Development of a Biocompatible In-Tube Solid-Phase Microextraction

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Development of a Biocompatible In-tube Solid Phase Microextraction Device: A Rapid and Sensitive Approach for Direct Analysis of Single Drops of Complex Matrices Hamed Piri-Moghadam, Sofia Lendor, and Janusz Pawliszyn Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03160 • Publication Date (Web): 20 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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Analytical Chemistry

Development of a Biocompatible In-tube Solid Phase Microextraction Device: A Rapid and Sensitive Approach for Direct Analysis of Single Drops of Complex Matrices Hamed Piri-Moghadam, Sofia Lendor and Janusz Pawliszyn* Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada * Corresponding author: Tel. 519-888-4641, Fax. 519-746-0435, E-mail: [email protected]

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Abstract The aim of the current study is to develop a sensitive solid phase microextraction (SPME) device for direct and rapid analysis of untreated complex matrices (i.e. single drop of the samples, V ≤ 2 µL). A thin layer of a biocompatible nano-structured polypyrrole (PPy) was electrochemically deposited inside a medical grade spinal needle, minimizing the matrix effect. Micro-sampling was facilitated by loading the sample inside the in-tube SPME device (withdraw of sample via plunger), where extraction was performed under static conditions. Two strategies were used for analysis of the compounds including offline desorption and running the extract to the LC-MS/MS or direct coupling of in-tube SPME device to MS. Given the high surface areato-volume ratio of the coating, a short equilibrium time (i.e. t ≤ 2 min) was obtained. The whole analytical procedure (i.e. extraction, rinsing, desorption, and LC-LC-MS/MS analysis) was performed within 10 minutes by LC-MS/MS, and 3 minutes by in-tube-MS/MS. Possible matrix effects for the prepared device were evaluated in whole blood samples at three levels of concentration, and encouraging results were achieved in the range of 83-120 %. The obtained results, no matrix effect, attributed to the smooth surface and small pore size of the biocompatible PPy coating, which was prepared in the presence of cetyltrimethylammonium bromide (CTAB) surfactant. The in-tube SPME device was shown to be very sensitive, with high total recoveries obtained for all compounds in PBS and urine samples owing to the large volume and capacity of the coating. Sub-ng mL-1 levels of detection were achieved for urine samples, and low ng mL-1 levels were found in whole blood samples for all studied compounds with a high protein binding index. Rapid analysis of whole blood samples was achieved without need of any pre-treatment or manipulation of sample, revealing the developed in-tube SPME device as an ideal probe for forensic application, drug monitoring and point of care diagnosis. Keywords: In-tube Solid Phase Microextraction, rapid analysis, biocompatibility, small volume, whole blood and urine, LC-MS/MS, in-tube MS/MS


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Introduction The development of bioanalytical tools for point-of-care (POC) diagnosis and drug monitoring has recently trended towards invention of miniaturized techniques and devices that enable direct analysis of small volumes of sample. Sensitive techniques providing non-invasive sampling with high accuracy and precision are required for effective monitoring and investigation of compounds of interest in biological fluids. In recent years, analysis of small volume/size of samples, (i.e. single drop whole blood analysis) was facilitated by high sensitivities of modern mass spectrometric instrumentation1–5. Ambient mass spectrometry techniques also provide rapid analysis of small volumes of complex samples with minimal or no preparation of samples.6,7 The main challenge of micro-sampling (e.g. V ≤ 10 µL) stems from the low quantities of compounds present within small volumes of sample. Dilutions during sample preparation and ionization suppressions also contribute towards decreases in the sensitivity of the method. Thus, an effective sample preparation technique is required to effectively isolate and extract target compounds from such small samples. Recently, solid phase extraction was integrated to paper spray (PS) technology for pre-concentration of drugs in 10-100 µL of plasma sample, and reduction of ionization suppression.8 However, extraction/concentration of compounds could not be performed when sample volumes lower than 10 µL were used.8 In addition, in order to lower the detection limits, enrichment of compounds and introduction of the extract into analytical instruments with the use of a short pulse were necessary to maximize the sensitivity of the method for trace analysis of matrices with high protein binding in whole blood and plasma samples. Solid phase microextraction (SPME) is a simple and solvent-free sample preparation technique introduced in the 1990s with the aim to overcome some of the limitations of conventional sample preparation techniques.9,10 SPME provides several geometrical configurations11,12 (e.g. fiber, miniaturized fiber/coated-tip, membrane, intube and particles), simplifying sampling and moving towards green and rapid analysis. Easy automation of SPME to analytical instrumentation via dedicated autosamplers and interfaces to gas chromatography (GC)10, liquid chromatography (LC),13 and direct-coupling to MS12,14–18 makes this a powerful and versatile technique for analysis of a wide range of compounds in several matrices (e.g. environmental,10,11,19,20 bio-fluids,21–24 food



