Nanofibrous sorbent for the determination of illicit substances in dried blood spot samples

Abstract

The invention relates to a nanofibrous sorbent for determining an illicit substance in dried blood spot samples.

Description

[0001] NANOFIBROUS SORBENT FOR THE DETERMINATION OF ILLICIT SUBSTANCES IN DRIED BLOOD SPOT SAMPLES

[0002] Technical Field of the Invention

[0003] The invention relates to a nanofibrous sorbent for the determination of illicit substances in dried blood spot samples, which enables the analysis of illicit substances in biological materials in the field of forensic toxicology under health sciences.

[0004] State of the Art of the Invention (Prior Art)

[0005] Illicit substance use is an important public health and public safety problem in the world and in Turkey. Geographically, Turkey is located between the production and consumption regions of illicit substances and is on an important transit route in terms of trade of these substances. In addition, the country is one of the countries directly affected by illicit substances due to its dense youth population.

[0006] The number of studies carried out on the detection of illicit substances is increasing day by day. The detection of illicit substances is in the form of the determination of the substances themselves or their metabolites formed as a result of metabolism in the tissues and samples taken from people suspected of substance use, and in seized substances. In recent years, scientific studies on the materials analyzed for these substances or their metabolites, the variety of products used, and the preferred methods have increased dramatically with advances in technology.

[0007] Although many different biological materials are used in forensic toxicological substance tests, blood is the most commonly used biological material due to reasons such as its wide range of analytical methods, the availability of a large number of published reference information, and easier pharmacological interpretation. Blood samples can be used to identify the substance the person was under the influence of, as well as to obtain data on the recent past (hours, days). Blood is one of the most preferred biological materials for qualitative and quantitative analysis, as it also gives an idea about pharmacological effects. However, taking blood samples is an invasive procedure and requires medical personnel to take the sample. In addition, blood is a biological material that is difficult to analyze and prepare. Hundreds of denatured proteins in the blood make analysis difficult and lead to the search for alternative techniques. Dried Blood Spot (DBS) is a term used to describe a technique involving the collection of a small volume of blood by piercing the fingertip or heel with a sterile disposable lancet and drying of the samples. In the analysis process, instead of venous blood drawn by healthcare personnel, capillary blood -DBS - that can be collected by any individual (such as laboratory staff, law enforcement, or traffic officers is a significant advantage. In order to avoid the limitations of substance analysis in blood, analysis techniques have been developed involving drying capillary blood samples using sorbents or cards.

[0008] These sorbents or cards have been developed since the 1900s and are not yet produced for this purpose in the country. When the general structure of these cards is examined, it is stated that they should be pH 5.7-7.5, contain 100% pure cotton fiber and 0.1% ash, and should not contain any other substance or solvent.

[0009] Certain standards have been introduced for sorbents developed to ensure accurate and reproducible absorption of blood samples and productions are made in accordance with these standards. Sorbents produced by brands such as Whatman Protein Saver 903, 226-1004 Perkin Elmer, Whatman BFC 180 filter paper, Guthrie Card, Ahlstrom are used for the determination of illicit substances in the blood. As an alternative to products developed for the purpose of collecting blood samples, there are also studies in which materials such as coarse filter paper and paper are used.

[0010] A major limitation of the dried blood spot technique is the quantitative determination phase of analytes. In this technique, a fixed area is usually determined on the commercial products sold for sampling. This approach is based on the assumption that blood spreads evenly when first used and provides a consistent area for a given sample volume. In some places, there may be unequal analyte / blood distribution.

[0011] Summary and Objects of the Invention

[0012] The primary object of the invention is related to the nanofibrous sorbent produced by the electrospinning method, which detects illicit substances in dried blood spot samples. The aim of the invention is to provide accurate, fast, and reliable results by developing nanofibers produced by electrospinning technique as an alternative to sample collection sorbents produced worldwide to determine the level of commonly abused illicit substances such as cannabis, amphetamine, morphine, cocaine in blood. In this way, selective and more cost-effective materials can be produced with the DBS technique for illicit substances, and the results obtained with the produced sorbents were compared with the results obtained with the sample collection sorbents on the market, and their suitability for use in illicit substance analysis was evaluated. It has been determined that the synthesized sorbents give results compatible with the commercially available sorbent (Whatman 903). A more effective, purpose-oriented, efficient product increases national competitiveness.

