Quantitative measurement of plasma hemoglobin by second derivative spectrophotometry

The development and validation of a second derivative spectrophotometric assay for hemoglobin in plasma is reported. Using the oxyhemoglobin absorbance peak at 578 nm, the method was found to be linear for standard samples in the range of 3-2,000 mg/l oxyhemoglobin. Because of one dilution step this range is doubled for plasma samples. Correlation with an enzymatic assay was excellent (r2 = 0.985). Within-run and between-run precision analysis showed CV values ranging from 0.5-6.5% for specified calculation conditions. Icteric or lipemic backgrounds did not influence the results. All calculations of derivative spectra were carried out for three different wavelength intervals. The largest interval (delta gamma = 6 nm) showed the best performance. The method is simple and rapid and it has been introduced successfully in our laboratory for routine clinical use.