Technical Field

The invention relates to a solid phase extraction column to be used in the enrichment of biological samples such as blood, urine, saliva, and low concentrations of analytes in food and environmental samples by solid phase extraction prior to chromatographic analysis.

State of the Art

Even though the sensitivity of chromatographic techniques is increased by using different detectors, because of the fact that the concentrations of the analytes to be determined in the environment, food and biological matrices (such as blood, urine, saliva, etc.) are low (in general ppb), some sample preparation and preconcentration (enrichment) techniques are required to be made before instrumental analysis. The main objectives of sample preparation methods are; (1 ) separation of the analyte from the matrix environment and (2) enrichment of the analyte at low concentrations and/or (3) removal of unwanted matrix components. The techniques that are generally used during sample preparation for enrichment are liquid-liquid extraction (LLE), liquid-liquid microextraction (pLLE), solidphase extraction (SPE) and solid-phase microextraction (pSPE).

SPE is the most widely used sample preparation method. The SPE method is based on the different affinity of the target analytes to two different phases. In the SPE procedure, firstly the adsorbent is conditioned with a solvent or solvent mixture in which the analytes dissolve. The liquid sample or a liquid sample extract is then interacted with the adsorbent. Typically, target analytes are adsorbed along with other components of the sample matrix. Finally, the analytes are desorbed with a small volume of a suitable solvent. The adsorbent can be used by filling in an SPE column or used as a batch. The operating principle of the solid phase extraction method is summarized in Figure 1 . Active inorganic adsorbents such as silica gel as well as activated charcoal, silica phases and polymers are used as adsorbent in SPE columns for liquid chromatography analysis. The most used adsorbents are commercially produced octadecyl (C18) silica, develosil (C30) silica, polystyrene-divinylbenzene copolymers, HLB (hydrophilic(N- vinylprolidone)/lipophilic (divinyl benzene)) cartridges and graphitized carbon black (Tadeo et al. 2008). A number of chemically modified adsorbents (e.g. silica gel, synthetic polymeric resins) are used in the enrichment of certain analyte groups through apolar, polar and ionic interactions. For example, reverse phase (C2, C8, C18, CH, PH, BP), normal phase (CN, SI, diol, alumina, florisil), anion exchange (SAX, DEA, NH2, PSA) and cation exchange (SCX, PRS, CBA) can be used. Interactions between the analytes and the adsorbent phase include hydrophobic interactions such as van der Waals forces and hydrophilic interactions such as dipole-dipole, hydrogen bond and TT-TT interactions. Appropriate adsorbent should be selected for the optimization of extraction. The sample solvent (water or organic solvent), analyte type (apolar, polar, or ionized), and the conditions under which ion formation occurs (strong or weak acid or base) provide guidance for the choice of method. Various SPE columns are commercially available, including diatomaceous earth Extrelut, Chem Elut, Bond Elut Certify, and Chromabond mixed mode columns. Mixed-phase extraction columns (e.g. Bond-Elut Certify and Chromabond) have good recovery values and allow for adsorption of all functional groups and analytes of different polarity. Bond-Elut Certify, which is a blend of C8 and SCX, has both hydrophobic and ion-exchange properties and is suitable for the extraction of basic analytes. N- vinylpyrrolidone increases the water-wettability of the hydrophilic polymer and lipophilic divinylbenzene (DVB) provides the reverse phase retention necessary to retain lipophilic analytes, for this reason hydrophilic-lipophilic balanced (HLB) copolymers provide good retention of both polar and non-polar compounds.

