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FUNDAMENTAL PRINCIPLE OF ALCOHOL ANALYSIS USING HS-GC-FID INSTRUMENT AS A PROTOTYPICAL METHOD THAT UTILIZES FOR ROUTINE WORK IN FORENSIC TOXICOLOGY
JWAN HASAN HUSSEIN, 2ALI H. DOSKY
lUniversity visiting lecturer and at Duhok Polytechnic University Research at the school of medicine, dentistry & nursing, University of Glasgow, Scotland Email: [email protected] Phone number: 07504933346 2Assistant Prof-Biochemistry Department of Medical Chemistry, College of Medicine, University of Duhok Corresponding author: Ali Hussein Dosky Email: [email protected] Phone number: 07504458584
Abstract Background
Gas chromatography (GC) is a significant method used widely to identify and quantify different types of analytes. The combination of high ability of separation power in GC with various types of detectors makes this method an important) .Gas chromatography is greatly used for volatile compounds that losses the analyte during sample preparation, have low molecular mass vapor pressure (under) less than 250 0C and decompose at 400°CThe aim of this theoretical and practical laboratory is to introduce fundamental principle of HS-GC-FID instrument as a prototypical method that utilized for routine work in forensic toxicology. Objective
• The aim of this theoretical and practical laboratory is to introduce fundamental principle of HS-GC-FID instrument as a prototypical method that utilized for routine
• Understand the foundation of headspace gas chromatography, explain the function of each major part in the instrument and determine the concentration of ethanol in blood as it is important factor in forensic toxicology using data obtained from analysis.
Method
Preparing calibration in different concentration (10,25,50,100,200,300,400) mg/dl, three controls 30, 80 and 300mg/dl and unknown sample. Each have DRF number by using compudil 300 diluter hook and tucker instrument to dilute 200mgl of calibrators, control and sample unknown with 500mgl of internal standard(n-propanol150mg/dl) and put in headspace vials then seal vial after that heat and shake vials to allow internal standard and any ethanol present to equilibrate between sample and headspace. Now the calibrators, controls and sample are ready to transfer to GC instrument CLARUS 500 Cofuigured with FID ionization and Turbomatrix 110 headspace sampler. The heater is 500 C inside the turbo and 150C is the oven temperature CALRUS. The sample stay minutes in the heater then injected to oven at 150C and the temperature is constant while all samples are going through column. Results
Shows that two out of three controls are within the range are lying between +2SD when compared with QC chart in the appendix and just control 80 is out of range due to consult technical manager. The R2 value from the graph (3) is 0.9999, according to criteria of acceptability is acceptable. In case of road traffic the CV for controls 80 and 300 and for the sample unknown is 0.8579,1.4217 and 0.7156 respectively are valid due to criteria of acceptability. Conclusion
The investigation of the unknown blood sample 13/008 that determined by GC-HS-FID have two
detectors each detector give result and both results tested positive for alcohol and the dependable result was from combine both detectors to ensure the result and that also tested positive for alcohol.
Key words: alcohol analysis, HS-GC-FID instrument, Gas chromatography
INTRODUCTION
Gas chromatography (GC) is a significant method used widely to identify and quantify different types of analytes. The combination of high ability of separation power in GC with various types of detectors makes this method an important (1) •
Gas chromatography is greatly used for volatile compounds that losses the analyte during sample preparation, have low molecular mass (2)> vapor pressure (under) less than 250 0C (3) and decompose at 4000C(4) .
Archer J. P. Martine and Richard L .M. Synge had a big role in GC development, when they anticipated that mobile phase could be gas instead of liquid and in this case the separation is notably shorter and the columns much more efficient. The first paper was published about gas chromatography in 1951 by Marrine and James (3). Traditionally, the identification of components based on peak retention time, while nowadays base on the nature of response get from detectors. The main target of analyst are to make compound appear in a distinct peak or band without overlap with other components, and the other target is to make these bands in one shape and narrow. These goals are achieved by careful choice of stationary phase column and by optimising the functioning conditions of the column. Furthermore, the introduction method of sample into the chromatograph, suitable type of detector and improve the volatility by chemical compound for modification, all these factors play role to changing a second-rate analysis into a first-class one (4) .
