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AN 9140 Blood Alcohol Analysis by Static Headspace with Dual FID/Megabore Capillary Columns
Terry Rankin Jessie Crockett Butler, Thermo Finnigan GC and GC/MS Division

Introduction
The analysis of blood and other body fluids for alcohol is most commonly performed using capillary columns. The selection of column size and injection technique depends on the hardware selected for sample analysis. The simplest method of analysis is to perform isothermal analyses of a headspace sample injected into a Megabore column attached to a split/splitless or packed injector. Confirmational analysis can be performed by injecting a single sample into a guard column and splitting the exit of the guard column into two different Megabore columns. The effluent of each column is connected to an FID. The analysis is performed in less than four minutes. The ThermoQuest TRACE™ GC 2000 gas chromatograph equipped with digital electronic flow control and dual FIDs with electronic gas controls provides rapid analysis of blood alcohols by static headspace injection. The Model HS 2000 Static Headspace Autosampler offers a gas-tight syringe injection technique that simplifies operation while maintaining sample integrity and analytical accuracy.

FID 1 Rtx -BAC1
®

FID 2

Restek "Y" press tight splitter Rtx®-BAC2 0.53 mm x 1 meter Phenylmethyl deactivated guard column
77001-0618 991218

Figure 1. Dual column/Dual FID blood alcohol analysis by static headspace

Experimental
The TRACE GC 2000 gas chromatograph was configured with a guard column and two Megabore columns for confirmational analysis into dual FIDs, Figure 1. The sample is placed into a 10 mL vial for analysis by static headspace, Figure 2. Then, 1.0 mL of headspace is removed from the vial with a gas-tight syringe and injected

into a split/splitless injector with a 5 mm I.D. liner. The HS 2000 headspace sampler was used for sample injection. The TRACE GC 2000/HS 2000 operating parameters and data collection was accomplished using the ChromQuest data station. The instrument parameters are listed below. TRACE GC FIDs: 250°C Range: x10 H2: 35 mL/min. Air: 350 mL/min Makeup: 30 mL/min. Split/Splitless injector: 200°C Guard Column: 0.53 mm x 0.5 m

(methylphenyl deactivated) Restek "Y" Splitter Column 1: Restek Rtx®-BAC1 0.53 x 30 meter, 3.0 film Column 2: Restek Rtx-BAC2 0.53 x 30 meter, 2.0 film Column Flow: DPFC at 12 mL/min for each column Column Temp: 40°C isothermal Inject volume: 1.0 mL, split 30:1 HS 2000 Syringe Temp: 85°C Incubation Temp: 80°C Incubation Time 20 min. ChromQuest data system
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AN 9140

7000 TRACE GC1-R-BAC1 Blood Alcohol Std. 0.1 gm/mL

6000

5000

Isopropanol

Acetone

4000 Mv 3000

Acetaldehyde

2000

1000
500

0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Time, minutes 2.0 2.2 2.4 2.6 2.8 3.0

Methanol

Ethanol

N-Propanol
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Figure 3. Blood alcohol standard in blood on the Rtx-BAC1 column

7000

TRACE GC1-L-BAC2 Blood Alcohol Std. 0.1gm/100 mL

1.365 Acetone 1.502 Isopropanol

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6000

Mv

Sample Preparation
The samples were placed in 10 mL headspace vials by adding 0.1 mL of blood, 200 mg of NaCl, 0.1 mL of Saponin, and 0.1 mL of the internal standard (N-Propanol or t-Butanol at 0.1 gr/100 mL of water.) Saponin is a powerful hemolytic used to dissolve red corpuscles increasing the extraction efficiency of the alcohols.1 The samples were sealed using crimptop vial caps with septa.

3000

2000

1000

0 0.00

0.25

0.50

0.565

0.75

0.960 Methanol

4000

1.00

1.25

1.272 Ethanol

Figure 2. Static headspace

5000

0.805 Acetaldehyde

1.50

1.75

2.00

2.25

2.410 N-Propanol
2.50

2.75

3.00

Time, minutes

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Figure 4. Blood alcohol standard in blood on the Rtx-BAC2 column

450

The two Megabore capillary columns evaluated demonstrated excellent resolution for all analytes with no distortion of peak shape due to overloading, Figures 3 and 4. The FIDs showed more than adequate linearity and sensitivity over a working range from 0.01-0.5 gr/100 mL of blood, Table 1. The lowest calibration standards are shown in Figures 5 and 6. Detection limits of <0.005 gr/100 mL are easily achievable. Blanks run after the 0.5 gr/100 mL standard showed no carryover, Figure 7.
1. The Merck Index, Tenth Edition, 1983, pg 1204. AN 9140

