OR WAIT null SECS
In Part II of a three-part article, the authors examine impurities from chiral molecules, polymorphic contaminants, and genotoxic impurities.
The public and the pharmaceutical industry are placing greater attention on impurities in drug as evidenced by the attention given to pharmaceutical impurities in books, journal articles, and national and international guidelines (1–10). The health implications of impurities can be significant because of their potential teratogenic, mutagenic, or carcinogenic effects. Controlling and monitoring impurities in APIs and finished drug products, therefore, is a crucial issue in drug development and manufacturing.
Adam Gault/ OJO Images/Getty Images
Part I of this article, which appeared in the February 2012 issue of Pharmaeceutical Technology, discussed the various types and sources of impurities with specific case studies (11). This article, Part II, discusses chiral, polymorphic, and genotoxic impurities (12, 13). Part III, to be published in the April 2012 issue of Pharmaceutical Technology, will examine various degradation routes of APIs, impurities arising from API–excipient interaction during formulation, metabolite impurities, various analytical methodologies to measure impurity levels, and measures to control impurities.
Impurities can be present in the enantiomers of chiral compounds. Differences in pharmacological and toxicological profiles have been observed with chiral impurities in vivo (14, 15). The significance of stereochemical purity may be illustrated by formoterol, a selective β2-adrenoceptor agonist (16). This compound contains two chiral centers. Initial investigations indicated that the β2-agonist activity resided in the stereoisomer with the (R, R) absolute configuration with a rank order of potency (R, R) > (R, S) > (S, S) > (S, R). Subsequent investigation reported much greater difference with the eudismic ratio R, R/S, S increasing from 50 to 850 when the impurity of the eutomer in the diastereomer decreased from approximately 1.5 % to < 0.1% (17). Similar examples of stereochemical isomers can be found in the stereospecific drugs of the (S)-enantiomer of a-methyldopa, picenadol, (R)-sopromidine, (+)-(S)-apomorphine, and sertraline (18–24).
Another example is asenapine maleate, an antipsychotic belonging to the dibenzo-oxepino pyrroles class. Based on its receptor pharmacology, the efficacy is thought to be mediated by its antagonist activity on dopamine (D)-2 and serotonin (5-HT)–2A receptors (25). Asenapine shows geometric isomerism and is a racemate of (+) and (-) enantiomers. It shows comparable binding affinities, meaning trans-asenapine showed higher affinity at D4 receptors than (+)/cis-asenapine (26).
Differences in pharmacological and toxicological profiles have been observed with chiral impurities in vivo, suggesting that chiral impurities should be monitored carefully. Although development of chiral drugs as single stereoisomers is a preferred approach, consideration must be given to unwanted stereoisomers, which may be present as impurities or degradants in the drug substance or drug product or generated through metabolism in biological systems. Chiral impurities in pharmaceutical samples may occur as side-products of the synthetic process as a result of an inversion of chiral centers due to chemical degradation of the drug substance or both. Similarly, inversion of the chiral center may occur in vivo as a result of metabolism, chemical degradation, or both.
Guidelines on the development of chiral compounds are published by regulatory authorities around the world, but they can be general and leave room for interpretation. The issues involved in chiral drug development are complex, and a coordinated approach among the many R&D groups is necessary. A multidisciplinary approach serves as a guide to the development of chiral compounds by coordinating research efforts in the various phases of development (22–36).
Polymorphism, the ability of a compound to exist in more than one crystalline form, affects the physical, chemical, and biological properties of a compound in question (37). These properties may influence several issues in pharmaceutical systems, such as processing characteristics, drug stability, and bioavailability. Demonstrating an understanding of the polymorphs in a given drug is an area of regulatory scrutiny in new drug applications (38).
The International Conference on Harmonization's Q6A guideline, Specification: Test Procedure and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, outlines when and how polymorphic forms should be monitored and controlled (39). For stability concerns, the most stable form is normally used in the formulation. The metastable polymorphic form, however, may be inadvertently generated due to temperature, mechanical treatment, and moisture during processing or storage of the drug product (40).
Contamination of polymorphic impurities can adversely influence the stability and performance of the final drug product. Moreover, FDA requires development of validated methods for analysis of the proportion of crystalline forms throughout the drug's retest period and shelf life (41).