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samples,25,26 and on-site sampling27). Fabrication of matrix-compatible coatings also provides suitable probes for in-vivo sampling28–30 and analysis of complex matrices with no or minimum cumbersome pre-treatment.25 The recent development of miniaturized SPME platforms such as coated-tip SPME and coated blade spray (CBS)12 have enabled SPME analysis of low volumes of samples (V ≤ 10 µL) down to the single cell level. The main goal of the current study is to provide another geometrical configuration of miniaturized SPME, intube SPME, to facilitate convenient automation31 and enable analysis of sample small volumes, while increasing the sensitivity of the method. The current study is continuation of our recent trend to develop devices for rapid analysis of small volume of sample. The developed technique is envisioned as complementary to previously developed miniaturized techniques, taking into account the goal of the study, analytical needs, sample matrix and the available instruments. Although several publications of in-tube SPME for analysis of target compounds can be found in the literature, time consuming and cumbersome pretreatment and manipulation steps, including dilution, protein precipitation, centrifugation, and filtration, are necessary prior to extraction so as to avoid clogging of the capillary tube.32,33 For instance, up to 10 fold dilution and filtration had to be performed for certain applications, even when urine samples were analyzed, 32,34–37

while proteins had to be precipitated in instances where more complex matrices such as plasma were

used.38 While in-tube SPME analysis of whole blood samples could potentially provide a powerful and convenient tool for POC diagnosis, clogging of the tube, associated with the high content of red blood cells and proteins in the matrix, have hindered the application of the in-tube SPME technique for direct analysis of whole blood. To overcome the aforementioned shortfalls, herein, a biocompatible in-tube SPME device (both coating and needle itself) was developed by electrochemical deposition of a thin layer of a nano-structured polypyrrole (PPy), with a pore size lower than 80 nm, inside a medical-grade spinal needle. Quantitation was performed by LC-MS/MS, and sensitive and precise results were achieved for compounds present in complex matrices. To the best of our knowledge, there are no reports in the literature regarding application of in-tube SPME for direct, sensitive and rapid extraction (i.e. t ≤ 2 min) of compounds present in small volumes (i.e. V ≤ 2 µL) of complex biological matrices, including analysis of whole blood samples that do not require pre-treatment or 4 ACS Paragon Plus Environment

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manipulation of samples. In addition, to shorten the time of analysis, a strategy of direct coupling of SPME to MS was proposed, based on the developed in-tube device. Herein, we present the strengths and advantages of the developed device compared to previous in-tube SPME studies.

Reagents and materials All chemicals and reagents used in this study were obtained at the highest available purity, > 97 %, and were used without further purification. Riboflavin, caffeine, dexamethasone, pindolol, carbamazepine, diazepam, thiabendazole, testosterone, propranolol, formic acid (LC-MS grade), trifluoroacetic acid, and pyrrole were purchased from Sigma-Aldrich (Oakville, ON, Canada). The LC-MS grade solvents acetonitrile, methanol, and water were obtained from Fisher Scientific (Oakville, ON, Canada). Pooled whole blood from healthy donors was obtained in potassium (K2) ethylenediaminetetraacetic acid (EDTA) from Bioreclamation IVT (Baltimore, MA, U.S.A.). Urine samples were collected from a healthy male. Collection of urine from healthy volunteers was conducted under the approval of the Office of Research Ethical Board of University of Waterloo. Properties of the studied compounds are shown in Table S1 of supplementary information. The phosphate buffered saline solution (PBS) (pH 7.4) was prepared by addition of KH2PO4, Na2HPO4, NaCl and KCl to nanopure water.

