This document describes Shimadzu's Nexera Method Scouting System for efficiently developing chiral separation methods using HPLC. The system allows up to 96 combinations of columns and mobile phases to be tested on three chiral compounds: Bromacil, α-Methyl-α-Acetyl-γ-Butyloractone, and Methylclothiazide. Data processing software quickly identifies the best separation conditions by comparing resolution and peak symmetry. Using the system and iCHIRAL columns, optimized methods for each compound were successfully determined. The method scouting system is useful for method development in pharmaceutical, chemical, food, and drug discovery industries.

1. Rapid Method Scouting of
Chiral Compounds
Kenichiro Tanaka, William Hedgepeth, Miho Kawashima,
Tadayuki Yamaguchi, Hidetoshi Terada and Yoshihiro Hayakawa
Shimadzu Scientific Instruments, Inc., Columbia, Md., USA

2. Introduction
It is important that the efficacy and safety of compounds are evaluated as enantiomers,
especially in pharmaceutical formulations and related industries.
Chiral separation with HPLC is one of the typical methods for purifying enantiomers
from racemic mixtures.
l In this method, the suitable column and mobile phase for targeted chiral
separation have to be determined before starting the analysis.
To determine the optimized analytical conditions:
l A large number of candidate conditions have to be examined.
l Extensive method development is required.
Today, a more prompt and simplistic system for determining the optimized
analytical conditions is needed.
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3. Introduction
On the other hand:
l
High-throughput processing is employed for the purpose of efficiency.
l
Recent advances in analytical throughput have been significantly improving
efficiency and productivity in the analytical operation.
l
The use of Ultra High-Performance Liquid Chromatography (UHPLC) has also
gained much attention.
l
Hence, in the field of method scouting, UHPLC technology is also increasing.
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4. System
Shimadzu has made a method scouting system with UHPLC for the purpose of making
method development more efficient.
Shimadzu applied this technology to the method development of:
l Non-steroidal anti-inflammatory drugs (NSAIDs)
l Cefem antibiotics
Here, we report on the process of high-efficiency method development of
three chiral compounds:
l Bromacil
l α-Methyl-α-Acethyl-γ-Butylrolactone
l Methylclothiazide
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5. System
Figure 1: Flow diagram of the Nexera Method Scouting System
developed in this experiment
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6. Nexera Method Scouting System
System consists of:
l Nexera Ultra High-Performance Liquid Chromatograph (UHPLC)
l Solvent switching valve
l Column switching valve
With this system, up to 96 combinations of columns and mobile phases
can be analyzed.
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7. Sample
Three standards of chiral compounds were analyzed:
l Bromacil
l α-Methyl-α-Acethyl-γ-Butylrolactone
l Methylclothiazide
Bromacil
α-Methyl-α-Acethyl-γ-Butylrolactone
Methylclothiazide
Figure 2: Structures of Chiral Compounds
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8. Chiral Columns
i CHIRAL-6 series (Daicel Corp.)
l CHIRALPAK® IA
l CHIRALPAK® IB
l CHIRALPAK® IC
l CHIRALPAK® ID
l CHIRALPAK® IE
l CHIRALPAK® IF
These columns can be used with various organic solvents and are
suitable for method scouting of chiral compounds.
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9. Analytical Conditions
Eight mobile phase conditions were suggested and mixed with solvents at given ratios.
With 8 mobile phases and 6 columns, a total of 48 analytical conditions were examined with
each substance.
Mobile
phase
No.
Flow
rate
Time
1
Hexane
/
2-­‐Propanol
=
9
/
1 （v/v）
3
mL/min
9
min
2
Hexane
/
2-­‐Propanol
=
6
/
4 （v/v）
3
mL/min
9
min
3
Hexane
/
Ethanol
=
8
/
2 （v/v）
3
mL/min
14
min
4
Ethanol
1
mL/min
18
min
5
Hexane
/
Dichloromethane
=
9
/
1 （v/v）
3
mL/min
4
min
6
Dichloromethane
/
Ethanol
=
100
/
2 （v/v）
3
mL/min
4
min
7
Hexane
/
Methyl
tert-­‐Butyl
Ether
=
9
/
1 (v/v）
3
mL/min
4
min
8
Methyl
tert-­‐Butyl
Ether
/
Ethanol
=
9
/
1（v/v）
3
mL/min
Remarks
4
min
Temp.:
40
°C
Inj.
vol.:
10
µL
Det.:
230
nm
Table 1: Analytical Conditions for Chiral Analyses
Note: Diethylamine was added to each mobile phase at 0.1% in the Methylclothiazide analysis
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10. Data Processing
Data processing software “CLASS-Agent Report” (Shimadzu) quickly picked the best
separation chromatogram by comparing the resolutions, number of detected peaks, and so on.
Ranking
Figure 3: Graph of analysis result with Excel
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11. Data Processing
The “CLASS-Agent Report” software can compare the data both visually and quantitatively,
helping to make data processing more efficient.
Methylclothiazide_ID_n-Hex_EtOH_3_analysis_B20%_14min
Methylclothiazide_IF_MC_EtOH_6_analysis_B2%_4min
Methylclothiazide_IB_MC_EtOH_6_analysis_B2%_4min
Methylclothiazide_IC_n-Hex_EtOH_3_analysis_B20%_14min
Methylclothiazide_IF_n-Hex_EtOH_4_analysis_B100%_18min
Table 2: Analysis result with Excel
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12. Results
The optimized methods of each chiral compound are shown in Figure 3.
Figure 4: Chromatograms of Bromacil, α-Methyl-α-Acetyl-γ-Butyloractone and Methylclothiazide
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13. Conclusions
With the Nexera Method Scouting System and Chiral columns “i CHIRAL-6 series,”
analytical conditions suitable for each chiral compound could be quickly determined.
Furthermore, the data processing software “CLASS-Agent Report” was able to
evaluate each chromatogram visually and quantitatively by comparing resolution or
symmetry factor numerically, which achieved higher data processing efficiency.
The Nexera Method Scouting System and Chiral columns used in this presentation are
considered to be useful in:
l pharmaceutical CMC;
l method development departments in the chemical or food industry; and
l drug discovery departments of active pharmaceutical ingredients
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