Application Note

Forensic and Toxicology

- Criminalistics

Separation of Enantiomers of
Amphetamine-Related Drugs and
Their Structural Isomers
Using the Agilent 1260 Infinity II SFC and Detection
by Coupled Mass Spectrometry

×106
2.5

2.0

Counts
1.5

1.0

0.5

0
0 1 2 3 4 5 6 7
Acquisition time (min)

Authors Abstract
Melanie Muelek,
Herbert Godel, and
This Application Note demonstrates the separation of enantiomers of amphet-
Edgar Naegele
amine-related drugs and their positional isomers as well as enantiomers of the
Agilent Technologies, Inc.
same compounds. This separation was performed using chiral phase columns with
the Agilent 1260 Infinity II SFC System. The qualitative detection and quantitative
determination of both the structural isomers and enantiomers was achieved using
the Agilent 6495 triple quadrupole MS and the Agilent 6150 single quadrupole
MSD.
 O
Introduction CH3
O N CH3
The isomeric compounds 4-, 5-, and H
6-EAPB are psychedelic drugs structur-
CH3 1-(Benzofuran-5-yl)-N-ethylporpan-2-amine
ally related to amphetamine, and belong
(5-EAPB)
chemically to the class of benzofuran HN
compounds (Figure 1)1. The three com-
CH3 H
pounds are structural isomers compris- N CH3
O
ing one chiral center; therefore, each 1-(Benzofuran-4-yl)-N-ethylporpan-2-amine
exists in two enantiomeric forms. CH3
(4-EAPB)
This Application Note demonstrates the 1-(Benzofuran-6-yl)-N-ethylporpan-2-amine
separation of these structural isomers (6-EAPB)
and their enantiomers by chiral chroma- Figure 1. Formulae of 4-, 5-, and 6-EAPB. Each of the isomeric compounds also has a stereo center
tography. This separation was achieved (asterisk), and thus exists in two enantiomeric forms.
using the Agilent 1260 Infinity II SFC
System coupled to mass spectrom-
etry for qualitative and quantitative SFC Method for the separation of all six isomers SFC Method for the separation of the three
determination. (enantiomers and structural isomers) on Column 1 structural isomers on Column 2

Experimental Parameter Value Parameter Value

SFC Flow 3 mL/min SFC Flow 2.5 mL/min

Instruments Modifier Methanol + 0.1 % NH3 aq. Modifier Methanol + 0.1 % NH3 aq.

Agilent 1260 Infinity II SFC/MS System Isocratic 10 % modifier Isocratic 11 % modifier

comprises: Column temperature 20 °C Column temperature 30 °C

BPR Temperature 60 °C BPR Temperature 60 °C

• Agilent 1260 Infinity II SFC Control
Module (G4301A) BPR Pressure 200 bar BPR Pressure 200 bar

Total run time 7 minutes Total run time 3.5 minutes

• Agilent 1260 Infinity II SFC Binary
Injection Injection
Pump (G4782A) 5 µL 1 µL

Feed speed 400 µL/min Feed speed 400 µL/min

• Agilent 1260 Infinity II SFC
Overfeed volume 4 µL Overfeed volume 4 µL
Multisampler (G4767A)
Needle wash 3 seconds methanol Needle wash 3 seconds methanol
• Agilent 1260 Infinity II DAD with
high‑pressure SFC flow cell
(G7115A)
• Agilent 1260 Infinity II Multicolumn
Thermostat (MCT) (G7116A) with
• Agilent InfinityLab Quick Change
4-position/10-port four-column
selection valve (p/n 5067-4287)
• Agilent 1260 Infinity II Isocratic
Pump (G7110B) and SFC/MS Splitter
kit (G4309-68715)
• Agilent 6495 Triple Quadrupole MSD
with Agilent Jet Stream and iFunnel
Technology
• Agilent 6150 Single Quadrupole MSD
with Agilent Jet Stream

2
 MS Method for triple quadrupole MS and single quadrupole MS
Instrumental setup
The recommended configuration of the Parameter Value
Agilent 1260 Infinity II Analytical SFC Make up composition Methanol/water (95/5) + 0.2 % formic acid
System with Agilent LC/MS Systems was Make up flow 0.4 mL/min
described previously2. Electrospray Ionization with Agilent Jet Stream Ion Source
Drying gas 150 °C, 11 L/min
Software
Sheath gas 350 °C, 12 L/min
• Agilent OpenLAB CDS ChemStation
Nebulizer
Edition for LC and LC/MS Systems, 45 psi

