Flow-injection MS/MS for gas-phase chiral recognition and enantiomeric quantitation of a novel boron-containing antibiotic (GSK2251052A) by the mass spectrometric kinetic method.

The present work demonstrates, for the first time, the application of the mass spectrometric kinetic method for quantitative chiral purity determination by automatic flow-injection MS/MS. The particular compound analyzed is GSK2251052A, a novel boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Chiral recognition and quantitation of GSK2251052A was achieved based on the competitive dissociation kinetics of the Cu(II)-bound trimeric complex [Cu(II)(A)(ref*)2-H](+) (A = GSK2251052A or its R-enantiomer, ref* = L-tryptophan) that gives rise to Cu(II)-bound dimeric complexes. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow chiral purity determination of pharmaceutical compounds during enantioselective synthesis. By using flow-injection MS/MS, enantiomeric quantitation of GSK2251052A by the kinetic method proved to be fast (2 min for analysis of each sample) and to have accuracy comparable to chiral LC-MS/MS and LC-UV methods as well as the method using chiral derivatization followed by LC-MS/MS analysis. This flow-injection MS/MS method represents an alternative approach to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful for rapid screening of chiral drugs during pharmaceutical development.

NMR spectroscopy as a characterization tool enabling biologics formulation development.

This review highlights recent advancements in using high resolution nuclear magnetic resonance (NMR) spectroscopy as a characterization tool to expedite biologics formulation development, meeting a current need in the biopharmaceutical industry. Conformational changes of protein therapeutics during formulation development can result in various protein-protein and protein-excipient interactions, which can lead to physical aggregation and/or chemical degradation. Innovative analytical techniques that allow studying protein integrity with high specificity during formulation development are urgently needed in order to assess protein formulation stability and mitigate product quality risks. Solution NMR spectroscopy is emerging as a powerful analytical tool for biophysical characterization of protein therapeutics. For instance, one-dimensional (1D) NMR has been employed in high sensitivity monitoring of monoclonal antibody (mAb) structural changes and protein-excipient interactions in parenteral formulations, demonstrating it as a potential tool for formulation screening. 2D NMR, such as ALSOFAST-[1H-13C]-HMQC experiments, on the other hand, offer superior capability to detect higher order structural (HOS) changes of mAbs in formulated solutions and their interactions with excipients. These determinations need to be achieved in actual formulations, where proteins of natural abundance are typically at low concentrations depending on the actual dose regimen. Studying proteins with natural abundance in the presence of hundredfold more concentrated excipients makes NMR studies of proteins in formulations extremely difficult considering the sample matrix interferences. Thus, successfully suppressing buffer signals while enhancing the protein signals of interest by optimizing the instrument specific parameters is critically important. Given the large size of typical mAbs, with a molecular weight (MW) ranging from 100 to 240 kDa, coupled with low protein concentrations, data collection becomes a demanding task in terms of NMR instrument time. As such, the biopharmaceutical industry is facing the common challenge of developing innovative NMR approaches to enhance signal detection (sensitivity and selectivity) and reduce experimental/instrument time. XL-ALSOFAST -[1H-13C]-HMQC was recently developed for tackling high MW proteins (up to 240 kDa) with much improved sensitivity and selectivity. We at BMS have implemented the XL-ALSOFAST experiment utilizing its high sensitivity and superior artifact suppression to successfully analyze formulations of several investigational proteins. In this manuscript we will discuss the general utility of this superior tool for studying therapeutic proteins across a range of molecular sizes and buffers. We envisage that this manuscript will serve as a primer to expand the role of NMR spectroscopy as a characterization tool supporting biologics formulation development.

A generic approach for the determination of trace hydrazine in drug substances using in situ derivatization-headspace GC-MS.

