Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Homoaromaticity wikipedia , lookup
Kinetic resolution wikipedia , lookup
Polythiophene wikipedia , lookup
Baylis–Hillman reaction wikipedia , lookup
Asymmetric hydrogenation wikipedia , lookup
Petasis reaction wikipedia , lookup
Strychnine total synthesis wikipedia , lookup
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.