Procedures for Analysis of Dried Plasma Using Microsampling

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Novel procedures for analysis of dried plasma using microsampling devices to detect sulfur mustardalbumin adducts for verification of poisoning Harald John, Sophia Willoh, Philipp Hörmann, Markus Siegert, Antje Vondran, and Horst Thiermann Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.analchem.6b02199 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Fig. 1

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A Mitra devices

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ACS Paragon Plus Environment

E Noviplex DUO card

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plasma blank A

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OPCW sample B

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filter paper

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Noviplex DUO card F

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Retention time

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Fig. 3

climate IV (hot and humid, 30°C, 70% RH) 10 µM SM

Relative peak area HETE-CP/IS [-]

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13 11 9 7 5 1 µM SM 1.3 1.1 0.9 0.7 0.5 0.1 µM SM 0.13 0.11 0.09 0.07 0.05 0

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Devices for dried plasma

µLC MS/MS for verification

proteolysis of albumin-adduct with sulfur mustard


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Novel procedures for analysis of dried plasma using microsampling devices to detect sulfur mustard-albumin adducts for verification of poisoning

Harald John1*, Sophia Willoh2, Philipp Hörmann3, Markus Siegert4, Antje Vondran2, Horst Thiermann1

1

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

2

University of Applied Sciences and Arts Coburg, Department of Applied Sciences, Coburg, Germany

3

University of Applied Sciences Weihenstephan-Triesdorf, Department of Biotechnology and Bioinformatics, Weihenstephan, Germany 4

Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany

* Address correspondence to this author. Bundeswehr Institute of Pharmacology and Toxicology Neuherbergstrasse 11, 80937 Munich, Germany Phone: +49-89-992692-2311. Fax: +49-89-992692-2333. E-mail: [email protected]

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ABSTRACT Incorporation of the chemical warfare agent sulfur mustard (SM) produces a covalent adduct with human serum albumin (HSA) representing an established plasma biomarker of poisoning. Bioanalytical verification requires both plasma generation from whole blood and shipping to specialized laboratories following strict guidelines for complex packaging. These needs often push the infrastructural boundary in crisis regions and war zones. Therefore, we herein originally introduce different reliable bioanalytical procedures using filter paper as well as novel volumetric microsampling tools (Mitra devices and Noviplex DUO cards) to generate dried plasma samples not liable to the shipping constraints. In addition, the Noviplex device enables in-transit separation of plasma from whole blood without the need of a centrifuge. Plasma-loaded and dried devices were subjected to pronase treatment yielding the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP) derived from the HSA-SM adduct, that was detected by µLC-ESI MS/MS. For all devices, samples exposed to SM yielded excellent linearity (0.025-50 µM SM) and good precision (≤ 13%) and fulfilled forensic quality criteria for ion ratios of qualifying and quantifying product ions. Stability of the HSA-SM adduct in dried and liquid plasma is shown under conditions of 3 climatic zones (temperate climate, hot and dry climate, and hot and humid climate) for at least 9 days simulating the period of delayed sample shipping. Our results originally document that dried plasma is appropriate for storage and shipping at ambient temperature and that novel microsampling tools are of essential benefit when targeting the HSA-SM adduct for verification analysis.

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KEYWORDS: Albumin adducts, dried blood, sample storage and shipment, microsampling, verification analysis

INTRODUCTION Sulfur mustard (SM, bis(2-chloroethyl)sulfide, CAS No 505-60-2) is a vesicant banned as chemical warfare agent (CWA) by the Chemical Weapons Convention that is supervised by the Organisation for the Prohibition of Chemical Weapons (OPCW, Nobel Peace Prize laureate of 2013). Depending on the dose SM causes e.g. erythema and blisters and evokes complicated and delayed wound healing.1 According to current press releases2-5 SM has recently been used in the ongoing Syrian Arab Republic conflict thus demonstrating an actual and serious threat to military personnel and civilians especially in asymmetric or terroristic scenarios. For evidence of an alleged deployment of SM modern complex analytical methods requiring advanced laboratory infrastructure are mandatory to investigate environmental and biological samples. In vitro and in vivo SM forms covalent adducts with nucleophilic moieties of endogenous biomacromolecules thereby typically attaching a hydroxyethylthioethyl (HETE)-group.1 The adduct found with Cys34-residue of human serum albumin (HSA) belongs to the most stable markers of exposure known so far being detectable in plasma samples even weeks after exposure.1,6,7 Very recently, we have introduced a HSA-derived alkylated dipeptide (HETE-CysPro, HETE-CP) as a novel biomarker for SM poisoning.8,9 According to our initial approach HSA was isolated from plasma followed by pronase-catalyzed proteolysis yielding HETE-CP that was detected by microbore liquid chromatography-electrospray ionization tandem-mass spectrometry (µLC-ESI MS/MS). Optimizing this procedure in a successive study we relinquished the time-consuming and cost-intensive HSA extraction step and instead performed direct liquid plasma proteolysis documenting applicability of this simplified method to real samples.10

