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European Biopharmaceutical Review

Compound X

Container closure systems for substances and components used in the drug manufacturing process may release unwanted chemicals into pharmaceutical products. These compounds can impact the safety of the product, affect its efficacy or stability, or even influence analytical tests used in its release. Therefore, determining the identity of these chemical entities and toxicologically assessing their impact on the patient is extremely important.

Extractables and Leachables

Extractables are compounds that can migrate from the material into a solvent under exaggerated conditions of time and temperature. They can result from the use of functional additives, solvent residues or catalysts utilised in the manufacturing process, and can also stem from the degradation of additives or polymers.

Leachables, meanwhile, are compounds which migrate into the drug product or bulk solutions under normal storage or processing conditions. Typically, they are a subset of extractables – however, not all leachables are extractables. They can be the outcome of an interaction between an extractable compound and the product, or be a degradation product from an extractable compound (1).

An extractable study should be performed on final packaging, delivery devices and manufacturing components that come into contact with the product formulation. This involves using stressing conditions which reflect a worst-case scenario – compared to typical conditions of use – in order to increase the rate of migration and facilitate detection and identification. Structural identification – and subsequent quantification – is crucial, since the actual toxicity of a molecule and its permitted daily intake amount can only be evaluated if the chemical structure is known. Several analytical techniques can be applied to these studies as extractable compounds can be either organic or inorganic in nature. Typical instrumentation utilised includes liquid chromatography (LC) and gas chromatography (GC), both coupled with mass detectors – highperformance liquid chromatographymass spectrometry (HPLC-MS) and GC-MS (2). These are used to monitor the presence of organic entities, while inductively-coupled plasma and ion chromatography can be applied to determine the level of inorganic compounds present.

Overcoming Hurdles

LC-MS poses the greatest challenge in an extractables study as HPLCMS libraries for the identification of unknown compounds are not commercially available. This is due to the fact that the generation of the mass spectrum is strictly dependent on the analytical conditions used. For this reason, the creation of a customised extractables database, which will permit identification of unknown compounds by retention time and mass, is critical to the success of these studies. The identification of extractable compounds is first required to build such a database.

Compound characterisation is undoubtedly a very complex analytical field that often requires data from several instruments such as high-resolution mass spectrometers (HR-MS), ion trap or triple quadrupole MS systems, ultraviolet absorption, nuclear magnetic resonance, infrared and elemental analysis. This thorough approach does not fit easily with the relatively high number of unknown chemicals frequently detected in an extractables study.

An alternative approach relies on HR-MS spectral data evaluation. Combining an analyst’s expertise with state-of-the-art instrumentation, knowledge of the packaging materials and online compound databases – for example, Chemspider – often provides sufficient tools to allow for formulation of an identification hypothesis.

Case Study

The transformation of an unknown extractable detected by HPLC-MS into an identified compound is demonstrated by the following example:

Drug Product in Polypropylene Primary Packaging
LC-MS data analysis (using MassHunter Qualitative Analysis Software) revealed an unknown compound detected at a significant concentration in an extraction solution placed in contact with the polypropylene primary packaging of a liquid drug product at 70°C for 24 hours.

Since the analysis was performed using a quadrupoletime- of-flight mass spectrometer, based upon its high-resolution performance, it was possible to determine the most probable molecular formula: C40H60N2O8 (MW 696.434). In particular, in electrospray ionisation (ESI) positive mode, the most representative positive-charged ions of C40H60N2O8 were the (NH4+)-adduct (m/z 714.469) and (Na+)-adduct (m/z 719.424). In ESI negative mode, the characteristic negative-charged ion was the corresponding deprotonated molecule C40H60N2O8(-H+) (m/z 695.427).

Unfortunately, it was not possible to identify the exact molecular structure of the compound, despite knowing the probable empirical formula. Searching the molecular formula in the Chemspider online database, a useful list of known molecules can be obtained; however, it is still unclear whether this unknown extractable was one of them (3).

Compound Identification

One candidate listed in the Chemspider search possessed functional groups related to common plastic additives from the Irganox family, and a quick search by CAS number identified it to be Naugard XL-1. Further evaluation showed that this is used in plastic manufacturing, serving as an antioxidant. In order to strengthen the case for identifying the unknown compound as Naugard XL-1, further fragmentation studies need to be performed using LC-MS/MS. This technique allows for generation of daughter ions of the unknown, providing information on the fragmentation pattern of the parent compound. Assessment of this information allows an experienced analyst to determine if the fragmentation pattern observed, along with the accurate mass of the parent ion, is consistent with the tentatively identified compound.

The (NH4+)-adduct of C40H60N2O8 was the most prominent positive-charged ion. When fragmented using two different collision energies (CE 15-25V), it showed a characteristic fragmentation pattern based firstly on the loss of NH4+ ion (C40H60N2O8(+H+) (m/z 697.442)) and then on the progressive loss of the four tert-butyl functional groups (C4H8; m/z 56.062).

