Application of 2-Aminothiazoline-4-carboxylic Acid as a Forensic

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APPLICATION OF 2-AMINOTHIAZOLINE-4-CARBOXYLIC ACID AS A FORENSIC MARKER OF CYANIDE EXPOSURE Monika Ru#ycka, Joanna Giebu#towicz, Marcin Fudalej, Pawe# Krajewski, and Piotr Wroczy#ski Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00219 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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Chemical Research in Toxicology

APPLICATION OF 2-AMINOTHIAZOLINE-4-CARBOXYLIC ACID AS A FORENSIC MARKER OF CYANIDE EXPOSURE

Monika Rużycka†, Joanna Giebułtowicz†*, Marcin Fudalej‡, Paweł Krajewski‡, Piotr Wroczyński† †

Bioanalysis and Drugs Analysis Department, Faculty of Pharmacy, Medical University of

Warsaw, 1 Banacha Street, 02-097 Warsaw, Poland ‡

Forensic Medicine Department, Medical University of Warsaw, 1 Oczki Street, 02-007

Warsaw, Poland

*Corresponding author: Joanna Giebułtowicz, Department of Bioanalysis and Drugs Analysis, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02-097 Warsaw, Poland, [email protected], phone +48 22 572 09 49, fax +48 22 572 09 76

Keywords: forensic, cyanide, sample preparation, bioanalysis, liquid chromatography coupled to mass spectrometry

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Abstract Cyanides are infamous for their highly poisonous properties. Accidental cyanide poisoning occurs frequently, but occasionally, intentional poisonings also occur. Inhalation of fumes generated by fire may also cause cyanide poisoning. There are many limitations in direct analysis of cyanide. 2-Aminothiazoline-4carboxylic acid (ATCA), a cyanide metabolite, seems to be the only surrogate that is being used in the detection of cyanide because of its stability and its cyanide-dependent quality in biological matrix. Unfortunately, the toxicokinetic study on diverse animal models suggests significant interspecies differences; therefore, the attempt to extrapolate animal models to human model is unsuccessful. The aim of the present study was to evaluate the use of ATCA as a forensic marker of cyanide exposure. For this purpose, postmortem materials (blood and organs) from fire victims (n=32) and cyanide-poisoned persons (n=3) were collected. The distribution of ATCA in organs and its thermal stability were evaluated. The variability of cyanides in putrid sample and in the context of their long-term and higher temperature stability was established. The presence of ATCA was detected by using LC-MS/MS method and that of cyanide was detected spectrofluorimetrically. This is the first report on the determination of ATCA distribution in tissues of fire victims and cyanide-poisoned persons. It was found that blood and heart had the highest ATCA concentrations. ATCA was observed to be thermally stable even at 90°C. Even though the cyanide concentration was not elevated in putrid samples, it was unstable during long-term storage and at higher temperature, as expected. The relationship between ATCA and cyanides was also observed. Higher ATCA concentrations were related to increased levels of cyanide



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in blood and organs (less prominent). ATCA seems to be a reliable forensic marker of exposure to lethal doses of cyanide.


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1 Introduction According to the American Association of Poison Control Centers (AAPCC), in 2014, 187 humans were exposed to cyanide in the United States. Of these, 4.8% were fatal.1 Cyanide is a compound of more interest in forensic toxicology studies because of its usage in suicide and homicide. Data from the National Forensic Service in Seoul, Korea, reported 255 cases of cyanide poisoning from 2005 to 2010. Approximately 97.3% of all cyanidepoisoning deaths were because of suicide.2 In 2014, AAPCC reported that of the total number of cyanide exposure, as much as 10% of cases were of intentional poisoning.1 Another alarming source of cyanide poisoning is fire smoke. The latest data for 2012–2014 from the US Fire Administration National Fire Data Center show that 47% of the fatalities are caused by the combined effect of thermal burns and smoke inhalation. Smoke inhalation alone accounted for 37% of the residential fire fatalities, whereas thermal burns by itself accounted for 6% of the fire victims.3 Another source of data state that smoke inhalation by itself caused 50%–80% of the fatalities.4 The scientist and US fire service authorities have published a number of articles regarding the changes in the composition of fire smoke. They have reported frequent occurrences of cyanide in the composition of fire smoke over the last 30 years.5 The National Fire Protection Association has underlined the significant decrease in the amounts of fires in time and fire deaths; however, the rate of civilian fire deaths is not proportional and does not differ much from the previous years.6 According to Agency for Toxic Substances and Disease Registry, cyanides refer to chemicals that contain a cyanide group (CN).7 They can exist in solution as molecular acid (HCN), cyanide ion (CN−), or metal complexes. HCN is a volatile molecule with a short halflife (t1/2=0.34–1.28 h).8 It has a pKa of 9.2, which means that it is a weak acid.7 According to the Henderson–Hasselbalch equation, cyanide that enters an organism would mostly appear in protonated form (HCN) because of the high buffer capacity of blood (pH=7.4).9 It disappears 5 ACS Paragon Plus Environment


