Fixed-Oil Excipient Monographs: Development of USP Fixed-Oil Reference Standards

This article summarizes the development and modernization of the United States Pharmacopeia–National Formulary (USP−NF) fixed-oil excipient monographs. Fats and fixed oils are processed from natural sources and have complex chemical compositions. As part of the public standards-setting processes, USP staff and the Excipients Expert Committee have formulated a strategic analytical testing plan for fixed-oil excipients. The plan balances modernization with ease of adoption and method simplicity. Modernization introduces more specific compositional methods that can identify as well as quantify the analyte(s) of interest. A combination of simple orthogonal methods uses existing instrumentation that can be applied to a multitude of fixed-oil monographs and encourages ease of adoption. This plan also has helped make possible the development of USP Reference Standards for such complex excipients because the proposed analytical methods provide a comprehensive understanding and characterization of fixed oils. This is an extended version of “Fixed-Oil Excipient Monographs” published in Pharmaceutical Technology’s April 2013 issue.

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The US Pharmacopeia (USP) Monographs—Excipients Expert Committee (EXC EC) for the current 2010–2015 revision cycle is responsible for the 31 United States Pharmacopeia–National Formulary (USP−NF) monographs with “oil” in the monograph title (see Table I) in USP 35−NF 30 through the Second Supplement (1). The 31 oil excipients include vegetable oils (edible), petrochemical oils, and essential oils. All vegetable oils are termed “fixed oils” in USP−NF. The term fixed oils distinguishes them from the relatively volatile petrochemical oils and essential oils. Fixed oils are obtained by expression or extraction. Their consistency varies with temperature. Some are liquid (oils), others are semisolid (fats), and still others are solid (tallows) at ambient temperature. Most fixed-oil excipients included in USP−NF are refined oils.

Table I: Excipient monographs that have “oil” in the titles and that fall under the responsibility of the excipients expert committee.Twenty-four of the 31 USP–NF oil excipients are fixed oils or fixed-oil derivatives. Most fixed oils consist mainly of triglycerides (or triacylglycerols). The four USP−NF mineral oils in Table I are classified as petrochemical oils: each is defined as a purified mixture of liquid hydrocarbons obtained from petroleum. The three volatile essential oils (see Table I) are composed primarily of acyclic monoterpenoids, terpenes, and their derivatives or aromatic compounds and their derivatives and contain lesser quantities of alcohols, aldehydes, esters, and/or phenols.

Three of the 22 USP−NFfixed oils—castor oil, soybean oil, and safflower oil—are designated as USP, and the remaining fixed oils are designated as NF. The USP−NFfixed oils can have different functions. The majority are used as oleaginous vehicles or solvents in drug formulations. Some, however, are used as emulsifying agents, flavors and perfumes, ointment bases, plasticizers, stiffening agents, tablet or capsule lubricants, wetting or solubilizing agents, or coating agents.

Because of the complex and multicomponent nature of fixed oils as well as the lack of a thorough understanding of these materials, the monograph definition usually identifies the source as a means of defining the excipient. In contrast, the monograph definition for a single-component, well-characterized, and highly purified excipient indicates the acceptance criteria for the assay that is reflective of its content and purity. The use of any permitted additives also is included in the monograph’s definition (2) and labeling sections.

Need for modernization

Incidences of intentional adulteration have increased in recent years, and adulteration has become a major concern as supply chains continue to expand globally. Intentional adulteration can include the dilution of expensive fixed-oil products with cheaper substances.

Because of incidences of intentional adulteration in almond oil, in 2004 stakeholders provided USP with supporting data and requested the inclusion of a sterol composition specification in the Almond Oil NF monograph to combat adulteration. The supporting data demonstrated that compliance with the existing NF specifications in USP 28–NF 23 (2005) (3) did not guarantee the purity and authenticity of almond oil. In 2005, USP staff and the Excipient Monographs 2 (EM2) Expert Committee (EC) started to revise the Almond Oil NF monograph. Historical data in the Almond Oil NF monograph revision files indicated that this monograph had not been updated since the 1960s. Reports included in the revision files showed that Almond Oil NF adulterated with 10% to 25% of a cheaper oil or almond oil mixed with persic oil (1:1) still could pass NF 23 (2005) tests and meet all acceptance criteria, and thus the material erroneously could be considered to be in compliance with NF 23 specifications.

During the previous 2005−2010 revision cycle, EM2 EC concluded that the fixed-oil NF 23 compendial specifications required revisions because they had not kept pace with industry practices, specifically with respect to oil manufacturers’ current quality control practices in releasing and characterizing oil substances (4). USP−NF standards not only facilitate trade by establishing a baseline for product quality that is internationally agreed upon, but, most importantly, the public standards also ensure performance of drug formulations and add assurance of drug and excipient safety, thereby protecting public health.