Instrumentation Preliminary experiments and optimization were performed by a Shimadzu (LC-10 AD) high-pressure liquid chromatography (HPLC) system coupled to an Applied Biosystems API 4000 (Concord, ON, Canada) triple quadrupole mass spectrometer (equipped with Turbo Ion Spray source). Experiments designed to obtain quantitation information and calibration curves were performed using a LC-MS/MS Thermo TSQ Vantage (Thermo Scientific, San Jose, USA). Sample volumes of 10 µL of both standards and extracted analytes were injected into the LC-MS/MS system using an autosampler from CTC-Combi-PAL (Zwingen, Switzerland). A



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Discovery HS F5 column, 2.1 mm × 5 cm, 3 µm particle size (Supelco, Bellefonte, PA, USA), using gradient conditions and a flow rate of 400 µL min−1, was used for chromatographic separation of carbamazepine, propranolol, testosterone, thiabendazole, riboflavin, caffeine, dexamethasone, diazepam, and pindolol. In the chromatographic method, mobile phase A consisted of 0.1 % v/v formic acid, and mobile phase B was acetonitrile/formic acid (99.9:0.1, v/v). The applied chromatographic gradient was set in the following manner: started at 0-1 min, 0% B; 1-5:30 min, linear gradient to 100% B; 5:30-6 min, held at 100% B; sharply dropped to 0% B; 1 min for re-equilibration by 0% B (total time was 7 min). MS/MS analysis was performed in positive mode under selected reaction monitoring (SRM) conditions. A summary of the MS/MS parameters used for the API 4000 instrument can be found in Table S2, in supplementary information. Mass spectrometry conditions for the API 4000 instrument were set in the following manner: ion source gas 1, +40; ion source gas 2, +60; curtain gas, +30; collision gas, 10; spray ionization voltage, +4500 V; and temperature, 500 °C. The following conditions were applied for use of the TSQ instrument: Spray voltage: +3000 V; vaporizer temperature: 300 ºC; sheath gas pressure: 45; Aux gas pressure: 30; and capillary temperature: 300 ºC. Mass spectrometry conditions and MS/MS parameters of the TSQ instrument are shown in Table S3, in supplementary information.

Fabrication of the biocompatible in-tube SPME device: A medical grade spinal needle (22-gauge Quincke cutting beveled needle, O.D. 0.41 mm, length of 9 cm) was selected for the in-tube SPME device. Polypyrrole was electrochemically deposited inside the spinal needle (length of coating 2.5 cm). Distilled pyrrole, 0.35 M, was used as monomer, and 0.45 M of trifluoroacetic acid (TFA) and 0.01 M of cetyltrimethylammonium bromide (CTAB) were added as counter ions and electrolytes. Previous research12 has demonstrated that addition of CTAB can contribute towards the stability and extraction efficiency of the coating, while also providing a homogenous and smooth coating that is obtained by decreasing the particle and pore size of the coating, less than 80 nm. The stainless steel and spinal needle acted as cathode and anode, respectively, in the electrodeposition of PPy inside the spinal needle, with a voltage of


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1.2 V applied for 15 minutes. A septum was used outside of the needle to limit the PPy deposition just inside the needle. Optical images of the device are provided in supplementary information (Figure S1).

