Rapid and Robust Quantification of p-Xyleneselenocyanate in Plasma

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Rapid and Robust Quantification of pXyleneselenocyanate in Plasma via Derivatization Wenyi Zheng, Fadwa Benkessou, Brigitte Twelkmeyer, Siyao Wang, Tobias Ginman, Håkan Ottosson, Manuchehr Abedi-Valugerdi, Maria Angels Subirana, Ying Zhao, and Moustapha Hassan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01426 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 25, 2017

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Analytical Chemistry

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Rapid and Robust Quantification of p-Xyleneselenocyanate in Plasma via Derivatization

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Wenyi Zheng1, Fadwa Benkessou1, Brigitte Twelkmeyer2, Siyao Wang3, Tobias Ginman4, Håkan Ottosson5, Manuchehr Abedi-Valugerdi1, Maria Angels Subirana6, Ying Zhao1,7, Moustapha Hassan1,7,*

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1. Experimental Cancer Medicine, Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, 141 86 Stockholm, Sweden

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2. Department of Anesthesiology and Intensive Care, CLINTEC, Karolinska Institutet and Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden

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3. School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia

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4. Sprint Bioscience, Huddinge, 141 86 Stockholm, Sweden

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5. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, 141 86 Stockholm, Sweden

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6. Universitat Autònoma de Barcelona. Department of Chemistry. Centre GTS. 08193 Bellaterra Barcelona Spain

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7. ECM, Clinical Research Center, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden

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*. Corresponding Author: Moustapha Hassan.

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Fax: +46-8-58583800

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Telephone: +46 8 585 838 62.

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E-mail address: [email protected]

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Abstract

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p-Xyleneselenocyanate (p-XSC) is one of the most investigated selenium compounds in cancer-prevention and -therapy. Despite the potent anti-cancer property, there is still no proper method to perform the quantitative analysis of p-XSC in plasma. In this investigation, we aimed at developing a method based on liquid chromatography-mass spectrometry (LCMS) for the measurement of p-XSC in plasma. Direct deproteinization was first used to extract parent p-XSC from plasma, but failed to achieve high recovery rate (< 2%) due to formation of selenium-sulfur bond between p-XSC and plasma protein. To overcome this problem, we modified the extraction method to three steps: 1) break the selenium-sulfur bond by tris(2-carboxyethyl)phosphine; 2) stabilize the newly formed intermediate selenol by Nethylmaleimide; 3) deproteinization. This three-step method efficiently recovered bound pXSC by more than 75%. In in vivo study, p-XSC was injected intravenously into mice and plasma was collected for LC-MS analysis. Consistently, p-XSC was undetectable in its parent form, whereas the bound form was readily quantified employing the modified extraction method. In summary, we describe a novel, robust and sensitive method for quantification of pXSC in plasma. The present method will enable pharmacokinetic and pharmacodynamics studies of p-XSC in both clinical and preclinical settings.

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Keywords: Selenocyanate, Liquid chromatography, Derivatization, Pharmacokinetics, p-Xyleneselenocyanate

Mass

Spectrometry,

Plasma,

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Selenium (Se) was discovered in Sweden in the early nineteenth century. Although selenium is similar to sulfur in many aspects, selenium compounds exhibit stronger acidity, nucleophilicity, electrophilicity, and leaving group ability.1 The biological activities are best demonstrated by smart insertion of selenocysteine into many key enzymes.2 Point mutation of selenocysteine in these enzymes diminishes their catalytic activity, and replacement of cysteine into selenocysteine increases the activity.3 Furthermore, many selenocysteinecontaining enzymes, such as thioredoxin reductase and glutathione peroxidase, are primary components of the antioxidant defense system.4 Under this rationale, selenium containing yeast has been marketed as a supplement designed for healthy individuals to enrich the intracellular selenium pool and strengthen the anti-oxidant system. However, selenium has also been known to exhibit a dose-related toxicity due to induction of reactive oxygen species (ROS), thought to be initiated by the formation of selenol (or selenolate anion) and subsequent one electron-transfer from selenol to molecular oxygen.5

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Due to the dual function of selenium, selenium compounds have been explored in the cancer field based on two opposite concepts. On the one hand, at a lower dose selenium is used as an anti-oxidant to prevent cancer initiation where ROS could play a detrimental role. On the other hand, those selenium compounds are applied at a higher dose to generate ROS and induce cancer cell-specific death. This concept is largely supported by the well-characterized phenotype of cancer cells, i.e. that they suffer from higher oxidative stress compared to their Page | 2 ACS Paragon Plus Environment

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normal counterparts. 6,7 Thus, in sharp contrast with the conventional chemotherapeutics, selenium compounds are believed to achieve cancer-targeted cytotoxicity.

