Quantitative Determination of Heme for Forensic Characterization of Bacillus Spores Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 76; no. 10; pp. 2836 - 2841
• Main Authors: Whiteaker, Jeffrey R., Fenselau, Catherine C., Fetterolf, Dean, Steele, Darin, Wilson, David
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-05-2004
• Subjects: Agar - chemistry; Analytical chemistry; Bacillus; Chemistry; Coumaric Acids - chemistry; Exact sciences and technology; Forensic hematology; Forensic Medicine - methods; Heme - analysis; Heme - chemistry; Hemin - analysis; Hemin - chemistry; Humans; Lasers; Mass spectrometry; Plasma - chemistry; Reference Values; Reproducibility of Results; Research methodology; Spectrometric and optical methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - instrumentation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Spores, Bacterial - chemistry; Forensic science; Internal standard; Agar; Matrix assisted laser desorption ionization; Bacillaceae; Optimization; Blood; Spores; Ammonium hydroxide; Repeatability; Protoporphyrin; Sample preparation; Bacillales; Time of flight method; Reproducibility; Detection limit; Bacillus; Heme; Standard curve; Bacteria; Mass spectrometry; Cobalt compound; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac034959r

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 ∼0.4 (200 fmol) and 0.8 μM (400 fmol), respectively. Spore samples prepared on blood agar and nonblood agar were analyzed using the method. Heme was detected at a concentration of ∼0.3 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.