Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC \u2044 NPD). In the former, hydrogen cyanide (HCN) was quantitatively
determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC \u2044NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC\u2044NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5\u201310 and 0.05\u20135 lg \u2044 mL, respectively. Detection limits were 0.2 lg \u2044mL for VIS and 0.05 lg \u2044mL for HS-GC\u2044NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases

ARNOLDI S

CASAGNI E

DELL'ACQUA L

GAMBARO V

PECORARO C

R. FROLDI

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC ⁄ NPD). In the former, hydrogen cyanide (HCN) was quantitatively
determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC ⁄NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC⁄NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5–10 and 0.05–5 lg ⁄ mL, respectively. Detection limits were 0.2 lg ⁄mL for VIS and 0.05 lg ⁄mL for HS-GC⁄NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases

FROLDI, Rino

Archivio istituzionale della ricerca - Università di Macerata

TECHNICAL NOTEVeniero Gambaro,1 M.Sc.; Sebastiano Arnoldi,1 M.Sc.; Eleonora Casagni,1 M.Sc.; Lucia Dell'Acqua,1M.Sc.; Chiara Pecoraro,1 M.Sc.; and Rino Froldi,2 M.Sc.Blood Cyanide Determination in Two Cases ofFatal Intoxication: Comparison BetweenHeadspace Gas Chromatography and aSpectrophotometric Method*ABSTRACT: Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automatedheadspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC ⁄NPD). In the former, hydrogen cyanide (HCN) was quantitativelydetermined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC ⁄NPDmethod, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liber-ated as HCN. The spectrophotometric (VIS) and HS-GC ⁄NPD methods were validated on postmortem blood samples fortified with potassium cya-nide in the ranges 0.5–10 and 0.05–5 lg ⁄mL, respectively. Detection limits were 0.2 lg ⁄mL for VIS and 0.05 lg ⁄mL for HS-GC ⁄NPD. This workshows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable forrapid diagnosis of cyanide in postmortem cases.KEYWORDS: forensic science, cyanide, headspace gas chromatography, Conway cellCyanide (CN) is a powerful, rapidly acting poison, death occur-ring within minutes of ingestion. Cyanide exerts its toxic effects byreacting with the trivalent iron of cytochrome oxidase, thus inhibit-ing electron transport and preventing cells from using oxygen(hypoxia), which results in rapid loss of vital functions (1,2).Cyanide exposure is relatively common (2,4). Apart from sodiumnitroprusside therapy (as a hypotensive agent) and ingestion of cya-nide salt in the context of suicidal or homicidal attempts, the mainsources of exposure are smoke from fires or cigarette smoking,accidental inhalation of hydrocyanic acid in the metal and plasticindustries, and ingestion of various types of food such as cassava,cherry, or almond (2,3).HCN, not being ionized and readily diffusible, is rapidlyabsorbed through biological membranes and diffuses throughout thebody. Most of the cyanide concentrates in erythrocytes, presumablybound to methemoglobin. The major pathway of cyanide detoxifi-cation is by enzymatic conversion to thiocyanide and subsequentexcretion by the kidney (1,4).The postmortem specimen most frequently analyzed for cyanidein forensic toxicology is blood. Other sources are spleen, liver, andbrain. Blood cyanide concentrations lower than 0.25 lg ⁄mL areconsidered normal, and those above 0.25 lg ⁄mL but below2–3 lg ⁄mL as elevated, but not ordinarily causing death. Concen-trations above 3 lg ⁄mL are consistent with death in the absence ofother relevant and ⁄or toxicological findings (4).In forensic toxicology, cyanide exposure is preliminarily deter-mined by classical spectrophotometric techniques and then con-firmed by chromatographic methods (2,5–9).The aim of the present work was quantitative determination ofblood cyanide in two cases of fatal intoxication. Blood