310APPLICATIONS TO SMALL MOLECULES TABLE 7-5. Isotopic Abundances for Ions Containing Different Numbers of Sulfur, Chlorine, and Bromine Atoms Number Number Number Number Number Number Number Number of Br of Br of Br of Br of Br of Sulfur of Sulfur of Cl Atoms Atoms Atoms Atoms Atoms Atoms Atoms Atoms Mass (0) (1) (2) (3) (4) (1) (2) 0 A 100 51.5 34.3 17.6 A+2 97.3 100 100 68.5 A+4 48.7 97.3 100 A+6 31.6 64.9 A+8 15.8 1 A 100 77.3 44.2 26.5 14.4 A+2 32 100 100 85.8 60.8 A+4 24.1 69.3 100 100 A+6 13.4 48.5 79.4 A+8 7.8 30 A+10 4.1 2 A 100 62 38.7 20.8 12.1 A+2 64 100 100 74 54.8 A+4 10.2 45 88.8 100 100 A+6 6.2 31.2 63.1 93.3 A+8 3.8 18.3 46.4 A+10 2 11.5 A+12 1.1 A 100 100 A+1 0.8 1.6 A+2 4.4 8.8 A+3 0.07 A+4 0.19 molecular ions at m/z 521 ([M + H + ]), 523, and 525, of relative abundances 100 : 70 : 14. The isotopic intensity pattern is in good agreement with that of two chlorine atoms (Table 7-5), due to the ions C 27 H 31 O 6 35 Cl 2 ,C 27 H 31 O 6 35 Cl 37 Cl, and C 27 H 31 O 6 37 Cl 2 . The characteristic isotopic patterns resulting from combi- nations of the isotope peaks can be used to ascertain elemental composition of the corresponding ion. 7.6.1.3 Nitrogen Rule. If the nominal molecular weight of an analyte appears to be an even mass number, the compound contains an even number of nitrogen atoms (or no nitrogen atoms). On the other hand, if the nominal molecular weight of an analyte appears to be an odd mass number, the com- pound contains an odd number of nitrogen atoms. This so-called “Nitrogen Rule” is very useful for determining the nitrogen content of an unknown com- pound. In the case of Verapamil (Scheme 1), the molecular weight of the com- pound is 420 Da, indicating an even number of nitrogen atoms in the molecule. 7.6.1.4 Hydrogen/Deuterium (H/D) Exchange. The exchange of hydrogen for deuterium in organic molecules has been used in mass spectrometry for structural studies in both solution phase and gas phase. It also has wide appli- cations in the structural studies of proteins [24].This method measures the dif- ference in molecular weight of a compound before and after the deuterium exchange to determine the exchangeable hydrogens in a molecule for struc- tural elucidations. For example, one can determine the number of labile hydro- gen atoms from the mass shift X of [M + H + ] in H 2 O to [M + D + ] in D 2 O as X − 1. The exchangeable hydrogens are usually bound to N, O, or S atoms in func- tional groups such as OH, NH, NH 2 , or COOH. In the case of mometasone furoate (structure shown in Figure 7-1), there is only one exchangeable hydro- gen atom from the OH group in the molecule. There are two general approaches to set up H/D exchange LC/MS experi- ments. One is to use deuterium oxide as sheath liquid to introduce it to the ESI/APCI source as a post-column addition. The actual exchange takes place in the ion source. This method can enable some degree of H/D exchange without change of chromatographic separation (i.e., retention time). However, back-exchange can occur and contribute to incomplete exchange due to the presence of H 2 O in solvents or inadequate amounts of D 2 O. The other approach is to couple ESI/APCI-MS with deuterated mobile phases such as D 2 O or CH 3 OD (less commonly used) for on-line LC/MS analysis of mixtures. The change of chromatographic retention time due to the use of deuterated mobile phases should not be an issue because of the use of mass identifica- tions. This approach provides accurate measurements of exchangeable hydro- gens in a molecule to assist structural elucidation (examples will be presented in later section on identification of drug metabolites). 7.6.1.5 Accurate Mass Measurement. Mass accuracy measurement,typically reported as parts per million (ppm), is essential for elemental-composition MS INTERPRETATION 311 assignment. Traditional accurate mass measurements are carried out in a mag- netic sector mass spectrometer, requiring relatively large quantities of materi- als.The development of modern MS instrumentation (i.e., QTOF, FT-ICR, etc.) has allowed accurate mass determinations of small molecules as well as bio- molecules that are present at very low levels. An internal mass calibration is generally needed to achieve mass measure- ment accuracy of 5 to 10ppm with a Q-TOF MS analysis [62–64]. Internal cal- ibration is based on mixing one or several internal standards or calibrants