure 2) show higher slopes for the use of MIP-QDs concentrations of 200 and 400 mg L-1, thus a concentration of 200 mg L-1 was selected. Finally, the interaction time between the analytes (COC and BZE) and MIP-QDs was studied by recording the fluorescence of 1.5 mL of 200 mg L-1 of MIP-QDs and COC/BZE solutions at 0.50 mg L-1 (all solutions prepared in KH2PO4/NaOH buffer, pH 5.5) after every one minute (from 10 min to 20 min). Results show a constant fluorescence emission within the 14-20 min range, thus an interaction time (delay time) of 15 min was selected. Imprinting effect and selectivity studies Experiments were performed for studying the binding affinity properties of specific imprinted binding sites (MIP-QDs) and nonspecific sites (NIP-QDs) for several template (COC) and metabolite (BZE and EME) concentrations under optimized operating conditions. The quench amount of binding COC by MIP-QDs, expressed as the (F.I.0 – F.I.)/F.I.0 ratio (F.I.0 is the fluorescence in absence of quencher, and F.I. is the fluorescence when using a fixed quencher concentration) at several COC concentrations, is given in Figure 3. It can be seen that the


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ratio is increased with the concentrations of COC in solution; whereas, significant differences were not observed when using NIP-QDs and COC mixtures. Similar results could be observed when using BZE and EME solutions (Figure 3). As shown in Figure 3, the quench amount of binding analyte (COC, BZE and EME) by MIP-QDs and the analyte concentration can be fit to a polynomial regression model (order two). The Stern–Volmer constants (KSV), defined as the slope of the linear curves F.I.0/F.I ratio versus the quencher (COC, BZE and EME) concentration, were 0.0728, 0.0354, and 0.0309 for COC, BZE and EME, respectively, when using the MIP-QDs probe. However, lower KSV constant values were obtained when using the NIP-QDs (0.0032, 0.0045, and 0.0034 for COC, BZE, and EBME, respectively), which also confirms specific interaction between analytes and MIP-QDs. The imprinting factor (IF), expressed as the KSV,MIP/KSV,NIP ratio (KSV,MIP and KSV,NIP are the Stern–Volmer constants obtained for MIP-QDS and NIP-QDs, respectively) was used to evaluate the imprinting effect. The high values for this ratio for COC, BZE, and EME (Table 1) prove the specific interaction of COC and its metabolites through the recognition cavities in the MIP layer. Regarding selectivity, other drugs of abuse/metabolites such as MOR, COD, and 6-MAM (heroin abuse); and ∆9-THC, ∆9-THC-OH, CBD, and CBN (cannabis abuse) within the 0.0 – 3.0 mg L-1 range were mixed with MIP/NIP-QDs under optimum conditions. As shown in Table 1, the selectivity factors, expressed as the ratio between the Stern–Volmer constant obtained for MIP-QDs using COC (template) as a quencher (KSV(MIP)COC) and the Stern–Volmer constants obtained for MIP-QDs using the other drugs/metabolites (KSV(MIP)Q) were lower for BZE and EME; whereas, they were high for other drugs/metabolites. This confirms that the prepared material is highly selective for COC and its metabolites. Calibration. Matrix effect


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The existence of matrix effect was established by comparing external calibrations, and also standard addition calibrations. In the latter case, several dilution ratios were tested (1:10; 1:20; and 1:40), implying the use of 200, 100, and 50 µL of a drug-free urine sample, respectively (total volume of 2.0 mL using 1.5 mL aliquots of 200 mg L-1 MIP-QDs solutions). COC concentration levels cover the 0 – 1.0 mg L-1 range (each concentration level in triplicate), and the three calibration experiments were performed in three different days. A significant decrease on slope calibration respect to external calibration (a mean slope of 273 ± 37, for three independent external calibrations) was obtained when urine is present at high dilution ratios (143 ± 25 and 162 ± 30, also three independent standard addition calibration, for 1:20 and 1:40, respectively). The decrease on slope is higher when preparing standard addition calibrations by using lower dilution factors such as 1:10 (49 ± 15, n=3). These findings imply that matrix effect is important and analysis must be performed using the standard addition technique. A 1:20 dilution ratio was finally selected because slope calibration was relatively high (143 ± 25), and this dilution is adequate for assessing low concentrations COC and metabolites. To diminish matrix effect, an alternative procedure based on using an SPE stage (section 2.6.) before measurements was used. Drug-free urine aliquots (1.0 mL) were spiked with increasing COC concentrations and were subjected to the optimized SPE in triplicate. The eluates (100 µL) were mixed with 1.5 mL of MIP-QDs solution (200 mg L-1), and 0.4 mL of 0.1M/0.1M KH2PO4-NaOH (pH 5.5), and the mixtures were subjected to fluorescence determination (2.7.1) for obtaining the calibration curve. Three calibration curves after SPE were obtained in three different days, and the mean slope obtained (263 ± 37) is the same as the slope obtained when using the external calibration (273 ± 45). Therefore, direct urine analysis (1:20 dilution) requires the standard addition calibration to perform the


