PHARMACEUTICAL IMPURITY ANALYSIS SOLUTIONS. Primer

Transcription

1 PHARMACEUTICAL IMPURITY ANALYSIS SLUTINS Primer

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3 CNTENTS. PHARMACEUTICAL IMPURITY ANALYSIS VERVIEW AND REGULATRY SITUATIN The Three Major Categories of Pharmaceutical Impurities...4 rganic impurities...4 Inorganic (elemental) impurities...5 Residual solvents...5 Selected Publications and Guidelines for the Control of Pharmaceutical Impurities...7. ANALYTICAL TECHNLGIES FR IMPURITY PRFILING IN PHARMACEUTICAL DEVELPMENT Fourier Transform Infrared Spectroscopy (FTIR)...9 Preparative Liquid Chromatography (LC)...9 Liquid Chromatography and Ultraviolet Spectrometry (LC/UV)... Liquid Chromatography and Mass Spectrometry (LC/MS)... Capillary Electrophoresis (CE)... Supercritical Fluid Chromatography (SFC)... Nuclear Magnetic Resonance Spectroscopy (NMR)...3 Inductively-Coupled Plasma ptical Emission Spectroscopy (ICP-ES) and Inductively-Coupled Plasma Mass Spectrometry (ICP-MS)...3 Gas Chromatography (GC) A SELECTIN F AGILENT APPLICATIN SLUTINS FR THE THREE MAJR TYPES F IMPURITIES verview ANALYSIS F RGANIC IMPURITIES...6 Achieve precision, linearity, sensitivity, and speed in impurity analysis with the Agilent Infi nity Series HPLC/UV Solutions...6 Improve profi ling productivity for the identifi cation of trace-level impurities using Agilent LC/Q-TF solutions... Quantitative analysis of genotoxic impurities in APIs using Agilent LC/QQQ solutions... Agilent rganic Impurity Profi ling Publications ANALYSIS F INRGANIC IMPURITIES...4 Determination of elemental impurities in pharmaceutical ingredients according to USP procedures by Agilent ICP-ES and ICP-MS based solutions...4 Agilent Elemental Impurity Analysis Publications RESIDUAL SLVENT ANALYSIS...6 Faster analysis and enhanced sensitivity in residual solvent analysis as per USP <467> procedures using Agilent GC based solutions...6 Agilent Residual Solvent Analysis Publications...8 Appendix: Agilent Solutions for Pharmaceutical Impurity Analysis...9 3

4 PHARMACEUTICAL IMPURITY ANALYSIS VERVIEW AND REGULATRY SITUATIN Pharmaceuticals impurities are the unwanted chemicals that remain with active pharmaceutical ingredients (API) or drug product formulations. The impurities observed in drug substances may arise during synthesis or may be derived from sources such as starting materials, intermediates, reagents, solvents, catalysts, and reaction by-products. During drug product development, impurities may be formed as a result of the inherent instability of drug substances, may be due to incompatibility with added excipients, or may appear as the result of interactions with packaging materials. The amount of various impurities found in drug substances will determine the ultimate safety of the fi nal pharmaceutical product. Therefore, the identifi cation, quantitation, qualifi cation, and control of impurities are now a critical part of the drug development process. Various regulatory authorities focus on the control of impurities: the International Conference on Harmonization (ICH), the United States Food and Drug Administration (USFDA), the European Medicines Agency (EMA), the Canadian Drug and Health Agency, the Japanese Pharmaceutical and Medical Devices Agency (PMDA), and the Australian Department of Health and Ageing Therapeutic Goods. In addition, a number of offi cial compendia, such as the British Pharmacopoeia (BP), the United States Pharmacopeia (USP), the Japanese Pharmacopoeia (JP), and the European Pharmacopoeia (EP) are incorporating limits that restrict the impurity levels present in APIs as well as in drug formulations. The Three Major Categories of Pharmaceutical Impurities According to ICH guidelines, impurities related to drug substances can be classifi ed into three main categories: organic impurities, inorganic impurities, and residual solvents.. rganic impurities rganic impurities can arise in APIs or drug product formulations during the manufacturing process or during the storage of drug substances. They may be known, unknown, volatile, or non-volatile compounds with sources including starting materials, intermediates, unintended by-products, and degradation products. They may also arise from racemization, or contamination of one enantiomeric form with another. In all cases they can result in undesired biological activity. Recently, genotoxic pharmaceutical impurities, which may potentially increase cancer risks in patients, have received considerable attention from regulatory bodies and pharmaceutical manufacturers. In general, genotoxic impurities include DNA reactive substances that have the potential for direct DNA damage. Potential genotoxic impurities include process impurities or degradants, present at trace levels, which are generated during drug manufacturing and storage. As per FDA and EMA guidelines, potential genotoxic impurities are to be controlled at levels much lower than typical impurities. The recommended acceptable thresholds for genotoxic impurities in pharmaceuticals can be found in the guideline documents published by the USFDA and EMA (See the selected list of key publications provided at the end of this section). The ICH M7 guidance on genotoxic impurities is currently under 4