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p-XSC is a synthetic organoselenium species and has gained large attention in cancer prevention and therapy during the last two decades. Its cancer-preventive role has been widely studied by oral administration of p-XSC in diet. 8,9 Significant decrease in tumor incidence and multiplicity was observed in both azoxymethane induced colon carcinogenesis and 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung carcinogenesis models. In comparison to selenium in the form of inorganics (selenite), selenoamino acid (selenomethionine), and yeast, p-XSC exhibits promising superiority in terms of efficacy and safety.8 In terms of chemotherapy, p-XSC is used at high concentrations, usually in the micromolar range in vitro, to induce apoptosis (e.g. oral squamous and prostate cancer) 10,11 Our group recently also found its therapeutic potential in a mouse leukemia model without severe side effects (unpublished data).

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In cancer prevention, p-XSC is proposed to reduce carcinogen-induced DNA damage by reinforcing the anti-oxidant system in the pre-malignant stage.9,12 On the other hand, the ability to generate ROS and induce further oxidative stress is necessary for chemotherapy. 13 Our preliminary results in leukemic cells have shown that p-XSC of a lower concentration promoted cell proliferation while a higher concentration resulted in cell apoptosis. Therefore, depending on the function p-XSC is supposed to fill, different doses should be applied to achieve personalized drug exposure. The in vivo behavior has been reported previously in the rat using radioactive p-XSC[C14]. In that study, the authors only monitored radioactivity and total selenium exposure, which could not describe the pharmacokinetic behavior. 14,15 The fact that selenium toxicity is highly species-dependent further stresses the necessity to have species-specific analytic methods for selenium compounds. 16

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Since parent p-XSC was almost undetectable in plasma in our initial investigation, we developed, evaluated and validated a new analytical method for quantification of bound pXSC instead after derivatization in order to study the pharmacokinetics behavior. The present method was able to track p-XSC after intravenous administration in the mouse in a rapid and sensitive way.

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Materials and Methods

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Materials

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p-XSC was bought from Abcam (Cambridge, United Kingdom). N-Ethylmaleimide (NEM), tris(2-carboxyethyl)phosphine (TCEP) and nitro-phenyl selenocyanate (NPSC) were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). Formic acid (FA) and acetonitrile (ACN) were both of analytic grades and purchased from Merck (Darmstadt, Germany), while dimethyl acetamide (DMA) was from Fluka (Seelze, Germany). Fetal bovine serum (FBS), horse serum (HS) and bovine serum albumin (BSA) were from ThermoFisher (Waltham, Massachusetts, United States). The water used in this work was Page | 3 ACS Paragon Plus Environment


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obtained from Purelab Ultra system from ELGA Veolia with resistance higher than 18.2 MΩ. Blank human plasma was purchased from blood centers as pooled plasma from anonymous healthy individuals and stored at -20 °C until use.

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Synthesis of p-XSC-NEM Conjugate

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p-XSC-NEM conjugate was synthesized as follows: p-XSC and NEM (12 mg and 90 mg, respectively) were dissolved in 9 ml DMA, to which 1.5 ml aqueous TCEP (84 mg) was added in drop-wise under vortexing. The crude material was purified by preparative liquid chromatograph and characterized via 1H-NMR (400 MHz, CDCl3) δ ppm 1.11 (t, J=7.2 Hz, 6 H) 2.37 (ddd, J=19.0, 6.3, 3.3 Hz, 2 H) 2.96 (dd, J=19.0, 9.1 Hz, 2 H) 3.50 (q, J=7.2 Hz, 4 H) 3.59 (dd, J=9.1, 3.0 Hz, 2 H) 3.89 (dd, J=12.0, 3.5 Hz, 2 H) 4.23 (d, J=12.0 Hz, 2 H) 7.24 7.29 (m, 4 H).