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determination; whereas, an external calibration can be used if the urine sample is previously subjected to a conventional SPE process. Limit of detection/quantification The limit of detection (LOD) was established through the 3σ criterion (σ is the standard deviation of eleven measurements of a blank), while the limit of quantification (LOQ) was established through the 10σ criterion.35 A K2HPO4/NaOH buffer (pH 5.5) blank was measured twelve times, and three times (LOD) or ten times (LOQ) the standard deviation (n = 12) of the measurements were divided by the mean slope of the standard addition graph (direct method, 1:20 dilution), or by the mean slope of the calibration graph (SPE based procedure). Calculated LODs referred to the original urine sample were 76 µg L-1 (direct method) and 4.2 µg L-1 (SPE based procedure); whereas, LOQs were 250 µg L-1 (direct method) and 14 µg L-1 (SPE based procedure). Better sensitivity was obtained when using the SPE based method because a pre-concentration factor of 10 was achieved, and because the slope of the external calibration was 2 fold higher than the slope of the standard addition calibration. Regarding the direct method, only the LOD was lower than the cut-off values for confirmation analysis of cocaine abuse in urine (150 µg L-1 for BZE) established by the European Workplace Drug Testing Society,36 and the American Substance Abuse and Mental Health Services Administration.37 However, both the LOD and LOQ for the SPE based method were lower than established cut-off values.36,37 Precision and accuracy Intra–day precision and inter-day precision of both methods (direct method and SPE based method) were assessed. Intra-day assay (direct method) implied the preparation of three standard addition graphs (drug-free urine, dilution 1:20) in three different days. The first standard addition graph was obtained by replicating the lowest COC concentration level (0.25 mg L-1) seven times; whereas, the other COC concentration levels were replicated twice.


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Similarly, second and third standard addition calibrations were obtained by replicating the intermediate (1.0 mg L-1) and the highest (2.0 mg L-1) COC concentration seven times (the remaining COC concentration levels were replicated twice). Intra-day precision was good since RSD values (Table 2) are below 10%. Inter-day precision (direct method) was assessed by preparing seven standard addition calibrations in seven different days, replicating each COC concentration level twice. RSD values for all COC concentration levels are listed in Table 2, and RSDs are below 14%. Regarding the SPE based method, three different calibrations were performed in three different days (one of the COC concentration levels was replicated seven times, and the other concentration levels were replicated twice) for assessing intra-day precision. RSD values lower than 10% (Table 2) were obtained, which implies good intra-day precision. Similarly, inter-day precision was established by preparing seven calibrations in different days (each COC concentration level in duplicate). RSD values lower than 15% (Table 2) allow us to conclude that good inter-day precision was achieved. Table 2 also lists the analytical recovery (intra-day/inter-day accuracy). As can be seen, values were close to 100% for all cases (direct method and SPE based method), which implies good intra-day and inter-day accuracy. Finally, accuracy was also assessed by analyzing an FDT +25% - In vitro Diagnosticum control urine sample (referenced BZE concentration of 34.80-58.60 µg L-1) by using the SPE based method. The analysis of this control sample was not possible using the direct method because the certified concentration was lower than the LOQ offered by this method. After subjecting the control material to the SPE procedure (four times), analysis was performed using external calibrations prepared with COC and with BZE as calibrants. The COC (BZE) concentration obtained was 43.88 ± 4.3 µg L-1 when using COC as a calibrant, and 40.90 ± 5.5 µg L-1 when using BZE as a calibrant.


