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Supercritical fluid chromatography with MS detection
for the separation of novel chiral compounds
Schad, Gesa J.1, Van den Heuvel, Dennis2
1 Shimadzu
Europa GmbH, Duisburg, Germany
2 Shimadzu Benelux B.V., ‘s-Hertogenbosch, Netherlands
1. Introduction
Projects in drug discovery and safety are constantly aiming at the development of novel and
safer drugs, therapeutics and diagnostics. During API (active pharmaceutical ingredient)
development, drug stereoisomerism is recognized as an issue having clinical and regulatory
implications. Enantiomers have essentially identical physical and chemical properties, while
potentially showing large differences in toxicity [1].
Therefore, the stereoisomeric composition of a drug with a chiral center should be well
documented. To evaluate the pharmacokinetics of a single enantiomer or any mixture of
enantiomers, manufacturers must develop quantitative assays for individual enantiomers early
in drug development.
3. Results
Amylose-1
Amylose-2
Cellulose-1
Cellulose-2
Cellulose-3
Cellulose-4
Figure 3: Chromatograms of screening results for unknown API 1 on 6 different
columns using methanol as organic modifier
Figure 1: Nexera UC SFC-MS chiral screening system system
2. Method Development
For SFC chiral screening the Shimadzu Nexera UC Chiral Screening System was used,
consisting of a CO2 and a quaternary solvent pump, an autosampler with loop injection and a
column oven including a six column switching valve. The system was also equipped with a
photo diode array detector and a LCMS 8040 triple-quadrupole mass spectrometer. Method
scouting for five different chiral drugs was performed in an overnight sequence using 6 min
gradient runs at 40°C with a backpressure of 150 bar at a flow rate of 2 ml/min. Twelve
combinations of stationary and mobile phases were selected (six different columns, two
different organic modifiers). Methods and sequence were created using the dedicated Method
Scouting Solution Software (figure 2).
Figure 4: MS data of isomeric compound for identification of enantiomers
API 1
API 4
B
A
API 2
A
API 5
B
API 3
Figure 2: Graphical user interface of Method Scouting Solution
A
Separation of chiral compounds requires use of chiral columns. For the chiral screening of
unknown chiral compounds the following columns were used:
1. Lux amylose-1 (250 x 4.6 mm, 5 µm)
Isocratic separation on
A: Lux cellulose-3 (250 x 4.6 mm, 5 µm)
Modifier A: 30 % of 0.1 % Formic acid in methanol
and
B: Lux cellulose-4 (250 x 4.6 mm, 5 µm)
Modifier B: 30 % MeOH
Figure 5: Chromatograms of optimized separations of five unknown APIs
2. Lux amylose-2 (250 x 4.6 mm, 5 µm)
3. Lux cellulose-1 (250 x 4.6 mm, 5 µm)
4. Lux cellulose-2 (250 x 4.6 mm, 5 µm)
5. Lux cellulose-3 (250 x 4.6 mm, 5 µm)
6. Lux cellulose-4 (250 x 4.6 mm, 5 µm)
The two organic modifiers tested during the chiral screening runs were methanol and
0.1 % formic acid in methanol.
MS parameters are listed below:
 Ionization mode: positive ESI
 Measurement mode: Q1 SIM
 Nebulizing gas flow: 2 L/min
 Desolvation line temperature: 250°C
 Heat Block Temperature: 400°C
 Drying gas flow: 15 L/min
4. Conclusion
• SFC-MS was shown to be a reliable, robust and simple alternative to routine LC analysis.
• Use of SFC in terms of complexity of the instrument and method development was very
similar to the HPLC approach. However, SFC is known to be superior to HPLC for chiral
separations.
• A fast and simple SFC – PDA/MS method for the screening of chiral compounds on different
columns was developed within two days, using a column screening system with 6 columns
and 2 different organic modifiers.
• The method was optimized with regards to separation and sensitivity.
• Simultaneous detection of PDA signal and MS scanning was used for confirmation of the
isomer signals.
• Resolution of > 1.5 was obtained for all compounds of interest with RSD < 2.0 % for
retention time using PDA and MS detection (without splitting)
[1] L.A. Nguyen, et al.; Int J Biomed Sci. 2006 Jun; 2(2): 85–100.