The goal of many metabolomic studies is to identify the molecular structure of endogenous molecules that are differentially expressed among sampled or treatment groups. The identified compounds can then be used to gain an understanding of disease mechanisms. Unfortunately, despite recent advances in a variety of analytical techniques, small molecule (<1000 Da) identification remains difficult. Rarely can a chemical structure be determined from experimental "features" such as retention time, exact mass, and collision induced dissociation spectra. Thus, without knowing structure, biological significance remains obscure. In this study, we explore an identification method in which the measured exact mass of an unknown is used to query available chemical databases to compile a list of candidate compounds. Predictions are made for the candidates using models of experimental features that have been measured for the unknown. The predicted values are used to filter the candidate list by eliminating compounds with predicted values substantially different from the unknown The intent is to reduce the list of candidates to a reasonable number that can be obtained and measured for confirmation. To facilitate this exploration, we measured data and created models for two experimental features; MS Ecom(50) (the energy in electronvolts required to fragment 50% of a selected precursor ion) and HPLC retention index. Using a data set of 52 compounds, Ecom(50) models were developed based on both Molconn and CODESSA structural descriptors. These models gave r(2) values of 0.89 to 0.94 depending on the number of inputs, the modeling algorithm chosen, and whether neutral or protonated structures were used. The retention index model was developed with 400 compounds using a back-propagation artificial neural network and 33 Molconn structure descriptors. External validation gave v(2) = 0.87 and standard error of 38 retention index units. As a test of the validity of the filtering approach, the Ecom(50) and retention index models, along with exact mass and collision induced dissociation spectra matching, were used to identify 1,3-dicyclohexylurea in human plasma. This compound was not previously known to exist in human biofluids and its elemental formula was identical to 315 other candidate compounds downloaded from Pub Chem. These results suggest that the use of Ecom(50) and retention index predictive models can improve nontargeted metabolite structure identification using HPLC/MS derived structural features.

The objective of this chapter is to give guidance with degradation and impurity structure elucidation for pharmaceutical drug candidates. Stress testing knowledge is provided as guidance in developing and executing forced degradation experiments. Impurity and degradant structure elucidation is a collaborative effort involving the degradation chemist, analytical chemist, process chemist, and/or formulator, as well as the isolation chemist, mass spectrometry and NMR experts. Updates in this second addition include: (1) the use of quality by design (QbD) model as applied to stress testing practices, expanded oxidative experimental analysis, and expanded information on predictive programs. (2) The impact of supercritical fluid chromatography as a tool for impurity isolations, along with expanded information on process workflow. (3) Mass spectrometry: additional examples and a section on the use of accurate mass information in the role of structure elucidation. (4) Expansion on the use of NMR as applied to structure characterization and elucidation. (5) In‐depth case studies that illustrate collaborative efforts made between disciplines, as well as the impact that new technologies have on the structure elucidation process.

The carbon-hydrogen bond dissociation enthalpy (BDE) concept is evaluated as a potential computed indicator of stability of pharmaceutical drug substance candidates - specifically for oxidative stability of these molecules. Computational methods are discussed. Accuracy and validity of the methods are evaluated. BDEs are computed for several well-known molecules, for which stability and degradant identification information is known. Anecdotal correlations are noted between the lowest BDE energies of familiar molecules (sertraline, ezlopitant and related structures, ziprasidone, trovafloxacin, and varenicline), the sites of oxidative lability on these molecules and the identities of oxidative degradants. A low BDE may correlate in general with a reactive site on a molecule, not just an oxidatively susceptible one.

Abstract Mass spectrometry has become one of the most acceptable methods for determining organic structure, and is generally the first line of experimentation done for determining the structure of organic molecules. Mass spectrometry, coupled with liquid chromatography, acts as a foundation experimental technique in drug metabolism. This article discusses the importance of mass spectrometry in drug research.

This chapter discusses hyphenated chromatography–spectroscopy techniques and also the three areas of combined gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS), and liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR). The combination of separation techniques with spectroscopy has provided powerful tools. To fully utilize the power of combined chromatography–mass spectrometry techniques, it is necessary to understand the information and nuances provided by MS in its various forms. These novel systems have solved many of the problems associated with structure elucidation of low-level impurities. LC-NMR and its various derivatives are ideally suited for looking at simple regiochemical issues in relatively complex systems. In some cases, full structural elucidation of unknown compounds can be completed, although this will become more routine with some of the recent advances such as peak trapping in combination with cryogenic flow-probes. There are many examples of successful applications of LC–NMR and LC–NMR/MS to very complex systems. The principal advantage is that in most cases, NMR and MS data can be collected on an identical sample, thus eliminating the possibility of isolation-induced decomposition.

