A new and sensitive method for quantitative determination of helium in human blood by gas chromatography–mass spectrometry using naturally existing neon-21 as internal standard

Forensic Toxicology https://doi.org/10.1007/s11419-018-0437-6 ORIGINAL ARTICLE A new and sensitive method for quantitative determination of helium in human blood by gas chromatography–mass spectrometry using naturally existing neon‑21 as internal standard Akira Tsujita1,2 · Hidehiko Okazaki1 · Asami Nagasaka1 · Akinaga Gohda1 · Mitsushi Matsumoto1 · Toshiro Matsui2 Received: 18 May 2018 / Accepted: 28 July 2018 © The Author(s) 2018 Abstract Purpose In this study, we proposed a new sensitive quantitative method for detecting helium in human blood by gas chromatography–selected-ion monitoring (SIM)-mass spectrometry (GC–SIM-MS) using naturally existing neon-21 in air as internal standard (IS). Methods GC–SIM-MS analysis was performed on a double TC-Molsieve 5A capillary column (total length 60 m) for the separation of permanent gases by a single-run experiment. By using hydrogen as the carrier gas, the analyte (helium) and IS (neon-21) were separated on the double column, and detected at m/z 4 and 21, respectively. The ratio of the peak area of helium-to-neon-21 was used for obtaining the calibration curve for helium determination. Results The limits of detection and quantification of helium under the present GC–SIM-MS conditions were as low as 1.8 and 6.0 ppm, respectively. The proposed GC–SIM-MS method also showed high repeatability with relative standard deviation at 1.3–5.1%, indicating that the use of neon-21 as IS was valid for reliable helium assays. The successful quantification of helium in the headspace of vacuum blood collection tubes containing the whole blood from four humans who died of helium inhalation was achieved using the proposed neon-21-aided GC–SIM-MS method; the values obtained for helium were 24–497 ppm. Conclusions The proposed GC–SIM-MS method in combination with the naturally existing neon-21 as IS is most recommendable for quantitative assays of helium in biological samples because of its simplicity and extremely high sensitivity. Keywords Helium analysis · Neon-21 as internal standard · Whole blood · GC–MS in SIM mode · Asphyxia · Inhalation suicide Introduction Helium is considered as an inert gas that has no flammability. It has been reported that deaths due to suffocation by excess helium inhalation are becoming an increasing serious social issue [1–7]. However, the lack of an appropriate analytical assay for the helium in the biological samples results * Akira Tsujita 1 Forensic Science Laboratory, Fukuoka Prefectural Police Headquarters, 7‑7 Higashikoen, Fukuoka 812‑8576, Japan 2 Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, 744 Motooka, Fukuoka 819‑0395, Japan in the difficulty for clarifying the unequivocal cause of death in the helium-related cases. Gas chromatography–thermal conductivity detection (GC–TCD) has been commonly used as a convenient method for helium assays so far. Some studies have reported the detection of helium accumulated in the organs (lung, stomach, brain, liver, and trachea) and blood by GC–TCD [7–11]. However, the GC–TCD method suffers from poor sensitivity and selectivity for helium detection [12]. Although headspace analysis of the helium in the blood during autopsy may solve the problem of insufficient detection [8, 9], it still suffers from poor reproducibility for the helium assay due to the unstable volatility of the helium found in the blood matrix. Norimine et al. [10] improved the poor GC–TCD detection of helium by increasing the helium volatility in the headspace using a reduced-pressured vial. To date, gas chromatography–mass spectrometry (GC–MS) has been 13 Vol.:(0123456789) Forensic Toxicology extensively used for the selective detection of helium in biological samples including the lungs, stomach, trachea, and blood. Auwaerter et al. [13] and Musshoff et al. [14] qualitatively assayed helium using nitrogen and hydrogen as carrier gases, respectively, on a nonpolar capillary column by the selected-ion monitoring (SIM) GC–MS technique. Alternatively, Malbranque et al. [15] proposed a quantitative GC–SIM-MS assay of the helium in the organs with the aid of an external standard (nitrous oxide; N 2O). Although the use of N 2O as a standard in GC–SIM-MS analysis might be useful for quantitative helium assay, it required a mixing of the headspace gas with N2O in another vial owing to its high solubility in biological samples, causing difficulty in direct helium assay in the target headspace samples. Because of the aforementioned disadvantages of the reported GC–SIM-MS methods for complex biological samples, in this study, we tried to develop an internal standard (IS)-aided GC–SIM-MS method for quantitative helium assays. Blood was targeted for the present assay, since the blood of the deceased is the first priority matrix for judging the asphyxiation by helium inhalation. The application of the neon-21 naturally existing in air (0.049 ppm [16, 17]) as the IS, but not as the external standard, to the present GC–SIMMS assay is described in this article. Materials and methods Materials Helium (99.995% purity), argon (99.995%), and nitrogen (99.995%) were purchased from Taiyo Nissan Co. (Osaka, Japan). Standard helium gas at a concentration of 100 ppm in nitrogen was obtained from GL Sciences (Tokyo, Japan). Helium with concentrations of 10–1000 ppm were prepared by diluting pure helium with air in an aluminum bag (1 L, GL Sciences) using a gas-tight syringe (SGE Analytical Science, Melbourne, Australia). The sample gas (1.0 mL) was injected into a GC–SIM-MS system with a gas-tight syringe fitted with a push-pull valve (1 mL-volume of syringe, SGE Analytical Science). The commercially available human whole blood was purchased from Cosmo Bio Co. (No. 12081545; Tokyo, Japan). GC–SIM‑MS analysis GC–SIM-MS analysis was carried out on a Shimadzu GC–MS QP2010plus (Shimadzu, Kyoto, Japan). A TCMolsieve 5A capillary column (30 m × 0.32 mm i.d., film thickness 30 μm, GL Sciences) was used for the separation. In this study, either a single (Fig. 1a) or double column (two connected TC-Molsieve 5A capillary columns of total length 60 m; Fig. 1b) was used for the GC–SIM-MS analysis. In 13 single-column assay, a particle trap (2.5 m × 0.32 mm, GL Sciences) that prevents the mass detector from contamination was connected to the analytical column on the detector side using a capillary mini-union (GL Sciences; Fig. 1a). In double-column assay, two TC-Molsieve 5A capillary columns and the particle trap were connected in series (Fig. 1b). The GC conditions were as follows, except for the carrier gas: column temperature, 34 °C; split ratio, 2:1; purge flow rate, 3.0 mL/min; and injection temperature, 35 °C. The MS conditions were as follows: electron ionization (EI) mode; detector gain, 1.0 kV; ionization voltage, 70 eV; emission current, 150 µA; interface temperature, 150 °C; ion source temperature, 200 °C; monitoring ions, m/z 4 and 21 in the SIM (...truncated)