Simultaneous ESI/APCI Measurement with a Dual Ion Source

Every day, researchers synthesize and test large amounts of compounds in the chemistry laboratories of pharmaceutical and chemical manufacturing companies. In these labs, many LCMS instruments run simultaneously. Although software improvements can enhance the efficiency of analytical throughput, software alone can not improve the analysis.

The question concerning researchers is whether the LCMS really detects synthesized compounds. Addressing this issue requires the application of new technology linked to the LCMS hardware.

In LCMS measurement, the polarity of the target compounds determines the selection of the ionization mode. For example, electrospray ionization (ESI) generally is selected for analyzing high-polarity compounds typically found in drugs and pesticides. Atmospheric pressure chemical ionization (APCI) is selected for compounds having lower polarity. (Figure 1.)

When analysts know the properties of a substance, selecting the appropriate ionization mode is relatively easy. However, for synthesis researchers who handle a wide variety of compounds, the time required for selecting the best ionization mode for each compound can have a negative impact on research and development efficiency because it may be difficult to predict the best ionization method for a variety of samples. Additionally, because each ionization approach can produce a variety of background and adduct ions, the question remains as to whether the synthesized compound is actually detected.

For many analysts, the primary goal is to achieve detection of the target compounds as quickly as possible. However, acquiring data using both ESI and APCI ionization modes generally requires the use of an ion source (probe) specific to each of the modes. For a researcher looking for any way possible to shorten analysis time, installing a second probe and re-running an analysis seems like a waste of time.

A Single Analysis for ESI and APCI Acquisition

For the researcher looking for ways to shorten the time required for analysis and

Figure 1: Selection of Ionization Mode with Respect to Analyte Polarity and Molecular Weight

method development, a technique combining two different ionization modes would significantly improve laboratory efficiency and confidence in results. How might sample analysis be conducted using both ESI and APCI ionization concurrently without severely compromising either technique?

Due to the substantial differences in the ionization mechanisms of ESI and APCI, achieving this goal requires significant development. Ionization is produced in ESI by applying a high voltage to the capillary through which a stream of ionized analyte molecules exits in nebulized droplet form. In APCI, reaction ions are formed by the electrical discharge around a high voltage corona needle. These ions react in the gas phase with neutral analyte molecules to produce ionization.

Because ESI and APCI probes are each designed to optimize their respective technique, a new technology would be required to integrate both of these probes to achieve simultaneous measurement of ESI and APCI data.

The DUIS-2020 is a dual ion source (DUIS) consisting of an integrated probe for analysis using both ESI and APCI techniques. The ionization flow using the DUIS is as follows: a) Sample (including mobile phase) is introduced into the probe. b) In the ESI probe, the liquid is nebulized into a fine spray. c) As a drying gas is used to evaporate the nebulized spray, ionization by APCI is produced by the corona needle. d) The sample ionized by the ESI and APCI techniques passes through the desolvation line to enter the vacuum region.

ESI and APCI ionization are executed concurrently and continuously with the DUIS without relying on switching between modes. Software control of the individual

Figure 2: Number of Acquired Points Affects Peak Shape

Figure 3: Analysis of Water Soluble and Fat Soluble Vitamins (3-Vitamin Mixture)

No. Of acquired points: 20 points No. Of acquired points: 4-5 points

continued from page 18

ESI and APCI voltages allows the chemist to easily select ESI, APCI or DUIS ionization without changing hardware. Furthermore, the standard ESI probe is used in the DUIS interface, permitting analysts to achieve uncompromised ESI performance.

Optimizing for Ultra-High Speed Analysis

The introduction of ultra-high speed analysis has proven effective for improving research and development efficiency. However, along with the benefits of greater efficiency comes the importance of minimizing the loss of data points (digital points acquired during analysis) due to the narrow peak widths associated with fast LC.

Polarity switching between negative ion detection and positive ion detection is commonly necessary for unknown or complex samples. Modern improvements in chromatography demand that polarity switching times be only a few milliseconds because even a slight reduction in the number of acquired points can result in reduced sensitivity and distorted peak shapes. (Figure 2.)

The DUIS interface combines rapid polarity switching with continuous ESI and APCI ionization, allowing sufficient acquisition of data points across even the narrowest peaks. Whereas polarity switching was previously accomplished by switching positive and negative high-voltage power sources arranged in parallel, the Shimadzu LCMS-2020 adopts a new technology (patent pending) in which polarity switching is conducted using serially arranged positive and negative high-voltage power sources. This ultra-fast switching technology achieves 15 ms polarity switching.

Water Soluble and Fat Soluble Vitamins (3-Vitamin Mixture) 1. Thiamine: m/z 265, Cation, generated through ionization, water soluble vitamin 2. Riboflavin: m/z 377, Protonated molecular ion, water soluble vitamin 3. Calciferol: m/z 397, Protonated molecular ion, fat soluble vitamin

Improved Throughput

Figure 3 shows the MS chromatograms obtained from the analysis of a sample consisting of water-soluble vitamins, thiamine and riboflavin, and the fat-soluble vitamin calciferol. Analysis was first conducted by ESI and by APCI individually, and then by the DUIS in dual mode.

