HPLC for Pharmaceutical Scientists 2007 (Part 14)

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In the drug discovery area, a compound with desired therapeutic properties is identified, and its structure may be modified by synthetic alterations to enhance potency and specificity or to decrease toxicity and undesired side effects. The lead drug candidate is then transitioned into the drug development area. Only small amounts of drug (typically less than a gram) are required to support the required studies in the Drug Discovery area. However larger amounts are required to support the studies conducted in the Drug Development area. The amount required in the preclinical stage typically ranges from 20 to 2000 g....

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  1. 14 ROLE OF HPLC IN PROCESS DEVELOPMENT Richard Thompson and Rosario LoBrutto 14.1 RESPONSIBILITIES OF THE ANALYTICAL CHEMIST DURING PROCESS DEVELOPMENT In the drug discovery area, a compound with desired therapeutic properties is identified, and its structure may be modified by synthetic alterations to enhance potency and specificity or to decrease toxicity and undesired side effects. The lead drug candidate is then transitioned into the drug develop- ment area. Only small amounts of drug (typically less than a gram) are required to support the required studies in the Drug Discovery area. However larger amounts are required to support the studies conducted in the Drug Development area. The amount required in the preclinical stage typically ranges from 20 to 2000 g. This material is required to support studies includ- ing subchronic toxicity, genotoxicity, ancillary pharmacology, early animal pharmacokinetics (PK), salt/form selection, and formulation development. As the drug candidate progresses through the various clinical stages, the drug requirements typically range from 1 kg to 200 kg. This material supports the various clinical studies as well as chronic toxicity, carcinogenicity, develop- ment and reproductive toxicity, and formulation development. Finally, tons of drug may be required upon successful approval and commercialization (Figure 14-1). The synthetic pathway to the drug substance is likely to evolve during the various stages of development. It is highly unlikely that the synthetic process HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. 641
  2. 642 ROLE OF HPLC IN PROCESS DEVELOPMENT Figure 14-1. Stages of process development in the context of drug development. utilized in drug discovery will be the same as that used to provide commercial scale quantities. The discovery chemist may utilize a large number of synthetic steps, use a number of reagents that are expensive or not practical at scale-up, use a number of chromatographic steps for purification, and experience very low yields. For scale up, the process development chemist must factor in safety, economical, and ecological considerations while producing a robust and repro- ducible synthesis. He must consider operating limitations such as heat and mass transfer. Economic factors will dictate minimization of the synthe- tic steps, maximization of yield, and choice of raw materials. In addition, the process must meet environmental, occupational health, and safety requirements. Furthermore, the process development chemist must follow guidelines from the Food and Drug Administration (FDA) in relation to the control and iden- tification of impurities in drugs that will be used in humans. Regulatory bodies require that the maximum possible human exposure to an impurity in a drug substance be supported by toxicological studies in animals that indicate no sig- nificant adverse effects. Consequently, impurities that exceed a 0.1% tolerance limit in clinical material must first be qualified in animal toxicological studies. Scale up of a synthesis, however, may generate a different impurity profile than observed for the smaller quantities prepared to support the toxicological studies. Kinetic factors, changes in raw materials, or changes in reaction con- ditions may result in the introduction of new or elevated impurities. These new impurities may be qualified in additional chronic toxicity and genotoxicity studies, but this strategy is often not economically feasible and is undertaken more as a last resort. A better strategy is to identify and then control impuri- ties that are generated during the continuously evolving stages of process development.
  3. HPLC SEPARATION MODES 643 As a consequence of process evolution and regulatory requirements, the analytical chemist supporting process development is faced with a number of challenges. He must evaluate the purity and stability of raw materials, inter- mediates, and drug substance. He must evaluate yield and impurity generation across the various synthetic steps. Impurities in drug substance, intermediates, and raw materials may require identification. Analytical methods may have to be adapted to accommodate process changes. Finally he must set specifica- tions, validate analytical methods, provide regulatory documentation, and perform a technology transfer prior to drug approval and commercialization. To this end, HPLC is a critical tool to perform many of the above tasks. Most pharmaceutical compounds are amenable to analysis by HPLC. HPLC is a powerful technology that is capable of separating complex mixtures into indi- vidual components that can then be quantified. A well-developed HPLC method resolves and quantifies impurities from an analyte of interest in a reproducible, rugged, precise, and accurate fashion. 14.2 HPLC SEPARATION MODES The multitude of available separation modes, mobile phases, and columns provide a plethora of parameters that can be manipulated to meet the crite- ria for a well developed HPLC method. Conversely, it also creates a dilemma in choosing the optimal parameters from the myriad of possibilities. The com- monly utilized modes of HPLC in pharmaceutical development are reversed phase (RP) and normal phase (NP) for small organic molecules (
