Development of a Simple and Rapid Spectrophotometric Method for the Quantification of Carboxyhemoglobin

Carbon monoxide (CO) is produced when organic matter is burned in an inadequate supply of oxygen and is considered one of the most frequent causes of poisoning in the industrial and domestic settings. CO binds to hemoglobin, leading to the formation of carboxyhemoglobin (COHb) leading to hypoxia and subsequently to anoxia. A COHb concentration about 50–60% is usually considered fatal. Given these considerations, it is necessary the existence of validated, sensitive and reliable methods for the determination of COHb in the blood. A spectrophotometric method for COHb determination in blood was developed. The method is based on the production of a two-pigment mixture by reducing oxyhemoglobin and methemoglobin, but not COHb, with sodium hydrosulfite after haemolysis. The standards of 0 and 100% COHb were prepared by bubbling ultra-pure CO and O2 in the haemolysed and reduced solution. The bubbling time and flow for the preparation of the standards was tested and validated. Intermediated standards were prepared by mixing different proportions of each standard of 0 and 100% COHb. The ratio of the absorbances at 420 and 432 nm was calculated, for each % COHb. The method proved to be specific, accurate and precise across the calibration range (0–100% COHb). Also, plotting expected % COHb against the observed values, linear regression r2 = 0.99 was obtained. The proposed method can be successfully applied in the quantification of COHb in human blood samples resulting from CO intoxication