Analytical Evaluation Threshold Research Articles

The analytical evaluation threshold for inorganic metal extractables and leachables analysis of medical devices

Chemical characterization of medical devices uses the analytical evaluation threshold (AET) to determine reportable organic extractables, as these chemicals may be of toxicological concern and should be addressed via toxicological risk assessment. The AET is not applicable to metal extractables due to the exclusion of toxicity data on inorganics from the dataset used to derive dose-based threshold (DBT) values. This results in minimal guidance for reporting metal extractables. Herein, an AET for metals, or mAET, is proposed as a reporting threshold for individual metal extractables. The mAET can ensure metals are reported that are at quantities that may present a patient safety risk. This may reduce the number of metals reported in a chemical characterization report, improving the efficiency of the overall biocompatibility evaluation by removing unneeded effort and resource time. Conversely, an analytical method’s ability to report all metals at toxicologically relevant levels can be confirmed by comparing method sensitivity to mAET values. DBTs were developed for 70 metals, permitting mAET values to be determined. These mAET values were then compared to metal reporting limits from 13 previously conducted chemical characterization studies, which used varying extraction designs and analytical methods, to determine the impact of the mAET.

Determination of Response Factors for Analytes Detected during Migration Studies, Strategy and Internal Standard Selection for Risk Minimization.

Migration studies are one of the few domains of pharmaceutical analysis employing wide-scope screening methodologies. The studies involve the detection of contaminants within pharmaceutical products that arise from the interaction between the formulation and materials. Requiring both qualitative and quantitative data, the studies are conducted using Liquid Chromatography or Gas Chromatography coupled to a mass spectrometer (LC-MS and GC-MS). While mass spectrometry allows wide-scope analyte detection and identification at the very low Analytical Evaluation Threshold (AET) levels used in these studies, MS detectors are far from "universal response" detectors. Regulation brings the application of uncertainty factors into the picture to limit the risk of potential analytes detected escaping report and further evaluation; however, whether the application of a default value can cover any or all relevant applications is still debatable. The current study evaluated the response of species usually detected in migration studies, generating a suitable representative sample, analyzing said species, and creating a strategy and evaluation mechanism for acceptable classification of the detected species. Incorporating novel methodologies, i.e., Design of Experiments (DoE) for Design Space generation, the LC-MS-based methodology is also evaluated for its robustness in changes performed.

Biocompatibility of novel albumin-aldehyde surgical adhesive

Many medical procedures could benefit from the use of tissue sealants which allow for reduced surgery time, limited blood loss, easier tissue handling, and fewer postoperative complications. The safety and biocompatibility of surgical sealants are of paramount importance therefore, the aim of this study is to investigate the biocompatibility of NE’X Glue Surgical Adhesive. Chemical characterization (VOC and elements), cytotoxicity (MEM elution), genotoxicity (AMES and MLA), endotoxin contamination, sensitization potential, intracutaneous reactivity, acute and subchronic systemic toxicity with implantation as well as pyrogenicity were evaluated to investigate the biocompatibility of the NE’X Glue Surgical Adhesive. Studies were conducted according to ISO 10993 standards. The biocompatibility requirements with accordance to ISO 10993-1 for NE'X Glue were met. In vitro studies showed that NE'X Glue surgical adhesive is non-cytotoxic and non-mutagenic. Also, in vivo studies demonstrated that NE'X Glue shows no signs of toxicity, has no pyrogenic potential, and is non-sensitizing and non-irritating. The chemical characterization showed that no compounds were identified above Analytical Evaluation Threshold (AET), and no elements with concentrations higher than element-specific PDE (µg/day) were detected. NE'X Glue Surgical Adhesive is a versatile and promising new surgical sealant with a wide range of potential applications and very good*0* biocompatibility.