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Good agreement between found concentrations and the reference concentration range was therefore achieved. Analysis of urine samples Eight urine samples from poly-drug abusers were analyzed with the proposed fluorescent probe using the direct method (urine dilution and standard addition calibration), and the SPE based method (external calibration). In addition, samples were also analyzed using the COBAS INTEGRA 400 analyzer, and also by HPLC-MS/MS.33 Each sample was subjected to the proposed method in triplicate, except when using the COBAS INTEGRA 400 analyzer (screening results for one individual aliquot). Results (Table 3) show good agreement between total COC concentration after applying the two proposed fluorescent probe methods and the conventional screening analyzer. In addition, results after HPLC-MS/MS (concentration as a sum of COC, BZE, and EME concentrations) were also in good agreement with those obtained after applying the fluorescent probe and the COBAS INTEGRA 400 analyzer. These findings have been confirmed after using a t-paired test. As shown in Table 3, the calculated t-values for each pair of methods are lower than the tabulated t-value of 2.4 (95% confidence level, seven degrees of freedom), which confirms that results offered by the MIP-QDs fluorescent probe are statistically similar to those obtained after conventional screening (COBAS INTEGRA 400 analyzer) and confirmation (HPLC-MS/MS) procedures.

Conclusions The developed MIP-QD fluorescence probe has been found to be highly sensitive and selective for determining COC and metabolites in complex clinical samples such as urine. Quenching of the fluorescence emitted by the MIP-QD nanoparticles led to LOD values (0.076 and 0.0042 mg L-1 for the direct method and SPE based method, respectively) lower


15


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Page 16 of 27

than the cut off values for establishing cocaine abuse through urine analysis. Matrix effect was found to be important when performing direct analysis (1:20 urine dilution); nevertheless, the developed SPE based procedure avoids matrix effect and improves sensitivity. However, the developed direct analysis results simple and adequate when analyzing urine samples in cases of cocaine overdose. The developed procedures provide results similar to those obtained when using conventional screening methods, such as those based on immunoassays, and also similar to those obtained when using sophisticated techniques for confirmation analysis, such as HPLC-MS/MS. Molecular recognition combined with the sensitivity of the prepared MIP-QD composite therefore offers great versatility which must be fully investigated when analyzing other complex samples of forensic/clinical interest.

Acknowledgments The authors wish to thank the Dirección Xeral de I+D – Xunta de Galicia (Project number 10CSA209042PR) for financial support.


16

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Stern–Volmer constants (MIP-/NIP-QDs) for COC, BZE, and EME (and other drugs of abuse/metabolites), and imprinting and selectivity factors. Imprinting factor

Selectivity factor KSV,MIP(COC)/KSV,MIP(Q) KSV,MIP(COC)/KSV,NIP(Q)

KSV,MIP

KSV,NIP

KSV,MIP/KSV,NIP

COC

0.073

0.0032

23

-----

23

BZE

0.035

0.0045

7.9

2.1

16

EME

0.031

0.0034

9.1

2.4

21

MOR

0.00057

0.011

0.050

121

6.5

COD

0.00091

0.012

0.080

81

6.1

6-MAM

0.0088

0.011

0.81

8.3

6.7

∆9-THC

0.0047

0.015

0.31

16

4.8

∆9-THC-OH

0.0000

0.00066

0.00

∞

104

CBN

0.0038

0.0020

1.9

19

36

CBD

0.0076

0.0051

1.0

9.6

14

Q = BZE, EME, MOR, COD, 6-MAM, ∆9-THC, ∆9-THC-OH, CBN, CBD


17


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Page 18 of 27

Table 2. Intra–day precision and inter-day precision (RSD/%), and intra-day analytical recovery and inter-day analytical recovery (AR/%) of the method.

Direct method (1:20 dilution) Added concentration / mg L–1 RSD / %a RSD / %b AR / %a

AR / %b

0.25

9

14

97 ± 8

102 ± 14

0.50

---c

13

---c

100 ± 13

1.0

7

10

99 ± 7

103 ± 10

1.5

---c

9

---c

99 ± 9

2.0

3

6

104 ± 3

98 ± 6

SPE based method 0.25

7

12

99 ± 7

106 ± 11

0.50

---c

9

---c

103 ± 9

1.0

4

6

102 ± 4

101 ± 6

1.5

---c

4

---c

96 ± 4

2.0

2

4

100 ± 2

100 ± 4

(a) Intra-day assay (n = 7); (b) inter-day assay (n = 7); (c) Not evaluated


18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. COC plus metabolites concentrations in urine samples after MIP-QDs fluorescent probe, COBAS INTEGRA 400 analyzer, and HPLC-MS/MS. [COC + BZE + EME] mg L-1 MIP-QDs fluorescent probe Sample