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Such real patient samples of interest might be collected in a crisis region or supposed crime scene for further shipment to relevant laboratories for verification analysis. Transport of patient specimens is subject to strict restrictions concerning substantial safety prerequisites. According to the IATA (International Air Transport Association) dangerous goods regulations (DGR) for transport by air samples collected directly from humans and animals for the purpose of diagnosis and investigational activities have to be categorized as infectious substances (hazard class 6, division 6.2).11 Biological substances potentially containing pathogens that do not cause life-threatening effects belong to category B (shipping name UN 3373). This classification is considered as relevant for specimens obtained from most patients potentially being exposed to CWA only. Transport of that material requires a complex triple packaging consisting of a primary receptable containing the specimen, a secondary packaging containing an appropriate amount of absorbent and an outer package.11 If dry ice is used for shipping of frozen samples additional constraints have to be fulfilled.11 For transport on the street, railway and sea comparable rules are fixed by the ADR (European Agreement concerning the International Carriage of Dangerous Goods by Road).12 In contrast to liquid or frozen blood/plasma the dried material (dried blood, DB) is not subject to these regulations. Accordingly, shipping of DB is much less complex, more economic and convenient and thus more favourable as simple envelopes may be used. By definition dried blood spots (DBS) are collected by applying a drop of blood onto an absorbent material followed by drying.11,12 In the following the abbreviation DB is used to refer to dried blood as well as dried plasma or serum. This procedure is well established and routinely applied for clinical diagnosis e.g. for newborn screening13,14 and multiple diagnostic parameters.15-18 In principle, the DB technology is applicable if the analyte was of sufficient stability in the dried matrix at ambient temperature and no interferences from solid support occurred. Simple filter paper is an accepted and elaborated support for DB since the early 1960s13 applicable to numerous compounds of pharmacological and clinical relevance (Fig. 1C,D).15-17 Within the last 2 years novel alternative devices have been introduced on the market providing additional beneficial properties. The Mitra microsampling 4


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devices (Neoteryx) enable blood or plasma sampling with a defined volume of 10 µL prior to drying (Fig. 1A,B).19,20 In addition to collection of a specified plasma volume (7.6 µL), the Noviplex DUO cards (Shimadzu Novilytic) also allow separation of the plasma fraction from whole blood within 3 min by means of an integrated specific membrane (Fig. 1E,F).21 Generation of the pure plasma fraction represents an indispensable sample preparation step in each case components of whole blood deteriorate subsequent analysis. Furthermore, the capability for plasma separation on that card makes independent of the availability of centrifuges and is thus of essential advantage for sample collection under field conditions or in a rudimentarily equipped medical service facility. Simple blood or plasma spotting will allow the physician sending samples rapidly without the need for complex infrastructural and logistic requirements. Therefore, the different variants of the DB procedure will be also of great and important benefit for patient specimens intended for analysis of albumin-adducts to verify SM poisoning. Nevertheless, this methodical approach has never been described before. Accordingly, we addressed this issue in the present study for the first time to detect HETE-CP in plasma after sample drying.

EXPERIMENTAL Chemicals Common chemicals normally found in the laboratory were purchased from appropriate providers. Human serum albumin (HSA) was obtained from Sigma-Aldrich (Steinheim, Germany) and pronase from Streptomyces griseus from Roche (Mannheim, Germany, lot No. 70327222). Sulfur mustard (SM) was made available by the German ministry of defence and tested for integrity and purity in-house by NMR. Deuterated atropine (d3-Atr) used as internal standard for control of chromatography was delivered by CDN Isotopes (Pointe-Claire, Quebec, Canada) and human EDTA plasma by Dunn Labortechnik (Asbach, Germany). Human whole blood was provided by Sonnengesundheitszentrum (Munich, Germany). Mitra microsampling devices were purchased from Neoteryx (Torrance, CA, USA), Noviplex DUO cards from Shimadzu Novilytic (Columbia, MD, 5



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USA) and Munktell TFN filter paper (basis weight 180 g/cm2, 0.44 mm thickness, longitudinal absorptive height 120 mm/10 min) from Ahlstrom (Pont-Eveque, France).

µLC-ESI MS/MS analysis of HETE-CP For microbore separation (µLC-ESI MS/MS) a µLC system coupled to an API 4000 QTrap mass spectrometer (AB Sciex, Darmstadt, Germany) was used as described before.8 Chromatography of 20 µL sample was carried out at 60°C on an Atlantis T3 column (50 x 1.0 mm i.d., 3 µm, 100 A, Waters, Eschborn, Germany) protected by a precolumn (security guard cartridges, widepore C18 4 x 2.1 mm i.d., Phenomenex) at a flow of 30 µL/min. Solvent A (0.05 % v/v formic acid, FA) and solvent B (acetonitrile, ACN/H2O 80:20 v/v, 0.05 % v/v FA) tempered at 50°C were applied in gradient mode: t[min]/B[%]: 0/0; 12/30; 13/95; 18/95; 19/0; 20/0 including an initial 10 min equilibration period under starting conditions. Mass spectrometric detection was done in multiple reaction monitoring (MRM) mode using instrument settings described previously.8 MS/MS transitions were recorded from m/z 323.1 to m/z 105.0 (quantifying ion) and m/z 137.0 (qualifying ion) for HETE-CP and m/z 293.2 to m/z 127.1 for d3-Atr.

