## 3 Results
### Analytical Performance  
Volumes of 100 \(\upmu\)L of KCN solutions with concentrations ranging from 0 to 2.6 \(\upmu\)g mL\({}^{-1}\) reacted with ninhydrin (1 mL of NR) in the spectrophotometer cuvettes for 60 min at 25 \({}^{\circ}\)C (not shown). The reaction kinetics were measured at 493 nm against the reference made with the reagents. Then, we plotted the calibration curve with the absorbance values measured at 20 min after reagent mixing. The cyanide-ninhydrin system obeys [PERSON]'s law in the range from 0.26 \(\upmu\)g mL\({}^{-1}\) to 2.6 \(\upmu\)g mL\({}^{-1}\) KCN (R\({}^{2}\) = 0.9879) with molar absorptivity \(\varepsilon\) = 4.5 \(\times\) 10\({}^{4}\) L mol\({}^{-1}\) cm\({}^{-1}\) (Figure 2). However, keeping in mind that 100 \(\upmu\)L of cyanide solution was mixed with 1 mL of NR, the concentration of KCN in the resulted solution becomes 0.236 \(\upmu\)g mL\({}^{-1}\) and \(\varepsilon\) = 4.95 \(\times\) 10\({}^{5}\) L mol\({}^{-1}\) cm\({}^{-1}\); a value close to that was found by other authors when cyanogen compounds were determined in plants (\(\varepsilon\) = 1.4 \(\times\) 10\({}^{5}\) L mol\({}^{-1}\) cm\({}^{-1}\), in the range from 0.020 \(\upmu\)g mL\({}^{-1}\) to 0.800 \(\upmu\)g mL\({}^{-1}\)) [38]. Figure 2 shows the calibration curve for cyanide determination at 493 nm 20 min after the start of the reaction. We reported the absorbance values of the cyanide-ninhydrin adduct as a function of cyanide concentration. At the beginning, we used an excel sheet to first calculate the HCN concentration (expressed as KCN) in absorbent solution, then in 0.5 mL of blood, then the HCN concentration expressed as \(\upmu\)g mL\({}^{-1}\) HCN. In the meantime, we simplified the HCN calculation, using the following formula: C = 2.0063 A\({}_{493}\) + 0.5022, where C means HCN concentration in blood and A\({}_{493}\) is the absorbance value at 493 nm as measured against the reagents. In this case, we recovered only 1.555 mL of absorbent from 2 mL initially introduced into the Conway unit. Since the calibration curve is linear in the concentration range 0.1-2.6 \(\upmu\)g mL\({}^{-1}\), we considered the lowest concentration to be accurately measured under the experimental conditions of this method as 0.10 \(\upmu\)g mL\({}^{-1}\) KCN. However, considering that HCN in 1 mL of blood is captured in 2 mL of alkaline solutions and only 100 \(\upmu\)L of absorbent is used in the reaction with ninhydrin, the limit of quantification becomes much lower. Indeed, the limit of quantification was 0.052 \(\upmu\)g mL\({}^{-1}\); the detection limit was 0.012 \(\upmu\)g mL\({}^{-1}\) and the standard error was \(\pm\)2.7%. However, concentrations higher than 0.1 \(\upmu\)g mL\({}^{-1}\) are of interest for HCN determination in blood samples. This value is sufficiently satisfactory for the determination of HCN in the blood of fire victims and not for the very sensitive determination of blood cyanide for other purposes. However, we sought to improve the sensitivity of HCN determination by investigating the relationship between absorbance values and a wider range of KCN concentrations (Figure 3). Although a calibration curve of polynomial and not linear shape, in the range indicated in Figure 2, did not show a  
Figure 2: Calibration curve for cyanide determination: relationship between the absorbance of ninhydrin reagent at 493 nm and the cyanide concentration. higher correlation coefficient \(r\) (r = 0.994 vs. r = 0.993; R\({}^{2}\) = 0.9896 vs. R\({}^{2}\) = 0.9879), better results were obtained with a calibration curve of polynomial shape in the concentration range 0-2.6 \(\mu\)g mL\({}^{-1}\). Thus, linear correlation and abscissa interception, with the regression equation: y = 0.5071x, where y is the absorbance and x, the KCN concentration, led to a lower correlation coefficient, r = 0.979 (R\({}^{2}\) = 0.9592).