## 4 Discussion
### Applications for Real Samples  
The proposed micro method was applied for the determination of HCN in several blood samples. The results of the determinations were in good agreement with those made with a well-known spectrophotometric method [55].  
First, the HCN concentration was determined in post-mortem blood samples after its removal with 20% H\({}_{3}\)PO\({}_{4}\) and capture in a 2% potassium carbonate solution. The autopsy and chemical analyses of a 51-year-old man were performed 24 h after death [14]. Free HCN concentration was determined to be 1.043 \(\mathrm{\SIUnitSymbolMicro}\)g mL\({}^{-1}\), whereas the total HCN (free HCN plus HCN metabolized to thiocyanate) was 1.904 \(\mathrm{\SIUnitSymbolMicro}\)g mL\({}^{-1}\). In this case, the toxic concentrations of HCN and total HCN were seen as a vital sign (suggesting that the victim must have been still alive when the fire started), indicating a contributing effect of HCN in the mechanism of death. Both HCN in the blood and thiocyanate resulting from the partial metabolism of HCN (salts of thiocyanate acid, HSCN) were determined using 0.5-1 mL of femoral blood. The cause of death was the severe second-degree burns on about 50% of the body surface and acute alcohol intoxication. In addition, the amount of HCN metabolized to HSCN salts could also have a contributing effect. Therefore, a cause of death with three factors can be considered in the reported case [14].  
The second case: an 80-year-old man with locomotor disabilities died due to an open fire of vegetation debris and household waste in his yard [37]. Similarly, the HCN from 1 mL of femoral post-mortem blood was captured in 2 mL of 2% potassium carbonate solution and the resulted solution was collected with an automatic pipette; the Conway vial was rinsed, and the two solutions were transferred into a vial and completed with 2 mL of distilled water. Aliquots of 100 \(\mathrm{\SIUnitSymbolMicro}\)L were taken and treated with 1 mL of 5 mg mL\({}^{-1}\) ninhydrin reagent. A toxic concentration of 1.3 \(\mathrm{\SIUnitSymbolMicro}\)g mL\({}^{-1}\) HCN was found. Nevertheless, the carboxyhemoglobin (COHb) level was lethal (73.7%). Despite extremely severe burns and high levels of carbon monoxide, the additive effect of HCN in the mechanism of death was also highlighted [37].  
Preservation of blood samples with 1% sodium fluoride at postmortem collection has been recommended [39]. Therefore, we collected the femoral venous blood in tubes with sodium fluoride as an anticoagulant and froze it immediately. Prior to HCN measurement, frozen blood was thawed and stored at 4 \({}^{\circ}\)C. In fact, no significant difference was observed between cyanide concentrations in the blood of fire victims in samples treated with sodium fluoride and control samples of at least 9 days [40]. However, the samples treated with sodium fluoride were stable over a 25-day period when compared with the control samples, which showed significant increases in HCN concentrations. The authors recommended the addition of 2% sodium fluoride to blood samples from fire victims to reduce potential instability due to bacteriological activity [40].