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Sample Preparation for Analysis of p-XSC

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Samples were prepared by two extractions. In the first method, a direct deproteinization was performed. Briefly, 50 µl solvent was added to spiked plasma (10 µl p-XSC solution plus 10 µl blank plasma) or mouse sample (10 µl DMA plus 10 µl mouse plasma), followed by centrifugation. To clarify the role of thiol, blank plasma was pre-incubated with 10 µl NEM (0.4 mol/l in DMA) for 10 min prior to being mixed with p-XSC. In the second method, a derivatization procedure was performed. Basically, 10 µl NEM (0.4 mol/l in DMA) and 5 µl TCEP (0.2 mol/l in water) were successively added to spiked plasma (5 µl p-XSC solution plus 10 µl blank plasma) or mouse sample (5 µl DMA plus 10 µl mouse plasma). The mixture was vortexed and left for 10 min at room temperature before deproteinization with 50 µl ACN.

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Liquid Chromatography and Mass Spectrometry Parameters

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p-XSC or p-XSC-NEM dissolved in ACN was infused into electrospray ionization-tandem MS to obtain mass spectrum. For method tuning, either was continuously introduced by mixing mobile phase and syringe infusion via a T-piece. During analysis, 2 µl sample was injected into the Agilent 1100 HPLC system. TSQ Quantum Mass Spectrometer from ThermoFisher was operated in positive mode. Other information is listed in Table 1.

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Assessment of Plasma Binding via Ultrafiltration

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200 µl p-XSC was mixed with either water (as control) or blank plasma. The mixture was transferred to centrifugal ultrafiltration tubes and centrifuged to obtain the extract in accordance with appropriate instructions. 20 µl extract was mixed with 50 µl ACN and analyzed for p-XSC. Plasma binding efficiency was calculated using the following equation: Plasma Binding Efficiency = 100 −

peak area of − XSC in plasma group peak area of − XSC in control group

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Application in Mouse Plasma

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All animal procedures were approved by the Stockholm Southern Ethical Committee and performed in accordance with Swedish Animal Welfare Law. p-XSC (dissolved in 10% Page | 4 ACS Paragon Plus Environment

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DMSO) was injected intravenously to a C57BL/6 albino mouse at the dose of 0.5 mg/kg. Blood was taken after 20 min via cardiac puncture and placed in an EDTA-coated vial. pXSC were analyzed using both extraction methods as mentioned above.

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Statistics

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Data was analyzed by GraphPad-Prism software and presented as mean ± relative standard deviation (Mean ± RSD) where applicable.

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Result and Discussion

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p-XSC Strongly Bound to Plasma

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The fragment ion m/z 209.9 was chosen as the target for monitoring p-XSC, and its response was linear to the concentration with a regression coefficient of 0.999 (Figure S-1A & 1A). The initial attempt was to determine p-XSC after direct deproteinization of spiked plasma, but only gave a recovery efficiency of 2%. Other types of matrices, including aqueous albumin solution (BSA), fetal bovine (FBS) and horse (HS) serum, exhibited the same phenomenon, with the order of binding efficiency being BSA > human plasma > FBS > HS (Figure 1B). Considering the absorption profile of p-XSC, a UV detector was used for verification. Similarly, p-XSC was strongly sequestered by plasma as well as other biologic matrices with the same order (Figure S-2A & 2B). Only half of the amount could be recovered when 1 µg pXSC was mixed with 5 µl blank plasma, while no parent compound was retrieved when increasing plasma to 30 µl (Figure S-2A). In addition to ACN, methanol and acid-based deproteinizing solvents were used, either alone or in combination. However, none of them improved the recovery (Figure 2A & Figure S-2C). Ultrafiltration is one of the golden methods for assessing the plasma binding profile. In consistence with the findings using deproteinization method, almost all p-XSC bound to plasma (Figure 1C).

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It appeared that the binding of p-XSC towards plasma was rather robust, and this phenomenon held true for multiple protein-containing matrices although with varying binding efficiency. Results from application of other deproteinizing solvents, which operate by distinct mechanisms, further suggest that the binding was not mediated by non-covalent interactions, and was accordingly irreversible when using direct deproteinization strategy.