Rev. C.01.07 SR3 Capillary 2,500 V

Nozzle 0V
• MassHunter LC/TQ Acquisition
Software, Version B.08.02 iFunnel* High-pressure RF: 90
Low-pressure RF: 70
• MasHunter Optimizer Software, Triple quadrupole parameters
Version B.08.02 ESI Polarity positive

• MassHunter Source and iFunnel Scan type MRM

Optimizer Software, Version B.08.02 Transitions 2

• MassHunter Quantitative Software, Cycle time 502 ms

Version B.08.00 ΔEMV +200 V

• MassHunter Qualitative Software, Single quadrupole parameters

Version B.07.00 SP1 ESI Polarity positive

Scan type SIM (m/z 204.1)

Columns
Dwell time 590 ms
1. Chiral Technologies, CHIRALPAK
Fragmentor 70 V
AD‑H, 250 × 4.6 mm, 5 µm
Gain 1.0
2. Chiral Technologies, CHIRALPAK * Only for triple quadrupole
AD‑3, 150 × 4.6 mm, 3 µm
Precursor Product Dwell Fragmentor Collision Cell acc.
Chemicals Compound ion (m/z) ion (m/z) (ms) (V) energy (V) voltage (V)
All solvents were purchased from 4/5/6-EAPB 204.1 159.1 250 380 12 1

Merck, Germany. 4/5/6-EAPB 204.1 131.1 250 380 22 1

Samples
Separate stock solutions of 4-, 5-, and
6-EAPB (1 ppm in methanol) were used
in dilution, as outlined in the text.

3
Results and Discussion ×105

1.1
Separation of enantiomers, and
1.0
quantitative determination by triple
quadrupole MS 0.9
A method developed for the chiral sepa- 0.8
ration of D- and L-amphetamine3 was
0.7
chosen as the starting point for develop-

Counts
ing a separation method for the six iso- 0.6

mers of 4-, 5-, and 6-EAPB. This method 0.5

immediately led to a promising separa-
0.4
tion of all six possible stereoisomers
(Figure 2), with all six isomers being 0.3

partially separated between 2.8 and 0.2

6.0 minutes. This separation was then
0.1
further optimized due to the incomplete
separation. 0
0 1 2 3 4 5 6 7 8
Method parameters for Figure 2 Acquisition time (min)

Parameter Value Figure 2. Initial method for the separation of all six isomers of 4-, 5- and 6-EAPB.

Column Chiralpak AD-H,

4.6 × 250 mm, 5 µm
×105
Column temperature 20 °C 1.0
20 °C
10 %B 0.8
Mobile phase
Counts

(EtOH + 0.1 % NH3 aq.) 0.6

Flow rate 4 mL/min 0.4
Injection volume 5 µL
0.2
0
Sample 10 ppb each in MeOH ×105
1.2
1.0 30 °C
The ethanol content of modifier B was
Counts

0.8
varied in the next step, which did not 0.6
result in improved separation. Reducing 0.4
the content of modifier B led to higher 0.2
0
retention times with broader peaks and
×105
a lower resolution, especially of the later 1.2 40 °C
eluting peaks (data not shown). The 1.0
Counts

0.8
effect of column temperature on the
0.6
separation of the enantiomers was also 0.4
examined (Figure 3). 0.2
0
Method parameters for Figure 3 ×105
1.2 45 °C
Parameter Value 1.0
Counts

0.8
Column Chiralpak AD-H, 0.6
4.6 × 250 mm, 5 µm
0.4
Column temperature 20, 30, 40, and 45 °C 0.2
0
Mobile phase 10 %B 0 1 2 3 4 5 6 7
(EtOH + 0.1 % NH3 aq.)
Acquisition time (min)
Flow rate 4 mL/min
Figure 3. Separation of all six isomers of 4-, 5-, and 6-EAPB depending on the column temperature.
Injection volume 5 µL

Sample 10 ppb each in MeOH

4
The highest resolutions were achieved ×105
3.0
at the lower temperatures of 20–30 °C,
and the resolution decreased when
using higher temperatures. Different
solvents were also tested to improve 2.5
the resolution between the six isomers.
When methanol was used as a modi-
fier instead of ethanol, the peaks eluted
earlier, between 2.0 and 3.5 minutes, and 2.0
with better peak shape. However, the
peaks were still not completely resolved Counts
(Figure 4).
1.0
Method parameters for Figure 4

Parameter Value

Column Chiralpak AD-H,

4.6 × 250 mm, 5 µm 0.5

Column temperature 20 °C

Mobile phase 10 %B
(MeOH + 0.1 % NH3 aq.)
0
Flow rate 4 mL/min
0 1 2 3 4 5 6 7
Injection volume 5 µL Acquisition time (min)
Sample 10 ppb each in MeOH
Figure 4. Method for the separation of all six isomers of 4-, 5-, and 6-EAPB with
methanol as modifier.