  4. 644 ROLE OF HPLC IN PROCESS DEVELOPMENT 14.2.2 Normal-Phase Chromatography Normal-phase or adsorption HPLC utilizes a polar stationary phase and a less polar mobile phase. Retention occurs through polar interactions, such as hydrogen bonding and dipole interactions, between the solute and the sta- tionary phase. Retention is more predictable than for RP chromatography. Carboxylic acids tend to show the strongest retention followed by amines, ketones, ethers, aromatic hydrocarbons, and saturated hydrocarbons in decreasing order of retention. Good selectivity is often observed for positional and stereoisomers. The mobile phase is usually a mixture of a nonpolar solvent such as heptane or hexane(s) and a more polar solvent. The polar organic solvent can be chosen based on it physicochemical properties (hydrogen bonding capabilities, lipophilicity, and polarizability).There are a large number of options for eluent components, and extensive selectivity changes are observed with the use of various mobile-phase components. In addition, small changes of the polar organic solvent can cause large changes in retention, and this should be investigated during method development. Common solvents include ethanol, isopropanol, tetrahydrofuran, ethyl acetate, and dichloromethane. The level of water in the solvents needs to be controlled as well, since differences in retention may be observed. Note that heptane and methanol have limited miscibility and only a maximum of 5% methanol should be used. Mobile-phase miscibility should be checked prior to pumping a par- ticular composition on the HPLC. A simple mixture of the solvents in the beaker should allow the chromatographer to discern if the two components are miscible. Additives such as triethylamine and trifluoroacetic acid are rec- ommended to reduce retention and improve peak shape for the analysis of bases and acids, respectively, by reducing interactions of the solute with the highly active sites of silica. Commonly utilized stationary phases include cyano, diol, amino, and silica. However, unmodified silica possesses greater surface heterogeneity and is more retentive than the other three phases. Very little selectivity differences are observed as a function of the type of stationary phase. When using NP the chromatographer must remember to convert their system to NP mode if RP mode was used previously. Any aqueous/buffer left in the system could precipitate out when the normal-phase solvents are pumped than the system. Water contamination in the mobile phase lines can also lead to water absorption on the column and change the chromatography significantly. It is generally recommended, that if the system was previously in RP mode, to flush the system with pure water for about 15 minutes at 2 mL/min. Then use IPA to flush the system for an additional 10 minutes at 2 mL/min. The system should then be flushed with the desired NP mobile phase for 5 minute at 2 mL/min. Then the NP column can be installed and equili- brated with the NP mobile phase. Despite the popularity of RP chromatography, NP has its usefulness in the analysis of compounds during drug development. It can be used for polar
  5. HPLC SEPARATION MODES 645 solutes that are poorly retained in RP, nonpolar solutes that are strongly retained in RP, positional and stereoisomers, or solutes that are labile or possess poor solubility in RP mobile phases. An example of an RP-incompat- ible method involves reaction monitoring of a mesylation step: RCOOH + MeSO2Cl → RCOOSO2Me + RCOOOCR + HCl The anhydride was formed as a side product in this reaction impacting yield. However, in an RP mobile phase, both the mesylate and anhydride would revert back to the carboxylic acid. Derivatization would produce the same product for both the mesylate and the anhydride. The reaction components were separated and quantified under NP conditions using a diol column with a 0.1 v/v% TFA in heptane/THF mobile phase (Figure 14-2). This method was used monitor the reaction such that the level of the carboxylic acid inter- mediate was less than 0.5% in the reaction mixture. 14.2.3 Sub-/Supercritical Chromatography Sub-/supercritical fluid chromatography is essentially NP chromatography with the added advantage that the lower viscosity and higher diffusivity of the mobile phase results in higher column efficiencies allowing for rapid resolu- tions. The columns employed are the same as those utilized in conventional NP chromatography. Carbon dioxide is the most commonly used nonpolar eluent but requires a more polar modifier such as an alcohol for the elution of polar solutes. The modifier increases the polarity of the mobile phase and Figure 14-2. Normal-phase separation of a mesylate from corresponding acid. Chro- matographic conditions: YMC Pack Diol 150 × 4.6 mm, 90% 0.1% TFA in heptane/10% 0.1% TFA in THF.
  6. 646 ROLE OF HPLC IN PROCESS DEVELOPMENT occupies active sites on the stationary phase, leading to reduced retention of solutes. As with conventional NP chromatography, the use of triethylamine and trifluoroacetic acid as additives is recommended for the analysis of amines and acids, respectively. The polar nature of most drug substance requires the use of high levels of organic modifier, and thus the mobile phase is most often in the subcritical state. Retention characteristics are the same as in conven- tional NP chromatography. Subcritical fluid chromatography was applied for the resolution of a bromosulfone drug intermediate from various process-related compounds (Figure 14-3). Initial steps toward method development were performed in RP mode. However, significant fronting of the bromosulfone peak was observed, indicating on-column degradation that was later determined to occur through (a) nucleophilic substitution of the bromo group with a hydroxyl group to form Figure 14-3. Structures of bromosulfone and process-related impurities. (Reprinted from reference 1, with permission.)
