Kenichiro Tanaka, William Hedgepeth, Miho Kawashima, Tadayuki Yamaguchi, Hidetoshi Terada and Yoshihiro Hayakawa Shimadzu Scientific Instruments, Inc., Columbia, Md., USA �

Rapid Method Scouting of Chiral Compounds

2 / 14 �

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.

3 / 14 �

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.

4 / 14 �

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

5 / 14 �

System

Figure 1: Flow diagram of the Nexera Method Scouting System developed in this experiment

6 / 14 �

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.

Nexera Method Scouting System

7 / 14 �

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

8 / 14 �

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.

9 / 14 �

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.

No.   Mobile  phase   Flow  rate   Time   Remarks  

1   Hexane  /  2-­‐Propanol  =  9  /  1　（v/v） 3  mL/min   9  min  

Temp.:  40  °C  Inj.  vol.:  10  µL  Det.:  230  nm  

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 4  min

Note: Diethylamine was added to each mobile phase at 0.1% in the Methylclothiazide analysis Table 1: Analytical Conditions for Chiral Analyses

10 / 14 �

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.

Figure 3: Graph of analysis result with Excel

Ranking

11 / 14 �

Data Processing

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

The “CLASS-Agent Report” software can compare the data both visually and quantitatively, helping to make data processing more efficient.

12 / 14 �

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

13 / 14 �

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

Thank you for viewing this presentation. Should you have any questions or require additional information about our research, products or services, please visit our support page: www.ssi.shimadzu.com/support/

@shimadzussi

Follow us on Twitter

Need More Info?