Evaluating Impurities in Drugs (Part III of III)

Controlling and monitoring impurities in APIs and finished drug products is a crucial issue in drug development and manufacturing. Part I of this article, published in the February 2012 issue of Pharmaceutical Technology, discussed the various types of and sources of impurities with specific case studies (1). Part II, published in the March 2012 issue, examined chiral, polymorphic, and genotoxic impurities (2). In Part III, the authors examine various degradation routes of APIs, impurities arising from API–excipient interaction during formulation, metabolite impurities, various analytical methodologies to measure impurity levels, and ways to control impurities in pharmaceuticals.

Definition of impurity

The term impurity reflects unwanted chemicals that are present in APIs or that develop during formulation or upon aging of the API in the formulated drug product. The presence of such unwanted material, even in small amounts, could affect the efficacy and safety of pharmaceutical products. Several guidelines from the International Conference on Harmonization (ICH) address impurities in new drug substances, drug products, and residual solvents (3–6). As per the ICH guidelines on impurities in new drug products, impurities present below a 0.1% level do not need to be qualified unless the potential impurities are expected to be unusually potent or toxic (5). In all other cases, impurities should be qualified. If the impurities exceed the threshold limits and data are not available to qualify the proposed specification level, studies to obtain such data may be required. Several recent articles describe a designed approach and guidelines for isolation and identification of process-related impurities and degradation products using mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, high-performance liquid chromatography (HPLC), and Fourier transform infrared (FTIR) spectroscopy for pharmaceutical substances (7–9).

Degradation-related impurities

Degradation products are compounds produced by decomposition of the material of interest or active ingredient. Several impurities may result because of API degradation or other interaction on storage, so stability studies need to be conducted to ensure drug product safety (10). Hydrochlorothiazide (see Figure 1) is a classical example of a degradation impurity. It has a known degradation pathway through which it degrades to the starting material as disulfonamide in its synthesis.

Figure 1: Degradation of hydrochlorothiazide. (ALL FIGURES ARE COURTESY OF THE AUTHORS)

Degradation products could result from the synthesis itself, storage, formulation of the dosage form, and aging (11). These degradation pathways are further discussed.

Synthesis-related impurities. Impurities in a drug substance or a new chemical entity originate mainly during the synthetic process from raw materials, solvents, intermediates, and byproducts. The raw materials are generally manufactured to much lower purity requirements than a drug substance, and thus, it is easy to understand why they can contain a number of components that can in turn affect the purity of the drug substance.

1-Methyl-3-phenyl piperazine (see Figure 2) is present as an unreacted starting material that competes in all the stages eventually leading to the impurity keto-piperazine derivative of mirtazapine (see Impurity C, Figure 2).

Figure 2: Reaction scheme for mirtazapine impurity. Ph. Eur is the European Pharmacopoeia. DMF is dimethylformamide. EtOAc is ethyl acetate.

Formulation-relatedimpurities. Several impurities in a drug product or API can arise from interactions with excipients used to formulate the drug product. In the process of formulation, a drug substance is subjected to various conditions that can lead to its degradation or other deleterious reactions. For example, if heat is used for drying or for other reasons, it can facilitate degradation of thermally labile drug substances. Solutions and suspensions are potentially prone to degradation due to hydrolysis or solvolysis. These reactions also can occur in the dosage form at solid state, such as in the case of capsules and tablets, when water or another solvent has been used for granulation.

There are two typical conditions in solid- and solution-state degradation studies. Typical conditions for the API in a solid state might be 80 °C, 75% relative humidity (RH); 60 °C at ambient RH; 40 °C at 75% RH; and light irradiation. Typical conditions for an API in the solution state might be: pH 1–9 in buffered media; with peroxide and/or free-radical initiator; and light irradiation.

Figure 3 shows the degradation pathway of ketorolac in the solid and solution states (12–14).

Figure 3: Degradation pathway of ketorolac.

Dosage form-related impurities. Impurities related to the dosage form are significant because many times precipitation of the main ingredient requires various factors, such as pH or leaching, to be altered (15). For example, the precipitation of imipramine hydrochloride with sodium bisulfite requires a subsequent pH alteration of lidocaine hydrochloride solution in the presence of 5% dextrose in saline.

Method-related impurities. A known impurity,1-(2,6-dichlorophenyl)indolin-2-one is formed in the diclofenac sodium ampuls. Formation of this impurity depends on the initial pH of the preparation and the conditions of sterilization (i.e., autoclave method, 123 °C ± 2 °C) that enforces the intermolecular cyclic reaction of diclofenac sodium, forming indolineone derivative and sodium hydroxide (16).

Environmental-related impurities . Environmental-related impurities may result from the following:

• Temperature. Many heat-labile compounds, when subjected to extreme temperature, lose their stability. Keeping this in mind, extreme care should be exercised to prevent them from degradation.