5 preparation with the working title "M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk".. Inorganic (elemental) impurities Inorganic impurities can arise from raw materials, synthetic additives, excipients, and production processes used when manufacturing drug products. Several potentially toxic elements may be naturally present in the ingredients and these elements must be measured in all drug products. A further group of ingredients may be added during production and must be monitored for elemental impurities once they are known to have been added. Sources of inorganic impurities include manufacturing process reagents such as ligands, catalysts (e.g., platinum group elements (PGE)), metals derived from other stages of production (e.g., process water and stainless steel reactor vessels), charcoal, and elements derived from other materials used in fi ltration. The United States Pharmacopeia (USP) is in the process of developing a new test for inorganic impurities in pharmaceutical products and their ingredients. The current Heavy Metals Limit Test (USP<3>) is widely acknowledged to be inadequate in terms of scope, accuracy, sensitivity, and specifi city, and is due to be replaced with two new general chapters, Limits (USP<3>) and Procedures for Elemental Impurities (USP<33>), due to be implemented in 3. In parallel with the development of USP<3> and USP<33>, the USP is also introducing a related method <3> which is specifi c to dietary supplements. USP<3> defi nes new, lower permitted daily exposure (PDE) limits for a wider range of inorganic elemental impurities: As, Cd, Hg, Pb, V, Cr, Ni, Mo, Mn, Cu, Pt, Pd, Ru, Rh, s, and Ir. A complete list of regulated elements and PDEs can be found in Agilent publication EN and the references therein. USP<33> further defi nes the sample preparation and method validation procedures that should be used for system suitability qualifi cation of any instrumentation used for the analysis of elemental impurities in pharmaceutical materials. Validation of analytical instruments that are used for the new USP<3> and USP<33> methods will be performance based. USP<33> defi nes the analytical and validation procedures that laboratories must use to ensure that the analysis is specifi c, accurate, and precise. 3. Residual solvents Residual solvents are the volatile organic chemicals used during the manufacturing process or generated during drug production. A number of organic solvents used in synthesis of pharmaceutical products have toxic or environmentally hazardous properties, and their complete removal can be very diffi cult. In addition, the fi nal purifi cation step in most pharmaceutical drug substance processes involves a crystallization step which can lead to the entrapment of a fi nite amount of solvent which can act as a residual impurity or can cause potential degradation of the drug. Residual solvent levels are controlled by the ICH, USP, and EP. Depending on their potential risk to human health, residual solvents are categorized into three classes with their limits in pharmaceutical products set by ICH guidelines Q3C. The use of class I solvents, including benzene, carbon tetrachloride,,-dichloroethane,,-dichloroethylene, and,, trichloroethane, 5

6 should be avoided. Class II solvents, such as methanol, pyridine, toluene, N,N-dimethylformamide, and acetonitrile have permitted daily exposure limits (PDEs). A few examples of common organic solvents which are found as volatile impurities and have their limits set by ICH guidelines are depicted in Table. Class III solvents, such as acetic acid, acetone, isopropyl alcohol, butanol, ethanol, and ethylacetate should be limited by GMP or other quality-based requirements. Table. ICH limits for a selected list of common organic solvents found as volatile impurities. Volatile rganic Impurity Limit (ppm) PDE (mg/day) Acetonitrile 4 4. Chloroform 6.6,4-Dioxane Methylene chloride 6 6. Pyridine.,,-Trichloroethane 8.8 USP <467> 9 General Chapter contains a more comprehensive method for residual solvent analysis that is similar to the ICH guidelines developed in 997. Here, a limit test is prescribed for class and class solvents while class C solvents are usually determined by non headspace methods due to their higher boiling point. The limits of detection (LD) recommended for class 3 solvents are up to 5 ppm. When the levels of residual solvents exceed USP or ICH limits, quantitation is required. NTE: Regulatory limits for impurities mentioned in this document are given as examples and may not provide the complete information needed. For complete, current regulatory information and the latest updates, please check the websites of the various regulatory authorities. 6

7 Selected Publications and Guidelines for the Control of Pharmaceutical Impurities Key Topics Guidelines for the control of impurities Specific guidelines for the control of genotoxic impurities Guidelines relevant to analytical methods for the control of genotoxic impurities Title International Conference on Harmonization (ICH) Q3A (R) Impurities in New Drug Substances, 5 ctober 6 ICH Q3B (R) Impurities in New Drug Substances, June 6 Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended approaches; US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); Silver Spring, MD, USA, December 8 EMA/CHMP/SWP/43994/7 Rev. 3, Questions and answers on the guideline on the limits of genotoxic impurities, adopted September 3, Guideline on the Limits of Genotoxic Impurities, CPMP/SWP/599/, EMEA/CHMP/QWP/53446; Committee for Medicinal products (CHMP), European Medicines Agency (EMEA); London 8 June 6 Pharmeuropa, Vol, No. 3, July 8, Potential Genotoxic Impurities and European Pharmacopoeia monographs on Substances for Human Use ICH M7 Guideline (in preparation) for control of Mutagenic genotoxic impurities ICH Guidance for Industry: Pharmaceutical Development Q8, (R); US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); Aug, 9, ICH Guidelines, Q9: Quality Risk Management Q9; US Department of Health and Human Services. Food and Drug Administration, Center for Drug Evaluation and Research (CDER): Rockville, MD, Nov, 5, ICH SA: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, April 996 ICH SB: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, July 997 ICH S (R): DRAFT Consensus Guideline (Expected to combine and replace ICH SA and SB): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, March 6, 8 Guidelines for the control of elemental Elemental impurities Limits (Pharm. Forum, ), 37 (3), Chapter <3> impurities Elemental impurities Procedures (Pharm. Forum, ), 37(3), Chapter <33> Guidelines for the control of residual solvents ICHQ3C, International Conference on Harmonization, Impurities Guidelines for Residual Solvents. Federal Register, 6 (47), 997, International Conference on Harmonization, ICH Q3C (R3) Impurities: Guideline for Residual solvents, November 5 ICH Topic Q3C (R4) Impurities: Guideline for Residual Solvents, European Medicines Agency, USP Method 467, US. Pharmacopeia, updated June 7, USP 3 NF 8 NTE: This list is a limited selection of key, recent regulatory publications. For complete, current regulatory information and the latest updates, please check the websites of the various regulatory authorities. 7