Extraction procedure by in-tube SPME device All the extractions were performed under static conditions by pulling 2 µL of sample inside the spinal needle, instead of draw/eject repetitions, or passing several cycles reported in the previous applications of in-tube SPME.32,39 After each extraction from PBS and urine samples, the needle was rinsed with water, 3 times fill and refill the needle, to remove salts and avoid/minimize ionization suppression of mass spectrometry. For whole blood sample, an additional rinsing step was performed due to the complexity of the sample to remove any possible macromolecules attached to the surface of the coating. The in-tube SPME devices were rinsed 5 times with water by filling and re-filling the spinal needle, then passing 20 µL of water through the needle to ensure no blood remained inside the device. After extraction (2 minutes) and rinsing, desorption of compounds was performed in 15 µL of MeOH:H2O (80:20) for 2 minutes under sonication and then 10 µL of extract run into the LC-MS/MS instrument. For direct coupling to MS, a mixture of iPA:MeOH:ACN (50:25:25), with 0.1% FA, was used for the desorption.

Results and discussions

Evaluation of matrix effect of the in-tube SPME device: The use of biocompatible coatings can help circumvent the co-extraction of interferences (e.g. macromolecules and proteins) and suppression of analytical signals, which affect the accuracy, precision, and sensitivity of the method. For this purpose, biocompatible materials with high affinity to the target compounds can be used to isolate and pre-concentrate said compounds from the matrices of interest. In the current study, polypyrrole was used as a biocompatible coating for the in-tube devices. The biocompatibility of polypyrrole as an SPME fiber was already validated in our previous study.12Application of in-tube SPME towards bio-fluid analysis can be a 7 ACS Paragon Plus Environment


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challenging undertaking, as samples can precipitate and clog the capillary tube. To evaluate the biocompatibility of the developed in-tube SPME device, the matrix effect was evaluated for three prepared devices by sampling from 2 µL of untreated whole blood sample for 5 minutes at three levels of concentration. Matrix effect was calculated according to the procedure already reported by Matuszewski et al 40, by dividing the peak areas of standards spiked after extraction into the extract by the corresponding peak areas in neat solution standard (more detail can be found in section 5 of supplementary information). As Table 1 shows matrix effects in the range of 83-120 % were obtained demonstrating that there is no ionization suppression and therefore no matrix effect occurred. The biocompatibility of the PPy coating towards the matrices of interest, the smooth surface of the coating, with a pore size of less than 80 nm, and the short extraction time achieved by the method make the presented in-tube SPME device an ideal matrix-compatible probe for analysis of complex matrices.

Extraction efficiency of the developed device: Due to the low quantities of compounds present in few microliters of sample, a sensitive approach, preferably exhaustive, is required to extract and isolate significant amounts of compounds so as to meet detection limit requirements and effectively determine the target compounds (i.e. in therapeutic range). In our previous study12, the SPME technique was used for fast analysis of target compounds present in small volumes of complex samples (i.e. V ≤ 10 µL) by providing two geometrical configurations, the miniaturized SPME fiber, namely coated-tip, and CBS. Herein, in-tube SPME geometry was developed in order to increase the sensitivity of the method in instances where lower volumes of sample (i.e. V ≤ 2 µL) are required for analysis. To evaluate the sensitivity of the developed SPME device, a wide range of compounds with different polarities (See Table S1 of supplementary information) were spiked to a PBS solution. The advantage of the presented in-tube SPME device, compared to regular fiber and previously reported in-tube SPME devices,41,42 lies in its ability to completely desorb the extracted compounds in small amounts of solvent (i.e. V ≤ 2 µL), which subsequently eliminates sample dilution and the requirement for solvent evaporation and reconstitution. It is beneficial to introduce the analytes in a short pulse into the instrument to improve the sensitivity, particularly 8 ACS Paragon Plus Environment

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when direct coupling to mass spectrometry via nano-ESI/MS/MS is used. Table 2 shows the results achieved by running an extract of sample into LC-MS/MS, for which high total recoveries were achieved for all of the studied compounds in the PBS solution. In addition, total recoveries up to 18 % were also obtained in whole blood sample for the studied compounds with high protein binding. Considering that the amount of extracted analyte is directly proportional to the volume of the extraction phase in SPME applications, the large volume of the PPy coating (i.e. 2.5 cm length of PPy coating inside the narrow needle, significantly enhanced the extraction efficiency of the in-tube SPME device. In order to obtain a better understanding of the PPy coating efficiency, a comparison experiment was conducted to identify extraction differences between a needle without any coating (i.e. the bare spinal needle), and an electrodeposited PPy coating needle. PPy is a conductive polymer which can effectively extract compounds through several interactions (i.e. hydrogen binding, dipole-dipole forces and π-π stacking). Figure 1 shows the chromatograms obtained after extraction of the target compounds, spiked at 100 ng mL-1, from 2 µL of whole blood samples by in-tube SPME devices and 2 minutes as the extraction time. It should be noted that the same extraction conditions were used for evaluation of the both devices. As anticipated, the PPy coated device displayed greater sensitivity, up to 30-fold greater, when compared to the spinal needle without any coating. More information regarding total recoveries is provided in Table 2.