This resulted in the following consecutive fragments: C36H52N2O8(+H+) (m/z 641.381); C32H44N2O8(+H+) (m/z 585.318); C28H36N2O8(+H+) (m/z 529.256); C24H28N2O8(+H+) (m/z 473.193). The remaining positive-charged molecular fragment C24H28N2O8(+H+) (m/z 473.193) was further reduced by the loss of an hydroxyphenylpropionate functional group (C9H9O3-) to C15H18N2O5(+H+) (m/z 307.130). In the end, another covalent bond in the molecule was broken, and the loss of C9H8O2 produced the last characteristic fragment of Naugard XL-1 (C6H10N2O3(+H+); m/z 159.077).

The deprotonated Naugard XL-1 C40H60N2O8(-H+) (m/z 695.425) was fragmented into two characteristic molecules due to the loss of a 3-[4-Hydroxy-3,5-bis(2-methyl-2- propanyl)phenyl]propanoate group (C17H26O3(-H+); m/z 277.179), which left the remaining molecule: (C23H35N2O5(-2H+); m/z 417.237). This characteristic fragmentation was confirmed by using two different collision energies (CE 15-25V). The fragments detected theoretically agree with the potential daughter ions expected from Naugard XL-1.

Final Confirmation

In addition, MS2 spectra – both in ESI negative and positive mode – were selected and transferred from MassHunter Qualitative Analysis Software into MassHunter Molecular Structure Correlator (4). By using this software, it is possible to obtain all structures that match the selected formula – parent compounds plus confirmed fragments – from a selected database (like Chemspider), with all candidates being sorted by a percentage score. The correlation result with the highest score matched the Naugard XL-1 compound.

The last step in confirming the extractable was, in this case, purchasing the reference standard to allow for analysis by LC-MS to check the identification based on both retention time and mass spectra. If not commercially available, synthesis of the compound or isolation by preparative HPLC would have been necessary.

Utilising Expertise

Identification of unknown extractable compounds is usually not as simple as in the above example, with investigations often being time-consuming and potentially having an important economic impact on the overall project cost. Drawbacks and challenges that may be encountered during the process include compounds not fragmenting into significant daughter ions (for example, fatty acids), or the Chemspider output list showing hundreds of compounds matching an accurate mass.

In conclusion, these all underline the necessity of a systematic analytical approach; the need for a customised extractables database that houses a collection of all acquired knowledge; and human expertise and intuition – which often represent the most precious and efficient of resources.


1. Jenke Dennis, Compatibility of pharmaceutical products and contact materials, John Wiley & Sons, 2009
2. Du Zhenxia, Zhang Yun, Li Ailin and Lv Surong, Rapid identification of polymer additives by atmospheric solid analysis probe with quadrupole time-of-flight mass spectrometry, Rapid Commun Mass Spectrom 28: pp1-8, 2014
3. Keller BO, Sui Jie, Young AB and Whittal RM, Interferences and contaminants encountered in modern mass spectrometry, Analytica Chimica Acta 627: pp71-81, 2008
4. Hill AW and Mortishire-Smith RJ, Automated assignment of high-resolution collisionally activated dissociation mass spectra using a systematic bond disconnection approach, Rapid Communications in Mass Spectrometry 19: pp3,111-3,118, 2005

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Silvia Scotti is a Chemical Manager in the R&D and Extractables and Leachables department at Eurofins BioPharma Product Testing Italy. She is actively involved in optimising resources and services to provide complete technical offerings to meet clients’ requests in the field of chemical studies, such as extractables and leachables, or method development and validation. Silvia has 14 years of experience as Good Laboratory Practice Study Director and as an R&D analytical chemist in both Italian and Irish pharma companies. She has a degree in Chemistry from the University of Milan, Italy.

Simone Carrara
is a Project Leader in the R&D and Extractables and Leachables department at Eurofins BioPharma Product Testing Italy. Having spent 10 years as a bioanalytical researcher using LC-MS method development for pharmacokinetics analysis and ADMET profiling, Simone has led projects in a wide range of areas, including impurities characterisation, method development and validation by LC-MS and GC-MS, and cleaning validation. He holds a Biotechnology degree from the University of Milan, Italy.

Marco Giulio Rozio is a Chemical Laboratory Analyst in the R&D and Extractables and Leachables department of Eurofins BioPharma Product Testing Italy. During his 18-year career, he has held the positions of Senior Bioanalytical Researcher and Supervisor of Experimental Area, and has wide experience based on the set-up of bioanalytical techniques for qualitative and quantitative analysis of chemical compounds using LC-MS/MS instrumentation. Marco has a degree in Biological Sciences from the University of Milan, as well as a qualification in Biology from the University of Pavia and certification for the role of Professional Pharmacological Researcher from the Mario Negri Institute, all in Italy.
Silvia Scotti
Simone Carrara
Marco Giulio Rozio
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