The stationary phase in gas chromatography is a solid or polymeric liquid and called GSC and GLC respectively and the second one is the most popular. The most significant difference in GC with other method like HPLC is in the mobile phase, here mobile phase is gas as Martine and Synge suggested also called carrier gas to transport the analytes through the columns. Since Marrine and James work there have been many development in GC method as ability to separate complex sample of volatile analytes. The affinity of stationary phase with analyte and vapor pressure select the rate of partitioning Kc=[CS]/[CM]
Cs = concentration of analyte in the stationary phase, Cm = concentration of analyte in the mobile phase. Large KC effect to the analyte and have longer retention time, column temperature and chemical nature of stationary phase can control the KC (distribution coefficient) (3). structure of Gas chromatography
Gas Inlet: the main purpose is to filter gases to make sure of high purity of gas. Types of gases required in GC are carrier gas (N2He,H2),make up gas(N2He,H2)and detector fuel gas(Air,Ar,H2,Ar-CH4,N2).
Pneumatic controls: used to regulate the pressure or flow by regulate the gas inter into the instrument.
Injector: to inject the sample into the instrument and include many inlet types as the sample is volatilized to avoid decomposition such as split/pplitless,cool-on-column and programmed thermal vaporizing.
Columns: the column is responsible of separating analytes. Columns have different length and diameter, for packed column the standard dimension is 1.5m*4mm and for capillary column is 30m*0.32mm*0.1mm film thickness coated with immobilized liquid stationary and hallow(spread)with silica.
Column oven: is the responsible of temperature in the GC process and it heat to give good control and it is steady temperature during the GC process.
Detectors: from the physicochemical property of the analyte, the detectors can respond and by enlarge this respond can create an electronic signal to produce a chromatogram in data system, there are many types of detectors, such as Flame Ionization (FID), Flame Photometric (FPD), Electron
Capture (ECD), Mass Spectrometer (MS), Nitrogen Phosphorous (NPD) and Thermal Conductivity (TCD), the use of it is depend on application whether qualitative or quantitative data is needed. Data system: is final steps of the process and give result that received from detectors as signal and digitizes it to form chromatogram.(3) Headspace
Headspace gas chromatography (HS-GC-FID) is typical method has been used for identify and quantify alcohol in blood in forensic toxicology for many years (5) , the principle of headspace is depend on the release of volatile from sample into close space above the sample called headspace(6) figurel. Headspace gas chromatography is considered prototypical method for ethanol analysis because of the volatility, no need for extraction, sensitivity, specificity, accuracy and ease of automation (5). in general HS is a suitable technique for sample has very light volatiles, is mostly used for complex matrices that can be placed directly to HS (7)- An advantage of HS technique is that these analyses can be detected without interference'8' .
This method consists of gas chromatography technique with static headspace instrument. Usually use FID detectors type.
CG
®
CD
Figure 1(8 'principle of static headspace-gas chromatography.(A)equilibration and (B) sample transfer. GC=carrier gas, SV=sample vial, TH=thermostat, COL=GC column, D=detector.
Method
Using internal standard in GC make it the preferable method in forensic for testing drugs and alcohol (3). The internal standard is a drug has the same chemical characteristics to the analyte drug and it adds to a sample at beginning steps before prepared so the internal standard IS has the same for preparing and analyzing like a sample drug. The signal produce by IS and sample compare to each other to help to quantify the analyte. The ideal IS is a deuterated drug (9).
