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Simplify Alcohol Analyses with Application-Designed Chromatography Columns

On the surface, alcohol analyses appear relatively straightforward. They involve few target analytes, are not extremely sensitive, and have quick sample preparation. However, they are not as simple as they seem. Ethanol analyses present distinct chromatography challenges when assaying in biological samples. Compounds found in biological matrices can interfere with the identification and quantitation of ethanol and its associated analytes.

Restek has developed tailored columns to address the common analyte interferences that can hinder productive chromatography. Dual capillary column sets for GC are available for common blood alcohol analyses, and high-throughput analytical LC column and guard cartridges are available for urinary alcohol assays. Starting method development with tailored columns designed specifically to lessen interferences can help labs develop methods faster and keep instruments running.

Blood Alcohol Content (BAC)

Gas chromatography coupled with headspace sampling and a flame ionization detector (HS-GC-FID) is the most commonly used technique for the analysis of blood alcohol in biological specimens from both living and dead persons. HS-GC-FID is remarkedly simple, reliable, and linear over the analytical range needed for the quantitative detection of alcohol. The troublesome part of the chromatographic analysis is ensuring analyte specificity from endogenous compounds. This is extremely important when analyzing postmortem samples due to the risk that fermentation or putrefaction may produce volatile chemical byproducts that can interfere with the analysis. For this reason, it is always recommended to use a dual column method. By splitting the sample after the injection port and analyzing it simultaneously on two different stationary phases, we can both quantify and confirm the analyte's identity in a single run (Figure 1). The target analytes will elute in the calibrated retention time window on both columns, thus confirming a positive identification. To simplify column selection, Restek has paired two distinct sets of column phases: Rtx-BAC1 and Rtx-BAC2 columns and Rtx-BAC Plus 1 and Rtx-BAC Plus 2 columns for in vivo and postmortem analysis.

decorative — **Figure 1:** HS-GC-FID dual column setup is common for BAC testing.

Rtx-BAC1 and Rtx-BAC2 columns are designed for routine in vivo blood alcohol analyses (Figure 2). This column pair has been in use for decades and has proven to provide stable, defensible results for routine BAC testing. When using n-propanol as the internal standard, this is the industry standard.

cgarm-img — **Figure 2:** The industry standard for in vivo BAC analysis is the Rtx-BAC1 and Rtx-BAC2 column set.

	Peaks	t_R (min)	Conc. (µg/mL)
1.	Methanol	0.886	200
2.	Acetaldehyde	0.993	200
3.	Ethanol	1.123	200
4.	2-Propanol	1.381	200
5.	Acetone	1.709	200
6.	n-Propanol (IS)	1.873	200

Column	Rtx-BAC1, 30 m, 0.32 mm ID, 1.8 µm (cat.# 18003)
Standard/Sample	BAC resolution control standard n-P (cat.# 36010)
Diluent:	Water
Conc.:	200 µg/mL
Injection	headspace-loop split (split ratio 50:1)
Liner:	Topaz 1.0 mm ID straight inlet liner (cat.# 23333)
Inj. Port Temp.:	220 °C
Headspace-Loop
Instrument:	Tekmar HT3
Inj. Time:	0.5 min
Transfer Line Temp.:	200 °C
Valve Oven Temp.:	200 °C
Standby flow rate:	100 mL/min
Sample Temp.:	60 °C
Platen temp equil. time:	60 min
Sample Equil. Time:	5 min
Vial Pressure:	30 psi
Pressurize Time:	2 min
Pressure Equilibration Time:	0.2 min
Loop Pressure:	20 psi
Loop Fill Time:	1 min
Oven
Oven Temp.:	40 °C (hold 4 min)
Carrier Gas	He, constant flow
Linear Velocity:	80 cm/sec

Detector	FID @ 240 °C
Make-up Gas Flow Rate:	45 mL/min
Make-up Gas Type:	N₂
Hydrogen flow:	40 mL/min
Air flow:	400 mL/min
Data Rate:	50 Hz
Instrument	Agilent 7890A GC