[0013] With this invention, a new analytical system and nanotechnological material have been developed to detect illicit substances threatening the health of individuals and public safety in the country in blood samples and to perform forensic and clinical monitoring. New easy-to-implement, less costly processes and services for people working in this field can be produced on a large scale.

[0014] The developed nanofibers and analytical method will enable the use of dried blood spot samples in the analysis of substance users in the Addiction Toxicology Laboratory, which serves in the fight against substance addiction in the country. The results to be obtained will contribute to the development of domestic commercial dried blood sorbents. illicit substance analysis of dried blood spot samples is routinely applied abroad and has the potential for widespread use in the country. The use of nanofibers, which are widely used in different fields, as sorbents will introduce a practical, targeted, technological new product. Nanotechnology applications and nanofiber production, where intensive studies have been carried out in recent years, are gaining importance day by day in terms of functional textile material production. In today's tough competitive conditions, nanotechnology is also used in the production of technical and functional textile materials. The electrospinning or spinning method used in the production of nanofibers is of great interest in terms of ease of application and the richness of polymers that can be used to obtain fibers. The most important advantage of the fibers produced with this technique is that the diameters are nano-sized and the polymers used in this method can be used both as melt and spinning solution. Nanofibrous surfaces have very fine fiber diameters in nanometer sizes and therefore very high surface area and porosity. Thus, nanofibers, especially those produced from hydrophilic polymers or subsequently hydrophilized, have a high liquid absorption capacity. For example, in the tests conducted, the water holding capacity of commercially used paper-based Whatman 903 was found to be approximately 230 wt%, while cellulose-based nanofibers were found to be in the range of 280-320 wt% depending on the thickness. Moreover, these nanofibrous sorbents have a very low thickness such as 0.04-0.07 mm. Since nanofibrous sorbents have a higher liquid absorption capacity, they trap the liquid they absorb into the sorbent and reduce the contamination problem to zero. Again, in comparative tests conducted with Whatman 903 sorbent, dyed water was dripped onto the sorbents, then covered with filter paper and kept under certain pressure for 1 minute. It has been determined that Whatman 903 sorbent contaminates the filter paper, but nanofibrous sorbents do not cause any contamination of the filter paper by keeping the liquid inside, despite being very thin. In summary, nanofibrous sorbents have the advantages of higher liquid absorption capacity, trapping the liquid in the sorbent, and no contamination problem in contact with various surfaces.

[0015] The developed analytical method will be able to serve institutions that conduct illicit substance analysis (such as Forensic Medicine Departments, the Forensic Medicine Institute, Addiction Institutes, the Criminalistics Laboratories of the General Directorate of Security, and Medical Biochemistry Laboratories) as well as institutions that need to perform substance analysis in suspicious cases (such as blood centers and the traffic units of law enforcement).

[0016] Description of the Drawings of the Invention

[0017] Fig. 1. Schematic representation of DBS sample preparation procedure

[0018] Fig. 2. SEM image of sample 1 comprising 17.5% CA

[0019] Fig. 3. SEM image of sample 2 comprising 17.5% CA Fig. 4. SEM image of sample 3 comprising 17.5% CA Fig. 5. SEM image of sample 4 comprising 15% CA Fig. 6. SEM image of sample 5 comprising 15% CA Fig. 7. SEM image of sample 6 comprising 15% CA Fig. 8. SEM image of sample 7 comprising 12.5% CA