8 ANALYTICAL TECHNLGIES FR IMPURITY PRFILING IN PHARMACEUTICAL DEVELPMENT verview An impurity profi le is a description of the identifi ed and unidentifi ed impurities present in a new drug substance (Source: Guidance for Industry, Q3A Impurities in New Drug Substances). Impurity profi ling processes usually begin with the detection of impurities, followed by their isolation and characterization. For all three types of impurities, it is critical to develop a robust method during process development that can eventually be validated and transferred to QA/QC. Developing reliable methods for impurities regulated at very low levels, such as genotoxic impurities, adds further challenges to this process. To better detect, identify, quantify, and characterize the impurities present in drug substances and products, pharmaceutical scientists rely on fast analytical tools with high sensitivity and specifi city. Major analytical tools for impurity analysis include spectroscopy, chromatography, and various combinations of both, i.e. tandem techniques. The appropriate technique is selected based on the nature of the impurity and the level of information required from the analysis. There are various complex analytical problems in pharmaceutical development that require the use of more than one analytical technique for their solution. Analytical techniques such as LC/UV, LC/MS, GC/MS, CE/MS, and LC/UV provide the orthogonal detection and complementary information that can address these challenges in a time effi cient manner. As a result, they play a vital role in impurity profi ling of pharmaceuticals from identifi cation to the fi nal structure elucidation of unknown impurities. Table summarizes of some of the techniques used in impurity analysis. Further details on key single and tandem techniques for impurity profi ling are found in the section that follows. Table. Impurity analysis techniques. Type of Impurity rganic impurities Inorganic/elemental impurities Residual solvents See sections below for defi nitions of abbreviations. Technologies FTIR, Preparative LC, LC/UV, LC/MS (SQ, Q-TF, and QQQ), CE, SFC, and NMR ICP-ES and ICP-MS GC and GC/MS 8

9 Fourier Transform Infrared Spectroscopy (FTIR) FTIR is very helpful for identifying and confi rming the structure of an impurity or degradant because it provides a complex fi ngerprint that is specifi c to a particular compound. An FTIR spectrum of an organic molecule is determined by the functional groups present. The technique helps to identify the structure and measure the concentration of the compound under investigation. Changes in the structure can be correlated with the help of an FTIR spectrum of a patent drug compared to that of the impurity or degradant. Agilent Cary 63 FTIR Figure. Agilent MicroLab software displays analysis results for the level of ethylene glycol, an impurity in glycerol. The red color band shows that the level of impurity is outside specification range. See Agilent publication EN. The Agilent Cary 63 FTIR packs a powerful combination of precision and compliance, making it one of the best FTIR systems for routine analysis in pharmaceutical laboratories. Measuring contaminants, such as ethylene glycol and diethylene glycol in glycerol, is quick and easy with the 63 FTIR, because its DialPath accessory reduces the tedious process of fi nding the right path length and optimum measurement conditions. In addition, Agilent MicroLab software makes it easy to meet regulatory requirement CFR by alerting users when the impurity level is outside specifi cation range (Figure ), while proprietary liquid analysis technology simplifi es sampling and reduces the risk of user error. Preparative Liquid Chromatography (LC) Since the impurities in the drug substance are usually present at very low quantities, detailed analysis is only possible upon isolation of the impurities. However, this is a major challenge in pharmaceutical laboratories. Preparative LC helps isolate impurities (usually from impurity-enriched analytes, such as the solution remaining from the crystallization of APIs) in suffi cient quantities to carry out structural analysis, usually using techniques such as FTIR, NMR, LC/MS, or GC/MS. Agilent 6 Infinity Purification Systems 9

10 Liquid Chromatography and Ultraviolet Spectrometry (LC/UV) Agilent Infi nity Series LC Systems and columns A number of impurity analysis methods found in pharmaceutical quality control (QC) laboratories use high-performance liquid chromatography (HPLC) coupled with UV detection (HPLC/UV methods). UV spectrometry helps identify impurity or degradants in drug substances based on absorption maxima. This technique is one of the most important and versatile analytical methods available for impurity profi ling today due to its high selectivity (i.e., ability to quantitatively determine a number of the individual components present in a sample using a single analytical procedure), especially for routine analysis where standards are available. Newer, stationary phase systems are available which operate in several modes, such as ion pairing, increased hydrophobic interactions, and variable ph, allowing a variety of samples to be analyzed concurrently based upon their unique properties. High resolution is particularly helpful when using LC/UV analysis for impurity detection, because all impurities can be identifi ed with less chance of error. Figure demonstrates the results achieved using an Agilent LC system combined with Agilent.8 μm RRHD columns identifying and quantifying seven impurities. Isocratic Impurity Method Column: 4.6 x 5 mm, 5 µm mau min 4.6 x 5, 5 µm Rs =.5 G/N = x 5, 3.5 µm Rs =.37 S/N = x 5,.8 µm Rs =.8 (+57 %) S/N = 44 4 impurities baseline not separated for 7 impurities baseline not separated for 6 7 impurities baseline separated for all Figure. This data demonstrates the value of UHPLC systems, like the Agilent 9/6/ Infinity Series systems, for impurity analysis. When combined with Agilent.8 μm RRHD columns, it was possible to identify all seven impurities with good baseline separation for accurate quantification. Agilent Technologies, unpublished data.

