Driving under the influence of nitrous oxide A retrospective study of HS-GC-MS analysis in whole blood

Since 2020, our lab has received blood samples from traffic cases involving suspicion of driving under the influence of nitrous oxide (N 2 O). While N 2 O analysis by gas chromatography (GC) has been around for decades, quantitative results in blood from drivers have been only scarcely reported. We present a three-year (2020 – 2022) retrospective study of N 2 O from traffic cases in Eastern Denmark with suspected involvement of N 2 O intake. Whole blood samples from traffic cases were analysed for N 2 O using headspace-GC-MS. Freshly made calibration curves and additions of xenon gas as an internal standard were used for calculation of N 2 O concentrations. Positive samples have been defined as having concentrations greater than 0.1 mL N 2 O/L blood. Over a three-year period, we have tested 62 traffic case blood samples for the presence of N 2 O. Despite the technical challenges associated with the analysis of N 2 O, we have found N 2 O in 52 of the samples. Calculated concentrations were in the range 0.1 – 48 mL N 2 O/L blood, which are similar to the few cases previously found in the literature.


Introduction
Nitrous oxide (N 2 O) is an inert gas used medicinally as an analgetic and anaesthetic agent in dentistry and childbirth, and in culinary applications as a bacteriostatic, non-oxidising propellant for whipped cream and as a ripening agent for fruit [1][2][3].Cartridges and cylinders containing food grade N 2 O are readily and legally available in most of the world.Subanaesthetic concentrations of the gas can produce euphoria, mood changes, altered body awareness and relaxation, as well as dizziness, disorientation, fainting and loss of coordination and balance [4,5].Recreational abuse is common within the younger population, with 22.5 % of the 2021 Global Drug Survey respondents having used N 2 O recreationally at least once within their lifetime [6].Abuse idealization on social media and low risk perception further contributes to the prevalence of abuse amongst adolescents [7].As the popularity of recreational N 2 O rises, so does the littering of cartridges and cylinders on the streets and around highway exits, indicating frequent abuse in the traffic [5].Abuse of N 2 O related to impaired driving is more and more frequently being reported by national and international news media [8][9][10][11][12][13][14].Many media reports address a lack of on-site or laboratory testing for N 2 O to confirm the abuse.
N 2 O exposure and/or abuse is in general cumbersome to measure, as the physicochemical properties of the gas imply a rapid onset and elimination from the body.Occupational exposure of N 2 O in health care personnel has previously been measured in both urine, blood, and exhaled air by use of gas chromatography (GC) coupled with electron capture detection [15,16].Lethal intoxication cases have likewise been confirmed through GC analysis of post-mortem blood, brain, and lung tissue [17][18][19].
To our knowledge, only two reports with quantification of N 2 O in blood from drivers currently exist, presenting a total of five cases [18,20].We present a retrospective study of results from GC analysis on blood samples from Danish traffic cases with suspicion of driving under the influence of N 2 O during a three-year period, from 2020 to 2022.

Retrospective data
Data from all performed tests on traffic case samples with special request for N 2 O testing in Eastern Denmark in the period from January 1st 2020 to December 31st 2022 were extracted from the laboratory information management system (LIMS).Data included basic case information (police district, basic demographic information about the driver, date of sample receipt) and forensic toxicological findings (performed tests, quantitative results for N 2 O, and quantitative results for all other compounds listed on the final toxicological report).

Samples
Blood samples were provided by the police with requests for forensic analysis.Samples were drawn by trained medical staff and provided in 5.0 mL Vacutainer® tubes prefilled with 12.5 mg sodium fluoride and 10 mg potassium oxalate (Becton Dickinson, Oxford, UK).Samples were delivered to the laboratory within a few days and stored at -20 ̊C or colder until analysis.

N 2 O analysis
N 2 O of medical grade for preparation of calibration standards was purchased from Hydrogas Danmark (Fredericia, Denmark), while N 2 O for the preparation of quality control (QC) samples was purchased from Fluka (Buchs, Switzerland).Xenon gas was purchased from Sigma-Aldrich (St. Louis, USA).Filtered deionized water was obtained using an in-house Milli-Q unit (Millepore, Bedford, MA, USA) and blank human blood was obtained from the local blood bank.
The analytical method was adapted from Giuliani et al. [17].Analysis was performed on an Agilent (Santa Clara, CA, USA) system comprising a 7697A headspace sampler (HS), a 7890A gas chromatograph (GC) and a 5975C mass spectrometer (MS).Aliquots of 500 µL sample were transferred to 20 mL headspace vials (Agilent, Santa Clara, CA, USA) and immediately closed with PTFE/BUTYL aluminum crimp caps (PerkinElmer, Waltham, MA, USA).To each vial, 3 µL of pure xenon gas was added as an internal standard through the septum using a gastight syringe (Trajan, Victoria, Australia).The vials were equilibrated at 60 • C for 20 min before injection onto a HP-PLOT/Q column (15 m × 0.32 mm).The injector was set to split mode (10:1) with a split flow of app.60 mL/min.The temperature was programmed to start at 35 • C for 1 min before raising, first to 95 • C at 30 • C/min and then to 240 • C at 50 • C/min and being held at 240 • C for 2 min.
A series of aqueous calibration standards were prepared fresh for each analytical sample batch by adding internal standard and N 2 O using separate gastight syringes to closed headspace vials.The calibrators had final N 2 O concentrations of 0.1, 0.2, 1.0 and 10 mL/L.The N 2 O concentration was calculated by comparing the N 2 O/xenon response ratio to the calibration curve.Integrated SIM m/z 30 and 129 responses were used for N 2 O and xenon, respectively.Samples were analysed in duplicate and considered positive and reported as "detected" at mean measured concentrations above 0.1 mL N 2 O/L blood.Spiked quality control samples were analysed before and after the samples to allow for the detection of any batch non-conformances.

