Modified congener analysis: Quantification of cyanide in whole blood, other body fluids, and diverse beverages.

The congener analysis is routinely used for the determination of volatile compounds in body fluids and beverages for forensic investigations. Although intoxications with cyanide via smoke inhalation or ingestion of cyanide salts are frequently encountered in forensic medicine, the inclusion of hydrogen cyanide in this analysis was never studied in detail. In this work, a very simple, fast, and sensitive quantification method with headspace gas chromatography and flame ionization detection for the analysis of cyanide in whole blood-was developed and validated. In contrast to the standard sample preparation of the congener analysis, an acidification step with tartaric acid was added. A limit of detection of 50 ng/ml, good linearity (coefficient of correlation > 0.9997), high accuracy (101.5%-106.4%), and precision (relative standard deviation 1.8%-3.7%) were achieved. Authentic blood samples of 10 forensic cases were investigated with the new method. Furthermore, the method was used for the quantification of cyanide in other body fluids (serum and urine) and diverse beverages. Interferences were investigated, and the addition of aldehydes produced a clear concentration-dependent decrease of the cyanide signal. Besides, the method offers an economical use of limited sample material by the simultaneous determination of cyanide, ethanol, and congener alcohols.

Development of a UPLC-ESI-MS/MS method to measure urinary metabolites of selected VOCs: Benzene, cyanide, furfural, furfuryl alcohol, 5-hydroxymethylfurfural, and N-methyl-2-pyrrolidone.

We report on the development of an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously measuring eight biomarkers of volatile organic compound (VOC) exposure, with potential application to e-cigarette aerosol biomonitoring. Phenylmercapturic acid (PMA) and trans, trans-muconic acid (tt-MA) are metabolites of benzene; 2-aminothiazoline-4-carboxylic acid (ATCA) is a metabolite of cyanide; N-2-furoylglycine (N2FG) is a metabolite of furfural and furfuryl alcohol; 5-hydroxymethylfuroic acid (HMFA), 5-hydroxymethyl-2-furoylglycine (HMFG), and 2,5-furandicarboxylic acid (FDCA) are metabolites of 5-hydroxymethylfurfural; and 5-hydroxy-N-methylpyrrolidone (5HMP) is a metabolite of N-methyl-2-pyrrolidone. A pentafluorophenyl-modified silica column was used for chromatographic separation. The overall run time for the method is about 6 min per sample injection. The method has low to sub-nanograms per milliliter sensitivity, linearity over 3 orders of magnitude, and precision and accuracy within 15%. The method was used to measure human urine samples. Results showed that people with known benzene exposure (daily cigarette smokers) had higher levels of tt-MA and PMA compared with non-smokers. The method is advantageous for high-throughput analysis of selected VOC metabolites in large-scale, population-based studies such as the National Health and Nutrition Examination Survey (NHANES). Quantifying these urinary biomarkers is important to public health efforts to understand human exposure to VOCs from various sources, including tobacco products and electronic nicotine delivery systems.

Effectiveness of wetting method for control of konzo and reduction of cyanide poisoning by removal of cyanogens from cassava flour.

BACKGROUND: Konzo is an irreversible paralysis of the legs that occurs mainly among children and young women in remote villages in tropical Africa and is associated with a monotonous diet of bitter cassava. Konzo was discovered in 1938 by Dr. G. Trolli in the Democratic Republic of Congo (DRC). It also occurs in Mozambique, Tanzania, Cameroon, Central African Republic, and Angola. It was first controlled in Kay Kalenge village, DRC, in 2011 with the use of a wetting method to remove cyanogens from cassava flour. Fourteen months later, another visit was made to Kay Kalenge. OBJECTIVE: To determine whether Kay Kalenge women were still using the wetting method, whether there were new cases of konzo, and whether the wetting method had spread to other villages. METHODS: Meetings were held with chiefs, leaders, and heads of mothers' groups, women from 30 households were interviewed, and three nearby villages were visited. Total cyanide and thiocyanate were analyzed in cassava flour and urine samples, respectively. RESULTS: The women in Kay Kalenge village still used the wetting method. There were no new cases of konzo. The mean cyanide content of the flour samples was 9 ppm, and no child had a mean urinary thiocyanate content greater than 350 micromol/L. The use of the wetting method had spread naturally to three adjacent villages. CONCLUSIONS: The wetting method has been readily accepted by rural women as a simple and useful method to control konzo by removing cyanide from cassava flour, and its use has spread to nearby villages. The wetting method should be promoted by health authorities to control konzo and reduce cyanide poisoning from high-cyanide cassava flour.

