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
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).
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.
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.
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.
KeywordsHelium analysis Neon-21 as internal standard Whole blood GC–MS in SIM mode Asphyxia Inhalation suicide
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, 2, 3, 4, 5, 6, 7]. However, the lack of an appropriate analytical assay for the helium in the biological samples results 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, 8, 9, 10, 11]. However, the GC–TCD method suffers from poor sensitivity and selectivity for helium detection . 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.  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 extensively used for the selective detection of helium in biological samples including the lungs, stomach, trachea, and blood. Auwaerter et al.  and Musshoff et al.  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.  proposed a quantitative GC–SIM-MS assay of the helium in the organs with the aid of an external standard (nitrous oxide; N2O). Although the use of N2O 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–SIM-MS assay is described in this article.
Materials and methods
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).
Hydrogen, argon, and nitrogen were used as the carrier gases in this study. Pure hydrogen (99.9999%) was supplied by a hydrogen generator NMH-100 (Air Tech, Yokohama, Japan), and the column flow rate was set at 36 cm/s.
Quantitative neon-21-aided GC–SIM-MS analysis of the helium in whole blood specimens
For quantitative neon-21-aided GC–SIM-MS analysis of helium in whole blood, the whole blood (ca. 2.0–8.9 mL) taken from the heart of the deceased was transferred to a 10-mL vacuum blood collection tube containing sodium heparin (Terumo Co., Tokyo, Japan) and refrigerated at 4 °C until analysis. Prior to the analysis, the blood tube was stood for 30 min at 25 °C. After achieving the equilibrium of the headspace phase at 25 °C, 1.0 mL of the headspace was injected into the GC–SIM-MS system. For neon-21-aided GC–SIM-MS analysis as part of the calibration experiments, helium gas samples diluted with air were used; because air contains a constant concentration of neon-21 (0.049 ppm), the air dilution method indicates that the fixed amount of neon-21 can be automatically spiked into the headspace as IS. The commercially available blood sample was used as the blank specimen.
Results and discussion
Optimization of the GC–SIM-MS conditions for helium quantification
In order to separate and detect inert helium gas by GC–SIM-MS using a single column, a capillary GC column, TC-Molsieve 5A, which was suitable for inert gas retention , was used. A middle-bore column with a 0.32-mm i.d. was used for the present headspace GC analysis. The single-column-attached GC–SIM-MS system (Fig. 1a) allowed the detection of helium (100 ppm in air) at a retention time of 0.9 min at m/z 4 (Fig. 1c). However, an unstable baseline obtained in the GC–MS conditions may result in less reproducible detection of helium. It seems likely that the variable pressure observed in the capillary column and/or reduced vacuum pressure in the MS system owing to the low viscosity of the carrier gas hydrogen (at 65 cm/s) might be associated with the unstable baseline behavior. To overcome this problem, a double-column separation system using two TC-Molsieve 5A capillary columns (total 60 m × 0.32 mm) was applied for helium detection (Fig. 1b). As shown in Fig. 1d, stable baseline behavior, together with the longer retention of helium at 3.2 min, was achieved using the present GC–MS method, probably because of the increased back-pressure in the double-capillary columns. In addition, the double-column separation system improved the peak shape of helium for quantitative assays.
Reliability of the method
Naturally existing neon is present in air at a concentration of 18.18 ppm , including the stable isotopes neon-20, neon-21, and neon-22, with natural isotopic ratios of 0.9048, 0.0027, and 0.0925, respectively . In this study, neon-21 was selected as the appropriate IS among the neon isotopes because of its abundance in air that was compatible to the peak intensities of the target helium that ranged from 10 to 1000 ppm (Fig. 5). In addition, reproducible peak detection of neon-21 [peak area units: 70,834 ± 639; n = 7; relative standard deviation (RSD), 0.9%] supported the beneficial use of neon-21 as IS.
Intraday and interday repeatabilities of the present method in whole blood specimens
Concentration found (ppm)a
Repeatability (% RSD)b
Concentration found (ppm)c
Repeatability (% RSD)
Low concentration (100 ppm)
106 ± 4.5
105 ± 5.4
High concentration (500 ppm)
506 ± 8.8
506 ± 6.8
Application of the method to the quantitative analysis of helium in the blood of deceased humans
In the other three cases, the concentration of helium was determined to be 90, 138, and 497 ppm. The three blood samples of the deceased were obtained at day 1 or a few days after death from the heart postmortem. Each sample was refrigerated at 4 °C and assayed about 20–100 days after sample collection.
The helium level of ≥ 24 ppm in the blood of the helium-suffocated deceased was in the range of the reported levels of 27 ppm  and 400 ppm . According to Henry’s law , the ratio of helium-to-neon-21 in the headspace must be matched with that in the blood matrix for any reduced pressure of the vacuum blood collection tube, because both the inert targets were rapidly equilibrated between the headspace and blood matrix. Considering the method and results together, the proposed GC–SIM-MS method with the aid of neon-21 as the IS seems valid for quantitative helium assays in blood. In addition, the use of neon-21 showing a constant blood level by natural breath must be of great benefit in forensic science, because the immersed helium-to-neon-21 ratio in the tube is fixed at the time of blood sampling; then, the ratio in the vacuum tube is constant, without any influence of the storage conditions such as period and temperature.
In this study, we developed a novel quantitative blood helium assay method by GC–SIM-MS with the aid of neon-21 as IS. In order to determine both helium and neon-21 content, the GC–SIM-MS system was equipped with a double-TC-Molsieve 5A capillary column system. Under the optimized conditions, with hydrogen as the carrier gas at a flow rate of 36 cm/s and a 1.0-kV detector voltage, a highly sensitive and reproducible helium assay could be achieved, with an LOD of 1.8 ppm and RSD not greater than 5.1%. The amount of helium in the headspace of the vacuum blood collection tube containing each whole blood of the four deceased after helium inhalation was successfully determined to be 24–497 ppm based on the present method. It was thus concluded that the GC–SIM-MS method in combination with the naturally existing neon-21 as IS may be extensively applicable to the quantitative assays of stable gases in biological samples, without tedious procedures.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors. The analyses of the toxic substance(s) in the whole blood specimens collected from the deceased were requested by judicial authorities through official documentation.
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