ISO 10993 biological evaluation of novel hemostatic powder \u2013 4SEAL\xae

BackgroundHemostasis plays a crucial role during every surgery allowing for a bloodless operating field. Fast and effective surgery leads to a reduced risk of postoperative complications. One of the latest methods for achieving homeostasis is using natural polysaccharide-based hemostatic powders. The study aimed to evaluate the biocompatibility according to the ISO 10993 standards of 4SEAL® Hemostatic powder.MethodsChemical characterization (Headspace GC-MS, GC-MS, and ICP-MS), cytotoxicity, genotoxicity (MLA and AMES), endotoxin contamination, sensitization potential, intracutaneous reactivity, acute and subacute systemic toxicity with implantation, and pyrogenicity were evaluated to investigate the biocompatibility of the 4SEAL® Hemostatic powder. Studies were conducted according to ISO 10993 standards.ResultsThe biocompatibility requirements according to ISO 10993-1 for 4SEAL® Hemostatic powder were met. Based on the conducted in-vitro studies, 4SEAL® Hemostatic powder shows a non-cytotoxicity and non-mutagenic potential. Also, the results of in vivo studies of 4SEAL® Hemostatic powder shows no signs of toxicity, non-sensitizing, non-irritating, and no pyrogenicity potential. In the chemical characterization of 4Seal® Hemostatic Powder, no compounds were identified above Analytical Evaluation Threshold (AET) and no elements with concentrations higher than element-specific PDE [μg/day] were detected.Conclusions4SEAL® Hemostatic powder is a promising new hemostatic agent with a wide range of potential applications and excellent biocompatibility.

A Practical Derivation of the Uncertainty Factor Applied to Adjust the Extractables/Leachables Analytical Evaluation Threshold (AET) for Response Factor Variation.

The analytical evaluation threshold (AET) establishes which chromatographic peaks, produced during organic extractables/leachables (E&L) screening, require toxicological safety risk assessment because the peaks are associated with compounds of potentially unacceptable toxicity. Thus, the AET protects patient safety as its proper application ensures that all potentially unsafe E&L are necessarily assessed. Generally, application of the AET involves the presumption that all organic E&L have the same detector response factor, an assumption that is not valid for any of the detection methods commonly used in E&L screening. Thus, the AET's ability to be protective is compromised for poorly responding compounds, as they will appear to be below the AET when in fact they are not. This unacceptable outcome is addressed by adjusting the AET with an uncertainty factor (UF) whose value is dictated by the magnitude of response factor variation, with a larger variation resulting in a larger UF and a lower adjusted AET. Although the concept of the UF is straightforward, setting a generally accepted, scientifically valid, and practical value for the UF has been challenging. In this article, a database of relative response factors obtained for nearly 1200 E&L via the most commonly applied chromatographic screening methods (gas chromatography/mass spectrometry [GC/MS], liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization [LC/MS-APCI], and LC/MS with electrospray ionization [LC/MS-ESI]) is used to justify UFs for these methods, individually and as a combined practice, based on the practical principle of "the point of diminishing returns". Using this concept results in nearly 92% of the compounds in the database being properly flagged as above an AET adjusted with a UF = 3. Ninety-five percent (95%) coverage of the compounds can be achieved when a UF of 4 is applied to the combination of GC/MS and LC/MS methods or with other combinations of UF values applied to the various methods individually. Coverage is increased to 97% when a UF of 4 is individually applied to the GC/MS method and a UF of 10 is individually applied to the LC/MS methods. Furthermore, the available data suggest that application of both APCI and ESI ionization in LC/MS screening (as opposed to either method separately) provides the greatest coverage of E&L.

Identification of an Oxidizing Leachable from a Clinical Syringe Rubber Stopper.

Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis. In the syringe leachables solution, the carcinogenic 1,1,2,2-tetrachloroethane (TCE) was detected in concentrations above the ICH M7-derived analytical evaluation threshold. TCE was shown to be an oxidation product of dichloromethane used during sample preparation. Since TCE was only isolated from incubations with the contained rubber stopper, we hypothesized that a stopper-derived leachable acted as a reactive oxidant promoting this chemical reaction. Subsequently, the leachable was identified to be the polymerization initiator Luperox® 101. Combining different analytical approaches led to the structural elucidation of a chemical reactive oxidant, which has the potential to interact and alter drug products. We conclude that chemically reactive compounds, such as the newly identified rubber stopper leachable Luperox® 101, may be of concern and therefore should be routinely considered if a prolonged exposure of polymers with drug products can be anticipated.