In the United States under the Federal Food, Drug, and Cosmetic Act (FDCA), both USP and NF are recognized as official compendia. USP−NF is continuously revised. Compendial standards in existence for a long period of time may require updates to keep pace with current regulatory and safety requirements and to incorporate advances in analytical methodology and metrological science. The modernization replaces outdated and nonspecific methodology with analytical procedures that provide improved specificity, accuracy, precision, and sensitivity and are less laborious and less time-consuming. Such methods make it possible to demonstrate whether or not an excipient meets the acceptance criteria and is suitable for its intended use, particularly when the excipient must meet a stringent safety regulatory requirement (e.g., for parenteral or inhalation use).

Specifications for a particular excipient can change over time. Such changes usually occur with the introduction of improved quantitation methods that are universally adopted, typically because they are more sensitive and selective or can simultaneously detect multiple analytes. Changes to specifications also are necessary to enhance public standards’ usefulness in preventing intentional adulteration (5).

Table II: Specification for Almond Oil NF in USP 28−NF 23 (2005) (Ref. 3).Status of fixed-oil monographs before modernization. The Preface of USP−NF indicates that a USP−NF monograph for an official substance or preparation includes the article’s definition; packaging, storage, and other requirements; and a specification (1). The specification consists of a series of universal tests (description, identity/identification, impurities, and assay) and specific tests, one or more analytical procedures for each test, and acceptance criteria. Following the format defined in the Preface (similar to the current USP−NF monograph redesigned format), the Almond Oil NF specification in USP 28−NF 23 (2005) is shown in Table II.

Specifications for fixed-oil monographs that were developed several decades ago and included in USP 28−NF 23 (2005) were substantially similar to those of Almond Oil NF. Table III presents the Olive Oil NF specification in USP 32−NF 27 (2009) (6) by following the format defined in the Preface. EM2 EC proposed a revision to Olive Oil NF in 2009. The modernized Olive Oil NF monograph became official in USP 33−NF 28 Reissue (2010) (7). Like almond oil, olive oil is one of the most frequently adulterated fixed oils.

Monograph test deficiencies before modernization. An evaluation of the fixed-oil monographs in 2005 indicated that previous USP−NF monograph content had been developed using characterization methods that were available at that time, before global supply chains became the norm, and in some instances, with incomplete understanding of the fixed oils. Substances derived from natural sources historically were characterized mostly from a safety perspective using generally applicable but nonspecific tests to detect elemental impurities, residual process impurities, and pesticides. Because of the deficiencies in these fixed-oil excipient monographs (Table I), users were unable to relate the material’s chemical composition to its physical and chemical properties or to anticipate any potential degradation or decomposition during storage. A majority of the oil monographs, including petrochemical oil and essential oil monographs in USP 28–NF 23 (2005) did not contain identification tests. As a result, the existing compendial specifications in USP 28−NF 23 would conform with difficulty to cGMPs for finished pharmaceuticals, specifically Subpart E—Testing and Approval or Rejection of Components and Drug Product Containers and Closures, 21 CFR §211.84(d)(1) and (2) (8).

Table III: Specification for Olive Oil NF in USP 32−NF 27 (2009) (Ref. 6).Table II and Table III provide examples of the types of test procedures from NF 23 and NF 27, respectively, used to detect heavy metals and several fixed-oil substitutes (either contaminants or adulterants) in the Impurities sections of the monographs for Almond and Olive Oils. As shown in Tables II and III, no test procedure existed for the Identification and Assay. For Almond Oil shown in Table II, in the Specific Tests section, a few of the tests such as Specific gravity, Solidification range of fatty acids, Free fatty acids, Iodine value, and Saponification value were used as a rough measure of purity and quality. In fixed-oil excipient monographs before modernization, most procedures for testing fixed oil substitutes were wet chemistry–based methods with undefined or poor sensitivity and specificity (e.g., color reactions caused by the presence of particular nonfat ingredients). However, because these specific nonfat ingredients could be removed from oils by refining, refined oils would not test positive (9). Therefore, it was desirable to replace these wet chemistry tests with tests that could detect specific adulterants or contaminants. Incorporating such tests in USP−NF fixed-oil monographs could help ensure the authenticity of the fixed oils. However, most importantly, monograph modernization should introduce tests to determine fixed oil chemical composition specifications.

EM2 EC evaluated the fixed-oil substances with regard to their basic chemical composition, structure and properties, and manufacturing processes and investigated potential applications of current scientific knowledge to modernize and improve the compendial specification. New advances in analytical methodology allow scientists to profile the entire chemical composition of a substance derived from natural sources. Based on this evaluation and improved understanding, USP reference standards then could be developed for these complex mixtures.