11 Liquid Chromatography and Mass Spectrometry (LC/MS) 6 Series Single Quad 65 Series Q-TF 64 Series Triple Quad Agilent Mass Spectrometers LC/MS is a powerful analytical tool that is routinely used in pharmaceutical development to test and identify product impurities. The detection limit of a few hundred ppm is readily achievable, ensuring the identifi cation of all the impurities present at concentrations greater than. %. MS-based methods generally provide additional robustness and ruggedness compared to techniques such as UV alone, due to their high specifi city and sensitivity. While single quadrupole mass spectrometers work well as analytical tools for the confi rmation of known impurities and the preliminary structural assessment of unknown impurities, highly sensitive Q-TF mass spectrometers provide higher resolution and mass accuracy that enables the unambiguous identifi cation of unknown trace impurities, making them very useful for genotoxic impurity analysis. MS-based methods are often selected for the impurity profi ling of APIs during process development, while UV-based methods are generally used to test for genotoxic impurities in QC laboratories at manufacturing sites. Triple-quadrupole (QQQ) LC/MS/MS systems have become a standard platform for the quantitative analysis of organic impurities in pharmaceutical analytical laboratories. Combining multiple reaction monitoring (MRM) with a triple quadrupole tandem mass spectrometer, such as the Agilent 64 Series QQQ, enables extraordinary sensitivity for multi-analyte quantitative assays. MRM assays are particularly useful for the targeted analysis of compounds present in complex mixtures and matrices, such as blood. Capillary Electrophoresis (CE) Agilent 7 CE instrument The determination of drug-related impurities is currently the most important task for CE within pharmaceutical analysis because it achieves high separation effi ciencies compared to other chromatographic techniques. CE can be employed when HPLC techniques are not able to adequately measure impurities, especially in the case of very polar compounds. A detection limit of. % is widely accepted as a minimum requirement for a related impurities determination method and this can be achieved using CE. In addition, CE is very useful for the separation of closely related compounds, such as diastereomers and enantiomers. An example of the value of CE in impurity analysis can be demonstrated using heparin (a polymeric anticoagulant) as an example. In this case, standard chromatography failed to distinguish drug lots associated with adverse events while CE was easily able to identify an unknown impurity (Figure 3). As a result, the use of CE helped to solve this analytical challenge. mau min Figure 3. Capillary electrophoresis of heparin and related impurities using highly concentrated buffers in a 5μm bubble cell capillary. See Agilent publication EN.

12 Supercritical Fluid Chromatography (SFC) SFC, which uses supercritical C as mobile phase, is another orthogonal technique that can be used for impurity detection because it offers HPLC-level sensitivity with reduced organic solvent usage (Figure 4). SFC also offers the advantage of chiral impurity analysis enabling the determination of enantiomeric excess at very low impurity levels (Figure 5). A mau Caffeine Caffeine Main X Agilent 6 Infinity Analytical SFC System B mau 3 - Estriol min Estriol X Main min Figure 4. Isocratic separation of the impurity (.5 % w/w level) from the main component (A) caffeine and(b) estriol; the signal-to-noise for the impurity at the. % level is well above 3, which is usually the level of detection (LD). See Agilent publication EN. A mau 4 R - 3 R R =.5 S B mau 4 S S R =.7 min R min Figure 5. Determination of enantiomeric excess at impurity levels below.5 % using SFC. Chromatograms of R-, -bi--napththol (A) and S-, -bi--naphthaol (B) at 5 ppm. See Agilent publication EN.

13 Nuclear Magnetic Resonance Spectroscopy (NMR) NMR is a powerful analytical tool that enables the study of compounds both in solution and in the solid state. It has wide applicability because it provides specifi c information about bonding and stereochemistry within a molecule, which is particularly important in the structural characterization of drug impurities and degradants often present only in extremely limited quantities. The non-destructive, non-invasive nature of NMR spectroscopy makes it a valuable tool for the characterization of impurities and degradants present at very low levels. NMR can also provide quantitative output, an important aspect of impurity profi ling. 4-MR DD Magnetic Resonance System Inductively-Coupled Plasma ptical Emission Spectroscopy (ICP-ES) and Inductively-Coupled Plasma Mass Spectrometry (ICP-MS) Agilent 7 and 73 ICP-ES Agilent 77 Series ICP-MS The new draft elemental impurities procedure (USP<33>) requires that an instrument-based method is used to determine elemental impurities and that the reference methods are based on either ICP-MS or ICP-ES. With both methods, sample analysis can be accomplished in three ways: directly (unsolvated), following sample preparation by solubilization in an aqueous or organic solvent, or after acid digestion using a closed-vessel microwave system. ICP-ES ICP-ES provides parts per billion (ppb) detection limits for most regulated elements in pharmaceutical products, easily meeting the specifi ed limits in cases where direct sample analysis or small dilution factors are appropriate. It also provides extended dynamic range, robust plasma, and one-step measurement of major, minor, and trace elements. Therefore, ICP-ES addresses the needs of a wide range of users, including those seeking a cost-effective solution for the direct analysis of elemental impurities in bulk raw materials and pharmaceutical products. ICP-MS ICP-MS is a powerful and sensitive technique that delivers a reliable trace-level analysis of all 6 elements whose limits are defi ned in USP<3>. The low detection limits of ICP-MS ensure that all regulated elements in drug substances or drug products can easily be determined using the new method, at or below regulated levels, and even when large sample dilutions are required. ICP-MS can also be used in combination with a variety of separation techniques, such as HPLC, GC, and CE, providing several options for separation (or speciation) of the different chemical forms of the elements, and depending upon the nature of sample. ICP-MS achieves low detection limits for almost all elements, including those found in the more extensive analyte list proposed in the ICH Q3D, such as Au and Tl. 3

14 Gas Chromatography (GC) 789A/5975C GC/MS system with 7697A Headspace sampler In combination with fl ame ionization detection (FID), GC is the standard choice for the analysis of volatile organic impurities, such as residual solvents. The gas chromatography headspace method is used worldwide for residual solvent analysis in quality control laboratories because it closely follows ICH Q3C guidelines. Sample preparation and introduction is via a static headspace which facilitates the selective introduction of volatile solvents without contamination by mostly non-volatile drug substance or drug products. Therefore, the use of an FID detector helps preferentially identify and quantify residual solvents. More recently, the combination of gas chromatography and mass spectroscopy (GC/MS) has been successfully used for confi rmation and identifi cation purposes, highlighting the fl exibility of this technology. GC columns 4

15 A SELECTIN F AGILENT APPLICATIN SLUTINS FR THE THREE MAJR TYPES F IMPURITIES 3 verview This section includes a selection of detailed examples of Agilent applications solutions that have been developed to meet the challenges encountered when analyzing the three types of pharmaceutical impurities: the qualitative and quantitative analysis of trace level organic impurities, the determination of elemental impurities, and the analysis of residual solvents according to USP procedures. 3. ANALYSIS F RGANIC IMPURITIES Achieve precision, linearity, sensitivity, and speed in impurity analysis with the Agilent Infi nity Series HPLC/UV solutions Improve profi ling productivity for the identifi cation of trace-level impurities using Agilent LC/Q-TF solutions Quantitative analysis of genotoxic impurities in APIs using Agilent LC/QQQ solutions 3. ANALYSIS F INRGANIC IMPURITIES Determination of elemental impurities in pharmaceutical ingredients according to USP procedures by Agilent ICP-ES and ICP-MS based solutions 3.3 ANALYSIS F RESIDUAL SLVENTS Faster analysis and enhanced sensitivity in residual solvent analysis as per USP <467> procedures using Agilent GC based solutions 5