Evaluation of extraction time profile In order to meet the requirements of drug monitoring applications that necessitate trace analysis of compounds, the volume of the extraction phase was increased to improve the sensitivity of the method. However, when the thickness of coating is increased, the extraction time is traded-off. Thin film microextraction,43 as an alternative configuration of SPME, increases the sensitivity of the method without sacrificing extraction time by providing large surface area-to-volume ratio. By increasing the length of the PPy coating inside the tube to 2.5 cm, the volume of the extraction phase is increased, thus subsequently improving the sensitivity of the method without sacrificing extraction time. Figure 2 shows the studied extraction times for the target compounds spiked in PBS at 100 ng mL-1, ranging from 1 to 20 minutes. As shown in Figure 2, 9 ACS Paragon Plus Environment


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all studied compounds reached equilibrium within 2 minutes. Given the simplicity of micro-sampling of untreated samples , and considering its short extraction time, the developed in-tube SPME device can be said to be an ideal device for rapid analysis of small volumes of samples, as the whole analytical procedure can be performed within 10 minutes.

Determination of target compounds in urine sample by in-tube-LC-MS/MS The performance of the developed in-tube SPME device was evaluated by validation of calibration curves and linear dynamic range (LDR), limits of detection (LOD), limits of quantification (LOQ), method blank and carry-over, precision, and accuracy. As acceptance criteria, the coefficient of determination (R2) had to be higher than 0.99, and the intercept of the calibration curve higher than the LOD. LODs and LOQs were obtained as S/N of 3 and 10, respectively. For the method to be considered carryover-free, the blank signal had to be lower than the reporting limit. As a proof of concept, drug-free urine samples were used for evaluation of the studied compounds, with propranolol, pindolol, diazepam, and carbamazepine chosen as model compounds (See Figure S2 of supplementary information). To obtain the calibration curve, up to 8 urine samples spiked with standard mixture were prepared in the range of 0.1 – 100 ng mL-1, and a weighted calibration curve (1/x2) was used to calculate the standard line. As shown in Table 3, the developed device was demonstrated to be capable of detection of trace amounts of compounds. Sub-parts per billion levels of detection (LOD) for pindolol and carbamazepine, of 0.2 and 0.4 ng mL-1, respectively, were achieved (Table 3 and Figure 3). LODs of 1 and 2 ng mL-1 were also obtained for propranolol and diazepam, respectively. Limits of quantitation (LOQ) of 0.5, 1, 3, and 5 ng mL-1 were also attained for pindolol, carbamazepine, propranolol, and diazepam. The accuracy of the method was evaluated at a concentration of 10 ng mL-1, by dividing the concentration calculated based on calibration curve by the known spiked concentration, and high quantitation accuracies, in the range of 91-115 %, were achieved for the model compounds (carbamazepine 91%, pindolol 113 %, propranolol 115 %, diazepam 91 %). Inter-device repeatability was evaluated at three levels of concentration, with RSD% obtained in the range of 1% to 18%, demonstrating the precision of the method for 10 ACS Paragon Plus Environment