  7. HPLC SEPARATION MODES 647 Figure 14-4. Separation of bromosulfone from process-related impurities by SFC. Chromatographic conditions: 50% Carbon dioxide/50% (80/20 methylene chloride/acetonitrile), Zorbax silica 250 × 4.6 mm. (Reprinted from reference 1, with permission.) an alcohol and (b) addition to the ketone group to form a gem diol. Separa- tion of bromosulfone from seven process-related compounds was achieved under subcritical conditions using a silica column and a mobile phase of carbon dioxide and 50% modifier (80/20 methylene chloride/acetonitrile) within three minutes (Figure 14-4) [1]. 14.2.4 Hydrophilic Interaction Chromatography Another option to conventional NP chromatography is hydrophilic interac- tion chromatography (HILIC). This mode utilizes a polar stationary phase with aqueous/organic modifier but with very high percentages of organic mod- ifier. A simple acetonitrile/aqueous buffer mobile phase is commonly utilized in conjunction with a silica or amino stationary phase. Ammonium acetate is often used as a buffer salt in the mobile phase because it possesses good sol- ubility at high organic content. At lower pHs, phosphoric acid can be utilized. An adsorbed water layer on the silica substrate is formed under these chro- matographic conditions. Polar solutes partition from the highly organic bulk mobile phase into the adsorbed water layer where they can undergo polar interactions. In addition, positively charged solutes, such as amines, can undergo ionic interactions with charged silanol groups. As a consequence, retention of solutes increases with their increasing polarity. This mode is par- ticularly useful for the separation of very polar solutes drug substance inter- mediates and/or raw materials that show minimal or no retention under RP conditions and are very strongly retained under NP conditions. Figures 14-5 and 14-6 depict the separation of nine very polar pyridine derivatives [2]. In process research environment, for example, one of these pyridine derivatives
  8. 648 ROLE OF HPLC IN PROCESS DEVELOPMENT Figure 14-5. Structures of pyridine-related compounds. Figure 14-6. Separation of pyridine-related compounds by HILIC. Chromatographic conditions: Atlantis HILIC silica 3 µm, 150 × 4.6 mm. Mobile phase A: 0.1% phosphoric acid in D.I water. Mobile phase B: Acetonitrile. Gradient at 95% B to 60% B in 7 min and then hold 8 min. could be a key raw material in a synthetic process. The possible isomeric forms of the key raw material should be well-resolved from the key raw material and needs to be controlled (sometimes a certain set of acceptance criteria are set for both the overall purity of the key raw material and maximum amount of undesired impurity) to avoid undesired reactions in the downstream processing.
  9. HPLC SEPARATION MODES 649 14.2.5 Ion-Exchange Chromatography Ion-exchange chromatography is useful for the separation of ionic or ioniz- able solutes and resolves solutes based on the strength of their ionic interac- tions with ionic functional groups on the stationary phase. The mobile phase is aqueous.The solute and the functional group on the stationary phase possess opposite charges, and the mobile phase contains a counterion with the same type of charge as the solute and thus effectively competes with the solute ion for ion pair interactions with the stationary phase. The retention of the solute is dependent upon the ionic size, charge magnitude, and polarizability of the solute and mobile-phase counterion as well as the ionic strength of the mobile- phase counterion. Gradients of counterion concentration can be employed. Retention is also dependent upon the mobile-phase pH and the dissociation constants of protolytic solute and mobile-phase species. The stationary phase can be categorized as strong or weak ion exchangers. The capacity of strong ion exchangers is independent of pH, while the capac- ity of weak ion exchangers varies as a function of their protonated state. Strong ion exchangers include sulfonate functionalities for the analysis of cationic species and quaternary ammonium functionalities for the analysis of anio- nic species. Weak ion exchangers include carboxylate functionalities for the analysis of cationic species and amines for the analysis of anionic species. The functionalities are commonly attached to a polymeric matrix such as poly(styrene-divinylbenzene), polyacrylate, or polymethylacrylate. Ion chromatography can be applied for the quantitation of inorganic impu- rities, drug substance counterions, and ionic synthetic impurities and degrada- tion products. The most common forms of detection are by conductivity detection and indirect photometric detection (IPD), which allows for the use of conventional UV detectors. With IPD the mobile-phase anion possesses a significant chromophore. When a solute molecule, with a weaker chro- mophore, is eluted and passes through the detector cell, it is manifested as a negative peak. This form of detection can be used for analysis of ionic impu- rities in API [3–5]. Alendronate is a highly ionic bisphosphonate species that also possesses a primary amine functionality that can be derivatized with 9-fluorenylmethyl chloroformate (FMOC) and analyzed by conventional RPLC. However, alendronate does not possess a significant chromophore, and process-related impurities may also have low chromophores and may also not have an amine functionality that can be derivatized by FMOC. Such impuri- ties would not be detected in the conventional RPLC method. To address this issue, an ion exchange method was developed to separate alendronate from similar bisphosphonates, synthetic impurities, and inorganic impurities (Figure 14-7) [4]. The addition of a compatible organic solvent may also influence selectivity, particularly when the stationary phase has a polymeric substrate. With these types of phases, the solute can undergo both hydrophobic and ion-exchange interactions. The addition of an organic solvent will result in increased
