Development and Application of Zirconia Coated Paper Substrate for

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Development and Application of Zirconia Coated Paper Substrate for High Sensitivity Analysis of Therapeutic Drugs in Dried Blood Spots Yajun Zheng, Qian Wang, Xiaoting Wang, Ying Chen, Xuan Wang, Xiaoling Zhang, Zongquan Bai, Xiaoxiao Han, and Zhiping Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04732 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 18, 2016

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Analytical Chemistry

Development and Application of Zirconia Coated Paper Substrate for High Sensitivity Analysis of Therapeutic Drugs in Dried Blood Spots Yajun Zheng,‡,a Qian Wang,‡,a Xiaoting Wang,a Ying Chen,b Xuan Wang,a Xiaoling Zhang,a Zongquan Bai,a Xiaoxiao Han,a Zhiping Zhang*,a a b ‡

School of Chemistry and Chemical Engineering, Xi’an Shiyou University, Xi’an 710065 (China) Clinical Analysis Laboratory, Xi’an Mental Health Center, Xi’an 710061 (China) These authors contributed equally to this work.

ABSTRACT: Paper spray mass spectrometry has been demonstrated to be promising for direct analysis of therapeutic drugs in dried blood spots (DBS); however, the strong hydrogen bond and van de Waals interactions between paper substrate and analytes containing polar functional groups (e.g., therapeutic drugs) affect greatly the elution behavior and analysis sensitivity of compounds of interest during paper spray. Herein, we developed a one-sided ZrO2 coated paper substrate through a facile vacuum filtration approach using commercial ZrO2 particles as coating material and soluble starch as adhesive agent. Owing to the unique surface properties, asprepared ZrO2 paper substrate has been shown to have excellent performance for analysis of therapeutic drugs in DBS during paper spray mass spectrometry. In contrast to original cellulose paper substrates, improvements of 43 – 189 folds in lower limit of quantitation (LLOQ) were obtained for the tested drugs using ZrO2 coated paper for paper spray. In comparing with the previously reported grade SG81 paper and one-sided silica coated paper, the LLOQs of the tested drugs with as-prepared ZrO2 paper decreased 1.5 – 16.5 folds relative to those from the above two, revealing that ZrO2 coated paper is a good candidate for paper spray in high sensitivity analysis of therapeutic drugs in DBS.

INTRODUCTION Therapeutic drug monitoring (TDM) is the clinical practice of measuring specific drugs at designated intervals to maintain relatively constant concentrations in a patient's bloodstream. 1 By combining the knowledge of pharmaceutics,2 pharmacokinetics,3 and pharmacodynamics,4 TDM enables the assessment of the efficacy and safety of a particular medication in a variety of clinical settings. Many drugs that require therapeutic monitoring must be maintained at steady concentrations even for a lifetime while the person ages and goes through life events that may alter the individual's therapeutic level. In order to get a correct assessment whether drug concentrations are maintained in a target range or not, a patient’s blood sample should be collected at timed intervals. In the past decades, DBS sampling5-8 has been a prevalent way to collect and store blood samples for metabolic disorders,9 epidemiological,10 toxicokinetics,11 pharmacokinetic studies,12 diagnostic screening,13 and TDM14-17 due to its numerous advantages (e.g., less invasiveness, small blood volume, simple storage method, cheap sample shipment, less risk of blood borne pathogens) over conventional blood or plasma sampling.7,8,14 Using this technique, blood is obtained generally via a finger-prick with an automatic lancet, and then the drop of blood is applied to a sampling paper, dried, and transported to a laboratory for analysis.14 To date, a wide range of assay techniques, including high performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS),18-21 HPLC with UV22,23 and fluorescence detection,24,25 gas chromatography (GC)-MS,26 enzyme-linked

immunosorbent assay (ELISA), 27 polarization immunoassay,28 and radio-immunoassay,29 have been extensively applied in TDM. Among these methods, HPLC-MS/MS is preferable due to its high specificity, sensitivity and precision in quantitative analysis of therapeutic drugs in DBS, whereas this method requires tedious and time-consuming sample extraction and chromatographic separation prior to MS analysis.7,8,30 The recent appearance and development of ambient ionization technique 31,32 have greatly promoted the TDM in DBS with highly simplified procedures and acceptable sensitivity.7,33 After interfacing this technique to MS/MS instruments, rapid identification and quantitative analysis of therapeutic drugs can be achieved in a few minutes or even less, and direct MS analysis offers a promising future for high-throughput analysis of therapeutic drugs. Several ambient ionization approaches, including desorption electrospray ionization (DESI),34 liquid extraction surface analysis (LESA),35 direct analysis in real time (DART),36 atmospheric pressure thermal desorption chemical ionization (APTDCI),37 laser diode thermal desorption atmospheric pressure chemical ionization (LDTD–APCI) 38 and paper spray ionization (PSI),33,39,40 have been successfully applied in rapid analysis of target therapeutic drugs in DBS. Owing to the facile operation, low-cost and simple separation of PSI, this method has demonstrated the potential in high-throughput therapeutic drug screening in DBS,41 and has been commercialized by Prosolia Inc. As reported in many studies,7,33,42 however, the performance of PSI is highly dependent on the surface properties of the used paper substrate. To expand the advantages of PSI in a wide range of applications, diverse materials, ranging from inorganic oxides