How sensitive does chemical characterization of medical devices need to be? Calibration of analytical evaluation thresholds with the carcinogenic potency database

Chemical characterization is a component of the safety evaluation of medical devices. An analytical evaluation threshold (AET) is recommended to calculate the required analytical sensitivity. There is a lack of consensus whether to use 1.5 or 120 μg/day in calculating the AET with the lower value often requiring sensitivities beyond analytical capabilities. The Carcinogenic Potency Database (CPDB) was reviewed to compare risks associated with using either value to calculate an AET. The TD50s for non-Cohort of Concern (non-COC) substances in the CPDB were used to extrapolate the doses to an excess cancer risk of 10−5 and calculate the total doses. The number of non-COC substances that would exceed this risk using an AET calculated using 1.5 μg/day or 120 μg/day were then compared. From the 199 substances evaluated, only two posed an excess risk at an AET calculated with 1.5 μg/day and only seven more with 120 μg/day. Furthermore, over 95 percent of non-COC substances would not pose an excess cancer risk using an AET calculated with 120 μg/day. Based on our evaluation, an AET based on 120 μg/day is protective and practical for chemical characterization of short and long-term medical devices.

A Commentary on Several Study Design Considerations Pertaining to the Screening of Substances Extracted and/or Leached from Pharmaceutical Contact Materials.

The assessment of substances that can be extracted (extractables) or leached (leachables) from the materials that come in contact with pharmaceutical products often starts with a general screening of these substances in relevant samples. Although such screening studies may be of a qualitative and semi-quantitative nature, the data they generate are critical in ensuring the suitability of the system(s) used to manufacture, store, and/or administer the product. As such, it is important they are designed using appropriate analytical techniques and methodologies so that representative and reproducible data are obtained. The goal of this commentary is to provide insight into several, but by no means all, good practices for the preparation and analysis of samples in extractable and/or leachable screening studies in order to help ensure representative and reproducible results are ultimately obtained. The specific topics covered are extraction stoichiometry, the importance of verifying method performance, assessing the need for incorporating derivatization in gas chromatography-based analyses, and properly accounting for analytical uncertainty when calculating the analytical evaluation threshold.

An Analytical Strategy Based on Multiple Complementary and Orthogonal Chromatographic and Detection Methods (Multidetector Approach) to Effectively Manage the Analytical Evaluation Threshold (AET).

To address patient safety, a drug product is chromatographically screened for organic leachables. Similarly, medical device and packaging system extracts are chromatographically screened for organic extractables as probable leachables. To protect patient health, the screening methods must respond to all potentially unsafe substances. To be efficient, analytes determined to be below the toxicologically relevant threshold are removed from consideration before the subsequent analytical tasks of identification and quantitation are performed. The analytical evaluation threshold (AET) was established for use as a toxicologically relevant threshold to differentiate between chromatographic peaks that are unlikely to be unsafe (and thus do not need safety assessment) and those that are possibly unsafe (and thus require safety assessment). In practice, the instrumental response associated with the AET is determined using surrogate standards. It is then assumed that the response strength for an unknown extractable is equivalent to that for the surrogate standard at the AET concentration (i.e., relative response factor = 1). It is an unfortunate reality that response factors vary for different compounds on nearly all detector systems. This complicates the application of the AET and can result in a failure to include potentially toxicologically relevant compounds in the identification phase of the analysis. To ensure protection, an uncertainty factor was built into the AET equation that adjusts the AET downward to address response variation. Although this mechanism does increase the protectiveness of the AET, it assumes that the available methodology and instrumentation is sufficiently sensitive to reach the new lowered AET value. However, in some cases, the response variation is so great and the resulting uncertainty factor so large that the revised AET is below the achievable sensitivity specifications of even state-of-the art, expertly operated instrumental technologies. The only option then remaining is to concentrate the samples, which can result in adverse effects on analysis quality-counteracting the perceived benefit of lowering the AET. This article demonstrates how an analytical strategy based on methods with multiple complementary and orthogonal detection techniques (a multidetector approach) mitigates the problem of response factor variation and thus eliminates the need for large uncertainty factors and the resulting lower AET values. The primary concept is that all analytes only need to be effectively detected by at least one of the combination of detectors applied, and it is this effective technique (combination of all detectors and chromatographic methods utilized) that is used to perform the AET assessment.

The Implications of Chromatographically Screening Medical Products for Organic Leachables Down to the Analytical Evaluation Threshold Adjusted for Response Factor Variation.