16 3. ANALYSIS F RGANIC IMPURITIES Achieve precision, linearity, sensitivity, and speed in impurity analysis with the Agilent Infinity Series LC Agilent Infi nity Series LC/UV Systems are an ideal solution for impurity analysis in pharmaceutical quality control laboratories seeking to achieve the necessary precision, linearity, sensitivity, and speed required to meet the regulatory standards for impurity analysis. The example shown in Figure 6 is for the analysis of amoxicillin and its impurities. This analysis was completed within 7 minutes and detected impurities down to a level of. %. Excellent precision of retention times, peak areas, and linearity was achieved with a correlation coeffi cient of R >.999 (Figure 7) for fi ve impurities. mau 8 Impurity A Amoxicillin 6 4 Impurity C Impurity B Impurity D Impurity E min Figure 6. Analysis of amoxicillin and five impurities using the Agilent Infinity LC System and a gradient method in combination with UV detection, an Agilent ZRBAX SB-Aq column, and ChemStation software. See Agilent publication EN. Area Impurity A Correlation: Amount (ng/μl) Area Impurity B Correlation: Amount (ng/μl) Area Impurity C Correlation: Amount (ng/μl) Impurity D Area Correlation: Amount (ng/μl) Area Impurity E Correlation: Amount (ng/μl) Figure 7. The impurities in amoxicillin are measured with excellent linearity at six concentration levels. See Agilent publication EN. 6

17 Agilent s UHPLC/UV solutions help achieve higher sensitivity, faster sample throughput, and signifi cant cost savings in impurity profi ling. Since the 9 Infi nity LC can be operated at up to bar pressure, using a very sensitive DAD detector, signifi cantly faster methods can be developed for profi ling impurities in a highly productive manner. This leads to a signifi cant reduction in the cost per analysis. The Agilent Multi-Method solution for LC is ideally suited for testing experimental conditions, such as determining the ideal combination of stationary and mobile phases. It makes scouting stationary and mobile phases a simple, automated task, especially when short run times are used (e.g., a few minutes on an Agilent 6 or 9 Infi nity LC). The Agilent Intelligent System Emulation Technology (ISET) can be used when there is a need to transfer the fi nal method optimized on UHPLC to standard HPLC equipment and columns, especially in regulated QA/QC environments. ISET can be used to execute new or legacy HPLC methods, delivering the same chromatographic results without the need to change the original method or modify the instrument hardware. ther HPLC or UHPLC System Series LC Series LC 9 Infinity LC with ISET Method Transfer Infinity LC 6 Infinity LC Figure 8. Agilent s ISET system can be used to efficiently transfer methods from a range of systems to the final QC environment. See Agilent publication EN. 7

18 The advantage of using ISET s seamless method transfer for impurity analysis is demonstrated in Figure 9. After a method was developed for the analysis of paracetamol and its impurities on the Agilent 9 Infi nity LC, the ISET tool emulated the target LC, an Agilent Series Quaternary LC System, to determine whether the method that had been developed was suitable for that system. The method was then transferred to the Series LC System. The three chromatograms obtained on the 9 Infi nity LC System, with and without ISET, and those obtained on the Series quaternary LC System are compared in Figure 9. mau Agilent 9 Infinity LC System without emulation Agilent 9 Infinity LC System using ISET to emulate the Series Quaternary LC Agilent Series Quaternary LC System Time (min) Figure 9. verlay of chromatograms at 7 nm obtained for paracetamol and its impurities on the Agilent 9 Infinity LC System (blue), the Agilent 9 Infinity LC System with ISET (orange), and on the Agilent Series Quaternary LC System (black). See Agilent publication EN. In addition to LC systems, LC columns can signifi cantly impact the results achieved in organic impurity profi ling. For example, laboratories performing compendia analysis with conventional, long, 5 µm totally porous LC columns can benefi t from the increased speed, resolution, and sensitivity that superfi cially porous, Agilent Poroshell columns provide, without having to replace existing instrumentation. Since USP and EP guidelines allow for method fl exibility in reducing column length and particle size, transferring methods to shorter.7 µm Poroshell columns can save signifi cant time, while maintaining performance in the separation. This results in higher throughput and greater productivity with Agilent Poroshell columns than can be achieved with conventional 5 µm columns (Figure ). 8

19 mau mau mau mau ml/min.5 ml/min. ml/min. ml/min mm Agilent Poroshell EC-C mm Agilent Poroshell EC-C mm Agilent Poroshell EC-C mm Agilent Eclipse Plus C8 5 µm Figure. Rapid analysis of cefepime and related impurities on ZRBAX Eclipse Plus (5 μm) and Poroshell EC-C8 (.7 μm) columns. See Agilent publication EN min min min min Software can also assist in a number of key tasks required for impurity profi ling. For example, Agilent penlab ELN guides chemists through the complete workfl ow and documents all data in a central and secure repository that meets regulatory standards. Agilent penlab Chromatography Data Software (CDS) software also offers builtin peak purity evaluations (Figure ) and lets you generate your fi nal impurity profi le report right from the CDS. By comparing spectra from the upslope, apex, and downslope, impurities present at less than.5 % can be identifi ed. This can and should be done as a matter of routine to achieve reliable high-quality data. Custom calculation functionality in this analytical software helps calculate the total level of impurities for a complete run and includes a PASS/FAIL notifi cation against user-defi nable limits depending on the toxicity class of the impurities. 5 %.5 %. % Figure. ChemStation peak purity software can be used to determine impurities present at less than.5 %, based on spectral differences. See Agilent publication EN. 9