COBAS INTEGRA

Direct method (1:20

SPE based

HPLC-

code

400

dilution)

method

MS/MS

397

0.66

0.65 ± 0.02

0.61 ± 0.07

0.60 ± 0.08

410

0.79

0.73 ± 0.03

0.80 ± 0.09

0.81 ± 0.11

418

5.0

5.4 ± 0.03

6.1 ± 0.21

5.8 ± 0.3

445

5.0

6.5 ± 0.11

6.0 ± 0.17

6.4 ± 0.5

451

5.0

6.4 ± 0.21

6.4 ± 0.26

6.8 ± 0.4

454

0.67

0.68 ± 0.02

0.76 ± 0.06

0.65 ± 0.12

477

3.6

3.4 ± 0.06

4.0 ± 0.20

3.2 ± 0.3

488

0.49

0.51 ± 0.02

0.46 ± 0.05

0.46 ± 0.08

COBAS INTEGRA 400 / Direct method

tcal = 1.6

COBAS INTEGRA 400 / SPE based method

tcal = 2.3

COBAS INTEGRA 400 / HPLC-MS/MS

tcal = 1.6

Direct method / SPE based method

tcal = 0.79

Direct method / HPLC-MS/MS

tcal = 0.70

SPE based method / HPLC-MS/MS

tcal = 0.36


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Page 20 of 27

Figure captions

Figure 1. Effect of pH on fluorescence quenching by several COC (a) and BZE (b) concentrations

Figure 2. Effect of the concentration of MIP-QDs on fluorescence quenching by several COC (a) and BZE (b) concentrations

Figure 3. Effect of analyte concentration on the fluorescence quenching of MIP-QDs [COC (♦), BZE (■), and EME (▲)] and NIP-QDs [COC (◊), BZE (□), and EME (∆)]


20

Page 21 of 27

Figure 1 5500

(a)

pH 5.0 F.I = -292.3 [COC] + 5133 R² = 0.998 pH 5.5 F.I. = -305.4 [COC] + 5071 R² = 0.996 pH 6.0 F.I. = -219.5 [COC] + 4625 R² = 0.996 pH 6.5 F.I. = -211.5 [COC] + 4351 R² = 0.997

F.I.

5000

4500

4000 0.0

0.2

0.4

0.6

0.8

1.0

[COC] mg L-1 5500

(b)

pH 5.0 F.I. = -132.1 [BZE] + 5132 R² = 0.997 pH 5.5 F.I. = -124.0 [BZE] + 5051 R² = 0.997 pH 6.0 F.I. = -81.54 [BZE] + 4833 R² = 0.997 pH 6.5 F.I. = -90.28 [BZE] + 4655 R² = 0.995 5000

F.I.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4500

4000 0.0

0.2

0.4

0.6

0.8

1.0

[BZE] mg L-1


21


Figure 2 5000

(a) [MIP-QDs] 100 mg L-1 F.I. = -256.7 [COC] + 4641 R² = 0.997 [MIP-QDs] 200 mg L-1 F.I. = -272.5 [COC] + 4344 R² = 0.997 [MIP-QDs] 400 mg L-1 F.I. = -300.0 [COC] + 4480 R² = 0.995

4800

F.I.

4600

4400

4200

4000 0.0

0.2

0.4

0.6

0.8

1.0

[COC] mg L-1 [MIP-QDs] 100 mg L-1 F.I. = -126.7 [BZE] + 4148 R² = 0.997 [MIP-QDs] 200 mg L-1 F.I. = 142.7 [BZE] + 4024 R² = 0.998 [MIP-QDs] 400 mg L-1 F.I. = 137.5 [BZE] + 4007 R² = 0.996 -

4200 4150

(b)

4100 4050

F.I.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 27

4000 3950 3900 3850 3800 0.0

0.2

0.4

0.6

0.8

1.0

[BZE] mg L-1


22

Page 23 of 27

Figure 3 0.10

(F.I.0 –F.I.)/F.I.0 =0.015[COC]2 +0.066[COC]+0.007 r2 =0.938 (F.I.0 –F.I.)/F.I.0 =0.012[BZE]2 +0.063[BZE]+0.002 r2 =0.956 (F.I.0 –F.I.)/F.I.0 =0.006[EME]2 +0.039[EME]+0.000 r2 =0.992

0.08

(F.I.0 – F.I.)/F.I.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.06 0.04 0.02 0.00 0.0

0.5

1.0

1.5

2.0

2.5

3.0

[COC/BZE/EME ] mg L-1


23


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Page 24 of 27

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[35] US Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research (2001). Guidance for Industry: Bioanalytical method validation, Rockville, MD, USA [36] European Workplace Drug Testing Society (2002) European Laboratory Guidelines for Legally

Defensible

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http://www.ewdts.org/guidelines/EWDTSGuidelines. pdf. Accessed October 8th 2015. [37] U.S. Substance Abuse and Mental Health Services Administration (2004) Mandatory Guidelines

for

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for TOC only


27

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