Monograph modernization—a key initiative for the 2010–2015 USP revision cycle. Excipient monograph modernization is one of the key initiatives for the current 2010–2015 USP revision cycle. USP staff and the EXC EC collaborate with stakeholders in the USP public standards-setting processes (10). Unlike drug substances, so-called inactive ingredients (excipients) are sourced from several different industries, their production processes are different from pharmaceutical drug manufacturing, and usually they are less costly than drug substances. In general, sophisticated or technically advanced and costly analytical methods are reserved for research and development laboratories and are not routinely employed in quality control laboratories for excipient testing and release. EXC EC values input from stakeholders, especially from excipient producers, and recognizes the importance of advanced, highly reproducible, specific, and cost-effective analytical methods for characterizing excipients.

The development of USP general chapters to characterize fixed oils

Fixed oils. The characterization of fixed oils ideally should indicate their chemical composition by identifying and quantifying their components with a high degree of specificity and should determine the physical and chemical properties and potential transformations of the fixed-oil substances as a result of hydrolysis, oxidation, degradation, etc.

A fixed oil is formed from two simple building blocks: glycerin (or glycerol) and fatty acids. Unlike glycerin, which has a fixed structure, fatty acids can exhibit significant variation in their structures. In USP−NF, fatty acids in fixed-oil excipients traditionally are designated as carboxylic acids with hydrocarbon chain lengths ranging from 6 to 24 carbon atoms. The majority of fatty acid constituents in USP−NF fixed-oil excipients have unbranched chains, and they either may be fully saturated or may contain one or more double bonds. Glycerin has three hydroxyl groups with which fatty acids can react, and the resulting products are either monoglycerides (monoacylglycerols), diglycerides (diacylglycerols), or triglycerides (triacylglycerols).

Figure 1: Chemical structure for 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol-anexample of a specific triglycerideMost fats and oils are composed of triglycerides that differ in their fatty acid composition to a certain extent. Those containing the same fatty acids on all three positions are called simple triglycerides. Most naturally occurring triglycerides are of mixed composition and contain two or three different fatty acids. Other constituents that make up not more than (NMT) 3% of the fats and oils are unsaponifiable matter and a number of acyl lipids (e.g., traces of free fatty acids and monoclycerides and diglycerides).

The simplified nomenclature for unbranched fatty acids specifies the chain length and number of double bonds, separated by a colon. For example the triglyceride in Figure 1 contains the 16-carbon saturated palmitic acid (top), abbreviated as C16:0, the 18-carbon unsaturated oleic acid (middle) with 1 double bond, C18:1, and the 18-carbon unsaturated linoleic acid (bottom) with 2 double bonds, C18:2. The 18-carbon unsaturated linolenic acid with 3 double bonds, C18:3, also is known as an omega-3 polyunsaturated fatty acid.

Generally, the fatty acids in a triglyceride define the properties and characteristics of the molecule. Both the physical and chemical characteristics of fixed oils are influenced by component fatty acids, their proportions, and the way in which these are positioned on the glycerin molecule. In general, only the straight, even-numbered chains are present although fats and oils of animal origin can contain both odd-numbered and branched chain fatty acids as well.

Even though the building blocks are limited to glycerin and fatty acids, a large number of different triglycerides can be obtained from them. Thus, in general, a typical oil is a complex mixture of various triglycerides.

USP general chapter Fats and Fixed Oils <401>. ZUSP general chapter Fats and Fixed Oils <401> includes several test procedures to characterize and determine the properties of fats and fixed oils. During the 2005–2010 revision cycle, <401> underwent several revisions in PF (11–13). Before its proposed revision that appeared in 2008 (11), the chapter contained mostly simple wet chemistry–based methods to measure values characteristic of fats and fixed oils such as Acid Value (Free Fatty Acids), Ester Value, Hydroxyl Value, Iodine Value, and Saponification Value. Essentially, these tests used chemical reactions to quantitatively estimate the selected functional group(s) or to calculate—but not necessarily to identify—the constituents of a fat or oil. Thus Ester Value, Hydroxyl Value, Iodine Value, and Saponification Value traditionally are treated as oil and fat structure index tests. These indices, especially if combined, help to provide a rough idea of the identity of the sample. A triglyceride can be hydrolyzed to fatty acids and glycerin. Thus, Acid Value (Free Fatty Acids) is used as a measure of the degree of an oil’s hydrolysis.

In addition, Peroxide Value, Anisidine Value, and Total Oxidation Value (Totox) also were included in general chapter <401> before the 2008 revision (11). The tests for Anisidine Value and Total Oxidation Value (Totox) were proposed in 2003 (14) to support several monographs that contain polyunsaturated fatty acids, particularly some dietary supplement monographs. Fats and oils containing unsaturated fatty acids are prone to oxidation. Peroxide Value, Anisidine Value, and Total Oxidation Value (Totox) using wet-chemistry principles can reveal the extent of oxidative degradation of fats and fixed oils. Peroxide Value measures the amount of primary oxidation products, such as hydroperoxides, and the Anisidine Value measures secondary products, including aldehydes and ketones. Because the tests can determine the extent of oxidative deterioration, they are useful analytical tools to predict the expected shelf life of a fat or oil and to monitor an oil’s stability.

