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.

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.

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.

Quantitative determination of the chemical composition of silica-poly(norbornene) nanocomposites.

Nanoparticle hybrid materials consisting of a silica core surrounded by a poly(norbornene) brush have been prepared by ring opening metathesis polymerization (ROMP). A quantitative determination of each stage of composite formation has been accomplished, including a determination of the density of surface-bound functional groups, catalyst molecules, and polymer chains. This analysis has enabled the determination of the reaction efficiency between the catalyst and the surface-bound functional groups as well as the determination of the fraction of metal-mediating species that initiate a polymer chain. Control of the chain density was demonstrated by two methods: the use of controlled reaction times between the catalyst and the surface, and the variation of the surface functional group density. Polymer chain densities resulting from composites prepared with different tether structures will also be reported. The resulting brush densities were found to span a wide range, including those previously reported for polymer layers formed by adsorption, grafting of preformed polymer chains, and surface-initiated polymerization (SIP).