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inside the organism quickly, and this process causes the difficulty of establishing steady-state cyanide levels with time. Hence, during postmortem sample analysis, not only the time between sample collection and analysis but also the time between death and sample collection is considered. The cyanide concentration in a body exposed to heat depends on the amount of heat produced by the fire, the closeness of the exposure, and the length of time of exposure. The investigation of heated blood samples revealed the instability of cyanide content. Tissues subjected to heat above body temperature for a significant period result in greater and quicker decrease in HCN concentration.10 All these contribute to the unreliable outcomes of cyanide levels in all the victims of cyanide poisoning. After absorption into human organisms, cyanide converts into thiocyanate (SCN−) in ~80% and 2-aminothiazoline-4-carboxylic acid (ATCA) in ~15%.11 Thiocyanate conversion is primarily catalyzed by the mitochondrial enzyme rhodanese in the presence of sulfur donor (e.g., thiosulfate).8 ATCA is formed when cyanide reacts with

L-cystine

through an

intermediate β-thiocyanoalanine.12 Thiocyanate has been previously studied as a cyanide marker; however, some studies suggest that thiocyanate may be formed endogenously by bacteria in the colon or be elevated as a result of consumption of cabbage-like vegetables.13 Moreover, possibly the production of ATCA predominates over that of thiocyanate when sulfur donors become depleted or when rhodanese is sparse.8 ATCA possesses the qualities of an excellent cyanide poisoning marker mainly because of its stability in postmortem and putrid specimens.14 ATCA does not metabolize further, and therefore, it may be a lasting signature of cyanide exposure.8 Recently, an accurate, sensitive, and reliable method of determination of ATCA in postmortem materials was developed and validated, and therefore, new possibilities in forensic analysis have been created.14


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The aim of the present study was to examine whether ATCA is a forensic biomarker of cyanide intoxication caused by fire. Therefore, for this purpose, for the first time thermal stability of ATCA was determined. Simultaneously, long-term stability and thermal stability of cyanides in preserved blood samples were examined in order to prove their uselessness in the context of our studies. The effect of putrefaction on cyanide concentration was also evaluated. This is the first report on the determination of concentrations of cyanide and ATCA and their correlation in cyanide-poisoned and fire victims. Also, we are the first research group who present the established results of distribution of ATCA in human organs.

2 Materials and methods 2.1 Chemicals The reference standard for 2-aminothiazoline-4-carboxylic acid (ATCA; purity: 98.8%) was purchased from Chem-Impex International (Wood Dale, USA). The internal standard for 2aminothiazoline-4-carboxylic acid-13C,15N2 (chemical purity: 98%, isotopic purity: 99.1%) was purchased from TRC (Toronto, Canada). Taurine (purity: 99%) was purchased from ACROS Organics (New Jersey, USA). 2,3-Naphthalenedicarboxaldehyde (NDA; purity: 95%) was purchased from Avantor Performance Materials (Gliwice, Poland). Cyanide standard solution (K2[Zn(CN)4]; concentration 1000±7 mg L−1) was purchased from Merck (Darmstadt, Germany). The solvents HPLC gradient grade methanol, acetonitrile, and formic acid 98% were purchased from Merck. Ultrapure water was obtained from a Millipore water purification system (Milli-Q, Billerica, USA).