  10. 650 ROLE OF HPLC IN PROCESS DEVELOPMENT Figure 14-7. Separation of organophosphonates and process-related impurities. Chro- matographic conditions: Hamilton PRP-X100, 250 × 4.6 mm, 1 mM trimesic acid (pH 5.5). 1, Phosphonopyrrolidine; 2, alendronate; 3, phosphite; 4, chloride; 5, methane- sulfonate; 6, alendronate dimer; 7, etidronate; 8, clodronate. (Reprinted from reference 4, with permission.) retention for solutes such as inorganic ions that only undergo ion interactions. Ions such as acetate and alendronate, which can undergo both types of inter- actions, may be more or less strongly retained depending on the ratio of hydrophobic to ion exchange interactions [4, 5]. 14.2.6 Chiral Chromatography Chiral separations can be considered as a special subset of HPLC. The FDA suggests that for drugs developed as a single enantiomer, the stereoisomeric composition should be evaluated in terms of identity and purity [6]. The unde- sired enantiomer should be treated as a structurally related impurity, and its level should be assessed by an enantioselective means. The interpretation is that methods should be in place that resolve the drug substance from its enan- tiomer and should have the ability to quantitate the enantiomer at the 0.1% level. Chiral separations can be performed in reversed phase, normal phase, and polar organic phase modes. Chiral stationary phases (CSP) range from small bonded synthetic selectors to large biopolymers. The classes of CSP that are most commonly utilized in the pharmaceutical industry include Pirkle type, crown ether, protein, polysaccharide, and antibiotic phases [7]. Pirkle-type phases are amino acid derivatives possessing an aromatic entity which can undergo π–π interactions with the solute. The aromatic entity can be either a π donor or π acceptor. The CSP and the solute form a π donor/acceptor pair. This complex is then stabilized by additional interactions such as hydrogen bonding, dipole interactions, or steric repulsion [8]. The Pirkle-type phases are most commonly used in normal-phase mode in order to enhance the π–π and hydrogen bond interactions. Hexane with an alcoholic modifier, such as isopropanol, is the mobile phase of choice. These phases have
  11. HPLC SEPARATION MODES 651 also been utilized in the reversed-phase mode but with poorer enantioselec- tivity and in some cases different elution orders indicating a change in the chiral recognition mechanism. These phases can also be utilized in super-/ subcritical mode. Crown ethers are heteroatomic macrocycles possessing a hydrophobic exte- rior and a hydrophilic cavity. Crown ethers show a strong affinity for primary amines through a hydrogen bonding interaction. The introduction of bulky groups, such as binaphthyl or carboxylate groups, onto the exterior of the crown ethers provides steric barriers and induces enantioselective interactions with solute molecules. Separations are performed in reversed-phase mode. Retention and selectivity is controlled by the concentration and type of coun- teranion in the mobile phase and the percent of organic modifier. One com- mercially available stationary phase contains a crown ether phase, with binaphthyl appendages, that is dynamically coated onto a silica substrate. An aqueous mobile phase is recommended when using this column. Retention increases with the chaotropicity and concentration of the counteranion [9]. A second commercially available phase utilizes a crown ether with carboxylate appendages and is covalently bonded to a silica substrate. Organic solvents can be used in the eluent. In an in-house study for a series of amines (drug substance intermediates), retention increased with organic content opposite for what is expected from a reversed-phase system. This behavior can be explained due to the fact that the primary interactions are hydrogen bonding and ion pairing, both of which would increase in strength with decreasing polarity of the mobile phase. Retention also increases with increasing depro- tonation of the CSP’s carboxylate groups as a consequence of increased sites for ion pair interactions [10]. The antibiotic glycopeptides—vancomycin, teicoplanin, and ristocetin A— have been extensively utilized as chiral selectors [11]. These macrocyclic antibiotics possess several characteristics that enable them to stereoselectively interact with solutes. They contain an aglycon bucket consisting of three or four macrocyclic rings. They also possess multiple stereogenic centers and a number of functional groups including sugars, aromatic rings, phenol groups, amide linkages, amine, moieties, and acid/esters moieties. As a consequence, they can interact with a solute through hydrogen bonding, dipole interactions, π–π interactions, hydrophobic interactions, electrostatic interactions, and steric hindrance. The phases can be used in normal-phase, reversed-phase, polar organic, and sub-/supercritical modes. These columns show very good selec- tivity to amino acids and other carboxylic acids but also resolve many neutral and basic solutes. A number of proteins are commercially available as CSPs including α-acid glycoproteins (AGP, the major plasma binding protein for basic drugs), human serum albumin (HSA, the major plasma binding protein for weakly acidic drugs), bovine serum albumin (BSA), ovomucoid (OVM), and cellobiohydro- lase (CBH) [12]. The proteins are bonded to silica and utilized in reversed- phase mode with an aqueous buffer/organic modifier eluent. Mobile-phase
  12. 652 ROLE OF HPLC IN PROCESS DEVELOPMENT optimization is performed through variation of the pH, ionic strength, tem- perature, and organic modifier [13]. It is believed that chiral recognition occurs predominantly through hydrophobic interactions in an apolar calyx that is buried in the interior of the structure. In the calyx, additional interactions such as electrostatic interactions, hydrogen bonding, dipole interactions, and steric hindrance occur. The protein CSPs are very broad-based in the types of drugs that they can enantioseparate. Several variations of the triphenylesters and triphenylcarbamates of amylose and cellulose are commercially available from Diacel. These poly- saccharide phases show the broadest applicability of all of the commercially available CSP and are capable of resolving a large and diverse selection of chiral solutes [14, 15]. The more popular phases are the 3,5-dimethylphenyl- carbamates of amylose and cellulose (Chiracel OD and Chiralpak AD, respec- tively). For most of these phases, the polysaccharide is dynamically coated onto a silica substrate. A 3,5-dimethylphenylcarbamate derivative of amylose that is covalently bonded to silica was recently introduced (Chiralpak IA). The polysaccharide phases are very flexible in that they can be used in normal- phase, reversed-phase, polar organic, and sub-/supercritical mode. Chiral recognition on polysaccharide phases are attributed to shape-selective inclu- sion into the chiral grooves enhanced by additional interactions such as hydro- gen bonding, dipole interactions, π interactions, and van der Waal forces, depending upon the chromatographic mode [16, 17]. Enantioselectivity can vary as a function of amylose versus cellulose, ester derivative versus carba- mate derivative, mobile-phase components, temperature, and chromato- graphic mode. A more detailed discussion of the stationary phase types and mechanism of interaction and separation theory in relation to chiral compounds is given in Chapter 22. A large number