Incubation of plasma and neat HSA with SM Human EDTA-plasma (960 µL) was mixed with 5 mM SM in iso-propanol resulting in a SM concentrations of 200 µM. Incubation was carried out for 2 h at 37°C under gentle shaking followed by overnight storage at 4°C. Samples were stored at – 25°C until further processing if necessary. Incubated plasma was diluted with blank plasma to obtain SM concentration equivalents required (indicated below). For production of a biomarker reference compound (HETE-CP) neat HSA (40 mg/ml in saline) was incubated with SM (100 µM) under the same conditions prior to pronasemediated proteolysis and ultrafiltration (UF).8

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SM is a toxic chemical warfare agent potentially causing burns and blisters requiring working under a fume hood. Laboratory consumables e.g. reaction vials and pipette tips were decontaminated by storage in basic NaOCl solution prior to disposal.

Use of Mitra microsampling devices Sample preparation Mitra microsampling devices (Fig. 1A) were loaded with SM-incubated plasma (concentration see below) or blank plasma (10 µL) by contact to surface of the liquid phase as recommended by the manufacturer (Fig. 1B). After overnight drying sampler tips were removed and put into a 0.5 mL reaction vial prior to addition of 80 µL 50 mM NH4HCO3. Following sonication for 15 min and intense vortex mixing dissolved proteins were mixed with pronase solution (20 µL, 60 mg/mL 50 mM NH4HCO3) allowing incubation at 37°C for 4 h under gentle shaking. Afterwards, 200 µL ACN were added to precipitate remaining proteins and enzyme followed by centrifugation (5 min at 10,270 g). A portion of the supernatant (200 µL) was dried under a gentle stream of nitrogen or using an evaporator (RVC 2-18, Christ, Osterode am Harz, Germany) prior to dissolution in 70 µL 0.5% (v/v) FA containing d3-Atr (3 ng/ml) and subsequent µLC-ESI MS/MS analysis. Liquid plasma references were made from aliquots of the same (SM-incubated) plasma with which the Mitra devices were loaded also. Their preparation was performed accordingly: 10 µL plasma was mixed with 70 µL 50 mM NH4HCO3 and 20 µL pronase solution (60 mg/mL 50 mM NH4HCO3) for incubation. Precipitation, evaporation, dissolution and analysis were done as described above.

Recovery of HETE-CP Human plasma incubated with SM (0.1 µM, 1 µM, and 10 µM) was analysed by µLC-ESI MS/MS in triplicate each following the standard procedures described above for the use of Mitra

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microsampling devices and liquid plasma references (10 µL). Mean peak areas obtained from Mitra devices were compared to references for calculation of analyte recovery.

Linearity, precision and LOD of HETE-CP analysis SM-treated plasma was diluted with blank plasma resulting in SM concentration equivalents of 0; 0.01; 0.025; 0.05; 0.1; 0.5; 1.0; 5.0; 10 and 50 µM. Each concentration was loaded onto three Mitra microsampling devices each. For elaboration of linearity of HETE-CP peak areas and estimation of the limit of detection (LOD) samples were analysed by µLC-ESI MS/MS following the procedure described above. Linear regression was done by the common method of non-weighted least squares and precision was calculated as relative standard deviation (RSD) of triplicate measurements of HETE-CP peak areas. LOD was defined as the lowest concentration of SM, that was still met by the linear regression curve and resulted in unambiguous peaks of the quantifying ion (m/z 105.0) in all triplicates with a RSD of the peak area ≤ 20%. In addition, the ion area ratio of qualifying to quantifying ion (m/z 137/m/z 105) had to fit the corresponding ratio of a HETE-CP reference (50.3%) produced from pure HSA-SM within the tolerance intervals given by the OPCW (± 20%). Accordingly the corresponding ion ratio for HETE-CP was allowed to be between 40.3% - 60.3%.

Stability of albumin-SM adduct in liquid and dried plasma Mitra microsampling devices were loaded with SM-treated plasma (0.1 µM, 1 µM and 10 µM) as well as blank samples not exposed to SM and stored for up to 9 days under 3 different climatic conditions controlled in a stability test chamber (VP 500, Voetsch Industrietechnik, BalingenFrommern, Germany). As reference liquid aliquots (50 µL) of the same plasma samples were locked in 1.1 mL glass vials in triplicate and stored in parallel under the same conditions. According to the World Health Organization (WHO) technical reports22 the following standardized storage conditions were tested: climatic zone I (temperate climate) at 21°C and 45% relative humidity (RH), climatic zone III (hot and dry climate) at 30°C and 35% RH, and climatic zone IV (hot and 8


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humid climate) at 30°C and 70% RH. Samples of all SM concentration were analysed by µLC-ESI MS/MS on day 0, 1, 2, 4, 7, and 9 (n=3 each). Resulting peak areas of HETE-CP were compared to monitor climate-dependent storage stability using d3-Atr (3 ng/ml) as IS.