Preparing calibration in different concentration (10,25,50,100,200,300,400) mg/dl, three controls 30, 80 and 300mg/dl and unknown sample. Each have DRF number by using compudil 300 diluter hook and tucker instrument to dilute 200mgl of calibrators, control and sample unknown with 500mgl of internal standard(n-propanol150mg/dl) and put in headspace vials then seal vial after that heat and shake vials to allow internal standard and any ethanol present to equilibrate between sample and headspace figure 2. Now the calibrators, controls and sample are ready to transfer to GC instrument CLARUS 500 Cofuigured with FID ionization and Turbomatrix 110 headspace sampler. The heater is 50 0 C inside the turbo and 150C is the oven temperature CALRUS. The sample stay minutes in the heater then injected to oven at 150C and the temperature is constant while all samples are going through column. Material and Equipment (6)
Table 1 material and equipment
Materials Description
GC Instrument CLARUS 500 Configured with FID ionization
Headspace Sampler Turbomatrix 110
Diluter Compudil 300 hook and tucker instrument
Standards and controls Concentration Mg/dl Lot number Expire date
CAL 1 10 FN080612-04 Checked
CAL 2 25 FN081712-01 Checked
CAL 3 50 FN010912-01 Checked
CAL 4 80 FN042808-02 Checked
CAL 5 100 FN050312-01 Checked
CAL 6 200 FN032712-01 Checked
CAL 7 300 FN121510-01 Checked
CAL 8 400 FN040909-01 Checked
Control 30 02835-4 Checked
Control 80 10844-3 Checked
Control 300 CB547-1 Checked
N. Propanol IS 150 Checked
Unknown sample b l o o d s ample with an unknown concentration
Methanol solution to wash/clean the Compudil diluter
Sample, dillution solvent and matrix modifier Figure 2(7) Phases of the headspace vial
Criteria of acceptability (lecture)
• R2 is at least 0.99
• Control Minimum of 2 QCs must be within 2 SD for both left and right detectors. If not, consult technical manager.
• %CV (RT < 2 %, PM < 10 %).
• For RT If the result < 100 mg/dl, 6 mg/dl will be subtracted. If the result > 100 mg/dl, 6% will be subtracted, because of uncertainties of machine.
Result and Discussion
Results for right detector, table 1 provides the data of right detector for calibrations and table 2 provide result for controls and sample. The concentration of controls that is determined by the equation from graph (3)
y = 0.0046x - 0.0026 , y=PAR , X=concentration
Shows that two out of three controls are within the range are lying between +2SD when compared with QC chart in the appendix and just control 80 is out of range due to consult technical manager. The R2 value from the graph (3) is 0.9999, according to criteria of acceptability is acceptable. In case of road traffic the CV for controls 80 and 300 and for the sample unknown is 0.8579,1.4217 and 0.7156 respectively are valid due to criteria of acceptability, while the CV for control 30 is high therefore is not pass criteria of acceptability, the reason mostly for CV high refer to pipetting, sample poorly homogenised, and manufacture error, nevertheless, this criterion for other controls and sample for criteria passed, so this outlier did not influence the validation of the result because there were still to other control lying within the range. In road traffic case the cutoff is 80mg/dl(10)and the sample is 70.21mg/dl after subtracted therefore is not criminal offence and did not break any law. In case of post-mortem the all CVs for control and sample are pass criteria of acceptability because is less than limit of 10%.