of chiral stationary phases are currently avail- able to meet the needs of the pharmaceutical industry for determination of the enantiomeric purity of active pharmaceutical ingredients, raw materials, and metabolites. As a consequence, there are a multitude of options in terms of columns, separation mode, and separation conditions to explore in achiev- ing an enantioseparation. For chiral liquid chromatography method development, the first choice to be made is the separation mode. The popular options are reversed-phase and normal/subcritical mode. The reversed-phase mode generally offers the advan- tage of sensitivity. Peak efficiency tends to be greater in reversed-phase mode relative to the normal-phase mode because of faster mass transfer. Combined with the ability to use low-UV-cutoff mobile-phase solvents, one can gener- ally detect 0.1% of the enantiomeric impurity. Moreover, premixed solvents may be used to increase the detection limits as this will lead to a flatter base- line (no pulsation due to the pump mixing will be observed). Subcritical mode also offers the same level of sensitivity but is hampered somewhat by instru- mental limitations with respect to ruggedness and robustness. Normal-phase
  13. SAMPLE PREPARATION 653 and the subcritical modes allow the analysts to take advantage of interactions such as hydrogen bonding and dipole interactions that are strongest in apolar media. The polysaccharide phases are known to separate a large range of phar- maceutical compounds. Chiral screening should include at least the Chiralpak OD and AD columns. Other popular columns that can be utilized include protein and antibiotic columns in reversed-phase mode and crown ether sta- tionary phases for the separation of primary amines. Chirbase (http://chirbase. u-3mrs.fr/chirbase), a database specializing in chiral chromatographic sepa- rations, offers comprehensive structural, experimental, and bibliographic information on both successful and unsuccessful separations. It lists over 100,000 separations. This database indicates that polysaccharide-based sta- tionary phases are the most frequently utilized phases accounting for ∼40% of the separations. This database can be utilized as a starting point for method development. 14.3 SAMPLE PREPARATION Sample preparation is required for the removal of potential interferents, to increase or decrease the concentration of an analyte and to convert the analyte into a suitable form for separation and detection. Sample preparation can be performed manually or through automation. In the process support area, sample preparation is seldom more complex than a simple “dilute and shoot.” In some cases it may be required to dilute the sample in a solution that quenches an ongoing reaction for in-process samples. In other cases, solid- phase extraction (SPE) may be required for analysis of certain species in the presence of an interfering component. The SPE sorbent is chosen either to retain the analytes of interest while the interfering component is unretained, or the interfering component is retained while the analytes of interest are unretained. As an example, a method for determination of azide in the pres- ence of a triazole derivative utilized a cation-exchange SPE step prior to analy- sis on an anion-exchange column [18]. The triazole derivative was strongly retained on the cation-exchange cartridge. The sorbent for SPE can be normal- phase, reversed-phase, or ion-exchange packings. SPE can also be used for enrichment of low-level analytes. Derivatization is another form of sample preparation. It is utilized for the analysis of labile analytes or to enhance retention or detection with a pre- ferred type of detector. Derivatization can be performed to enhance detection by UV/Vis, fluorescence, or electrochemical detection. Consideration must be given to the stability of the derivatize to solvolysis and thermal degradation. In our labs alendronate, a bisphosphonate with a primary amine functionality, was derivatized with FMOC to enhance detection by UV/Vis as well as to increase retention in RPLC mode [19]. An acylchloride was derivatized with
  14. 654 ROLE OF HPLC IN PROCESS DEVELOPMENT aniline to form a stable anilide derivative prior to analysis in the RPLC mode to quantitate the content of the corresponding carboxylic acid and other impu- rities [20]. The triflation of a drug intermediate alcohol formed an active trifluoromethanesulfonyl ester. This active ester was derivatized with tetra- butylammonium bromide to form the bromo analog prior to analysis by reversed-phase LC [21]. 14.4 HPLC DETECTORS The detectors utilized for HPLC are designed to respond to the solute being eluted. HPLC detectors can be classified into two broad categories: universal and selective. Selective detectors respond to some physicochemical property of the solute, while universal detectors respond to all solutes independent of their physicochemical properties. The ideal detector would be highly univer- sal and highly sensitive, have a wide linear range, and not be affected by change in temperature or mobile phase composition. Commercially available detec- tors possess some of these characteristics but not all. The most commonly utilized detectors used in process development are the UV/Vis detectors that can be fixed-wavelength, variable-wavelength, or diode array. These detectors are sensitive, have a wide linear range, and are relatively unaffected by temperature or mobile-phase composition. They respond to solutes containing double bonds, and compounds with unpaired electrons such as bromine, iodine, and sulfur. Their response, however, is not equivalent. A variable-wavelength detector uses a deuterium or xenon lamp source, and the desired wavelength is isolated by a monochromator. A diode array detector performs a simultaneous measurement of absorption as a function of analysis time and over a chosen wavelength range. Thus a UV spectrum is obtained for each eluted peak. The main advantage of a diode array detector is for method development where wavelength maxima of the drug substance and its impu- rities may be unknown or where the UV spectra can be used to track peaks as operating conditions are changed. If the solute of interest does not possess a significant chromophore, then indirect photometric detection can be utilized. In this mode it is the mobile phase that possesses a chromophore and absorbs light. The detector still measures the difference in absorption between the mobile phase and the solute. When an analyte without a significant chro- mophore passes through the detector cell, the absorption of the mobile phase is decreased and is recorded as a negative peak. An ion-exchange method for the resolution of alendronate from other bisphosphonates, ionic synthetic impurities, and inorganic impurities utilized indirect photometric detection (Figure 14-7) [4]. Fluorescence occurs when a compound absorbs radiation then emits it at a longer wavelength. It is highly selective. Fluorescence is exhibited by rigid molecules possessing a large number of delocalized π electrons. Electron-