20 Improve profiling productivity for the identification of tracelevel impurities using Agilent LC/Q-TF solutions The Agilent 654 Q-TF delivers sensitive MS and MS/MS analysis of trace level impurities in drug substances with sub-ppm mass accuracy. The workfl ow shown in Figure 3 uses advanced MassHunter data analysis features like molecular feature extraction (MFE) and molecular formula generation (MFG), along with molecular structure correlator (MSC) software. The effective use of this novel workfl ow for impurity profi ling is demonstrated by the rapid identifi cation and structural elucidation of atenolol and eight impurities (present at >. % relative to the API s UV detection area) as shown in Figure 4. A unique feature of MSC software helps elucidate the structure of impurities in an effi cient manner. This workfl ow is streamlined to provide high confi dence, accurate identifi cation and faster structure elucidation compared to conventional impurity profi ling which requires multiple platforms and spreads analysis over multiple days. If the method is MS compatible HPLC Separation LC/MS analysis using Agilent 654 Q-TF with full MS scan followed by auto MS/MS If the method is not MS compatible Result Ex. m/z: Develop equivalent MS Compatible LC method Find and identify impurities by MFE and MFG based on the accurate mass MS data C 4 H N 4 MSC facilitates the structure elucidation of the impurities 3 Figure 3. Software-assisted workflow for impurity identification and profiling of pharmaceuticals on the Agilent Infinity Series LC combined with an accurate mass Q-TF, and MassHunter Qualitative Analysis and MSC software. Agilent publication in development.

21 CH3 CH3 NH H H H3C H3C CH3 CH3 NH NH H H H NH H3C CH3 NH H H H3C CH3 NH H3C CH3 H NH NH H H3C H H3C CH3 NH CH3 NH H H H NH x NH NH 4 3 H CH3 CH3 NH H H 3 C NH H 3 C x 3 5 H 4 3 H C Counts vs Mass-to-Charge (m/z) H 3 C NH H Figure 4. Structure elucidation of atenolol and impurity G demonstrating the wide usability of MSC software to assign structures for each fragment of atenolol (precursor m/z: 67.73) and impurity G (precursor m/z: ). Agilent publication in development. Quantitative analysis of genotoxic impurities in APIs using Agilent LC/QQQ solutions The Agilent Infi nity Series LC and Agilent 64 Series Triple Quadrupole (QQQ), in combination with Agilent columns and MassHunter software, provide a dependable solution for the quantitative analysis of genotoxic impurities at the lower detection limits required by current regulations. Variations in organic modifi er, and column stationary phases and dimensions, can be used to tune the selectivity, peak capacity, and peak resolution. This generic approach can be applied in early method development or used for potential genotoxic impurity screening procedures prior to method optimization. MRM-based quantitation of nine arylamine and aminopyridine potential genotoxic impurities (PGIs) at trace levels (well below ppm relative to the API) using an Agilent 9 Infi nity Series LC coupled to an Agilent 64 Series QQQ is demonstrated in Figure 5. Detection limits for these nine PGIs were below ppb (relative to the API) using MS/MS. Selectivity in the presence of related impurities was assured through the use of specifi c quantifi ers and qualifi ers for each PGI. All nine PGIs were well separated within 9 minutes using an Agilent 5 mm ZRBAX Eclipse Plus C8 RRHD column (. mm id,.8 μm). Analysis time can be further reduced to 3 minutes by using a 5 mm Agilent ZRBAX Eclipse Plus C8 RRHD column. ne of the PGIs (,6-dichloroaniline) was quantifi ed using a diode array detector (DAD) at a detection level of ppb relative to the API. The recoveries calculated by comparison of a standard solution of the PGIs provided accuracy levels of 7 %- 3 %, which are typical limits in pharmaceutical trace analysis procedures (e.g., limit tests).

22 A mau 4 Chlorhexidine, spiked with ppm PGIs DAD 6 nm min PGI 5, 66.>3. (8.4%) - PGI 4, 63.>. (98.3%) PGI 6, 5.>8. (3.8%, coelution with API) B PGI 5, 66.> PGI 7, 9.>93. (79.%) PGI 3, 36.>. (.7%) PGI 4, 63.>. PGI 6, 5.>8. PGI 3, 36.>. PGI 8, 8.>93. (Present in API, > ppm) 5 PGI 7, 9.>93. - PGI, 6.>. (96.%) PGI 8, 8.>93. - PGI 9,.>5. (98.5%) 5 PGI, 6.>. PGI 9,.>5. - PGI, 9.>9. (89.6%) Counts (%) vs. Acquisition Time (min) 5 PGI, 9.> Counts vs. Acquisition Time (min) Figure 5. DAD result and quantifier MRM transitions for the analysis of a chlorohexidine sample spiked with PGIs. A comparison is shown between results achieved with 5 mm column (A) and 5 mm column (B) Agilent ZRBAX Eclipse Plus C8 RRHD (. mm id,.8 μm) columns. Transitions and calculated recoveries are also indicated. See Agilent publication EN.