	Peaks	t_R (min)	Conc. (µg/mL)
1.	Acetaldehyde	1.011	200
2.	Methanol	1.199	200
3.	Ethanol	1.623	200
4.	Acetone	1.758	200
5.	2-Propanol	1.929	200
6.	n-Propanol (IS)	3.165	200

Column	Rtx-BAC2, 30 m, 0.32 mm ID, 1.2 µm (cat.# 18002)
Standard/Sample	BAC resolution control standard n-P (cat.# 36010)
Diluent:	Water
Conc.:	200 µg/mL
Injection	headspace-loop split (split ratio 50:1)
Liner:	Topaz 1.0 mm ID straight inlet liner (cat.# 23333)
Inj. Port Temp.:	220 °C
Headspace-Loop
Instrument:	Tekmar HT3
Inj. Time:	0.5 min
Transfer Line Temp.:	200 °C
Valve Oven Temp.:	200 °C
Standby flow rate:	100 mL/min
Sample Temp.:	60 °C
Platen temp equil. time:	1 min
Sample Equil. Time:	5 min
Vial Pressure:	30 psi
Pressurize Time:	2 min
Pressure Equilibration Time:	0.2 min
Loop Pressure:	20 psi
Loop Fill Time:	1 min
Oven
Oven Temp.:	40 °C (hold 4 min)
Carrier Gas	He, constant flow
Linear Velocity:	80 cm/sec

Detector	FID @ 240 °C
Make-up Gas Flow Rate:	45 mL/min
Make-up Gas Type:	N₂
Hydrogen flow:	40 mL/min
Air flow:	400 mL/min
Data Rate:	50 Hz
Instrument	Agilent 7890A GC

For labs looking to expand their HS-GC-FID platform beyond routine in vivo BAC testing, the Rtx-BAC Plus 1 and Rtx-BAC Plus 2 column set is a more suitable option. When analyzing postmortem blood alcohol, t-butanol is often used as the internal standard instead of n-propanol. N-propanol can be created by putrefaction, which can alter quantitation. When the internal standard, which exists to stabilize quantitation, is erroneously high, the results will lose accuracy. The Rtx-BAC Plus 1 and Rtx-BAC Plus 2 columns were designed to fully resolve the alternate internal standard, t-butanol, making it amenable to a fast, two-minute postmortem BAC analysis (Figure 3). With this column set, GC-FID workflows can also be expanded to include inhalants, glycols and GHB.

	Peaks	t_R (min)	Conc. (µg/mL)
1.	Methanol	0.859	200
2.	Acetaldehyde	0.910	200
3.	Ethanol	1.085	200
4.	2-Propanol	1.328	200
5.	Acetone	1.444	200
6.	t-Butanol (IS)	1.574	200

Column	Rtx-BAC Plus 1, 30 m, 0.32 mm ID, 1.8 µm (cat.# 18004)
Standard/Sample	BAC resolution control standard t-B (cat.# 36011)
Diluent:	Water
Conc.:	200 µg/mL
Injection	headspace-loop split (split ratio 50:1)
Liner:	Topaz 1.0 mm ID straight inlet liner (cat.# 23333)
Inj. Port Temp.:	220 °C
Headspace-Loop
Instrument:	Tekmar HT3
Inj. Time:	0.5 min
Transfer Line Temp.:	200 °C
Valve Oven Temp.:	200 °C
Standby flow rate:	100 mL/min
Sample Temp.:	60 °C
Platen temp equil. time:	1 min
Sample Equil. Time:	5 min
Vial Pressure:	30 psi
Pressurize Time:	2 min
Pressure Equilibration Time:	0.2 min
Loop Pressure:	20 psi
Loop Fill Time:	1 min
Oven
Oven Temp.:	40 °C (hold 4 min)
Carrier Gas	He, constant flow
Linear Velocity:	80 cm/sec

Detector	FID @ 240 °C
Make-up Gas Flow Rate:	45 mL/min
Make-up Gas Type:	N₂
Hydrogen flow:	40 mL/min
Air flow:	400 mL/min
Data Rate:	50 Hz
Instrument	Agilent 7890A GC