For example, olanzapine crystallizes in more than 25 crystalline forms, of which Form II has been designated the most stable form and is used in the dosage form (42, 43). Olanzapine discolors in the presence of air (44). Polymorphic Forms I and II show very minor differences in their diffractograms. Evaluating olanzapine Form I for the presence of Form II, therefore, becomes very important.
Salmeterol xinafoate is known to exist in two crystalline polymorphic forms, with Form I being stable and Form II being the metastable polymorph under ambient conditions (45). These polymorphs have been characterized using differential scanning calorimetry, X-ray powder diffractometry, thermogravimetric analysis, and inverse gas chromatography (46). Commercial salmeterol xinafoate is a micronized form with the same crystal structure as that of Form I. The commercial drug, however, can contain traces of the Form II polymorph that is formed during the micronization process.
Exceptional case impurities
When a new process is developed, such as to overcome patent issues, it generally begins with new key starting materials, intermediates, reagents, or solvents that may react differently to give byproducts or process impurities. For example, in the synthesis of linezolid and pemetrexed disodium, several process impurities can be formed due to different process approaches.
Pharmaceutical companies can develop new processes based on raw materials, solvents, reagents, process conditions (i.e., temperature), and new polymorphs. Using new materials or processes, they may encounter several impurities that may not have been not present in the basic or initial synthesis of an API. After publication of monographs in the United States Pharmacopeia, European Pharmacopoeia, British Pharmacopoeia, Indian Pharmacopoeia, and Japanese Pharmacopoeia, they may not have a control of those impurities that are formed due to different process approaches. After publication of the monograph, companies have to change the analytical method or control these impurities as nonpharmacopeial impurities, including genotoxic impurities, with separate analytical methods, such as high-performance liquid chromatography (HPLC) or gas chromatography (GC).
For example, during the synthesis of linezolid, impurities based on a bis-linezolid compound and a bis-benzyl impurity are formed due to the non-infringed patent process (47–49). Some published patents have different potential process impurities, which cannot be separated in a single HPLC method, and which result from synthetic routes different from the synthetic route in the basic patent (47–55).
Pemetrexed disodium heptahydrate, the API in Eli Lilly's Alimta, is a multitargeted antifolate used to treat mesothelioma and a second-line treatment for non-small-cell lung cancer. Alimta also is under investigation for multiple other cancers (56). Each non-infringed process patent has different potential impurities (see Figure 1, Process Impurities 1, 2, 3, and 4) (57–60). It may not be possible to analyze these impurities in a single HPLC method.
Figure 1: Reaction scheme for different process approaches for pemetrexed sodium impurities, respectively labeled as 1, 2, 3, and 4 (Refs. 57â60). Ph. Eur. is European Pharmacopoeia.
Impurities due to the piperazine ring
The piperazine moiety is present in the chemical structure of more than 200 drugs. The biotransformation of the piperazine ring involves several well-known metabolic reactions, including N-oxidation, hydroxylation, N-dealkylation, and ring cleavages to N-substituted as well as N,N'-disubstituted ethylenediamines. In addition, several unexpected metabolic pathways have been reported for the piperazine ring: N-glucuronidation, N-sulfonation, formation of carbamoyl glucuronide, and glutathione adducts (61). Some compounds containing the piperazine ring indicate that the ring is normally metabolically stable when both nitrogen atoms are substituted with groups larger than ethyl.
The lack of partial degradation of the piperazine ring to form ethylenediamine in olanzapine (2-methyl-4-(4-methyl-1-piperazinyl)10H-thieno[2,3-b][1,5]benzodiazepine) is slightly surprising. Some major metabolites were reported in humans plasma and urine, such as 4'-N-glucuronide and 4'-N-glucuronide (61, 62). Several other metabolites also were reported in mice, rats, monkeys, and dog urine (63). The ethylenediamine impurity, however, is not reported as a metabolite and a process impurity (see Figure 2).
Figure 2: The piperazine ring and metabolite impurities of olanzanpine. Ph. Eur. is European Pharmacopoeia. USP is US Pharmacopeia.
