Chiral compound purification using superposition injection and collection of the mass spectrometric Prep 100 SFC system

Chiral compound purification using superposition injection and collection of the mass spectrometric Prep 100 SFC system

Steve Zulli, Dan Rolle, Ziqiang Wang (Ph.D.), Timothy Martin, Rui Chen (PhD) and Harbaksh Sidhu

Waters Corporation, Milford, MA, US

Application benefit

The use of superimposed injection mode for chiral compound purification demonstrates the versatility and flexibility of the collection scheme provided by the mass spectrometric Prep 100 SFC system. The open bed collection platform at atmospheric pressure provides higher efficiency and success rate when using multiple detectors including mass spectrometers for trigger collection.

Waters Solutions

Mass Spectrometric Prep 100 SFC System, Model 2998 Photodiode Array (PDA) Detector, Model 3100 Mass Spectrometer Detector, Model 2767 Sample Manager

MassLynxTM software, FractionLynxTM application management program, overlay injection module

Key words

Chiral, Prep 100 SFC, superimposed injection, mass spectrometry, open bed collection

introduction

According to FDA regulations1, chiral chromatography has become the tool of choice for the accurate identification of single pure enantiomers and separation by pharmacology, toxicology and clinical information early in drug development.

Supercritical fluid chromatography (SFC) has proven to be a mainstream technology for the separation of chiral compounds due to its higher efficiency, greater throughput and wider applicability. Chiral SFCs are getting more and more attention and their applications are expanding, and in some cases they are becoming the preferred method.

Typically, enantiomeric mixtures contain a certain amount of impurities that can reduce the efficiency of the actual purification process for common superposition injections and signal threshold based collection strategies (eg UV/PDA detection). In most cases, a one-step pre-purification is necessary, but it is not practical due to the existence of funding and workload constraints. This requires a versatile detection scheme that distinguishes the enantiomer from other impurities. In addition to UV/PDA detectors, the Model 3100 Mass Spectrometer is an ideal choice for a wide range of chiral separations.

In this application note, the mass spectrometric Prep 100 SFC system and its function of superimposing injection and collection on an open bed platform are demonstrated and proved to be an effective tool for the purification of chiral compounds. System configurations and methods for chiral separation cases are reviewed and described below.

test

Chemicals

CO2 is supplied by Airgas (Salem, NH, USA) and supplied to the mass spectrometric Prep 100 SFC system via a built-in pipe at approximately 1100 - 1300 psi in the form of a pressurized liquid. Methanol and trans cerium oxide (T SO, MW: 196) were supplied by Sigma-Aldrich (St. Louis, MO, USA).

SFC column

ChiralPak AD-H and ChiralCel OD-H (both 21 mm x 250 mm, 5 μm) were supplied by Chiral Technologies, Inc. (West Chester, PA, USA).

SFC system

The MS-guided Prep 100 SFC system is equipped with an additional superposition injector.

The Model 2767 Sample Manager is configured as a simplified repeat fraction collector.

Method condition

SFC gradient and flow rate program

For all of the data described, the maximum total flow rate of 100 g/min was used in conjunction with various isocratic modifier procedures.

Mass spectrometer conditions

The standard ESI mode for the Model 3100 Mass Spectrometer for various tests uses the following key parameters:

Capillary voltage: 3.5 KV

Taper hole voltage: 40.0 V

Secondary cone voltage: 3.0 V

RF lens voltage: 0.1 V

Source temperature: 150 ÌŠC

Desolvation gas temperature: 350 ÌŠC

Desolvent gas flow rate: 400 L / hour

Cone gas flow rate: 60 L / hour

A 0.1% formic acid-methanol solution was used as a compensating stream to enter the mass spectrometer to improve ionization efficiency.

Data management

MassLynx/FractionLynx, version 4.1

Results and discussion

Purification amplification in superimposed injection mode

A common best practice in chiral separations is to use a superimposed injection mode for sample injection and fraction collection, which maximizes efficiency and reduces production costs.

In complex systems containing certain impurities, mass spectrometry-guided systems can identify and selectively collect target compounds of interest and properly ignore unwanted impurities. Therefore, the system has high efficiency and wide application range for SFC purification of chiral compounds, and has become a routine mainstream tool for chiral drug development.

We have made some modifications to the mass spectrometric-guided Prep100 SFC system to maximize the benefits of this system for the separation and purification of chiral compounds, including the addition of a dedicated injector and changing the layout of the collection bed to accommodate larger The container is such that the fraction of the enantiomer can be collected repeatedly.

Cascade injection/injector activation

The Prep 100 SFC system incorporates a Waters superimposed sampling module. The user selects the “injection type” and enters the total number of superimposed injections and other relevant parameters in the software program, as shown in Figures 1 and 2. Run a custom injection sequence in a superimposed injection mode that extracts multiple equal samples from a single sample container.

When the superimposed injection mode is not used, the Model 2767 Sample Manager can continue to inject a single sample one by one from the rack in the order defined by the Sample List.

Figure 3 shows a typical chromatogram obtained by superimposing a bimodal mixture. UV and mass spectrometry are correct for the desired material, ensuring reliable and successful fraction collection by UV or mass spectrometry triggering. In this case, the UV signal is used as a collection trigger; the mass spectrum signal can also be used if necessary.

Customize the collection bed layout for a single vial

The mass spectrometric Prep 100 SFC system uses the Model 2767 Sample Manager as a dedicated fraction collector. In chiral compound purification, because the fraction collection is two (or in some cases up to four), it is necessary to replace the conventional type of test tube in one-to-one mode with a larger container and a repeating front and back collection mode. frame.

Therefore, the Model 2767 Sample Manager can be customized by defining the location of the collection and larger containers. Thus, the results of all the superimposed injection sequences of the same enantiomer can be collected into the same collection bottle by repeated pre- and post-collection methods.

As shown in Figure 3, the two enantiomer fractions were collected into a No. 1 bottle (pink strip) and a No. 2 bottle (green strip). This is done in a repeating mode on the Model 2767 Sample Manager based on a single injection line within the sequence. This demonstrates that the process of sample collection using the Masslynx software and the Fractionlynx sample manager was successful and met the key criteria for proper identification and collection of signal intensity levels based on the pair of enantiomers.

Figure 4 shows an example of separation and selective collection of a system containing unrelated impurity peaks and enantiomeric pairs. As indicated by the colored bands, only two separated target compounds were collected by mass spectrometry of the target compound, while the third peak (unrelated impurities) was not collected.

MassLynx/FractionLynx The AutoPurifyTM platform has a number of advanced detection and collection algorithms for complex workflows, such as Boolean logic algorithms that use multiple detector signals for triggering. If the sample is pure enough, the user can choose to use UV/PDA for detection; if the sample contains a significant amount of impurities, the user can choose to use a combined signal and slope algorithm and a specific target molecular weight to ensure a purer collection of fractions. .

in conclusion

The mass spectrometric guided Prep 100 SFC system has been shown to be highly efficient, adaptable and versatile in the development of different drugs. The additional features of the mass spectrometric Prep 100 SFC system superimposed injection and collection described in this paper make it more customizable for chiral separations, which can benefit chromatography analysts in purification laboratories, such as:

â–  Multiple, multi-function detection mode achieves a higher success rate;

â–  The same superimposed injection and collection mode based on an open bed platform simplifies usage;

â–  Provides a safer laboratory environment that complies with industry and government regulations.

The Waters MS-guided Prep 100 SFC system is a powerful tool for chiral purification in drug discovery and other preparative chromatography to meet the needs for greater productivity and higher success rates.

references

[1] http://

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