Before the proposed revision in 2008, <401> also provided tests for Unsaponifiable Matter, Solidification Temperature of Fatty Acids, and Fatty Acid Composition (11). The latter employs a modern gas chromatographic (GC) test procedure to analyze the distribution of fatty acid moieties that are attached to the three hydroxyl groups of the glycerin backbone if the sample is a fat or fixed oil. Fatty Acid Composition yields more detailed and reliable information when compared to these oil and fat structure index tests and thus has improved the identity determination of fats and fixed oils. Because Fatty Acid Composition can determine the percentages of each fatty acid group, structure indices, such as Iodine Value and Saponification Value, can be calculated or estimated based on the Fatty Acid Composition profile (15–17). However, Fatty Acid Composition is subject to considerable variation and presents challenges, the details of which are discussed in a later section.

Through the 2005 and 2006 revisions (12,13), the test for Acid Value was revised to include another titrant, to provide a calculation formula, and to add an additional test procedure allowing use of a different solvent mixture. The 2008 revision (11) replaced all the descriptive texts used in the calculations under the test sections with the appropriate calculation formula. Additionally, three new test sections were proposed: Omega-3 Fatty Acids Determination and Profile, Trace Metals, and Sterol Composition. These methods were introduced using a modern instrumental analysis approach. These additions enhanced the quality of general chapter <401> by providing compendial users further analytical methods that help to better characterize and evaluate fats, fixed oils, and related substances and that ensure purity of fixed oils and absence of adulteration.

USP general chapter Injections <1>. In 2006, stakeholders indicated that the test procedure for Unsaponifiable Matter described in general chapter <1> in USP 28–NF 23 was unclear and created uncertainty with regard to reporting of results (3).

USP staff and EM2 EC concluded that the test criterion for Unsaponifiable Matter under Other Vehicles in Ingredients, Vehicles, and Added Substances in <1> was subjective and thus unsuitable for its intended purpose, but the EC acknowledged the importance of the test for Unsaponifiable Matter for injectable products because it measured impurities such as waxes, phospholipids, and proteinateous matter to which patients could be allergic.

In addition, the original Cottonseed Oil NF monograph did not provide quality specifications for use as an injection vehicle. As a result, the monograph required revision to reflect its use in an intramuscular injection product. The EC also considered additional specific tests and appropriate acceptance criteria for this specific grade of oil.

EM2 EC collaborated with the Parenteral Products—Industrial EC to revise the tests under Other Vehicles in Ingredients, Vehicles, and Added Substances in <1> by means of a proposed revision published in 2008 (18). A quantitative test procedure for Unsaponifiable Matter and a test for Acid Value replaced the previous test for Unsaponifiable Matter and the test for Free Fatty Acids. Thus quantitative specifications for Acid Value and Unsaponifiable Matter were introduced. Three additional tests and acceptance criteria also were introduced into <1>: Peroxide Value, Water, and Limit of Copper, Iron, Lead, and Nickel. All three tests are critical quality control measures for fixed oils used in parenteral drug formulations. Usually, specifications for Peroxide Value and Water proposed under the Other Vehicles in <1> are more stringent than those implemented in the oils used for oral and topical products. The atomic absorption spectroscopy tests for Limit of Copper, Iron, Lead, and Nickel, referred to as Trace Metals in general chapter <401> were preferable to the method of general chapter Heavy Metals <231>. The quantitative Trace Metals test satisfied the accuracy requirements for much lower specification limits for parenteral products. If the oil had not been subjected to hydrogenation or if a nickel catalyst had not been used in processing, a note was included in the test for nickel stating that the test for nickel was not required, thus preventing unnecessary testing.

For cottonseed oil and other vegetable oils that have parenteral applications in drug formulations, the updated quality specifications in the Other Vehicles section in general chapter <1> were introduced and referenced in the fixed oil monographs.

Monograph labeling. The monograph Labeling section may contain a labeling requirement to differentiate a specific grade or chemical composition of the excipient. If a highly purified fixed oil will be used in injectable dosage forms (i.e., qualified as a specific grade), this is indicated in the Labeling section. Additional quality specifications for the specific grade may be necessary and can be included in Other Requirements under the Additional Requirements section of the monograph.