Electrospray mass spectral observation directly on a sample of a derivatized protein, such as porcine somatotropin (pST), affords a method for evaluating the degree of substitution of this protein. Derivatization of the lysine residues and the terminal amino residue here by formation of a Schiff base with a small aromatic aldehyde (in this case, o-vanillin) affords stabilization of the protein so that it may be used in a controlled release veterinary pharmaceutical formulation. This method permits direct observation of substitutions, optimization of manufacturing procedures for producing a commercial product, and permits quality evaluation of material.

A multidisciplinary team approach to identify pharmaceutical impurities is presented in this article. It includes a representative example of the methodology. The first step is to analyze the sample by LC-MS. If the structure of the unknown impurity cannot be conclusively determined by LC-MS, LC-NMR is employed. If the sample is unsuitable for LC-NMR, the impurity needs to be isolated for conventional NMR characterization. Although the technique of choice for isolation is preparative HPLC, enrichment is often necessary to improve preparative efficiency. One such technique is solid-phase extraction. For complete verification, synthesis may be necessary to compare spectroscopic characteristics to those observed in the original sample. Although not widely practiced, an effective means of getting valuable structural information is to conduct a degradation study on the purified impurity itself. This systematic strategy was successfully applied to the identification of an impurity in the active pharmaceutical ingredient 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulphonylurea. Identification required the use of all of the previously mentioned techniques. The instability of the impurity under acidic chromatographic conditions presented an additional challenge to purification and identification. However, we turned this acidic instability to an advantage, conducting a degradation study of the impurity, which provided extensive and useful information about its structure. The following discussion describes how the information gained from each analytical technique was brought together in a complementary fashion to elucidate a final structure.

A multifaceted approach was successfully used to identify three of four unknown degradants in degraded low dose tablets. Accelerated solvent extraction (ASE) was found to be an invaluable tool in this multifaceted approach. ASE was capable of extracting four individual degradants of an active pharmaceutical component from 10 tablets into 15 mL of solvent with approximately 100% recovery for each degradant. Using ASE instead of manual extraction led to the extraction and isolation of the degradants in 1 day instead of 7 days. One of the degradants was extracted by ASE, isolated by semi-prep HPLC, and identified by LC-MS and NMR spectroscopy. The structures of two of the remaining three degradants were confirmed by synthesis of authentic samples, while the fourth degradant is yet to be identified.

Several chromanol drug substance candidates exhibit unconventional behavior under the soft ionization conditions of fast atom bombardment and electrospray ionization in the mass spectrometer. Under FAB, these compounds produce radical cation molecular ions rather than protonated molecular ions. Similarly, under acidic mobile phase conditions in an electrospray LC-MS experiment, they produce radial cation molecular ions. Upon changing to a neutral, ammonium acetate-containing mobile phase, the molecular ion species is an ammonium adduct. The two example compounds behave conventionally under negative ion detection, both being free carboxylic acids and forming abundant [M - H](-). Examination of structural analogs indicates that the chromanol, methoxyl and chroman compounds behave this way. Oxidation to a chromanone causes formation of a conventional [M + H](+). Oxidation to a chromene produces even more complex behavior-namely a mixture of [M - H](+), M(+') and [M + H](+). We propose that, for these compounds, elimination of a valence electron to form the radical cation is the more energetically favored reaction than attachment of a proton.

The chapter presents case studies related to solving impurity/degradation problems. The chapter presents guidance for isolating and identifying process-related impurities and degradation products from pharmaceutical drug candidates using actual case studies. Impurity and degradant structure elucidation is a collaborative effort involving the analytical chemist, process chemist and/or formulator, as well as the degradation, mass spectrometry, and nuclear magnetic resonance (NMR) experts. The process described in this chapter uses a designed approach for the impurity and/or degradant identification, which focuses on efficiency so that the success of data collection is maximized and project time lines are met. There are a number of activities other than collecting experimental data, even though the experiments are central to the process. Some of these key activities include collecting project background information prior to pursuing experimental work, asking the right questions, and meeting with project analysts and structure elucidation experts. The activities associated with the overall process are captured in the process flowchart presented. One of the most important aspects of the project that determines approach is where the pharmaceutical drug candidate is in the drug development time line. NMR spectroscopy is used as a complementary technique to liquid chromatography/mass spectrometry (LC/MS). The chapter presents several case studies related to scaled-up oxidative degradation and isolation using solid-phase extraction and preparative high-performance liquid chromatography (HPLC), scaled-up oxidative degradation, preparative HPLC, and characterization by LC/MS and NMR, scaled-up light degradation and LC/MS and NMR characterization, and others.