[0020] Fig. 9. SEM image of sample 8 comprising 12.5% CA

[0021] Fig. 10. SEM image of sample 9 comprising 12.5% CA

[0022] Fig. 11. SEM image of sample 10 comprising TPU-DMF

[0023] Fig. 12. SEM image of sample 11 comprising TPU-DMF

[0024] Fig. 13. SEM image of sample 12 comprising TPU-DMF

[0025] Fig. 14. SEM image of Whatman 903 filter paper before sample application

[0026] Fig. 15. Post-treatment SEM image of nanofiber comprising 15% CA shot for 24 hours Fig. 16. Post-treatment SEM image of nanofiber comprising 15% CA shot for 48 hours Fig. 17. Post-treatment SEM image of nanofiber comprising TPU-DMF shot for 24 hours

[0027] Fig. 18. Post-treatment SEM image of nanofiber comprising TPU-DMF shot for 48 hours

[0028] Fig. 19. Post-treatment SEM image of nanofiber comprising CA- TPU-DMF

[0029] Fig. 20. Post-treatment SEM image of Whatman 903 filter paper

[0030] Description of the References in the Drawings

[0031] 1001. Adding to 50 pL of sample taken from the blood pool 100 ng / mL of a stable, nondesiccant standard substance with a high equivalent weight

[0032] 1002. Applying 50 pL of blood sample on Whatman 903 and nanofibrous sorbents, drying at room temperature for 2 hours, protected from sunlight

[0033] 1003. Cutting the spots formed after the drying process with the help of scissors / puncher

[0034] 1004. Adding 1 mL of methanokacetonitrile (40:60 v / v) mixture and 25 ng / mL of internal standard mixture in methanol to blood samples

[0035] 1005. Mixing by vortexing for 10 seconds

[0036] 1006. Holding at room temperature in an ultrasonic bath for 30 minutes

[0037] 1007. Removing sorbents from Eppendorfs with the help of forceps

[0038] 1008. Centrifuging samples for 10 minutes at 4100 rpm

[0039] 1009. Transferring the entire supernatant (1 mL) to a clean Eppendorf

[0040] 1010. Evaporating samples at room temperature under nitrogen

[0041] 1011. Adding 150 pL of mobile phase mixture (Mobile phase A: Mobile phase B 85:15 (v / v)) to the samples containing residues and transferring these to Eppendorf. 1012. Centrifuging at 14000 rpm for 5 minutes, transferring samples to a vial and submitting to the LC-MS / MS device for analysis

[0042] Detailed Description of the Invention

[0043] The invention is nanofibrous sorbents to be used for taking dried blood spot samples and developed as an alternative to commercial paper product. In double layer nanofiber sorbents, nanofiber diameters can vary between 100-800 nm and said sorbents are produced as single or double layer according to the electrospinning method. Single-layer membranes are cellulose-based, while double layer ones contain thermoplastic polyurethane (TPU) nanofibers in the bottom layer and cellulose nanofibers in the top layer. In the invention, double layer nanofibrous sorbents contain hydrophilic cellulose-based nanofibers in the top layer and hydrophobic TPU nanofibers in the bottom layer. In this way, the liquid absorbed from the top layer is prevented from reaching the bottom surface. In the tests performed by dripping dyed water on the sorbents placed on the filter paper, it was observed that the liquid transfer to the bottom surface was high after liquid dripping to the surface in Whatman 903 and single-layer nanofibrous sorbents and the liquid reached the filter paper at the bottom. However, in double layered ones, it has been observed that the liquid cannot reach the bottom surface and does not contaminate the filter paper thanks to the TPU layer at the bottom. In addition, the nanofibrous structure of nanofibrous membranes is degraded during the extraction phase, and is disposable, thus preventing fraudulent interference in toxicological analyzes.

[0044] Selection, Collection, Storage of Blood Samples

[0045] Blood samples were selected from those that met the blood center sample collection criteria, were in protective packaging, and were close to disposal. Samples were collected in a blinded manner. If there is contamination or degradation in the blood samples, they were excluded from the study and destroyed under appropriate conditions. Samples were stored at+4°C. Method of Working with Blood Samples

