molecular polarities are retained in the instrument for different amounts of time; how long a molecule remains in the instrument is referred to as the retention time. After separation, the molecules are identified using mass spectroscopy. Briefly, mass spectroscopy works by fragmenting molecules into electrically charged pieces and then measuring the weight of those pieces very precisely. With knowledge of both the retention time and mass fragmentation patterns, VOCs can almost always be positively identified by comparison with large public or commercial databases. Purge and trap measurements differ from headspace only in the way compounds are sampled; first volatile organics are purged from the water by bubbling inert gas through the liquid and trapped in an adsorbent tube. VOCs are released from the tube into the GC/MS for analysis as with HS‐GC/MS.
SVOC measurement methods provide the single broadest source of information regarding the content of extracts and are amenable to both aqueous and nonaqueous extraction matrices. The term SVOC is ill defined in the medical device community but generally is considered to be those compounds most well suited for analysis by direct injection GC/MS. The distinction of this definition is important, as there are many molecules amenable to direct injection GC/MS that are considered to be NVOCs by every other definition. The methods used for SVOCs by GC/MS are mostly characterized by the details of their sample preparation and rigor of data analysis; instrumental details of the GC/MS remain largely harmonized. Water extracts are prepared for analysis by first doing a solvent exchange to a solvent compatible with GC/MS. Typically this is accomplished by repeatedly shaking the extract with methylene chloride under acidic, neutral, and basic conditions. The methylene chloride can then be concentrated and directly injected into the instrument. Organic solvents do not need a solvent exchange and are typically concentrated and then directly injected.
NVOCs not amenable for analysis by GC/MS are most clearly those compounds that have such a high molecular weight or polarity that they are not capable of vaporization without decomposition. For these compounds, liquid chromatography with mass spectroscopy (LC/MS) must be used. Unlike GC/MS analyses, which have more or less standardized instrument parameters, LC methods are highly variable. Because of this variability, large public databases are of limited utility, and effective interpretation of data relies much more on the level of expertise of the analyst and internal experience of the analyzing lab. LC techniques coupled with advanced mass spectroscopy tools providing high‐resolution accurate mass (HRAM) such as quantitative time of flight (qTOF) or Orbitrap can be a significant advantage, as these more sensitive methods can greatly narrow down the number of possible compounds in the identification process.
One of the key variables in chemical analysis for toxicological risk assessment and biocompatibility is the degree of certainty in the identification and quantification of compounds. Quality of identification can range from a fully automated comparison to a public database, without peer review of the results to fully confident identification. Fully automated identification can lead to scenarios where compounds with very low match scores are reported as compounds for which they are almost certainly not. On the other end of the identification spectrum is a fully validated identification where the compound in question has been injected using a standard on the same instrument and under the same conditions and under expert review. Of course, in practice, results can be a mix. It is not possible to inject standards for every compound that might occur from a biomaterial. With respect to quantification, results can vary based on the amount of evidence that is present to support the accuracy and precision of the presented results. On one end of the spectrum, results can be fully validated with calibration curves and precision and accuracy measurements. On the other end, results may be estimates based only on the concentration of an internal standard. Because patient safety may hinge on the result, often toxicologists want something more than a blind estimate of concentration of the compound is on the edge of being considered safe.
Chemistry results must be evaluated and assessed through the lens of toxicology to understand the possible systemic risks associated with the findings and the route of exposure of the device per ISO 10993‐17. This assessment should complement the results of traditional biocompatibility tests performed on biopolymeric device materials.
1.4 Specific Biological Endpoint Evaluations
For most biological endpoints per ISO 10993‐1, a biopolymer would be tested very similarly to any other polymer. The main concern with a biopolymer is the degradation profile and the impact of the degradation on the test system. The testing system that needs the most consideration for the individual degradation profile of a material is in cytotoxicity, systemic toxicity, implantation, and material/chemical characterization.
1.4.1 Cytotoxicity
In general, cytotoxicity tests are a broad range of assays that look for the impact of a substance on individual cells grown under in vitro conditions. The test can be performed on different cell lines and can look at (qualitatively) or assess (quantitatively) different cellular endpoints. The various internationally accepted cytotoxicity assays are summarized in part 5 of the ISO 10993 series (i.e. ISO 10993‐5). All the tests usually run using the L929 mouse fibroblast cell line. Although it is possible to use other cell lines for testing, the L929 cell line is the one that has historically been used and is therefore recommended for comparison. Additionally, despite the availability of many different versions of cytotoxicity tests, the standard testing for biocompatibility of medical devices consist of either MEM elution, MTT/XTT assays, or neutral red uptake assay. Each assay has different cytotoxicity evaluation endpoints and sensitivity, so comparing results from one assay to the other has proven to be difficult.
The cytotoxicity test is a very sensitive test and is the most likely test to cause trouble with any medical device, but specifically with biopolymers. This trouble comes from the fact that some biopolymers lack the mechanical properties and stability in the extraction fluid that is used to prepare a sample for the cytotoxicity test. This lack of stability may be caused a high concentration of ions in the extraction fluid that could result in a cytotoxic response in the assay. Crosslinking can be used in the attempt to improve the results, but this can also cause potential cytotoxicity as these crosslinking agents themselves can be cytotoxic (e.g. glutaraldehyde).
Therefore, the best approach for assessing cytotoxicity of biopolymers is a risk‐based approach. As mentioned before, the cytotoxicity test is historically the most sensitive test available and is thus often used as a screening test for materials, process residuals, and the final device configuration. In the ANSI/AAMI/ISO 10993‐5 Guidance section 10, it states “Any cytotoxic effect can be of concern. However, it is primarily an indication of potential for in vivo toxicity and the device cannot necessarily be determined to be unsuitable for a given clinical application based solely on cytotoxicity data.” When elevated cytotoxicity results are seen, a risk assessment should be performed to identify the source of observed cytotoxicity. Then on, the risk assessment should evaluate the toxic potential of the material or compound to determine the clinical impact. The investigation should include a review of the procedures to determine the effectiveness of the test system, additional testing to evaluate clinical risk of the results, and then a clinical risk assessment of the toxicity using additional animal testing along with chemical analysis and toxicological assessment of the detected compounds.
Based upon examination of the biopolymer, its history of use in medical industry, inherent surface properties of the device material, surface area in contact with the user, use and contact type, duration of contact, and the route of exposure, this cytotoxicity failure may not be clinically relevant, and subsequently it can be concluded that adverse effects in patients are unlikely to develop.
1.4.2 Systemic Toxicity (Acute, Subacute, Subchronic, and Chronic)
Systemic toxicity is a potential adverse generalized response including organ or organ system effects that can result from the absorption, distribution, and metabolism of leachates from the device or its materials to parts of the body that are not in direct contact with the device or material.