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analysis of real samples (Table S4 of supplementary information). Chromatograms of a blank of urine and two levels of concentration are provided in Figure S2 of supplementary information. While several publications and well-documented reviews regarding applications of in-tube SPME in urine samples32 have been published prior to this study, the unprecedented strength and novelty of the herein developed in-tube SPME device is presented. In previous studies, volumes of samples ranging from 30 µL to few mL were used by passing the sample through the tube or by drawing/ejecting (D/E) the sample by pump while herein, a single drop of sample, V ≤ 2µL was analyzed by pulling the sample inside the needle with no requirement for other apparatus, and fast extraction was achieved within 2 minutes under static extraction conditions. Direct and rapid analysis of untreated samples is one of the main strengths of the developed in-tube SPME device in comparison to previously reported in-tube SPME applications, which necessitate pretreatment and manipulation of samples such as dilution (e.g. urine samples were diluted 10 times and filtered34,35,37) and centrifugation of samples prior to extraction.36

Application of the in-tube SPME device for analysis of a single drop of whole blood by LC-MS/MS However whole blood is an easily accessible matrix for diagnostic routines, it is also one of the most complicated biological matrices available for analysis due to the high content of different blood cells, protein (i.e. hemoglobin and albumin), and macromolecules present in this matrix. Biocompatible SPME fiber, as an alternative technique, has been already used for direct analysis of whole blood samples.15,22 However, analysis of whole blood samples by in-tube SPME poses a critical challenge (i.e. clogging the tube) due to the presence of different blood cells, mainly erythrocytes (i.e. 40-50 % of content whole blood cell), which have hindered efficient application of the method in the past. As the diameter of red blood cells are in the range of 6-8 µm, a smooth and homogenous thin layer of coating is required to avoid clogging of the tube. Our previous study has demonstrated that addition of CTAB as a surfactant results in nano-structures and a homogenous coating with a pore size lower than 80 nm.12 In addition to the device biocompatibility and have a smooth surface, a short equilibrium time (i.e. t ≤ 2 min), facilitated by the geometry of the device, helps minimizing the possibility of clogging the needle by blood cells present in the matrix. 11 ACS Paragon Plus Environment


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To evaluate the sensitivity of the method, compounds characterized by high protein binding, including diazepam (protein binding ≥ 98 %), propranolol (protein binding 90 %), carbamazepine (protein binding 70-80 %), and pindolol (protein binding 40 %), were spiked onto drug-free whole blood samples. Chromatograms of a blank of blood and extraction at 25 ng mL-1 are provided in Figure S3 of supplementary information. LODs of 1, 3, 4, and 10 ng mL-1 were attained for pindolol, carbamazepine, propranolol, and diazepam. High quantitation accuracies, ranging from 95-101 %, were attained when the method was evaluated at 50 ng mL-1, using a drop of whole blood as sample volume (carbamazepine 98%, pindolol 95 %, propranolol 101 %, and diazepam 95 %). Table 3 shows the figures of merit of the studied compounds in the evaluated dynamic range. While previous in-tube SPME applications in complex matrices such as plasma36 and serum44 have necessitated protein precipitation, the successful direct analysis of untreated whole blood in the presented work showcases the developed in-tube SPME device as an ideal bioanalytical tool for therapeutic drug monitoring and POC diagnosis.

Direct coupling of in-tube SPME device to mass spectrometry Aiming to shorten the time of analysis, a strategy was developed by coupling the developed in-tube SPME device directly to MS, as the proof of concept. The PBS solution spiked at 50 ng mL-1 of carbamazepine, propranolol, pindolol and diazepam was used to evaluate the applicability of the developed in-tube-MS/MS configuration. After extraction and rinsing steps, the in-tube SPME device was assembled in the loop, Figure S4-a supplementary information, in the load position. Mixture of iPA:MeOH:ACN (50:25:25), with 0.1% FA, was used for on-line desorption and determination of the extracted compounds by switching the loop from load to inject position. To enhance the sensitivity of the method, compounds need to be introduced in the MS in a short pulse improving the S/N ratio. To do so, a guard column was attached before the electrospray probe to concentrate the compounds before introduction to MS. Several parameters were investigated (Section IX of supplementary information) to improve the peak shape and sensitivity of the method. Our finding based on the experimental results showed that desorption of the analytes at low flow rate (i.e. 20 µL min-1) for a short period of time and then increasing to 500 µL min-1 results in improving the sensitivity (See Figure S5c of the 12 ACS Paragon Plus Environment