The determining factor for the adsorbent type to be used in SPE columns is the analytes to be analyzed (targeted to be enriched). The most important objective is that the adsorbent must enrich as many analytes as possible with a single SPE procedure (same flow rate, elution solvent and elution volume, etc.). In this way, it is possible to enrich a large number of analytes in biological samples such as blood, saliva and urine, as well as food and environmental samples, at detectable concentrations by chromatographic analysis. The decisive factor for achieving this objective is the affinity and selectivity of the interaction between the analytes and the adsorbent. Therefore, the most important disadvantage of the presently used techniques is the limited number of analytes that can be enriched with a single adsorbent and the same SPE procedure (same flow rate, elution solvent and elution volume). In addition, if the adsorbent does not have sufficient selectivity, additional steps are required before enrichment because the matrix of food, environment, urine, saliva and blood is quite complex. The reason for this is that the matrix containing many components prevents the analyte(s) to be determined from binding to the adsorbent. This situation becomes a significant disadvantage, especially when complex matrices such as blood and food are used as samples and a large number of analyzes must be performed quickly. Another important disadvantage is that the adsorbed analyte or analytes cannot be desorbed at a high ratio from the adsorbent. Also, the determining factor in the desorption of analytes is the affinity of the analytes to the adsorbent.

The documents identified in the patent and literature research conducted for the state of the art are summarized below.

CN1 10314413A discloses a solid phase extraction apparatus and the solid phase extraction method by which this extraction apparatus is used in sample preparation. One of the adsorbents in the document is water-retaining resin and the other is polymeric material. HEMA and MA polymers are not included among the used polymeric materials.

EP3278100A4 discloses a method for detecting the presence of one or more analytes in a test sample. The document does not describe a new solid phase extraction adsorbent/adsorbent mixture.

Therefore, due to the presence of the problems described above and the inadequacy of the solutions to meet the needs, it is required to make an improvement in the relevant technical field

Brief Description of the Invention

The present invention relates to a solid phase extraction column that meets the requirements mentioned above, eliminates all the disadvantages and provides some additional advantages.

The main objective of the invention is to provide a solid phase extraction column prepared by mixing two polymeric adsorbents with hydrophilic and hydrophobic (lipophilic) characteristics. The most important problems in the current technical field in solid phase extraction are that a limited number of analytes can be enriched at one time with the same SPE procedure, and that the adsorbent does not have the appropriate affinity (which also allows desorption) against the analyte(s) desired to be enriched. The solid phase extraction column, which is the subject of the invention, is prepared by mixing two polymeric adsorbents with hydrophilic and hydrophobic (lipophilic) characters in order to solve the existing problems. Hydrophobic polymeric adsorbent comprises apolar N- methacryloyl-amido-L-tryptophan methyl ester (MA) residue (Osman et al, 2013; Ozer et al, 2017; Ozer et al, 2020; Ozer et al, 2015; Demir et al, 2020; KIIIQ et al, 2018), while hydrophilic polymeric adsorbent comprises polar hydroxyethyl methacrylate (HEMA) residue (Denizli et al, 1997; Osman et al, 2021 ). The polymer with hydrophobic (lipophilic) character functions as a reverse phase in the solid phase extraction column due to the hydrophobic (apolar) character of the MA residue in its structure and enables apolar analytes to be adsorbed. The polymer of hydrophilic character allows water molecules to enter the polymeric structure and adsorb polar analytes due to the hydrophilic character of the HEMA residue in its structure. Both polymeric adsorbents (hydrophilic and hydrophobic) have high swelling capacity and large surface area. These two features increase the interaction of the analyte(s) with the adsorbent, this, in turn, provides an increase in the adsorption capacity. Therefore, recovery increases and analytical performance indicators such as precision and repeatability of the results obtained in chromatographic analysis are also improved. The existence of polymeric adsorbents containing hydrophobic and hydrophilic residues together in the prepared SPE column ensures that a large number of analytes are bound to the adsorbent according to their polarity (hydrophilic and hydrophobic) and thus enriched at one time and with the same SPE procedure. In addition, since the SPE column contains two polymeric adsorbents or can be used as a mixture of two polymers in the batch system, for this reason it is also possible to mix the polymeric adsorbent in the sample (food, environmental or biological sample) in proportions that will provide appropriate affinity for the analyte or analyte group to be determined. Since the interaction between the adsorbent and the analytes occurs with weak intermolecular interactions, the interactions with the elution solvents used during desorption can be easily broken down and a high ratio of desorption can be achieved.