In situ derivatization-headspace GC-MS methodology has been developed for the determination of hydrazine in drug substance at low ppm levels. This general method uses acetone or acetone-d(6) as the derivatization reagent. The resulting acetone azine or acetone azine-d(12) can then be analyzed by headspace GC-MS. The method gives excellent sensitivity with a limit of quantitation (LOQ) as low as 0.1ppm when the API (active pharmaceutical ingredient) samples are prepared at 10mg per headspace injection vial. The spike recoveries of hydrazine at the 1ppm level were between 79% and 117% in various APIs tested. The precisions (%RSD) of six preparations of the hydrazine standards at the concentration of 1ppm level were typically between 2.7 and 5.6%. A linear range of concentrations from 0.1 to 10ppm has been demonstrated with R(2)> or =0.999. This general method has been tested in a number of API matrices and successfully applied to the determination of hydrazine in support of API batch releases and process chemistry at GlaxoSmithKline.

Fragmentation of aromatic sulfonamides in electrospray ionization mass spectrometry: elimination of SO(2) via rearrangement.

Arylsulfonamides are attractive pharmacophores for drug candidates. Fragmentation behaviors of selected aromatic sulfonamides were investigated using electrospray ionization mass spectrometry in the positive ion mode. Some of the sulfonamides afforded unique loss of 64 (loss of SO(2)) ions upon collision-induced dissociation followed by intramolecular rearrangements in the gas phase. This SO(2) elimination-rearrangement pathway leading to the generation of [M + H - SO(2)](+) ions appeared to be susceptible to substitutions on the aromatic (Ar) ring that would affect the Ar--sulfur bond strength and the stability of the partially positive charge developed at the ipso position upon bond dissociation. Electron withdrawing groups such as chlorine attached to the aromatic ring at ortho position seem to promote the SO(2) extrusion. Although this fragmentation pathway in atmospheric pressure ionization MS is less predictable than in electron impact MS, it is a frequently encountered reaction. The absence of this fragmentation pathway in some of the arylsulfonamides indicates that other factors such as nucleophilicity of the nitrogen may also play a role in the process. With respect to the site of attachment of the migrating NR'R'', ipso-substitution on the aromatic ring is evident since this fragmentation mechanism is operative in the fully ortho-substituted arylsulfonamides.

A novel GC-MS method for rapid determination of headspace oxygen in vials of pharmaceutical formulations.

A novel GC-MS method which requires small injection volumes was developed for fast and selective determination of headspace oxygen in pharmaceutical packages. This method does not require a specific GC column for separation of oxygen from other permanent gases such as nitrogen; instead it offers the advantage of using co-eluting nitrogen as the internal standard for quantifying oxygen in the headspace under electron ionization (EI, 70 eV) conditions. The relative ionization efficiency of oxygen to nitrogen, termed as ionization efficiency correction factor (IECF), can be measured using a control sample with known composition of oxygen and nitrogen such as the standard dry air used in this study. To avoid contamination, it is necessary to flush the syringe with pure helium. The measurements by the method are independent of the variations of sampling volumes. The determined headspace oxygen contents (R.S.D.<1%) in the containers of an investigational intravenous formulation using this method are consistent with the results obtained by an oxygen instrument at the manufacturing facility. The performance of the analytical approach was evaluated in the study of the container closure integrity at various storage conditions including upright and inverted orientations. The results suggest that there is no obvious oxygen penetration over 12 months. This method provides a convenient tool for measuring the levels of HS oxygen in vials of pharmaceutical formulations.

A practical derivatization LC/MS approach for determination of trace level alkyl sulfonates and dialkyl sulfates genotoxic impurities in drug substances.

Derivatization LC/MS methodology has been developed for the determination of a group of commonly encountered alkyl esters of sulfonates or sulfates in drug substances at low ppm levels. This general method uses trimethylamine as the derivatizing reagent for ethyl/propyl/isopropyl esters and triethylamine for methyl esters. The resulting quaternary ammonium derivatization products are highly polar (ionic) and can be retained by a hydrophilic interaction liquid chromatography (HILIC) column and readily separated from the main interfering active pharmaceutical ingredient (API) peak that is usually present at very high concentration. The method gives excellent sensitivity for all the alkyl esters at typical target analyte level of 1-2 ppm when the API samples were prepared at 5mg/mL. The recoveries at 1-2 ppm were generally above 85% for all the alkyl esters in the various APIs tested. The injection precisions of the lowest concentration standards were excellent with R.S.D.=0.4-4%. A linear range for concentrations from 0.2 to 20 ppm has been established with R(2)>or=0.99. This general method has been tested in a number of API matrices and used successfully for determination of alkyl sulfonates or dialkyl sulfates in support of API batch releases at GlaxoSmithKline.