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supplementary information). It should be noted that guard column was only used to introduce the compounds in the MS in a short pulse without having any significant retention and sacrificing the time of analysis. Provided the fast extraction and on-line desorption and determination, total analysis time about 3 minutes (e.g. 2 minutes extraction and 1 minute on-line desorption and determination) was achieved. The developed method was used for extraction of pindolol, propranolol and diazepam, as the model compounds, in 2 µL of urine sample. LOD values of 0.7, 2.0 and 2.0 ng mL-1 with R2 of 0.9927, 0.9992 and 0.9995 in the evaluated range, were achieved for pindolol, propranolol and diazepam, respectively, See Figure S9 of supplementary information. Accuracy of the method was also evaluated at the concentration of 10 ng mL-1, and high accuracy in quantitation including 101 %, 101 % and 102 % for pindolol, propranolol and diazepam were achieved with the RSD below 20 %. It should be noted that our group already used a GC capillary column as the in-tube SPME device, before LC loop, for direct coupling to MS45. However, lack of biocompatible coating limited its application to water samples as the capillary column is blocked by passing the complex matrices. Apart from advantages of the developed in-tube SPME device compare to the GC column including facilitating the rapid analysis, biocompatibility and on-site and in-vivo sampling, it can be used as the both sampling device and electrospray probe. As the future study the dedicated ion source can be built to insert the developed in-tube device in front of the MS to be used as extraction device and an electrospray probe for direct introduction of the compounds to MS without any loop and pump, by pre-loading in-tube device with an appropriate solvent. Moreover, by selection of the tubes with narrower diameter, sensitivity can be improved similar to nano-ESI approach. In addition, capability of desorption of the compounds in a few micro-liter of solvent, (i.e. V ≤ 2 µL)

makes the developed device an ideal probe for offline desorption and direct coupling to MS via nanoESI/MS/MS12. Owing to high extraction efficiency of the in-tube SPME along with enhanced sensitivity achieved by the nano-ESI probe, orders of magnitude improving of sensitivity are expected to be attained. While the in-tube SPME device is very robust and stable and can be used over 50 times, given the low cost of the spinal needle along with simple and rapid preparation of PPy coating, it can be used as a disposable device effectively in drug monitoring and point of care 13 ACS Paragon Plus Environment


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analysis. Given its high sensitivity, the developed in-tube SPME device offers a promising approach not only for target analysis of drugs for POC, but also, as the future direction, for untargeted metabolomics studies in instances where limited amounts of sample are available, or analysis of single cells to monitor heterogeneity populations of individual cells.

Conclusion Rapid analysis of untreated complex matrices (i.e. whole blood and urine) was achieved by development of a biocompatible in-tube SPME device. Preparation of PPy in the presence of CTAB as counter ion resulted in a nano-structure homogenous network with a pore size below 80 nm. Given the biocompatibility of both the PPy coating with a small pore size and the chosen spinal needle, analysis of a single drop of whole blood was attained without the need for any further sample modifications, such as dilution or protein precipitation. Provided the large surface area of the coating inside the narrow needle, low detection limits were obtained, even for compounds characterized by a high protein binding index (i.e. diazepam, more than 98 % binding). The extraction efficiency of the device can be further improved by coating a thin layer of hydrophiliclipophilic balance (HLB) inside the needle. In previous work, our research group developed miniaturized methods such as coated tip SPME and CBS for analysis of small volumes of complex matrices. The currently presented study offers yet another geometrical configuration of SPME towards miniaturization and greener, rapid analysis. These devices can be used in a complementary manner for a variety of applications

Acknowledgment The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for the financial support.


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Figure captions

Figure 1- Comparison of the chromatograms of a) spinal needle without PPy coating, bare spinal needle, and b) PPy coated spinal needle. Extraction was performed in 2µL of whole blood sample spiked at 100 ng mL-1 with caffeine, thiabendazole, carbamazepine, pindolol, testosterone, diazepam, and propranolol (in order of elution).