In the literature, there is no solid phase extraction column in which MA polymer (lipophilic) and HEMA polymer (hydrophilic) (two separate polymeric adsorbent) are combined. In this sense, the invention includes an innovation different from the state of the art. It is also possible to use the adsorbent developed with the invention in batch. Also, it is possible to prepare the most optimum SPE column for the analyte group to be analyzed by mixing the MA polymer and HEMA polymers filled in the column at different ratios. In addition, it is also possible to prepare the SPE column with microspheres in different diameter ranges (size ranges). By means of these features, it is possible to enrich a large number of analytes with a single SPE column and a single SPE procedure (same flow rate, elution solvent and elution volume, etc.).

The SPE column of the invention should be used directly with biological samples or after adsorbent (a mixture of MA polymer and HEMA polymer) has been conditioned with buffer solutions that will provide physiological pH. After conditioning with environmental samples directly or with distilled water, it should be used with food samples by conditioning with the solvent/solvent system used in the extraction of the food sample initially. All solvents used in reverse phase chromatographic analysis can be used as elution solvents.

The structural and characteristic features of the invention and all its advantages will be understood more clearly by means of the figures presented below and the detailed description written by reference to these figures. For this reason, the evaluation should be made by considering these figures and the detailed description.

Drawings to Help Understand the Invention

Figure 1 shows the schematic representation of the solid phase extraction method (Tadeo et al. 2008).

Figure 2 shows the SEM view of the MA polymer (microsphere).

Figure 3 shows the SEM view of HEMA polymer (microsphere).

Detailed Description of the Invention

This detailed description describes the preferred embodiments of the invention only for a better understanding of the subject.

The invention relates to a solid phase extraction column and production method for the enrichment of biological samples such as blood, urine, saliva, and low concentrations of analytes in food and environmental samples by solid phase extraction prior to chromatographic analysis.

In the method of the invention, firstly L-tryptophan methyl ester is dissolved in dichloromethane. Triethylenediamine and methacryloyl chloride are added to the L- tryptophan methyl ester solution and mixed. A magnetic stirrer is preferably used in the said process steps. NaOH solution is added to said reaction mixture, preferably in the extraction flask, and the dichloromethane phase is separated. The said dichloromethane phase is dried with Na2SC . At this stage, filter paper is used.

At the next step, N-methacryloyl-amido-L-tryptophan methyl ester monomer (MA) is obtained by evaporating the dichloromethane phase. In a preferred embodiment of N- methacryloyl-amido-L-tryptophan methyl ester monomer (MA) synthesis, preferably 3.08% by weight of L-tryptophan methyl ester is dissolved in a glass flask, preferably 82.05% by weight of dichloromethane (CH2CI2) for the synthesis of N-methacryloyl- amido-L-tryptophan methyl ester (MA) monomer. To the solution cooled in the ice bath, preferably 7.86 wt% triethylamine is slowly added at a stirring speed of 400 rpm in a magnetic stirrer. Then, preferably 3.3% by weight methacryloyl chloride is slowly added to the solution and stirred with a magnetic stirrer, preferably in an ice bath for 2 hours and preferably at room temperature for 24 hours. The solution is taken into the separation funnel and unreacted methacryloyl chloride is extracted 4 times, preferably with 3.08% by weight of NaOH. The dichloromethane phase is separated and filtered through ordinary filter paper containing anhydrous Na2SC . Then, MA monomer is obtained by evaporating the dichloromethane phase taken into the balloon in the evaporator (preferably 50-60 ^< C). The obtained MA monomer, tol uene, EGDMA and AIBN are preferably mixed in an ultrasonic bath.

In the said method, PVA solution is prepared by dissolving the PVA polymer in distilled water. The solution containing MA monomer, toluene, EGDMA and AIBN is added to the PVA solution and polymerized. At this stage, preferably a circulating water bath with glass reactor, magnetic stirrer is used. The obtained MA polymer is filtered, washed with ethyl alcohol and distilled water and dried. Then, the obtained MA polymer is sieved and polymers with different diameter ranges are grouped.