Table 2 data for right detector calibration
Level mg/dl Mean PA Mean IS PAR
BLK 0
CAL 1 10 1064391.5 24781412.5 0.0429512
CAL 2 25 2799729.5 24870751 0.11257117
CAL 3 50 5747005 25258306.5 0.22752931
CAL 4 80 9157578 25093258 0.36494177
CAL 5 100 11399661.5 24947342 0.45694894
CAL 6 200 23340450.5 25135710 0.92857733
CAL 7 300 34647549.5 25180648 1.37595941
CAL 8 400 46371807.5 25177431 1.8418006
Table 3 result for controls and sample for right detector
Sample mg/dl PAR CV SD
CON 30 31.6603724 0.14303771 4.94802577 179196.415
CON 80 82.2329489 0.37567157 0.85798614 82399.8603
CON300 298.842281 1.37207449 1.42177678 490483.205
Unknown 76.2126788 0.34797832 0.71562859 60703.703
Figure 3 calibration curve for right detector
Results for left detector,table 3 and 4 provide data for left detector for calibrations, controls and sample unknown. From the graph 4 calculated the concentration of controls and sample by equation y = 0.0047x - 0.01 ,Y=PAR, X=concentration
show that the quality control for all controls and sample are valid and within the range of+ 2SD of QC chart in appendix, the R2 value from the graph 4 is 0.9999 and is acceptable according to criteria. The CV for control 30 for left detector is 6.11434866% is not acceptable for road traffic case as is more than 2% but is valid for PM case, and CV for other controls and sample is undergo within the
criteria of acceptability in both case RT and PM. The concentration of alcohol is still less than cutoff limit in RT case is 70.19mg/dl.
Table 4:result for calibrations from left detector
Level mg/dl Mean PA Mean IS PAR
CAL 1 10 1064430 27465703.5 0.03875488
CAL 2 25 2976070.5 27576921 0.10791888
CAL 3 50 6326071 28010049 0.22585005
CAL 4 80 10118332.5 27746943.5 0.36466476
CAL 5 100 12732185.5 27604131 0.46124203
CAL 6 200 26206758 27892328.5 0.93956867
CAL 7 300 39115441.5 27857665 1.40411774
CAL 8 400 52412308.5 27892123.5 1.87910786
Table 5:result for controls and sample from left detector
Sample mg/dl PAR CV SD
CON 30 31.6201391 0.13861465 6.11434866 236811.475
CON 80 81.7925106 0.3744248 0.43658292 46340.95
CON 300 299.102128 1.39578 1.75450924 681838.32
Unknown 76.192178 0.34810324 0.43650161 40749.1496
Alcohol calibration curve left
detector y = 0.0047x - 0.01
Axis Title
Figure 4 calibration curve for left detector For both detectors combine, due to the fact that each sample ran in duplicate and was measured
by two detectors, four-peak area and internal stander peak area was received there should be calculate the mean for all data of both detectors to confirm the result. From table 8 shows the mean of concentrations for controls30, 80,300 and sample unknown for both detectors are 31.6402558, 82.2329489, 298.972469 and 76.2024284mg/dl respectively. The control 80 is not lying between +2SD while other do as show in table 9 and the result is still valid as two controls are within the range and for criteria of acceptability minimum 2 controls must be within the range.
For CV of control 30 is an outlier with CV of 5.53118722% so this control do not pass the criteria of acceptability if the case is road traffic, and for other controls 80, 300 and sample unknown have CV% acceptable so one outlier sample can not effect the validation of result because there are still two other controls within the range, so the result is valid do depend on in our investigate. As known that is If the sample is part of a road traffic 6gm/dl must be subtracted for the sample concentration because of uncertainties of machine the therefore the final result is 70.202mg/dl, this concentration of ethanol in road traffic case is normal concentration as is under cutoff limit. In post mortem case the presence of ethanol is refer to many factors, in appropriate condition ethanol can be produced in concentration up to, and afar, ethanol can also help as a substrate for many microorganisms such that ethanol concentration in blood and tissues may increase and then decrease. Production of post mortem ethanol not related with the degree of putrefaction. Many strictly decomposed sample may contain no ethanol, whereas other less strict decomposed may contain of 80mg/dl or higher. Other factors are when the stomach contains large amount of ethanol and this amount may diffuse through the stomach wall and diaphragm and enter into the heart and central blood. Sever trauma, adequate to rupture the stomach and diaphragm and may allow gastric contents to pass into the chest cavity. In such cases it may be difficult to find blood from the peripheral vessels.