Figure 2- Evaluation of the extraction time profile of the developed in-tube SPME device in LCMS/MS. PBS solution spiked at 100 ng mL-1 of the studied compounds.

Figure 3- Evaluation of the calibration curve of the developed device; a) carbamazepine in 2 µL of urine sample. Accuracy of the method towards extraction of analyte was evaluated in urine spiked at 10 ng mL-1. b) pindolol in 2 µL of whole blood sample. Accuracy of the method towards extraction of analyte was evaluated in whole blood sample spiked at 50 ng mL-1. Extraction time: 2 minutes; desorption volume: 15 µL of MeOH:H2O (80:20 v/v); 10 µL of desorption solvent run into the LCMS/MS.



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Figure 1


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Figure 2



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Figure 3. a)

b)


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Table 1- Matrix effect of the in-tube SPME device in whole blood sample by LC-MS/MS Matrix effect (n=3*) Compounds

Low

Mid -1

RSD % (n=3*) High

Low -1

High

(5 ng mL )

(50 ng mL )

(0.5 ng mL )

(5 ng mL )

(50 ng mL-1)

Riboflavin

120%

109%

83%

11%

8%

6%

Carbamazepine

116%

109%

91%

10%

8%

6%

Propranolol

93%

111%

92%

9%

10%

6%

Testosterone

97%

112%

94%

19%

7%

8%

Dexamethasone

103%

111%

85%

20%

10%

4%

Thiabendazole

98%

117%

93%

14%

6%

6%

Pindolol

103%

109%

93%

10%

10%

1%

113%

89%

10%

8%

2%

102% * Three independent SPME devices

-1

Mid

(0.5 ng mL )

Diazepam

-1


-1


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Table 2- Total recovery of the studied compounds obtained by in-tube SPME-LC-MS/MS of 2 µl of PBS and whole blood sample, extraction time of 2 minutes Compound

Total recovery in PBS

Total recovery in whole

Total recovery in whole

sample by PPy coating

blood by PPy coating

blood (no coating)

7%

0.8%

10%

0.4%

12%

1.7%

13%

1.2%

8%

0.6%

-

-

18%

0.6%

8%

0.4%

7%

1.0%

Log P

Riboflavin

-1.46

Carbamazepine

-0.07

Propranolol

1.75

Testosterone

1.83

Dexamethasone

2.45

Thiabendazole*

2.47

Caffeine

2.82

Pindolol

3.32

Diazepam

3.48

31% 51% 41% 40% 31% 55% 42% 48% 43%

* Thiabendazole is a pesticide and not a compound of interest in whole blood sampling for drug monitoring purposes.


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1 2 Table 3- Figures of merit of the in-tube-SPME-LC-MS/MS in 2 µL of urine and blood samples, extraction time of 2 minutes 3 4 5 Urine Blood 6 7 Compound Accuracy at Accuracy at LOD LOQ LDR LOD LOQ LDR 8 Equation R2 Equation 9 10 ng mL-1 50 ng mL-1 ng ml-1 ng ml-1 ng ml-1 ng ml-1 ng ml-1 ng ml-1 10 11 y = 0.2992x+ y = 0.1087x 12 Carbamazepine 0.4 1 1-100 91 % 0.9986 3 10 10-200 98 % 0.0911 0.3016 13 14 y = 0.3418x y = 0.0871x 15 0.2 0.5 0.5-100 113 % 0.9994 1 3 3-200 95 % Pindolol 16 0.0674 0.0558 17 18 y = 0.3892x y = 0.0315x 2 5 5-100 91 % 0.9965 10 25 25-200 104 % Diazepam 19 0.0222 0.0043 20 21 y = 0.3446x y = 0.1056x 1 3 3-100 115 % 0.9913 4 10 10-200 95 % Propranolol 22 0.6265 0.0158 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 23 45 46 ACS Paragon Plus Environment 47 48

Protein R2 binding 0.9983

0.9977

0.9952

0.9931

70-80 %

40 %

≥ 98 % ≥ 90 %


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For TOC only


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Development of a Biocompatible In-Tube Solid-Phase Microextraction

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