The Benefits of Combining UHPLC-UV and MS for Peptide Impurity Profiling

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BioPharm International, BioPharm International-02-01-2019, Volume 32, Issue 2
Pages: 35–37

Determining a peptide’s purity is challenging because impurities are often structurally similar to each other and the API and can be present at very low concentrations. New approaches offer a solution.

In the manufacture of synthetic peptides, accurately identifying and quantifying impurities is essential to ensure product safety and quality. However, determining a peptide’s impurity profile can be challenging, because impurities are often structurally similar to each other and to the active ingredient, and can be present at very low concentrations in the product. 

Highly sensitive and selective analytical techniques are needed in order to identify and quantify potentially unknown impurities in peptides. Recent advances in high- performance liquid chromatography (HPLC) and mass spectrometry (MS) can enable accurate and reliable peptide impurity profiling in routine quality control (QC) workflows. This article discusses how combining these technologies can improve impurity analysis and optimize peptide production.

The importance of impurity profiling

Impurity profiling is a vital step in the production of any pharmaceutical or biotherapeutic product because it enables manufacturers to confirm product quality. Impurities include any substance that is not the API or an intentionally added excipient with a known safety profile. Potentially generated from the API and/or the excipients, impurities can be trace components of the starting materials, substances produced as a result of synthetic processes (e.g., intermediates or by-products of side reactions), or degradation products resulting from the product’s interaction with light, moisture, oxygen, or the excipients themselves (1).

Because many impurities are derived from or closely related to the API, they may have pharmacological effects, which can reinforce or decrease product efficacy, or can even have toxic effects on patients taking the product. Furthermore, because the impurity profile often depends upon the method of synthesis and the environmental conditions, the type, number, and volume of impurities present will typically vary between batches. If impurities are physiologically active, the side effect profile may be inconsistent and difficult to predict. It is crucial, therefore, to minimize this risk by measuring and characterizing impurities accurately to ensure that their levels fall within strict regulatory limits. Monitoring the product impurity profile also allows companies to reduce long-term risks by optimizing reaction conditions so that impurities will not form in the first place. 

Improved synthetic peptide impurity profiling, therefore, allows companies to better assure ... [product] safety and efficacy.

To ensure regulatory compliance, pharmaceutical and biotechnology companies must confirm that impurities do not exceed strict levels. Regulatory limits will be set for ‘specified impurities’ including major degradants, starting materials, intermediates and synthetic by-products. In addition to these limits, drug developers must comply with additional guidelines such as those set by the International Council on Harmonization (ICH) (2). Pharmaceutical and biopharmaceutical manufacturers are accountable for ensuring that product impurity levels do not exceed regulatory limits, and that the product will be stable and will not degrade. Synthetic peptide manufacturers must ensure that they can meet these regulatory requirements by employing highly accurate, precise, and reliable analytical methods to determine product purity, and identify and quantify all impurities present. Underestimating the impact of those impurities can have obvious consequences for consumer safety, while overstating their risk can result in safe and effective products being rejected. 

Challenges of analyzing impurities in peptides

However, analyzing impurities in synthetic peptide production presents specific challenges for analytical technologies to overcome:

  • Impurities in synthetic peptide products are often structurally similar, both to the active ingredient and to each other, so techniques must have sufficient resolution to distinguish between molecules of very similar size and mass.

  • Product-related impurities are often unknown, so advanced analytical techniques are needed in order to reliably identify them.

  • Impurities may be present at very low concentrations, so analytical techniques must be sufficiently sensitive to be able to detect and quantify analytes at trace levels.

HPLC for impurity profiling: advantages and limitations

HPLC coupled to ultraviolet (UV) detection is one of the most commonly used separation techniques for impurity profiling (3). The high throughput and reproducibility of HPLC suits the technique well for separating structurally similar impurities, while the high sensitivity offered by UV detection allows trace levels of impurities to be quantified with confidence.  

While HPLC-UV is widely used for impurity profiling because it is easy to use, reliable, and robust, it suffers from the following limitations:

  • The chromatographic separation achieved using conventional HPLC-UV systems may not be sufficient to distinguish between similar impurities, particularly when analytes coelute. 

  • If analytes elute with the same or similar retention times, it is not possible to reliably distinguish and identify them by UV detection alone. As a result, unknown impurities cannot be identified solely with UV detection. 

  • The sensitivity of conventional HPLC-UV analysis methods is not always sufficient to detect and quantify trace levels of impurities. Some analytes also have poor UV absorption and are not reliably detected by this technique.

Novel workflows that combine HPLC-UV and MS have been developed to overcome these challenges (4). Using the powerful separation capability of HPLC, and the high sensitivity and selectivity of MS, these workflows offer improved analytical sensitivity and specificity, and can facilitate the identification of unknown analytes, thus helping to overcome the challenges of peptide impurity analysis. The ability to use UV and/or MS to quantify impurities can also give the user more flexibility and confidence within the workflow, and the ability to react to unexpected circumstances, such as the detection of additional impurities or contaminants.

To improve synthetic peptide impurity profiling, single quadrupole MS can be used to analyze the compounds eluting from HPLC-UV, so that the analytes are separated based on their mass-to-charge ratios. MS analysis allows a mass to be assigned to each peak on the HPLC chromatogram, and this information can be used along with retention time to allow for more reliable identification. Employing MS data in this way allows analysts to distinguish between coeluting compounds.