Control of konzo by detoxification of cassava flour in three villages in the Democratic Republic of Congo.

Three villages in Boko Health Zone, Bandundu Province, Democratic Republic of Congo (DRC), had 61 konzo cases and konzo prevalences of 2.5%, 4.1% and 7.5% respectively. Konzo cases occurred every year for 10 years and every month, peaking in July. The high mean cyanide content of cassava flour of 50 ppm was due to short soaking of cassava roots for 1-2 days instead of 3-4 days. Konzo cases were examined and village women taught the wetting method that removes cyanogens from flour. The villages were visited every month for 1 year following previous methodology. No new konzo cases occurred during the intervention, mean flour cyanide levels reduced from 50 to 14 ppm and mean urinary thiocyanate levels of school children reduced from 930 to 150 µmole/L. The percentage of children with urinary thiocyanate levels of >350 µmole/L was reduced from a maximum of 80 in Ikialala before the intervention to 0 in Ikusama, Ikialala and 3 in Imboso Mwanga 1 year later. This is the second time that konzo has been controlled and success depends on regular use of the wetting method by village women. The methodology is now being used in other villages in DRC with financial support of AusAID.

Normative concentrations of urine thiocyanate in cassava eating communities in Nigeria.

Exposure to cyanide is a major public health problem where highly cyanogenic cassava foods are consumed. Thiocyanate (SCN), the biomarker of exposure to cyanide is present in several foods, and produced endogenously. Concentrations of urine SCN were measured in endemic and non-endemic areas of ataxic polyneuropathy in Nigeria. Cassava food consumption in the endemic area was twice that of non-endemic areas. Geometrical mean (95% CI) urine SCN was 20 µmol/l (18-24) for no consumption of cassava foods, 56 µmol/l (49-64) for daily consumption, 56 µmol/l (48-65) for twice daily consumption and 85 µmol/l (62-117) for thrice daily consumption. 95th percentile reference limit was 125 µmol/l for no consumption of cassava food, but 360 µmol/l for thrice daily consumption. Urine SCN is a useful biomarker of exposure to cyanide from cassava foods. There is strong ecological association of exposure to cyanide and endemicity of ataxic polyneuropathy.

Comparison of cyanide exposure markers in the biofluids of smokers and non-smokers.

Cyanide is highly toxic and is present in many foods, combustion products (e.g. cigarette smoke), industrial processes, and has been used as a terrorist weapon. In this study, cyanide and its major metabolites, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid (ATCA), were analyzed from various human biofluids of smokers (low-level chronic cyanide exposure group) and non-smokers to gain insight into the relationship of these biomarkers to cyanide exposure. The concentrations of each biomarker tested were elevated for smokers in each biofluid. Significant differences (p < 0.05) were found for thiocyanate in plasma and urine, and ATCA showed significant differences in plasma and saliva. Additionally, biomarker concentration ratios, correlations between markers of cyanide exposure, and other statistical methods were performed to better understand the relationship between cyanide and its metabolites. Of the markers studied, the results indicate plasma ATCA, in particular, showed excellent promise as a biomarker for chronic low-level cyanide exposure.

Single-drop microextraction as a powerful pretreatment tool for capillary electrophoresis: A review.

Single drop microextraction (SDME) is a convenient and powerful preconcentration and sample cleanup method for capillary electrophoresis (CE). In SDME, analytes are typically extracted from a sample donor solution into an acceptor drop hanging at the inlet tip of a capillary. The enriched drop is then introduced to the capillary for CE analysis. Since the volume of the acceptor drop can be as small as a few nanoliters, the consumption of solvents can be minimized and the preconcentration effect is enhanced. In addition, by covering the acceptor phase with an organic layer or by using an organic acceptor phase, inorganic ions such as salts in the sample solution can be blocked from entering the acceptor phase, providing desalting effects. Here, we describe the basic principles and instrumentation for SDME and its coupling with CE. We also review recent developments and applications of SDME-CE.