samples ofthe two cases were analyzed in two different laboratories, first by aspectrophotometric method (VIS), performed in the laboratory ofFroldi et al., and then by headspace gas chromatographic methodwith nitrogen-phosphorus detector (HS-GC ⁄NPD), in the laboratoryof Gambaro et al.Case HistoriesCase 1A 26-year-old woman was taken to a hospital emergency depart-ment, presenting with slowed respiration, gasping breath and brady-cardia. She died more than 1 h later. Symptomatology suggestedcyanide poisoning.Qualitative analysis by the Gettler and Goldbaum (10) techniquerevealed cyanide in stomach contents. Blood samples collected atautopsy were analyzed by VIS and results were confirmed by agaschromatographic method.Toxicological analysis on postmortem specimens was negativefor common drugs and volatile compounds.Case 2The body of a 30-year-old man was found in his closed car inan isolated area. A bottle containing a colorless liquid was foundnearby. Laboratory analysis of the liquid revealed cyanide. Forensicevaluation could not establish the time interval between ingestionand death.1Istituto di Chimica Farmaceutica e Tossicologica, Universit degli Studidi Milano, Viale Abruzzi 42, 20131 Milano, Italy.2Istituto di Medicina Legale, Universit di Macerata, Via Don Minzoni 9,62100 Macerata, Italy.*Presented at the 11th Meeting on Recent Developments in Pharmaceuti-cal Analysis, Rimini, Italy, September 25–28, 2005.Received 4 Nov. 2006; and in revised form 4 Mar. 2007; accepted 18Mar. 2007; published 21 Dec. 2007.J Forensic Sci, November 2007, Vol. 52, No. 6doi: 10.1111/j.1556-4029.2007.00570.xAvailable online at: www.blackwell-synergy.com 2007 American Academy of Forensic Sciences 1401The stomach contents, collected during autopsy, were analyzedby the Gettler and Goldbaum (10) technique. Postmortem bloodsamples were analyzed by VIS and results confirmed by a gaschro-matographic method. Toxicological analysis was positive for cya-nide and negative for common drugs and volatile compounds.Blank Sample Collection and StorageSamples were collected in hermetically sealed vials and stored at)20C until analysis.The concentrations of cyanide in blood decay rapidly in the firstfew hours after exposure. Literature data confirm that CN dis-appears quickly from blood, but does remain long enough to bedetected in forensic or clinical samples which may not be availablefor analysis immediately after exposure (9).The blood specimens used as blanks in this study were obtainedfrom a pool of postmortem blood samples of nonsmoking adults orsubjects who had not been injured by smoke inhalation. These speci-mens had been previously screened and confirmed to be devoid ofcyanide, according to sensitivity of both analytical methods used.Blank blood samples, fortified with cyanide, were used as workingstandards for the validation and development of both methods.Materials and MethodsChemicals and ReagentPotassium cyanide (KCN) standard was purchased from CarloErba Reagents (Rodano, Italy). Acetonitrile (IS), Chloramine T,H2SO4, NaOH, and HCl were purchased from J.T. Backer (Deventer,Holland).Other chemicals used in this study were: Pyridine, Glacial aceticacid, and Barbituric acid, all purchased from Fluka (Buchs,Switzerland).Pyridine-barbituric acid reagent was prepared by adding 15 mLof pyridine, 3 mL of concentrated HCl and 7 mL of H2O to 3.0 gbarbituric acid.Water was obtained from a Milli-Q ultrapurifying system,18.2 MW⁄cm (Millipore SA, Molscheim, France). All solvents andreagents were of analytical grade.Spectrophotometric Method (VIS)Glass Conway microdiffusion cells were used (18 · 70 mm o.d.;8–10 · 41 mm o.d., inner chamber).Adsorbing solution (2 mL, 0.1 M NaOH) was added to the innercompartment of each Conway cell, and the liberating solution (2 mL,50% H2SO4) was added to the outer compartment. Blood samples(1 mL) were added to the opposite part of the outer chamber, asmixing had to be avoided. The cell was then quickly closed by aTeflon-lined screw cap