  15. HPLC DETECTORS 655 donating groups enhance while electron-withdrawing groups decrease fluo- rescence. Few drugs possess natural fluorescence but for those that do, fluo- rescence detection is an option that offers increased specificity and sensitivity over UV/Vis detectors. Fluorescence is more sensitive than UV/Vis detection, particularly for laser-induced fluorescence. Care must be taken in choosing a compatible mobile phase because fluorescence can be quenched by highly polar solvents or halide ions. Fluorescence efficiency is also dependent upon pH of the mobile phase. Some solutes may not have a significant chromophore, and alternate detec- tors must be utilized. These detectors include refractive index, evaporative light-scattering, element-specific, electrochemical, and mass spectrometric detectors. Refractive index (RI) detectors monitor changes in the refractive index of the mobile phase that occur due to the presence of solute molecules. Detection is universal but less sensitive than UV detectors. It is suitable for solutes without significant chromophores. The refractive index of the mobile phase must be constant, and thus this mode of detection is not amenable to gradient elution. Slight variations in temperature will also change the refrac- tive index of the mobile phase. Therefore, very good temperature control is required. Evaporative light-scattering detectors (ELSD) require nebulization of the eluent after which the aerosol is transported through a heated tube allowing the mobile phase to be evaporated. The residual particles pass through a light beam, and scattered light is then detected at a fixed angle from the incident light. Volatile mobile-phase components such as trifluoroacetic acid, formic acid, acetic acid, and ammonium hydroxide must be used. The ELSD is a uni- versal detector as long as the solute is less volatile than the mobile phase. The linear range is not wide. It is intermediate between UV and RI detectors in terms of sensitivity and can be utilized with gradient elution. ELSD is useful for detection of solutes that do not possess a significant chromophore [22] but should not be used for thermolabile solutes. A recently commercialized alter- native to ELSD is a corona discharge detector. The HPLC effluent is similarly converted to an aerosol, the aerosol particles are then charged by a positive corona discharge, and the current from the charged particle flux is then mea- sured. This detector is generally regarded as more sensitive than ELSD [23]. Chemiluminescent detectors (CLND) are very selective and sensitive. If a solute contains at least one nitrogen atom, it can be detected. The effluent is nebulized, and then it is oxidized by combustion in a high-temperature furnace. Nitrogen-containing solutes are converted into nitric oxide, which is then passed into a chamber where it reacts with ozone to produce excited- state nitrogen dioxide that emits a photon upon relaxation. The photon flux is then measured by a photomultiplier tube [24]. The signal generated is pro- portional to the number of nitrogen atoms in the solute molecule. This detec- tion mode requires volatile mobile phases that are free of nitrogen-containing molecules (no acetonitrile). CLND have been determined to have a wider linear range and greater sensitivity than ELSD [24].
  16. 656 ROLE OF HPLC IN PROCESS DEVELOPMENT Electrochemical detection can be utilized for compounds that are ionic or readily oxidizable or reducible. Thus, this form of detection can be used for the analysis of inorganic ions, protolytic organic compounds such as amines and carboxylic acids, and other compounds such as phenols, thiols, and alcohols. Conductivity detectors measure differences in the equivalent conductance of the solute and ions in the mobile phase. The conductivity response is maxi- mized through the use of ion suppressors that effectively eliminate the con- ductivity of the mobile-phase ions through chemical removal or electronic subtraction. The linearity range is wide, and detection is highly sensitive. In our labs, conductivity with ion suppression was utilized to detect residual levels (∼0.1%) of choline (quaternary saturated amine) in drug substance [25].Amper- ometric detection is less commonly utilized and is suitable for compounds that can be electrolytically oxidized such as phenols. This mode is not generally applied in the reductive mode due to interference from dissolved oxygen in the mobile phase. Amperometric detection is highly sensitive and selective. Mass spectrometric detection is close to being a universal detector. Ioniza- tion techniques such as atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are routinely employed. These techniques allow the transfer of the LC effluent into the gas phase. With APCI, the eluent is converted to an aerosol by a sheath gas. The aerosol is then subjected to a chemical ionization plasma created by a corona discharge, leading to forma- tion of solute ions. These ions are then transferred into the mass spectrome- ter. With ESI, the eluent is converted to charged droplets. ESI is preferred for compounds that are ionized in solution. APCI is better for compounds of medium polarity. Both techniques can be used in positive or negative ion mode. Positive ion mode is commonly used. Negative ion detection is useful for negatively charged ions such as acids. Nonpolar compounds are difficult to analyze with these atmospheric ionization techniques due to their soft ioniza- tion mechanisms. Atmospheric pressure photoionization is an emerging tech- nology for the analysis of these nonpolar compounds. This technique is similar to APCI; however, a gas discharge lamp that emits photons in the vacuum UV region is utilized. Sensitivity can be increased by the use of dopants such as toluene or acetone added post-column to the eluent. The dopant is first ionized and then ionizes the analytes through further reactions [26]. Mass spectrometric detection in the process development area is generally performed with a single-quadropole, triple-quadrupole, or ion trap mass spec- trometer. Other options include sector and time of flight spectrometers. A single quadrupole provides information pertaining to the mass to charge ratio (m/z) of the solute. Ion traps and triple quadrupoles provide additional infor- mation through tandem MS, allowing for a more definitive structural elucida- tion of the solute. Volatile buffers such as ammonium acetate or ammonium formate and low-pH mobile phases such as 0.1% formic or acetic acid are rec- ommended to prevent blockages of sample cones or capillaries. The relative sensitivity of MS versus UV/Vis detection may differ by many orders of mag- nitude in either direction, depending upon the chromophoric properties and