[0046] A pool was created from samples taken from the blood center. Illicit standard substances (morphine, amphetamine, 3,4-methylenedioxy-N-methylamphetamine (MDMA), cocaine metabolite benzoylecgonine and cannabis metabolite 11 -nor-9- carboxy-THC (D9-THC-COOH) were added onto 50 pL of the sample taken from the pool at 100 ng / mL. 1 mL methanokacetonitrile (40:60 v / v) mixture and 25 ng / mL internal standard mixture in methanol were added to the blood samples and mixed by vortexing for 10 seconds. Samples were centrifuged for 10 minutes at 4100 rpm and the entire supernatant (1 mL) was transferred to a clean tube. Samples were evaporated at room temperature under nitrogen. 150 pL of mobile phase mixture (Mobile phase A: 0.01% formic acid, 5 mM ammonium formate buffer in water and Mobile phase B: acetonitrile 85:15 (v / v)) was added to the samples containing residues and the samples were transferred to an Eppendorf. After centrifugation at 14000 rpm for 5 minutes, the samples were transferred to a vial and submitted to liquid chromatography sequential mass spectrometry (LC-MS / MS) device for analysis.

[0047] Method of Working with Dried Blood Spot Samples

[0048] A method for use in the determination of illicit substances from dried blood spot samples comprises the process steps of;

[0049] • adding to 50 pL of sample taken from the blood pool 100 ng / mL of a stable, non-desiccant standard substance with a high equivalent weight (1001),

[0050] • applying 50 pL of blood sample on Whatman 903 and nanofibrous sorbents, drying at room temperature for 2 hours, protected from sunlight (1002),

[0051] • cutting the spots formed after the drying process with the help of scissors / puncher (1003) and adding 1 mL methanokacetonitrile (40:60 v / v) mixture and 25 ng / mL internal standard mixture in methanol to the blood samples (1004) and mixing by vortexing for 10 seconds (1005),

[0052] • holding at room temperature in an ultrasonic bath for 30 minutes (1006),

[0053] • removing sorbents from Eppendorfs with the help of forceps (1007) • centrifuging samples for 10 minutes at 4100 rpm (1008) and transferring the entire supernatant (1 mL) to a clean Eppendorf (1009),

[0054] • evaporating samples at room temperature under nitrogen (1010),

[0055] • adding 150 pL of mobile phase mixture (Mobile phase A: Mobile phase B 85:15 (v / v)) to the samples containing residues and transferring these to Eppendorf (1011),

[0056] • centrifuging at 14000 rpm for 5 minutes, transferring samples to a vial and submitting to the LC-MS / MS device for analysis.

[0057] 100 ng / mL of a stable, non-desiccant standard substance with a high equivalent weight was added to 50 pL of sample taken from the blood pool (1001), 50 pL of blood sample was applied on Whatman 903 and nanofibrous sorbents, and dried at room temperature for 2 hours, protected from sunlight (1002), The spots formed after the drying process were cut with the help of scissors / puncher (1003) and 1 mL methanol :acetonitrile (40:60 v / v) mixture and 25 ng / mL internal standard mixture in methanol were added to the blood samples (1004) and mixed by vortexing for 10 seconds (1005). It was held at room temperature in an ultrasonic bath for 30 minutes (1006). The sorbents were removed from Eppendorfs with the help of forceps (1007). The samples were centrifuged for 10 minutes at 4100 rpm (1008) and the entire supernatant (1 mL) was transferred to a clean Eppendorf (1009). The samples were evaporated at room temperature under nitrogen (1010). 150 pL of mobile phase mixture (Mobile phase A: Mobile phase B 85:15 (v / v)) was added to the samples containing residues and these were transferred to Eppendorf (1011). It was centrifuged at 14000 rpm for 5 minutes, the samples were transferred to a vial and submitted to the LC-MS / MS device for analysis (1012).