[Measurement of acetonitrile in blood and urine by head-space gas chromatography].

OBJECTIVE: To establish the method for measurement of acetonitrile in blood and urine by head-space gas chromatography. METHODS: DB-ALC1 (30 m x 320 microm x 1.8 microm) and DB-ALC2 (30 m x 320 microm x 1.2 microm) capillary column were used to measure the acetonitrile in blood and urine with the isopropanol as internal standard reference. RESULTS: The limits of detection of acetonitrile in both blood and urine were 0.5 microg/mL, with a linear range of 5-1000 microg/mL (r = 0.999).The accuracy of this method was 93.2%-98.0%. The RSD for the intra-day and inter-day were less than 3.7%. CONCLUSION: The method is capable for measurement analysis of acetonitrile in blood and urine.

Fire fighting trainers' exposure to carcinogenic agents in smoke diving simulators.

It is well known that fire fighters are potentially exposed to various carcinogenic agents at a fire scene. An almost unheeded issue, however, is fire fighters' exposure to carcinogenic agents in smoke diving simulators. Biomonitoring (urinary muconic acid, 1-naphthol and 1-pyrenol), dermal (polycyclic aromatic hydrocarbons) and occupational hygiene measurements (cyanides, hydrogen cyanide, polycyclic aromatic hydrocarbons, volatile organic compounds and formaldehyde) were used to determine how the burning material, the type of simulator and protective clothing used affect fire fighting trainers' exposure. The highest excretion of 1-pyrenol (sampled 6h after end of exposure, in average 4.3-9.2nmol/L) and emissions of benzene (1.0-2.5mg/m(3)) and hydrogen cyanide (0.2-0.9mg/m(3)) were measured during the burning of conifer plywood and chipboard, and the lowest when pure pine and spruce wood (1.5nmol/L, 0.6mg/m(3), and 0.05mg/m(3)) was burned. However the safest burning material seemed to be propane (1.0nmol/L, 0.2mg/m(3), and not measured). The type of simulator used affected trainers' exposure very clearly. The highest dermal whole body exposures to polycyclic aromatic hydrocarbons were measured in the fire house simulator (in average 1200ng/cm(2)). Clearly lower exposure levels were measured in container training sessions (760ng/cm(2)), where the average dermal exposure level was 35% lower than in the fire house. The exposure levels (30ng/cm(2)) in the gas simulator in turn, were only 4% of the levels in container training sessions. The amount of polycyclic aromatic hydrocarbons decreased by 80% on trainers' hands when they used under gloves (in average 8.7ng/cm(2)) compared to those (48.4ng/cm(2)) who did not. There was not difference in protection efficiency against polycyclic aromatic hydrocarbons between tested fire suits (Brage and Bristol).

Determination of cyanide, in urine and gastric content, by electrospray ionization tandem mass spectrometry after direct flow injection of dicyanogold.

A rapid and sensitive electrospray ionization tandem mass spectrometric (ESI-MS-MS) procedure was developed for the determination of cyanide (CN(-)). CN(-) in biological fluids was reacted with NaAuCl(4) to produce dicyanogold, Au(CN)(2)(-), which was extracted with methyl isobutyl ketone (MIBK). One microliter of the extract was injected directly into an ESI-MS-MS instrument. Quantification of CN(-) was performed by selected reaction monitoring of the product ion CN(-) at m/z 26 that derived from precursor ion Au(CN)(2)(-) at m/z 249. CN(-) could be measured in the quantification range of 10(-7) to 5x10(-5) M with the limit of detection at 4x10(-8) M using 10 microL of urine within 10 min. A victim's urine and gastric content were diluted with water to 4-fold and 500-fold and measured, respectively.

Rapid determination of cyanide in human plasma and urine by gas chromatography-mass spectrometry with two-step derivatization.