In a preferred application of the invention, in the preparation of the MA polymer, preferably 13.44% by weight toluene, 8.15% by weight EGDMA and 0.22% by weight MA monomer are mixed in a beaker and 0.15% by weight AIBN is added into it and mixed in an ultrasonic bath for 15 minutes, preferably at room temperature. A glass reactor connected to a circulating water bath is placed on a magnetic stirrer. The temperature of the circulating water bath is preferably set to 70 ^< C. To prepare the PVA solution preferably, 77.7% by weight distilled water and 0.31 % by weight PVA are mixed in a glass reactor, preferably at 70 <0, at a mixing speed of 400 rpm, for 20 minutes. Then, preferably solution containing toluene, EGDMA, MA monomer and AIBN is added to the PVA solution in the glass reactor. The resulting mixture is first polymerized at a mixing rate of 400 rpm at 70 <0 for preferably 2 hours and then pr eferably at 80 <0 for 8 hours at a mixing rate of 400 rpm. After cooling the glass reactor, ethyl alcohol is added to the mixture and taken to a beaker and mixed in a magnetic stirrer at room temperature, preferably for 24 hours. The resulting MA microspheres are then filtered using a stony funnel and vacuum pump, washed preferably with ethyl alcohol and distilled water, and dried at 50 <0 in a vacuum oven. Preferably screens in diameter ranges of 53 pm, 106 pm, 150 pm, 180 pm and 212 pm are lined up in the sieve shaker. The MA microspheres are poured onto the sieve at the top, preferably 212 pm in diameter, and kept in the sieve shaker for 8 hours. MA microspheres are preferably screened into diameter ranges of 53- 106 pm, 150-180 pm and 180-212 pm. MA microspheres, preferably in the diameter range of 150-180 pm, are used in the preparation of the SPE column. Figure 2 shows the SEM images of the MA polymer (microsphere).

At the next step, HEMA monomer, toluene, EGDMA and AIBN are mixed, preferably in an ultrasonic bath. PVA solution is prepared with PVA polymer dissolved in distilled water and the solution containing HEMA monomer, toluene, EGDMA and AIBN is added to the PVA solution and polymerized. In a preferred embodiment of the invention, during the preparation phase of the HEMA polymer, preferably 10.53% toluene by weight, 6.39% EGDMA by weight and 6.52% HEMA monomer by weight, are mixed in beaker and 0.15% AIBN by weight is added and mixed in the ultrasonic bath at room temperature, preferably for 15 minutes. A glass reactor connected to a circulating water bath is placed on a magnetic stirrer. The temperature of the circulating water bath is preferably set to 70 ^< C. PVA solution is prepared by stirring preferably 76.1% distilled water by weight and 0.3% PVA by weight in the glass reactor, preferably at a mixing rate of 400 rpm at 70 <0, preferably for 20 minutes. The solution, preferably containing toluene, EGDMA, HEMA monomer and AIBN, is then added to the PVA solution in the glass reactor. The resulting mixture is first polymerized, preferably at a mixing rate of 400 rpm at 65 <0, preferably for 4 hours, and then preferably at 80 <0, for a mixing rate of 400 rpm, preferably for 2 hours. After cooling the glass reactor, ethyl alcohol is added to the mixture and taken to a beaker and mixed in a magnetic stirrer at room temperature, preferably for 24 hours. The resulting HEMA microspheres are filtered using a stony funnel and vacuum pump, preferably with ethyl alcohol and distilled water, and dried at 50 <0 in a vacuum oven. Preferably screens in diameter ranges of 53 pm, 106 pm, 150 pm, 180 pm and 212 pm are lined up in the sieve shaker. HEMA microspheres are poured onto a 212 pm diameter sieve at the top and preferably kept in the sieve shaker for 8 hours. HEMA microspheres are preferably screened into diameter ranges of 53-106 pm, 150-180 pm and 180-212 pm. HEMA microspheres, preferably in the diameter range of 150-180 pm, are used in the preparation of the SPE column. Figure 3 shows the SEM image of the HEMA polymer (microsphere).

