Ph.D. Thesis RESIDUAL SOLVENTS DETERMINATION IN DRUGS BY STATIC HEADSPACE AND SPME BASED TECHNIQUES: ADVANTAGES AND DRAWBACKS COSTIN C.

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1 Since 1950 IVAX DRUG RESEARCH INSTITUTE LTD. Pharmacokinetics and Metabolism Budapest University of Technology and Economics General and Analytical Chemistry Department Ph.D. Thesis RESIDUAL SOLVENTS DETERMINATION IN DRUGS BY STATIC HEADSPACE AND SPME BASED TECHNIQUES: ADVANTAGES AND DRAWBACKS COSTIN C. CAMARASU SUPERVISOR: DR. JÓZSEF BALLA 2004

2 7. C. Camarasu, J. Balla; "GC-MS-SIM SPME analysis of dithiocarbamate derivatives", Balaton Conference on High- Performance Separation Techniques, Siófok, Articles not related to the thesis 1. J. Balla, C. Camarasu; "The GC-MS technique importance in environmental analysis", Anyagvizsgálók Lapja, 1996/1. 2. A. Juhász, P. Buchwald, C. Camarasu, A. Csárdi, M. Pátfalusi, P. Kovács, N. Bodor, The Effects of a High-Calorie, High-Fat Diet on the Oral Bioavailability of Talampanel among Healthy Volunteers from Different N-Acetylation Polymorphism Groups Determined by Genotyping, J. Clin Pharm., under publication. 1. Challenges and study aims Residual solvent analysis from pharmaceutical products is one of the most difficult tasks in pharmaceutical analysis. The main challenges with residual solvents determination in pharmaceutical products and at the same time the aims of our study were as following: - Pharmacopoeia methods are suitable for residual solvent determination from articles soluble in water. For articles soluble in organic solvents these methods are not enough sensitive and selective for the limits set with the ICH Q3C(M) guideline. Our first aim was to develop, optimize and validate a methodology for sensitive and selective quantitative determination of residual solvents in pharmaceutical articles soluble in organic solvents. - Pharmacopeias give no methodologies for residual solvents identification or confirmation from From our and other laboratories experience we found that static/dynamic headspace is not compatible with modern GC-MS instrumentation, as brings along large amounts of water/high boiling point organic vapors generating contamination and severe corrosion of EI ion sources. It was needed that other sample preparation techniques, which have better compatibility (if possible solventless extraction) with the GC-MS technical requirements, to be explored. Our second aim was to introduce Solid Phase Microextraction techniques (SPME) related techniques in the pharmaceutical analysis and explore their compatibility with GC-MS for residual solvents determination, confirmation and identification tasks for pharmaceutical articles soluble in water or organic solvents. In this way, our aim was to develop and, where applicable, validate a complete analytical package capable of solving all

3 challenges related with residual solvent determination from 2. Experimental techniques and instrumentation In our experiments we used for residual solvent determination from pharmaceutical products soluble in organic solvents a static headspace sample preparation device coupled to a gas chromatograph with flame ionization and electron capture detection (HS-GC-FID/ECD). The developed methods were optimized and validated corresponding to relevant ICH and GLP guidelines. At the same time, for residual solvent confirmation or unknown residual solvents identification from pharmaceutical products soluble in water or organic solvents, we used headspace and gastight solid phase microextraction as sample preparation procedure coupled to gas chromatography with mass spectrometric detection (Headspace- SPME-GC-MS). The GC-MS instrument used in our study was an ion trap system. All experimental parameters, optimization and validation procedures are presented in detail in my publications and in the Ph.D. thesis. 3. Results and discussion 3.1 Two static headspace gas chromatographic methods for residual solvent determination in pharmaceutical products were developed, optimized and validated. The methods are sensitive (allowing detection limits of as low as 0.1ppm for benzene), accurate and precise. The developed method were applied to residual solvent determination for the release of proprietary substances 1 and 2 at Gedeon Richter Ltd., and offered unattended performance for more than 5 years under continuous operation, Posters not related to the thesis: 1. C. Camarasu, J. Balla, T. Halasi; "GC-MS study of some dithiocarbamate derivatives", 9th Danube Symposium on Chromatography, Budapest, 1993, Th-p A. Halász, C. Camarasu, J. Balla; "Application of the internal standard method in routine GC analysis", 9th Danube Symposium on Chromatography, Budapest, 1993, Th-p N. Németh, C. Camarasu, J. Balla; "Pesticide analysis by GC-MS-SIM technique", XIII Conference on High- Performance Separation Techniques, Balatonszéplak, Hungary, 1994, p C. Camarasu, J. Tóth, G. Bagyinski, J. Balla; "Waste water analysis by GC-MS ", XIII Conference on High-Performance Separation Techniques,Balatonszéplak, Hungary., 1994, p A. Halász, C. Camarasu, J. Balla; "Volatile aromatic trace analysis from industrial waste waters", XIII Conference on High-Performance Separation Techniques, Balatonszéplak, Hungary, 1994, p J. Balla, C. Camarasu, N. Németh; "High performance analytical techniques in organic trace analysis", Scientific workshop of the Hungarian Academy of Sciences, Nyiregyhaza, Hungary, 1994, p