Recommendations and criteria for modernization

EM2 EC recommendations for fixed-oil monographs. Fixed-oil excipients are used in a large number of drug products and are essential to product safety and performance. Thus, the successful manufacture of a robust product requires the use of well-defined excipients and processes that together yield consistent product quality. Typically, excipients are manufactured and supplied to comply with compendial standards. USP−NF excipient monograph specifications are not designated to explicitly test for material functionality, except for co-processed excipients (19). A greater understanding of the chemical composition and the physical and chemical properties of excipients is necessary to set compendial specifications in USP−NF monographs. The specificity of identification methods and specificity, precision, and accuracy of assay methods help prevent intentional adulteration and ensure a quality excipient. Such understanding provides the basis for well-defined and well-characterized excipients so that additional testing for product functionality and performance consistency can be employed by end users to yield consistent product quality.

Based on a literature review, comparative analysis of compendial specifications for fixed-oil articles in NF 23, and studies of fixed oils using modern analytical technologies, USP staff and EM2 EC made the following recommendations to modernize fixed oil monographs:

• Introduce identification tests that are specific for chemical composition. A fingerprint or chemical profile—not all components or constituents must be individually identified—should be considered. Where possible, test for a greater number of nonoverlapping attributes by introducing a greater number of relatively simpler tests. In best cases, these tests can be performed orthogonally. Introduce tests that are generic and can be applied to most (if not all) fixed oils.
• Introduce an oil chemical composition specification specific for relative compositional constituents such as fatty acids.
• Introduce tests that identify and quantify the ingredient itself (e.g., component triglycerides rather than a surrogate).
• Introduce specifications to identify natural or deliberate transformation and stability.

These recommendations are discussed below.

Recommendations for test procedures. EXC EC proposes several criteria for test procedures for fixed-oil excipient monograph modernization:

• Universality of application
• Ease of adoption—uses existing equipment and methodology to the greatest extent possible
• Simplicity of procedure—a greater number of individual simpler tests is preferable to a smaller number of tests whose methodology is more complex and challenging
• Specificity—any test or combination of tests must be able to uniquely identify the fixed oil and exclude the presence of other oils
• Sufficient specificity to differentiate between an authentic fixed oil and an oil adulterated with the same oil of inferior quality
• Preferably identify as well as assay a fixed oil using one method.

Modernization strategy

Table IV: A strategic analytical test plan recommended in fixed oil monographs.USP staff and EM2 EC have developed an analytical test plan to fulfill the current regulatory requirements and keep abreast of current industry developments.

The analytical test plan for fixed oils is summarized in Table IV. The tests recommended in Table IV are critical control measures that ensure the identity, strength, quality, and purity of fixed oils. Further explanation and rationale to justify these recommendations are presented below.

Figure 2: Fourier transform infrared (FTIR) spectrum for olive oil.Identification tests. Spectroscopic identification and laboratory results. In the USP 2005–2010 revision cycle, USP staff and EM2 EC identified excipient monographs that lacked an identification test and recommended introducing an infrared (IR) spectroscopy or similar spectroscopic identification test in preference to wet chemistry or colorimetric tests. Generally, spectroscopic procedures provide a good identification for highly purified single-molecular compounds. This approach is similar to that adopted in a number of existing USP monographs for highly purified drug substances. These monographs usually specify a high-performance liquid chromatography (HPLC) identification test—HPLC peak agreement between the drug substance sample and a corresponding USP Reference Standard—in addition to the use of IR identification as an orthogonal test. An HPLC analytical procedure alone, depending on the separation mechanism of the compounds studied, is unable to provide information about the chemical structure of the components separated, whereas IR does provide information about the basic chemical structure of the components studied or their functional groups. In general, spectroscopic methodology and separation science are complementary. Separately, neither spectroscopic nor separation procedures is likely to be sufficient for unique identification, but when used together they provide a greater assurance of uniquely identifying an excipient.

Figure 3: 1H nuclear magnetic resonance (1H NMR) spectra for seven oils.The USP Research and Development laboratory performed suitability studies for inclusion of an IR identification test in several fixed-oil monographs such as cottonseed oil, olive oil, peanut oil, safflower oil, and soybean oil. The results indicated that IR could not be used as a suitable identification test for these fixed oils because their IR spectra were indistinguishable from each other. IR was unable to uniquely identify fixed oils by visual comparison because of the similarities in their functional groups. For example, general functional group assignments for IR analyses of olive oil (Figure 2) and peanut oil are presented in Table V, and their IR spectra are similar. The IR spectrum shown in Figure 2 is representative of a fatty ester. In contrast, the five fixed oil spectra could be differentiated from those of other oils, (e.g., mineral oil [a petrochemical oil] and polyoxyl 35 castor oil [a derivative of fixed oil]).