The resulting HEMA polymer is filtered, washed and dried with ethyl alcohol and distilled water. Then, the screening of the HEMA polymer and the grouping of polymers in different diameter ranges are started. At this stage, preferably screens and sieve shakers are used. Finally, the MA and HEMA polymer is filled into the SPE column in a homogeneous mixture. In a preferred embodiment of the invention, during the preparation of the solid phase extraction column, the MA polymer (preferably 150-180 pm diameter range; 0.0500 g) and HEMA polymer (preferably 150-180 pm diameter range; 0.0500 g) are weighed with a precision analytical balance. The empty SPE column is placed in the vacuum manifold and a frit is attached to the bottom to prevent the adsorbent from leaking out of the column. First, the MA polymer (0.0500 g) and then the HEMA polymer (0.0500 g) are transferred into the SPE column and mixed with a thin metal rod for 10 minutes in circular motions. Then, the upper frit is placed by applying vacuum.

N-methacryloyl-amido-L-tryptophan methyl ester (MA) is an amino acid monomer and is synthesized by the reaction of L-tryptophan methyl ester with methacryloyl chloride. N- methacryloyl-amido-L-tryptophan methyl ester has an apolar structure. When incorporated into the polymeric structure, apolar molecules and residues of N- methacryloyl-amido-L-tryptophan methyl ester easily combine with hydrophobic interactions in aqueous solution.

Chemical structure of MA monomer Ethylene glycol dimethacrylate (EGDMA) is one of the most widely used cross-linking monomers in radicalic addition polymerization. It has a good hydrophilicity by means of its oxygen-containing groups.

Chemical structure of EGDMA monomer

Hydroxyethyl methacrylate is a monomer with a hydrophilic structure. Its hydrophilic property is due to the presence of the hydroxyl group (-OH) in its chemical structure. When incorporated into a polymeric structure, it easily make hydrogen bond with water molecules, allowing water molecules to enter the polymeric matrix.

Chemical structure of HMA monomer

The MA polymer is synthesized by polymerization of EGDMA and MA monomer by radicalic addition polymerization. Since it is prepared by the suspension polymerization method, it has the form of a microsphere. It is the hydrophobic (lipophilic) component of the adsorbent in the solid phase extraction column. By means of its hydrophobic (apolar) structure, the MA residue in the polymeric structure acts as a reverse phase in the solid phase extraction column and takes on the function of retention of the apolar analytes in the sample (environment, food, biological sample). By means of its hydrophobic structure, it especially adsorbs apolar analytes depending on the polarity.

HEMA polymer is synthesized by polymerization of EGDMA and HEMA polymer by radicalic addition polymerization. Since it is prepared by the suspension polymerization method, it has the form of a microsphere. It is the hydrophilic component of the adsorbent in the solid phase extraction column. By means of its hydrophilic (polar) structure, the HEMA residue in the polymeric structure easily bonds with water molecules and assumes the function of entering water molecules into the polymeric matrix and retention (adsorption) of polar analytes. The preferred parameter ranges in the method of the invention and the effect of these parameters on the invention are listed below:

• The mixing time of the solution containing L-tryptophan methyl ester, triethylenediamine and methacryloyl chloride at room temperature is 6-24 hours. This time is the reaction time applied to obtain the monomer. The MA monomer is the raw material of the MA polymer. If obtained in large amounts, more MA polymers can be obtained. MA monomer can be obtained with high efficiency in 6 hours. But the reaction can be continued for up to 24 hours to increase the yield a little more. However, even if it is done in 6 hours, the MA polymer is obtained and there is no situation that prevents the invention.

• The polymerization time of MA microspheres is 8-10 hours. This time is the reaction time applied after the first 2 hours for the synthesis of the MA polymer. In 8 hours, the MA polymer is obtained in sufficient quantity. However, if the amount of MA polymer obtained is to be increased a little more, the time can be increased to 10 hours. 8 hours is sufficient for the MA polymer to be obtained. There is no situation that prevents the invention.

• The polymerization time of HEMA microspheres is 6-8 hours. This time is the total reaction time applied for the synthesis of the HEMA polymer. In a total of 6 hours, HEMA polymer is obtained in sufficient quantity. However, if the amount of HEMA polymer obtained is to be increased a little more, this time can be increased to a total of 8 (2 + 6) hours. A total of 6 hours is sufficient for the HEMA polymer to be obtained. There is no situation that prevents the invention.