  17. METHOD DEVELOPMENT 657 the ionizability of the analyte. It can be very selective when used in selected ion monitoring mode where it is detecting one specific mass/charge ratio. The use of MS is extremely valuable in identifying by-products of reactions, impu- rities in intermediates that may react further in downstream processing, and impurities that are formed during stability testing. 14.5 METHOD DEVELOPMENT The approach to method development is dependent upon the physicochemi- cal properties of the solute and any known potential impurities and the purpose of the method. The method may be required for an impurity profile, assay, in-process monitoring, or chiral/isomeric evaluation. Method develop- ment is usually dynamic.As more knowledge about the properties of the solute and potential impurities is gained, the method can be further optimized. Ana- lytical laboratories supporting process development should be stocked with a variety of columns for RPLC, NPLC, ion-exchange, and chiral separations. Column switching capability is also an asset for method development. Column switching allows for analysis of the same sample with as many as six differ- ent columns in an overnight run to help speed method development. Initial development can be performed empirically, based on the chromatographer’s experience, or through the use of simulations with one of the commercially available method development software packages. The parameters to explore for method development include separation mode, column selection, mobile- phase optimization, temperature, detection wavelength, sample diluent and concentration, injection volume, and sample preparation procedure. The use of an orthogonal chromatographic method, with the developed method as a check, is recommended. Having an orthogonal method minimizes the possibility of peak co-elution, particularly in cases where there is limited information available regarding the nature of impurities. An orthogonal method may be employed once the final synthesis is set during the develop- ment of a drug. The final synthesis is usually set for preparation of the clinical material used for Phase II clinical studies. When a method has been developed that is deemed appropriate for the purpose, system suitabil- ity parameters should be implemented and some degree of validation should be performed to ensure that the method meets the needs of the chromatographer. For developing an impurity profile for raw materials, intermediates, or drug substance, communication with the process chemists regarding potential reac- tion by-products is always the best start. This information plus any garnered knowledge of the physicochemical properties of the solute and potential impu- rities such as pKa, log P (octanol/water partition coefficients), solubility, and UV spectrum will determine the selection of the appropriate mode, column, mobile-phase, and other separation parameters. Given the potential for gen- eration of impurities that are unanticipated by the process chemists, it is
  18. 658 ROLE OF HPLC IN PROCESS DEVELOPMENT recommended that for early development a gradient method be employed. A gradient method will allow for coverage of a wide range of polarity and thus be able to capture early and late eluting impurities in the same run. For this reason, a reversed-phase method is the first choice. Most components can be eluted in a 10–90% gradient of organic modifier as long as there are no mis- cibility issues with the aqueous mobile phase. An isocratic hold at 90% organic should be performed especially in early development to detect the presence of any extremely hydrophobic impurities. Ideally the peaks of interest should be eluted with a capacity factor between 1 and 10. The choice of a column is dictated in large part by the hydrophobicity (eval- uated as log P when available) of the solute. C18 or C8 columns are commonly utilized in reversed-phase mode. Retention and selectivity for these phases can vary, depending upon whether they are conventional, polar end-capped, polar embedded, or hybrid silica. A high-carbon-load C18 or a graphitic column can be used to increase retention. A low-carbon-load C8 or a phenyl or cyano column can be used to decrease retention. Alkyl, phenyl, and cyano phases may offer different selectivities. Selectivity may also vary as a function of the substrate: silica versus polymer versus zirconia. The sheer volume of com- mercially available reversed-phase columns makes selection of the best column, for a particular separation, anything but a simple task. Much research has been performed toward the classification of reversed-phase columns. Approaches include regression of log k versus log P, thermodynamic mea- surements of retention, and quantitative structure–retention relationships (QSRR) using experimentally determined or calculated molecular descriptors [27–35]. For example, classifications in terms of efficiency, hydrophobicity, silanol activity, and steric selectivity were used in the evaluation by principal component analysis of 69 columns differing in type of silica, pore size, end- capped/not end-capped, base deactivated/not base deactivated, and polar embedded [31]. Based on this classification, one can select four columns, which fall into separate categories ensuring selectivity differences, for initial method development. Similarly, classification of 28 columns in terms of selectivity based on hydrophobicity, steric selectivity, efficiency, and silanol activity using chemometric approaches led to the selection of eight columns of low, inter- mediate, and high hydrophobicity that were highly efficient and showed good steric selectivity [35]. One could then choose one column from each hydropho- bic class for method development. One should also ensure that the selected columns are stable within the intended pH and temperature regions that they will be employed. A good understanding of the chemical stability of the sta- tionary phases is essential. An additional variable for varying selectivity is column temperature. Sig- nificant changes in selectivity may be observed when comparing separations at 10°C and 50°C. This depends on the nature of the analyte and its interac- tion with the stationary-phase and mobile-phase components. Elevated tem- peratures, however, may lead to unwanted compound degradation and should be avoided for labile components.