Use of filter paper Plasma volume-dependent spot size Plasma of 11 different volumes ranging from 5 µL up to 100 µL was spotted onto filter paper in triplicate to measure the diameter of the resulting dried plasma spots.

Sample preparation Human plasma blank and plasma incubated with SM (100 µL each, concentration see below) was spotted onto filter paper for air drying (Fig. 1C,D). A quadratic piece of paper just containing the entire circular plasma spot was cut off and cut into small pieces of about 13 mm x 2 mm each, that were transferred into a 2 mL reaction vial. NH4HCO3 buffer (500 µL, 50 mM) was added followed by sonication (15 min), intense vortex mixing and addition of 100 µL pronase solution (60 mg/mL in 50 mM NH4HCO3). After incubation (4 h at 37°C under gentle shaking) the entire mixture including the paper pieces was transferred into an ultrafiltration (UF) device (cut-off 10 kDa, Vivaspin 500 centrifugal concentrator, Sartorius Stedim, Göttingen, Germany) for subsequent UF (20 min at 10,270 g) yielding a filtrate of about 350 µL. The filtrate (40 µL) was diluted 1:3 with 0.5% (v/v) FA containing d3-Atr (3 ng/ml) for µLC-ESI MS/MS analysis. For preparation of the filter paper reference 100 µL plasma were mixed with 400 µL 50 mM NH4HCO3 and 100 µL pronase solution (60 mg/mL 50 mM NH4HCO3) prior to incubation, UF and final dilution as described above.

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Recovery of HETE-CP Human plasma incubated with SM (0.1 µM, 0.5 µM, 5 µM) was prepared in triplicate each by the standard filter paper procedure to compare HETE-CP peak areas with references obtained from liquid plasma.

Linearity, precision and LOD of HETE-CP analysis Human plasma standards corresponding to 0; 0.01; 0.025; 0.05; 0.1; 0.5; 1.0; 5.0; 10 and 50 µM SM were prepared and analysed in triplicate following the filter paper procedure described above. Criteria for the determination of LOD were as described above for Mitra devices.

Use of Noviplex DUO cards Sample preparation Whole blood (760 µL) was mixed with 40 µL of SM-treated plasma (resulting SM concentration equivalents given below) prior to triplicate application of 100 µL on two Noviplex DUO cards per sample each (Fig. 1E). In addition, whole blood mixed with plasma not treated with SM was prepared accordingly as blank sample. Following the manufacturer´s recommendations blood was allowed staying on the membrane for 3 min before the top layer was peeled off. Collection discs (two per card containing 7.6 µL plasma in total) were air dried overnight (Fig. 1F) prior to removement with tweezers and transfer into a 2 mL reaction vial thus yielding 4 discs per sample. Proteins were dissolved by addition of 120 µL 50 mM NH4HCO3 and subsequent sonication for 15 min and intense vortex mixing. Pronase solution was added (30 µL, 60 mg/mL 50 mM NH4HCO3) for proteolysis (4 h at 37°C under gentle shaking) followed by addition of 300 µL ACN for precipitation of remaining proteins and pronase. Part of the supernatant (300 µL) was evaporated to dryness under a gentle stream of nitrogen prior to reconstitution in 105 µL 0.5% (v/v) FA containing d3-Atr (3 ng/ml) for final µLC-ESI MS/MS analysis. 10


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Preparation of liquid plasma references was carried out by mixing 15.2 µL plasma with 104.8 µL 50 mM NH4HCO3 and 30 µL pronase solution (60 mg/mL 50 mM NH4HCO3) for incubation. Subsequent precipitation, evaporation, dissolution and analysis were done as described above.

Recovery of HETE-CP Whole blood spiked with SM-incubated plasma yielding SM concentration equivalents of 0.1 µM, 1 µM and 10 µM was spotted onto the Noviplex DUO cards in triplicate each. Peak areas obtained from µLC-ESI MS/MS analysis were compared to those of liquid plasma references prepared as described above to calculate recovery.

Linearity, precision and LOD of HETE-CP analysis Whole blood was mixed with SM-incubated plasma resulting in SM concentration equivalents of 0; 0.01; 0.025; 0.05; 0.1; 0.5; 1.0; 5.0; 10 and 50 µM SM. Samples were analysed in triplicate each using the Noviplex DUO card procedure as described above. Principle criteria for LOD determination were as described for Mitra devices.

Application of different procedures to samples sent by the OPCW Plasma samples potentially exposed to SM were sent by the OPCW within the course of the 1st biomedical proficiency test (1st BioPT) asking for verification analysis targeting SM-protein adducts. Samples were prepared in our laboratory using Mitra devices, filter paper and Noviplex DUO cards following the procedures described above after drying and storage for 24 h at room temperature in the laboratory. As corresponding whole blood samples had not been sent, plasma was directly applied to the Noviplex DUO cards instead.