When one of the nitrogen atoms is substituted by hydrogen on the piperazine ring, whether its methyl or ethyl, ethylenediamine formation is normally observed. An example is levofloxacin, S-(-)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid, which is the (S)-isomer of ofloxacin. In levofloxacin, the piperazine nitrogen atom is substituted with methyl due to several photodegradation impurities (see P 2 to P 10, Figure 3) (64–67). Some process impurities also are observed (see Figure 3). If the levofloxacin process involved methylenedichloride as a solvent, a chloro methyl impurity may form, and after isolation of the final product, the same impurity may convert to a di-quaternary cyclic piperazine impurity.
Additionally, when the ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl) quinoline-3-carboxylic acid) nitrogen atom is substituted by hydrogen on the piperazine ring, several metabolites and process impurities are formed (see Figure 3) (68–74). When nitrogen is substituted with hydrogen during the reaction, two dimer impurities (F-F dimer ciprofloxacin and F-Cl dimer ciprofloxacin) also are observed (75).
Figure 3: The piperazine ring and metabolite impurities of levofloxacin and ciprofloxacin. Ph. Eur. is European Pharmacopoeia and USP is US Pharmacopeia. CAS No. refers to Chemical Abstracts Service (CAS) number.
There was no specific document on control of genotoxic impurities before 2000. ICH guidelines made passing references to compounds of unusual toxicity. Genotoxic impurities are chemical compounds that may be mutagenic and could potentially damage DNA (76). Non-monoalkylated agents are classified as genotoxic due to the nature of the functional groups they possess and also of related aniline derivatives. Additionally, salt-forming steps can introduce genotoxic impurities. Some examples include formation of methyl chloride as a side reaction of hydrochloric acid in methanol or esters of methanesulfonic acid as byproducts from the methanesulfonic acid salt-formation step in alcohol-based solvents (77, 78).
EMA issued guidelines on the threshold of toxicological concern (TTC) that recommended limits for exposure to potential genotoxic impurities to be 1.5 mcg per day for commercially approved drugs (79). As per the guidelines, testing will be required for all potential impurities from an API's synthetic route containing structural elements that are the cause of concern for genotoxicity potential using the well-established Salmonelle Ames test. The Ames test is a screening test that is used to help identify chemicals that affect the structure of DNA. The test exposes Salmonella bacteria to chemicals and looks for changes in the way bacteria grow. These changes result from mutations that occur when the structure of DNA is altered in certain places and the micronuclei test for mutagenicity (80, 81). Recommended qualification thresholds based on the maximum daily dose for drug substances and for drug products are provided in ICH Q3A and ICH Q3b (7, 8). The TTC data set was conducted from the perspective of an organic chemist who develops process technology for APIs (82). As part of the EMA guidance, API process designers are instructed to avoid all possible situations that could lead to the presence of impurities possessing genotoxic potential at any level in APIs.
During the establishing of the control mechanism, other factors, such as reactivity, solubility, and volatility, should be considered. Action should not be based only on the presence of alerting structures. It is important to make evaluations on a case-by-case basis, and precedence data should be considered, such as the stage of impurity formation, reactivity and carryover to the API, the intake of other routes, Ames test results, and data of closely related structures.
During process development, a genotoxic impurity may be introduced as a starting material, reagent, intermediate, catalyst, byproduct, isomer, or degradation product. (83). Alkyl halides used as reagents in synthesis are genotoxins (84). The same also was generated during chemical synthesis when a salt counter ion (e.g., hydrogen halide) of a drug substance reacts with alcohols when used as a solvent media.
The genotoxins ethyl chloride, methyl chloride, and isopropyl chloride were generated during the preparation of the hydrochloride salts of ethanol, methanol, and isopropyl alcohol (ICH listed solvents), respectively, at lower temperature (< 5 °C) as the key parameter of these impurities. In alcohol solvents, when HCl was 37% aqueous HCl or gas, it creates the maximum chance to form these alkyl halide impurities at trace levels. These impurities are detectable in GC at ppm level. Methane sulfonic acid (mesylate), benzene sulfonic acid (besylate) and p-toluenesulfonic acid (tosylate) are commonly used as counter ions to form API salts (85–87). Interactions of these acids with residual alcohols may lead to the generation of genotoxic impurities. Alkyl methane sulfonates, alkyl benzene sulfonates, and alkyl para-toluene sulfonates may combine with imatinib mesylate, amlodipine besylate, and denagliptin tosylate, respectively (88, 89).
The emphasis on genotoxic impurities is increasing, which creates challenges for both synthetic and analytical chemists, to develop sensitive and efficient methods to detect impurities at low levels (i.e., below TTC < 1.5 mcg/per day), which sometimes is not feasible and which increases the time and cost of drug development.