Fast atom bombardment mass spectral examination of molecules containing a 1,2-benzisothiazole ring, using a thiol reducing agent matrix, promotes reductive ring opening of the benzisothiazole ring, giving an [M+H]+ two daltons higher than expected. Measurements using a non-reducing matrix produce the expected [M+H]+. This is a general phenomenon, observed with a number of molecules containing the benzisothiazole ring. The ring-opened structure has been confirmed by chemical synthesis and observed in metabolic studies.

Photostability challenge of ziprasidone in solution shows that the benzisothiazole moiety undergoes isomerization to the corresponding benzthiazole. A model compound, 3-piperazinyl-1,2-benzisothiazole, also undergoes this photoisomerization. Identification of the products has been confirmed by synthesis of the proposed molecules.

The chapter presents the comprehensive profile for sertraline (L)-lactate. The chapter discusses the nomenclature, formulae, elemental analysis, and appearance of sertraline (L)-lactate. Sertraline is a selective serotonin reuptake inhibitor (SSRI) used to treat depression, obsessive-compulsive disorder, panic disorder, and posttraumatic stress disorder. Sertraline (L)-lactate can be prepared from sertraline hydrochloride, sertraline mandelate, or sertraline free base. The chapter describes the physical properties of sertraline (L)-lactate. The equilibrium solubility of sertraline (L)-lactate in water and other solvents was determined by preparing saturated solutions. These solutions were subsequently filtered, diluted, and assayed by reversed phase high-performance liquid chromatography (HPLC). The particle morphology of sertraline (L)-lactate was studied using optical microscopy and scanning electron microscopy. Sertraline (L)-lactate was characterized by electron impact (EI) and fast atom bombardment (FAB) mass spectrometry. The identity of a sample of sertraline (L)-lactate is established through the use of infrared spectroscopy, reversed-phase HPLC, and thin-layer chromatography (TLC). Potentiometric titration is used to assay samples of sertraline (L)-lactate. Reversed-phase HPLC with UV detection at 210 nm is used to determine the amount of lactic acid in a sample of the drug substance. Thin-layer chromatography is used to establish the identity of sertraline (L)-lactate, and to estimate the levels of potential process-related impurities and degradation products in the drug substance. Degradation studies were performed on sertraline (L)-lactate under strongly acidic, strongly alkaline, and oxidative conditions to identify the potential degradation products of the drug substance.

The objective of this two-part review article is to provide guidance for isolating and identifying process related impurities and degradation products from pharmaceutical drug candidates. The identification of degradation products can provide an understanding of impurity formation and define degradation mechanisms. If the identification process is performed at an early stage of drug development, there is adequate time for improvements in the drug substance process and drug product formulation to prevent these impurities and degradants long before the filing stage. Impurity and degradant structure elucidation is a collaborative effort involving the analytical chemist, process chemist and/or formulator as well as the degradation, mass spectrometry and NMR experts. The process described in this two-part article uses a designed approach for the impurity and/or degradant identification, which focuses on efficiency so that the success of data collection is maximized. There are a number of activities other than collecting experimental data, even though the experiments are central to the process. Part I of this article describes a process for isolating unknown impurities and degradants, while Part II will illustrate the role of mass spectrometry and NMR in the identification process.

A combination of high-performance liquid chromatography (HPLC)–mass spectrometry and 1H and 13C NMR spectroscopy was utilized to characterize the photodecay products of droloxifene, a potent new estrogen receptor agonist. Structurally similar to tamoxifen, droloxifene demonstrates a complex and unique decay scheme, including the formation of two naphthalene derivatives which were unexpected decay products and previously unreported for this class of compound. Elucidation of the decay products was assisted by the use of computational chemistry, namely by correlating simulated UV spectra and aqueous solvation free energies with actual UV spectra and HPLC retention data. In addition to describing the photodecay scheme of droloxifene, the present work demonstrates the utility of computational chemistry in providing support for the identification of unknown compounds. Copyright © 1999 John Wiley & Sons, Ltd.