Cyanide (CN) is a powerful poison and rapidly toxic agent. Because of its wide availability and high toxicity, quantification of CN in blood and urine is frequently required in clinical and forensic practice. We present a sensitive and less time consuming method based on solid-supported liquid-liquid extraction (SLE) and gas chromatography-mass spectrometry (GC-MS) with two-step derivatization for determination of CN in plasma and urine. Buffer solution, 1,3,5-tribromobenzene (internal standard) and benzaidehyde were added to sample to complete the first-step derivatization. Then the analytes were poured onto the column of diatomaceous earth, eluted with n-hexane containing 0.4% of heptafluorobutyryl chloride (HFB-Cl) to complete the second-step derivatization forming the final analyte, heptafluoro-butyric acid-alpha-cyanobenzyl ester. This method was linear (r(2)=0.9988, 0.9993), reproducible (intra-day RSD=4.37-7.24%, 3.19-5.74%; inter-day RSD=5.13-7.63%, 4.31-6.69%), accurate (recoveries=90.58-115.56%, 93.01-114.6%) and sensitive (LOD=0.04, 0.01microg/mL) for plasma and urine, respectively. The total time was about 25min. This method was successfully applied to the analysis of blood sample and urine sample collected from a victim who died as a result of ingestion of potassium cyanide.

Simultaneous derivatization and extraction of free cyanide in biological samples with home-made hollow fiber-protected headspace liquid-phase microextraction followed by capillary electrophoresis with UV detection.

A new method is reported for the simultaneous derivatization and extraction of free cyanide in biological samples using home-made hollow fiber-protected headspace liquid-phase microextraction (HF-HS-LPME) followed by capillary electrophoresis (CE) determination. The acceptor phase containing Ni(II)-NH(3) (as derivatization agent), sodium carbonate and ammonium pyromellitate (as internal standard) is held within a hollow fiber membrane, affixed to a syringe needle and immersed in the headspace of sample container. The extracted cyanide from the neutral samples forms a stable Ni(CN)(4)(2-) complex which is determined by CE. Parameters affecting extraction efficiency were investigated and optimized. For optimum peak shapes, the capillary was coated with cetyltrimethylammonium bromide (CTAB). The calibration curve was linear for concentrations of CN(-) in the range from 0.1 to 20 micromol L(-1) (R(2)=0.9987). The LOD (S/N=3) was estimated to be 0.01 micromol L(-1) of CN(-) concentration. Such a detection sensitivity is high enough for free cyanide determination in common environmental and biological samples. Excellent repeatability of the extraction (RSD < or = 5.6%, n=5) was achieved. The feasibility of this method was demonstrated by analyzing human urine and saliva samples with spiked recoveries in the range of 92-103.4%. This work provided an efficient alternative to the present headspace microextraction techniques such as headspace solid-phase microextraction (HS-SPME) and headspace single-drop microextraction (HS-SDME).

A novel biamperometric biosensor for urinary oxalate determination using flow-injection analysis.

A biosensor for determination of oxalate concentration in urine has been developed by immobilisation of oxalate oxidase and peroxidase on the surface of an interdigitated gold electrode. Enzyme immobilisation was performed using BSA and glutaraldehyde. Biamperometric measurements were made in flow conditions both in aqueous oxalate solutions (tested concentration range between 50 microM and 10 mM) and in real urine samples (tested measuring range between 5 and 100 microM). Optimal working conditions were examined for flow-injection analysis, and good correlation was achieved between added oxalate quantity and the one measured by biosensor in urine matrix (R(2)=0.9983). The influence of some interferences (ascorbic acid, uric acid, paracetamol, acetylsalicylic acid) was also studied using biamperometric measurement mode.

Headspace single-drop microextraction with in-drop derivatization and capillary electrophoretic determination for free cyanide analysis.