Linezolid (S)-N-[[3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide, has genotoxic structural alerts and represents a new class of antibiotics, oxazolidinones. Forced-degradation studies are an important part of the drug-development process and are used increasingly in testing new molecules. These studies may give different impurities that may not be formed during process optimization and manufacturing validation, but these impurities must be controlled as per ICH guidelines. The authors have observed two impurities during a forced-degradation study in peroxide and alkaline conditions, Compounds 7, 8, and 9 (see Figure 4), which are structural alerts for genotoxicity, and which should be controlled so that the exposure to it is less than 1.5 mcg/day based on the maximum daily dose of the linezolid.
Figure 4: Process, genotoxic, and metabolite impurities of linezolid.
Linezolid's key starting material (A) shows genotoxicity alert and it contains five other intermediates, Compounds 1, 2, 3, 4, and 5 (see Figure 4). Compound A converts to the final drug, and it contains, Mesyl Impurity 6, Amine Impurity 12, Des Fluoro Impurity 13, Chloro Impurity 14, and O-Acetyl Impurity 15; these are the process impurities and have genotoxicity alert (49). During human studies, from the total amount of linezolid administered, only 30% was eliminated through the kidneys. Its major part was metabolized by oxidation of its morpholine ring, which resulted in the formation of two metabolites (see Figure 4): amino ethoxy acetic acid metabolite and hydroxy ethyl glycine metabolite (i.e., a major urinary metabolite) (90–92).
Part II of this article examined impurities that are associated with drug molecules having one or more chiral centers, APIs existing in various crystalline forms, drug substances with the piepererazine moiety, and APIs developed by new processes. Part II also looked into the extended application of the TTC to pharmaceuticals. To guarantee the quality and safety of pharmaceuticals during drug development, a quality concept has been proposed that adapts the ICH guidelines and which is focused on qualified impurity profiles.
Call for Papers
Part III, to be published in the April 2012 issue of Pharmaceutical Technology, will examine various degradation routes of APIs, impurities arising from API–excipient interaction during formulation, metabolite impurities, various analytical methods to measure impurity levels, and measures to control impurities. Part I, published in the February issue of Pharmaceutical Technology, examined the types and sources of impurities (11).
Kashyap R. Wadekar, PhD,* is a research scientist (II), 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, E. Balasubrahmanyam is a research chemist, and Ponnaiah Ravi, PhD, is senior vice-president of R&D, all with Neuland Laboratories, 204 Meridian Plaza, 6-3-854/1, Ameerpet, Hyderabad, India, tel. 91 40 30211600, firstname.lastname@example.org.
1. FDA, Guidance for Industry—ANDAs: Impurities in Drug Products (Rockville, MD, Aug. 2005).
2. FDA, Guidance for Industry—ANDAs: Impurities in Drug Substances (Rockville, MD, Jan. 2005).
3. S. Görög, Identification and Determination of Impurities in Drugs (Elsevier Science, Amsterdam, 2000).
4. S. Ahuja, Impurities Evaluation of Pharmaceuticals (Marcel Dekker, New York, 1998).
5. S. Hovorka and C. Schöneich, J. Pharm.Sci. 90 (3), 253–269 (2001).
6. J. Roy, AAAPS PharmSciTech3 (2), 1–8 (2002).
7. ICH, Q3A(R) Impurities in New Drug Substances (Feb. 2003).
8. ICH, Q3B(R) Impurities in Drug Products (Nov. 2003).
9. ICH, Q3C (R5) Impurities: Guideline for Residual Solvents (March 2011).
10. ICH, Q1A(R2) Stability Testing of New Drug Substances and Products (Nov. 2003).
11. K. R. Wadekar et al., Pharm. Technol. 36 (2), 46–51 (2012).
12. B.C. Allen, K.S Crump, and A.M. Shipp, Risk Anal.8 (4), 531–544 (1988).
13. S.W. Baertschi and D.W. Reynolds, "Introduction" in Pharmaceutical Stress Testing: Predicting Drug Degradation, J. Swarbick, Ed. (Taylor & Francis, New York, 2005), pp. 4–8.