	Peaks	t_R (min)	Conc. (µg/mL)
1.	Acetaldehyde	0.771	200
2.	Methanol	0.818	200
3.	Ethanol	0.979	200
4.	Acetone	1.060	200
5.	2-Propanol	1.116	200
6.	t-Butanol (IS)	1.229	200

Column	Rtx-BAC Plus 2, 30 m, 0.32 mm ID, 0.6 µm (cat.# 18006)
Standard/Sample	BAC resolution control standard t-B (cat.# 36011)
Diluent:	Water
Conc.:	200 µg/mL
Injection	headspace-loop split (split ratio 50:1)
Liner:	Topaz 1.0 mm ID straight inlet liner (cat.# 23333)
Inj. Port Temp.:	220 °C
Headspace-Loop
Instrument:	Tekmar HT3
Inj. Time:	0.5 min
Transfer Line Temp.:	200 °C
Valve Oven Temp.:	200 °C
Standby flow rate:	100 mL/min
Sample Temp.:	60 °C
Platen temp equil. time:	1 min
Sample Equil. Time:	5 min
Vial Pressure:	30 psi
Pressurize Time:	2 min
Pressure Equilibration Time:	0.2 min
Loop Pressure:	20 psi
Loop Fill Time:	1 min
Oven
Oven Temp.:	40 °C (hold 4 min)
Carrier Gas	He, constant flow
Linear Velocity:	80 cm/sec

Detector	FID @ 240 °C
Make-up Gas Flow Rate:	45 mL/min
Make-up Gas Type:	N₂
Hydrogen flow:	40 mL/min
Air flow:	400 mL/min
Data Rate:	50 Hz
Instrument	Agilent 7890A GC

Urinary Alcohol Metabolites

When ethanol metabolites are being monitored in urine, which is commonly performed in treatment programs, high-throughput LC-MS/MS methods are employed. This technique is highly sensitive and fast. Since mass spectrometry is being used, analyte identification is not as significant a concern compared to when GC-FID is used. However, matrix interferences are still problematic during chromatographic assays. The presence of ethanol is determined using two urinary metabolites—ethyl glucuronide and ethyl sulfate. These analytes are highly water-soluble and, therefore, can be difficult to retain using traditional reversed-phase columns. Because of the inherent variability of urine samples and the common presence of early eluting matrix components, not retaining the target analytes beyond the zone of matrix interferences can cause ionization suppression, resulting in low sensitivity and problematic quantitation. Restek has developed a novel stationary phase to retain and separate the polar analytes EtG and EtS from common urine matrix interferences. As part of our Raptor column line, the Raptor EtG/EtS LC column features an SPP particle for improved cycle times and stability. It effectively separates EtG and EtS from potentially interfering matrix, ensuring sensitivity and accurate quantitation, in a fast, four-minute analysis.

CFAR3698-UNV

	Peaks	t_R (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
1.	Ethyl-β-D-glucuronide-d5 (EtG-d5)	1.21	200	226.2	85.0	-
2.	Ethyl-β-D-glucuronide (EtG)	1.23	500	221.2	75.1	85.1
3.	Ethyl sulfate-d5 (EtS-d5)	2.36	50	130.1	98.0	-
4.	Ethyl sulfate (EtS)	2.38	500	125.1	97.1	80.0

Detector	MS/MS
Ion Source:	Electrospray
Ion Mode:	ESI-
Mode:	MRM
Instrument	HPLC
Sample Preparation	A 500 ng/mL standard was prepared in urine. A 50 μL aliquot was mixed with 10 μL of internal standard (20 μg/mL EtG-d5 and 5 μg/mL EtS-d5 in water) and 150 µL of acetonitrile by vortexing at 3000 rpm for 10 seconds and centrifuged at 4300 rpm for 10 minutes at 10 °C. After centrifugation, 100 µL of the supernatant was diluted with 900 µL (40x dilution) of 0.01% formic acid in water. The sample was then vortexed at 3000 rpm for 10 seconds and injected for LC-MS/MS analysis.