A drug product is chromatographically screened for organic leachables, derived from the product's packaging system, as leachables might adversely impact the health of a patient to whom the drug product is administered. Similarly, medical device and packaging system extracts are chromatographically screened for organic extractables as probable leachables. To be protective of patient health, the screening methods must produce recognizable responses for all potentially unsafe substances. To be efficient, the screening methods should provide a means of differentiating between the responses linked to likely to be safe substances and to potentially unsafe substances. The analytical evaluation threshold (AET) was established as a means of differentiating chromatographic peaks, based on concentration, that are unlikely to be unsafe (and thus do not need safety assessment) and that are possibly unsafe (and thus require safety assessment). Thus, the AET manages the competing objectives of protection and efficiency. Although the AET is based on concentration, it is applied based on response. As no chromatographic detection method applied to extractables and leachables screening produces a uniform response to all potential analytes (thus, the magnitude of the response differs across analytes), the objectives of protection or efficiency can be compromised by false negatives and positives. To ensure protection at the expense of efficiency, the AET can be adjusted to address response variation. This article addresses the practical issue that the protectiveness of the AET is affected both by response factor bias and variation and thus correction for only variation is incomplete and ineffective. The article illustrates the proper adjustment of the AET for bias and variation.

Reducing relative response factor variation using a multidetector approach for extractables and leachables (E&L) analysis to mitigate the need for uncertainty factors

Characterization of Extractables and Leachables (E&Ls) is an important aspect of product quality in important fields such as pharmaceuticals, medical devices and food contact materials. The main goal of an E&L study is identification and quantification of those species which may leach from packaging materials used to contain pharmaceuticals or which may leach directly out of a medical device or food contact material and thus may result in patient exposure. It is common practice to perform relative quantitation of extractables and leachables using surrogate standards due to the large diversity of species observed and the lack of available reference standards. A key problem in obtaining accurate E&L results arises due to response factor (RF) variation. Different compounds at the same concentration give different signal intensities and thus have different RF values. Two key aspects of study quality are affected by this problem. First, the evaluation of the number of compounds which are above the toxicologically relevant threshold (analytical evaluation threshold, (AET)) can be affected (RF Problem 1: AET Underreporting). Second, quantitative accuracy is affected which can reduce the reliability of the margin of safety (MOS) calculations which serves as the basis of the toxicological evaluation (RF Problem 2: Quantitative Error). RF databases have been the main solution proposed for solving these problems but do not reduce the underlying RF variation and lack the scope required to address quantitative error for compounds not contained in the database. In the absence of other solutions, large uncertainty factors (UF) have been applied in the AET calculations to account for RF Problem 1: AET Underreporting. These UF factors have been assigned values of 4 for GCMS and up to 10 for LCMS. Large uncertainty factors have a number of unintended negative consequences including the need for large amounts of sample concentration (>10X) prior to analysis resulting in potential compound loss or degradation and increased matrix effects. To overcome these problems, this publication demonstrates a multidetector approach using an HPLC system coupled with a Quadrupole Time of Flight Liquid Chromatography Mass Spectrometer (QTOF-LCMS), Charged Aerosol Detector (CAD) and an Ultraviolet-Visible Detector (UV) and a dual detection Gas Chromatography Mass Spectrometry (GCMS) system using a Polyarc Reactor system with Flame Ionization Detection (FID). Herein, it is demonstrated that this combination of methods (the multidetector approach) allowed detection and accurate surrogate standard quantitation of 217 unique extractables spanning a wide range of chemical properties (Mw, logP, pKa and boiling point). The combination of optimized detector selection with appropriate standard selection was verified to provide positive detection for 94% of the compounds at the AET level and a high level of quantitative accuracy (± 20% for 85% of the compounds and ±40% for 91% of the compounds) while using only a UF of 2. Unlike the RF database approach, the multidetector approach is not limited to only those compounds contained in the database but is applicable to the majority of extractables.

Correcting the Analytical Evaluation Threshold (AET) and Reported Extractable's Concentrations for Analytical Response Factor Uncertainty Associated with Chromatographic Screening for Extractables/Leachables.