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RESULTS AND DISCUSSION For legal and medical reasons analysis of biological specimens is essential to identify the agent and prove poison incorporation. Liquid samples like plasma and serum have to be transported typically under freezing or at least cooling conditions by e.g. air or on the road taking some days to specialized laboratories. Strict and complex safety rules for packing have to be followed.11,12 In contrast, DB samples represent an exceptional case not demanding such package conditions. For CWA verification analysis the use of the DB technology is still not established. To the best of our knowledge, only one report published in open literature so far presents an analytical procedure for detection of butyrylcholinesterase-adducts to verify nerve agent poisoning making use of DBS.23 Accordingly, no data on the stability of adducts, especially the albumin-adduct with the vesicant SM in dried material as well as applicability of DB to enzymatic proteolysis were available. Therefore, we addressed detection of the HSA-SM adduct in blood and plasma samples prepared as DB. To consider current progress and up to date development of sample taking and storage tools not only conventional filter paper was tested but also novel devices including Mitra and Noviplex DUO cards. Initial studies using whole blood spiked with SM-treated plasma that was subjected to direct proteolysis resulted in insufficient reproducibility of peak areas, retention times (tR) and ion ratios (data not shown). Furthermore, tremendous effects of analyte ion suppression minimized peak intensity and thus proved whole blood to be an inappropriate matrix for our approach. Application of whole blood samples to Mitra devices and filter paper does not allow separation of the plasma fraction. In contrast, Noviplex DUO cards enable that separation and thus purification of the sample matrix. Therefore, matrices subjected to direct proteolysis by incubation with pronase would be highly different. The entire diversity of all components of whole blood including dried cells and their cytosols would be present for Mitra devices and filter paper, whereas the plasma components only (without cells and cytosols) were present when using Noviplex DUO cards. Accordingly, the present study focussed on the use of plasma for Mitra devices and filter paper but made use of whole blood for Noviplex DUO cards. 12


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µLC-ESI MS/MS analysis of HETE-CP In principle, µLC-ESI MS/MS analysis was performed as described recently9 with some modifications. A smaller particle size (3 µm instead of 5 µm) of the same stationary phase material (Atlantis T3) as well as a higher chromatographic temperature (60°C instead of 50°C) were used to improve HETE-CP peak shape (peak width at the base 0.8 min instead of 1.5 min). A shorter column (50 mm instead of 150 mm), reduced equilibration time (10 min instead of 15 min) and a modified steeper gradient were applied to reduce tR (7.9 min instead of 19.8 min), total run time (20 min instead of 45 min), and cycle time (30 min instead of 60 min). Accordingly, the modified conditions allowed improved sample throughput in combination with better peak characteristics when compared to our initial approach.9 In contrast to our previous procedure for direct proteolysis of 100 µL liquid plasma10 , the Mitra and Noviplex DUO cards provide much smaller volumes of 10 µL and 2 x 7.6 µL, respectively. Therefore, volumes used for individual sample preparation (e.g. buffer for enzymatic cleavage and solvent used to dissolve residuals after evaporation) were adopted to yield maximum analyte concentrations in the injection solutions sufficient for duplicate analysis of 20 µL each. At the tR of the analyte blank plasma samples were free of interferences independent on the support used: Mitra microsampling devices (Fig. 2A), filter paper (Fig. 2C), Noviplex DUO cards (Fig. 2E) or liquid plasma itself (Fig. 2G). No cross talk or carry over effects were observed even when analyzing higher concentrated samples.

Use of Mitra microsampling devices The Mitra microsampling device represents a novel volumetric absorptive technology, that was launched in 2014 for collection and shipping of blood, plasma, serum or any other biological liquid (Fig 1A,B). This support combines the advantage of easy DBS shipping with volumetric taking of fixed sample volumes (10 µl) from as little as 15 µl body fluid soaked in a highly hydrophilic 13



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sampler tip.24 Handling of numerous sampling devices might be automated in a 96-well plate format thus enhancing sample throughput. In addition, the Mitra device was shown to make independent on the hematocrit bias found on punched filter paper and is thus suitable for analyte quantification in general.19,25,26 However, our method presented herein was designed for qualitative verification of SM-exposure only.

Sample preparation Sampler tips of Mitra devices soaked plasma within seconds after simple touching the drop´s surface (Fig. 1B). After air drying, disposal of the sampler tip containing the dried plasma, addition of buffer, sonication and subsequent addition of pronase a clear and colourless solution was obtained yielding a white precipitate after mixing with ACN and subsequent centrifugation. Residuals of both the Mitra device preparation as well as the liquid plasma reference were soluble completely in 70 µL 0.5% FA v/v corresponding to a plasma equivalent of 1.90 µL when injecting 20 µL onto the column for chromatography (Table 1).

Recovery of HETE-CP Sonication and subsequent intense vortex mixing allowed reliable and reproducible dissolution of dried proteins. After proteolysis ACN was added for protein precipitation thus requiring a drying step of the analyte-containing supernatant prior to µLC-ESI MS/MS analysis. The supernatant may be evaporated to dryness either by using a gentle stream of nitrogen or by means of a vacuum centrifuge (evaporator). Both methods showed good precision (1.2% - 3.7%, n=3 each) and yielded identical results. Mean peak areas derived from plasma-loaded Mitra devices were about 10% higher than those of the plasma references of all 3 concentrations tested yielding recoveries of: 106.9% ± 7.7% (0.1 µM SM), 112.8% ± 8.1% (1 µM SM) and 109.2% ± 2.4% (10 µM) (Table 1).