[0058] Validation Studies

[0059] In the method validation phase for blood and DBS; selectivity, accuracy, lower limit of detection (LOD), lower limit of quantification (LOQ), linearity, precision, accuracy, reproducibility, stability and carry over effect parameters were systematically performed with a plan in accordance with the “Standard Practices for Method Validation in Forensic Toxicology” (SWGTOX) guidelines published in the field of forensic toxicology (Scientific Working Group for Forensic T oxicology (SWGTOX), 2017). Obtaining Nanofibrous Sorbents by Electrospinning Method

[0060] The electrospinning method used in the production of nanofibers is of great interest in terms of ease of application and the richness of polymers that can be used to obtain 5 fibers. The most important advantage of the fibers produced with this technique is that the diameters are nano-sized and the polymers used in this method can be used both in melt and solution form in the spinning process. In this study, cellulosic and cellulosic / synthetic fiber blended nanofibrous surfaces were developed by using the electrospinning method to synthesize micro / nano-structured papers proposed for the 0 formation of a dried blood spot with appropriate standards. When the produced sorbents were compared to other sorbents produced in accordance with the standards in the analysis of illicit substances in the blood, compatible results were obtained and it was seen that they could be used as an alternative. The sampling performance of the developed nanofibrous sorbents is the same as that of cellulosic paper-based 5 commercial sorbents. It has been observed that the taken samples spread more evenly over a smaller area in nanofibrous sorbents.

[0061] All nanofiber synthesis studies were carried out at room temperature. Firstly, studies were carried out to obtain cellulose acetate (SA) and thermoplastic polyurethane (TPU) 0 based nanofibers.

[0062] SA solutions are prepared by dissolving an appropriate amount of SA in Dimethylacetamide (DMAC) / Acetone mixture (2:1). SA concentrations were selected as 12.5%, 15%, and 17.5%. For TPU, a fixed polymer concentration of 10% was used 5 and an electrospinning solution was prepared by dissolving TPU in DMF.

[0063] The parametric study plan applied for electrospinning solutions and working conditions is shown in Table 1 . 0 Table 1. T rials with electrospinning method

[0064] SA: Cellulose acetate, TPU: Thermoplastic polyurethane, DMF: Dimethylformamide,

[0065] DM AC: Dimethylacetamide

[0066] 5 Electrospinning was performed at these parameters and the morphology of the obtained nanofibrous surfaces was examined by SEM analysis. Optimum SA and TPU nanofiber spinning conditions with bead-free and smooth nanofibers were determined. From the images obtained, the diameters of the fibers were measured 50 times using the Image J program and the averages were calculated. It was decided to use the parameters of sample no. 5 for SA and sample no. 11 for TPU in Table 1 .

[0067] According to the electrospinning parameters determined as a result of the preliminary trials, electrospinning was performed for 24 and 48 hours and SA and TPU nanofibrous membranes / sorbents at sufficient thicknesses were produced. Furthermore, in addition to these single-layer nanofibers, a nanofibrous membrane has also been produced with SA on the bottom surface and TPU on the top surface. For this, firstly, electrospinning was done with SA solution for 24 hours, and then spinning was done with TPU solution for 24 hours without removing the produced SA nanofibers from the cylinder collector. The expression "mix" is used in the text for this double layer membrane / sorbent obtained.

[0068] After 24 and 48 hours of electrospinning, deacetylation was performed to convert the SA in the obtained membranes into cellulose. For this purpose, the membranes were kept in 0.05 M NaOH solution in 1 L of water for 30 hours. They were then washed with distilled water until pH 7 and dried in an oven at 50-60 °C for 5 hours.

[0069] Sorbent Comparison

[0070] The results of the analytical methods developed for blood and DBS were compared statistically. Real samples and samples taken from the blood pool were added with the standard substance and analyzed using the Whatman 903 filter paper and 5 different synthesized nanofiber dried blood spot methods. The results of the standard are proportioned to the internal standard. The results were statistically evaluated by using Spermann correlation coefficients from nonparametric tests with SPSS 25.0 program.

[0071] Using the developed analytical methods, the suitability of the interchangeable use of blood and DBS matrices was statistically analyzed. For this, the results of the comparison study were used. Results are negative for all analytes known to be negative. Therefore, positive results for all analytes were evaluated. Correlation tests were performed for a total of 58 positive substance results. Since added standard substance values did not fit the normal distribution theoretically, Sperman correlation coefficient, one of the nonparametric tests, was evaluated; r= 0.949 was found for blood and DBS samples. This value being positive and close to 1 indicates that when one variable increases, the other one also increases and that there is a strong positive correlation.