A new method involving headspace single-drop microextraction (SDME) with in-drop derivatization and CE is developed for the preconcentration and determination of free cyanide. An aqueous microdrop (5 microL) containing Ni(II)-NH(3) (as derivatization agent), sodium carbonate and ammonium pyromellitate (as internal standard) was used as the acceptor phase. The extracted cyanide forms a stable Ni(CN)(4) (2-) complex which is then determined by CE. Common experimental parameters (sample and acceptor phase pH, extraction temperature, extraction time and sample ionic strength) affecting the extraction efficiency were investigated. Using headspace SDME, free cyanide can be effectively extracted from the neutral solutions, i.e. without the acidification of the sample which often is prone to errors due to incomplete liberation and artefactual cyanide production. Proposed SDME-CE method provided about 58-fold enrichment in 20 min. The calibration curve was linear for concentrations of CN(-) in the range from 0.25 to 20 micromol/L (R(2) = 0.997). The LOD (S/N = 3) was estimated to be 0.08 micromol/L of CN(-). Such a detection sensitivity is high enough for free cyanide determination in common environmental and physiological samples. Finally, headspace SDME was applied to determine free cyanide in human saliva and urine samples with spiked recoveries in the range of 91.7-105.6%. The main advantage of this method is that sample clean-up, preconcentration and derivatization procedures can be completed in a single step. In addition, the proposed technique does not require any sample pretreatment and thus is much less susceptible to interferences compared to existing methods.

An assessment of the release of inorganic cyanide from the fragrance materials benzyl cyanide, geranyl nitrile and citronellyl nitrile applied dermally to the rat.

Organonitriles are widely used as components of fragrances that are incorporated into consumer products, many of which are for human topical use. Some organontriles are readily broken down metabolically to potentially toxic inorganic cyanide. Studies were therefore undertaken to assess whether this occurs with three representative fragrance nitriles, namely, benzyl cyanide, geranyl nitrile and citronellyl nitrile when applied dermally to the rat. The nitriles (benzyl cyanide, 150 mg/kg; geranyl and citronellyl nitriles, 400 mg/kg) were applied to the shaved backs of rats and maintained under occlusion for 24 h. Urine samples were collected for 0-24 h, 24-48 h and 48-72 h from the time of first application. These samples were analysed for thiocyanate, a biomarker for cyanide formation in vivo, as described previously (Potter, J., Smith, R.L., Api, A.M., 2000. Urinary thiocyanate levels as a biomarker for the generation of inorganic cyanide from benzyl cyanide in the rat. Food and Chemical Toxicology 39, 141-146). In the case of benzyl cyanide, there was a marked increase in urinary thiocyanate levels attributable to the release of cyanide in vivo. The amount of thiocyanate recovered was equivalent to 37% of the dose for males and 32% for females. For geranyl nitrile there was no significant increase in urinary thiocyanate excretion and there was only a marginal increase in the case of citronellyl nitrile that was equivalent to 0.40% of the applied dose for males and 0.29% for females.

Cyanide determination in biological fluids using a microdiffusion method with a flow system and polarographic detection.

The report describes a method for the automated polarographic determination of cyanide as tetracyanonickelate (II) anion complex in a gas-diffusion flow system. The volatile cyanide, existing in whole blood, plasma and urine samples, was measured after gas-diffusion using 8 x 10-5 mol l-1 hexaaminenickel solution as acceptor. The linear range of calibration, for measurements at the hanging mercury-drop electrode (HMDE), was from 0.1 to 2.0 micrograms cyanide with r = 0.998. The RSD was, respectively, 3.4 and 1.2% (n = 5) for 0.4 microgram cyanide measured with and without the flow-system configuration. Detection limits of 7.4 microgram l-1 were calculated using the flow system and the method was compared with the classical method using Cavet flasks. Parameters that affect the cyanide determination in the proposed method, such as acceptor solution, pH, flow rate and temperature, were investigated.

Geographical and seasonal association between linamarin and cyanide exposure from cassava and the upper motor neurone disease konzo in former Zaire.