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Selenium-Sulfur Bond Bridged the Binding

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The aforementioned results imply a covalent interaction between p-XSC and plasma protein; however, the possible bond remained to be illustrated. Moreover, it was reported that benzyl selenocyanate could be conjugated with glutathione at rather mild conditions via formation of a selenium-sulfur bond. 17 Since p-XSC contains two selenocyanate moieties, it’s likely that this compound interacts with free thiol groups presented on plasma proteins, the most likely candidate of which is albumin. 18



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To exclude the involvement of non-protein components of plasma, we analyzed the recovery of p-XSC after mixing with the extract of blank plasma. As shown in Figure 2B, comparable recovery was obtained after mixing p-XSC with ACN or plasma extract. This strongly indicates that plasma proteins sequester p-XSC. To further validate the likelihood of thiol groups present on proteins causing sequestration, excessive NEM was used to block the thiol groups on protein. As expected, no binding of p-XSC was found towards thiol-scavenged plasma (Figure 2C). In addition, another proof-of-concept experiment was conducted using an analogue of p-XSC, nitro-phenyl selenocyanate (NPSC), which has the thiol-reactive selenocyanate group (Figure S-3). Results demonstrate that plasma efficiently sequestered NPSC and higher plasma/NPSC ratio inversely correlated with recovery, while such effects were completely abolished when applying NEM-treated plasma. Together, these observations suggest that the covalent binding, as a result of high reactivity between selenocyanate and the thiol group, might be responsible for low recovery of p-XSC upon direct deproteinization.

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Our group recently tested p-XSC in leukemic mice and demonstrated its therapeutic effect (unpublished data). However, after examining the recovery of p-XSC from a different amount of plasma (Figure S-2A), the dose employed to treat leukemic mice was thought to give little p-XSC in parent form. Therefore, an alternative form should be in place to confer the therapeutic effect. In addition, bound p-XSC is still capable of generating a selenol group, which centers on the toxicity machinery of selenium compounds, after reduction by the endogenous glutathione system. 18,19 From this perspective, bound p-XSC maintains its therapeutic potential and should be monitored.

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Derivatization of p-XSC with TCEP/NEM

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Since the selenium-sulfur bond stabilized the bound p-XSC, several reducing reagents were used to cleave it. Among them, TCEP was more efficient than others including dithiothreitol, glutathione and N-acetylcysteine (data not shown). The minimal amount of TCEP was calculated basing on plasma albumin concentration (6% herein). Notably, besides one thiol group, each albumin molecule has 17 disulfide bonds that are substrates of TCEP as well. The reduced intermediate, selenol, is quite reactive per se 19,20 and necessitates immediate stabilization by excessive NEM (see graphic abstract and Figure 3A).

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In order to confirm the reaction route, the derivative p-XSC-NEM was synthesized and characterized by 1H-NMR (Figure S-4). The peaks at ppm of 1.11 and 3.50 were assigned to the ethyl group of NEM, which verified our proposal. The fragment ion m/z 309.9 was selected from the mass spectrum for quantification (Figure S-1B).

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The derivatization method was optimized from the following four aspects:

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I. The order in adding TCEP and NEM (Figure 3B); adding TCEP prior to NEM yielded much lower response than the other way around (50% vs 100%), in line with the short halftime of selenol.

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II. Derivatization duration (Figure 3C); the response reached the maximum in less than 1 min, suggesting an efficient extraction procedure. Page | 6 ACS Paragon Plus Environment

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III. TCEP amount (Figure 3D); when increasing the amount of TCEP while keeping NEM amount constant, an initial increase, intermediate plateau and final decline in response was observed, indicating that p-XSC-NEM was also decomposed by excess TCEP.

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IV. NEM amount (Figure 3E); the response increased in the front and reached a plateau when NEM exceeded 1 µmol, marking complete stabilization of selenol intermediates.

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After optimization, to recover p-XSC from 10 µl plasma (approximately containing 9 nmol free thiol group and 153 nmol disulfide bond), 4 µmol NEM was added prior to 1 µmol TCEP (24.7-fold and 6.2-fold than total disulfide groups on albumin, respectively).