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This phenomenon might be due to different reasons. It might be possible that a larger volume than the 10 µL declared by the manufacturer were loaded by the sampler tip. Comparable findings were published by Spooner et al. determining an averaged blood volume of 10.6 µL per tip when loading whole blood.25 Furthermore, higher HETE-CP peak intensities found in Mitra-derived samples might have also been due to adsorption of some ion-suppressing matrix components to the tip surface thus reducing ion suppression effects and yielding higher intensities. In addition, slight influence of the sampler tip surface on the catalytic activity of some endo- or exopeptidases present in the pronase mixture27 cannot be excluded which might favour HETE-CP production instead of possible but still unknown Cys34-containing side products. However, independent of potential reasons mentioned above excellent reproducibility and peak intensity favour the use of Mitra devices for verification analysis.

Linearity, precision and LOD of HETE-CP analysis Excellent linearity was obtained in the concentration range from 0.025 µM to 50 µM SM including standards of 0.025; 0.05; 0.1; 0.5; 1.0; 5.0; 10 and 50 µM (Y=33845X - 991, r2 0.9999) thus demonstrating highest reproducibility of the volume soaked by the sampler tips as well as proteolysis. RSD values were between 2.7-11.6% for all standards (Table 1). Therefore, the Mitra devices are also appropriate for comparative quantitative studies. Ion ratios of both product ions (m/z 137.0/m/z 105.0) derived from standards ≥ 0.1 µM SM (50.9% ± 1.3%) were identical to liquid plasma references (50.4% ± 2.4%) and the pure HSA-SM reference (50.3% ± 2.1%) indicating the absence of interferences (Table 1). In contrast, standards ≤ 0.05 µM SM did not fulfil that criterion as intensity of the quantifying ion was too low to be detected. Therefore, LOD was fixed at 0.1 µM thus being well suited for verification fulfilling the OPCW quality criteria and allowing highly sensitive analysis of concentrations with toxicological relevance (Table 1). Despite the small sample volume the presented Mitra device procedure enabled slightly improved sensitivity

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when compared to procedures established previously working up sample volumes being 10-times larger.8,10

Stability of albumin-SM adduct in plasma For storage of dried samples defined climatic conditions were selected following the WHO recommendations for stability testing of pharmaceutical ingredients and finished pharmaceutical products.22 Climates chosen considered conditions that may be relevant for e.g. Europe (temperate climate I, 21°C, 45% RH), Northern Africa and Near East (hot and dry climate III, 30°C, 35% RH) and the tropical equatorial region (hot and humid climate IV, 30°C, 70% RH). The test period of 9 days was considered to be sufficient simulating the time for shipping when samples are sent to a relevant laboratory for analysis. Excellent stability of the HSA-SM adduct was found for each concentration tested in triplicate for all 3 climates over the entire 9 days test period (RSD values ≤ 12.5%). Exemplarily, Figure 3 (blue diamonds) illustrates the results of climate IV. The adduct was shown to be also stable in liquid plasma (Fig. 3, red squares) underlining the favourable robustness of that biomarker. Results obtained from Mitra devices stored at ambient temperature with contact to aerial oxygen were quite surprising as the HSA-SM adduct contains 2 sulfur atoms –one of the cysteine-thiol group and one of the SM molecule- that both should be sensitive to oxidation in general.1,28,29 However, resistance to degradation is of important benefit for reliable and robust verification analysis thus tolerating potentially delay in shipping or sample preparation in the laboratory.

Use of filter paper Since the 1960s filter papers represent the common and established support to prepare DBS13 suitable for qualitative and quantitative diagnostic screening by subsequent immunoassays or LCMS/MS.15-18 For numerous commercial clinical approaches small blood volumes –often only some drops obtained by pricking e.g. a patient´s finger or neonate´s foot with a lancet - are spotted onto a 16


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marked circular area of a filter paper. This card is sent by mail after the blood spot has dried. Prior to analysis a disc of defined area is punched using punch pliers followed by appropriate extraction procedures.15-18 Numerous diverse kinds of filter paper, that are also available as larger sheets, may differ by e.g. pore size and thickness determining loading capacity and spreadability of the liquid sample. For our study we used pure cotton linters without any chemical impregnation (Munktell TFN) commonly applied in newborn screening and approved by the Clinical Standards Laboratory Institute (CLSI).

Plasma volume-dependent spot size A linear relation of spotted plasma volume and resulting spot area was found (Y=0.0514 X + 0.116, r2 0.998) indicating reproducible liquid loading properties of the filter paper. Aliquots of e.g. 100 µL, 50 µL and 10 µL resulted in spot diameters of 2.55 cm (area 5.11 cm2, Fig. 1D), 1.85 cm (area 2.69 cm2) and 0.85 cm (area 0.57 cm2), respectively. As the disc size produced by conventional punch pliers is quite small (diameter 0.3-0.6 cm corresponding to 1.4-3.0 µL) insufficient assays sensitivity was expected for our method thus recommending the use of numerous small discs or of larger volumes. Even though the dried spot of 100 µL plasma was quite large (Fig. 1D) it was found well suited for sample preparation providing sufficient and comfortable sample amounts identical to our previous method introduced for direct liquid plasma proteolysis.10 Therefore, this large volume was applied for further characterization of the DBS approach. Nevertheless, down-scaling would easily be possible, if necessary.

