Chapter 5
High-Performance Liquid Chromatography in Pharmaceutical Development of Antibiotic Products L. J. Lorenz Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285
The analytical properties and the preparative abilities of HPLC make it a very important tool in the development of antibiotic products. The drug development process requires evaluation of drug stability and of the types of degradation that may arise in the product. Both isocratic and gradient screening techniques are routinely used for these types of studies. HPLC and hyphenated HPLC techniques offer many handles for this type of work. HPLC, coupled with mass spectrometry or diode array detectors, can often offer some insight into the nature of the degradation product or impurity. Unfortunately, NMR information is usually required to definitively assign the structure of the compound. Preparative HPLC techniques offer the capability for obtaining suitable samples of degradation products for more sophisticated structural studies. HPLC is an invaluable tool in the development of antibiotic products. HPLC has become the technique of choice for determining the potency and dose uniformity of a drug product or the purity of a bulk drug raw material. This technique also allows a chemist to establish the factor composition of a multifactored product. In addition, HPLC offers the ability to isolate and monitor impurities and degradation products. Once isolated, these compounds can be studied with modern spectroscopic techniques to establish structure and confirm the entity's identity. Cefaclor, shown in Figure 1, is an important β-lactam antibiotic that has undergone extensive development effort. Cefaclor capsules and tablets contain cefaclor as a dry crystalline powder in a monohydrate form. Cefaclor suspensions contain cefaclor as a dry crystalline powder until the product is constituted and dispensed to the patient. While the hydration of the molecule affects the stability and solubility properties of cefaclor, the actual solid form of cefaclor has little or no impact on the HPLC results. Antibiotics are often labile compounds. This stability issue often dictates using special or very conservative sample handling procedures rather than those used for common stable pharmaceutical substances. Elaborate refrigerated sampling equipment and automated robotic sample processing are commonly used to accommodate these special needs for antibiotic substance evaluation. 0097-6156/92/0512-0054$06.00/0 © 1992 American Chemical Society
5. LORENZ
HPLC in Pharmaceutical Development of Antibiotic Products 5
Assay and Evaluation of Antibiotic Products Simple, rugged assays are desired for the assay and evaluation of antibiotic products. Reversed phase HPLC systems are preferred for such applications. Most β-lactam antibiotics can be chromatographed using several different separation mechanisms. Figure 2 shows a typical HPLC chromatogram for cefaclor. This assay is run in an acidic phosphate system containing pentanesulfonic acid as an ion pair reagent. Small amounts of triethylamine are also added to improve and maintain good peak shape over extended column use in the assay procedure. Bulk Drug and Formulated Products. This same chromatographic system is used to assay both bulk drug materials and formulated products. The assays are primarily designed as a "dilute-and-shoot" type of application. The drug substance is dissolved in an aqueous acidic buffer containing an internal standard that provides suitable drug solubility and reasonable solution stability for the drug substance. The resulting solution may then be directly injected onto the chromatographic system with or without the use of a membrane filtration step before the injection. Formulated products are handled in a similar manner to provide the simplest preparation procedure possible. Suspensions are handled by taking an aliquot of the suspension. For best results, the aliquots are weighed and converted to a volumetric with incorporation of the measured sample density. This procedure allows a dose per volume result to be calculated and eliminates problems associated with dispersed air bubbles in the suspension. When dealing with capsule or tablet products, the assay sample may be prepared either from a composite sample or by an individual dose approach. Procedures for suspensions or capsules and tablets involve a dissolution process that may incorporate a secondary dilution to provide an assay solution containing the appropriate concentration of the analyte and internal standard. After preparation of the assay solution, the solution is passed through a membrane filter and injected onto a chromatographic system. If the samples are prepared in a batch mode, the assay solutions are usually queued in a refrigerated autoinjector. Internal Standard vs. External Standard. The cefaclor assay described here incorporates an internal standard that accounts for the second large peak in the assay chromatogram. Experiences in our laboratories have shown that the actual assay precision is usually slightly better when using an external standard approach. There are still several good reasons why internal standards are routinely incorporated into the assay procedure. Several of these reasons are listed in Figure 3. One major reasons stems from the fact that our laboratory develops procedures that will be used in many different facilities in several different countries. Internal standards are still preferred in many foreign countries and in the laboratories of our foreign affiliates. Also, there are technical reasons for incorporating the internal standard into an assay procedure. The resolution between peaks is a useful monitor to show that the method performs properly throughout the sample run. Additionally, internal standards aid in deciding if an instrument malfunction occurred during the run and which data should be suspect. With an external standard approach, rejection of an assay result can be difficult. The rejection of an individual piece of datafroma large run requires the assignment of a reason for the deletion of the information from the data set. This is especially a problem when a very small bubble may have been present in the injected sample causing a low result. With the internal standard approach, it is easy to see that both peaks are low. In many situations, the internal standard corrects for the problem. In other cases, the internal standards provide a mechanism for rejecting an individual
56
CHROMATOGRAPHY OF PHARMACEUTICALS
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Figure 1. Cefaclor structure.