23 Agilent rganic Impurity Profiling Publications Publication Number EN EN EN EN EN 599-5EN EN EN EN EN EN EN EN EN EN EN Title Analysis of potential genotoxic arylamine and aminopyridine impurities in active pharmaceutical ingredients by UHPLC and UHPLC-MS/MS using the Agilent 9 Infi nity LC system and the Agilent 646A Triple Quadrupole MS system Method development on the Agilent 9 Infi nity LC using Intelligent System Emulation Technology (ISET) with subsequent transfer to an Agilent Series LC - analysis of an analgesic drug Agilent 9 Infi nity LC with Intelligent System Emulation Technology Fast analysis of cefepime and related impurities on Poroshell EC-C8 Analysis of amoxicillin and fi ve impurities on the Agilent Infi nity LC system Single-run assay and impurity testing of fi xed-dose combination drugs using the Agilent Infi nity Series High Dynamic Range Diode Array Detector Solution Quantifi cation of genotoxic "Impurity D" in atenolol by LC/ESI/MS/MS; with Agilent Series RRLC and 64B Triple Quadrupole LC/MS Direct analysis by LC/MS speeds up determination of potential genotoxins in pharmaceutical drug candidates: AZ success story Impurity profi ling with the Agilent series LC system: part 4 method validation of a fast LC method Impurity profi ling with the Agilent Series LC System: part 5 QA/QC application example using a fast LC Increasing productivity in the analysis of impurities in metoclopramide hydrochloride formulations using the Agilent 9 Infi nity LC System Application compendium: analysis of pharmaceuticals and drug related impurities using Agilent instrumentation Isolation of Impurities with Preparative HPLC Peak purity analysis in HPLC and CE using diode-array technology Highly sensitive UV analysis with the Agilent 9 Infi nity LC System for fast and reliable cleaning validation Quality verifi cation of incoming liquid raw materials using the Agilent 55 DialPath FTIR spectrometer 3

24 3. ANALYSIS F INRGANIC IMPURITIES Determination of elemental impurities in pharmaceutical ingredients according to USP procedures by Agilent ICP-ES and ICP-MS based solutions The new methodology for the preparation and analysis of pharmaceutical samples described in the draft General Chapters USP<3> and <33> provides an opportunity for pharmaceutical laboratories to update their methodology and instrumentation to address the limitations of the current heavy metals limit test (USP<3>). The robust plasma system on the Agilent 7 Series ICP-ES is capable of analyzing the most challenging samples, such as undiluted organic solvents and concentrated salt solutions, to enable fast, accurate analysis which is free of complex sample digestion procedures (See Figure 6). 6 4 PPM As Cr Ba Mn 57.6 Co Se 96.6 Sr Zn Time (min) Figure 6. The robust plasma system of the Agilent 7 Series ICP-ES ensures the stable analysis of difficult samples, such as the 5 % NaCl brine solution shown here. Agilent Technologies, unpublished results. In combination with closed-vessel microwave digestion and sample stabilization using HCl, the Agilent 77x ICP-MS has been shown to be capable of determining all regulated elements at low levels in typical pharmaceutical sample digests (See Agilent publication EN). Simple method development and routine operation are provided by the standard helium (He) mode method, which uses a single set of consistent instrument operating conditions for all analytes and samples. As required in USP<33>, unequivocal identifi cation and verifi cation of analyte results is also provided by the secondary (qualifi er) isotopes measured in He mode. Low limits of detection are particularly important for potentially toxic trace elements defi ned in USP<3>, notably As, Cd, Hg, and Pb. Calibrations for these elements in He mode are shown in Figure 7, together with Pd and Pt, which are representative members of the platinum group elements (PGEs) that must be monitored when added as catalysts as per USP<3>. 4

25 System performance validation of the 77x ICP-MS delivered data that was easily within method requirements for accuracy, stability, and spike recovery at detection limits that were all several orders of magnitude lower than the levels at which the trace elements are currently controlled. This provides the reassurance that the Agilent 77x will be able to meet the regulatory requirements for pharmaceutical materials regulated under USP methods, even if control limits are made more sensitive in the future. The Agilent 77x also provides a full mass spectrum screening capability, is tolerant of all commonly-used organic solvents, and can be linked to a chromatography system to provide integrated separation and analysis of the different forms of As and Hg, as required under USP<3>. 75 As [He] ISTD: 45 Sc [He] Cd [He] ISTD: 59 Tb [He] Hg [He] ISTD: 9 Bi [He] x R =.9998 x 3 R =.9999 x R = Ratio Ratio Ratio.5.5 As Cd Hg Conc (ppb) Conc (ppb) Conc (ppb) 8 Pb [He] ISTD: 9 Bi [He] 5 Pd [He] ISTD: 59 Tb [He] 95 Pt [He] ISTD: 9 Bi [He] x R =.9999 x R =.9999 x R = Ratio.5 Ratio Ratio 5.. Conc (ppb) Pb 5.. Conc (ppb) Pd 5.. Conc (ppb) Pt Figure 7. Calibration curves for As, Cd, Hg, Pb, Pd, and Pt in He mode, demonstrating limits of detection of ng/l or below, and good sensitivity and linearity for all elements including Hg, Pd, and Pt, which require stabilization in HCl. See Agilent publication EN. Agilent Elemental Impurity Analysis Publications Publication Number EN EN Title Pharmaceutical analysis by ICP-MS: new USP test for elemental impurities to provide better indication of potentially toxic contaminants Validating the Agilent 77x ICP-MS for the determination of elemental impurities in pharmaceutical ingredients according to draft USP general chapters <3>/<33> EN Proposed new USP general chapters <3> and <33> for elemental impurities: The application of ICP-MS for pharmaceutical analysis EN Regulatory compliance for ICP-MS 5

