Determination Of Cyanide In Blood Research Articles

Determination of Cyanide in Blood for Forensic Toxicology Purposes-A Novel Nci Gc-Ms/Ms Technique.

One of the recently evolving methods for cyanide determination in body fluids is GC-MS, following extractive alkylation with pentafluorobenzyl bromide or pentafluorobenzyl p-toluenesulfonate. The aim of this study was to improve previous GC methods by utilizing a triple quadrupole mass spectrometer, which could enhance selectivity and sensitivity allowing for the reliable confirmation of cyanide exposure in toxicological studies. Another purpose of this study was to facilitate a case investigation including a determination of cyanide in blood and to use the obtained data to confirm the ingestion of a substance, found together with a human corpse at the forensic scene. The blood samples were prepared following extractive alkylation with a phase transfer catalyst tetrabutylammonium sulfate and the PFB-Br derivatization agent. Optimal parameters for detection, including ionization type and multiple reaction monitoring (MRM) transitions had been investigated and then selected. The validation parameters for the above method were as follows—linear regression R2 = 0.9997 in the range of 0.1 µg/mL to 10 µg/mL; LOD = 24 ng/mL; LOQ = 80 ng/mL and an average recovery of extraction of 98%. Our study demonstrates the first attempt of cyanide determination in blood with gas chromatography-tandem mass spectrometry. The established method could be applied in forensic studies due to MS/MS confirmation of organic cyanide derivative and low matrix interferences owning to utilizing negative chemical ionization.

Rapid point of care analyzer for the measurement of cyanide in blood.

A simple, sensitive optical analyzer for the rapid determination of cyanide in blood in point of care applications is described. HCN is liberated by the addition of 20% H(3)PO(4) and is absorbed by a paper filter impregnated with borate-buffered (pH 9.0) hydroxoaquocobinamide (hereinafter called cobinamide). Cobinamide on the filter changes color from orange (λ(max) = 510 nm) to violet (λ(max) = 583 nm) upon reaction with cyanide. This color change is monitored in the transmission mode by a light emitting diode (LED) with a 583 nm emission maximum and a photodiode detector. The observed rate of color change increases 10 times when the cobinamide solution for filter impregnation is prepared in borate-buffer rather than in water. The use of a second LED emitting at 653 nm and alternate pulsing of the LEDs improves the limit of detection by 4 times to ~0.5 μM for a 1 mL blood sample. Blood cyanide levels of imminent concern (≥10 μM) can be accurately measured in ~2 min. The response is proportional to the mass of cyanide in the sample: smaller sample volumes can be successfully used with proportionate change in the concentration LODs. Bubbling air through the blood-acid mixture was found effective for mixing of the acid with the sample and the liberation of HCN. A small amount of ethanol added to the top of the blood was found to be the most effective means to prevent frothing during aeration. The relative standard deviation (RSD) for repetitive determination of blood samples containing 9 μM CN was 1.09% (n = 5). The technique was compared blind with a standard microdiffusion-spectrophotometric method used for the determination of cyanide in rabbit blood. The results showed good correlation (slope 1.05, r(2) 0.9257); independent calibration standards were used.

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN(-)) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN(-) from methemoglobin. CN(-) was then reacted with NaAuCl(4) to produce dicyanogold, Au(CN)(2)(-), that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN(-) was performed by selected reaction monitoring of the product ion CN(-) at m/z 26, derived from the precursor ion Au(CN)(2)(-) at m/z 249. CN(-) could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN(-) in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN(-) in the blood of a victim who ingested NaN(3) was confirmed using MS-MS for the first time, and the concentrations of CN(-) in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.

Determination of cyanide in blood by reaction head-space gas chromatography

A method describing determination of cyanide in blood by head-space gas chromatography with electron capture detector was reported. The method involves transformation of cyanide into cyanogen chloride by reacting hydrogen cyanide with chloramine-T on a stick of filter paper in the space above the blood in the head-space vial. The recovery was 84-96% and the coefficient of variation was 3.3-7.2%. The limit of quantitation was about 0.01 mg cyanide/l.

