7 LC/MS: THEORY, INSTRUMENTATION, AND APPLICATIONS TO SMALL MOLECULES Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik 7.1 INTRODUCTION The discovery of a new drug is a challenging task that includes (a) identifica- tion of a biochemical target for certain diseases and (b) screening of a large number of compounds from libraries of compounds arising from synthetic chemistry, combinatorial chemistry, and natural product isolation for lead gen- eration. The lead compound is then optimized based on biological activity, selectivity, pharmacokinetic property, and metabolism. This process produces a large volume of samples requiring rapid and accurate analysis, with the speed of analysis contributing directly to the drug discovery cycle time. As one of primer analytical techniques, mass spectrometry (MS) developed from nineteenth-century physics, starting with the pioneering work of J. J. Thomson on the electrical discharges in evacuated tubes. In 1913, Thomson wrote “I feel sure that there are many problems in Chemistry which could be tackled with far greater ease by this than any other method. The method is surprisingly sensitive—more so than that of Spectrum Analysis—requires infinitesimal amount of material, and does not require this to be specially puri- fied. . . .” Indeed, MS offers speed, high sensitivity and isotopic specificity. This technique separates mixtures of ions on the basis of mass-to-charge ratios, 281 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. providing the molecular weight of a compound and its structural information from fragment ions. It is widely used for identification and quantification of known/unknown organic compounds. Rapid development of MS in recent decades has further expanded its role in the structural characterization of small molecules in the drug discovery process [1]. In combination with chromatographic separation techniques, principally in the form of high-performance liquid chromatography (HPLC) / MS (LC/MS), mass spectrometry has become the principal method of mixture analysis in pharmaceutical research and development [2]. Early discovery research often involves library compounds analysis using high-throughput LC/MS methods. Identification and quantification of drug metabolites is essential in drug metabolism and pharmacokinetic studies. Structural characterization of impu- rities and decomposition products in bulk drug substances is an integral part of pharmaceutical product development. LC/MS combines the high-resolution separation capability of HPLC with MS detection and characterization ability, playing important roles in all these aspects of drug discovery process. 7.2 IONIZATION METHODS AND LC/MS INTERFACES Different physical principles can be used to separate and measure ions (charged particles) with different mass-to-charge ratios under high vacuum conditions, and this has resulted in a variety of mass spectrometers [3]. In prin- ciple, the functioning of all mass spectrometers in generating mass spectra involves four steps: (1) introduction of the sample; (2) ionization of the sample molecule to convert the neutral molecules to ions in the gas phase (ionization method); (3) sorting of the resulting gas-phase ions by mass-to-charge ratios (mass analyzer); and (4) detection of separated ions. Critical components of a mass spectrometer include ion source and mass analyzer. Depending on desired applications and sample types, different mass spectrometers can be uti- lized to perform specific analytical tasks. 7.2.1 Ionization Methods An ideal ionization source for a mass spectrometer should provide a high ion- ization efficiency and a high stability of ions for subsequent mass analysis by mass analyzers. In addition, control of internal energy deposited on ions during ionization should be achievable in the ionization source to control the degree of fragmentation. It is also desirable to couple ionization source with chro- matographic separation techniques, especially with HPLC. Various ionization methods have been developed over the years, including electron impact (EI), chemical ionization (CI), desorption ionization (DI), matrix-assisted laser des- orption/ionization (MALDI), desorption electrospray ionization (DESI), elec- trospray ionization (ESI), and atmospheric pressure chemical ionization (APCI). Note that ESI and APCI are part of LC/MS interfaces that will be 282 APPLICATIONS TO SMALL MOLECULES discussed in separate sections. Table 7-1 summarizes some characteristics of different ionization methods. EI and CI are two early developed ionization methods. They are extremely useful for ionizing volatile compounds. In EI process, molecules are ionized by collisions with energetic electrons (typically 70 eV) produced from a heated filament. It produces highly reproducibly mass spectra with extensive frag- mentation of molecular