and gently rotated to mix blood and liberatingsolution. After 30 min contact at 38C, 1 mL of the inner chambercontents from each cell was taken up and transferred into a 10-mLvolumetric flask. To each flask 3 mL of 1 M NaH2PO4 and 1mL ofChloramine-T (2.5 g ⁄L) were added, mixed, and allowed to stand for2–3 min. Pyridine-barbituric acid reagent (3 mL) was then added andthe solution diluted to 10 mL with H2O.Absorbance was determined at 586 nm against a blank.Spectrophotometric AnalysisVIS was Performed on a Varian CARY50 (Torino, Italy) Spectro-photometer. Standard cyanide solution was prepared by placing25.0 mg of KCN in a 100-mL volumetric flask, to yield a solutionwith a concentration of 100 lg ⁄mL of CN; 0.1 N NaOH was usedas diluent. In another volumetric flask, 10 mL of this solution wastransferred and added with 90 mL of 0.1 N NaOH, to yield a solu-tion with a concentration of 10 lg ⁄mL.The standard cyanide solution was further diluted to yield sixworking solutions at concentrations in the range of 0.5–10.0 lg ⁄mL.Automated HS-GC ⁄NPD MethodApparatus—Automated headspace GC analysis was carried outon a ThermoFinningan Trace TG, equipped with an NP detector850, and interfaced with an autosampler (all from ThF, Rodano,Italy). A 10-mL vial and a 2.5-mL Hamilton 1002 NTL headspacesyringe (Hamilton Co, Reno, NV, U.S.A.) were used.After addition of acetic acid to the sample vial, the autosamplermoved the vial from the vial tray holder to the sample heater,where the vial was heated at 60C with continuous agitation for40 min. Using a thermostated syringe, 750 lL of headspace vaporwas injected into the GC inlet at a split rate of 40:1. Injector tem-perature was 100C.Gas chromatographic separation took place in a PoraBOND Ufused-silica capillary column (30 m · 0.32 mm i.d., 7 lm filmthickness) (Varian, Torino, Italy). Purified helium was used as car-rier gas, at constant pressure to assure a steady column flow rate of2.0 mL ⁄min. Detector gas flow rate was set at 60 and 3 mL ⁄minfor hydrogen and air, respectively.Column temperature was programmed at 90C for 5 min andincreased by 10C ⁄min to a final temperature of 140C. Detectortemperature was 300C.In these chromatographic conditions, retention times (tR) wereabout 1.9 and 4.2 min for cyanide and IS, respectively. (Fig. 1).HS-GC ⁄NPD AnalysisStandard cyanide solution was prepared by placing 25.0 mg ofKCN in a 100-mL volumetric flask to yield a solution with a con-centration of 100 lg ⁄mL of CN; 0.1 N NaOH was used as diluent.In another volumetric flask, 5 mL of this was transferred and addedwith 95 mL of 0.1 N NaOH to yield a solution with a concentra-tion of 5 lg ⁄mL.Standard cyanide solution was further diluted to yield six work-ing solutions at a concentration in the range of 5.0–0.1 lg ⁄mL.Approximately, 0.5 mL of each solution was transferred into a 10-mL vial together with 0.5 mL of IS and 0.5 mL of blank blood.Lastly, 50 lL of glacial acetic acid was then introduced throughthe silicone rubber with a microsyringe.Internal standard solution was prepared by placing 1 mL aceto-nitrile in a 100-mL volumetric flask and diluting with water. Thissolution was then further diluted to yield a solution with a finalconcentration of 0.005 lg ⁄mL.1,870 4,197CNIS(mVolt)(Minutes)FIG. 1—GC chromatogram of blank blood fortified with cyanide and IS.1402 JOURNAL OF FORENSIC SCIENCESPreliminary analysis of samples revealed high concentrationsof cyanide, so that the blood samples had to be suitably dilutedto yield concentrations in a calibration range. 