Finally, it was found that 10 % methanol ×105

(with 0.1 % NH3 aq.) as modifier gave
the best separation at a flow rate of
3 mL/min (Figure 5).
2.0
Method parameters for Figure 5

Parameter Value

Column Chiralpak AD-H, 1.5

4.6 × 250 mm, 5 µm
Counts

Column temperature 20 °C

Mobile phase 10 %B
(MeOH + 0.1 % NH3 aq.) 1.0

Flow rate 3 mL/min

Injection volume 5 µL

Sample 10 ppb each in MeOH 0.5

0
0 1 2 3 4 5 6 7
Acquisition time (min)

Figure 5. Optimized separation of all six isomers of 4-, 5-, and 6-EAPB.

5
The compounds eluted between 3.2 ×106
2.5
and 5.5 minutes. The compounds 4-EAPB
were identified by comparison to 5-EAPB
6-EAPB
racemic single standards of 4-, 5-, and
6-EAPB (Figure 6). Under the optimized
2.0
conditions:
• 4-EAPB elutes between 3.1 and
3.7 minutes
• 5-EAPB elutes between 3.6 and 1.5

4.6 minutes
Counts
• 6-EAPB elutes between 4.4 and
5.4 minutes
1.0
In particular, 5- and 6-EAPB were suc-
cessfully separated by the developed
method, which was the aim for the devel-
opment of this method. 0.5

After optimization of the MS source

and all related MS parameters, calibra-
tion curves were created for 4-, 5-, and
0
6-EAPB between 100 ppt and 100 ppb 0 1 2 3 4 5 6 7
(Figure 7), with three replicates for each Acquisition time (min)

calibration level. The linearity, R2, showed Figure 6. Identification of the enantiomeric compounds 4-, 5-, and 6-EAPB by injection of their
values of 0.9998, 0.9993, and 0.9990 for racemic single standards.
4-, 5-, and 6-EAPB, respectively. The limit
of quantification (LOQ) was determined
at 100 ppt for a signal-to-noise ratio
(S/N) of 10. The limit of detection (LOD)
was determined at 30 ppt for an S/N
of 3.

6
 204.1 → 131.1 204.1 → 131.1
×107 ×106 204.1 → 159.1 ×102 204.1 → 159.1
4-EAPB 9
1.0 100 ppb 100 ppt
1.0 y = 110454.344390*x – 7897.069371 8
R2 = 0.9998
7
Responses

Counts

Counts
0.5 5
0.5
4
3
2

0 0 1

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 3.1 3.2 3.3 3.4 3.5 3.1 3.2 3.3 3.4 3.5
Concentration (ng/mL) Acquisition time (min) Acquisition time (min)

204.1 → 131.1
×107 ×106 204.1 → 159.1 204.1 → 131.1
×102 204.1 → 159.1
5-EAPB
y = 165535.846950*x – 30697.003155 1.0 100 ppb 9 100 ppt
1.5
R2 = 0.9993 8
7
Responses

1.0 6
Counts

Counts
0.5 5

0.5 4
3
2
0 0 1
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 3.7 3.8 3.9 4.0 4.1 4.2 3.7 3.8 3.9 4.0 4.1 4.2
Concentration (ng/mL) Acquisition time (min) Acquisition time (min)

204.1 → 131.1 204.1 → 131.1

×107 ×105 204.1 → 159.1 ×102 204.1 → 159.1
6-EAPB 6.5
7
y = 138334.441119*x – 14907.984008 100 ppb 6.0 100 ppt
6 5.5
R2 = 0.9990
5 5.0
1.0
4.5
Responses

4
Counts

Counts

4.0
3.5
3
0.5 3.0
2 2.5
2.0
1
1.5
0 0 1.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1
Concentration (ng/mL) Acquisition time (min) Acquisition time (min)

Figure 7. Calibration curves for 4-, 5-, and 6-EAPB between 100 ppt and 100 ppb. The quantifier and qualifier transition at 100 ppb and at
100 ppt (LOQ) are shown next to the individual calibration curves.