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to carbon nanotubes,33,43-51 have been coated on the surfaces of commercially available paper substrates. Although these coated papers played a crucial role in protein and therapeutic drug analyses,33,46 and lowering the spray voltage of PSI,45,47 the analysis sensitivity of this technique in TDM is still needed to be improved. Metal oxides, as a type of exceptionally important inorganic materials, have been widely applied in many fields ranging from catalysis,52 separation,53 biosensor,54 semiconductor,55 ceramics56 to insulator57 due to their unique surface properties and thermal stability. In the previous studies, a number of metal oxides (e.g., MgO, Al2O3, TiO2 and ZrO2) as the packing materials for HPLC have demonstrated superior efficiency in separation of basic, neutral and acidic compounds over silica.58,59 As is well-known, the paper substrate for PSI not only serves as a carrier for loading samples and electrospray, but also has the capability of facile separation of target analytes from sample matrices.17,39,44,60,61 In order to fully capitalize the advantages of these materials for PSI, herein we coated different metal oxides, including MgO, Al2O3, TiO2, ZnO and ZrO2, at the surface of filter paper substrates through a facile vacuum filtration method,44 and explored the performances of different coated paper substrates in PSI for TDM in DBS. The results demonstrated that ZrO2 coated paper substrate exhibited superior performance to others in TDM. More importantly, we illustrated how to incorporate different commercially available materials onto the surface of filter paper through a facile process, and applied the coated paper substrates into TDM, which paves the way for high throughput and high sensitivity analysis of therapeutic drugs in DBS.

EXPERIMENT SECTION Chemicals and materials. The used filter paper for coating was from Hangzhou Special Paper Co. (Fuyang, China). The commercially available grade 1 chromatography paper and silica coated paper, namely grade SG81 paper, were purchased from GE Healthcare Bio-Sciences Corp. (Westborough, MA, USA). The irregular MgO, Al2O3, TiO2, ZnO and ZrO2 and silica particles with diameters of around 1 μm were from Shanghai STNano Science & Technology Co. Ltd (Shanghai, China). The used adhesive agents were ordered from Henan Yuzhong BioSciences Corp. (esterified starch and crosslinked starch, Zhengzhou, China), Jilin Zhiyou Technology Co., Ltd (corn starch, Changchun, China), Qingzhou Beilian Starch Co. Ltd (cassava starch, Weifang, China), Zhengzhou Yuanda Food Additives Co. Ltd (oxidized starch, Zhengzhou, China), Henan 1000Trading Company (pregelatinized starch, Zhengzhou, China), Tianjin Kemiou Chemical Reagent Co. (soluble starch, Tianjin, China), respectively. The used therapeutic drug standards including amitriptyline, D3-amitriptyline, clozapine, D8-clozapine, quetiapine, D8-quetiapine, risperidone, D4-risperidone, aripiprazole, D8-aripiprazole, verapamil and D6-verapamil were from Sigma-Aldrich (St. Louis, USA) or Toronto Research Chemicals Inc. (Toronto, Canada). The used bovine whole blood was purchased from Lanzhou Institute of Biological Products Co., Ltd. (Lanzhou, China). The used 0.9% (w/v) NaCl physiological saline was from Cisen Pharmaceutical Co., Ltd. (Jining, China). The human blood samples were collected from some patients treated with the drugs taken into account in Xi’an Mental Health Center (Xi’an, China). Preparation of metal oxides and silica coated paper substrates. The detailed procedures for preparing metal oxides