4 . Posters related to the thesis: 1. C. Camarasu, M. Mezei, G. Bertók, "Residual solvents determination in pharmaceutical products by GC-HS and SPME- GC-ITMS" Drug Analysis 98 Conference, May 1998, Brussels, Belgium, P C. Camarasu, M. Mezei, "Residual solvents determination in pharmaceutical products by GC-MS-SPME, 8 th International Meeting on Recent Devlopments in Pharmaceutical Analysis (RDPA 99), June 29-July 3, 1999, Rome, Italy, P C. C. Camarasu, "Residual solvents determination in pharmaceutical products by HS - GC and SPME-GC-MS" Balaton Symposium 01 on High-Performance Separation Methods, September 3-5, 2002 Siófok, Hungary, best poster award. 4. C. C. Camarasu, Residual solvents determination in pharmaceutical products by SPME-GC-MS 24th International Syposium on Chromatography, September 15-20, 2002, Leipzig, Germany, COUP-019. with more than samples analyzed without any method adjustment. The method development and validation documentation was inspected by regulatory agencies (OGyI, FDA, EU) and found to be conforming to modern GXP regulations and guidelines. We found that dissolving solvent have to be chosen with great care, as for certain drugs solute-solvent interactions can occur, generating in certain cases underestimation of residual solvents content in During method optimization we found that sample volume, extraction temperature, solvent composition and pharmaceutical product s water content (reported for the first time by us) have a critical influence on method sensitivity and accuracy. From our data is beneficial to choose low sample volume, if sample solubility in the organic solvent allows it, the optimum sample volume is between ml. For drug products with water content greater than 7%, the artifactual increase in sensitivity produced by water presence should be taken into consideration, otherwise inconsistent recovery data and underestimation of residual solvent content will happen. Great care should be taken when optimizing extraction temperature, as pharmaceutical products are heat sensitive materials, and overheating the sample can generate artifact peaks. We reported for the first time in literature that making slight adjustments in solvent polarity (by mixing to the solvent used to dissolve the sample other solvents with different polarities) can efficiently improve headspace extraction of residual solvents, and in this way to finely tune headspace extraction efficiency. For instance by adding 40 µl of dimethylsulfoxide (solvent used for solvent polarity tuning ) to 100 µl of benzyl alcohol (dissolving solvent) generated 5 time increase in N,N-dimethylformamide sensitivity

5 Significant pressures into the headspace vial could have detrimental effects on sensitivity and reproducibility of analytical data, therefore care should be taken on choosing headspace vial volume. For the first time we applied Risk Management approaches to method development and showed that care should be taken when choosing dissolving solvent and that drug product water content must be monitored and taken into consideration. The developed methodologies are currently under consideration of being introduced as official methods for residual solvent determination for drug products soluble in organic solvents in the United States Pharmacopoeia. 3.2 We pioneered headspace-spme and gastight-spme extraction techniques for residual solvent determination and identification in the pharmaceutical analysis of drug products and pharmaceutical preparations soluble in aqueous solutions. We followed the development of commercially available fibers and studied the main parameters influencing efficiency of headspace-spme based techniques. Between the investigated sample preparation techniques gastight-spme proved to be more sensitive and headspace-spme proved to be more precise. The most important difference between the two techniques is that the gastight-spme showed better behaviour towards very volatile impurities, thus allowing lower detection/quantitation limits. Compared with the static headspace technique both SPME methods showed superior results in terms of sensitivity, being from all points of view compatible with the pharmaceutical samples, demonstrated by successful validation of one developed method. 6. Publications Articles related to the thesis: 1. C. Camarasu, M. Mezei, G. Bertók, "Residual solvents determination in pharmaceutical products by GC-HS and SPME- GC-MS", J. Pharm. Biomed. Anal. 18 (1998) C. Camarasu, M. Mezei, A. Szabo, "Headspace solid phase microextraction method optimization for residual solvent analysis" Acta Pharmaceutica Hungarica, 69 (1999) C. C. Camarasu, "Headspace SPME method development for the analysis of volatile polar residual solvents by GC-MS", J. Pharm. Biomed.Anal., 23 (2000), C. C. Camarasu, "Unknown Residual Solvents Identification in Drug Products by Headspace Solid Phase Microextraction Gas Chromatography Mass Spectrometry", Chromatographia, 56 (Suppl.) 2002, C. C. Camarasu, "Residual Solvents Determination in Drug Products by Static Headspace-Gas Chromatography", Chromatographia, 56 (Suppl.) 2002,

