Quantitative determination of heme for forensic characterization of Bacillus spores using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A quantitative method was developed for the determination of heme (ferriprotoporphyrin IX) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method was designed for forensic characterization of the use of blood agar in preparation of Bacillus spores. An alkali wash of 0.3 M ammonium hydroxide was used to solubilize heme from spore samples. The wash was concentrated and analyzed by MALDI-TOFMS. Experimental parameters were optimized to obtain the best signal intensity, maximize signal reproducibility, and improve day-to-day repeatability of the measurement. Sinapinic acid was found to be the best matrix. A sandwich sample preparation protocol was determined to increase the shot-to-shot and point-to-point reproducibility of the measurement. Cobalt(III) protoporphyrin was used as an internal standard and the analyte/ internal standard ratio responses from solutions of known concentrations were used to construct a calibration curve (R-2 = 0.993). Limits of detection and quantitation for heme were calculated to be similar to0.4 (200 fmol) and 0.8 muM (400 fmol), respectively. Spore samples prepared on blood agar and nonblood agar were analyzed using the method. Heme was detected at a concentration of similar to0.3 ng/mg of spore on samples prepared on blood agar and purified by extensive washing. Heme was not detected on spore samples prepared without blood.