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(e.g., MgO, Al2O3, TiO2, ZnO and ZrO2) and silica coated paper substrates were similar to our recent studies. 43,44 For a typical preparation, 0.7 g ZrO2 particles were dispersed into 100 mL deionized water containing 0.1 g soluble starch as adhesive agent. In order to get a uniform solution for coating, the mixture solution was sonicated for 20 min, and the obtained suspension solution was directly transferred to a Buchner funnel covered by a piece of blank filter paper with 11 cm in diameter for coating. When the aqueous solution was completely penetrated through the filter paper, around 20 mL absolute ethanol was applied for washing in order to get rid of the remaining water at the surface of the coated paper. The papers were then hung in a hood to dry for hours and were pressed between glass plates overnight for use. Characterization of paper substrates. The surface structures of as-prepared ZrO2 coated paper and uncoated filter paper were examined by using a JEOL JSM-6390A scanning electron microscope (SEM). The photographic images of the paper substrates with DBS were taken with a Canon digital camera. Temperature-programmed desorption of CO2 (CO2-TPD) was carried out to determine the basicity of the metal oxide particles (e.g., MgO, Al2O3, TiO2, ZnO and ZrO2) using a Micromeritics ChemiSorb 2750 reactor. In the process, 600 mg metal oxide was heated to 300 oC at a rate of 10 oC min−1 under an Ar gas flow (50 mL min−1) and maintained at that temperature for 2 h to remove the surface impurities. After cooling down to 50 oC under an Ar gas flow (50 mL min−1), the sample was exposed to CO2 atmosphere (50 mL min−1) for 3.5 h. Thereafter, the sample was purged with helium gas (50 mL min−1) for 1.5 h and then heated to 800 oC at a rate of 10 oC min−1. The N2 adsorption-desorption isotherm was determined on a Micromeritics ASAP 2020HD88 apparatus at the temperature of liquid nitrogen, in which the samples were degassed at 350 °C for 4 h before measurement. The surface area was calculated by the Brunauer-Emmett-Teller method. Preparation of DBS samples. The standard samples used in the current study were prepared as follows: the solutions of therapeutic drugs, including amitriptyline, clozapine, quetiapine, risperidone, aripiprazole and verapamil and their corresponding internal standards, were prepared by dilution of stock solutions into 1:1 methanol/water with a concentration of 1 mg mL−1. The standards were then spiked into bovine whole blood samples by pipetting 1 μL of the standard into 999 μL of blank blood with a therapeutic drug concentration of 1 μg mL−1. The samples with lower concentrations of therapeutic drugs were prepared with a series of dilutions, each with a small volume of the therapeutic drug sample at a higher concentration and a large volume of blood. For example, the blood sample with 100 ng mL−1 verapamil was prepared with 30 μL of the prepared 1 μg mL−1 blood sample and 270 μL of blank blood. The concentrations of the therapeutic drugs in the final blood samples were 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, and 100 ng mL−1. For the precise quantification of these therapeutic drugs in DBS, the corresponding internal standard with a final concentration of 20 ng mL−1 was also added into the mixed blood samples as an internal standard. The dried blood spots were prepared by spotting a fixed volume (4 μL or unless otherwise specified) of the blood sample onto the as-prepared paper substrates, uncoated filter paper, and commercially available grade SG81 paper and drying for 6 h at room temperature. The samples were then stored at room temperature in a sealed bag for use.


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Paper spray analysis. All experiments on paper spray were the fragment ion m/z 165 from protonated verapamil m/z 455 in carried out with a TSQ Quantum Access Max mass spectromeDBS. It is apparent that the analysis sensitivity of verapamil ter (Thermo Fisher Scientific, San Jose, CA, USA). For paper with these metal oxides coated papers was 2 – 12-fold better spray, the DBS substrate was cut into a triangle (around 11 mm than that from uncoated filter paper. Among these coated papers, height and 7 mm base width). A copper clip was used to hold ZrO2 coated paper demonstrated a superior performance to oththe paper triangle and to apply the high voltage needed for the ers, and the analysis sensitivity with the papers coated with spray. The distance between the tip of the paper triangle and the MgO, ZnO, Al2O3 and TiO2 presents a gradual decreasing trend. inlet to the mass spectrometer was about 5 mm. Mass spectra To our knowledge, the difference might be ascribed to the varwere recorded in the positive ion mode with a capillary temperious surface properties of metal oxides in adsorption of veraature of 270 °C. The identification of analyte ions was conpamil. A weak adsorption interaction between them would be firmed by tandem mass spectrometry (MS/MS) using collisionfavorable to the elution of verapamil from metal oxide during induced dissociation (CID). Argon gas (99.995% purity) was PSI, and vice versa. To get a direct evidence into both interacused as collision gas. The SRM and instrumental parameters tions, the following adsorption experiment was carried out. First, used for the therapeutic drugs are as follows: amitriptyline, m/z 5 mmol metal oxides were, respectively, added into 10 mL aqueous solution containing 10 μg mL-1 verapamil followed by 278→84; tube lens, 76 V; collision energy, 23 V; D3-amitriptystirring for 30 min. After that, the solution was allowed to mainline, m/z 281→87; tube lens, 76 V; collision energy, 25 V; tain for 5 min, and 100 μL of the upper clear solution was exclozapine, m/z 327→270; tube lens, 79 V; collision energy, 21 actly pipetted, which was then diluted with 900 μL acetonitrile. V; D8-clozapine, m/z 335 → 275; tube lens, 79 V; collision energy, 24 V; quet100 100 (a) (b) iapine, m/z 384→253; tube lens, 94 V; collision energy, 22 V; D8-quetiapine, 80 80 m/z 392→258; tube lens, 94 V; collision energy, 23 V; risperidone, m/z 411 → 60 60 191; tube lens, 75 V; collision energy, 27 V; D4-risperidone, m/z 415 → 195; 40 40 tube lens, 85 V; collision energy, 26 V; aripiprazole, m/z 448→285; tube lens, 20 20 102 V; collision energy, 24 V; D8-aripiprazole, m/z 456→293; tube lens, 102 V; collision energy, 26 V; verapamil, 0 0 ZnO ZrO MgO Uncoated TiO MgO Solution ZrO Al O Al O TiO ZnO m/z 455→165; tube lens, 91 V; collision Different Adsorpion Systems Different Metal Oxides Coated Paper Substrates energy, 26 V; m/z 455→303; tube lens, (c) 92 V; collision energy, 24 V; D6-verapamil, m/z 461→309; tube lens, 93 V; collision energy, 24 V. Ethical conduct of research. The auTop Side Top Side Top Side Top Side Top Side Top Side thors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Xi'an Back Side Back Side Back Side Back Side Back Side Back Side Shiyou University for all animal or biological sample experimental investigaZrO2 Al2O3 Filter Paper TiO2 ZnO MgO tions. Relative Intensity