Results & discussion
In the 2020-2022 period, our lab has received 62 traffic case blood samples with a request for N 2 O analysis.Of the 62 N 2 O traffic cases, 52 (84 %) were positive for N 2 O; concentrations in positive samples ranged from 0.1 to 48 mL N 2 O/L blood.The distribution of the measured N 2 O concentrations was skewed towards the lower range, as shown in Fig. 1.
The analytical method has proved to be effective in detecting N 2 O in regular whole blood samples without the need for any special or extended sample preparation.The chromatogram of a spiked blood sample is shown in Fig. 2.
The results presented here are quantitative; the N 2 O concentration in each sample has been calculated using a freshly made calibration curve, the addition of internal standard and evaluation of quality controls.The reporting is however done qualitatively, as the measured value is likely only a small fraction of the actual concentration in the moment of police apprehension because of the biological half-life being in the range of minutes.The magnitude of the total pre-analytical loss of N 2 O from physiological elimination, sample storage and opening of the vials for sample transfer is yet unknown.
Our threshold for reporting a case as positive is set to 0.1 mL/L blood, which is an approximately 10 times higher response than obtained from atmospheric levels of N 2 O (Fig. 3), and thus indicates a recent exposure to the gas.Use of the m/z 30 signal for N 2 O quantification, corresponding to the NO fragment, eliminated interference from the high concentrations of CO 2 in the blood samples.While the overall response is lower at m/z 30 than at m/z 44, the atmospheric levels of N 2 O still produces clearly visible peaks (Fig. 3).
Our results are similar to the previously reported quantifications of N 2 O in ante-mortem traffic cases; D'Orazio et al. measured 2.3-8.0 µg/ mL (equivalent to app.1.4 -5.0 mL N 2 O/L blood) from four cases [20].Concentrations are generally lower for ante-mortem cases compared to post-mortem cases [20], with Wagner et al. measuring 14.6 mg/dL (equivalent to app. 90 mL N 2 O/L blood) from the deceased driver responsible for a car crash [18].This difference between case types was expected, as the N 2 O concentration in living individuals will decrease by exhalation during any transport and waiting time experienced before a blood sample can be drawn [28].In contrast to this, N 2 O detection has been demonstrated in tissues up to 31 days after asphyxial deaths [19].
were positive.52 cases were tested for traffic-impairing drugs, of which 26 (50 %) were positive, with concentrations of compounds above the legal limit.Detected drugs mainly included tetrahydrocannabinol (n = 13), alprazolam (n = 7) and cocaine (n = 6), but tramadol, benzoylecgonine, amphetamine, fentanyl, oxazepam, gammahydroxybutyrate (GHB), and ketamine were also found.Concomitant intake of other drugs of abuse are common with N 2 O, as it can heighten or alter the user experience [7].
Samples were primarily received from the Greater Copenhagen area (60 cases, 97 %) with a few cases from the Rural Zealand area (2 cases, 3 %).The number of samples has been rising during the period, with more than 66 % being received in 2022 alone.This trend does not necessarily represent an increase in the number of N 2 O affected drivers in Danish traffic over the period; the analysis of N 2 O was only performed when the police suspected a driver of recent intake of N 2 O. On the other hand, the police has reported increased findings of empty containers at the roadside in the same period, according to local news.
High rates of N 2 O traffic cases are also seen in the Netherlands, although without the forensic confirmation from laboratory analysis.According to police sources cited by the Dutch youth road safety organization TeamAlert, N 2 O abuse was involved in almost 1800 Dutch traffic accidents from mid-2019 to the end of 2021 [29].Denmark and the Netherlands are in many ways comparable traffic-wise, with similar regulations, road conditions and percentages of drivers aged 18-24 years who were killed in road traffic in 2016 compared to their percentages of the total population [30].Male drivers accounted for most (57 cases, 92 %) of our cases, while few drivers (5 cases, 8 %) were female.Ages ranged from 15 to 37 years, with a median age of 22.This demographic profile is similar to that of impaired drivers previously documented in Eastern Denmark, though notably younger [31].This is consistent with the apparent popularity among adolescents and students.

Conclusion
Recent intake of N 2 O has been confirmed in 52 out of 62 blood samples from apprehended drivers analysed for N 2 O from Eastern Denmark in 2020-2022.Measured concentrations of N 2 O in blood ranged from 0.1 to 48 mL/L blood.Concomitant intake of other drivingimpairing drugs and alcohol was confirmed for 26 and 3 of the cases, respectively.This study shows that detection of N 2 O in whole blood samples is possible by HS-GC-MS with a minimum of sample prep.

Fig. 1 .
Fig. 1.Distribution of N 2 O concentrations in the 52 positive samples.

Fig. 2 .
Fig. 2. Full chromatogram of a blind blood sample spiked to 1.3 mL N 2 O/ L blood.

Fig. 3 .
Fig. 3. Chromatograms taken at m/z 30.A, double blank sample containing purified water and atmospheric air.B, negative authentic blood sample.C, positive authentic blood sample with calculated concentration of 0.12 mL N 2 O/L blood.