14. S. Ahuja, Chiral Separations by Chromatography (Oxford University Press, New York, 2000).
15. S. Ahuja, Chiral Separations by Liquid Chromatography, ACS Symposium Series 471 (American Chemical Society, Washington, DC, 1991).
16. J. Trofast et al., Chirality3 (6), 443–450 (1991).
17. B. Waldeck, Chirality5 (5) 350–355 (1993).
18. L. Gillespie et al., Circulation25, 281–291 (1962).
19. H. Kubota et al., Chem. Pharm. Bull. 40, 1619–1622 (1992).
20. R.B. Carter, J. Pharmacol. Exp. Ther. 234 (2), 299–306 (1985).
21. Chiral Agonists of Histamine in Fornitier in Histamine Research (Oxford, 1985), pp.39-46.
22. M.E. Goldman et al., J. Mol. Pharmacol. 25 (1), 18–23 (1984).
23. W.M. Welch et al., J. Med. Chem. 27 (11), 1508–1515 (1984).
24. B.K. Koe et al., J. Pharmacol. Exp. Ther. 226 (3), 686–700 (1983).
25. T. de Boer et al., Chromatogr.26 (2), 156–165 (2012).
26. TGA, Australian Public Assessment Report for Asenapine (Woden, Australia, April 2011).
27. S.G. Allenmark, Chromatographic Enantioseparation: Methods and Applications (Ellis Horwood, Chichester, 1991).
28. G. Gubitz, Chromatographia30, 555–564 (1990).
29. D.E. Drayer, Clin. Pharmacol. Ther.40 (2), 125–133 (1986).
30. F.J. Jamali, J. Pharm. Sci. 78 (9), 695–715 (1989).
31. "FDA's Policy Statement for the Development of New Stereoisomer Drugs," Chirality4 (5), 338–340 (1992).
32. H. Wilson et al., J. Pharm. Biomed. Anal.11 (11–12), 1167–1173 (1993).
33. A.G. Rauws, Chirality 6 (2), 72–75 (1994).
34. M. Gross et al., Drug Inf. J. 27 (2), 53–457 (1993).
35. W.L. Heydorn, Pharm. News2, 19–21 (1995).
36. ICH, Q6A Document, Specification for New Drug Substances and Products: Chemical Substances, Step 2 version (1992).
37. J. Haleblian et al., J. Pharm. Sci. 58 (8) 911–929 (1969).
38. G.A. Stephenson et al., Adv. Drug. Deliv. Rev.48, 67–90 (2001).
39. ICH, Q6A Specification: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances (1999).
40. G.G. Z. Zhang et al., Adv. Drug Deliv. Rev.56 (3), 371–390 (2004).
41. S.R. Vippagunta et al., Adv. Drug Deliv. Rev.48 (1), 3–26 (2001).
42. P.B. Molinoff, The Pharmacological Basis of Theraputics (McGraw Hill, New York, 1996), pp. 399–430.
43. S.M. Reutzel-Edens, Cryst. Growth Des. 3 (6) 897–907 (2003).
44. Eli Lilly, "Olanzapine Polymorph Crystal Form,"US Patent 5,736,541 (April 1998).
45. H.Y. Tong et al., Pharm. Res. 18 (6), 852–858 (2001).
46. H.H. Tong et al., Pharm.Res.20 (9), 1423–1429 (2003).
47. D. Clemett et al., Drugs 59 (4), 815–827 (2000).
48. Teva Pharmaceutical Industries, "Isolated Bis-Linezolid, Preparation Thereof and Its Use as a Reference Standard, WO 2006/091848 A2, Aug. 2006.
49. Neuland Laboratories, "A Process for the Preparation of (5S)-(N)-[[3-[3-fluoro-4-4(4-morpholinly)phenyl-2-oxo-5-oxazolidinyl]methyl] acetamide," WO2010084514A2, July 2010.
50. Symed Labs, "Novel Process for the Preparation of Linezolid and Related Compounds," US Patent 2007/0032472, Feb, 2007.
51. B. A. Peralman, "Process to Produce Oxazolidiones," US Patent 2002/0095054, July 2002.
52. Pfizer, "Process for Preparing Linezolid," WO 2007/116284, Oct. 2007.
5 3. Pharmacia & Upjohn, "Process to Prepare Oxazolidinones," US Patent 5837870, Nov. 1998.