2.2 Materials Postmortem blood samples were collected, depending on accessibility, from intracranial sinuses or peripheral veins of upper and lower extremities from corps (a) with signs of putrefaction, (b) from fire victims, and (c) from cyanide-poisoned persons (obtained from



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Forensic Medicine Department in Warsaw). Blood samples and organs from cadavers were collected within 12 hours after corps delivery to the Forensic Medicine Department. For intensive therapy unit (ITU) patients and fire victims, the corpses were delivered to Forensic Medicine Department within 6 hours after death, a time frame for the corpses with signs of putrefaction was estimated between 5 and 14 days. Approximately 2% sodium fluoride was added at the time of collection in order to preserve blood before the determination of cyanide. Cyanide concentrations were quickly estimated after sampling. Blood samples from ITU were pooled, fortified, and used for the assessment of cyanide and ATCA stability at elevated temperatures of 27°C–90°C and long-term cyanide stability. The materials from fire victims (21 males and 11 females, aged 21–95 years), cyanide-poisoned persons (2 males and 1 female, aged 31–75 years), and ITU patients (3 persons, unknown age) were collected and analyzed using the new method of determination of ATCA.14 Blood samples for ATCA measurement were stored in a freezer (−80°C) until analysis up to 1 month. On the basis of our unpublished data, we decided not to use preservative because the ATCA concentration seemed to be at the same level in samples with preservative and in those without it. Moreover, we proved long-term ATCA stability in non-preserved blood, which has been described in our previous study.14 Moreover, organs (liver, heart, kidneys, spleen, brain, and lungs) of fire victims and cyanide-poisoned persons were also collected. They were placed into plastic 20 mL tubes and stored in the freezer (−80°C) until they were thawed for analysis. Putrid blood was collected from seven persons who possessed signs of putrefaction (6 males and 1 female, aged 30–85 years and 1 person of unknown age at death).

2.3 Standard solutions, calibration standards, and quality control samples 2.3.1 ATCA measurements The stock solutions of the analyzed compounds of ATCA and ATCA-13C,15N2 were prepared by an accurate weighting of an appropriate amount of each compound and dissolving it in 8 ACS Paragon Plus Environment

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water and methanol, respectively, in order to obtain a concentration of 1 mg mL−1. The working standard solutions were prepared before use by dilution of the appropriate stock solutions with water in order to obtain the required concentrations. All stock solutions were stored at −20°C. 2.3.2 Cyanide measurements The cyanide standard solution K2[Zn(CN)4] (CCN-=1000 mg L−1) was bought from Merck (Darmstadt, Germany) and was stored at 4°C. The working standard solutions were prepared before use by dilution of the appropriate amount of cyanide standard solution K2[Zn(CN)4] with deionized water in order to obtain the required concentrations. Subsequently, it was used to fortify the blood (1:39 v/v). The calibration standards for cyanides were prepared before cyanide analysis. The concentrations of the standards ranged from 0.25 to 1.00 µg mL−1. The protocol of cyanide measurement follows the UK implementation of ISO 27368:2008 by The British Standards (Section 9.5: CN- by spectrophotofluorimetry or high-performance liquid chromatography using a fluorescence detector).15 It states that calibration curve should be prepared for human whole blood, in our case all the calibrations curves were done in postmortem whole blood.

2.4 Instrumentation 2.4.1 ATCA measurements Instrumental analysis was performed by using Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA), equipped with a degasser, autosampler, and binary pump coupled with hybrid triple quadrupole/linear ion trap mass spectrometer QTRAP 4000 (SCIEX, Framingham, MA, USA). The Turbo Ion Spray source was operated in positive mode. The curtain gas, ion source gas 1, ion source gas 2, and collision gas (all high purity nitrogen) were set at 345 kPa, 345 kPa, 276 kPa, and “high” instrument units (4.6×10−5 Torr), 9 ACS Paragon Plus Environment