On-line H/D exchange LC-MS strategy for structural elucidation of pharmaceutical impurities.

Structural elucidation of pharmaceutical impurities in drug substances and drug products is an important task in pharmaceutical analysis in various phases of drug development. Liquid chromatography-mass spectrometry (LC-MS) technologies play a key role in this task owing to their general attributes of superior selectivity, sensitivity and speed. Full scan and product ion scan analysis, providing molecular weight information and fragmentation data, respectively, offer rich structural information and allow proposal of candidate structures rather quickly. However, these proposed structures often lack certainty especially when dealing with structural isomers. On-line hydrogen/deuterium (H/D) exchange by LC-MS using D2O as the mobile phase component is a powerful tool for identifying active hydrogen atoms, thus constituting a simple strategy for distinguishing between isomeric structures which are sometimes difficult by product ion spectral data or accurate mass data. This review describes the typical experimental setup we use routinely in the laboratories for performing H/D exchange LC-MS experiments in conjunction with representative applications of the strategy in structural elucidation of pharmaceutical impurities.

Simultaneous quantitation of trace level hydrazine and acetohydrazide in pharmaceuticals by benzaldehyde derivatization with sample 'matrix matching' followed by liquid chromatography-mass spectrometry.

Hydrazine and acetohydrazide are potential genotoxins and therefore need to be controlled in APIs and drug products to ppm levels for patient safety in cases where there is a reasonable probability of either of them being present. They are structurally related and could both be formed in the same chemical process under certain circumstances. However, no previous studies have reported simultaneous trace level quantification of these two compounds. Herein, a chemical derivatization scheme using benzaldehyde followed by LC-MS analysis is presented to address that need. During method development, unexpectedly high recoveries were encountered and presented a major challenge. A systematic investigation was undertaken to understand the benzaldehyde derivatization reaction and determine the underlying causes of the unacceptable recovery. It was found that this was due to the presence of the counter ion of the API in the sample matrix. Employing a 'matrix matching' sample preparation strategy, which involved acidifying the derivatization reaction medium with benzoic acid, gave similar reaction rates for the chemical derivatization in the presence and absence of the API salt and accordingly more consistent recoveries. Resultantly, a robust method for simultaneous quantification of hydrazine and acetohydrazide (1-100ppm) was successfully developed and validated.

Strategies for characterization of drug metabolites using liquid chromatography-tandem mass spectrometry in conjunction with chemical derivatization and on-line H/D exchange approaches.

Strategies using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with techniques such as chemical derivatization and on-line hydrogen/deuterium (H/D) exchange for structural elucidation of drug metabolites in crude samples are reviewed. Useful mass spectrometric scan techniques discussed include product ion scan, constant neutral-loss scan, precursor ion scan, multistage MS(n), and accurate mass measurements. In biological systems, xenobiotics are transformed into metabolites, which usually involves introduction of one or more polar functional groups or removal or blockage of such structural moieties. Therefore, chemical derivatization strategies for determination of functional groups and on-line H/D exchange approaches for probing number of exchangeable hydrogens are powerful tools for structural elucidation of drug metabolites in drug metabolism studies. More importantly, these experiments can be carried out on crude samples in microscale, providing sufficient material for LC-MS/MS analysis. Therefore, labor intensive and technically challenging purification of low levels of drug metabolites from complex biological matrices can be avoided. It is the authors' conclusion that strategies such as chemical derivatization and on-line H/D exchange should be used more routinely in drug metabolism studies in order to facilitate metabolite identification.

LC-MS/MS and density functional theory study of copper(II) and nickel(II) chelating complexes of elesclomol (a novel anticancer agent).