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Figure 2. Typical HPLC chromatogram for cefaclor. Condition of run: Mobile phase: 1 gram pentane sulfonic acid sodium salt, 780 mL water, 10 mL triethylamine. Adjust the pH to 2.5 with phosphoric acid. Add 220 mL of methanol. Column: Beckman Ultrasphere ODS (25 cm). Flow rate: 1 mL/min. Detection: Ultraviolet at 265 nm.
• Laboratory preference •Verification of system performance •Verification of injection performance • Provide justification for data rejection • Verify process of automated sample processors
Figure 3. Justification for internal standards.
5. LORENZ
HPLC in Pharmaceutical Development of Antibiotic Products 5
injection. If an injection problem occurs and both peaks in the chromatogram are low, there is a legitimate reason for rejecting the data from the assay average. Robotic Sample Processors. The advent of robotic sample processors is yet another compelling reason for having internal standards present as a running check of the assay performance. Here the internal standard provides a valuable piece of information to validate or to ensure that the sample preparation and injection were properly performed. Even though internal standards have fallen somewhat into disfavor for sample quantitation in HPLC, one can see a resurgence in the need for internal standards in assay systems for validation purposes. Internal standards provide a good monitor of system performance and measurement criteria that aid in decisions about data acceptability. This need for an internal standard becomes even more critical when dealing with automated sample processing systems that will become the norm for performing assays in the future. Determination of Impurities and Degradation Products A very important part of antibiotic development programs is the determination of impurities, synthetic precursors, and degradation products for the drug substance. Again, researchers rely heavily upon HPLC techniques for these determinations. Sophisticated gradient HPLC procedures are frequently used during compound development efforts. Figure 4 shows a typical gradient chromatogram for cefaclor. These gradient procedures allow a sample to be monitored over a broad range of polarities to separate the different types of materials that may be present in a sample. Synthetic precursors and some degradation products are often much less polar than the drug entity itself, leading to the presence of late eluters in the chromatographic system. Additionally, many degradation products are much more polar than the drug itself, thus this set of polar compounds elutes early in the chromatogram. The examination of the entire range of potential sample components requires gradient HPLC. Detection of Extraneous Substances. Selecting the means to detect extraneous substances is an important part of HPLC assay development. Ideally, diode array detectors can be used through some of the early developmental phases. These detectors can monitor many different wavelengths and can potentially detect many different compounds. Figure 5 is a typical chromatogram that can be obtained from this type of program. In this example, several compounds can be observed at lower wavelengths. Typically, the ultraviolet absorption maximum for cephalosporins lies near 260 nm. In this example, there are also many sample entities that are nondetectable at or near 260 nm. Therefore, monitoring at a lower wavelength provides more information than at higher wavelengths. Figure 6 shows an actual chromatogram monitored at 220 nm. The chromatography system chosen for this work is a buffered system with acetonitrile serving as the modifier for the gradient. In this application for cefaclor, the separation of several of the major degradation products is very pH sensitive. This is shown in Figure 7, which is the pH retention profiles for several substances related to cefaclor. Several compounds cross and undergo reversal of retention as a function of change of pH. Therefore, good pH control is essential to maintain acceptable assay performance. Identification and Quantitation of Impurities. Often, a major problem in drug development is identifying and quantitating impurities in a drug substance. Again, HPLC plays a very important role in this phase of drug development In this phase of the drug development program, the drug product is stressed using heat, light, peroxide, acid, and base to establish the most likely degradation compounds that
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Figure 4. Typical gradient chromatogram for cefaclor. Mobile phase: A solvent, 6.9 g/ L sodium phosphate monobasic adjusted to pH of 4 with phosphoric acid. Β solvent, 550 mL of solvent A with 450 mL of acetonitrile. Gradient: Start at 5% of solvent Β with a linear program increasing to 25% at a rate of 0.67% change/ min. The second step programs to 100% Β solvent at a rate of change of 5% change/min. Flow rate: 1 mL/ min. Detection: ultraviolet at 220 nm.
