by Brandon Sharp, Ph.D.
Impurities such as residual solvents, extractables, and leachables may contaminate drug products at any point during manufacturing. There are a variety of analytical techniques that can provide information about the chemical identity of these impurities, but they are not all equally effective for every type of analyte. For example, pure chromatography-based techniques such as headspace gas chromatography with flame ionization detection (HS-GS-FID) can detect residual solvents in pharmaceuticals, but unknown peaks are sometimes found. Coupling gas chromatography with mass spectrometry, i.e., GC-MS, provides an enhanced method for qualitatively analyzing and identifying such unknown peaks compared with chromatography alone. However, there are many variables that will affect the success of this analysis. Here are some of the most important “dos” and “don’ts” to help ensure the success of your GC-MS analysis.
Do Perform Derivatization if Necessary
Headspace analysis GC-MS can be performed directly to analyze the volatiles content of a sample. However, a more complex sample preparation procedure involving derivatization may be necessary to examine extractables and leachables when organic extractables from aqueous extracts have poor thermal stability or when they are non-volatile or semi-volatile. Often, this involves a step-wise extraction procedure first involving aqueous extraction, followed by extraction using an organic solvent such as dichloromethane. The organic extract is then derivatized, often using either trimethylchlorosilane (TMCS) or N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).
Don’t Use Wildly Different Polarities
As most analytes are typically dissolved in a solvent prior to analysis, a large amount of solvent is also typically injected. When the polarity of the solvent is significantly different from that of the stationary phase, the solvent may form “beads” instead of evenly wetting the stationary phase. This affects the retention time and may lead to peak tailing, splitting, or even the generation of multiple peaks for the same target analyte. In this case, the best option will be to dissolve the analyte in a solvent with a polarity more similar to that of the column.
For example, if you initially dissolved your analyte in hexane and are using a moderately polar stationary phase (e.g., 6% cyanopropyl-phenyl, 94% dimethyl polysiloxane), consider switching to a more polar solvent such as ethyl acetate. Solvent mixtures (such as hexanes/ethyl acetate) can also be used to achieve a different polarity if the analyte is insoluble in the new solvent.
Do Consult USP to Provide Guidance for Developing a Procedure
USP chapters <1663> (extractables) and <1664> (leachables) provide general frameworks for designing, performing, and validating extractables and leachables assessments for pharmaceutical containers. USP also updated its chromatography chapter <621> in 2022 to provide harmonized standards that specify adjustments that are allowed to chromatographic systems while still maintaining compliance with standard procedures. Users should verify that changes to a method by assessing analysis performance under the “System Suitability” section of chapter <621>. The USP-NF provides a list of brand names of columns used to validate chromatographic procedures and their possible alternatives at www.uspchromcolumns.com.
Don’t Analyze Thermally Degradable Compounds
Because GC-MS is performed at higher temperatures (150–300°C), compounds that undergo thermal degradation at analysis temperatures may be difficult or impossible to analyze. Sometimes, thermal degradation may be unavoidable, such as when using high-temperature extraction for headspace-GC-MS analysis, which may induce the thermal degradation of some polymeric components. This often produces complex chromatograms that are notoriously difficult to analyze.
Do Use Dry Solvents
GC-MS is highly sensitive to water, which can affect retention times, peak shapes, and can even cause peak splitting of the target compounds, resulting in inaccurate results. Care should be taken to use dry solvents such as HPLC-grade solvents, or solvents that have been double-distilled or otherwise dried to remove water. It is also recommended to use dried glassware during sample preparation, as ambient water can adsorb onto the glass surface.
Don’t Damage the Column
Although this might seem like obvious advice, the forms of damage to GC columns might not be immediately apparent. For example, scratching the column during use or storage may generate stresses that cause the column to prematurely crack. Impurities and non-volatiles can also damage the column. Despite your best efforts to remove them these impurities may ultimately be injected into the GC column, where they may clog the column or strongly and irreversibly bind to the stationary phase, decreasing the column’s efficiency and leading to a lower resolution.
Properly storing the column and using a guard column prevents non-volatile compounds and impurities from entering GC columns. It is comparatively much easier and cheaper to replace a guard column instead of a GC column. Users should also remember to replace the guard column periodically, often when there is a notable increase in system pressure, which indicates guard column clogging.
Do change the column length to improve the resolution
The number of theoretical plates increases proportionally with increasing column length. Keep in mind that this will also increase the backpressure of the column and may exceed the pressure limit of the instrument and will also increase the run time of the separation. Furthermore, the resolution increases according to the square root of the column length, according to the Purnell equation, so doubling the column length will not provide twice the resolution.
Don’t Forget to Conduct a Literature Review
Each sample will require a different preparation method for GC-MS analysis depending on the compounds being analyzed. There is a wealth of information available in the published literature about how to prepare samples, and there will be identical or similar samples or standard methods available in the literature to guide your sample preparation and analysis procedure. Extractables and leachables are typically well-known compounds, so consulting with a mass spectra database, such as the one maintained by the NIST Mass Spectrometry Data Center or the Polymer Additives Library, will also help with peak assignment.
GC-MS analysis is affected by numerous variables, many of which may be unintuitive. Following the above “dos” and “don’ts” can provide users with some basic considerations to enhance the performance of their extractables and leachables assessments, as well as any other GC-MS analysis.
References:
- GCMS Qualitative Analysis of Impurities Detected in Tests for Residual Solvents in Pharmaceuticals; Shimadzu; https://www.shimadzu.com/an/industries/small-molecule-pharmaceutical/development/gcms-qualitative/index.html (accessed 1-15-2024).
- Avoiding Solvent Phase/Polarity Mismatch; Phenomenex; https://discover.phenomenex.com/0521-gc-technical-tip-en?elqTrackId=2315b27f1a814371a7c1fca912e02f4e&elq=00000000000000000000000000000000&elqaid=2098&elqat=2&elqCampaignId (accessed 1-15-2024).
- Pharma materials study: GC-MS identification of extractables and leachables from elastomer material; Thermo Scientific; https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/AN-10398-GC-MS-Identification-Extractables-Leachables-Elastomer-AN10398-EN.pdf (accessed 1-15-2024).
- Jenke D.; Christaens P.; Verlinde P.; et al.; Good Identification Practices For Organic Extractables & Leachables Via Mass Spectrometry; Nelson Labs; https://www.nelsonlabs.com/wp-content/uploads/2020/12/Good-ID-Practices_Part4-002.pdf (accessed 1-16-2024).
- Jones, J.; Stenerson, K.; The Use of Derivatization Reagents for Gas Chromatography (GC); https://www.sigmaaldrich.com/TW/en/technical-documents/technical-article/analytical-chemistry/gas-chromatography/the-use-of-derivatization (accessed 1-16-2024).
- USP; USP General Chapter <621>, “Chromatography”; https://doi.org/10.31003/USPNF_M99380_06_01.
- Steven A. Zdravkovic, Solid phase extraction in tandem with GC/MS for the determination of semi-volatile organic substances extracted from pharmaceutical packaging/delivery systems via aqueous solvent systems, Journal of Pharmaceutical and Biomedical Analysis, Volume 112, 2015, Pages 126-138, ISSN 0731-7085, https://doi.org/10.1016/j.jpba.2015.04.031.
About the author
Brandon Sharp, Ph.D., is a freelance technical content writer with hands-on experience designing photoresists and other organic materials for advanced lithography applications.