[0072] The synthesized 5 nanofibers were studied by DBS method and compared with the results of the analysis using Whatman 903 filter paper. It was observed that the most compatible results to the filter paper used as standard were cellulose-based (singlelayer) and cellulose / thermoplastic polyurethane-based (double-layer) nanofibers synthesized in 48 hours.

[0073] Fig. 1 , 2 and 3 show SEM images of CA nanofibers spun with 17.5% CA solution and with flow rates of 3, 4 and 5 mL / h, respectively (Samples no. 1 , 2 and 3 from the nanofiber sorbent development studies shown in Table 1). The diameters of the obtained nanofibers were measured as 480.6, 589.9 and 403.2 nm for the feed flow rate of 3, 4, and 5 mL / s, respectively. Due to the high concentration of polymers, thicker nanofibers were obtained compared to other samples. Fig. 4, 5 and 6 show SEM images of CA nanofibers spun with 15% CA solution and with flow rates of 3, 4 and 5 mL / h, respectively. (Samples no. 4, 5, and 6 from the nanofiber sorbent development studies shown in Table 1). The fibers obtained were observed to be smooth and bead-free, and the fiber diameters were measured as 217.0, 358.6, and 357.9 nm, respectively. On the other hand, in sample no. 4 (low feed flow rate), fine- thick fiber formation was more common. Fig. 7, 8 and 9 show SEM images of CA nanofibers spun with 12.5% CA solution and with flow rates of 0.5, 0.6, and 0.7 mL / h, respectively. (Samples no. 7, 8, and 9 from the nanofiber sorbent development studies shown in Table 1). The diameters of the obtained fibers were measured as 125.8, 118.4, and 117.4 nm respectively. Although the fibers are thin, bead formation has been observed in places. For this reason, sample no. 5, which does not have bead formation and has a more homogeneous and thin fiber diameter, was selected as the most suitable from cellulose acetate studies and it was decided to perform spinning for 24 and 48 hours for the study of DBS samples. SEM images of TPU nanofibers are shown in Fig. 10, 11 and 12. SEM images of CA nanofibers spun with flow rates of 0.5, 0.6, and 0.7 mL / h, respectively, are shown. (Samples no. 10, 11 , and 12 from the nanofiber sorbent development studies shown in Table 1). The diameters of fibers were measured as 443.7, 233.9, and 275.9 nm respectively. In sample no. 10, the fibers are thicker and their surfaces are rough due to the high flow rate, and in sample no. 12, although the fibers are thin, it was observed that there were irregularities and jumps during fiber spinning. For this reason, since smooth and fine nanofibers could be obtained and fiber spinning was smoother, sample no. 11 was chosen to be spun for 24 and 48 hours for the study of DBS samples.

[0074] In order to produce membranes of two different thicknesses, nanofibrous membranes were produced with SA and TPU fiber spinning for 24 and 48 hours to study DBS samples. In addition, a double layered membrane referred to as "mix", which is SA in the bottom layer and TPU in the top layer, is also produced. The membranes containing SA were deacetylated and the conversion of SA into cellulose was ensured. Here, cellulose acetate is hydrophobic due to the acetate group in its structure and has low water absorption. For this reason, by deacetylation, the structure is converted into cellulose and thus a hydrophilic structure capable of absorbing a high amount of water is obtained. In addition, since cellulose does not dissolve in almost all commonly used organic solvents, conversion to cellulose by deacetylation is necessary to maintain the structure and prevent contamination in further testing stages. As a result, the 5 final membranes to be used in DBS trials are listed below:

[0075] - SA nanofibrous membrane - 24-hour spinning

[0076] - SA nanofibrous membrane - 48-hour spinning

[0077] - TPU nanofibrous membrane - 24-hour spinning

[0078] - TPU nanofibrous membrane - 48-hour spinning

[0079] - TPU / SA nanofibrous membrane - 48-hour spinning (mix)