7
 2.074
Separation of position isomers
of 4-, 5-, and 6-EAPB and 40,000 1.871 2.296
qualitative determination by single
35,000
quadrupole MS
Another possibility for the determina- 30,000
tion of 4-, 5-, and 6-EAPB is the nonchiral
separation of the structural isomers. 25,000

Unfortunately, there was no existing sep-

20,000
aration method found that worked on an
achiral stationary phase and separates 15,000
all three structural isomers. Typically, the
4-EPAB is separated, but the other two 10,000

isomers, 5- and 6-EPAB, coelute com- 0.5 1.0 1.5 2.0 2.5 min
pletely or elute with insufficient separa- Figure 8. Separation of 4-, 5-, and 6-EAPB structural isomers with 15 % methanol as modifier.
tion. To solve this problem, it has been
attempted to separate only the isomers
2.873 3.248
without separation of the enantiomers, 2.495
30,000
but on a chiral stationary phase.
The initial experiment, which showed 25,000

some separation of the three structural

20,000
isomers without showing a separation
of the enantiomers was performed
15,000
on a CHIRALPAK AD-3 column with
methanol as mobile phase (15 % with 10,000
0.1 % NH3 aq.) (Figure 8). To improve the
partial separation of the three isomers, 5,000
the methanol content was decreased in
steps of 1 %. A sufficient separation with 1 2 3 4 5 min
acceptable peak width and run time for
Figure 9. Separation of 4-, 5-, and 6-EAPB structural isomers with 11 % methanol as modifier.
the three compounds was found for a
modifier content of 11 % methanol (with
0.1 % NH3 aq.) (Figure 9).
Method parameters for Figure 8 Method parameters for Figure 9

Parameter Value Parameter Value

Column Chiralpak AD-3, Column Chiralpak AD-3,

4.6 × 150 mm, 3 µm 4.6 × 150 mm, 3 µm

Column temperature 30 °C Column temperature 30 °C

15 %B Mobile phase 11 %B
Mobile phase
(MeOH + 0.1 % NH3 aq.) (MeOH + 0.1 % NH3 aq.)

Flow rate 2 mL/min Flow rate 2 mL/min

Injection volume 1 µL Injection volume 1 µL

Sample 100 ppb each in MeOH Sample 100 ppb each in MeOH

8
 2.510
The final method was achieved by 30,000 2.847
2.159
increasing the flow rate to shorten the
run time to 3.5 minutes (Figure 10). 25,000
The identity of the compounds was
confirmed by injection of the single 20,000
standards of the three achiral isomers
(Figure 11). The 4-EAPB elutes at 15,000
2.15 minutes, 5-EAPB at 2.50 minutes,
and 6-EAPB at 2.83 minutes. This 10,000

method could be used as quick qualita-

tive detection of the EAPBs by a combi- 5,000

nation of SFC and a single quadrupole

MS in SIM mode. For a proper quantifica- 0.5 1.0 1.5 2.0 2.5 3.0 min
tion, the method shown for the combina- Figure 10. Final method for the separation of 4-, 5-, and 6-EAPB structural isomers.
tion of the SFC and a triple quadrupole
with its additional selectivity can be used. Norm.
4-EAPB 2.152 2.503 2.838
30,000 5-EAPB
Method parameters for Figure 10
6-EAPB
Parameter Value 25,000

Chiralpak AD-3,
Column 20,000
4.6 × 150 mm, 3 µm

Column temperature 30 °C 15,000

Mobile Phase 11 %B
(MeOH + 0.1 % NH3 aq.) 10,000

Flow rate 2.5 mL/min

5,000
Injection volume 1 µL

Sample 100 ppb each in MeOH 0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 min

Figure 11. Overlay of the separation of individual samples of 4-, 5,- and 6-EAPB structural isomers
with the final method.

9
Conclusion References
This Application Note demonstrates the 1. Taschwer, M.; Hofer, M. G.;
use of the Agilent 1260 Infinity II SFC for Schmid, M. G. Enantioseparation
the separation of either all six possible of benzofurys and other novel
isomers of 4-, 5-, and 6-EAPB, or the psychoactive compounds by CE
separation of only the three respective and sulfobutylether β-cyclodextrin
structural isomers. The detection and as chiral selector added to the
quantitative determination have been BGE. Electrophoresis 2014, 35, 19,
done by coupling the SFC either to a 2793–2799.
single quadrupole or a triple quadrupole 2. The use of the SFC-MS Splitter Kit
MS. The calibration curves for quantita- G4309-68715. Agilent Technologies
tive analysis were performed on the Technical Note, publication number
triple quadruple MS and showed excel- G4309-90130, 2015.
lent linearity (R2 >0.9990) and sensitivity.
The LOQs were found at 100 ppt and the 3. M. Muelek, H. Godel,
LODs at 30 ppt. E. Naegele. Development
of a Method for the Chiral
Separation of D/L‑Amphetamine.
Agilent Technologies Application
Note, publication number
5991‑8262EN, 2017.

Acknowledgement
Thanks to Martin Josefsson and Markus
Roman from the National Board of
Forensic Medicine, Linkoping, Sweden
for providing the samples.

www.agilent.com/chem

For Forensic Use.

For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc. 2017

Printed in the USA, November 1, 2017
5991-8416EN