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RESULTS AND DISCUSSION Screening of coating materials. In order to take a survey of the performance of different metal oxides coated paper substrates for PSI, some common packing materials for HPLC (e.g., Al2O3, TiO2 and ZrO2) and MgO and ZnO were, respectively, coated onto the surfaces of filter papers through our recently developed vacuum filtration strategy.44 Figure 1a compares the performance of different metal oxides coated paper substrates for PSI in analysis of 1 μg mL-1 verapamil in DBS. The performance of different metal oxides coated papers was evaluated through the peak intensity of

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Figure 1. (a) Comparison of the performance of different paper substrates coated with metal oxides in paper spray. Note: The coated amount of metal oxides was 0.7 g, and the solution volume for coating was 100 mL with 0.1 g soluble starch. The quantitative analysis was evaluated by the peak intensity of the fragment ion m/z 165 from protonated verapamil m/z 455 in DBS by using paper spray mass spectrometry (verapamil concentration in dried blood spot: 1 μg mL−1; blood volume: 2 μL; spray solvent: 25 μL acetonitrile; applied voltage: 3.5 kV). Uncoated represents uncoated filter paper; (b) Comparison of the adsorption performance of different metal oxides. Note: The experiments were carried out by adding 5 mmol different metal oxides into 10 mL aqueous solution containing 10 μg mL-1 verapamil followed by stirring for 30 min. After that, the solution was maintained still for 5 min, and then 100 μL of the upper clear solution was pipetted, which was then diluted with 900 μL acetonitrile for subsequent analysis. The remaining content of verapamil in solution was analyzed with paper spray through the peak intensity of the fragment ion of m/z 165 from verapamil. The used paper substrate was grade 1 chromatography paper, and the applied amount of sample solution was 25 μL. Solution in Figure 1b represents 1 μg mL-1 verapamil solution (9:1 acetonitrile/water); (c) Photographic images of top and back sides of uncoated filter paper and different metal oxide coated paper substrates spotted with 2 μL whole blood samples containing 1000 μg mL−1 methylene blue, and the diameters of the blood spots were around 4.5 mm.



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The content of verapamil in the prepared solution was analyzed subsequently with (a) (b) paper spray. As shown in Figure 1b, with variation of the metal oxides ranging from ZrO2 to TiO2, the peak intensity of m/z 165 from verapamil changed dramatically. In the system containing ZrO2, the concentration of verapamil presented a close value as that from the pure verapamil solution (labelled as Solution in X 500 50 μm X 500 50 μm Figure 1b), which suggests that ZrO2 has a very weak adsorption ability to vera(c) (d) (e) (f) pamil. For the solution systems of MgO, ZnO and Al2O3, about 20 – 40% verapamil was adsorbed at the surface of these metal oxides. When TiO2 was added into the solution, the level of veraTop Side Back Side Top Side Back Side pamil decreased sharply to around 8%, which means that 92% verapamil in the Figure 2. SEM images of (a) uncoated filter paper and (b) ZrO2 coated paper, and photographic aqueous solution system has been ad- images of [(c) and (e)] top and [(d) and (f)] back sides of [(c) and (d)] uncoated filter paper and sorbed on its surface. Notably, the ad- [(e) and (f)] ZrO2 coated paper with DBS (Note: The coated amount of ZrO2 was 1.0 g, which sorption ability of these metal oxides was the optimal value after series of experimental optimization as discussed below, and the demonstrated a similar trend as the per- solution volume for coating was 100 mL with 0.1 g soluble starch; Sample volume: 2 μL; Diformance of metal oxides coated paper ameters of the blood spots: around 6.0 mm). substrates in paper spray for analysis of back side. But when the ZrO2 coated paper was used, it is notaverapamil in DBS (Figure 1a). These results suggest that during ble that methylene blue is mainly distributed at the top side of the paper spray, the elution efficiency of verapamil from the the coated paper, and it is hardly observed at the back side. It metal oxides coated papers are related closely to the adsorption should be pointed out that when other chromogenic agents such ability of these metal oxides. A weaker adsorption ability would as indigo was mixed with whole blood sample, such a phenomlead to a higher analysis sensitivity of the resulting paper subenon was also observed (Figure S2). These results suggest that strate in PSI. More importantly, it can be learned from the above during the spotting of the blood sample containing methylene experiment that if an optimal material was desired for coating blue at the ZrO2 coated paper, an obvious separation event bepaper substrate for PSI in analysis of a target compound, the tween the blood sample and methylene blue occurred, presumabove adsorption experiment would be helpful in rapidly ably due to the stronger interaction between methylene blue and screening the coating material. For other tested drugs including the coated ZrO2 particles than that with other metal oxides (e.g., amitriptyline, clozapine, quetiapine, and risperidone, they TiO2, Al2O3, ZnO and MgO). In general, the capabilities of demonstrated a similar trend as verapamil described above exmetal oxides are closely related to its surface properties, cept for aripiprazole (Figure S1). whereas no direct correlation was found between the perforIn addition to the variation in the adsorption behavior of the mance of these metal oxides in paper spray and their surface target analyte with metal oxides, we also found that the distriacid-base properties (Figure S3) and texture properties (Table bution of the compound of interest at the surfaces of the coated S1). The detailed reason is not clear yet and needs to be further paper substrates also varied. To demonstrate this case, methinvestigated. ylene blue, instead of the colorless therapeutic drug verapamil, was first mixed with whole blood sample followed by spotting Surface properties of ZrO2 coated paper substrate. Figure at the surface of the coated papers, and the concentration of 2a and b shows the typical SEM images of uncoated filter paper methylene blue in blood sample was 1000 μg mL-1. As shown and ZrO2 coated paper. It is obvious that for uncoated filter pain Figure 1c, it is apparent that with variation of the employed per (Figure 2a), its surface structure is composed of cellulosic paper substrates, methylene blue illustrated different distribufibers with diameters in the range of 10 - 28 μm, in good agreetion patterns. For the top sides of these papers, the color of ment with the commercial grade 4 chromatography paper,33 and methylene blue at the surfaces of the paper substrates coated each fiber is connected to each other through hydrogen bonding with Al2O3, ZnO, MgO and ZrO2 is much lighter that those from interaction resulting from the polysaccharides at its surfaces. 62 the uncoated filter paper and TiO2 coated paper, and no obvious In the construction of these fibers, it leaves behind some gaps rule could be obtained from the former. However, careful obbetween fibers. After the filter paper with a diameter of 11 cm servation can be found that for the back sides of the papers, the was coated with 1.0 g ZrO2 particles by using 0.1 g soluble color of methylene blue appears a steadily lighting trend rangstarch as adhesive agent, its surface structure underwent a great ing from uncoated filter paper to ZrO2 coated paper, in good change (Figure 2b). The cellulosic fibers and the gaps between agreement with variation in the performance of the different pathem were covered by the coated ZrO2 particles with diameters per substrates coated with metal oxides for paper spray (Figure of around 1 μm. Apparently, there was no obvious aggregation 1a). Namely, when the blood sample containing methylene blue phenomenon between the coated particles, indicating that ZrO 2 was spotted at the surface of uncoated filter paper, methylene particles could be well coated at the filter paper using the develblue uniformly penetrated through the paper substrates although oped vacuum filtration method.44 The uneven surface structure its color was a little darker at the top side than that from the of the resulting paper could be attributed to the irregular shapes