Sample preparation Applied plasma (100 µL) was soaked immediately and yielded circular spots (Fig. 1D) that could easily be cut off after drying to prepare small strips. These strips sponged up a larger volume of the buffer added prior to sonication. However, the paper was still covered by the remaining buffer allowing successful solubilisation of dried plasma proteins. The filter paper was resistant against 17



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pronase treatment not causing any interference in HETE-CP analysis (Fig. 2C). To minimize loss of analyte-containing solution after proteolysis wet filter paper stripes were centrifuged in UF devices thereby allowing both separation of remaining proteins and enzyme as well as recovering sufficient buffer volumes of at least 350 µL. Otherwise ACN-mediated precipitation followed by centrifugation would have caused higher loss of liquid phase absorbed by the filter material. Finally, a plasma equivalent of 1.11 µL was injected onto the column for chromatography (Table 1).

Recovery of HETE-CP The ratio of mean peak areas (filter paper/reference) defining recovery was between 112.9% (0.1 µM) and 94.6% (5 µM) documenting appropriate reproducibility of the sample preparation procedure allowing quantitative investigations and application to verification analysis (Table 1).

Linearity, precision and LOD of HETE-CP analysis Similar to the Mitra device approach excellent linearity ranging from 0.025 µM – 50 µM SM including standards of 0.025; 0.05; 0.1; 0.5; 1.0; 5.0; 10 and 50 µM was found (Y=28890 X + 272, r2 0.9999) thus underlining applicability of the DBS technology for quantitative purposes. The LOD was fixed at 0.05 µM SM with regard to ion ratio criteria as no peak was detected for the qualifying ion trace (m/z 137.0) of the 0.025 µM standard. RSD values were in the range from 0.8% - 8.7%. The mean ion ratio calculated from standards was 48.2% ± 2.5% thus indicating high suitability for verification of SM exposure (Table 1).

Use of Noviplex DUO cards Noviplex DUO cards were introduced onto the market in 2015 and represent a unique tool for intransit separation and collection of plasma from whole blood (Fig. 1E,F). Simple non-volumetric spotting of 60 µL - 100 µL whole blood onto the top of a size filtration membrane allows hematocrit-independent volumetric microsampling of plasma (7.6 µL) within only 3 min. 18


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Accordingly, no power supply is required for operating a centrifuge to generate plasma from whole blood samples. The plasma volume is collected precisely by two cellulose discs positioned under the membrane that can easily be shipped after removing the covering overlay and subsequent drying (Fig. 1F). These features are of essential benefit for scenarios where laboratory infrastructure is not accessible to generate and prepare plasma samples ready for shipping to specialized analytical facilities. Therefore, our study aims an important situation often found in crisis regions and war zones demanding verification of an alleged use of CWA. Furthermore, we present a rare original application using this brand-new technology21 and it is the first addressing CWA verification analysis.

Sample preparation Whole blood samples spiked with SM-incubated plasma were applied to obtain a dark red blood fraction on the top (Fig. 1E) and brownish-yellow collection discs underneath within 3 min (Fig. 1F). Dried proteins could be dissolved from the discs in small buffer volumes yielding a clear solution after sonication ready for proteolysis. Due to the small volume of the incubation mixture precipitation by ACN was carried out allowing subsequent evaporation and concentration prior to analysis. Finally, a plasma equivalent of 1.93 µL was injected onto the column for chromatography of 20 µL. No interferences hampering HETE-CP detection were observed (Fig. 2E) favoring this microsampling support for verification analysis of HSA-SM adducts.

Recovery of HETE-CP Similar to other supports tested excellent recovery ranging from 83.7% (5 µM) to 100.6% (0.5 µM) was obtained from replicate measurements (Table 1) recommending its use for proving SMexposure by protein adducts.

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Linearity, precision and LOD of HETE-CP analysis Excellent linearity was found between 0.025 µM and 50 µM SM (Y=35413X – 7807, r2 0.9995). RSD values between 1.7% and 5.0% for all standards analysed and appropriate ion ratios (48.1% ± 2.5%) underline the usability of Noviplex DUO cards for verification analysis (Table 1). In addition, the LOD determined at 0.05 µM SM was identical to the other approaches very well suited for intended analysis.

Application of different procedures to samples sent by the OPCW As illustrated in Figure 2B,D,F the Mitra device as well as filter paper and Noviplex DUO cards proved applicability to the OPCW samples resulting in unambiguous positive detection of the HSASM adduct. HETE-CP was detected at tR 7.9 min fulfilling the OPCW ion ratio tolerance criteria (± 20%) of qualifying ion/quantifying ion when compared to the HSA-SM derived reference compound (40.3%-60.3%). Following the individual procedures comparable peak intensities of HETE-CP as well as corresponding ion ratios were obtained. As these samples were sent to define facilities being part of the international OPCW network of designated laboratories correct results underline their potential future benefit for verification analysis.