• The amount of adsorbent (mixture of MA polymer and HEMA polymer) filled into the SPE column is 50-200 mg. It is one of the features that define the invention. It is the total amount of polymer filled into the column (MA polymer + HEMA polymer). It affects the detectability and recovery values of the analyzed compounds. The amount of adsorbent to be used allows the preparation of columns that will provide high recovery according to the number and chemical structure (polarity, etc.) of the compounds to be analyzed.

• The ratio of MA polymer in the adsorbent is 10%-90%. It is one of the defining features for the invention. Regardless of the ratio, the invention performs its function. But the MA polymer is the hydrophobic component of the column. If the compounds to be analyzed are predominantly hydrophobic and higher recovery values are desired in addition to hydrophilic analytes, the MA polymer ratio can be kept high and HEMA polymer ratio can be kept low for filling into the column. In the case study, the efficacy for hydrophobic and hydrophilic analytes was proven by using the SPE column prepared in the most optimum condition of 50% + 50%.

• The ratio of HEMA polymer in the adsorbent is 10%-90%. It is one of the defining features for the invention. Regardless of the ratio, the invention performs its function. But the HEMA polymer is the hydrophilic component of the column. If the compounds to be analyzed are predominantly hydrophilic together and higher recovery values are desired in addition to hydrophobic analytes, the HEMA polymer ratio can be kept high and MA polymer ratio can be kept low for filling into the column. In the case study, the efficacy for hydrophobic and hydrophilic analytes was proven by using the SPE column prepared in the most optimum condition of 50% + 50%.

• The diameter range of the MA polymer in the adsorbent is 53-106 pm - 180-212 pm. It is one of the defining features for the invention. The adsorbent size affects the recovery of analytes. In our studies, considerably high recovery values have been obtained in the diameter range of 150-180 pm. However, it may be possible to achieve higher recovery values with different sizes of MA polymer for different analytes, elution solvents and extraction parameters. Because it is not possible to experimentally test all possibilities for a column that can be used to determine thousands of possible analytes, such as biological, environmental, and food samples, such a range is foreseen to cover smaller and larger values.

• The diameter range of the HEMA polymer in the adsorbent is 53-106 pm - 180-212 pm. It is one of the defining features for the invention. The adsorbent size affects the recovery of analytes. In our studies, considerably high recovery values have been obtained in the diameter range of 150-180 pm. However, it may be possible to achieve higher recovery values with different sizes of HEMA polymer for different analytes, elution solvents and extraction parameters. As it is not possible to experimentally test all possibilities for a column that can be used to determine thousands of possible analytes, such as biological, environmental, and food samples, such a range is foreseen to cover smaller and larger values. In the method, which is the subject of the invention, hydrophilic monomers can be prepared with the mixtures of the adsorbents prepared with (2-dimethylamino ethyl methacrylate, oligo(ethylene glycol) monomethyl ether methacrylate, 2-hydroxypropyl methacrylate, N-isopropilacrylamide, N,N-dimethylilacrylamide, 2-methacrylooxyethyl phosphorylcholine, glycidyl methacrylate) (alternatives to the HEMA monomer or hydrophilic monomers as a general name), hydrophobic monomers (methyl acrylate, methyl methacrylate, styrene, butyl acrylate, vinyl, vinyl acetate) (alternatives to the MA monomer or as a general name hydrophobic monomers) and different cross-linkers (divinyl benzene, trimethylolpropane trimethacrylate) (alternatives to EGDMA monomer).

In the invention, polar and polar organic solvents can be used as general names instead of toluene. Instead of AIBN, radicalic initiators perform the same function as the generic name.