• Light (ultraviolet light). Exposure to light results in a photolytic reaction. Several studies reported that ergometrine and ergometrine injections are unstable under tropical condition such as light and heat (17–19).

• Humidity. Humidity is one of the important factors when working with hygroscopic compounds. Humidity can be deleterious to bulk powders and formulated solid dosage forms. Well-know examples are ranitidine and aspirin (19).

Impurities on aging. Generally, a longer stay on the shelf increases the possibility that impurities will occur. Such impurities can be caused by several interactions as further described.

• Interaction among ingredients. Vitamins are highly prone to instability after aging. For example, the presence of nicotinamide containing four vitamins (nicotinamide, pyridoxine, riboflavin, and thiamin) caused the degradation of thiamin to a substandard level during a one-year shelf life (20). Table I lists some examples of interactions among ingredients.

• Hydrolysis. Many drugs are derivatives of carboxylic acids or contain functional groups susceptible to acid–base hydrolysis (e.g., aspirin, atropine, and chloramphenical).

• Oxidation. In pharmaceuticals, the most common form of oxidative decomposition is auto-oxidation through a free-radical chain process. Drugs that are prone to oxidation include methotrexate, adinazolam, catecholamine, conjugated dienes (i.e., vitamin A), and nitroso and nitrite derivatives. Olanzapine is especially prone to oxidative degradation in the presence of oxygen (see Figure 4) (21).

• Photolysis. Photolytic cleavage on aging products occurs with APIs or drug products that are prone to degradation on exposure to UV light. For example, the ophthalmic formulation of ciprofloxacin drops 0.3%, when exposed to UV light and undergoes photolysis to form ethylene diamine, an analog of ciprofloxacin (22).

• Decarboxylation. Carboxylic acid (–COOH) tends to lose carbon dioxide from carboxyl groups when heated. For instance, a photoreaction of a rufloxacin enteric tablet coated with cellulose acetate phthalate and subcoated with calcium carbonate causes hydrolysis of cellulose acetate phthalate. This reaction liberates acetic acid, which on reacting with calcium carbonate, produces carbon dioxide as a byproduct.

• pH. It is well understood that pH, particularly extreme levels of pH, can encourage hydrolysis of the API when ionized in an aqueous solution. This situation necessitates buffer control if such a dosage form is required.

• Packaging materials. Impurities may result from packaging materials (i.e., containers and closures (23).

Table I: Effect of interactions among ingredients

Two impurities in olanzapine have been identified as 1 and 2 (see Figure 4) (24). The structures indicate that the two impurities are degradation products resulting from oxidation of the thiophene ring of olanzapine.

Figure 4: Olanzapine impurities due to air, heating, and formulation.

From a regulatory perspective, forced degradation provides data to support the following (25):

• Identification of possible degradants

• Degradation pathways and intrinsic stability of the drug molecule

• Validation of stability for indicating analytical procedures

• Facilitation of the development of analytical methods to evaluate stability

• Understanding the degradation of the API to a rational product.

• Screening for possible formation of potential genotoxins.

Various issues are addressed in regulatory guidance (3–6). Some key issues are:

• Forced degradation is typically carried out using one batch of material.

• Forced-degradation conditions are more severe than accelerated stability testing, such as 50 °C; ≥ 75% RH; light conditions exceeding ICH standards; high and low pH; and oxidation.

• Photostability should be an integral part of forced-degradation study design (10).

• Degradation products that do not form in accelerated or long-term stability may not have to be isolated or have their structure determined.

• Mass balance should be considered.

Various issues are not addressed in regulatory guidance (3-6). Some key issues not addressed are:

• Exact experimental conditions (temperatures, duration, and extent of degradation)

• Experimental design (left to the applicant's discretion).

Metabolite impurities

Metabolite impurities are byproducts formed in the body after a drug substance is ingested. During metabolism, the API and drug product in the body are exposed to various enzymes, from which metabolite impurities can be formed (26–34). Drug metabolism is traditionally divided into two phases: metabolic (i.e., hepatic) clearance and the Phase I and Phase II process. The division is based on the observation that a drug substance first undergoes oxidative attack (e.g., benzene to phenol), and the newly introduced hydroxyl function will undergo glucouronidation (e.g., phenol to phenyl glucouronic acid). Some metabolites are formed as impurities during the development of a process. Control of these process-related metabolite impurities in the final API may not be necessary if control of other metabolites has already occurred and taken into consideration. Tightening the limits, therefore, may not be needed.

Examples are asenapine N-oxide, asenapine desmethyl, and ciprofoxacin ethyl diamino impurity, which are formed as process impurities, but are also metabolites of the same process (see Figure 5). It put forth a question whether limiting such a metabolite impurity in the final API is still required.

Figure 5: Process impurities, thermal decomposition impurities, and metabolites of asenapine. CAS No. is Chemical Abstracts Service number.