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Method Validation

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The novel extraction method was validated in human plasma. Representative chromatographs from blank and spiked plasma are shown in Figure 4, and no interference from plasma was observed. The response was linear to concentration within the range of 0.2-5 µg/ml (Regression Coefficient = 0.999). The limit of detection was around 0.04 µg/ml (defined as S/N = 3). Although containing two steps in derivatization, it held high accuracy and recovery within the linear range (82.8-103.3% for accuracy, 75.1-88.3% for recovery). Both intra-day and inter-day precision were less than 2.6% (Table 2). Stability of processed sample and spiked plasma was monitored as well (Table 3). p-XSC-NEM adduct was stable after 24 hours of incubation at room temperature (about 22°C), 4°C or -20°C, or 5 cycles of freeze-thaw procedure, with almost full signal remaining in all cases. In comparison, leaving the spiked plasma at room temperature for 24 hours compromised the response with about 10% signal loss, indicating diminished stability of p-XSC in plasma. However, neither immediate cooling-down (4 or -20 °C for 24 hours) nor freeze-thaw treatment (5 cycles) of spiked plasma affected the stability as demonstrated by a signal loss of ≤ 2.6%.

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Analysis of p-XSC from Mouse Sample

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Figure 5A shows the extraction efficiencies from human plasma and that from mouse plasma were similar (84.1% and 84.6%, respectively) compared to that from aqueous solution. In the following studies, p-XSC was analyzed in mouse plasma collected 20 min after intravenous injection. Parent compound was undetectable using direct deproteinization (Figure 5B), while bound p-XSC was readily quantified after derivatization (0.59 ± 0.01 µg/ml, Figure 5C). This further reconciles with the conclusion drawn from the experiment on spiked plasma where almost all p-XSC exists in bound form. Notably, when analyzing p-XSC after mixing with plasma extract (final p-XSC concentration of 1 µg/ml); only a tiny peak was seen from the chromatograph insert in Figure 5B. Nevertheless, p-XSC-NEM peak was much more prominent despite a lower concentration (Figure 5C).

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The similarity in extraction percentage between mouse and human plasma is most probably due to the comparable composition of thiol groups on proteins, especially albumin. 21 We assume that more TCEP/NEM would be needed to extract p-XSC from matrices which contain a high amount of proteins or thiol groups. In summary, this present method not only recovered bound p-XSC, but also improved the sensitivity and enhanced limit of detection. Page | 7 ACS Paragon Plus Environment


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Conclusion

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In the present study, a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS) based method for analysis of p-XSC in plasma was developed. We showed that p-XSC held strong affinity to plasma proteins via a covalent selenium-sulfur bond, which hindered extraction with conventional deproteinizing solvents. TCEP/NEM derivatization strategy was utilized to break the covalent bond and stabilize the selenol intermediate. The present method enabled the detection of bound p-XSC in plasma and improved sensitivity, which could facilitate pharmacokinetic, pharmacodynamics and biodistribution studies in animals and humans. Although ESI-MS was used in this study, the application field of our method could be further expanded to encompass multiple detection models, such as ICP-MS and UV. Since several other selenium compounds, e.g. ebselen and diphenyl diselenide, have been assumed to be highly protein-bound via the selenium-sulfur (Se-S) bond, we believe that our method can potentially be adapted to analyze a wider range of such compounds to serve a variety of biological, chemical, and pharmacological applications.

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Supporting Information

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Experimental details and figures for analysis of p-XSC and NPSC by HPLC/UV;

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Mass spectra of p-XSC and p-XSC-NEM;

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1

H-NMR spectrum of p-XSC-NEM.

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Acknowledgement

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All authors contributed to conduction of experiments and manuscript writing. We acknowledge Jonathan Forsberg and Jennifer Usterud for critical linguistic editing and Fredrik Rahm for enthusiastic arrangement in 1H-NMR. This study was supported by grants from the Swedish Cancer Society (CAN2014/759, to M.H.). Wenyi Zheng is a recipient of a PhD student scholarship from China Scholarship Council.

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Conflict of Interest

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The authors declare no competing financial interests.

