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  • Published: Oct 17, 2016
  • Source: Shimadzu Europa GmbH
  • Categories: Gas Chromatography / Base Peak
thumbnail image: <font size=3>Shimadzu Europa</font><br />Explore Shimadzu´s wide range of solutions to your separation problem

Supercritical fluid chromatography with MS detection
for the separation of novel chiral compounds

Authors: Schad, Gesa J.1, Van den Heuvel, Dennis2
1Shimadzu Europa GmbH, Duisburg,Germany
2Shimadzu Benelux B.V., 's-Hertogenbosch, Netherlands

www.shimadzu.eu, shimadzu@shimadzu.eu

Introduction

Projects in drug discovery and safety aim constantly 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].

Stereoisomeric composition of a drug with a chiral center should therefore be well documented. To evaluate the pharmacokinetics of a single enantiomer or any mixture of enantiomers, manufacturers must develop quantitative assays in the early stages of drug development for individual enantiomers.

Figure 1: Nexera UC SFC-MS chiral screening system

Figure 1: Nexera UC SFC-MS chiral screening system

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 an 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 flowrate 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).

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Figure 2: Graphical user interface of Method Scouting Solution

Figure 2: Graphical user interface of Method Scouting Solution

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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)
  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

Results

Figure 3: Chromatograms of screening results for unknown API 1 on 6 different columns using methanol as organic modifier

Figure 3: Chromatograms of screening results for unknown API 1
on 6 different columns using methanol as organic modifier

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Figure 4: MS data of isomeric compound for identification of enantiomers

Figure 4: MS data of isomeric compound for identification of enantiomers

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Figure 5: Chromatograms of optimized separations of five unknown APIs

Figure 5: Chromatograms of optimized separations of five unknown APIs

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Conclusions

  • 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 in terms of 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 alInt J Biomed Sci. 2006 Jun; 2(2): 85–100. Chiral Drugs. An Overview.

For more information please download the Shimadzu HPLC application handbook – free of charge

Free download of the poster application here.

Shimadzu Europa GmbH
Albert-Hahn-Str. 6–10, 47269 Duisburg, Germany
www.shimadzu.eushimadzu@shimadzu.eu

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