<1663> ASSESSMENT OF EXTRACTABLES ASSOCIATED WITH PHARMACEUTICAL PACKAGING/DELIVERY SYSTEMS 

CHARACTERIZING THE EXTRACT

Once an extract has been generated, the next objective is to perform a thorough chemical characterization of the extract. Setting a threshold (as described above), which is a specified level of an individual extracted chemical entity which requires characterization, can be based on safety considerations such as the SCT; functional considerations including nominal levels of known chemical additives in the formulation of an extracted component or material; or technological considerations such as the known or determined sensitivity of an analytical technology, instrument, or method. The extract characterization phase of the extraction study must enable the realization of the overall goals of the extractables assessment.

The ultimate objective of thorough extract characterization as defined above cannot be realized in all cases, even when state-of-the-art analytical chemistry is practiced with best available skill and diligence. It is a reality that there is no analytical technique or combination of analytical techniques that is capable of the discovery, identification, and quantitation of any and all organic and inorganic extractable chemical entities known to science. In some cases, authentic reference compounds for organic extractables may not be available for confirmation of identifications, or for quantitative instrument calibration. Thus, the practical objective of extract characterization must therefore be an exercise of due diligence in the discovery, identification, and quantitation to a reasonable degree of scientific certainty of all individual extractable chemical entities present in an extract above a specified level or threshold.

• Significant levels of nonvolatile residue determined by gravimetric analysis could suggest the presence of significant levels of inorganic chemical entities in the extract. This suggestion would be reinforced if significant mass remained after ashing the extracted nonvolatile residue (residue on ignition).

• Significant UV absorbance of an extract suggests that organic chemical entities are present which contain UV chromophores within their molecular structure, such as phenolic antioxidants.

• Characteristic features in an infrared spectrum of this extract could provide more detailed insights into the chemical classes of organic extractables present. These insights could be used to develop and apply analytical methods for discovery and identification that would detect the chemical classes of extractables suggested by the scouting process.

• For aqueous extracts, total organic carbon provides a measure of the total amount of organic extractables present.

The process of discovery involves testing an extract and thereby producing one or more analytical results that are attributable to individual extractables. The process of discovery is accomplished by detecting instrumental responses from the individual organic and inorganic extractables that are proportional to the levels of these individual extractables within the extract. It is in the discovery process that analytical techniques typically associated with trace organic and inorganic analysis are first required for extract characterization. 

Trace organic analysis typically involves the use of chromatographic techniques, particularly gas chromatography (GC) and high-performance liquid chromatography (HPLC). GC has enormous separating capability for volatile and semi-volatile organic compounds while HPLC is most applicable to semi-volatile and relatively nonvolatile organic compounds, making the two separation techniques complementary and orthogonal for application to the significant chemical diversity of extractables. A discussion of the principles of both gas and liquid chromatography is available in Chromatography <621>.

The chemical diversity of extractables with respect to polarity and volatility can require alternative sample introduction techniques or sample modification, particularly for GC. Relatively volatile extractables like methanol are most amenable to headspace sampling of aqueous-based extracts into a GC. Organic acids and bases can often be analyzed more effectively by GC after chemical derivatization, such as methylation or silylation for organic acids. Both GC and HPLC can employ detection systems with different specificities (Table 3) which take advantage of unique structural properties of various chemical classes of extractables.

The analytical techniques useful for organic extractables discovery can also be applied to identification as well as quantitation. Analytical techniques such as gas chromatography/mass spectrometry (GC/MS), that are most often applied to identification, can also be used for both discovery and quantitation (Table 3). Inorganic extractables such as trace elements and metals are typically discovered, identified, and quantitated by the same suite of analytical techniques, such as atomic emission spectroscopy. 

Analytical techniques designed to study inorganic speciation, particularly in aqueous extracts, are considered beyond the scope of this chapter.It is important to state that the overall goals of an extraction study always require the identities and quantitative amounts of individual organic and inorganic extractables, and so the mere discovery of extractables does not achieve the ultimate objectives of an extraction study.

Identification of an extractable can be accomplished either by structural analysis or qualitative analysis. Structural analysis is the process by which the molecular structure of an unknown analyte is elucidated from compound-specific data, and therefore requires compound-specific detection of the unknown analyte. A compound-specific detector is one that provides information specific to the molecular structure of the individual unknown analyte (not just its chemical class). Qualitative analysis is the process by which an unknown analyte is matched with an authentic reference compound via one or more analytical techniques. 