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of the coated ZrO2 particles. After 2 μL of bovine blood was, respectively, deposited on the surfaces of both uncoated filter paper and ZrO2 coated paper for preparation of DBS, it is notable that significant difference was observed between the top and back sides of both paper substrates. For the filter paper (Figure 2c and d), the applied bovine blood penetrated uniformly through the paper substrate, and the diameters of the DBS on the top and back sides illustrates a close value. The tiny difference between them demonstrates that the color of the back side of the paper substrate within the DBS area was a little darker than that from the top side. When bovine blood was deposited on the ZrO2 coated paper, the blood sample was completely blocked at the top side of the paper substrate (Figure 2e), and no obvious blood sample could be observed at the back side (Figure 2e), which is different from the performance of the commercially available silica coated paper SG81.33 To our knowledge, this phenomenon could be attributed to the surface property of the one-sided ZrO2 coated paper. Namely, after the blood sample was deposited on the coated paper, it would interact directly with ZrO2 particles covered at the top side of the cellulosic fibers of filter paper. Due to the plenty of capacity of the coated ZrO2, the blood was adsorbed at the surfaces of these particles and, therefore, was difficult to penetrate through the paper substrate to another side, which might be favorable to the analysis of target compounds in DBS during PSI. At the same time, from the photographic image of the DBS at the ZrO2 coated paper (Figure 2e), a “volcano” effect (higher concentrations of blood sample between the edge and the center of the spot, resembling the mouth of a volcano) was observed, which might be an issue when a sub-punching strategy is adopted and a non-whole spot is analyzed due to the non-uniform distribution of blood sample on the ZrO2 coated paper. From Figure 1c, however, it is obvious that the compound of interest was mainly distributed at the center of the coated paper substrates rather than illustrating a “volcano” effect similar to the blood sample. More importantly, the results from different sizes of punched ZrO2-coated paper bearing DBS suggests that the “volcano” effect had little effect on the subsequent analysis of therapeutic drugs (Figure S4).

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Determining factors in preparation of ZrO2 coated paper substrate. In the preparation of ZrO2 coated paper substrate, it was found that the type and amount of adhesive agent and the amount of ZrO2 particles played crucial roles in determining the performance of the resulting paper. Figure 3a shows the effect of the type of adhesive agent on the capability of ZrO 2 coated paper, and the investigated adhesive agents included esterified starch, corn starch, cassava starch, oxidized starch, crosslinked starch, pregelatinized starch, and soluble starch. The performance of the prepared paper was evaluated by the peak intensity of the product ion m/z 165 from verapamil in DBS using PSI. From this figure, it can be seen that with variation of the type of the used adhesive agent, the analysis sensitivity of PSI changed dramatically. When esterified starch was employed, the resulting paper demonstrated the poorest performance, whereas soluble starch gave the optimal paper substrate among the prepared ones, which was around 4-fold better than that from esterified starch. These results illustrate much difference from the preparation of one-sided silica coated paper for analysis of pesticides in milk, in which corn starch demonstrated the optimal performance rather than soluble starch.43 It reveals that in order to prepare a coated paper substrate for PSI, it is necessary to optimize