Determination of Cyanide in Blood by Isotope-Dilution Gas Chromatography–Mass Spectrometry

Determination of Cyanide in Blood by Isotope-Dilution Gas Chromatography–Mass Spectrometry

Determination of Cyanide in Blood by Isotope-Dilution Gas Chromatography–Mass Spectrometry

Cyanide (CN) is a lethal toxin. Quantification in blood is necessary to indicate exposure from many sources, including food, combustion byproducts, and terrorist activity. We describe an automated procedure based on isotope-dilution gas chromatography-mass spectrometry (ID GC/MS) for the accurate and rapid determination of CN in whole blood. A known amount of isotopically labeled potassium cyanide (K13C15N) was added to 0.5 g of whole blood in a headspace vial. Hydrogen cyanide was generated through the addition of phosphoric acid, and after a 5-min incubation, 0.5 mL of the headspace was injected into the GC/MS at an oven temperature of -15 degrees C. The peak areas from the sample, 1H12C14N+, at m/z 27, and the internal standard, 1H13C15N+, at m/z 29, were measured, and the CN concentration was quantified by ID. The analysis time was 15 min for a single injection. We demonstrated method accuracy by measuring the CN content of unfrozen whole blood samples fortified with a known amount of CN. Intermediate precision was demonstrated by periodic analyses over a 14-month span. Relative expanded uncertainties based on a 95% level of confidence with a coverage factor of 2 at CN concentrations of 0.06, 0.6, and 1.5 microg/g were 8.3%, 5.4%, and 5.3%, respectively. The mean deviation from the known value for all concentrations was <4%. The automated ID GC/MS method can accurately and rapidly quantify nanogram per gram to microgram per gram concentrations of CN in blood.

Determination of blood cyanide by HPLC-MS.

An original high-performance liquid chromatographic-mass spectrometric (HPLC-MS) procedure was developed for the determination of cyanide (CN) in whole blood. After the addition of K13C15N as internal standard, blood was placed in a microdiffusion device, the inner well of which was filled with a mixture of taurine (50mM in water)/naphthalene-2,3-dicarboxaldehyde (NDA, 10mM in methanol)/methanol/ concentrated (approximately 20%) ammonia solution (25:25:45:5, v/v). Concentrated H2SO4 was added to the blood sample, and the microdiffusion chamber was sealed. After 30 min of gentle agitation, 2 microL of the contents of the inner vial were pipetted and directly injected onto a NovaPak C18 HPLC column. Separation was performed by a gradient of acetonitrile in 2mM NH4COOH, pH 3.0 buffer (35-80% in 10 min). Detection was done with a Perkin-Elmer Sciex API-100 mass analyzer with an ionspray interface, operated in the negative ionization mode. MS data were collected as either TIC or SIM at m/z (299 + 191) and (301 + 193) for the derivatives formed with CN and 13C15N, respectively. Inspired by previous works dealing with the complexation of CN by NDA + taurine to form a 1-cyano [f] benzoisoindole derivative analyzed by HPLC-fluorimetry, this method appears simple, rapid, and extremely specific. Limits of detection and quantitation for blood CN are 5 and 15 ng/mL, respectively. The use of 13C15N as internal standard allows the quantitation of CN with elegance and accuracy in comparison with previously reported methods.