Elesclomol (N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide)), which is a novel anticancer agent, can form chelating complexes with Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) in the gas phase during electrospray ionization (ESI) mass spectrometry. In the solution phase with acidic medium during chromatographic separation, however, only Cu(II) and Ni(II) to a lesser degree favor the formation of chelating complexes with elesclomol. The Cu(II)-chelating complex [Cu(II)+elesclomol-H]+· exhibits more complicated MS/MS fragmentation pathways than the Ni(II)-chelating complex [Ni(II)+elesclomol-H]+. One significant difference is the ready occurrence of the electron transfer upon collision-induced dissociation (CID) of [Cu(II)+elesclomol-H]+·. This leads to the reduction of Cu(II) to Cu(I). However, such phenomenon was not observed upon CID of [Ni(II)+elesclomol-H]+. On the basis of the density functional theory (DFT) calculations at the B3LYP/6-31+G(d)/LANL2DZ level, the Cu(II)- and Ni(II)-chelating complexes of elesclomol exist in the keto-form with tetra-coordinated trapezoid geometry in the gas phase but at different levels of distortion. As compared to the Ni(II)-elesclomol complex, the Cu(II)-elesclomol complex is more stable (by -55.25 kcal/mol). This relative stability of the chelating complexes of elesclomol is consistent with the Irving-Williams series of bindings to ligands.

Gas-phase derivatization via the Meerwein reaction for selective and sensitive LC-MS analysis of epoxides in active pharmaceutical ingredients.

A gas-phase derivatization strategy is reported by using the gas-phase Meerwein reaction for rapid and direct LC-MS analysis of epoxides, which are potential genotoxic impurities (GTIs) in active pharmaceutical ingredients (APIs). This class-selective ion/molecule reaction occurs between epoxides and the ethylnitrilium ion (CH(3)-C≡NH↔CH(3)-C=NH) that is generated by atmospheric pressure ionizations (when acetonitrile is used as the mobile phase). Density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Commonly used atmospheric pressure ionization techniques including ESI, APCI and APPI were evaluated for optimal formation of the Meerwein reaction products. APCI appears to be the method of choice since it offers better sensitivity and more robust detection under typical LC-MS instrumentation conditions. Quantitative analysis of epoxides can be achieved by either single ion monitoring (SIM) or multiple reaction monitoring (MRM) of the Meerwein reaction products. We demonstrate herein quantitative analysis of two potential GTIs of SB797313 and SB719133 in APIs. The validated methods afford excellent linearity (r(2)≥0.999), sensitivity (LOD≤1 ppm by w/w in 10 mg/mL APIs) and recovery (ranging from 92% to 102%), as well as accuracy (≤2.8% difference) and precision (≤2.2% RSD) based on injections of six prepared standards. This novel strategy is particularly useful when a target analyte is difficult to be directly analyzed by LC-MS (e.g. due to poor ionization) or unstable in the course of solution-phase derivatization.

Gas-phase meerwein reaction of epoxides with protonated acetonitrile generated by atmospheric pressure ionizations.

Ethylnitrilium ion can be generated by protonation of acetonitrile (when used as the LC-MS mobile phase) under the conditions of atmospheric pressure ionizations, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) as well as atmospheric pressure photoionization (APPI). Ethylnitrilium ion (CH(3)-C≡N+H and its canonical form CH(3)-C+=NH) is shown to efficiently undergo the gas-phase Meerwein reaction with epoxides. This reaction proceeds by the initial formation of an oxonium ion followed by three-to-five-membered ring expansion via an intramolecular nucleophilic attack to yield the Meerwein reaction products. The density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Collision-induced dissociation (CID) of the Meerwein reaction products yields the net oxygen-by-nitrogen replacement of epoxides with a characteristic mass shift of 1 Da, providing evidence for the cyclic nature of the gas-phase Meerwein reaction products. The gas-phase Meerwein reaction offers a novel and fast LC-MS approach for the direct analysis of epoxides that might be of genotoxic concern during drug development. Understanding and utilizing this unique gas-phase ion/molecule reaction, the sensitivity and selectivity for quantitation of epoxides can be enhanced.

Eliminating pharmaceutical impurities: Recent advances in detection techniques.