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Figure 3. (a) Effect of starch type on the performance of as-prepared ZrO2 coated paper including esterified starch, corn starch, cassava starch, oxidized starch, crosslinked starch, pregelatinized starch and soluble starch; (b) effect of the applied amount of soluble starch on the paper performance; and (c) effect of the applied amount of ZrO2 on the paper performance. The solution volume for coating was 100 mL, and the evaluation was based on the analysis of 1 μg mL−1 verapamil [(M + H)+, m/z 455, product ion, m/z 165] in DBS by using paper spray mass spectrometry (spray solvent: 25 μL acetonitrile; applied voltage: 3.5 kV).

the suitable adhesive agent according to the properties of target analytes. Figure 3b shows the effect of the amount of soluble starch on the performance of as-prepared paper. It is apparent that with increase in the amount of soluble starch, the capability of the resulting paper demonstrates a first increasing trend followed by decreasing. When the used amount for coating was in the range of 0 – 0.10 g, the peak intensity of m/z 165 appeared a gradual increasing trend, and 0.10 g starch resulted in the optimal performance. Further increasing the amount of soluble starch from 0.10 to 0.30 g led to progressively decrease. This case might be ascribed to the influence of the ratio between the




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amounts of soluble starch 100 100 100 (a) (b) (c) and ZrO2 particles. When the amount of soluble 80 80 80 starch was too little, ZrO2 could not be well coated 60 60 60 at the surface of filter pa40 40 40 per, and serious powder nature would occur, 20 20 20 which was not favorable to the subsequent paper 0 0 0 spray analysis. Excess DCM ACN IPA EtOH MeOHWater 1 2 3 4 5 6 7 Pure Blood 1:1 1:2 1:4 1:8 amount of soluble starch Different Raitos of Blood Samples Volume of Blood Sample (L) Different Solvents would make its nature Figure 4. (a) Effect of spray solvent on the analysis of verapamil [(M + H)+, m/z 455, product ion, m/z 165] more dominant than that in DBS, in which DCM, ACN, IPA, EtOH and MeOH mean dichloromethane, acetonitrile, isopropanol, ethfrom ZrO2 at the surface of anol and methanol, respectively; (b) Effects of haematocrit level and (c) blood sample volume on the perforthe coated paper substrate mance of paper spray by using ZrO2 coated paper (spray solvent: 25 μL acetonitrile; applied voltage: 3.5 kV). although superfluous starch could lead to a tight by diluting the whole blood sample with 0.9% (w/v) NaCl physcombination of ZrO2 particles onto the surface of filter paper. iological saline because it approximates physiologically normal The amount of coated ZrO2 particles was also crucial in decondition and is a neutral solution. The volume ratios between termining the capability of as-prepared paper substrate. As blood sample and physiological saline were, respectively, 1:1, shown in Figure 3c, with increase in the amount of coated ZrO2 1:2, 1:4 and 1:8 (v/v). Figure 4b shows the effect of haematocrit ranging from 0 to 0.8 g by fixing the amount of soluble starch level on the analysis sensitivity of verapamil in DBS. It is clear (0.1 g), the signal abundance (m/z 165) of verapamil from the that with decrease of the haematocrit level in blood sample, the obtained paper substrates demonstrates a gradual increasing analysis sensitivity of verapamil demonstrates a gradually intrend followed by maintaining almost constant (0.8 – 1.0 g). creasing trend followed by keeping almost constant. Namely, Further increasing the amount from 1.0 to 1.2 g led to the dewhen the blood samples diluted with 1:4 and 1:8 physiological crease of the signal. This trend is similar to our recent reports saline, they demonstrate the highest analysis sensitivity, which on the preparation of silica coated paper substrate, which was was around 2.8-fold higher than that from the whole blood samclosely related with the ratio between the amounts of adhesive ple. From these results, we can conclude that a higher haematoagent and coating material.43,44 By analysis of the ratio derived crit level would lead to a lower analysis sensitivity of the target from Figure 3b and c, as the ratio was in the range of 10 – 12.5%, analyte in DBS using paper spray. the ZrO2 coated paper demonstrated the optimal performance, As reported in the literature,64,65 the loading amount of blood which was a little bit lower than that from the SiO2 coated paper sample had a great influence on PSI. In the present study, the (ca. 17%) for analysis of pesticides in milk.43 These results sugeffect of blood volume on the analysis performance of PSI using gest that in the preparation of different materials coated paper as-prepared ZrO2 coated paper substrate was investigated. As substrates, the ratio between the amounts of adhesive agent and shown in Figure 4c, when the volume of the loaded blood samcoating material should be well controlled besides the choice of ple ranged from 1 to 4 μL, the peak intensity of m/z 165 from their types as described above for the purpose of obtaining a verapamil in DBS illustrated a gradual increasing trend. Further favorable coated paper. enhancing the amount of blood sample (4 – 7 μL) resulted in a tiny decreasing of the signal. In our opinion, this phenomenon Spray solvent, haematocrit level and blood amount. Figure could be due to the fact that at a smaller volume of blood sample, 4a shows the effect of spray solvent on the analysis of verapamil the larger volume (25 μL) of spray solvent acetonitrile would in DBS, and the tested solvents included dichloromethane dilute the blood sample and lower the signal of the target ana(DCM), acetonitrile (ACN), isopropanol (IPA), ethanol (EtOH), lyte in blood.65 When the blood volume exceeded a certain methanol (MeOH) and water. It is clear that for as-prepared amount, it would spread over a larger area after depositing onto one-sided ZrO2 coated paper substrate, both the lowest (DCM) the surface of paper substrate. During PSI, the applied solvent and highest (water) polar solvents demonstrated the poorest an33,43 acetonitrile was hard to dissolve the DBS in a short time, which alytical results, in consistent with the previous studies. not only would influence a thorough interaction between the apWhen IPA, EtOH and MeOH were used as the spray solvents, plied solvent and target analytes in DBS, but also would prevent the signal illustrated a comparable value and was improved by the migration of analytes to the tip of paper for spray analysis. a factor of around 17 – 60-fold relative to those from DCM and Therefore, the signal presented a tiny decrease at a large sample water. As the spray solvent was ACN, the peak intensity of m/z volume. 165 presented the most intensive, which was around 5-fold better than those using different alcohols, presumably due to the Quantitative analysis. Figure 5a displays the full mass specfavorable solubilities and/or spray formation in PSI.63 trum recorded by dropping 4 μL of bovine whole blood spiked In the analysis of real blood samples, the level of haematowith 1 ng mL-1 clozapine onto as-prepared ZrO2 coated paper crit varies dependent on the individual discrepancy, which may and applying 3.5 kV DC voltage and 25 μL acetonitrile for pahave a pronounced effect on the interaction between the haeper spray. Among these peaks observed, it is hard to distinguish matic matrix and paper substrate. To get an insight into the efthe protonated clozapine (m/z 327) due to its low concentration fect of haematocrit level on the analysis of therapeutic drugs in and sample matrix interference. The tandem mass spectrum of DBS using as-prepared ZrO2 paper for paper spray, a series of clozapine is much simpler and shows the characteristic fragblood samples with different haematocrit levels were prepared ment ion m/z 270 with few impurities (Figure 5b). Quantitative Relative Intensity