It is generally acknowledged that quantitation in extractables and leachables (E&L) can be variably reproducible and accurate, depending on the quantitation approach taken. This is especially true for "simple" quantitation, which is the practice of estimating an analyte's concentration based on its response relative to that of an internal standard that has been added to the sample in a known amount. Simple quantitation is prone to error and variation as it is based on the largely false premise that the response factors for all extractables, leachables, and internal standard candidates are the same. It has been proposed that this uncertainty (inaccuracy and variation) be accounted for by adjusting two key parameters in E&L assessment, the reported concentrations themselves and the analytical evaluation threshold (AET) via an uncertainty factor (UF). This paper examines quantitation variation and discusses the means of establishing and utilizing the UF to adjust the AET to lower values and to adjust reported concentrations to higher values, enabling an impact assessment performed with this data to be more protective of patient safety. Although adjustment of the AET lower with the UF is supported, flaws in the concept of using the UF to adjust reported concentrations upward are considered, and it is recommended that the UF not be used in this manner. Rather, E&L quantitation should be based on compound-specific relative response factors, collected and collated in an E&L database.

Development of an HPLC/UV method for the evaluation of extractables and leachables in plastic: Application to a plastic-packaged calcium gluconate glucoheptonate solution

Calcium gluconate glucoheptonate (GGCa) is known to interact with glass containers, leading to the leaching of aluminum from the glass into the solution at toxic level. Therefore, plastic containers seem to be a preferable packaging alternative. Nevertheless, plastics contain potentially toxic additives which could be released into the solution. In order to study content container interaction between GGCa and two plastic containers (polypropylene PP and polyethylene PE containers), an HPLC-PDA method was developed to separate, detect and quantify eleven additives commonly found in plastic materials, with good limit of detection and quantification. This method was then applied to evaluate the compatibility between GGCa and the two plastic containers. After 3 months of storage at 25 °C, none of the eleven additives were detected in GGCa solutions. The safety concern threshold (SCT) and of the analytical evaluation threshold (AET) were evaluated to discriminate the need to identify and qualify unknown peaks.

Qualitative assessment of extractables from single-use components and the impact of reference standard selection

The advent of single-use bioprocess systems used for the delivery, storage or manufacture of biopharmaceuticals has introduced a new potential source for extractables and leachables (E&L) as these systems are comprised of polymeric materials. Several industry working groups, the FDA and USP have issued guidance and draft guidance on E&L analyses for a variety of applications. These documents typically indicate that mass spectrometry should be applied for discovery of E&L’s but provide little guidance as to the exact analytical methodology which should be used. We investigated the extractable profiles of a model single-use bioprocessing system consisting of a single-use bioprocess bag, connector tubing, and a hydrophilic disk filter including filter housing. Extractions were performed in water, ethanol, ethanol/water (50:50) and saline solutions. Extracts were analyzed using a stepwise analytical methodology including a variety of screening and mass spectrometry methods We then used this model system to demonstrate the use of recursive feature finding to automatically detect unique extractables followed by statistical filtering to focus on differentially present extractables which were above the analytical evaluation threshold (AET). We further show the significant affects of standard selection on the number of compounds determined to be above AET when reducing liquid chromatography–mass spectrometry (LC/MS) data. A relative response factor database consisting of 14 structurally diverse commercially available polymer additives was used to arrive at an LC/MS identification threshold. The results of this study demonstrate that significant care should be taken when selecting standards for LC/MS analysis to avoid under reporting of extractables and leachables.

Meta-analysis of preclinical studies of mesenchymal stromal cells for ischemic stroke

To evaluate the quality of preclinical evidence for mesenchymal stromal cell (MSC) treatment of ischemic stroke, determine effect size of MSC therapy, and identify clinical measures that correlate with differences in MSC effects. A literature search identified studies of MSCs in animal models of cerebral ischemia. For each, a Quality Score was derived, and effect size of MSCs was determined for the most common behavioral and histologic endpoints. Of 46 studies, 44 reported that MSCs significantly improved outcome. The median Quality Score was 5.5 (of 10). The median effect size was 1.78 for modified Neurological Severity Score, 1.73 for the adhesive removal test, 1.02 for the rotarod test, and 0.93 for infarct volume reduction. Quality Score correlated significantly and positively with effect size for the modified Neurological Severity Score. Effect sizes varied significantly with clinical measures such as administration route (intracerebral > intra-arterial > IV, although effect size for IV was nonetheless very large at 1.55) and species receiving MSCs (primate > rat > mouse). Because many MSC mechanisms are restorative, analyses were repeated examining only the 36 preclinical studies administering MSCs ≥ 24 hours poststroke; results were overall very similar. In preclinical studies, MSCs have consistently improved multiple outcome measures, with very large effect sizes. Results were robust across species studied, administration route, species of MSC origin, timing, degree of immunogenicity, and dose, and in the presence of comorbidities. In contrast to meta-analyses of preclinical data for other stroke therapies, higher-quality MSC preclinical studies were associated with larger behavioral gains. These findings support the utility of further studies to translate MSCs in the treatment of ischemic stroke in humans.