The elimination of organic impurities to produce highly pure drug substances is an important goal of process chemistry. For the detection of general impurities, hyphenated techniques (eg, liquid chromatography-mass spectrometry [LC-MS]) play a critical role in rapid structural identification (qualitative detection) and in understanding the mechanisms of formation of the impurities, enabling informed decisions to control and eliminate the impurities resulting from the chemical process where possible. Concern regarding genotoxic impurities (GTIs), which must typically be controlled at low parts-per-million limits, continues to increase, and significant advances have been achieved in recent years for the selective and sensitive quantitation (quantitative detection) of such impurities. Conventional detection techniques, such as ultraviolet (UV) detection, are often inadequate for the detection of potentially minute quantities of GTIs; therefore, various advanced MS-based detection strategies, either stand-alone or in conjunction with chemical approaches, are playing an increasing role in this field. The primary aim of this review is to highlight recent advances in qualitative and quantitative detection of impurities at trace levels, with a particular focus on GTIs.

Recent advances in trace analysis of pharmaceutical genotoxic impurities.

Genotoxic impurities (GTIs) in pharmaceuticals at trace levels are of increasing concerns to both pharmaceutical industries and regulatory agencies due to their potentials for human carcinogenesis. Determination of these impurities at ppm levels requires highly sensitive analytical methodologies, which poses tremendous challenges on analytical communities in pharmaceutical R&D. Practical guidance with respect to the analytical determination of diverse classes of GTIs is currently lacking in the literature. This article provides an industrial perspective with regard to the analysis of various structural classes of GTIs that are commonly encountered during chemical development. The recent literatures will be reviewed, and several practical approaches for enhancing analyte detectability developed in recent years will be highlighted. As such, this article is organized into the following main sections: (1) trace analysis toolbox including sample introduction, separation, and detection techniques, as well as several 'general' approaches for enhancing detectability; (2) method development: chemical structure and property-based approaches; (3) method validation considerations; and (4) testing and control strategies in process chemistry. The general approaches for enhancing detection sensitivity to be discussed include chemical derivatization, 'matrix deactivation', and 'coordination ion spray-mass spectrometry'. Leveraging the use of these general approaches in method development greatly facilitates the analysis of poorly detectable or unstable/reactive GTIs. It is the authors' intent to provide a contemporary perspective on method development and validation that can guide analytical scientists in the pharmaceutical industries.

Gas-phase Smiles rearrangement in structural analysis of a pseudo-oxidative impurity generated in the pharmaceutical synthesis of S-(thiobenzoyl)thioglycolic acid.

Several mass spectrometry (MS) techniques including accurate MS and MS/MS, as well as hydrogen/deuterium (H/D) exchange, were utilized to characterize a pseudo-oxidative reaction by-product (impurity I) in the pharmaceutical synthesis of S-(thiobenzoyl)thioglycolic acid. The negative ion MS/MS data provided complementary structural information to the positive ion MS/MS data. An understanding of the gas-phase Smiles rearrangement upon collision-induced dissociation (CID) in the negative ion MS/MS mode played an important role in structural elucidation of impurity I. The theoretical calculations by density functional theory (DFT) at the B3LYP/6-311G(d,p) level provided insights into the thermochemistry of the Smiles rearrangement reaction. This pseudo-oxidative impurity is proposed to be generated via the base-catalyzed hydrolysis in solution.

Enhancing the detection sensitivity of trace analysis of pharmaceutical genotoxic impurities by chemical derivatization and coordination ion spray-mass spectrometry.

Many pharmaceutical genotoxic impurities are neutral molecules. Trace level analysis of these neutral analytes is hampered by their poor ionization efficiency in mass spectrometry (MS). Two analytical approaches including chemical derivatization and coordination ion spray-MS were developed to enhance neutral analyte detection sensitivity. The chemical derivatization approach converts analytes into highly ionizable or permanently charged derivatives, which become readily detectable by MS. The coordination ion spray-MS method, on the other hand, improves ionization by forming neutral-ion adducts with metal ions such as Na(+), K(+), or NH(4)(+) which are introduced into the electrospray ionization source. Both approaches have been proven to be able to enhance the detection sensitivity of neutral pharmaceuticals dramatically. This article demonstrates the successful applications of the two approaches in the analysis of four pharmaceutical genotoxic impurities identified in a single drug development program, of which two are non-volatile alkyl chlorides and the other two are epoxides.

