Department of Forensic Sciences, Oslo University Hospital, Lovisenberggt. 6, Oslo 0456, Norway.
J Anal Toxicol. 2023 Mar 24;47(3):311-316. doi: 10.1093/jat/bkac099.
A comparative evaluation of two methods used for carboxyhemoglobin (COHb) determination in postmortem whole blood was performed: carbon monoxide (CO)-oximetry measuring at 128 wavelengths and headspace gas chromatography with flame ionization detection (HS-GC--FID) where CO was determined after catalytic reduction of CO to CH4 and Fe was determined by atom absorption spectrophotometry (AAS, 248.3 nm). An aliquot of 100 µL whole blood was loaded into the CO-oximetry module. In the HS-GC--FID analysis, to 1.0 mL of whole blood, 3.0 mL of saponin solution was added, mixed and centrifuged. To 20 mL HS vials, 400 µL of the supernatant was added and the vials were immediately sealed. One milliliter of potassium hexacyanoferrat (III) solution was added through the HS septum and mixed. The samples were incubated at 70°C for 5 min. CO was separated using He as carrier gas and a CP-Molsieve 5 Å PLOT capillary column. Fe was determined using 400 µL of the saponin supernatant diluted to 10 mL by water. During a period of ∼3 years, 124 postmortem whole blood samples were analyzed. Bland-Altman method comparison showed satisfactory agreement and no significant bias between the methods for the whole saturation range (5 to 85% COHb). Five samples, all with %COHb >40, showed deviations of more than 10% COHb in absolute terms. One sample, in the lower COHb range <10%, was false negative on the CO-oximetry method. The between-assay accuracy, reported as bias, at 60% COHb was -0.8% and -9.0%, and precision, reported as relative standard deviation, was 1.6% and 7.7%, for the CO-oximetry and HS-GC--FID-AAS methods, respectively. Both methods obtained satisfactory results in proficiency testing rounds, with z-scores <±2 (n = 11). This study showed that the CO-oximetry method based on the 128-wavelength principle and the HS-GC--FID-AAS method are comparable and satisfactory for %COHb determination in postmortem whole blood.
比较了两种用于检测死后全血中碳氧血红蛋白（COHb）的方法：测量 128 个波长的一氧化碳（CO）比色法和顶空气相色谱-火焰离子化检测（HS-GC--FID），其中 CO 在 CO 催化还原为 CH4 后测定，Fe 通过原子吸收分光光度法（AAS，248.3nm）测定。将 100µL 全血等分试样装入 CO 比色法模块。在 HS-GC--FID 分析中，向 1.0mL 全血中加入 3.0mL 皂素溶液，混合并离心。向 20mL 顶空瓶中加入 400µL 上清液，立即密封顶空瓶。通过顶空隔垫加入 1 毫升六氰合铁（III）钾溶液并混合。将样品在 70°C 下孵育 5 分钟。使用 He 作为载气和 CP-Molsieve 5Å PLOT 毛细管柱分离 CO。使用 400µL 皂素上清液稀释至 10mL 的水，测定 Fe。在大约 3 年的时间里，分析了 124 例死后全血样本。Bland-Altman 方法比较表明，在整个饱和度范围内（5%至 85%COHb），两种方法之间具有良好的一致性，没有显著的偏差。五个样本，所有 COHb%均>40，在绝对值上有超过 10%COHb 的偏差。一个样本在 COHb 低值<10%时，CO 比色法呈假阴性。在 COHb 为 60%时，CO 比色法和 HS-GC--FID-AAS 法的检测准确性（表示为偏差）分别为-0.8%和-9.0%，精密度（表示为相对标准偏差）分别为 1.6%和 7.7%。两种方法在能力验证试验中均取得了满意的结果，z 分数均<±2（n=11）。本研究表明，基于 128 波长原理的 CO 比色法和 HS-GC--FID-AAS 法在死后全血中 COHb 测定方面具有可比性和良好的效果。
