Analysis of Ethanol in Blood using Master SHS Static Headspace Sampler and Master GC Gas Chromatograph APPLICATION NOTE Authors: DANI Instruments SpA viale Brianza, 87 Cologno Monzese Milano Italy Key Words: Headspace Blood Alcohol Analysis HS-GC Dual Column Introduction The determination of ethanol content in blood is one of the most important analysis in forensic toxicology, either in samples from corpses, or in drivers suspected of driving under the influence; in any case, precise, reliable and defensible ethanol determination is necessary. In the US limit is higher than 0.08% or 80 mg ethanol per 100 ml of whole blood. The average BAC permitted across the majority of European countries, including Spain, Italy, and Ireland, is 0.05%, with some countries such as the United Kingdom allowing 0.08%. Limits can be more stringent depending on the age of the suspect. Blood Alcohol Content (BAC) represents one of the most important and largest volume analysis in forensic toxicology. The number of samples run in a day by a forensic lab includes specimen and quality control standards and can easily reach several hundreds. Consequentely, it is paramount that the system will be able to run many unattended analysis and safely classify and store results. Most of the forensic laboratories performing BAC analysis use different technologies among which gas chromatography may not be the main applied technology. For this reason instruments need to require minimum maintenance, easy serviceability, and safety performance. The results obtained can be used in court during a legal dispute with possible serious repercussions on people s life. Data has to be, therefore, defendable. Currently, to address all the above-mentioned needs, the headspace extraction technique is preferred due to the minimal contamination produced to the injector and column of the gas chromatograph; this technique for the determination of ethanol has been refined over time, to the extent that is now possible to perform these tests quickly and accurately. Blood is a very complex biological matrix, which includes salts, proteins, lipids and other metabolites. As such, a direct analysis of blood would result in a very frequent contamination of the GC injector, leading to frequent stops in the working routines and as such very low efficiency. Forensic Toxicology
In the present work the DANI BAC system is presented to provide fast and accurate analysis of blood alcohol content to be confidently used in legal disputes and whenever defendable data are required. System Configuration Typical system configuration is static headspace hooked to a gas chromatograph equipped with dual FID detectors. [The analysis can also be performed using one separation system (column) and one FID. In this latter case, results in term of separation, linearity, and reproducibility are usually valid. However, since this data can be used in court, it is always critical to be able to defend this data using information acquired from another chromatographic channel, used for confirmation and identification (and full separation) of the ethanol s chromatographic peak.] In the present work the analysis is performed with DANI Master SHS Static Headspace Sampler equipped with Master VH, Vial Handler with 120 positions, coupled with DANI MAster GC Fast Chromatograph. In the Master SHS the chemical inertness of the entire sample flow path, along with the uniform and constant temperature of the heated zones eliminate analytical carryover and mantain sample integrity. Improved repeatability is guaranteed by the Valve & Loop technique. Moreover, the highest sample capacity with the 120-position vial tray and the unmatched capacity of the oven to lodge 18 vials simultaneously allow sample overlapping and consequently maximize productivity. Sample throughput is also enhanced by the use of the DANI Master GC Gas Chromatograph that permits to obtain the results in a very short time. This is fundamental, as BAC analysis is a high throughput analysis which requires many different controls for each sample. Therefore, short analysis times are required and the system needs to be able to handle many unattended samples. Master GC GC Oven Temperature 35 C Time 6 min Rate 0 C/min Injector Split/Splitless Temperature 150 C Pressure 18.8 Split 1:30 Purge 5 ml/min Detector FID Temperature 300 C Columns DB-ALC1 30m, 0.32mm, 1.8um DB-ALC2 30m, 0.32mm, 1.2um Table 1: Master GC Analysis Conditions Master SHS Oven Control 70 C Manifold 90 C Transfer Line 90 C Vial Eq. Time 15 min Shaking High Pressurization mode Pressure Aux pressure 1 bar Pressure Equilibration Time 0.2 min Loop fill mode Pressure Loop pressure 0.2 bar Loop Eq. Time 0.1 min Injection TIme 0.5 Injection mode Standard Vial Venting ON Table 2: Master SHS Analysis Conditions Sample The standard mixtures for the evaluation of the linearity are Ethanol and n-propanol as Internal Standard in water at: 50, 100, 250, 500, 1000 and 2000 mg/l for Ethanol and 500 ppm for n-propanol, three repetitions for each point. Repeatability has been calucalted using the standards of Ethanol and n-propanol at 500 ppm and the Restek standard solution (1mL at 1000ppm) at 50 ppm. The volume for each sample is 0.5mL in 20mL vials.