Perspectives on the PQRI Extractables and Leachables "Safety Thresholds and Best Practices" Recommendations for Inhalation Drug Products

In 2006, the Product Quality Research Institute's (PQRI) Leachables and Extractables Working Group released a comprehensive and detailed recommendation document related to leachables and extractables for inhalation drug products. The document includes best pharmaceutical development practice recommendations regarding container closure/delivery system component composition and selection, controlled extraction studies, drug product leachables studies, and routine extractables testing for component release. Also included in the document are two safety-based thresholds for leachables in inhalation drug products, the qualification threshold (QT) and the safety concern threshold (SCT), the first such safety-based thresholds for leachables in any drug product type. A process was described for converting the SCT into an analytically useful threshold for leachables/extractables characterization, the analytical evaluation threshold (AET), with consideration of individual drug product dosing parameters and container closure system component characteristics. This commentary presents the history and evolution of this recommendation document starting from the propellant changeover (chlorofluorocarbons to hydrofluorocarbons) in metered dose inhaler drug products, which helped prompt interest in inhalation drug product leachables, through the work process of the PQRI group. The overall positive acceptance of the PQRI recommendations is discussed, along with a brief summary of regulatory initiatives influenced by the recommendations. Also presented and discussed are certain key issues and questions that have arisen since the recommendation document was released. The extension and application of best practice recommendations to other high risk drug product types (e.g., large and small volume parenterals, ophthalmics), led by the PQRI Parenteral and Ophthalmic Drug Product Working Group, is introduced and considered. The recommendation document released by the Product Quality Research Institute's (PQRI) Leachables and Extractables Working Group in 2006 includes the first safety-based thresholds for leachables in any drug product type, along with comprehensive best practice recommendations for inhalation drug product pharmaceutical development related to extractables and leachables. The best practice recommendations encompass a number of important functional areas, including container closure/delivery system component composition and selection, controlled extraction studies, drug product leachables studies, and routine extractables testing for component release. This commentary presents the history and evolution of this recommendation document starting from the propellant changeover (chlorofluorocarbons to hydrofluorocarbons) in metered dose inhaler drug products, which helped prompt interest in inhalation drug product leachables, through the work process of the PQRI group. The overall positive acceptance of the PQRI recommendations is discussed, along with a brief summary of regulatory initiatives influenced by the recommendations. Also presented and discussed are certain key issues and questions that have arisen since the recommendation document was released. The extension and application of best practice recommendations to other drug product types (e.g., large- and small-volume parenterals, ophthalmics), led by the PQRI Parenteral and Ophthalmic Drug Product Working Group, is introduced and considered.

Utilization of Internal Standard Response Factors to Estimate the Concentration of Organic Compounds Leached from Pharmaceutical Packaging Systems and Application of Such Estimated Concentrations to Safety Assessment

Substances from packaging systems that are leached into packaged medical products may have a safety impact on patients to whom such medical products are administered. The potential safety impact depends on the identity and concentration of the leached substances. The concentration above which a leachable must be identified in order to assess its safety impact is frequently estimated using an internal standard to "calibrate" the analytical response of a chromatographic system. Such an estimate is accurate to the extent that the responses of the internal standard and leachables are similar. To establish the accuracy of the internal standard approach, a database of gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) responses was generated for thirty-eight leachables and eight internal standard candidates. Although the FID and MS responses of many of the leachables and internal standards fell within a narrow band, acidic and basic compounds produced responses that were discernibly different from those of neutral analytes. While most of the internal standards were suited for concentration estimation, three of the candidates, dimethylphthalate, triphenylphosphate and 4,4-dibromobiphenyl, produced the smallest mean error in estimated concentration for the analytes examined. As the FID and MS responses were linear, internal standards could be used to estimate leachables concentrations even when the difference in leachable versus internal standard concentrations was as great as a factor of 25. A multiplier may be appropriate to adjust an estimated concentration to its greatest possible value, and it is this value that is used to convert an estimated Analytical Evaluation Threshold (AET) into a working or final AET.