1mL of each bloodsamples was diluted in a 10-mL volumetric flask with water.Therefore, 0.5 mL each of diluted sample, IS, and 0.1 N NaOHwere placed in a 10-mL glass vial, which was then capped withteflon-coated silicone rubber and sealed by crimping an alumin-ium cap. An aliquot (50 lL) of glacial acetic acid was intro-duced through the silicone rubber with a microsyringe. Aftereach injection, the syringe was flushed many times with air toensure no carry-over of residual cyanide. Blank air injectionswere also routinely run between samples to avoid this problemcompletely.Results and DiscussionFor UV ⁄VIS measurements, the usual linear relationship followsthe Lambert–Beer Law, which states that the absorbance of an ana-lyte is directly proportional to its concentration.In the VIS method, three calibration lines were constructed usingsix calibrators in the cyanide range 0.5–10 lg ⁄mL. The concentra-tions for the first regression line were 0.525, 1.050, 2.625, 5.250,and 7.875 lg ⁄mL, those of the second 0.515, 1.030, 2.575, 5.150,and 7.725 lg ⁄mL, and those of the third 0.555, 1.110, 2.775,5.550, and 8.325 lg ⁄mL.The regression lines for determination of linearity werey = 0.2109x ) 0.059, y = 0.1419x ) 0.049, and y = 0.2059x )0.051, respectively. The correlation coefficients (R2) of the threecurves were 0.9989, 0.9994, and 0.9991. Method precision wasevaluated by measuring intra-day and inter-day precision for cya-nide. The intra-day coefficient of variation (CV%) was determinedby performing three replicate analyses of each concentration on thesame day. Inter-day CV% was determined by analyzing each con-centration on three different days. Intra-day CV% was 8.1% andinter-day CV% 12.3%.The limit of detection (LOD) was evaluated analyzing threeblank samples and calculating the standard deviation of theseresponses. In our case, it was 0.2 lg ⁄mL. The limit of quantitation(LOQ) is the lowest point of concentration on the curve, so thatthe Lambert–Beer law is still valid. In our case, the value was0.5 lg ⁄mL.In the HS-GC ⁄NPD method, sample cyanide concentration wascalculated by the following ratio:CCN ¼ ðACN=AISÞ=RRMEAN;where ACN is cyanide peak area and AIS is IS peak area in theanalytical sample and RRMEAN is the mean of the RR calculatedin the linearity study:RRMEAN ¼ ðACN=AISÞ=CCN;where ACN is cyanide peak area and AIS is IS peak area inworking standard samples and CCN is cyanide concentration(lg ⁄mL) in working standard samples.No interference at the retention time of the examined substances(CN and IS) was found on analysis of blanks (Figs. 2 and 3).Calibration lines were constructed using at six calibrators over aconcentration range of 0.05–5.0 lg ⁄mL, and three independentdeterminations were made at each concentration (n = 18). The con-centrations of the first and second calibration lines were 0.056,0.112, 0.558, 1.116, 2.790, and 5.588 lg ⁄mL, and those of thethird line were 0.054, 0.108, 0.542, 1.084, 2.710, and5.420 lg ⁄mL.Linear regression lines were obtained by plotting normalizedpeak areas (ratios of cyanide peak areas to IS peak areas) versuscyanide concentration by means of the least-squares method.The regression lines for determination of linearity werey = 1.2142x ) 0.0282, y = 1.2597x ) 0.0371, and y = 0.8374x )0.0627. Correlation coefficients in the range 0.9754–0.9990 wereobtained. The use of RR is suitable because the intercept of eachcalibration line was close to zero.Method precision was ascertained by measuring intra-day andinter-day precision for cyanide. The study was carried out over aperiod of three days at six concentrations: intra-day precision wasevaluated by RRMEAN and coefficient of variation (CV%) of threereplicate analyses of each working standard sample on the sameday, inter-day precision was determined by analyzing six workingstandard samples on three different days. The resulting intra-dayCV% was 13.8% and the inter-day CV% 18.6%.Accuracy is the agreement between the measured and the truevalue. The percentage recovery of a standard samples provide themeasure of method accuracy. The percentage recovery is calculatedusing the following formula:Rec% ¼ experimental value=theoretical value 100;where experimental value is the measured of standard sample;theorical value is the theorical concentration of standardsample.Accuracy data were obtained by performing three replicate anal-yses of standard samples at six concentrations (n = 18) and theRec% (mean) was 91.4%.Limits of