Select analytical methodologies

The development of a new drug mandates that meaningful and reliable analytical data be generated at various steps of drug development. The drug also should exhibit excellent stability throughout its shelf-life. To meet these requirements, methodologies need to be developed that are sensitive enough to measure low levels of impurities. This need has led to analytical methods that are suitable for determining trace and ultra-trace levels (i.e., submicrogram) quantities of various chemical entities (35–39). Various methods are available for monitoring impurities.

Spectroscopic methods . Various spectroscopic methods can be used for characterization of impurities, such as UV-visible spectroscopy, FTIR spectroscopy, NMR spectroscopy, and mass spectrometry (MS).

Separation methods . Various separation methods can be used, including thin-layer chromatography (TLC), gas chromatography (GC), HPLC, capillary electrophoresis (CE), and supercritical fluid chromatography (SFC). A review of these methods is provided in the literature (39). CE is an electrophoretic method that is frequently lumped with chromatographic methods because it shares many of the common requirements of chromatography. A broad range of compounds can be resolved using TLC by using different plates and mobile phases. GC is a useful technique for quantification. It can provide the desired resolution, selectivity, and ease of quantification. This technique is useful for organic volatile impurities. SFC offers some of the advantages of GC in terms of detection and HPLC in terms of separation.

Hyphenated methods. The following hyphenated methods can be used effectively to monitor impurities: GC–MS; liquid chromatography (LC)–MS; LC–diode-array detection (DAD)–MS; LC–NMR; LC–DAD–NMR–MS; and LC–MS–MS.

Isolating impurities . It is often necessary to isolate impurities because the instrumental methods are not available or further confirmation is needed. The following methods have been used for isolation of impurities: solid-phase extraction, liquid–liquid extraction, accelerated solvent extraction, supercritical fluid extraction, column chromatography, flash chromatography, TLC, HPLC, CE, and SFC.

Impurity profiling

Ideally, an impurity profile should show all impurities in a single format to allow monitoring of any variation in the profile. The driving forces for studying an impurity profile are quality considerations and regulatory requirements.

Samples to be profiled . Impurity profiling should be done for APIs, process check of the synthesis or formulation, and final drug product.

Components in an impurity profile. Ideally, an impurity profile should show synthesis-related impurities, formulation-related impurities, degradation products, and interaction products.

Crucial factors for controlling impurities in API s. Several factors are important in controlling impurities in APIs as further outlined.

Crystallization . The size of crystals not only determines the quality, but also the stability of the drug. During crystallization, fine crystals should be formed to prevent entrapment of minute amounts of chemicals from the mother liquor, which in turn causes degradation of the drug.

Wet-cake washing. Many unwanted chemicals, including residual solvents, could be removed by thorough washing of the wet cake, which if not done correctly, could lead to retention of solvents and impurities in the cake.

Drying. Use of vacuum dryer or a fluid-bed dryer is always advisable in comparison to a tray dryer. Use of the former reduces drying time and brings about uniform drying, which is helpful in drying sensitive drug substances.

Appropriate packaging. The packing of bulk drugs should be based upon their nature and sensitivity. Light-sensitive products should be packed in light protective packing. Use of opaque containers for ciprofloxacin eye-drops preparations protects the active ingredients from photodegradation (22). Use of ampuls with either black carbon paper or aluminum foil for ergometrine produced negligible degradation (40). It is important to determine the most appropriate container-closure system.

Production methods based on stability studies . A manufacturer of a bulk drug should perform a detailed investigation of the process, including stability studies while finalizing the method of preparation. For example, for producing diclofenac sodium injections, the aseptic filtration process is better than the autoclave method that produces the impurity (16).

Measures by pharmacopoeias . Pharmacopoeias should take steps to incorporate impurity limits for drug substances made from a raw material in which that particular impurity is controlled. It becomes convenient for the users if the impurity limit is mentioned in the dosage forms.

Conclusion

Parts I, II, and III of this article discussed the types, origin, causes, chemistry, and impact of impurities in APIs and drug products (1, 2). Parts I and II explained how, when, and why impurities are formed. This article, Part III, highlighted the degradation-related, formulation-related, and metabolite impurities, the various analytical techniques available for their identification and separation, and crucial factors that are to be controlled while preparing bulk drugs.

Kashyap R. Wadekar , PhD ,* is a research scientist (II), Ponnaiah Ravi , PhD , is senior vice-president of R&D, Mitali Bhalme , PhD , is an associate research scientist, S. Srinivasa Rao is a research associate, K. Vigneshwar Reddy is a research associate, L. Sampath Kumar is a research chemist, and E. Balasubrahmanyam is a research chemist, all with Neuland Laboratories, 204 Meridian Plaza, 6-3-854/1, Ameerpet, Hyderabad, India, tel. 91 40 30211600, kashyapwadekar@neulandlabs.com.

*To whom all correspondence should be addressed.

Submitted: Sept. 19, 2011; Accepted Nov. 28, 2011.

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