  19. METHOD DEVELOPMENT 659 Mobile-phase composition is another major parameter for affecting selec- tivity in a separation. Points to consider include choice of organic solvent, mobile-phase pH, and use of additives. The three most commonly utilized organic solvents are acetonitrile, methanol, and THF. Acetonitrile is usually a good starting organic solvent as a consequence of its lower viscosity and UV cutoff. Method development can be performed with each of these three organic solvents or with mixtures of them. The pH of the mobile phase is also critical. Low pH protonates acids and bases, resulting in neutral acids and charged bases. Conversely, high pH deprotonates acids and bases, resulting in charged acids and neutral bases. In general, retention decreases with increas- ing charge on the solute. Buffers are recommended. Phosphate is a commonly utilized buffer with pKa values of 2.1 and 7. Buffers such as acetate and formate are useful for detection modes requiring volatilization of the mobile phase. Care should be taken to avoid working within ±1.5 units of the pKa of the solute, because this may result in poor retention precision. Many pharmaceu- tical compounds are acidic or basic, and a good starting point for method development is low pH. A low pH suppresses the ionization of acid solutes and the silanol sites of the stationary phase. High pH can be used to increase retention of bases (neutral form) or to take advantage of ion-exchange inter- actions (with bases in ionized form) to improve selectivity (however, bad peak shapes sometimes are the result due to strong silanophilic interactions). Retention can also be enhanced by the use of additives such as chaotropic anions (perchlorate, hexafluorophosphate) or by ion-pairing agents (hexanesulfonate) [36]. Knowledge of the UV spectra of the solute of interest can be applied to choice of wavelength for UV/Vis is detection where amenable. One can choose a wavelength near or at the UV maxima for detection. This choice suffers the disadvantage that unknown impurities in the intermediates and/or drug sub- stance may not exhibit strong extinction coefficients at the chosen wavelength and may go undetected. An alternative is to work at a low wavelength such as 210 or 220 nm where most solutes possessing a chromophore will have signif- icant absorption (π–π* bands for double bonds and n–σ* bands for amines and halogens). The choice of wavelength is also dictated by the UV cutoff of the mobile-phase components. Knowledge of the solubility of the solute as well as its compatibility directs the choice of diluent. A combination of adequate sol- ubility and injection volume should be chosen such that ideally 0.05% of the solute can be detected with a signal-to-noise ratio of greater than 10 to 1. For development of a weight percent assay, a short isocratic method can be implemented based on observations from the gradient method used for the impurity profile. One can use a shorter column such as a 5-cm column and keep retention of the solute of interest to around a capacity factor of 3 as long as it is still resolved from impurities observed in the impurity profile. Addi- tionally, the elution of more hydrophobic species should not co-elute with the drug substance in later injections. During the method development of an iso- cratic method, the compound should be injected and then a suitable number
  20. 660 ROLE OF HPLC IN PROCESS DEVELOPMENT of blanks injected to ensure that more hydrophobic impurities do not elute at the same time as the analyte peak in later injections. A similar approach using an isocratic method can be applied to in-process monitoring, where the goal is to monitor the disappearance of the starting material and appearance of the product. In-process methods will be discussed in greater detail in Section 14.6. The use of computer simulations is an alternative approach to method development. Computer-based expert systems are designed to mimic the thought processes of an experienced chromatographer. These systems contain a database that can used to evaluate chromatographic data and provide opti- mized conditions. Variables such as solute structure, column type, mobile- phase components, pH, and temperature can be inputted, and proposed optimized chromatographic conditions are outputted. This approach is gener- ally faster and cheaper than performing all of the experiments necessary for method development. Systems with artificial intelligence can plan experi- ments, collect and evaluate data, and adjust chromatographic conditions in real time according to predefined decision schemes until a satisfactory separation is achieved. Further discussion of the different automated method develop- ment software available is given in Chapter 10. 14.6 IN-PROCESS MONITORING In-process monitoring is implemented to maximize yield and minimize impu- rity generation during the various synthetic steps. An ideal in-process method should quickly evaluate a specific sample and provide results in a timely fashion such that changes may be triggered to maintain the reaction condi- tions at the optimal level required to secure production with high purity and maximum yield. Process analytics using on-line spectroscopic analysis can provide instantaneous feedback; however, the reaction mixture is often too complex to provide accurate results. Oftentimes, separation is required to eval- uate levels of several components. Chromatography can provide the necessary separation, but the time lag of the analysis must be short enough to monitor the actual state of the reaction. An emphasis should be placed on providing near-real-time feedback by using methods with short run times. Ideally, this would be accomplished by reducing the run time without a concomitant loss in column efficiency or resolution. One approach to achieving near-real-time feedback with chromatography is through the use of short columns with smaller particles. Small particles result in higher column efficiency, but with increased backpressure limiting the work- able column length. Short columns (10 cm or less) with smaller particle sizes (1.5 to 3.5 µm) can result in comparable separations to longer columns (25 cm, 5-µm particles) but with one-half to one-fifth the run time. The efficiency of these shorter columns is equivalent to, and often superior to, the longer con- ventional columns. Shorter columns, however, are susceptible to instrumental
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