of known molecular weight with the analyte and then using the known masses to calibrate the mass measurements of unknowns that coexist in the sample mixture. Currently, FT-ICR MS provides the highest mass resolving power and mass accuracy among all the mass spectrometric methods. Using external calibra- tion, FT-ICR MS is capable of achieving mass measurement accuracies of 1 ppm or better. Internal calibration can provide an order-of-magnitude greater mass accuracy than external calibration for the FT-MS analysis. One example is the high-resolution ESI-FT-ICR MS analysis of a mixture of Verapamil and peptide A. The following MS data were obtained for the mono-isotopic molecular ion and the isotopic molecular ions of peptide A using an external calibration (calculated mass; mass error in ppm): (829.5393, 0.1 ppm), (830.5423, 1.3 ppm), (831.5450, 1.0 ppm), and (832.5476, 1.2 ppm). Similar results were obtained for the Verapamil (data not shown). Accurate mass measurement is important in establishing compound iden- tity. For example, an unknown with a mass of m/z 122.0606 ([M + H + ]) can be C 7 H 8 NO; it cannot be C 5 H 13 NCl (m/z 122.0731), C 3 H 8 NO 4 (m/z 122.0447), C 4 H 12 NO 3 (m/z 122.0811), C 4 H 9 NOCl (m/z 122.0367), or C 8 H 12 N (m/z 122.0964).A mass measurement accuracy of 102ppm is required to distinguish these elemental compositions. Thus, an unequivocal elemental composition of a compound can be obtained with sufficient high mass measurement accuracy (i.e., <5 ppm) along with other information. 7.6.1.6 Double Bond Equivalency (DBE). To evaluate whether a formula, C x H y N z O n , is a reasonable elemental composition for a certain mass, one can calculate the DBE (numbers of rings and double bonds) of the formula. The calculation is based on the valences of elements involved, as shown in equa- tion (7-1). For example, pyridine (C 5 H 5 N) has a calculated DBE of 4 (= 5 − 5/2 + 1/2 + 1), indicating the ring and three double bonds in this molecule. For benzene (C 6 H 6 ), its calculated DBE is 4 (= 6 − 6/2 + 1), suggesting the ring and three double bonds in the molecule as well. A more general case, I y II n III z IV x , was suggested by McLafferty [65], where I = H, F, Cl, Br, I; II = O, S; III = N, P; and IV = C, Si. (7-1) DBE + Rd b xy z () =− + +221 312 APPLICATIONS TO SMALL MOLECULES The calculated value 12 found for mometasone furoate (C 27 H 30 O 6 Cl 2 ) repre- sents five rings and seven double bonds of this molecule [equation (7-2)]. Note that although an oxygen atom is present in the formula of mometasone furoate, it does not contribute to the calculation of DBE. The intensity of the molecular ion usually parallels the chemical stability of the molecule, and com- pounds with high numbers of rings and double bonds (DBE) often show higher molecular-ion abundance than those with low DBE. This is consistent with the abundant molecular ion peak observed in the CI-MS spectrum of mometasone furoate (Figure 7-2). (7-2) 7.6.2 Fragmentation Pattern Fragmentation pattern of a molecule, after ionization in the mass spectrome- ter, can be used to obtain structural information.The fragment ions from singly charged parent ions are the ions observed at low-mass range of a MS/MS spec- trum. The fragmentation chemistry and mechanisms are reasonably under- stood. The prominent abundant fragment ions are the most stable fragments that tend to be formed. The fragmentation processes also depend on the sta- bility of the transition states by which the ions are produced. Many of the fragment ions observed in the product-ion spectra are formed by collision-induced heterolytic cleavage. For example, the formation of a product ion, [M + H + − HX], can be explained by a 1,4 hydrogen rearrange- ment mechanism (Scheme 3). The product ion is formed by the neutral loss of HX, where X can be a heteroatom or a more electronegative group. A less common fragmentation mechanism by homolytic cleavage is also observed in tandem MS experiments. In this case, the driving force for the fragmentation of an ion is dependent on the stabilities of the resulting ion and the radical species relative to the energy of the initial ionic species. For instance, the for- mation of stable product ions, including acylium ion, benzylic ion, and allylic carbonium ion, are able to promote homolytic cleavage. DBE + Rdb () =− +=27 32 2 1 12 MS INTERPRETATION 313 Scheme 3. Charge-remote