Table V: IR spectral analyses for fixed oils.The laboratory further investigated liquid ¹H nuclear magnetic resonance (NMR) spectroscopy for identification of seven vegetable oils—corn oil, cottonseed oil, olive oil, palm oil, peanut oil, safflower oil, and soybean oil—using a 300-MHz NMR instrument. Their ¹H NMR spectra were distinguishable from each other, and analysts observed significant differences between 0.8 and 2.8 ppm (Figure 3). Liquid ¹H NMR could uniquely identify each of the seven oils studied. These results confirmed published findings (20). General information about ¹H NMR signals vs. functional protons (Figure 3) for ¹H NMR analysis of the seven fixed oils is summarized in Table VI. Table VI lists notable differences observed in the four ¹H NMR signal ranges for the oils studied.

Figure 4: Chemical structure of ricinoleic acid.Although ¹H NMR was capable of distinguishing the seven fixed oils studied, EM2 EC did not recommend the ¹H NMR test be included as a compendial identification test because no assurance could be provided to ensure that mixtures of fixed oils could not unequivocally be identified as such and thus the samples could be identified—erroneously—as pure oils. In this regard, it should be emphasized that distinguishing between oils was entirely different from identification of a particular oil. Furthermore, there were concerns related to cost, lack of NMR instruments in most quality-control laboratories in industry, and a lack of personnel training for the NMR procedure. Currently, ¹H NMR use typically is limited to research and development laboratories. It is, however, a necessary analytical tool used to qualify USP reference standards.

Table VI: NMR analysis for seven fixed oils studied (NMR signal 0.8–2.8 ppm).Some non-oil excipient monographs do incorporate ¹H NMR testing, although not explicitly for identification testing, and have done so for several decades (e.g., the Polyoxyl 10 Oleyl Ether and the Polyoxyl 20 Cetostearyl Ether monographs that use ¹H NMR to determine the average polymer chain length). In the Poloxamer NF monograph, ¹H NMR test helps to determine the percentage of oxyethylene in the polymer sample. More recently, the ¹H NMR test increasingly is being introduced into NF monographs for complex excipients such as Hydroxypropyl Betadex, Chitosan, hydroxypropyl starches of different botanical origins, and corresponding pregelatinized species because it is the only suitable method to appropriately characterize these complex excipients.

Table VII: Compendial specifications of Fatty Acid Composition (%) of fixed oils in USP 35−NF 30 (Ref. 1).Fatty acid composition and melting range or temperature. Ideally, a robust specification should be independent of genetic modifications or seasonal or geographic variations that tend to change the composition of the substance over time and place. The Fatty Acid Composition of a fixed oil, however, varies with natural, seasonal, and geographical changes. Thus, the specification commonly defines the widest possible compositional range that is acceptable from the viewpoints of safety, functionality, and effective extraction. Even so, the specification limits for the Fatty Acid Composition should be narrow enough so that there is reasonable assurance that the substance is pure, not adulterated, not processed improperly or incompletely, and suitable for its intended use. The compendial specifications for Fatty Acid Composition for various fixed oils (not hydrogenated) in USP 35−NF 30 are summarized in Table VII.

Based on current practices employed by the fat and fixed oil industry, Fatty Acid Composition commonly is recommended as an identification test. The use of the Fatty Acid Composition to establish the purity of an oil has been criticized because of large variations permitted for certain fatty acid ranges (see Table VII). Thus, Fatty Acid Composition used alone as an identification test may not be sufficient to guarantee identity because some oils, such as Coconut Oil NF and Palm Kernel Oil NF, may exhibit similar fatty acid composition profiles, and some overlap exists in fatty acid ranges for different fixed oils. These two oils do possess different melting ranges. Therefore, both tests, Fatty Acid Composition and Melting Range or Temperature, used together may be able to achieve adequate differentiation. However, most of the fixed oils are liquids at ambient temperature, so application of Melting Range or Temperature specification is quite limited but can be found in several hydrogenated fixed-oil monographs.

Thin-layer chromatography. In recent years, thin-layer chromatography (TLC), specifically high-performance TLC (HPTLC), increasingly has been used for lipid analysis (21,22). TLC/HPTLC is a specific, reproducible, cost-effective, and routine quality-control analytical tool that provides a fingerprint identity for complex excipients such as fats, oils, and phospholipids. EXC EC has worked with the General Chapters—Chemical Analysis EC and the USP laboratory to propose a new general chapter, Identification of Fixed Oils by Thin-Layer Chromatography <202> (23). USP plans to release 11 USP Fixed-Oil Reference Standards in support of <202> (23). USP’s Dietary Supplements department is planning to add three new fixed oil dietary supplements to <202> to support new monograph development.

Unique patterns of TLC bands from specific triglycerides can distinguish fixed oils of different botanical origins, so it can be used orthogonally in conjunction with Fatty Acid Composition. The procedure is harmonized with European Pharmacopoeia general chapter 2.3.2. Identification of Fixed Oils by Thin-Layer Chromatography (24).