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References

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(1) Reich, H. J.; Hondal, R. J. ACS Chem. Biol. 2016, 11, 821-841. (2) Low, S. C.; Berry, M. J. Trends Biochem. Sci. 1996, 21, 203-208. (3) Bar-Noy, S.; Moskovitz, J. Biochem. Biophys. Res. Commun. 2002, 297, 956-961. (4) Becker D, Dickman M, Gladyshev V, Ragsdale S. Redox biochemistry; John Wiley & Sons: Hoboken, 2007. (5) Spallholz, J. E. Free Radicals Biol. Med. 1994, 17, 45-64. (6) Schumacker, P. T. Cancer cell 2006, 10, 175-176. (7) Trachootham, D.; Zhou, Y.; Zhang, H.; Demizu, Y.; Chen, Z.; Pelicano, H.; Chiao, P. J.; Achanta, G.; Arlinghaus, R. B.; Liu, J. Cancer cell 2006, 10, 241-252. (8) Reddy, B. S.; Rivenson, A.; Kulkarni, N.; Upadhyaya, P.; el-Bayoumy, K. Cancer Res. 1992, 52, 5635-5640. (9) Richie, J. P., Jr.; Kleinman, W.; Desai, D. H.; Das, A.; Amin, S. G.; Pinto, J. T.; El-Bayoumy, K. Chem.-Biol. Interact. 2006, 161, 93-103. (10) Ghose, A.; Fleming, J.; El-Bayoumy, K.; Harrison, P. R. Cancer Res. 2001, 61, 7479-7487. (11) Pinto, J. T.; Sinha, R.; Papp, K.; Facompre, N. D.; Desai, D.; El‐Bayoumy, K. Int. J. Cancer 2007, 120, 1410-1417. (12) Emmert, S. W.; El-Bayoumy, K.; Das, A.; Sun, Y. W.; Amin, S.; Desai, D.; Aliaga, C.; Richie, J. P., Jr. Free Radicals Biol. Med. 2012, 52, 2064-2071. (13) Brozmanova, J.; Manikova, D.; Vlckova, V.; Chovanec, M. Arch. Toxicol. 2010, 84, 919-938. (14) El-Bayoumy, K.; Upadhyaya, P.; Sohn, O.; Rosa, J.; Fiala, E. Carcinogenesis 1998, 19, 16031607. (15) Sohn, O. S.; Desai, D. H.; Das, A.; Rodriguez, J. G.; Amin, S. G.; El-Bayoumy, K. Chem.-Biol. Interact. 2005, 151, 193-202. (16) Marschall, T. A.; Bornhorst, J.; Kuehnelt, D.; Schwerdtle, T. Mol. Nutr. Food Res. 2016, 60, 2622-2632. (17) Kawamori, T.; El-Bayoumy, K.; Ji, B. Y.; Rodriguez, J. R.; Rao, C.; Reddy, B. S. Int. J. Oncol. 1998, 13, 29-34. (18) Yamasaki, K.; Chuang, V. T. G.; Maruyama, T.; Otagiri, M. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 5435-5443. (19) Prigol, M.; Nogueira, C. W.; Zeni, G.; Bronze, M. R.; Constantino, L. Chem.-Biol. Interact. 2012, 200, 65-72. (20) Mitchell, N. J.; Malins, L. R.; Liu, X.; Thompson, R. E.; Chan, B.; Radom, L.; Payne, R. J. J. Am. Chem. Soc. 2015, 137, 14011-14014. (21) Zaias, J.; Mineau, M.; Cray, C.; Yoon, D.; Altman, N. H. J. Am. Assoc. Lab. Anim. Sci. 2009, 48, 387-390.



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Table 1. Liquid chromatography and mass spectrometry parameters for determination of p-XSC and p-XSC-NEM Item Column Mobile Phase LC Flow Rate Gradient Spray Voltage Sheath Gas Auxiliary Gas MS Ion Sweep Gas Skimmer Offset Capillary Temperature Tube Lens Offset

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p-XSC p-XSC-NEM YMC AQ12S05-1546WT ACN/H2O + 0.1% FA 0.2 ml/min 70% ACN 5000 V 5000 V 50 Arb* 40 Arb 5 Arb 50 Arb 2 Arb 1.5 Arb -15 V -15 V 375 °C 277 °C 100 V 186 V

*

: Arbitrary Unit.