fragmentation is defined as a class of gas-phase decompo- sitions that occur physically remote from the charge site [66–70]. Although the mechanism of charge-remote fragmentation is still debatable (Scheme 4) [67], It is possible to derive structural information from the fragmentation pattern in a spectrum. The appearance of prominent peaks at certain mass numbers is empirically correlated with certain structural features. For example, the mass spectrum of an aromatic compound is usually dominated by a peak at m/z 91, corresponding to the tropylium ion. Structural information can also be obtained from the differences between the masses of two peaks in a spec- trum. For instance, a fragment ion occurring 20 mass numbers below the mol- ecular ion strongly suggests a loss of a HF moiety. Thus, a fluorine atom is likely to be present in the substance analyzed. In addition, the knowledge of the principles governing the mode of frag- mentation of ions makes it possible to confirm the structure assigned to a com- pound. This information is often used to determine the juxtaposition of structural fragments and thus to distinguish between isomeric substances. Rea- sonable guesses can be made as to which fragment ions to be expected in a mass spectrum if the isomeric substances are known. The molecular ions formed in ESI/APCI and their fragment ions are usually even electron ions, that is, [M + H + ]. In some cases, radical cations M +• can be formed in ESI-MS [24], depending on their structures and ESI conditions. In the case of Florfenicol (SCH 25298, Scheme 5, I), an antibacterial agent in Animal Health, its product ion mass spectrum only displays a very low abun- dant molecular ion peak at m/z 358 (data not shown). An abundant peak cor- responding to the loss of water is formed by heterolytic fragmentation as illustrated in Scheme 5 (II, m/z 340). The II likely decomposes to III (m/z 320) by a neutral loss of HF, and this fragmentation is promoted by the formation of a substituted tropylium ion (Scheme 5). The fragmentation pattern of Flor- fenicol is characterized by an unusual feature of a most abundant peak occur- ring at odd mass, that is, m/z 241. The IV, a radical cation, is likely formed by homolytic cleavage of the sulfur–carbon bond in III, with loss of the methane- sulfinic radical (Scheme 5). 314 APPLICATIONS TO SMALL MOLECULES Scheme 4. it has been proven useful in the structural determination of long-chain or poly- ring molecules, including fatty acids, phospholipids, glycolipids, triacylglycerols, steroids, peptides, ceramides, and so on. 7.7 PRACTICAL APPLICATIONS Mass spectrometry is a powerful and effective technology in drug discovery and development. This section will concentrate on the practical applications of LC/MS in problem solving, including high-throughput LC/MS analysis for combinatorial chemistry, structural characterization of impurities and decom- position products in bulk drug substances, and identification and quantifica- tion of drug metabolites. 7.7.1 High-Throughput LC/MS for Combinatorial Chemistry The application of combinatorial chemistry to the synthesis of potential ther- apeutic agents has received increasing attention such that combinatorial chem- istry is now an important tool in modern drug discovery [71]. Automated approaches capable of screening large libraries of small molecules have resulted in the successful application of LC/MS in combinatorial chemistry. Current trends for further integration of LC/MS techniques with new instrumental development have generated structure-based assays for drug discovery. To assess the quality of a combinatorial chemistry library, it is essential to determine the purity and quantity of the expected products. Commercial soft- ware, developed by instrument manufacturers, has made possible the unat- tended and rapid analysis of tens of thousands of individual components of a specific library. The application of LC/MS in high-throughput screening of combinatorial libraries has been reviewed by several authors [72–78]. An important application of LC/MS in relation to combinatorial synthesis is the introduction of open-access LC/MS instrumentation. The dedicated PRACTICAL APPLICATIONS 315 Scheme 5. open-access software packages are available from most instrument manufac- turers. Using an open-access LC/MS system, organic chemists can readily obtain the molecular weight