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Iclozapine /ID8-clozapine

Relative Intensity

Relative Intensity

270 318 100 100 analysis of clozapine in DBS was m/z 327 274 (a) (b) achieved using the ratio of this characteristic fragment ion abundance to that 80 80 of the corresponding fragment ion generated from D8-clozapine, which was 60 60 also added into the blood as an internal 327 standard. To quantify the amount of clozapine in DBS, the blood samples 391 40 40 spiked with clozapine at different levels 362 (0.01, 0.05, 0.1, 1, 5, 10, 20, 50, 100 ng 20 20 mL-1) were prepared. Figure 5c shows 327 296 the dependence of clozapine signal on 0 0 its concentration in DBS using un200 240 280 320 360 400 200 240 280 320 360 400 coated filter paper for paper spray. m/z m/z 1 1 These data demonstrate a linear rela10 10 tionship in the concentration range of (c) Uncoated Paper (d) ZrO2 Coated Paper 0.5 – 100 ng mL-1 with a linear regres2 0 0 sion coefficient of R = 0.9856 covering 10 10 2 2 R = 0.9998 two orders of magnitude. A limit of R = 0.9856 -1 quantitation (LOQ) of 0.5 ng mL was -1 -1 derived from the lowest concentration 10 10 within the set of linear responses observed in the sensitivity plot. When as-1 0.5 ng mL prepared ZrO2 coated paper was used -2 -2 10 10 for paper spray, an LOQ of 0.05 ng mL-1 1 0.05 ng mL was achieved, which was one order of magnitude better than that from the un-3 -3 10 10 coated paper substrate, and the R2 value -2 -1 0 1 2 -2 -1 0 1 2 10 10 10 10 10 10 10 10 10 10 was as high as 0.9998 (Figure 5d). It is -1 -1 Concentration of Clozapine in Blood (ng mL ) Concentration of Clozapine in Blood (ng mL ) also worth noting that the relative Figure 5. (a) Full mass spectrum and (b) tandem mass spectrum of 1 ng mL-1 clozapine in DBS standard deviation (RSD) varied from using ZrO2 coated paper for paper spray; Comparison of the quantitative analysis of DBS 5.2% to 23.3% (n = 4 per concentration spiked with clozapine (0.01 - 100 ng mL-1) and its isotopomer [D ]clozapine (20 ng mL-1) using 8 point) in the investigated linearity range (c) uncoated filter paper and (d) as-prepared ZrO2 coated paper for paper spray (Note: 4 μL -1 (0.05 – 100 ng mL ). The RSD values blood sample was used, and the product ion m/z 270 of clozapine was monitored; spray solvent: were a little high (19.7 – 23.3%) at a 25 μL acetonitrile; applied voltage: 3.5 kV). lower concentration range (0.05 – 0.10 ng mL-1), and it could be well controlled different drugs. To get a better understanding on the perforin the range of 15% when the concentration of clozapine was mance difference between these papers and ZrO2 coated paper more than 0.50 ng mL-1, which meets the required standard of for PSI, the quantitation analysis of the above drugs in DBS was inaccuracy and imprecision set by the U.S. Food and Drug Adcarried out. As listed in Table 1, the LLOQ values of the tested ministration for bioanalytical method validation. 66 drugs from grade SG81 paper were in the range of 0.26 – 10.21 ng mL-1, which was as a whole comparable to the performance From the above discussion, it is obvious that the perfor(0.65 – 4.57 ng mL-1) from one-sided SiO2 coated paper. In mance of as-prepared ZrO2 coated paper is much better than that comparison with grade SG81 paper and SiO2 coated paper, imof the uncoated paper in quantitation of clozapine in DBS. Due provements of 1.5 – 16.5-fold were obtained in LLOQs for to this case, other therapeutic drugs in DBS such as amitriptythese drugs using ZrO2 coated paper except for verapamil. line, quetiapine, risperidone, aripiprazole, verapamil were also These results suggest that the present developed ZrO2 coated analyzed, and an LOQ in the range of 0.05 – 0.5 ng mL-1 was paper is a promising candidate for high sensitivity analysis of achieved for the tested drugs using as-prepared ZrO2 coated patherapeutic drugs in DBS. per for paper spray. An improvement of 10 - 100-fold relative to those from uncoated filter paper was obtained. Table 1 lists the obtained estimated lower limit of quantitation (LLOQ) valCONCLUSIONS ues for the examined drugs in DBS with the two paper subA novel one-sided ZrO2 coated paper substrate has been develstrates. It is apparent that the LLOQs from ZrO2 coated paper oped for high sensitivity analysis of different therapeutic drugs was as 43 – 189-fold better as those of uncoated filter paper, in DBS. To gain a better understanding into the effects of coatwhich could be attributable to the more favorable elution being conditions on the performance of the resulting paper subhaviors of the tested drugs (Figure 1a and Figure S5) and the strate for PSI, the type and amount of adhesive agent and the lower matrix effect of ZrO2 coated paper than those from the amount of ZrO2 particles for coating have been extensively exuncoated filter paper(Figure S6). These results indicate that the amined. We also found that the type of spray solvent and the present developed ZrO2 coated paper substrate is promising in applied volume of blood sample played significant roles in dehigh sensitivity analysis of therapeutic drugs in DBS. In the pretermining the performance of ZrO2 coated paper for quantitative 33,44 vious reports, however, the commercially available silica analysis of therapeutic drugs in DBS. Under the optimal condicoated paper (namely grade SG81 paper) and one-sided SiO2 tions, the LLOQs of the tested drugs achieved with the ZrO2 coated paper also demonstrated their potential in quantitation of coated paper has an improvement of 43 – 189 folds compared