300 250
Mv

Acetaldehyde

200 150 100 50 0 -50 0.0

0.2

0.4

0.6

0.8

1.0

1.2

Methanol

1.4

Ethanol

1.6

1.8

2.0

2.2

2.4

Acetone
2.6

350

Isopropanol

400

TRACE GC1-R-BAC1 Blood Alcohol Std. 0.01gm/100mL

N-Propanol
2.8

Results

3.0

Time, minutes

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Figure 5. 0.01 g/mL std in blood on the Rtx-BAC1 column
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900 800

TRACE GC1-L-BAC2 Blood Alcohol Std. 0.01gm/100mL

Acetone

700 600 Mv 500 400 300 200 100 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Acetaldehyde

Methanol

Ethanol

Isopropanol

N-Propanol

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

Time, minutes

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Figure 6. 0.01/100mL std in blood on the Rtx-BAC2 column

500

1000

2000

7000 TRACE GC1-R-BAC1 Blank 6000 TRACE GC1-R-BAC1 Blood Alcohol Std. 0.1gm/100mL

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Figure 8. Syringe flush
Isopropanol

5000

N-Propanol

Acetone

140000000

Acetaldehyde

3000

Ethanol

4000 Mv

120000000

100000000

Methanol Acetaldehyde Ethanol Isopropanol Acetone

Methanol

80000000

2000

60000000

1000

40000000

20000000

0 0.0

0.2

0.4

0.6

0.8

1.0

1.2 1.4 1.6 Time, minutes

1.8

2.0

2.2

2.4

2.6

2.8

3.0

0 0 0.1 0.2 0.3 0.4 0.5 0.6
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Figure 7. No carry-over found in blank

Figure 9. Blood alcohol linearity (0.0125 to 0.5 grams/100 mL) BAC1 column

Conclusion
The TRACE GC 2000 and HS 2000 headspace autosampler has been proven to be a reliable instrument system for the analysis of blood alcohols. Confirmational analysis is accomplished using a single injection into a standard split/splitless injector equipped with a guard column, "Y" connector, two dissimilar Megabore columns and dual FIDs. The HS 2000 uses no sample loops which may require cleaning or transfer lines that create active sites for

these polar compounds. The sample is injected with an automated headspace analyzer using a Hamilton 2.5 mL gastight syringe. The HS 2000 features a heated syringe and an incubator with a programmable mixer to enhance the equilibration of the vapor between the gas and liquid phase of the sample. To eliminate carryover the syringe is cleaned automatically between injections with a nitrogen purge for 1 minute.
Copies of this and other applications as well as technical information can be obtained from our website:

250000000 Acetaldehyde Methanol Ethanol Acetone Isopropanol

200000000

150000000

100000000

50000000

0 0 0.2 0.4 0.6
77001-1642 981120

www.thermofinnigan.com
AN 9140

Figure 10. Blood alcohol linearity (0.0125 to 0.5 grams/100 mL) BAC2 column

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BAC1 (area counts)
Rtx-BAC2 Area Reproducibility at 0.1 gm/100mL Vial 1 2 3 4 5 6 7 8 9 10 Average STDEV % RSD Acetaldehyde 142866148 140179897 138294611 139631947 134439545 137718041 139452548 141327403 138899148 139684344 139249363 2239932 1.609% Methanol 62567033 61076728 62873192 61517780 62314123 62840209 62010360 60895853 62041285 62183715 62032028 684595 1.104% Ethanol 150065987 149185186 150790854 149162082 150502389 151308788 149763317 148709492 149933712 149891957 149931376 792434 0.529% Acetone 370811655 368449176 365961157 375269443 364817044 371036399 378890713 380694507 378067501 380639839 373463743 6025991 1.614% Isopropanol 336142626 334659762 333149558 335878278 333712840 332579336 336021830 336679829 334800132 336043304 334966750 1418786 0.424% N- Propanol 349524145 333968554 325950398 318262167 336796445 324278447 326981236 332041064 324757559 327954487 330051450 8660031 2.624%
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(IS)

BAC2 (area counts)
Rtx-BAC1 Area Reproducibility at 0.1 gm/100mL

Vial 1 2 3 4 5 6 7 8 9 10 Average STDEV % RSD

Methanol 36034380 35284058 36237211 35449006 36090220 36762467 35744577 35312512 36008255 35928255 35885094 456335 1.272%

Acetaldehyde 89392419 87634657 86555797 87348436 84171076 86186092 87284115 88402380 86939866 87278833 87119367 1378272 1.582%

Ethanol 97381265 96777892 97897395 96863003 97607041 98459578 97234238 96591973 97549914 97221157 97358346 558391 0.574%

Isopropanol 204353246 203775845 202774952 204125272 203451596 202873387 204555921 205156326 204220713 202325079 203761234 893905 0.439%

Acetone 240746733 235459480 232578289 238238401 233292272 236041827 242466572 245668728 241851003 244114340 239045765 4588470 1.919%

N- Propanol 217512300 207925470 202979384 198609698 209942658 202426257 204202480 207160771 202634243 205175436 205856870 5214075 2.533%

(IS)

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Table 1. Repeatability on Rtx

®

AN 9140 January 99

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