detection and LOQ were calculated from linear regres-sion analysis. The LOD was 0.011 lg ⁄mL and the LOQ0.055 lg ⁄mL. The calculated LOD value for our assay is belowthe lethal cyanide blood concentration, thus making the methoddescribed here suitable for detecting accidental or self-induced cya-nide poisoning.Blood samples from the two cases were first analyzed by VISand subsequently confirmed by HS-GC ⁄NPD.In case 1, cyanide concentrations, determinated by the two meth-ods, were 7.0 and 7.5 lg ⁄mL, respectively. For case 2, they were30.1 and 30.8 lg ⁄mL. The results obtained by the two techniquescompared favorably with each other.The chromatographic procedure is easier to perform than theVIS procedure, and also has several other advantages:4,197(mVolt)(Minutes)1,870FIG. 2—GC chromatogram of blank blood.(mVolt)(Minutes)1,870 4,197ISFIG. 3—GC chromatogram of blank blood fortified with IS.GAMBARO ET AL. • BLOOD CYANIDE DETERMINATION IN TWO CASES OF FATAL INTOXICATION 1403sample preparation and analysis are easier and quicker; and thesample, once in the vial, does not require extraction or derivatiza-tion steps. Analysis performed with the fused-silica PLOT columnsshowed a very good separation for both substances, and no interfer-ence with endogenous blood components was observed.Spectrophotometric data were confirmed by automated HS-GC ⁄NPD results, showing that the gaschromatographic procedureis an effective alternative to VIS in determining cyanide in case ofintoxication.The automated HS-GC ⁄NPD method is not linear for cyanideconcentrations of >5 lg ⁄mL, because high concentrations produceresiduals in the headspace system, so that it was validated in thecyanide range 0.05–5 lg ⁄mL.As one important advantage of the VIS is the possibility of ana-lyzing samples with high concentrations of cyanide, this method issuitable for rapid preliminary analysis. Instead, the automated HS-GC ⁄NPD method is more sensitive, and is therefore suitable fordetermining cyanide in blood samples of fire victims with smokeinhalation and for confirming cyanide exposure in forensictoxicology.References1. Admur MO, Doull J, Klassen CD, editors. Casarett and Doull's toxicol-ogy: The basic science of poisons. 4th ed. New York: Pergamon Press,1991.2. Baselt RC. Disposition of toxic drugs and chemicals in man, 5th ed.Foster City, CA: Chemical Toxicology Institute, 2000;221–5.3. Guatelli MA. The toxicology of cyanide. In: Curry AS, editor. Methodsof forensic science, Vol. 3. New York: Academy Press, 1964;233–65.4. Levine B, editor. Principles of forensic toxicology. Washington: AACC-Press, 2003.5. Holzbecher M, Ellenberger HA. An evaluation and modification of amicrodiffusion method for the emergency determination of bloodcyanide. J Anal Toxicol 1985;9:251–3.6. Cardeal ZI, Gallet JP, Astier A, Pradeau D. Cyanide assay: statisticalcomparison of a new gas chromatographic calibration method versus theclassical spectrophotometric method. J Anal Toxicol 1995;19:31–4.7. Moriya F, Hashimoto Y. Potential for error when assessing blood cya-nide concentration in fire victims. J Forensic Sci 2001;46:1421–5.8. McAuley F, Reive DS. Rapid quantitation of cyanide in blood by gaschromatography. J Anal Toxicol 1983;7:213–5.9. Calafat AM, Stanfill SB. Rapid quantitation of cyanide in whole bloodby automated headspace gas chromatography. J Chromatogr B2002;722:131–7.10. Gettler AO, Goldbaum L. Detection and estimation of microquantities ofcyanide. Anal Chem 1947;19:270–1.Additional information and reprint requests:Prof. Veniero Gambaro, M.Sc.Istituto di Chimica Farmaceutica e TossicologicaFacolt di FarmaciaUniversit degli Studi di MilanoViale Abruzzi42-20131 MilanoItalyE-mail: veniero.gambaro@unimi.it1404 JOURNAL OF FORENSIC SCIENCES

Blood cyanide determination in two cases of fatal imtoxication: comparison between headspace gas chromatography and a spectrophotometric method

Abstract

Similar works

Full text

Available Versions

Archivio istituzionale della ricerca - Università di Macerata