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Table 1. Comparison of the estimated lower limit of quantitation (LLOQ) values (ng mL−1) of different therapeutic drugs in DBS by using different paper substrates Drug

Uncoated Paper

Amitriptyline

Grade SG81 Paper SiO2 Coated Paper

ZrO2 Coated Paper

Therapeutic Range67,68

241.65

10.21

4.57

1.38

50 -200

Clozapine

27.31

0.82

1.93

0.27

300 - 800

Quetiapine

15.24

0.44

0.61

0.10

20 - 400

Risperidone

20.13

0.51

1.02

0.21

3 - 20

Aripiprazole

5.18

0.26

1.98

0.12

150 - 500

79.58

0.30

0.65

0.42

20 - 250

Verapamil

Note: The coated amount of ZrO2 for preparation of ZrO2 coated paper was 1.0 g, and the solution volume for coating was 100 mL with 0.1 g soluble starch. For paper spray analysis, 4 μL blood sample was used, spray solvent: 25 μL acetonitrile; applied voltage: 3.5 kV. Estimated LLOQ was calculated through the equation LLOQ = 10 × sb/m, in which sb is the standard deviation of the response from the blank, m is the slope of the calibration curve. Four replicates for each sample. The procedure for preparation of SiO 2 coated paper was same as that in reference [44].

to uncoated filter paper. In comparison with the previously reported grade SG81 paper and SiO2 coated paper, improvements of 1.5 – 16.5 folds were obtained in LLOQs for tested drugs except for verapamil. To the best of our knowledge, the present developed paper substrate enables the highest sensitivity in analysis of different drugs in DBS using paper spray mass spectrometry, which paves the way for high efficiency and high throughput analysis of different therapeutic drugs in complex biological samples such as urine (Table S2) and human blood samples (Table S3).

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ASSOCIATED CONTENT

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Supporting Information Supporting Information Available: Surface and texture properties of different metal oxides, elution behaviors of the tested drugs, matrix effect of different paper substrates and the contents of different therapeutic drugs in real patents’ blood. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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Corresponding Author * Phone: +86-(29) 8838 2694. Fax: +86-(29) 8838 2693. E-mail: [email protected].

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Author Contributions ‡ These

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authors contributed equally. (16)

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT

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We are grateful for funding from the National Natural Science Foundation of China (21205093 and 21575112) and Shannxi S&T Research Development Project of China (Nos. 2014K13-16 and 2016GY-231)..

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REFERENCES (1)

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Concentration of Clozapine in Blood (ng mL )


Development and Application of Zirconia Coated Paper Substrate for

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