Assay. Content of Triglycerides. In recent decades, advances in chromatography and spectrometric methods (including mass spectrometry) have provided significant advancements in the understanding of complex excipient mixtures.

Some fixed oils from different plant sources yield characteristic patterns with distinct triglycerides that predominate (9). In many cases, the triglyceride pattern is more representative of the fixed oil identity than the fatty acid composition. This pattern can be determined with the help of HPLC or GC. A test for Triglyceride Composition based on an HPLC procedure using a refractive index detector was included in Sesame Oil NF in USP 28−NF 23 (2005) (3). The test has an additional advantage because it can be performed on an intact sample, and it directly assesses the chemical composition of the oil and is recommended as an Assay or Composition test. If the test for Triglyceride Composition is introduced into fixed-oil monographs, it also can be used as a stand-alone identification test.

In recent years, instrument manufacturers have developed and introduced more universal and sensitive detectors such as the evaporative light scattering detector (ELSD) and the charged aerosol detector (CAD) to test nonchromophoric analytes. When analytical instruments’ reliability, availability, suitability, and affordability substantially improve, public standards using these analytical tools can be developed and published. During the modernization of the Lecithin NF monograph in recent years, EXC EC for the first time introduced a test for Content of Phospholipids based on HPLC/ELSD to determine and quantify each individual phospholipid in lecithin (22).

Castor oil is one of the few naturally occurring triglycerides with high purity because the fatty acid portion comprises nearly 90% of the unique hydroxylated fatty acid, ricinoleic acid C₁₈H₃₄O₃, shown in Figure 4.

Ricinoleic acid is a major constituent of castor oil, and triricinolein is a major triglyceride in the product (25). Advances such as HPLC/ESLD and HPLC/CAD may provide opportunities to develop a test for Content of Triglycerides for castor oil and the other fixed oils.

High-performance size-exclusion chromatography (HPSEC) is a useful analytical tool to identify and quantify triglycerides, diglycerides, and monoglycerides, as exemplified in Glyceryl Monostearate NF, Glyceryl Distearate NF, and Glyceryl Tristearate NF, monographs in USP 36–NF 31 (2013) (26). In the Lipid Injectable Emulsion USP monograph, HPSEC is used to determinate soybean oil or other relevant oils used in the emulsion. Reports indicated that all triglycerides from soybean oil or other relevant oils are eluted as one peak under the test conditions, without separation. Although HPSEC cannot be used to differentiate fixed oils of different botanical origin, the amount of triglycerides in a fixed oil can be determined using the HPSEC procedure.

Table VIII: Iodine value and saponification value for fixed oils included in USP 31–NF 26 (Ref. 27).Specific tests. Sterol Composition. The composition of fatty acids traditionally has been used as an indicator of purity, although the wide variation in the composition of edible oils from different geographical origins or different cultivars is a limiting factor in the interpretation of data with regard to adulteration. Sterols, which comprise a major portion of the unsaponifiable matter, are found in almost all fats and fixed oils, and they also are characteristic of the authentic fixed oil.

Sterols are highly specific for each oil source, and a sterol composition analysis can reveal the identity of the components in a mixture. The determination of the composition of sterols from unsaponifiable matter serves as a further test for purity. Such general tests for selected attributes—when used together—provide a robust and unique identification for the fixed oil.

USP staff and EM2 EC recommended that the tests of Fatty Acid Composition (from the saponifiable portion) and Sterol Composition (from unsaponifiable matter) be used orthogonally for certain fixed oils to determine their identity and quality. Because of characteristic sterol profiles and the high economic values of almond oil and olive oil, comprehensive Sterol Composition specifications have been implemented in both monographs. The Sterol Composition specifications in Soybean Oil USP and Corn Oil NF serve to exclude any other contaminating oils such as canola oil when these fixed oils are manufactured in the same facilities.

Test specifications: Iodine Value, Saponification Value, and some other physical methods. Iodine Value and Saponification Value. Iodine Value and Saponification Value are used to measure the degree of unsaturation and the relative proportions of fatty acids and glycerin in a sample, respectively. Introducing the Fatty Acid Composition test into the monograph makes the Iodine Value and Saponification Value redundant because both values can be estimated from the Fatty Acid Composition profile (see Appendix 1). Therefore, when the Fatty Acid Composition test is part of the specifications of a fixed-oil monograph, the Iodine Value and Saponification Value usually are not included or have been deleted from the monograph.

The iodine and saponification values for each USP−NF fixed-oil included in USP 31–NF 26 (2008) (27) are summarized in Table VIII.

Other Physical Tests. Other tests such as Specific Gravity and Refractive Index that previously were included in several fixed oil monographs have been moved into the Description and Solubility section of USP−NF for the fixed oils. With the proposed modernization for the fixed oils, these physical tests provide added optional assurance of identification and purity of the fixed oils.

