A new method may speed the process of separating and quantifying anions and cations.
The separation and quantification of anions and cations is critical during the pre-formulation phase of drug development when the correct salt form is identified; it is important in other stages as well. The most popular current method, ion chromatography (IC) with conductivity detection, uses relatively expensive non-standard chromatography instruments and consumables and requires two runs with different columns to measure anions and cations. A new method uses hydrophilic interaction chromatography (HILIC) with a charged aerosol detector (CAD) on standard instruments with standard consumables to measure anions and cations in a single run. The new method offers excellent sensitivity, more consistent response, wide dynamic range, and superior reproducibility.1
Importance of ion analysis
Approximately half of all active pharmaceutical ingredients (APIs) are administered as salts.2 During drug development, the selection of the correct salt form early in the development process can prevent repeating toxicology, biological, and stability studies. Quantifying the ionic ingredients of pharmaceutical products plays a critical role in the process of discovering, developing, and manufacturing these drugs. IC, a common current approach for measuring ions, uses weak ionic resins for its stationary phase and an additional neutralizing suppressor column to remove background eluent ions. Measuring both anions and cations requires a lengthy changeover time or duplicate systems. The operator must receive special training and may require special certification. IC consumables can be quite costly.
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HILIC is a variation of normal phase chromatography that uses very polar stationary phases such as diol (neutral), silica (charged), amino (charged), or zwitterionic (charged). The mobile phase is highly organic but contains a small amount of aqueous/polar solvent. This establishes a stagnant, enriched water layer around the polar stationary phase allowing analytes to partition between the two phases based on polarity. Water or polar solvent is the strong eluting solvent. With zwitterionic columns and chromatographic conditions, the partition function between the two phases permits easier access for electrostatic interaction of anionic analytes to the positively charged group, enhancing anion retention. Risley and Pack used a zwitterionic stationary phase operating in the HILIC mode for the separation and quantification of 33 commonly-used pharmaceutical counter-ions, 12 cations, and 21 anions.3
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Risley and Pack used evaporative light scattering detection (ELSD) to detect these pharmaceutically-relevant salts. However, ELSD requires optimizing the nebulizer and evaporation tube, which makes this method less than ideal. ELSDs also do not have the high sensitivity, dynamic range, reproducibility, and robustness desirable in a workhorse instruments in critical pharmaceutical applications. While mass spectrometers are typically found in drug discovery research labs, some companies have found that their cost, complexity and fragility makes them difficult to deploy widely as routine analysis tools for product formulation and production. HPLC configured with ultraviolet (UV) detection is a relatively easy technology to use but it cannot detect ions.
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Innovation with the charged aerosol detector
A scientist in the analytical research and development department of one of the world’s leading pharmaceutical companies combined the separation advantages of HILIC with a new detection method. The charged aerosol detector (CAD) method is applicable to both the pharmaceutical development laboratory and to the QA/QC process. The HILIC/CAD-based method measures anions and cations in a single run, exceeds the measurement accuracy and reproducibility of IC for this application, and offers much higher sensitivity and dynamic range than HILIC/ELSD methods. Besides excellent sensitivity, the CAD shows consistent inter-analyte response independent of chemical structure, enabling quantification across a range that exceeds four orders of magnitude.
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The CAD detector works as illustrated in Figure 1. The HPLC column effluent is pneumatically-nebulized, typically with nitrogen. The largest of these droplets impinge upon an impactor where they collect and drip to waste. This results in only the smaller droplets passing into a drying tube where the mobile phase evaporates, thus producing analyte particles. The higher the amount in the chromatographic peak, the larger the particles. A secondary stream of nitrogen gas acquires positive charge as it passes a high voltage corona wire. The stream of charged nitrogen passes through an orifice into a mixing chamber forming a jet that collides with the opposing jet of analyte particles. As the two streams collide and are mixed, the charge is diffusionally-transferred to the analyte particles. The larger the particle the greater the number of charges it accepts. After leaving the mixing chamber, high mobility excess nitrogen ions are removed by a negatively-charged, low voltage ion trap. The charged analyte particles pass by the ion trap and they impinge on the collector. The charged particles transfer their charge to the collector. The current to neutralize this charge is measured by a highly sensitive electrometer, which in turn generates a signal that is recorded as the detector output. Table 2 shows a HILIC/CAD method for measuring Cl- and Na+ ions in a single run. Figure 2 shows how six different injection concentrations were done in triplicate. Table 3 shows that the correlation coefficient for linearity of response was found to be >0.998 for each ion. Figures 3 and 4 show the calibration curves for Cl- and Na+, respectively.
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Measuring Inorganic Ions
Figure 5 Chromatograms showing the separation of a variety of anions and cations in a single run as presented by Huang et. al. of Bristol-Myers Squibb.4 The separation can be modified to enhance the separation buy changing organic solvent to methanol. Table 3 shows the HPLC conditions used to produce the chromatograms. Figure 6 shows counter-ion analysis of chloride ion in two proprietary APIs. Table 4 shows the CAD results compared to results by other methods. The %RSD for API-2 was 2.2 with the other method and 0.32 with CAD. The %RSD for API-2 was 2.2 with the other method and 0.32 with CAD. The method precision was also better with CAD than the other method for API-4 and API-6.
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Excipient analysis
Huang et. al. also performed excipient analysis on a magnesium stearate standard solution and a table containing 1% magnesium stearate as shown in Figure 7. The HPLC conditions used in this analysis are shown in Table 5. Table 6 shows that the injection repeatability for 6 replicates was 1.15 %RSD. Table 7 shows the precision of the method was 2.30% RSD for a 1% magnesium stearate ground powder mixture, 2.30% RSD for 1% magnesium stearate tablets, 1.21 %RSD for 1% magnesium stearate placebo powder, and 0.99 % RSD for 1.25% magnesium stearate in DP-1 tablet.
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Conclusion
These results demonstrate the advantages of the HILIC/CAD technique for ion analysis. Both anions and cations can be quantified at the same time. Accuracy and dynamic range are comparable or better than with IC. This technique uses a standard HPLC system. The response is consistent and reproducible from low nanograms to micrograms. A strong correlation has been demonstrated between theoretical ion levels and those obtained using the CAD method.
About the Author
Jerry Fireman is a technical writer based in Lexington, Mass., who has published many articles in the fields of drug discovery and development, pharmaceutical manufacturing, and medical device design, and manufacturing.
1. ESA Biosciences, Application Note: “Counter Ions in Pharmaceutical Preparations.”
2. Lokesh Kumar, Aeshna Amin, Arvind Bansal, Pharmaceutical Technology. 2008;March 2.
3. D.S. Risley, B.W. Pack, LCGC North America. 2006;August 1.
4. Huang et. al. Bristol-Myers Squibb. ESA Biosciences Corona CAD User Forum. September 2007.
5. Brian Forsatz, Nicholas H. Snow, LCGC North America. 2007;September 1
| Table 6: Injection Repeatability – magnesium stearate (0.0825 mg/mL) | ||
| Mean Peak Area | %RSD | |
| 6 Replicate Injections | 2772946 | 1.15 |
| (Source: Huang et. al.) | ||
Filed Under: Drug Discovery