High cyanide intake from consumption of insufficiently processed cassava has been advanced as a possible aetiology of the upper motor neurone disease konzo. However, similar neurodamage has not been associated with cyanide exposure from any other source. With an ecological study design, we compared 22 cases of konzo, 57 unaffected household members and 116 members from unaffected households, a total of 195 subjects, in konzo-affected savanna villages with 103 subjects in adjacent non-affected forest villages in the Paykongila area in the Bandundu Region, Zaire. In the dry season, the mean value (+/- SEM) of urinary thiocyanate, the main cyanide metabolite, was higher in the three groups in konzo-affected villages (563 +/- 105, 587 +/- 44 and 629 +/- 47 micromol/l) than in unaffected villages (241 +/- 17 micromol/l). In affected villages in the dry season when konzo incidence was high, mean urinary thiocyanate was also higher than the levels found in the wet season when incidence was low. The wet season values (mean +/- SEM) were 344 +/- 60, 381 +/- 35 and 351 +/- 27 micromol/l. Urinary levels of inorganic sulphate were low in all groups, indicating low intake of the sulphur amino-acids which provide a substrate for cyanide detoxification. These findings support an aetiological role for cyanide in konzo. However, urinary linamarin, the cyanogenic glucoside and source of cyanide in cassava, was more closely associated with the occurrence of konzo. The mean value (+/- SEM) of urinary linamarin in the konzo cases was 632 +/- 105 micromol/l and in their household members 657 +/- 52 micromol/l, which was significantly higher than in members of control households in the same village (351 +/- 28 micromol/l) and in unaffected villages (147 +/- 18 micromol/l). This suggests that a specific neurotoxic effect of linamarin, rather than the associated general cyanide exposure resulting from glucoside breakdown in the gut, may be the cause of konzo.

Analysis of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine by high-performance liquid chromatography.

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATC) was converted to N-carbamylcysteine and analyzed by high-performance liquid chromatography (HPLC) and fluorometric detection. ATC was first separated from interfering substances by passing through the cation exchanger AG 50W-X8. Interfering disulfides were converted to thiols by reduction of the eluate with thiopropyl-Sepharose 6B. The thiols were then absorbed to the organomercurial absorbent p-acetoxymercurianiline-Sepharose 4B while ATC was obtained in the effluent. ATC was then converted to N-carbamylcysteine by ring opening at high temperature in an alkaline environment. Derivatization of N-carbamylcysteine was with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. The imprecision (CV) of the method was 3.6% and the analytical recovery was quantitative. The detection limit was 0.3 microM. ATC in urine was stable for at least 3 months when stored at -196 to +20 degrees C. The ATC concentration in urine was below the limit of detection in health nonsmokers. The utility of the method was demonstrated by the finding of up to 10.5 microM ATC concentrations in urine samples from human subjects in Mozambique living on a high dietary intake of cyanide from the cassava root. In a metabolic study rats were exposed to acetonitrile in the drinking water. After 4 weeks very high ATC levels were found in the urine, representative values from two rats being 195 and 525 microM, respectively.

Fate in humans of dietary intake of cyanogenic glycosides from roots of sweet cassava consumed in Cuba.

We studied if consumption of boiled fresh roots from sweet cassava varieties grown in Cuba resulted in exposure to cyanogenic glycosides and their final breakdown product, cyanide. When adult, nonsmoking subjects consumed 1-4 kg cassava over 2 days, their urinary levels of the main cyanide metabolite, thiocyanate, only increased from a mean +/- SEM of 12 +/- 2 to 22 +/- 2 mumol/l, indicating a negligible cyanide exposure. Their mean urinary linamarin, the main cyanogenic glucoside in cassava, increased from 2 +/- 1 to 68 +/- 16 mumol/l. In a second experiment 5 subjects consumed one meal of 0.5 kg boiled cassava that contained 105 mumol linamarin and 8 mumol hydrogen cyanide (HCN). Quantitative urine collections prior to and after intake showed that 28% of linamarin was excreted during the following 24 hours, whereas a modest increase of urinary thiocyanate (SCN) only corresponded to the small amount of free HCN ingested. These results indicate that the dominant cyanogen in boiled cassava is glycosides that pass through the human body without causing cyanide exposure. It remains to be studied whether humans occasionally possess intestinal or tissue beta-glucosidases that can hydrolyse cyanogenic glycosides from cassava.