26 3.3 RESIDUAL SLVENT ANALYSIS Faster analysis and enhanced sensitivity in residual solvent analysis as per USP <467> procedures using Agilent GC-based solutions Quality assurance laboratories routinely use United States Pharmacopeia (USP) method <467> for residual solvent analysis. The Agilent 7697A Headspace Sampler coupled to an Agilent 789 GC offers a very effi cient solution for the analysis of UPS<467> class and class residual solvents at their limit concentrations in aqueous solutions. USP<467> specifi es three procedures for class and class residual solvents:. Procedure A: identifi cation and limit test. Procedure B: confi rmatory test (if solvent is above limit) 3. Procedure C: quantitative test Procedure A uses G43 phase Agilent 64 columns (VF-64ms or DB-64) and Procedure B uses a G6 phase (HP-INNWax) column. In general, analytes that co-elute in one of these phases do not co-elute in the other. As demonstrated in Figures 8 and 9, the Agilent 7697A Headspace sampler is capable of outstanding repeatability for the analysis of residual solvents. Repeatability is better than.5 % relative standard deviation (RSD) for class, class A, and class B solvents. An inert sample path, thermal zones with set point stability of better than +/-. C, and EPC-controlled vial sampling using absolute pressure, all contribute to system performance. Carryover is essentially non-existent in all confi gurations. User programmable fl ow rates and times, needle/loop purges, and vent line purges are effectively used to clean the system between runs. Laboratories should perform system suitability studies and validate their proposed methods according to USP or ICH guidelines. For new drug development and quality control, a dual-channel confi guration using both FID and a mass selective detector (MSD) is a powerful tool for residual solvent determinations, especially when unknown identifi cation or confi rmation is needed. This system is particularly well-suited for the development of generic methods that do not need to follow USP<467> guidelines. MSD analysis also helps avoid ambiguity, as over 6 solvents are currently used in pharmaceutical manufacturing. When unknown peaks or solvents are present, this system may be the best solution for confi rmation and quantitation. 6

27 A 4.,-dichlorothene.,,-trichloroethane 3. carbon tetrachloride 4. benzene 5.,-dichloroethane 5 3 B. methanol. acetonitrile 3. dichloromethane 4. Trans-,-dichloroethene 5. Cis-,-dichloroethene 6. tetrahydrofuran 7. cyclohexane 8. methylcyclohexane 9.,4-dioxane. toluene. chlorobenzene. ethylbenzene 3. m-xylene, p-xylene 4. o-xylene C hexane. nitromethane 3. chloroform 4.,-dimethyoxyethane 5. trichloroethene 6. pyridine 7. -hexanone 8. tetralin Figure 8. Class (A), class A (B), and class B (C) solvents at USP<467> limit concentrations. See Agilent publication EN. TIC FID Figure 9. Class A solvents at limit concentrations with FID-MSD. See Agilent publication EN. 7

28 Agilent Residual Solvent Analysis Publications Publication Number EN EN EN EN EN EN Title Analysis of USP <467> residual solvents with improved repeatability using the Agilent 7697A Headspace Sampler Simultaneous dual capillary column headspace GC with fl ame ionization confi rmation and quantifi cation according to USP <467> A generic method for the analysis of residual solvents in Pharmaceuticals using static headspace GC-FID/MS Fast analysis of USP <467> residual solvents using the Agilent 789A and low thermal mass (LTM) system Improved retention time, area repeatability and sensitivity for analysis of residual solvents The determination of residual solvents in pharmaceuticals using the Agilent G888 headspace/689n GC/5975 inert MSD system 8

29 Appendix: Agilent Solutions for Pharmaceutical Impurity Analysis Agilent leads the industry with a wide range of instrumentation, LC and GC column choices, and software and informatics solutions for impurity analysis. Instrumentation Category of Impurity Application Agilent Instrumentation rganic impurities Impurity detection and rapid method scouting/development Infinity Series LC + Diode-array Detector SL Detection of impurities not easily separated by HPLC (e.g., 7 CE System highly polar compounds) Detection of chiral impurities 6 Infinity Analytical SFC System Isolation of impurities 6 Infinity Preparative-scale Purification System Identification of impurity structure 6-IR series FTIR + 4-MR DD Magnetic Resonance System + Infinity Series LC + 6 Series Single Quadrupole or 6 Series Accurate-Mass TF or 65 Series Accurate-Mass Q-TF LC/MS Systems (for trace level genotoxic impurities) Quantitation of genotoxic impurities Infinity Series LC + 64 Series Triple Quadrupole LC/MS Systems Inorganic impurities Analysis of elemental impurities in pharmaceutical 7 Series ICP-ES ingredients - basic requirements of USP that do not necessitate the lowest detection limits Analysis of all 6 regulated elements at and below the 77 Series ICP-MS regulated levels in the new USP <33> method, even when large sample dilutions are required Speciation of certain regulated elements (As and Hg) Infinity Series LC + 77 Series ICP-MS Residual solvents Analysis per USP <467> procedures 789A GC A Headspace sampler Analysis involving unknown peaks/solvents 789A GC C GC/MS system A Headspace sampler Columns and Supplies Agilent offers a comprehensive portfolio of GC and LC columns, and supplies for chromatography, spectrometry, and spectroscopy, all meeting IS 9 standards to ensure maximum instrument performance and reproducible results. Agilent leads the LC industry with column choices that meet a wide range of analytical needs and support the pharmaceutical lifecycle with maximum scalability across laboratory development settings, and around the world service and support. For example Poroshell columns can save signifi cant analysis time, and Rapid Resolution High Defi nition (RRHD) columns offer maximum fl exibility in solvent selection and fl ow rates. Agilent also has the broadest portfolio of GC columns available, including innovative options like our ultra inert GC columns. Agilent s comprehensive portfolio of supplies including vials, syringes, gas management, fl ow meters, leak detectors, fi ttings, tools, and standards, all engineered or selected by our instrument design teams, manufactured to our demanding specifi cations, and tested under a variety of conditions. Software and Informatics Agilent s industry leading software and informatics portfolio is continuously expanding to cover a broader range of analytical workstations, data and laboratory management solutions, and applications to satisfy the growing needs of the life sciences and chemical industries. Agilent software solutions are integrated to address the complete life cycle of scientifi c data, including experimental design, data acquisition, knowledge management, and analysis in an open system architecture. The Agilent penlab Software Suite includes penlab Chromatography Data System (CDS), penlab Enterprise Content Manager (ECM), and penlab Electronic Lab Notebook (ELN). Laboratory Qualification and Testing Solutions You can count on Agilent to provide the system qualifi cation services or proof of calibration that you need to support your GLP/GMP or IS/IEC 75 quality initiatives. Agilent has been ranked # in compliance since 995. With the delivery of over, successful instrument qualifi cations and over a decade of practical experience in quality testing, you can trust Agilent to deliver confi dence in your analytical results. 9