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respectively. The ion spray voltage and source temperature were 5500 V and 400°C, respectively. The target compounds were analyzed in MRM mode (Table 1). Chromatographic separation was achieved by using a SeQuant® Zic®-HILIC column (50 mm × 2.1 mm, 5 µm; Merck, Darmstadt, Germany) equipped with a security guard. The column was maintained at 40°C with a flow rate of 0.5 mL min−1. The mobile phases consisted of HPLC grade water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as eluent B. The gradient (%B) was as follows: 0 min 90%; 1 min 90%; 7 min 50%. The re-equilibration of the column to the initial conditions lasted 8 min. The volume of injection was 5 µL. 2.4.2 Cyanide measurements The method of cyanide detection follows the UK implementation of ISO 27368:2008 by The British Standards and is based on the availability of equipment in the laboratory.15 Instrumental analysis was performed using Hitachi F7000 spectrofluorimeter (Hitachi High Technology, Tokyo, Japan), equipped with a thermostat. This technique is based upon the transformation of cyanide ion by acidification, from blood to HCN and then the subsequent reaction of cyanide ion in HCN with 2,3-naphthalenedicarboxaldehyde and taurine in a selfcontained system. The reaction product 1-cyano-2-benzoisoindole [1-cyano[f]benzoisoindole] derivative is then measured fluorometrically (λex = 418 nm; λem = 460 nm).

2.5 Sample preparation 2.5.1 ATCA determination The concentration of ATCA in blood and tissue homogenates was measured by using the method described in a previous study.14 The validation parameters of the method met the acceptance criteria of FDA16 and EMA.17 The calibration curve was linear in the range of 30– 900 ng mL−1. The accuracy ranged 86%–107% (RSD = 1%–13%) within 1 day and 91%– 104% (RSD = 6%–11%) between three runs. The limit of detection (LOD) and limit of 10 ACS Paragon Plus Environment

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quantification (LOQ) of the method for postmortem blood were calculated as 0.43 and 1.5 ng mL−1, respectively. For organ homogenization, samples were thawed, chopped, and weighed in homogenizing tubes. After rinsing blood from tissue, 10 mM phosphate buffer (pH 7.4) was added to make a concentration of 0.15 g organ mass per mL−1 in each tube. The samples were homogenized using a glass Dounce homogenizer, and the homogenates were prepared manually. The solution was vortexed and centrifuged for 10 min at 9300 × g, and 100 mL of the supernatant was used for the liquid–liquid extraction. Equal volumes of fortified blood samples or tissue homogenates (100 mL) and water (100 mL) were mixed and deproteinized with acetonitrile (1:4 v/v). Then, the sample was kept at −20°C for 20 min and centrifuged at 9300 × g at 25°C for 5 min. A total volume of 750 mL of the supernatant was placed into glass test tubes, evaporated under nitrogen (45°C, 5 min), and reconstructed with 50 mL of water. Then, 35 mL of sodium tetraborate buffer (0.15 M; pH=9.4) and 50 mL of (S)-N -(4nitrophenoxycarbonyl) phenylalanine methoxyethyl ester (S-NIFE) (2.5 mg mL−1) were added. The mixture was incubated for 10 min at room temperature. The reaction was stopped by adding 12.5 mL of 3M HCl. Then, the sample was diluted with 355 mL of water, and the interfering compounds were removed by using 2.5 mL of ethyl acetate (1:7 v/v). The water residues were air dried under nitrogen (45°C, 8 min) and reconstructed with mobile phase.

2.5.2 Cyanide determination The concentration of cyanide in blood was measured by using the method from BSI standard publication (BS ISO 27368:200815). In order to prepare the calibration curve, 50 µL of appropriate concentration of working standard solutions of cyanide was added to 2 mL of blood that was pipetted into a 20-mL vial. Then, the 2-mL tube containing 200 µL each of the diluted NDA solution (1 mM) and the taurine solution (5 mM) was immediately placed into



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this vial. A total of 0.5 mL of the 10% sulfuric acid solution containing 200 mg NaHSO4 was carefully added to the 20-mL vial. The 20-mL vial was sealed using aluminum crimp-top caps with teflon-faced butyl-rubber septum. The contents of the vials were gently vortexed for 5 s. The sealed vial was incubated for 90 min at 35°C in order to allow the diffusion of HCN from the sample into the mixture of NDA and taurine solutions in the 2-mL tubes. After incubation, the fluorescence intensity of 200 µL of an aliquot from the 2-mL tubes was measured using microplate.

