Impurities Analysis in Active Pharmaceutical Ingredients Comparison of Cold EI with Standard EI

Aviv Amirav, Tel Aviv University and Aviv Analytical, Tel Aviv Israel.

Introduction

Active pharmaceutical ingredients (APIs) in drug formulations need to have impurity levels < 0.1% according to the FDA or else the impurities need to be characterized via lengthy and expensive clinical toxicology procedures. Current impurities in APIs are typically analyzed by LC-MS. However, such LC-MS analysis is confronted by ion suppression effects for impurities that elute near the API, non-polar impurities are not ionized, those impurities that are discovered exhibit mostly protonated molecular ions without structural information and since Electrospray LC-MS has highly non-uniform ionization yields there is no information on the concentration of the discovered impurities. Thus, those API impurities that are observed need to be fully identified, synthesized and follow compound specific concentration calibration.

GC-MS with Cold EI is ideal for analysis of API impurities because:

• It has uniform compound independent response, thus detected impurity concentrations can be assessed, and those below 0.1% can be neglected 
• It often provides EI-based library identification, which is usually improved by the presence of an enhanced molecular in Cold EI plus structural information from the full display of fragment ions 
• Cold EI ionizes non-polar as well as polar analytes 
• It does not suffer from any ion suppression effects 
• Total ion mass chromatograms in Cold EI often provides greater sensitivity than ESI-LC-MS 
• Cold EI has much greater range of compounds amenable for analysis than any other GC-MS. 

Thus, Cold EI seems ideal for API impurities analysis.

In this application note we compare GC-MS with Cold EI with GC-MS with standard EI in the analysis of impurities in haloperidol drug powder. Haloperidol is used for the treatment of schizophrenia and other psychotic symptoms. Its elemental formula is C21H23ClFNO2 and its molecular ion mass is m/z 375.1396.

We show that Cold EI provides useful impurities analysis information and can thus serve for this application with major benefits over LC-MS. While GC-MS with standard EI is incompatible with the requirements of API impurities analysis, Cold EI meets the needs well.

Analytical conditions summary

GC-MS with Cold EI System: An Aviv Analytical 5975-SMB GC-MS with Cold EI system was employed, based on the combination of an Agilent 7890A-5975 GC-MSD with the Aviv Analytical supersonic molecular beam interface and its fly-through ion source. The system used has a six years old fly-through ion source that was never serviced and which still uses its original filament and ion cages.
GC-MS with Standard EI System: Agilent 7890B-5977B GC-MS with extractor ion source.

Samples: Haloperidol powder provided by AstraZeneca several years ago that was stored at 4ºC without use. The Haloperidol was dissolved in methanol at 900 ppm concentration

Injection: 1 µL split 10 at 250ºC in both Cold EI and Standard EI (90 ng on-column)     

Column: In standard EI 30 m 0.25 mm ID DB5MS-UI 0.25 µ film and in Cold EI the same column type but 5 m long.

He column flow rate: 1.2 ml/min in standard EI and 16 ml/min in Cold EI.     

Oven: In Cold EI 60ºC initial temperature immediately ramped at 30ºC/min to 300ºC and held until 9 min. In standard EI 50ºC initial temperature immediately ramped at 10ºC/min to 320ºC and held until 30 min. 

Cold EI Source: 7 mA emission, 70 eV electron energy and 60 mL/min combined He makeup gas plus column flow.

Standard EI Ion Source Temperature: 250ºC

Transfer line temperatures: For Cold EI 250ºC for 4 min followed by temperature program at 15°C/min to 280°C. For standard EI it was 280ºC 

Mass spectral range and scan speed: m/z 50-500 at 3.2 Hz scan frequency.

Summary of the results

As clearly demonstrated in Figures 1 and 2 Cold EI is superior to standard EI in all the performance aspects including:

1. Enhanced Molecular Ions: Cold EI provided abundant molecular ions to all the impurities as well as to Haloperidol while standard EI did not provide a molecular ion to any of the impurities. For Haloperidol itself Cold EI provides 18% molecular ion abundance while in standard EI is was 0.14% and masked by an equally abundant m/z=374 (M-1) (As shown in Figure 2). Typically, large and labile compounds (on the GC-MS scale) such as drugs and their impurities have weak or no molecular ions in standard EI MS. Thus, the feature of enhanced molecular ion in Cold EI is a must in impurities analysis since the impurities are not typically found in the library hence without having trustworthy molecular ions they can-not be identified.  
2. Improved Identification: The combination of enhanced molecular ions, low baseline and column bleed noise plus clear peaks resulted in far better impurities characterization. As an example shown in Figure 1, the arrow indicated impurity was identified with Cold EI by the NIST library as haloperidol – water (M-H2O, possibly via water elimination) as confirmed by the Cold EI molecular ion and fragment ions. We found molecular ions for all the observed impurities of m/z= 189, 327, 341, 355, 357, 371, 375 and 387. For example, the impurity with m/z = 371 exhibited the same fragmentation pattern as haloperidol thus it is concluded to be haloperidol minus 4 hydrogen atoms.            
3. Superior Chromatography: Cold EI exhibits much better chromatographic separation despite its use of a shorter column and higher column flow rate. The main reason for this is the elimination of ion source peak tailing for the polar impurities in the fly-through contact-free Cold EI ion source. The high column flow rate further increases the column sample capacity and thus reduces chromatographic peak fronting of the API.   
4. Superior TIC sensitivity: Cold EI exhibits over an order of magnitude better total ion mass chromatogram sensitivity. In addition, the ratio of impurities peaks to baseline noise was far better for Cold EI. 
5. Superior quantitation: Cold EI exhibits far better quantitation and enabled the quantitation of all the eight observed impurities while in standard EI no impurity could be quantified. The arrow indicated impurity was measured by the Agilent percent report as having 0.29% abundance.   
6. Faster Analysis: Cold EI resulted in three times faster analysis. 

In conclusion: GC-MS with Cold EI can effectively serve for API such as haloperidol impurities analysis while GC-MS with standard EI cannot serve for this type of analysis. Thus, API impurities analysis joins the list of over 50 types of analyses that are unique for Cold EI that as a result can significantly extend the range of compounds and applications amenable for GC-MS analysis and thereby increase the total GC-MS market.

Figure 1. A comparison of total ion mass chromatograms of Haloperidol (90 ng on-column) obtained by the Aviv Analytical 5975-SMB GC-MS with Cold EI (upper trace) and by Agilent 5977B GC-MS with standard EI (lower trace). In both cases the Haloperidol peaks were zoomed about 120 times. The arrows indicate the area from which the mass spectra at right of the most abundant impurity were obtained.

 

Figure 2. A comparison of Haloperidol mass spectra obtained with Cold EI (upper trace) and standard EI (lower trace). While the NIST identification probability was high in both cases Cold EI exhibits trustworthy molecular ion which is 130 times more abundant than of standard EI which is obscured. The Cold EI molecular ion further provides isotope abundance analysis matching factor of 996, which can serve to provide the elemental formula of haloperidol, while in standard EI it is completely distorted.