Determination of carboxyhemoglobin in the presence of other blood hemoglobin pigments by visible spectrophotometry

Abstract

The convenience of the spectrophotometric method for the determination of carboxyhemoglobin has been tempered by the observation that the analysis of postmortem bloods is often biased by the presence of pigments other than oxyhemoglobin, carboxyhemoglobin, and reduced hemoglobin. These other pigments include most prominently methemoglobin and sulfhemoglobin. Using a microprocessor-controlled spectrophotometer, a method was developed depending on absorbance difference measurements at isosbestic points for oxyhemoglobin, carboxyhemoglobin, and reduced hemoglobin that is accurate down to 2% carboxyhemoglobin in fresh blood. A correction for the error caused by methemoglobin is part of the method. Qualitative confirmation of carboxyhemoglobin by examination of spectra details, sodium dithionite reduction, and first derivative spectra is described. The analysis of denatured and autolyzed bloods is examined in the context of postmortem case reports. A number of spectra are shown in detail, including methemoglobin, sulfhemoglobin, alkaline hematin, acid hematin, and mixtures of blood pigments containing varying concentrations of carboxyhemoglobin. The method has been shown to be precise, accurate, and reliable for fresh bloods. While accuracy for denatured bloods is diminished, reliability of carboxyhemoglobin identification is maintained. The analysis time is about 5 min for routine blood samples and the method is easily implemented with a precise microprocessor-controlled spectrophotometer.