information of reaction products and monitor the progress of chemical synthesis. The chemist just needs to log-in to the com- puter system, assign an identification code to the sample, and select the type of LC/MS experiments to be performed. The automatic sample analyses can be performed rapidly using short HPLC columns and fast gradients, with run times typically of 5 min or less. Multichannel ESI inlets have been developed to enhance sample through- put for large combinatorial library analysis. A multiplexed electrospray inter- face (MUX), which enables four- and eight-channel parallel introduction from four or eight LC systems into a multiplexed ESI source, was introduced in 1999. These systems, when coupled with TOF-MS, can provide not only the high-throughput capacity needed for library analysis, but also accurate mass determination of drug candidates and their synthetic by-products. Fang et al. [79] reported that they had coupled a nine-channel MUX-TOF MS system to conduct eight parallel high-throughput accurate mass LC/UV/TOF MS analy- sis. They used one of the nine channels to introduce reference standard as the lock mass to calibrate the instrument. The mass accuracies were found to be better than 5 and 10 ppm for 50% and 80% of the samples, respectively, from a single batch analysis of 960 samples [79]. The average root mean square (RMS) errors of the accurate mass measurements of the molecular weight of the combinatorial library samples were determined to be 10 ppm [79]. The use of FTMS has been growing rapidly in drug discovery and pharma- ceutical development [80]. One opportunity for utilizing the ultrahigh mass resolving power of FTMS is in characterization of complex mixtures, such as combinatorial libraries. Burton et al. [81] conducted multiple accurate mass measurements for 41 compounds, using three different approaches. The absolute mean errors were 5.2 (σ=7.4 ppm), 0.7 (σ=0.9 ppm), and 0.8 (σ= 1.0 ppm) ppm for the external, conventional internal, and dual ionization inter- nal calibrations, respectively. In another application, Nawrocki et al. [82] employed a 4.7-T external-source ESI FT-ICR mass spectrometer to analyze small-peptide libraries, demonstrating the feasibility of analyzing several com- binatorial libraries containing 100 to 10,000 small peptides. Furthermore, by comparing the FTMS data with computer-simulated combinatorial library mass spectra, the authors were able to monitor the diversity and degeneracy of the library syntheses. A well-established method for drug discovery is the utilization of a biolog- ical assay to screen a large library of small organic molecules for their ability to bind target biopolymers (i.e., protein) in a specific assay [83, 84]. In general, the MS-based technologies have the advantage that only small amounts of protein reagent are required. Recently, Annis et al. [85, 86] reported a high- throughput affinity selection–mass spectrometry assay to screen mass-encoded 2500-member combinatorial libraries. A schematic representation of the method is shown in Figure 7-12. Combination of the protein and a small 316 APPLICATIONS TO SMALL MOLECULES molecule library leads to the formation of a complex of the protein with any suitable library member. Size-exclusion chromatography (SEC) is then employed to rapidly separate the protein target, along with any small mole- cules bound to the target, from any unbound small molecules. The SEC band containing the complex is immediately transferred to a reversed-phase chro- matography column (60°C and pH 2). This step serves to denature the target, thereby dissociating the previously bound small molecules from the complex. The unbound small molecules are directly introduced into a high-resolution mass spectrometer for analysis. By using the affinity selection–mass spectro- metry method, Annis and co-workers discovered a bioactive ligand for the anti-infective target Escherichia coli dihydrofolate reductase (DHFR) [85, 86]. 7.7.2 Characterization of Impurities and Decomposition Products in Bulk Drug Substances One of the major applications of LC/MS in pharmaceutical analysis is the iden- tification of impurities and degradation products in pharmaceuticals. Often, impurities are synthetic by-products, starting materials, or degradation prod- ucts. Drug regulatory agencies require the purity of a pharmaceutical to be fully defined. Impurities that are present at a level of 0.1% or greater relative to the active ingredient need to be characterized to comply with the regula- tory requirements. This is important to ensure that the pharmacological and toxicological effects are truly those of the drug substances and not due to the impurities. 