A quantitative method was developed for the determination of heme (ferriprotoporphyrin IX) using matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOFMS). The method was designedfor forensic characterization of the use of blood agar inpreparation of
Bacillus spores. An alkali wash of 0.3 Mammonium hydroxide was used to solubilize heme fromspore samples. The wash was concentrated and analyzedby MALDI-TOFMS. Experimental parameters were optimized to obtain the best signal intensity, maximize signalreproducibility, and improve day-to-day repeatability of themeasurement. Sinapinic acid was found to be the bestmatrix. A sandwich sample preparation protocol wasdetermined to increase the shot-to-shot and point-to-pointreproducibility of the measurement. Cobalt(III) protoporphyrin was used as an internal standard and the analyte/internal standard ratio responses from solutions of knownconcentrations were used to construct a calibration curve(
R2 = 0.993). Limits of detection and quantitation forheme were calculated to be ~0.4 (200 fmol) and 0.8
M(400 fmol), respectively. Spore samples prepared onblood agar and nonblood agar were analyzed using themethod. Heme was detected at a concentration of ~0.3ng/mg of spore on samples prepared on blood agar andpurified by extensive washing. Heme was not detected onspore samples prepared without blood.