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Figure 5. Typical HPLC diode array chromatogram. (See Figure 4 for conditions.)
LORENZ
HPLC in Pharmaceutical Development of Antibiotic Products
Figure 6. Chromatogram for cephalosporin monitored at 220 nm (See Figure 4 for conditions.)
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Figure 7. pH rétention profiles for several cefaclor related substances. (Conditions are the same as given in Figure 4 except for the pH of the mobile phase. The pH was adjusted with phosphoric acid or sodium hydroxide to provide the appropriate pH.)
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5. LORENZ
HPLC in Pharmaceutical Development of Antibiotic Products
might form. When possible, these stress-generated compounds are compared to products that form during the actual storage life of the drug substance or product. Antibiotics are usually very sensitive to these types of conditions and form many different products. The detection of these compounds, again often with diode array detection devices, offers a way to identify and quantitate the major products that are formed under each of the conditions. These studies also serve as a basis for the selection of preparative HPLC systems for processing these samples. Preparative HPLC Systems. During the development of rugged assays, one normally searches for good buffering agents to use in the reverse phase chromatographic systems. These buffers are usually selected to be ultraviolettransparent to allow maximum flexibility in the selection of monitoring ultraviolet wavelengths. Typically, inorganic salts are selected as the normal HPLC buffer systems. However, salt-free systems are desired in preparative applications. Therefore, systems utilizing acetic acid, trifluoroacetic acid, and triethylamine are frequently used in the developmental phase that concentrates on the isolation and characterization of impurities. These substances can normally be removed by lyophilization techniques as part of the sample work-up procedure. These criteria for preparative applications often dictate the development of additional systems for isolation and characterization work. Hyphenated systems such as HPLC-diode array systems and HPLC-mass spectrometry systems may also be used in this phase of the developmental program. Often these systems have unique requirements about buffers and additives present in the HPLC mobile phase. Figure 8 is a chromatogram of the degradation products formed when cefaclor is exposed to heat and acid for the heat-induced and acid-induced degradation of cefaclor. Hyphenated techniques such as the HPLC-diode array device and HPLCMS can often provide valuable information about sample impurities and degradents. Figure 9 is an HPLC diode array chromatogram of a portion of the cefaclor chromatogram showing the UV spectra for the individual components in the sample. This information helps to determine which compounds might be structurally related. This is shown vividly by the late eluters in this chromatogram where there are a whole series of degradents having similar spectra which are different from the spectrum of the parent drug substance. The addition of mass spectral data with this information can be an important part of the structural elucidation for such compounds. Identification of Unknown Entities. These techniques might be sufficient to positively identify a known compound in the sample. Unfortunately, this information is not always sufficient to establish the identity of an unknown entity. Thus, the preparative features of HPLC must usually be applied to generate authentic isolated impurities that can be examined by additional techniques to allow the assignment of structure. Semi-preparative techniques using columns up to two inches in diameter and sometimes larger are used in antibiotic development programs. Volatile buffers can be used in a gradient or isocratic mode. Several major chromatographic cuts are obtained from the degraded drug material. These initial chromatographic cuts are then lyophilized and rechromatographed one or more times until a single component entity is obtained. Following verification that one has a chromatographically pure entity, the material is submitted for sophisticated spectral characterization. The determination of impurities and degradation products in an antibiotic drug such as cefaclor can become a very complex issue for the analytical chemist. One often must quantitate many materials. Some of the impurities are structurally similar to the drug substance. These entities will have spectral response characteristics similar to those of the parent drug material. For these types of substances, quantitation can be done versus the response of the parent drug substance. Another set of materials may
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62
CHROMATOGRAPHY OF PHARMACEUTICALS
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Figure 8. Chromatogram of heat -induced and acid-induced degradents of cefaclor. (Conditions are equivalent to those provided in Figure 4.)
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