Another factor is that the movement of gastric contents into the trachea and lungs and this could lead to raised blood ethanol concentration mainly in the central pulmonary and cardiac vessels and consequently to erroneous interpretation, for all these reasons a second test in post mortem cases in different specimen is required (4). In general, the concentration in ethanol in sample 76.2024284 mg/dl is not fatal is just lead to Slight impairment of balance, speech, vision, reaction time, hearing and Euphoria. Reduced judgment and self-control. Impaired reasoning and memory (11).
Table 6:CV mean for both detectors
level mg/dl cv1 cv2 mean
CAL 1 10 0.5994246 0.03560678 0.31751569
CAL 2 25 1.04189623 1.38911353 1.21550488
CAL 3 50 0.20458955 0.16511641 0.18485298
CAL 4 80 1.78878925 1.8648123 1.82680078
CAL 5 100 1.58662726 2.01182055 1.79922391
CAL 6 200 0.39063109 0.76503255 0.57783182
CAL 7 300 1.90951314 2.11574482 2.01262898
CAL 8 400 2.47034449 2.43339925 2.45187187
CON 30 4.94802577 6.11434866 5.53118722
CON 80 0.85798614 0.43658292 0.64728453
CON 300 1.42177678 1.75450924 1.58814301
unkown 0.71562859 0.43650161 0.5760651
Table 7:SD mean for both detectors
level mg/d l SD1 SD mean
CAL 1 10 6380.22449 379.009235 3379.61686
CAL 2 25 29170.276 41340.998 35255.637
CAL 3 50 11757.7716 10445.3814 11101.5765
CAL 4 80 163809.771 188687.909 176248.84
CAL 5 100 180870.136 256148.724 218509.43
CAL 6 200 91175.0555 200490.228 145832.642
CAL 7 300 661599.51 827582.927 744591.219
CAL 8 400 1145543.39 1275400.72 1210472.06
CON 30 179196.415 236811.475 208003.945
CON 80 82399.8603 46340.95 64370.4052
CON 300 490483.205 681838.32 586160.763
unknown 179196.415 236811.475 208003.945
Table 8:PAR mean for both detectors
level mg/dl PAR1 PAR2 mean 1+2
CAL 1 10 0.0429512 0.03875488 0.04085304
CAL 2 25 0.11257117 0.10791888 0.11024503
CAL 3 50 0.2275293 0.22585005 0.22668968
CAL 4 80 0.36494176 0.36466476 0.36480326
CAL 5 100 0.45694892 0.46124203 0.45909548
CAL 6 200 0.92857729 0.93956867 0.93407298
CAL 7 300 1.37595935 1.40411774 1.39003855
CAL 8 400 1.84180053 1.87910786 1.8604542
CON 30 - 0.14303771 0.13861465 0.14082618
CON 80 - 0.37567155 0.3744248 0.37504818
CON 300 - 1.37207444 1.39578 1.38392722
Unknow - 0.34797831 0.34810324 0.34804078
n
Table 9:concentration means
Sample Concentration R Concentration L Mean concentration
CON 30 31.6603724 31.6201391 31.6402558
CON 80 82.2329489 81.7925106 82.0127298
CON 300 298.84281 299.102128 298.972469
unknown 76.2126788 76.192178 76.2024284
Table 10 acceptability criteria for controls
sample Right detector Left detector Combine
CON 30 2SD 2SD 2SD
CON 80 >2SD 2SD >2SD
CON 300 2SD 2SD 2SD
Conclusion
The investigation of the unknown blood sample 13/008 that determined by GC-HS-FID have two detectors each detector give result and both results tested positive for alcohol and the dependable result was from combine both detectors to ensure the result and that also tested positive for alcohol, the sample is considered to be 76.2024284mg/dl. In road traffic case the sample concentration was determined as 70.202mg/dl. Hence, at the same time of sample collection the person was under the limit of 80mg/dl which consider not above illegal limit with consider the sample just measured in duplicate not in quadruplicate so the result is not 100% valid. If the sample is belonged to a postmortem case, all criteria of acceptability were valid.
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