6 4. Significance of presented results We succeeded to introduce in the pharmaceutical analysis a complete analytical package capable of solving all challenges related with residual solvent determination from pharmaceutical products, which is internationally recognized and broadly employed by pharmaceutical industry all over the world. 5. Acknowledgements I am indebted to the staff at Gedeon Richter Ltd., QC department for their help and support, especially to Ms. Mária Mezei and Éva Berzsenyi. They provided me with the instrumentation for this work, technical skills, material support, and possibility to participate at international conferences and have my data published. I appreciate the financial support I got from the Varga József Alapítvány, which make possible my studies at Technical University of Budapest, General and Analytical Chemistry Department, and especially to Prof. Dr. Sándor Gál. I m very grateful to my supervisor, Dr. József Balla for his support, constructive discussions and patience. I m also grateful to all my colleagues from IVAX Drug Research Institute, DMPK Department for their active help and support in finishing writing my thesis, especially to Dr. István Hazai. I m very grateful to my wife and my children, as without their constant help and support I would never been able to finish my thesis. I m indebted to my friend Iulian Dragomir, who continuously supported me and encouraged in my work. Between the investigated polymeric films the polydimethylsiloxane/divinylbenzene coated fiber and later on Carboxen/polydimethyl siloxane showed by far the best sensitivities for all compounds. The fibers were able to extract compounds with different polarity and volatility. Extensive optimization is necessary each time a headspace SPME method is developed. We found that the extraction time, total volatile content, headspace volume and the pressure inside the headspace vial are very important parameters. These parameters need to be reoptimized each time a new component is added to the analytical method. At the same time, we found that chromatographic conditions (low starting temperature of the column, 30 C; narrow bore injector liner, 1 min splitless time), together with the optimum desorbtion parameters (optimum injection depth into the injector of 2.5 cm; optimum desorbtion temperature for CX/PDMS of 300 C, and for all other fibers of 250 C) are not influenced by the type of the extracted compounds and do not need to be reoptimized. We also found that sensitivity and reproducibility are inversely related parameters. When maximizing sensitivity (using low headspace volumes with low total volatile organic contents), the reproducibility worsens. For routine purposes, larger headspace volumes and higher (around 0.1%) total volatile contents should be used. At the same time, when routine measurements are done, care should be taken that sample solutions and test solutions have similar total volatile organic contents, in the range %. The headspace-spme GC-MS proved to be a powerful technique in the identification and determination of unknown solvent residues

7 in With this technique, we were able to identify residual solvents in our proprietary Even if SPME techniques are not yet accepted as sample preparation methods by Pharmacopoeias, taking into consideration their precision, accuracy and speed of analysis, we can state that they are suitable for quantitative residual solvent determination in 3.3 We applied for the first time the headspace-spme technique for the extraction of residual solvents from organic solutions, thus being able to identify and determine residual solvents from pharmaceutical products soluble in organic solvents. We developed a method for release type activities, but we found that Carboxen/dimethyl polysiloxane have low fiber-to-fiber reproducibility, which made the method unsuitable for validation. The phenomenon is related to immature fiber production technologies, and important breakthroughs are to be expected in this area. We found that each time a Headspace SPME method is developed extensive optimization is necessary. We found that headspace volume, added organic solvent volume, and in some cases the pressure inside the headspace vial are very important parameters. These parameters need to be reoptimized each time when a new component is added to the analytical method. We found that sensitivity and reproducibility are inversely related parameters. When maximizing sensitivity (using low headspace volumes with low total volatile organic contents) the reproducibility worsens. For routine purposes, larger headspace volumes and more organic solvent should be used. Other researchers, who applied and adapted the methodology to their practical needs, followed our methodology and approaches. Headspace-SPME using Carboxen/dimethylpolysiloxane fibers showed to be a powerful sample preparation method for residual solvents extraction from organic solvents. We developed a method for identification type activities, in order to support the static headspace methods developed and validated previously. Using the method we were able to identify unknown components from drug products. The headspace-spme GC-MS proved to be a powerful technique in the identification and determination of unknown solvent residues in pharmaceutical products, and was applied for more than 7 years at Gedeon Richter with excellent results. We developed a method for confirmation purposes, in order to allow determination of Class I solvents at much more lower levels than the static headspace methods developed and validated in Chapter 1. In this method we employed deuterated internal standards also, in order to improve method precision and accuracy. The publicized approaches and methodologies were followed by other research groups and successfully applied to their needs. Our publications are constantly cited as reference studies in residual solvents determination, confirmation and identification from