The purity of specific chromatogram peaks can be confirmed by running a full MS scan and checking for additional mass peaks that would indicate coelution. Full-scan MS can also be used to detect unknown impurities, which can be identified by searching spectral libraries. Searches can be narrowed down depending on the nature of peptide products and excipients, giving consideration to possible starting materials, intermediates, degradants and by-products.

In addition to simplifying analyte identification, using MS alongside HPLC-UV can also improve confidence in trace analyte quantification. As a rule, UV detection is preferred for quantification, where possible, because the strength of the MS signal can vary depending on the ionization efficiency of the individual analytes, which can be affected by the coelution of matrix components. However, when analytes are present at trace levels, their concentrations can be so low that they approach the limit of UV detection. In these cases, MS can provide a more sensitive technique for quantifying analytes that may not be detected by HPLC-UV alone. Workflows coupling the benefits of HPLC-UV and MS can therefore allow the most appropriate technique to be used to quantify each analyte.


Increased accuracy 

Because HPLC-UV and MS provide complementary information, combining these techniques can result in very accurate synthetic peptide impurity profiling. MS provides the mass-based information used for the identification of impurities, while HPLC-UV is used to quantify those impurity levels, where possible.

The extended mass range of newer single-quadrupole mass spectrometers permits better detection of both small and large molecules, and enables the detection of the complete charge state profile for peptides. As a result, quadrupole-based mass spectrometers can resolve isotopic masses of large peptides, providing extra information to support analyte identification.  

Analyte identification is also being improved thanks to the advent of advanced software solutions. The latest software platforms have improved spectra analysis features, such as improved peak picking algorithms, different noise reduction algorithms, adduct selection options and adjustable mass accuracy thresholds.

Employing optimized settings for matching against reference mass spectra increases confidence in identification and minimizes false positives. Reference spectra can be selected from a library or from an existing sample, and matched against data obtained for unknown analytes. This extra information permits better target identification, allowing the method to be optimized. Using these reference spectra also enables the software to compensate for shifting retention times, allowing for correct identification even in situations where experimental conditions change. 

The system can identify the closest match to the reference spectrum at an approximate retention time, whereas previous software programs that only used retention time had a higher risk of mis-identification. Using reference spectra also saves time when screening for unknowns, as tentative identification can be confirmed by retention times after injecting a standard. 

With ‘intelligent’ software programs, user-defined variables can be monitored and tested, with different actions automatically taken depending on whether the test is passed or failed. This means the system can optimize its efficiency, reducing the requirement for analyses to be re-run. Injections are also automatically synchronized with the pump cycle, resulting in higher retention time reproducibility. 

Calculating settings

Finally, some software solutions can calculate the required settings to adapt parameters when switching from HPLC to ultra-high performance LC (UHPLC), enabling the system to be upgraded to the highest chromatographic resolution. Taken together, the advantages of the latest software platforms are increasing the reliability of impurity profiling. 

Optimized electrospray ionization (ESI) source settings result in stable spray and hence stable signal throughout the whole analysis and high mass stability over the whole sequence of analyses, be it hours or days, generating more reliable data. These advances in MS technology, in addition to the software improvements, are helping to provide highly accurate quantification at trace levels.

Convenient software platforms are enabling combined HPLC-UV and MS workflows in manufacturing QC. Routine QC workflows must be simple to use and must deliver rapid results, and offer high run-to-run reproducibility. In the past, the complexity of MS-based systems limited their use in these applications (4). However, software advances have reduced complexity so that a single software package can now control the whole workflow, streamlining the process of HPLC-UV and MS into a fully automated and efficient process. Experimental set-up and reporting of results can easily be achieved by interfacing to a laboratory information management system, and software can be configured to confirm regulatory compliance.

The latest impurity profiling workflows based on HPLC-UV and MS allow fast, accurate and reliable analyte quantitation for routine QC applications. Modern MS technology, coupled with software solutions, now allow analysts to quantify trace levels of impurities and determine their identity accurately and with confidence. A better understanding of the impurity profile also allows manufacturers to refine their manufacturing processes to increase purity. Improved synthetic peptide impurity profiling, therefore, allows companies to better assure, and ultimately improve, the safety and efficacy of treatment.


1. S. Ingale, C. Sahu, et al., Int. J. Pharm. Life Sci., 2 (7), pp. 955–962, 2011.
2. ICH Guidelines, Quality Guidelines, Q3A-Q3D Impurities.
3. R. Sharma and A. Goyal, Int. J. Adv. Res. Pharm. Bio Sci., 4 (3) pp. 1–7, 2014.
4. M. D’Hondt, B. Gevaert, et al., J. Pharm. Anal., 6(1), pp. 24–31, 2016. 

About the Authors

Sylvia Grosse is application technician; Stephan Meding is product manager; Martin Samonig is manager of product management; and Mauro De Pra is application manager, all with Thermo Fisher Scientific.

Article Details

BioPharm International
Vol. 32, No. 2
February 2019
Pages: 35–37


When referring to this article, please cite it as S. Grosse, S. Meding, M. Samonig, and M. De Pra, “The Benefits of Combining UHPLC-UV and MS for Peptide Impurity Profiling," BioPharm International 32 (2) 2019.