The analytical techniques used for qualitative analysis can, but do not need to be, compound specific.The analytical techniques most applicable to structural analysis, and to trace organic analysis problems in general, involve the combination of chromatography with mass spectrometry. These are the so-called “hyphenated” techniques of GC/MS and high-performance liquid chromatography/mass spectrometry (LC/MS). A discussion of the principles of mass spectrometry (including both GC/MS and LC/MS) is available in Mass Spectrometry <736>.Both GC/MS and LC/MS are capable of generating extractables profiles in the form of chromatograms. 

However, since LC/MS includes a relatively high chemical background of HPLC mobile phase ions, it is typical to include a non-destructive UV detector in series with the mass spectrometer to assist in locating peaks of individual extractables. The compound-specific data available from mass spectrometry include:

• The monoisotopic molecular weight of the extractable based on confirmation of the molecular ion from one or more ionization processes

• The molecular formula of the extractable based on accurate mass measurements, and/or accurate isotope ratio measurements, of the molecular ion

• The fragmentation behavior of the extractable based on in-source fragmentation or tandem mass spectrometry

1. Mass spectrometric fragmentation behavior/expert mass spectrum interpretation
2. Confirmation of molecular weight
3. Confirmation of elemental composition
4. Mass spectrum matches automated library or literature spectrum
5. Mass spectrum and chromatographic retention index match authentic reference compound
6. Supporting spectral information from an orthogonal method (e.g., NMR)

   
   
   

   • A Tentative identification means that data have been obtained that are consistent with a class of molecule only. This is typically the case when only information such as a or d is available.
   • A Confident identification means that the tentative identification has been bolstered by additional and sufficient confirmatory information to preclude all but the most closely related structures. This would be the case, for example, if the tentative information (a and/or d) were augmented by b, c, or f. The more confirmatory information obtained, the greater the level of confidence.
   • A Confirmed identification means that the preponderance of evidence confirms that the entity in question can only be the identification that is provided. Although it is possible that a highly confident identification may meet the standard implied by the preponderance of evidence (for example, having a, b, c, e, and f), the only means of providing a confirmed identification is via mass spectral and retention time match with an authentic reference compound (item e).

   
   

Extracts can often be analyzed directly without significant preparation or concentration. Many organic solvent extracts (e.g., dichloromethane, ethyl acetate, hexane) can be directly injected into a gas chromatograph, while others (e.g., methanol, ethanol, isopropanol) are either too reactive in the heated GC injection port or too high boiling. 

Organic solvent extracts with inappropriate physical/chemical properties for direct analysis by GC can be switched to more appropriate solvents. Certain extractables, such as fatty acids (e.g., palmitic acid, stearic acid) perform better in gas chromatographic analysis when they are derivatized to either methyl esters or trimethylsilyl esters.It is usually considered inappropriate to directly analyze aqueous extracts by gas chromatography, due to the reactivity and high boiling point of water. In addition, pH-buffered aqueous extracts contain nonvolatile salts which are not suitable for GC injection. 

Aqueous extracts are typically back extracted with an organic solvent to remove organic extractables from the water, with the resulting organic extract being injected into the GC. Unlike GC, liquid chromatography (HPLC, LC/MS) is perfectly suited to the direct analysis of aqueous extracts, since most HPLC methods include water and water-miscible mobile phases. Water-immiscible organic solvents (e.g., hexane) cannot be injected onto these reversed-phase HPLC systems, so these must be dried and the resulting extractable residue taken up in a solvent suitable for HPLC (e.g., acetonitrile, methanol, or mixtures of these with water).

Organic or aqueous extracts with insufficient levels of extractables for analysis can be concentrated by various techniques. Many organic solvents can be dried down under inert gas, a rotary evaporator, or a Kuderna-Danish concentrator. Aqueous extracts can be lyophilized, concentrated under vacuum, or back extracted into an organic solvent which is then further concentrated.

The final concentration at which an extract is analyzed depends on the goal(s) of the extractables assessment and the inherent sensitivities of the analytical techniques applied. A good “rule of thumb” is that in order to accomplish a complete structural analysis of an unknown extractable, a GC/MS requires approximately 5 ng injected into the instrument. This suggests a concentration in the injected extract of 5 ng/µL or 5 µg/mL. In a 200-mL dichloromethane extract, this converts to a total of 1 mg of this particular extractable recovered from the extracted test article. If this analyte concentration is insufficient to meet the goal of the extractables assessment, then the following parameters can be optimized:

• Extraction stoichiometry (i.e., extract more material or use more extracting solvent)

• Extraction conditions (i.e., use higher temperatures, longer times, solvents with greater extraction power, more aggressive extraction technique, etc.)

• Extract processing (i.e., concentration of the extract)