7.7.2.1 Characterization of Impurities by LC/MS. The impurities in phar- maceuticals are mainly formed during the synthetic process from starting materials, intermediates, and by-products. Generally, the impurities in staring materials and intermediates are not required to be characterized by the PRACTICAL APPLICATIONS 317 Figure 7-12. Diagram of the automated ligand identification system (ALIS) affinity selection–mass spectrometry method. (Reprinted from reference 85, with permission of Elsevier Science.) regulatory agencies. These impurities, however, are likely to contain compo- nents that affect the purity of the final manufactured pharmaceutical. By- products are often generated during synthesis and are one of the major sources of pharmaceutical impurities. The identification of the by-products often allows the Development Operations to refine the manufacturing process to minimize impurities and, thus, to maximize yield. Traditionally, the impurities are isolated and purified by off-line HPLC and then characterized by using FT-IR, NMR, MS, and X-ray crystallography, among others. The main limitation associated with this approach is that relatively large sample quantities are needed for analysis, and the process can be very labor-intense. In contrast, LC/MS and LC/MS/MS are highly sen- sitive techniques requiring typically less than 1 µg of material for analysis. In certain cases, if the impurities are found at very low levels in the drug substance, extraction procedures are used to concentrate them to detectable levels. The capabilities of separating a mixture containing highly varied concen- trations of analyte and structural characterization of impurities have led to the increased use of LC/MS. Nicolas and Scholz [87] illustrated the characteriza- tion of a number of DuP 941 (Scheme 6) impurities by LC/MS(/MS). The five unknown impurities were labeled A, D, E, F, and G along with the known impurities B and C in the total ion chromatogram of DuP 941 obtained from LC/MS analysis (Figure 7-13B). Because the UV-visible absorption and response factor of related compounds tend to be similar, while their MS ion- ization efficiencies can be significantly different, it is always useful to record the UV chromatogram (Figure 7-13A) as well as the mass spectra for the iden- tification of impurities. The protonated molecular ions ([M + H + ]) of the impu- rities A, D, E, F, G were found to be at m/z 392, 339, 324, 482, and 558, respectively. The molecular ions of the impurities were selected for tandem MS analysis in order to identify the unknown structures (Figure 7-13C). The product-ion spectra for the five unknown impurities were shown in Figure 7- 14. The impurity A was found to be a by-product (Figure 7-15A) during the synthetic process. Major fragment ions were observed at m/z 319, 305, and 261 318 APPLICATIONS TO SMALL MOLECULES Scheme 6. Structure of DuP 941. (Reprinted from reference 87, with permission of Elsevier Science.) (Figure 7-14A). The base peak at m/z 305 might arise from the neutral loss of a 2-vinylamino-ethanol. The product ions formed by the neutral loss of 73 (2-methyleneamino-ethanol) and 44 (Ethenol) Da from the precursor ion of m/z 392 were also consistent with the proposed structure (Figure 7-15A). The production spectra of impurities D and E were similar in two ways. Both have a base peak at m/z 88 which was produced when an N-(2-hydroxyethyl) aminoethyl group was cleaved from the molecule. Both produce a less intense product ion that was 61 Da less than the precursor ion (Figure 7-15; m/z 278 in D, and m/z 263 in E). This 61-Da loss was found to be the loss of a 2- aminoethanol neutral. Further studies suggested that impurities D and E are photo-decomposition products of DuP 941[87]. Based on the LC/MS/MS data, PRACTICAL APPLICATIONS 319 Figure 7-13. DuP 941 lot 3 chromatograms: (A) LC/UV; (B) LC/MS; (C) LC/MS/MS. A and B were acquired from a single injection using an HP 1090. UV and MS detec- tors were in series. C was acquired from a subsequent injection. (Reprinted from ref- erence 87, with permission of Elsevier Science.) [...]