Matrix deactivation: A general approach to improve stability of unstable and reactive pharmaceutical genotoxic impurities for trace analysis.

Trace analysis of unstable and reactive pharmaceutical genotoxic impurities (GTIs) is a challenging task in pharmaceutical analysis. Many method issues such as insufficient sensitivity, poor precision, and unusual (too high/low) spiking recovery are often directly related to analytes' instability. We report herein a matrix deactivation approach that chemically stabilizes these analytes for analytical method development. In contrast to the conventional chemical derivatization approach where the analytes are transformed into stable detectable species, the matrix deactivation approach chemically deactivates the hypothetical reactive species in the sample matrix. The matrix deactivation approach was developed on the premise that the instability of certain analytes at trace level is caused by reactions between the analytes and low level reactive species in the sample matrix. Thus, quenching the reactivity of the reactive species would be a key to stabilizing the unstable and reactive analytes. For example, electrophilic alkylators could be destabilized by nucleophiles or bases through either nucleophilic substitution or elimination reactions. One way to mask those reactive species is via protonation by adding acids to the diluent. Alternatively, one can use nucleophile scavengers to deplete reactive unknown species in the sample matrix completely, in analogy to the use of antioxidants and metal chelators to prevent oxidation in the analysis of compounds liable to oxidation. This paper reports the application of the matrix deactivation to the analyses of unstable and reactive pharmaceutical genotoxic impurities. Some of the methods have been used to support development of manufacturing processes for drug substances and a recent regulatory filing.

Analytical control of process impurities in Pazopanib hydrochloride by impurity fate mapping.

Understanding the origin and fate of organic impurities within the manufacturing process along with a good control strategy is an integral part of the quality control of drug substance. Following the underlying principles of quality by design (QbD), a systematic approach to analytical control of process impurities by impurity fate mapping (IFM) has been developed and applied to the investigation and control of impurities in the manufacturing process of Pazopanib hydrochloride, an anticancer drug approved recently by the U.S. FDA. This approach requires an aggressive chemical and analytical search for potential impurities in the starting materials, intermediates and drug substance, and experimental studies to track their fate through the manufacturing process in order to understand the process capability for rejecting such impurities. Comprehensive IFM can provide elements of control strategies for impurities. This paper highlights the critical roles that analytical sciences play in the IFM process and impurity control. The application of various analytical techniques (HPLC, LC-MS, NMR, etc.) and development of sensitive and selective methods for impurity detection, identification, separation and quantification are highlighted with illustrative examples. As an essential part of the entire control strategy for Pazopanib hydrochloride, analytical control of impurities with 'meaningful' specifications and the 'right' analytical methods is addressed. In particular, IFM provides scientific justification that can allow for control of process impurities up-stream at the starting materials or intermediates whenever possible.

Analytical control of genotoxic impurities in the pazopanib hydrochloride manufacturing process.

Pharmaceutical regulatory agencies are increasingly concerned with trace-level genotoxic impurities in drug substances, requiring manufacturers to deliver innovative approaches for their analysis and control. The need to control most genotoxic impurities in the low ppm level relative to the active pharmaceutical ingredient (API), combined with the often reactive and labile nature of genotoxic impurities, poses significant analytical challenges. Therefore, sophisticated analytical methodologies are often developed to test and control genotoxic impurities in drug substances. From a quality-by-design perspective, product quality (genotoxic impurity levels in this case) should be built into the manufacturing process. This necessitates a practical analysis and control strategy derived on the premise of in-depth process understanding. General guidance on how to develop strategies for the analysis and control of genotoxic impurities is currently lacking in the pharmaceutical industry. In this work, we demonstrate practical examples for the analytical control of five genotoxic impurities in the manufacturing process of pazopanib hydrochloride, an anticancer drug currently in Phase III clinical development, which may serve as a model for the other products in development. Through detailed process understanding, we implemented an analysis and control strategy that enables the control of the five genotoxic impurities upstream in the manufacturing process at the starting materials or intermediates rather than at the final API. This allows the control limits to be set at percent levels rather than ppm levels, thereby simplifying the analytical testing and the analytical toolkits to be used in quality control laboratories.