[0080] The nanofibrous sorbent of the invention comprises thermoplastic polyurethane nanofibrous membrane and / or cellulose acetate nanofibrous membrane. In an embodiment of the invention, said nanofibrous sorbent comprises a 10% thermoplastic polyurethane nanofibrous membrane and / or a 15% cellulose acetate nanofibrous membrane. In another embodiment of the invention, said nanofibrous sorbent comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 48 hours and a cellulose acetate nanofibrous membrane produced by electrospinning for 48 hours. In another embodiment of the invention, said nanofibrous sorbent comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 24 hours or a cellulose acetate nanofibrous membrane produced by electrospinning for 24 hours. In an embodiment of the invention, said nanofibrous sorbent comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 48 hours or a cellulose acetate nanofibrous membrane produced by electrospinning for 48 hours.

[0081] COMPARISON RESULTS IN DEVELOPED NANOFIBROUS SORBENTS

[0082] For the comparison study; 4 real samples, negative samples, and samples added with different concentrations of standard substance mixture were prepared. Samples were studied by being applied to nanofibers synthesized by validated blood method and DBS method. Blood samples obtained from substance users and living persons were collected in yellow-capped, vacuum-sealed, plastic tubes containing separator gel. After centrifugation, serum samples were obtained and these samples were studied. Tables 2-6 show the results of the study of illicit substances morphine, amphetamine, 3,4-methylenedioxy-N-methylamphetamine (MDMA), benzoylecgonine, and 11 -nor-9- carboxy-THC (A9-THC-COOH) analytes by blood method, the result of the study of dried blood spot samples with standard paper (Whatman), and the result of the study of dried blood spot samples with 5 different nanofiber sorbents synthesized. Said results show the results of the study of illicit substances morphine, amphetamine, 3,4- methylenedioxy-N-methylamphetamine (MDMA), benzoylecgonine, and 11 -nor-9- carboxy-THC (D9-THC-COOH) analytes by blood method, the result of the study of dried blood spot samples with standard paper (Whatman), and the result of the study of dried blood spot samples with 5 different nanofiber sorbents synthesized.

[0083] Table 2. Comparison results with developed nanofibrous sorbents-Morphine (MOR)

[0084] No answer (n / a): The sample could not be submitted to the device, no analysis was performed. Table 3. Comparison results with developed nanofibrous sorbents-Amphetamine

[0085] (AMP)

[0086]

[0087] No answer (n / a): The sample could not be submitted to the device, no analysis was performed. Table 4. Comparison results with developed nanofibrous sorbents-3,4-methylenedioxy-

[0088] N-methylamphetamine (MDMA)

[0089] No answer (n / a): The sample could not be submitted to the device, no analysis was performed. Table 5. Comparison results with developed nanofibrous sorbents-Benzoylecgonine

[0090] (BZG)

[0091] No answer (n / a): The sample could not be submitted to the device, no analysis was performed. Table 6. Comparison results with developed nanofibrous sorbents-11 -nor-9-carboxy-

[0092] THC (THC-COOH)

[0093]

[0094] No answer (n / a): The sample could not be submitted to the device, no analysis was performed. The suitability of using blood and DBS matrices interchangeably was statistically analyzed. For this, the results of the comparison study were used. Results are negative for all analytes known to be negative. Therefore, positive results for all analytes were evaluated. Correlation tests were performed for a total of 58 positive substance results. Since added standard substance values did not fit the normal distribution theoretically, Sperman correlation coefficient, one of the nonparametric tests, was evaluated; r= 0,949 was found for blood and DBS samples. This value being positive and close to 1 indicates that when one variable increases, the other one also increases and that there is a strong positive correlation.

[0095] The synthesized 5 nanofibers were studied by DBS method and compared with the results of the analysis using Whatman 903 filter paper.

[0096] Table 7. Sperman correlation coefficients obtained after application of synthesized nanofibers and Whatman filter paper

[0097] It was observed that the most compatible results to the filter paper used as standard were cellulose synthesized in 48 hours and double layer nanofibrous membranes comprising CA and TPU, referred to as mix. The results of the nanofibers and blood samples that gave the most compatible results were compared. Correlation coefficients of Blood-Whatman903 r=0.949, Blood-CA 48h r= 0.941 and Blood-Mix r= 0.965 were obtained.

