## 4 Discussion  
### Basis of the Process  
The kinetics of cyanide reaction with ninhydrin have not previously been fully investigated. Here, we reported on the specificity of kinetic curves and reached the conclusion that kinetic measurements may improve cyanide determination in blood. As a result, a kinetic-spectrophotometric method for determining the concentration of hydrogen cyanide in the blood of fire victims was developed based on the reaction between HCN and ninhydrin with the measurement of absorbance at 493 nm. HCN is removed from blood samples with 20% H\({}_{3}\)PO\({}_{4}\) and absorbed in a 2% potassium carbonate (K\({}_{2}\)CO\({}_{3}\)) solution or, better, 0.01 N KOH. Other authors also reported that HCN derivatives in the blood can be decomposed using a citrate/phosphoric acid mixture or phosphoric acid only at 40 \({}^{\circ}\)C, and the resulting HCN is captured in an alkaline solution [44]. However, in our method, different amounts of HCN trapped in the absorbent react with a ninhydrin reagent to form a red-type complex, which indicates the mass concentration of cyanide ions in the sample. Determinations performed using the Conway vial and a ninhydrin-containing reagent require less space, sample material (0.5-1 mL), reagents, and cleaning. Spectrophotometric determination with ninhydrin prevents the user from having to deal with unpleasant substances, such as pyridine and other bad smelling compounds. Finally, the selectivity of both the cyanide reaction with ninhydrin and the HCN captured in an alkaline solution make this method extremely specific and suitable for medico legal as well as clinical applications. The standard method is sensitive to cyanide but the complexation with chloramines-T and barbituric acid-pyridine is difficult and expensive for the determination of cyanide in blood [45]. The proposed method has similar specificity, accuracy, and limit of detection. HCN from 0.5-1 mL of blood is captured in 2 mL of absorbent and 0.1 mL only was used in the reaction with ninhydrin. Therefore, this micro method can be greatly improved for use in forensic toxicology where analytical requirements are very high. We compared the kinetic spectrophotometric determination of HCN in the blood samples of the fire victims with the fixed-time spectrophotometric measurement. Furthermore, a comparison of this method with the chloramine T spectrophotometric method showed a significant advantage in terms of simplicity, less cost, and the use of common reagents and instruments. Figure 6: Calibration curves in the cyanide determination by the spectrophotometric method with chloramine T and by the kinetic spectrophotometric method with ninhydrin (**a**). The dose correlation relationship between the values for absorbance (standard method) and the slope in the kinetic measurements (**b**). Indeed, some methods are based on gas chromatography and measure cyanide as cyanogen chloride after conversion with chloramine-T. Such a method involves a number of chemical manipulations and requires an electron capture detector and a specific column [46]. On measuring cyanide with this method, [PERSON] et al. [18] found a linear detection response from 0.005 to 1.0 \(\upmu\)g mL\({}^{-1}\) cyanide. The blood cyanide was measured by isotope dilution with a detection limit of 0.3 \(\upmu\)M [47]. The addition of phosphoric acid liberates the cyanide as HCN, which is determined by headspace gas chromatography coupled with by mass spectrometry. A linear calibration graph over a wide concentration range of cyanide, and the limit of determination of 1 ng mL\({}^{-1}\) was reported, when head-space capillary gas chromatography with nitrogen-phosphorus detection was applied [48]. However, thiocyanate showed a positive interference. The results of this method agree with those obtained by the microdiffusion spectrophotometric method for blood samples from fire victims [48]. Moreover, [PERSON] et al.