The results show that good sensitivity is obtained for thiamine and riboflavin using ESI, but that they are scarcely detected using APCI. Additionally, calciferol is detected using APCI, but ionization is insufficient using ESI. When DUIS is employed, however, well-balanced MS spectra are obtained.

In conventional systems, the same analysis must be repeated after exchanging the probe, but by using DUIS, the analysis is completed in a single run. This demonstrates the power of DUIS in providing laboratories with a tool for faster method development and higher sample throughput.

Conclusion

To achieve a rapid and thorough mass spectral analysis of complex or unknown samples, it is imperative to develop a system that results in a high probability of detection. Because compounds preferentially ionize based on both polarity and ionization methods, it is critical to develop techniques that support multiple methods.

Traditional dedicated ionization sources can hinder high-throughput analysis such as metabolic screening by limiting the number of samples that can be examined within a set timeframe. By combining ESI and APCI into a single run, researchers can reduce instrument time by half, allowing a greater number of samples to be analyzed.

Chris Gilles is the LCMS Product Manager at Shimadzu Scientific Instruments

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The biosimilar company must ultimately justify that all the patents are invalid, non-infringed or expired. If this is not done, then the biosimilar will not receive an NOC until the patents expire.

continued from page 15

If the patent owner and biosimilar manufacturer disagree about freedom to operate, a court case known as an NOC Proceeding will assess evidence about patent infringement and validity. The court will then decide whether to prohibit Health Canada from issuing the biosimilar a NOC because of the risk of patent infringement. No NOC can be issued while the court is considering the issues, which can take up to 24 months. If the biosimilar company wins, it gets its marketing authorization. If the biosimilar company loses, it has to wait until patent expiry to get its NOC. The biosimilar litigation landscape is complex because a conventional patent infringement or invalidity lawsuit may be initiated concurrently with, or after, a NOC Proceeding.

The NOC Regulations have been extensively litigated in the context of conventional, small molecule pharmaceuticals. No NOC Proceeding has been completed for a biosimilar yet. However, the NOC Regulations will be an important biosimilar patent protection tool once biosimilar development increases.

Europe does not have patent regulations linked as a precondition to a marketing authorization. Biosimilar manufacturers can get their marketing approval without addressing patents. In the US, a complex patent clearance system has been implemented as a precondition to biosimilar approval. The biosimilar manufacturer must disclose its regulatory submission to the reference product sponsor. The reference product sponsor then provides a list of patents it believes could be infringed. There are opportunities during the process for settlement negotiations and a patent infringement lawsuit.

Regulatory Exclusivities

Canada, the US and Europe all have data exclusivity. During the exclusivity period, the pioneer biologic data cannot be relied on by a biosimilar company seeking approval of its own drug. In Canada, innovative biologic drugs may be eligible to receive an 8 year period of data exclusivity. Slightly modified versions of previously-approved biologics will not be eligible. An additional six months of data exclusivity is available for biologics that have been the subject of pediatric studies. The intent is to allow the brand name company a period of exclusivity to recoup its R&D investments in the biologic. A biosimilar manufacturer cannot even have Health Canada review its drug submission during the first six years of the data exclusivity period.

Conclusions

With the arrival of the first SEB in Canada, SEBs are undoubtedly here to stay. Health Canada has created guidance documents to facilitate approval of SEBs, which will increase competition in the biologic marketplace. The extent to which a SEB will be allowed to rely on the pioneer biologic data will vary considerably depending on the nature of the product. Awareness of patent and regulatory exclusivity strategy will become increasingly important for both pioneer companies and SEB manufacturers. Lastly, it is important to ensure that all stakeholders are educated to make well-informed decisions with the goal of patient safety as the top priority.

References:

1. Schachter H. The ‘yellow brick road’ to branched complex N-glycans. Glycobiology 1991;1(5):453-61. 2. Impact of manufacturing changes. Biotechnology Advances 2007;3(25):325-31 3. Schellekens H. Biosimilar epoetins: how similar are they? Eur J Hosp Pharma Sci 2004;3:43-47 4. Health Canada. Information and submission requirements for subsequent entry biologics (SEBs) and related documents, 2010. Available at: http://www.hc-sc. gc.ca/dhp-mps/brgtherap/applic-demande/guides/seb-pbu/notice-avis_sebpbu_2010-eng.php (accessed May 10, 2011). 5. Health Canada. Fact sheet: Subsequent entry biologics. Available at: http://www. hc-sc.gc.ca/dhp-mps/brgtherap/activit/ fs-fi/fs-fi_seb-pbu_07-2006-eng.php (accessed May 10, 2011). 6. BIOTECanada. SEBs, Biosimilars, FOBs:

Understanding the clinical implications and international experience. Available at: http://www.biotech.ca/uploads/ sebs/belin-110909.pdf (accessed May 10, 2011). 7. European Medicines Agency. Guideline on similar biological medicinal products, 2005.

Noel Courage is a partner in the intellectual property law firm, Bereskin & Parr LLP.

Eva Furczon is freelance consultant specialized in biotechnology, D5Pharma Consulting and oncology specialist at Roche Canada.

Acknowledgements:

The scientific content of this paper has benefited from the input and support provided by Dr. Leigh Revers, University of Toronto.

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