Claims

CLAIMS1. A nanofibrous sorbent, characterized in that it comprises a thermoplastic polyurethane nanofibrous membrane and / or a cellulose acetate nanofibrous membrane.

2. A nanofibrous sorbent according to claim 1 , characterized in that it comprises a 10% thermoplastic polyurethane nanofibrous membrane and / or a 15% cellulose acetate nanofibrous membrane.

3. A nanofibrous sorbent according to claim 1 or 2, characterized in that it comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 48 hours and a cellulose acetate nanofibrous membrane produced by electrospinning for 48 hours.

4. A nanofibrous sorbent according to claim 1 or 2, characterized in that it comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 24 hours or a cellulose acetate nanofibrous membrane produced by electrospinning for 24 hours.

5. A nanofibrous sorbent according to claim 1 or 2, characterized in that it comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 48 hours or a cellulose acetate nanofibrous membrane produced by electrospinning for 48 hours.

6. A nanofibrous sorbent according to any one of the preceding claims, characterized in that said nanofiber diameters are 100-800 nm.

7. A nanofibrous sorbent according to claim 1 or 2 for use in the determination of illicit substances from dried blood spot samples.

8. A nanofibrous sorbent according to claim 7, characterized in that said illicit substance is morphine, amphetamine, 3,4-methylenedioxy-N- methylamphetamine (MDMA), cocaine metabolite benzoylecgonine, or cannabis metabolite 11 -nor-9-carboxy-THC (D9-THC-COOH).

9. A method for use in the determination of illicit substances from dried blood spot samples, characterized in that it comprises the process steps of;• adding to 50 pL of sample taken from the blood pool 100 ng / mL of a stable, non-desiccant standard substance with a high equivalent weight (1001),• applying 50 pL of blood sample on Whatman 903 and nanofibrous sorbents, drying at room temperature for 2 hours, protected from sunlight (1002),• cutting the spots formed after the drying process with the help of scissors / puncher (1003) and adding 1 mL methanokacetonitrile (40:60 v / v) mixture and 25 ng / mL internal standard mixture in methanol to the blood samples (1004) and mixing by vortexing for 10 seconds (1005),• holding at room temperature in an ultrasonic bath for 30 minutes (1006),• removing sorbents from Eppendorfs with the help of forceps (1007)• centrifuging samples for 10 minutes at 4100 rpm (1008) and transferring the entire supernatant (1 mL) to a clean Eppendorf (1009),• evaporating samples at room temperature under nitrogen (1010),• adding 150 pL of mobile phase mixture (Mobile phase A: Mobile phase B 85:15 (v / v)) to the samples containing residues and transferring these to Eppendorf (1011 ),• centrifuging at 14000 rpm for 5 minutes, transferring samples to a vial and submitting to the LC-MS / MS device for analysis.

10. A method according to claim 9, characterized in that said nanofibrous sorbent comprises a thermoplastic polyurethane nanofibrous membrane and / or a cellulose acetate nanofibrous membrane.11 . A method according to claim 9, characterized in that said nanofibrous sorbent comprises a 10% thermoplastic polyurethane nanofibrous membrane and / or a 15% cellulose acetate nanofibrous membrane.

12. A method according to claim 9, characterized in that said nanofibrous sorbent comprises a thermoplastic polyurethane nanofibrous membrane produced byelectrospinning for 48 hours, and a cellulose acetate nanofibrous membrane produced by electrospinning for 48 hours.

13. A method according to claim 9, characterized in that said nanofibrous sorbent comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 24 hours or a cellulose acetate nanofibrous membrane produced by electrospinning for 24 hours.

14. A method according to claim 9, characterized in that it comprises a thermoplastic polyurethane nanofibrous membrane produced by electrospinning for 48 hours or a cellulose acetate nanofibrous membrane produced by electrospinning for 48 hours.

Images