There is a synthesis method of MA polymer in the technical field. The MA polymer was synthesized by the inventors with differences in the synthesis recipe and used for SPE purposes alone. HEMA is a commercial monomer and has thousands of applications. HEMA polymer was also synthesized by the inventors with different synthesis recipes and its use for different purposes is described. However, HEMA polymer has never been used and explained for the purpose of SPE. However, the approach of mixing MA polymer and HEMA polymers as specified in the invention and filling them into the SPE column and using them for simultaneous enrichment of hydrophilic and hydrophobic components is a new method.

The preferred usage rates and usable rate ranges of the components used in the invention are given in the table below.

In a sample study carried out within the scope of the invention, narcotic I stimulant I drug active ingredient analysis was performed in whole blood. The sample study was carried out with a solid phase extraction column prepared by mixing 50% MA and 50% HEMA polymer (in the diameter range of 150-180 pm).

Experimental Conditions for Sample Analysis

In the said example, a whole blood sample is used, which is often used in forensic toxicology cases. The LC-MS/MS data in the blood samples were generated using the following conditions. Unless otherwise specified, analyte concentrations, properties and abbreviations are given below. LC Conditions

LC System: Shimadzu Nexera XR LC-20ADXR, Shimadzu SIL-20ACXR

Detection: LCMS-8050 Triple Quadrupole Mass Spectrometer

Analytic Column: Poroshell 120 EC-18 (4.6 x 150 mm x 2.7 pm)

Mobile Phase A: distilled water containing % 0.1 (v/v) formic acid and 2 mM ammonium acetate

Mobile Phase B: 100 % Methanol (v/v)

Flow Rate: 0.6 mL/min

Analysis Time: 15 min

The gradient table for the mobile phase is shown in Table 1 .

Table 1. Mobile phase gradient used in LC-MS/MS system

Column Temperature: 40 <C

Sample Temperature: 15 <C

Injection Volume: 10 pL

MS Conditions

MS System: LCMS-8050, ESI+ and ESI- Desolvation Temperature: 526 <C Nebulized Gas Flow: 3 L/min

Heating Gas Flow: 10 L/dk

Interface Temperature: 300 <C Interface Voltage: 4,0 kV

DL Temperature: 250 ‘C

Heat Block Temperature: 400 <0

MRM transition observed: See the following table The sample MRM transition monitored by ESI is shown in Table 2.

Table 2. Retention Times and MS parameters for Narcotics /Stimulants/ (Drug Active Substances)

Narcotic/Stimulant/ (Drug Active Substances) Standards

Standards were purchased from Cayman Chemical and Chiron AS. Single standard stock solutions (1 mg/mL) were prepared in methanol, or acetonitrile. A stock solution containing a mixture of all compounds (10 pg/mL) in methanol was prepared. The calibration and quality control (QC) samples were prepared by adding working standards at various concentrations and into the matrix (whole blood). The calibration concentrations were in the range of 0.5-100 ng/mL for all analytes. Quality control samples were prepared by adding a mixture of standards at 1 , 10 and 100 ng/mL to human whole blood.

Sample preparation

The samples were extracted using a column combination comprising 100 mg MA polymer and 100 mg HEMA polymer. A 0.5 mL human whole blood sample was diluted with 4.5 mL of distilled water and internal standards were added. The column containing 200 mg sorbent was preconditioned with 2 mL of ethyl acetate, 2 mL of methanol and 2 mL of distilled water. The sample is loaded into the column. The column was then washed with 2 mL of methanol: water (5:95) and dried. The analytes were then eluted from the column with 2 x 500 pL methanol and 2 x 500 pL ethyl acetate. The elution solvent was then evaporated in a sand bath (50 <0) and the residue w as dissolved again with 500 pL methanol: water (20:80) and injected into the LC system in a volume of 10 pL.

Recovery of analytes was calculated according to the following equation:

Present concentration

%Recovery = — — — - xlOO

Added concentration

Results

With this extraction protocol, the recovery values of 33 active substances were evaluated for the concentration of three different standard addition to human blood samples. The obtained results are given in Table 3. A total of 31 compounds with different polarity and functional groups were recovered. The overall average recovery values for concentrations of 1 , 10 and 100 ng/mL were calculated as 59.5%, 56.9% and 57.6%, respectively.

Table 3. Extraction protocol results

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