... derivatization, were well-established approaches for characterization of small 336 APPLICATIONS TO SMALL MOLECULES molecules, including drug metabolites [121, 140–142] Historically, the H/D exchange for structural elucidation has been used for a number of years [143], including determination of the affinity constants for protein–ligand interactions and for quantifying the conformational changes associated with ligand... (Scheme 11) [131] Other metabolites, which might be the intermediates for formation of the keto acid, were also observed in the radiochromatogram of rat plasma (spectrum not shown) [131] Scheme 11 Mechanism for the formation of metabolite J via oxidative deamination (Reprinted from reference 131, with permission of the American Society for Pharmacology and Experimental Therapeutics.) One of the goals of... are formed [108, 121, 127, 132] It is generally accepted that toxicities can stem from drug bioactivation in vivo, thus identifying the potential toxic metabolites is crucial in the lead optimization process [108, 121, 127, 132] The generation of acyl glucuronide and glutathione (GSH) metabolites are important biotransformation pathways for many drugs and xenobiotics [127, 132, 133] For example, the formation... drug candidates to forced degradation conditions such as acid, base, heat, oxidation, and exposure to light A successful identification of the degradation products can help formulation scientists to understand the degradation mechanism of drug candidate and improve the clinical formulation development There had been numerous reports in the literature that involved LC/MS and LC/MS/MS for characterization... 545–557 93 Y Wu, The use of liquid chromatography–mass spectrometry for the identification of drug degradation products in pharmaceutical formulations, Biomed Chromatogr 14 (2000), 384–396 94 P A Shipkova, L Heimark, P L Bartner, G Chen, and B N Pramanik, Highresolution LC/MS for analysis of minor components in complex mixtures: Negative ion ESI for identification of impurities and degradation products of a... candidate for clinical trials Drug degradation in formulations is highly complex and often unpredictable The degradation products usually arise from the ingredients used in dosage formulation and/or in the process of formulation where temperature, humidity, and light may all play a role The degradants can be generated from hydrolysis, oxidation, adduct formation, dimerization, rearrangement, and often... characterized as degradant 1 to 10, respectively (Figure 7-17) A mass measurement accuracy of 0.4 to 1.9 ppm was achieved for the exact mass measurements, using PEG sulfates as internal calibration substance Based on the high-resolution MS information, empirical formulae were obtained for all components (data not shown) The MS/MS spectra of source-produced fragment ions were used to characterize the unknown... products of SCH 27899, whereas degradant 6 was hydrolysis and oxidation products of SCH 27899 For example, degradant 2 was formed via ring opening of ortho-ester C, whereas degradant 5 was formed by loss of a terminal aromatic group (2) from SCH 27899 The unique ability of FTMS to provide an exact-mass measurement for each of the ions produced in multiple-stage tandem MS (MSn) assists greatly in the structure... determination of the number of exchangeable hydrogen atoms in a structure can provide additional information for structural characterization, such as the differentiation between N- or S-oxide formation and hydroxylation in drug metabolism studies Ohashi et al [142] demonstrated the on-line H/D exchange for characterization of metabolites of promethazine (MW 284 Da) (Scheme 12) M1 and M2, metabolites... Schug and H M McNair, Adduct formation in electrospray ionization Part 1: Common acidic pharmaceuticals, J Sep Sci 25 (2002), 760–766 60 K Schug and H M McNair, Adduct formation in electrospray ionization mass spectrometry II Benzoic acid derivatives, J Chromatogr A 985 (2003), 531–539 61 X.-G Zhao, J Ma, H Feng, J Wu, and Z.-M Gu, Loss of hydroxyl radical in CAD MS/MS for identification of oxidized . of a compound before and after the deuterium exchange to determine the exchangeable hydrogens in a molecule for struc- tural elucidations. For example, one. other information. 7.6.1.6 Double Bond Equivalency (DBE). To evaluate whether a formula, C x H y N z O n , is a reasonable elemental composition for a certain