54. Jubilant Life Sciences, "Process for the Preparation of Linezolid," WO 2011/114241, Sept. 2011.
55. Pharmacia and Upjohn, "Substituted Oxazine and Thiazine Oxazolidinone Antimicrobials," US Patent 5688792, Nov. 1997.
56. D.C.M Chan et al., Curr. Med. Chem.13 (4), 377–398 (2006).
57. Fortress Metro Hainan Tianyuan Pharmaceutical Technology Co., Ltd., "Nitro Compounds and the Preparation of Pemetrexed CN1827604A, Sept. 2006.
58. Wu Torrent, "Pemetrexed Intermediates and Preparation Methods, CN101085775A, Dec. 2007.
59. Dr. Reddy's Laboratories, "Process for Preparing Pemetrexed, WO 2011/019986 A2, Feb. 2011.
60. D.P. Kjell et al., Org. Proc. Res. Dev.9 (6), 738–742, 2005.
61. K. Kassahun et al., Drug Metab. Dispos. 25 (1), 81–93 (1997).
62. K. Kassahun et al., Drug Metab. Dispos.26 (9), 848–855 (1998).
63. E. Mattiuz et al., Drug Metab. Dispos. 25 (1), 573–583(1997).
64. M. Lalitha Devi et al., J. Pharm. Biomed. Anal.50 (5), 710–717 (2009).
65. Y. Yoshida et al., Arzneimittelforschung43 (5), 601–606 (1993).
66. J. Sunderlanda et al., J. Antimicrob. Chemother.47 (3), 271–275 (2001).
67. A. V. Polishchuk et al., High Energy Chemistry42 (6), 459–463 (2008).
68. W. Gau, et al., Arzneimittel Forschung36 (10), 1545–1549 (1986).
69. P. Mojaverian et al., J. Pharm. Biomed. Anal.16 (3), 439–445 (1997).
70. A. Taicheng et al., Applied Catalysis B: Environmental94 (3–4), 288–294, 2010.
71. M. Mella et al., Helvetica Chimica Acta84 (8), 2508–2519 (2001).
72. K.A. Thabaj et al., Polyhedron26 (17), 4877–4885 (2007).
73. T.G. Vasconcelos et al., Chemosphere76 (4), 487–493 (2009).
74. K. Torniainen et al., J. Pharm. Biomed. Anal.15 (7), 887–894 (1997).
75. K. Tovarna Zdravil, "Process for Preparing Purified Ciprofloxacin," WO 2005/075430, Aug. 2005.
76. R. J. Islam M et al., J. Pharm. Sci.90 (5), 541–544 (2001).
77. H.V Hogerzeil et al., British Medical Journal 304, 210–214 (1992).
78. R. J. Bhuiyan K et al., Indian Drugs34 (11), 634-636 (1997).
79. EMA, Guideline on the Limits of Genotoxic Impurities (London, June, 2006).
80. B.N. Ames and L.S. Gold, Proc. Nat. Acad. Sci. USA87 (19), 7772–7776 (1990).
81. B.N. Ames and L.S. Gold, Proc. Nat. Acad. Sci. USA 87 (19), 7777- 7781 (1990).
82. M.A. Cheeseman et al., Food Chain Toxicol.37, 387–412 (1999).
83. D.A. Pierson et al., Org. Proc. Res. Dev.13 (2), 285–291 (2009).
84. D.P. Elder. et al., J. Pharm. Biomed. Anal. 48 (3), 497–507 (2008).
85. D.P. Elder. et al., J. Pharm. Biomed. Anal. 46 (1), 1–8 (2008).
86. D.P. Elder. et al., J. Pharm. Sci. 99 (7,) 2948-2961 (2010).
87. G.E. Taylor. et al., J. Chromatogr A1119 (1–2), 231–237 (2006).
88. K. Ramakrishna. et al., J. Pharm. Biomed. Anal. 46 (4), 780–783 (2008).
89. N.V.V.S.S. Raman. et al., J. Pharm. Biomed. Anal.48 (1), 227–230 (2008).
90. J. G. Slatter et al., Drug Metab. Dispos.29 (8), 1136–1145 (2001).
91. N. Plock et al., Drug Metab. Dispos. l35 (10), 1816–1823 (2007).
92. Wynalda et al., Drug Metab. Dispos. 28 (9), 1014–1017 ( 2000).