2.6 Long-term cyanide stability Long-term stability of cyanide in blood spiked with a solution of K2[Zn(CN)4] to obtain cyanide concentration levels of 5 and 1 µg mL−1 was determined in the samples after storage of up to 13 weeks under refrigeration (−80°C±2°C). Postmortem blood was collected and preserved with 2% sodium fluoride. The cyanide concentrations were chosen on the basis of their associated symptoms. Concentrations >3 µg mL−1 are associated with death; 1 µg mL−1 of cyanides results in serious symptoms of cyanide intoxication without the consequences of death.

2.7 Thermal stability Thermal stability tests were performed by using a temperature-controlled water bath. In order to determine the stability of ATCA and cyanide, heating was performed in Eppendorf test tubes and 20-mL vials, respectively. Postmortem blood was separately fortified with ATCA and cyanide at two concentration levels: 350 and 900 ng mL−1 (ATCA); 1 and 5 µg mL−1 (K2[Zn(CN)4]). Prepared samples were then incubated at constant temperatures: 27, 50, 63, 75, 90°C for 1 hour. Thermal stability of each sample was determined by repeated analyses (three repetitions). Similar to long-term stability experiment, concentrations of both the compounds were selected on the basis of the concentrations of cyanide in blood and 12 ACS Paragon Plus Environment

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associated symptoms: 1 µg mL−1 of cyanide and 350 ng mL−1 ATCA concentrations that may be found during toxic but non-lethal cyanide intoxication and 5 µg mL−1 of cyanides and 900 ng mL−1 ATCA that may be found during lethal cyanide poisoning. Blood samples that were collected were not stabilized against coagulation to preserve natural conditions similar to corpses’ exposure to fire.

2.8 Statistical analysis Normal distribution and homogeneity of data variance were tested using Shapiro–Wilk test and Levene’s test, respectively. When the data were not normally distributed and/or their variance was not homogeneous (p50°C and in the blood samples at temperatures >63°C. He found the highest total cyanide (bound plus free) production in both hemoglobin and blood at 75°C. Seto established that the increase in cyanide concentration of ~0.2 mmol of cyanide was formed from 1 mol of hemoglobin at 75°C.22 The ATCA results obtained in our study showed thermal stability at 350 ng mL−1 (RSD 4.1%) and 900 ng mL−1 (RSD 6.3%) concentrations at all tested temperatures. We did not observe statistically significant differences between any points of the curve for 350 and


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900 ng mL−1. Thus, it can be concluded that the stability of ATCA at both the levels correspond in the same fashion (Figure 3). In summary, after heating, spontaneous cyanide production is more likely than ATCA production. Cyanides may be released from decomposition products of amino acids and their derivatives. Cyanides are also highly volatile which may also show ATCA as a better marker of cyanide intoxication at elevated temperatures.

3.4 ATCA concentration in postmortem samples and in relation to cyanide level Measurement of cyanide occurrence in the blood samples from fire victims showed that 9 fire victims had undetectable cyanide concentration. Eighteen of the tested blood samples had cyanide concentrations 3 µg mL−1 was not found in fire victims. Cyanidepoisoned persons have greatly elevated cyanide concentrations (>3 µg mL−1), which resulted in significantly elevated ATCA concentrations with an average value: 1430 ng mL−1 (SD=640 ng mL−1). Statistically significant differences between ATCA concentrations depending on cyanide intoxication levels (i) 3 µg mL−1 CN−, p=0.0369 were also observed (Figure 5). Moreover, the Spearman test revealed the correlation between cyanide and ATCA concentrations in blood (R=0.47, p

Application of 2-Aminothiazoline-4-carboxylic Acid as a Forensic

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