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Table 2. Precision, accuracy and recovery for analysis of p-XSC in plasma afterderivatization (N = 3). Precision

p-XSC (µg/ml)

Intra-Day

0.2 1 5

0.17 ± 0.67 1.03 ± 2.48 4.94 ± 1.80

Inter-Day

Accuracy

Recovery

0.17 ± 4.38 82.76 ± 0.68 75.10 ± 0.70 0.98 ± 2.55 103.23 ± 2.48 80.33 ± 2.49 4.81 ± 1.49 98.81 ± 1.81 84.55 ± 1.81

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Table 3. Stability of p-XSC in processed sample and plasma (N = 3). Treatment

Bench-top

4 °C

-20 °C

Freeze-Thaw

Processed Sample 103.48 ± 3.24 107.58 ± 2.75 104.64 ± 0.77 101.26 ± 0.70 Spiked Plasma 89.06 ± 1.43 97.46 ± 0.94 97.37 ± 1.41 99.72 ± 1.49 328


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Figure 1. Protein binding property of p-XSC.(A) Regression line between p-XSC peak area as determined by LC-MS and concentration of p-XSC. (B) Analysis of parent p-XSC in water (Control), blank human plasma, BSA solution (60 mg/ml in water), fetal bovine serum (FBS) or horse serum (HS) after deproteinization. Recovery rate is shown as the percentage of pXSC peak area in indicated group to that in control. (C) Plasma binding profile of p-XSC assayed by ultrafiltration method. In all experiments related to panel B&C, p-XSC concentration in spiked matrices was 30 µg/mL (N = 3).

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337

Figure 2. Plasma binding mechanism of p-XSC.

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(A) Analysis of parent p-XSC in water (control) or plasma after deproteinization with different solvents. Recovery rate is shown as the percentage of p-XSC peak area in indicated group to that in control. p-XSC concentration in spiked matrices was 30 µg/ml. (B) Analysis of parent p-XSC after being mixed with ACN or plasma extract (obtained from deproteinization of 10 µl blank plasma). (C) Analysis of parent p-XSC after being mixed with water or NEM-treated plasma. X-axis in panel B&C stands for p-XSC concentration in spiked matrices. (N = 3).

345


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Figure 3. Optimization of the derivatization method

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(A) Schematic reaction route between bound or parent p-XSC and TCEP/NEM. (B) Yield of the derivative, p-XSC-NEM, upon changing the order in adding TCEP (1 µmol) and NEM (4 µmol): TCEP before NEM (□, TCEP/NEM) or NEM before TCEP (■, NEM/TCEP). The reaction time was 10 min. (C) Yield of p-XSC-NEM after different reaction time. TCEP and NEM amount were 1 µmol and 4 µmol, respectively. (D) Yield of p-XSC-NEM at different TCEP concentration. NEM concentration was 4 µmol and reaction time was 10 min. (E) Yield of p-XSC-NEM at different NEM concentration. TCEP concentration was 1 µmol, and the reaction time was 10 min. In experiments related to Figure 3B-3E, yield of p-XSC-NEM was shown as the percentage of its peak area at indicated settings to that at optimal condition (N = 3).

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Figure 4. Representative chromatographs of p-XSC in blank and spiked plasma after derivatization.

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Blank and spiked plasma were treated with NEM (4 µmol) and TCEP (1 µmol) for 10 min. The supernatant was collected after ACN deproteinization and subjected to LC-MS analysis.

362


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Figure 5. Analysis of p-XSC in mouse plasma.

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(A) Analysis of bound p-XSC in water (Control), human or mouse plasma after derivatization. . Recovery rate is shown as the percentage of p-XSC-NEM peak area in plasma to that in control. p-XSC concentration in spiked matrices was 20 µg/ml. (B&C) C57BL/6 albino mouse was injected with p-XSC intravenously at the dose of 0.5 µg/g. Blood was collected at 20 min post injection for analysis of parent (B) and bound p-XSC (C). The insert in panel B stands for the spiked extract of blank mouse plasma (corresponding to mouse plasma containing 1 µg/ml p-XSC). N = 3.

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372

For TOC only

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Rapid and Robust Quantification of p-Xyleneselenocyanate in Plasma

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