Determination of Cyanide in Blood by High Performance Liquid Chromatography with Fluorescence Detection

Simultaneous determination of inorganic ions including cyanide by photometric ion chromatography was useful for a cyanide analysis in drinks, but not applicable to that in blood, because of its poor resolution for cyanide and chloride. In this report, to determine cyanide in blood, we adopted a selective and sensitive method for cyanide based on a fluorometric reaction with 2,3-naphthalenedialdehyde (NDA) and taurine to afford 1-cyanobenz[f]isoindole derivative. Cyanide was extracted from blood by adding water and methanol to whole blood, and then derivatized with NDA and taurine. The cyanide derivative was analyzed on a reversed-phase high performance liquid chromatograph system with fluorescence detector. In the analysis of standard solutions, the reagent blank showed a minor peak of cyanide corresponding to ca. 0.04 ng/ml. Thus the lower detection limit for cyanide standard solution was 0.1 ng/ml as 2.5-fold concentration of the reagent blank peak. The peak seemed to be due to trace cyanide in reagents, however, it was so minor peak that it didn't interfere with cyanide determination in blood. The calibration curve for cyanide standard solution was linear in the range 0.1-200 ng/ml. In the blood analysis, the method enabled us to determine cyanide from healthy persons level (ca. 10 ng/ml) to fatal level (ca. 3000 ng/ml) employing the same treatment.

Specific determination of cyanide in blood by headspace gas chromatography.

An improved method has been developed for the quantitative determination of cyanide in human blood by headspace gas chromatography with electron-capture detection. In this novel method, cyanide was detected after conversion of hydrogen cyanide into cyanogen chloride by a reaction with chloramine T. The advantage of this new procedure lies in the fact that hydrogen cyanide formation and chlorination are carried out in a single step and in the same reaction medium. This method is simple, rapid, and specific for cyanide and does not suffer from any interference by cyanate and thiocyanate. The detection limit is 5 micrograms/L. The detection response is linear from 5 to 1000 micrograms/L, and the within-run coefficient of variation in this range is 8% or less. This method is particularly useful for routine diagnostic analysis of biological samples in case of acute cyanide poisoning.

Determination of blood cyanide by headspace gas chromatography with nitrogen-phosphorus detection and using a megabore capillary column

A method for the determination of cyanide in blood using head-space capillary gas chromatography (HS-GC) with nitrogen-phosphorus detection (NPD) was developed. The separation efficiency and sensitivity for hydrogen cyanide (HCN) were improved by using a GS-Q megabore capillary column and by injection of 500 μl of HS gas with a splitting ratio of 5. No interference or column deterioration was observed. Optimum HS conditions were found to be incubation of 50°C for 30 min in the presence of 10% phosphoric acid as an acidifying reagent. The distribution coefficient ( k) was 75.7 and 158.9 for HCN acetonitrile (CH 3CN; candidate for internal standard), respectively. HCN showed a different vaporization behaviour to CH 3CN with respect to temperature and matrix effect. The salting-out effect was relatively small for HCN and CH 3CN. An increase in the blood content in the liquid phase resulted in a significant increase in k for HCN. The calibration graph was linear over a wide concentration range of cyanide, and the limit of determination was 1 ng ml −1 (R.S.D. 10%). Because a considerable amount of CH 3CN was detected in control blood, the direct calibration method could be used instead of the internal standard method using CH 3CN. The detectable amount of cyanide was significantly decreased during the preincubation of cyanide and blood at 2°C. Thiocyanate in blood showed a positive interference. Contents determined by this HS-GC method agreed closely with those obtained by the microdiffusion-spectrophotometric method for blood samples from fire victims.

Rapid and sensitive quantitation of cyanide in blood and its application to fire victims.

We developed a head-space method for the determination of blood cyanide by gas chromatography with electron-capture detection. In this technique, a reaction precolumn packed with chloramine-T was used for the conversion of hydrogen cyanide into cyanogen chloride. Since the reaction precolumn eliminated the necessity of trapping hydrogen cyanide from biological samples, blood cyanide could be analyzed quickly by acidification only. Using this method, blood cyanide levels of fire victims were determined at autopsy. The serum values of cyanide ranged from 0.11 micrograms/ml to 18.12 micrograms/ml. However, a significantly higher cyanide content was detected in the left ventricular blood than in the right. This indicates that death was caused by the fire and suggests that the collecting point of the blood sample is an important factor in the determination of inhaled cyanide. There was a positive correlation between blood cyanide and carboxyhemoglobin contents.