CONCLUSION HSA-SM proved to be a highly stable long-termed biomarker even in dried matrix thus underlining its use for robust verification analysis. Even though the absolute volume of plasma sample was quite low (≤ 15.2 µL) for the Mitra and Noviplex approach, procedures were shown to result in excellent reproducibility and appropriate sensitivity. Essential characteristics concerning sample preparation, precision, recovery, linear range, and LOD were elaborated individually and underlined suitability of the microsampling devices. Respective LOD values of all tools are highly

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appropriate for toxicological concentrations as documented by the analysis of patients´ plasma in a previous study.7 The presented procedures take into account that the infrastructure of sample drawing facilities in crisis regions is often inappropriate to generate plasma from whole blood (lack of centrifuge) or to pack samples adequately following IATA and ADR guidelines.11,12 Therefore, especially the Noviplex DUO card represents a highly valuable tool for more effective and less complex sample drawing and shipping. Nevertheless, future developments of the manufacturers should aim collecting larger and thus more comfortable sample volumes possibly in the range of 50-100 µL. In addition, adaption of the diverse microsampling procedures to target the alkylated tripeptide HETE-CPF derived from albumin after direct proteolysis with proteinase K was realized in a pilot study also showing excellent RSD values (≤ 6%) (results not shown). Targeting HETE-CPF considered the original analytical approach for HSA-SM detection introduced by Noort et al.30 and thus documented its applicability to alternative established verification methods. Method modifications to target additional adducts of SM with e.g. hemoglobin or DNA or BChEadducts with nerve agents and pesticides will be addressed in future studies.

AUTHOR INFORMATION Corresponding Author Phone: +49-89-992692-2311. Fax: +49-89-992692-2333. E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We gratefully thank B. Klaubert, R. Bogan, and G. Preis (Central Institute of the Bundeswehr Medical Service Munich) for helpful support and placing the climatic exposure test cabinet at our disposal. 21



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LEGENDS TO FIGURES Figure 1 Microsampling devices appropriate for HSA-SM adduct analysis

Mitra microsampling device prior to (A) and after (B) blood loading (10 µL); filter paper while loading (C) and after drying (D) of 100 µL plasma; Noviplex DUO card while loading whole blood (E) and after drying of 7.6 µL plasma fraction (F).

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Figure 2 µLC-ESI MS/MS analysis of HETE-CP after use of microsampling devices

Chromatograms of blank plasma and an OPCW sample of the 1st bioproficiency test using Mitra microsampling devices (A, B), filter paper (C, D) and Noviplex DUO cards (E, F) as well as of liquid plasma blank (G) and a reference made from neat HSA-SM adduct (H). Devices loaded with dried plasma were subjected to pronase treatment yielding the biomarker HETE-CP (tR 7.9 min, marked with a star) if liquid samples had been exposed to SM before. Separation at 30 µL/min was carried out on Atlantis T3 (50 x 1.0 mm i.d., 3 µm, 100 A) at 60°C applying an acetonitrile gradient. Detection was done after positive ESI in MRM mode. For reasons of clarity only the trace of the quantifying ion (m/z 323.1 > m/ 105.0) is illustrated.

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Figure 3 Stability of HSA-SM adduct in dried and liquid plasma

Human plasma exposed to sulfur mustard (SM) concentrations indicated was either loaded onto Mitra microsampling devices and dried (blue diamonds) or present as liquid (red squares) during storage for 9 days under controlled climatic conditions (climate IV, hot and humid, 30°C, 70% relative humidity, RH). Samples were analysed by µLC-ESI MS/MS targeting HETE-CP as biomarker of SM exposure. Results obtained for climatic zone I (temperate climate) and climatic zone III (hot and dry climate) were of equal good quality. Data representing the peak area ratio of HETE-CP and d3-atropine used as internal standard are shown as the mean ± SD (n=3). Lines indicate the mean calculated over the 9 days test period.

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Table captions

Table 1. Assay characteristics of different supports for plasma sample taking and storage

LOD, limit of detection, a whole blood was applied in contrast to other supports using plasma and 2 cards were combined for one sample. b data according to direct plasma analysis as described previously by John et al.10 .c recovery was defined as relative ratio of the mean HETE-CP peak area to liquid plasma references (quantifying ion at m/z 105.0). d by definition. e linear range found for the peak areas of quantifying ion (m/z 105.0) with r2 ≥ 0.999. f RSD (relative standard deviation) was determined from standards (0.05 µM SM – 50 µM SM) analysed in triplicate each. g ion ratio was defined as quotient of peak area of qualifying (m/z 137.0) and quantifying ion (m/z 105.0) averaged from all standards. Ion ratio of a reference compound derived from pure HSA-SM adduct was found at 50.3%.

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Procedures for Analysis of Dried Plasma Using Microsampling

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