Table IX: Newly developed and modernized oil monographs.Monograph development and modernization status. From May 2005, USP staff and EM2 EC worked with FDA, USP Research and Development laboratories, and stakeholders during the 2005–2010 revision cycle to develop and modernize many fixed-oil monographs. Except for the newly proposed general chapter <202>, EM2 EC followed a strategic analytical plan for fixed oils (see Table IV) and developed eight new oil monographs and modernized six existing ones. All became official during the previous revision cycle (2005–2010) and were adopted into USP–NF. A list of the oil monographs developed and modernized during the 2005−2010 revision cycle is provided in Table IX.

Table X: Modernized Olive Oil NF in USP 33–NF 28 Reissue (Ref. 7).Table X displays the Olive Oil NF monograph modernized by following the guidelines in Table IV, except for the newly proposed chapter <202>.

With the introduction of new test chapter <202>, EXC EC will re-evaluate fixed oils and will continually revisit the modernization projects for fixed oil monographs. Recently, stakeholders requested USP to improve the Sterol Composition for Soybean and Olive Oil. The oil manufacturer recommended that the procedure for separation of sterol by TLC be replaced by a separation procedure using preparative HPLC to avoid cross-contamination by the TLC plate coating. The preparative HPLC method improves precision and reproducibility. Fixed oil modernization proposals will be published in future PFs (see Table XI for a list of on-going modernization projects).

Table XI: Continued monograph modernization for fixed oils.Conclusion and future development and modernizationAs shown in Table XI, excipient monograph modernization is a continuing endeavor that requires a consistent approach within a family of excipients. Such an approach is necessary to streamline analytical testing across multiple monographs as well as to keep the number of specific analytical tests to a minimum. This decreases the analytical burden on industry and allows methods to be referenced in monographs (e.g., from a general chapter).

USP continues to update USP−NF to provide standards for articles, including revised specifications based on advances in analytical and metrological science. USP ECs also rely on stakeholders’ and sponsors’ comments to keep monographs current. Emerging methodologies such as carbon number testing, C¹³/C¹²/ratio testing, and chemometrics can be considered as they become commonly adopted by stakeholders.

Acknowledgments

The authors would like to thank USP visiting scientists Hua Yin, MS (Chinese Pharmacopoeia) and Cheetham Mingle, MS (Food and Drug Board, Ghana) for their contributions to the monograph modernization projects for almond oil and castor oil, respectively. USP laboratory staff (Samir Z. Wahab; Patricia White (retired); Shane X. Tan; MinLi Liu; Johanna M. Smeller; Zarema K. Kassymberk; Eduardo R. Lim; Kornepati V. Ramakrishna; David C. Parmelee; Karen V. Gilbert; and Nadejda V. Soukhova) are acknowledged for their support in updating the documentary standards. The authors thank Stefan Schuber, PhD, MS, ELS, of USP for editorial assistance.

REFERENCES

1. USP, USP 35–NF 30 through First Supplement (US Pharmacopeia Convention, Rockville, MD, 2012).

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3. USP, USP 28–NF 23, (USP, Rockville, MD, 2005).

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8. 21 CFR 211.84.

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27. USP, USP 31–NF 26 (US Pharmacopeia Convention, Rockville, MD, 2008).

Appendix

Iodine Value

The formula to estimate the iodine value from the Fatty Acid Composition profile (15–17) is:

where

IV_mixture = iodine value

P_FA = percentage of a fatty acid determined from the Fatty Acid Composition (%)

M_W(2I) = atomic weight of two iodine atoms that are theoretically added to one double bond, 253.81

N_db = number of double bonds

M_W(FA) = molecular weight of the fatty acid.

The calculated iodine values for oleic acid, linoleic acid, linolenic acid (each with purity < 99%) using the equation above are 89.9, 181.0, and 237.5, respectively.

Saponification Value

The formula to estimate the saponification value from the Fatty Acid Composition profile (15,17) is:

For triglycerides

where

SV_mixture = saponification value

P_FA = percentage of a fatty acid determined from the Fatty Acid Composition (%)

M_W(KOH)= molecular weight of potassium hydroxide, 56.11

M_W(FA) = molecular weight of the fatty acid

M_W(Glycerin) = molecular weight of glycerin, 92.09

M_W(H2O) = molecular weight of water, 18.02.

About the Authors

Hong Wang* is senior scientific liaison at USP, Rockville, MD, hw@usp.org. Catherine Sheehan is director, excipients, USP. Lawrence H. Block (chair), Richard C. Moreton (vice-chair), Richard H. Wendt, Shireesh P. Apte, and Eric J. Munson are members of the USP 2010–2015 Monographs—Excipients Expert Committee.

*To whom all correspondence should be addressed.