DISCUSSION Calibration An internal standard calibration curve with 3 repetitions for point at 50, 100, 250, 500, 1000 and 2000 ppm has been calculated. Repeatability Repeatability is evaluated on 10 points at 500 ppm of Ethanol and Internal standard, n-propanol. Repeatability DB-ALC1 Repeatability DB-ALC1 Area Ethanol (mv*s) Area n-propanol (mv*s) Area Ethanol (mv*s) Area n-propanol (mv*s) 1 202.35 379.49 1 206.20 390.10 2 203.65 384.41 2 207.32 395.01 3 198.39 374.14 3 202.10 384.56 4 195.03 371.50 4 198.39 381.47 5 192.57 366.54 5 196.03 376.36 6 201.06 377.21 6 204.64 387.33 7 201.70 381.11 7 205.36 391.29 8 198.13 379.46 8 201.7 389.73 9 198.97 378.15 9 202.63 388.51 10 193.95 370.81 10 197.55 381.11 Average 198.58 376.28 Average 202.19 386.55 DEV.ST 3.75 5.44 DEV. ST 3.84 5.62 RSD% 1.89% 1.45% RSD% 1.90% 1.45%
Repeatability of Restek Standard Solution evaluated on 10 points at 50ppm and calculated with and without (U2) normalization internal standard. Repeatability ALC1 Methanol Acethaldehyde Ethanol Isopropanol Acetronile/ Acetone N Propanol IS MEK Ethyl Acetate Ethanol norm IS 1 13.12 87.84 25.93 47.14 131.78 49.05 141.91 141.11 0.53 2 12.19 91.07 24.94 46.76 133.32 47.37 147.45 149.05 0.53 3 12.40 89.88 24.90 46.44 132.17 47.25 144.70 147.09 0.53 4 12.39 90.86 24.95 46.85 133.05 47.55 146.88 147.75 0.52 5 12.39 91.77 25.90 48.08 135.95 48.97 148.30 149.34 0.53 6 12.57 92.01 25.51 47.76 135.46 48.32 148.83 149.44 0.53 7 12.58 87.96 25.35 47.44 132.97 47.96 144.40 140.53 0.53 8 12.26 88.95 24.89 46.55 131.72 46.98 144.35 144.88 0.53 9 12.28 89.20 24.94 46.74 132.41 47.30 145.04 144.97 0.53 10 12.20 89.48 24.70 46.14 131.39 46.68 144.97 145.55 0.53 MEDIA 12.49 89.89 25.20 46.99 133.02 47.74 145.68 145.97 0.53 DEV. ST 0.32 1.50 0.45 0.61 1.55 0.81 2.13 3.21 0.0015 RSD% 2.53% 1.67% 1.77% 1.30% 1.17% 1.70% 1.46% 2.20% 0.29% Repeatability ALC2 Acethaldehyde Methanol Ethanol Acetone Isopropanol Acetonitrile N PROPANOL IS Ethyl Acetate MEK Ethanol norm IS 1 90.34 13.76 26.81 86.55 50.10 47.35 50.20 145.26 150.07 0.53 2 93.662 12.80 25.62 88.45 49.47 47.06 48.40 153.60 155.36 0.53 3 92.60 13.12 25.80 87.42 49.41 46.91 48.12 151.20 153.20 0.54 4 93.63 13.03 25.87 88.17 49.76 47.12 48.61 152.59 154.83 0.53 5 94.55 13.61 26.84 89.86 51.23 48.40 49.98 153.17 157.02 0.54 6 94.69 13.18 26.32 89.72 50.56 47.94 49.35 153.66 157.25 0.53 7 90.69 13.24 26.25 87.84 50.03 47.36 49.01 144.67 152.60 0.54 8 91.61 12.94 25.70 87.26 49.22 46.71 48.01 148.76 152.85 0.54 9 91.92 12.98 25.79 87.73 49.17 46.83 48.30 148.83 153.60 0.53 10 92.10 12.80 25.54 87.09 49.05 46.47 47.71 150.07 152.77 0.54 MEDIA 92.57 13.15 26.05 88.01 49.80 47.21 48.77 150.18 153.95 0.53 DEV. ST 1.52 0.32 0.48 1.08 0.69 0.58 0.84 3.30 2.19 0.0023 RSD% 1.64% 2.44% 1.83% 1.23% 1.39% 1.23% 1.73% 2.20% 1.42% 0.42%
RESULTS The above described configuration allows very short analysis time. Analysis of Ethanol and n-propanol can be performed in less than 2 minutes as shown in Chromatogram 1 here below, and less than 6 minutes for the Standard Solution Chromatogram 1: Separation of Ethanol and n-propanol from columns DB-ALC1 and DB-ALC2 [mv] Voltage 60 50 40 30 20 10 Acetaldehy de Methanol Acetaldehy de Methanol Ethanol Ethanol Acetone Isopropan ol Isopropan ol Acetone/ Acetonitrile Acetonitril e n-propanol Ethyl acetate n-propanol MEK MEK DB-ALC1 DB-ALC2 Ethyl acetate 1 2 3 4 5 Time [min.] Chromatogram 2: Separation of all the compounds from the Standard Solution
Conclusions The number of samples run in a day by a forensic laboratory includes specimen and quality control standards and can easily reach several hundreds. It is therefore paramount that the system will be able to run many unattended analysis and safely classify and store results. DANI Master SHS, with its 120 vials positions and the unlimited number of priority samples, provides the highest samples number capacity in the market. The DANI solution for Blood Alcohol Analysis can be fed with samples without stopping the workflow, providing the ultimate throughput in the forensic laboratories. Moreover, multiple cycles can be pre-defined in a sequence and the analyst can keep adding samples without virtually stopping the workflow during the day. The Master SHS hosts up to 18 vials in the secondary oven providing overlapping of thermostatting and greatly enhancing system s throughput. The barcode allows proper confirmation and traceability of the sample. All these features provide workflow flexibility and high throughput. Considering the criticality of the analysis, accuracy is paramount. At this regard, it is useful to highlight that the DANI system is capable to automatically run suitability tests and provide pass/fail reports that are very useful to understand the status of the system and facilitate compliance adherence. Furthermore, the DANI headspace not only uses a thermostatted loop, but also allows to control pressure in the injection loop greatly enhancing the precision and the accuracy of sample amount injected. The use of electronically controlled pneumatics allows indeed obtaining high precision in the injection volume and, as such, high precision in the peak area results. This is fundamental to obtain accurate and precise results. Moreover, the analytical path is fully inert to avoid secondary interactions with specific solvents that might affect precision and accuracy. www.dani-instruments.com The contents of this publication are for reference and illustrative purposes only. Information, descriptions and specification are subjected to change without notice. DANI Instruments assumes no responsability and will not be liable for any errors or omissions contained herein or for incidental, consequential damages or losses in connection with the furnishing, performance, or use of this material. AN_166