ions. Thus, library searching with existing EI mass spectra is possible for unknown identifications. The nature of fragmentation in EI often leads to lower abundances or absence of molecular ions. On the other hand, CI is a soft ionization method that generates mainly molecular ions by ion/molecule reactions of regent ions with analyte molecules [4]. IONIZATION METHODS AND LC/MS INTERFACES 283 TABLE 7-1. Summary of Ionization Methods Ionization Method Ionization Agent Strengths Limitations EI Electrons (∼70 eV) Extensive Limited to fragmentation, volatile/ reproducible nonpolar spectra, searchable molecules large reference compound EI libraries CI Gaseous ions Abundant molecular Limited to ions with nonpolar and controllable moderately fragmentation polar molecules, limited fragmentation APCI Corona discharge/ Operative at Limited to low gaseous ions atmospheric to moderately pressure, easy polar interface to molecules HPLC, abundant molecular ions DI Energetic particles Abundant molecular Difficult to (atoms, ions), ions from high- interface to photons mass compounds HPLC ESI Electrical/thermal/ Operative at Poor results for pneumatic atmospheric nonpolar energy pressure, easy molecules, interface to HPLC, limited multiple-charged fragmentation ions for large biomolecules Commonly used reagent gases include methane, ammonia, and isobutane. The internal energy deposition or fragmentation of ions determines the appear- ance of mass spectra. It can be controlled by selecting appropriate reagent gases. In terms of proton transfer reactions in CI-MS, the relative proton affin- ity between the analyte and the reagent gas determines whether the analyte will be ionized. The analyte with a higher proton affinity than that of the reagent gas will be ionized, while the analyte with a lower proton affinity than that of the reagent gas will not be ionized. Furthermore, the difference in proton affinities of reagent gas and analyte is largely responsible for the extent of fragmentation if ionization of the analyte occurs. EI and CI methods are complementary to each other, providing molecular weight and structural information. As an illustration, Figure 7-1 shows an EI- MS spectrum of mometasone furoate, an anti-inflammatory steroid drug. A very low abundant molecular ion at m/z 520 is visible. However, the base peak in the spectrum is the fragment ion at m/z 295 corresponding to the loss of furoate ring, HCl, and a moiety of [COCH 2 Cl]. Other fragment ions in the spectrum yield structurally characteristic fragmentations for this molecule. In contrast, a CI-MS spectrum of the same compound exhibits the protonated molecular ion as the most abundant peak at m/z 521 along with some frag- ment ions (Figure 7-2). The appearance of these two spectra clearly demon- strates the utility of EI-MS and CI-MS methods. An inherent limitation for EI and CI methods is the requirement that the sample analyzed must be volatile. Both methods do not produce MS data for 284 APPLICATIONS TO SMALL MOLECULES Figure 7-1. EI-MS spectrum of mometasone furoate . polar compounds. One solution to this limitation is to employ DI methods to ionize nonvolatile samples with high molecular weights [5]. In the DI process, energetic particles or photons impact onto samples on a surface and result in the liberation of intact molecular ions via selvedge region without direct trans- fer of the energy to the sample molecules. The particle bombardment includes keV atoms (e.g., Ar, fast atom bombardment [6]), keV ions (e.g., Cs + , liquid secondary ion MS [7]), and MeV ions (e.g., plasma desorption [8]). Both fast atom bombardment (FAB) and liquid secondary ion MS (LSI) utilize large excess matrix for absorption, excitation, and relaxation of energetic particles, producing mainly molecular ions of interest. They are well-suited for studies of natural products and small polar compounds with molecular weights of a few thousand daltons (Da). Another important DI method is MALDI, which employs a UV or IR absorbing matrix in large excess with samples (5000 :1) to absorb the photon energy from laser irradiation [9]. This method generates mostly singly charged molecular ions with molecular weight as high as 500 kDa. MALDI has a high ionization efficiency for large biomolecules with supersensitivity in the range of low-femtomole level. It has become one of most widely used ionization methods in biological mass spectrometry. The latest addition to DI methods is DESI [10]. It directs an aqueous spray from an electrospray apparatus onto the sample on a surface. In the process, the fast nebulizing gas jet transports the charged droplets and impacts the IONIZATION METHODS AND LC/MS INTERFACES 285 Figure 7-2. CI-MS spectrum of mometasone furoate using NH 3 as CI reagent gas. surface in the absence of matrix, carrying away analyte molecules. This approach has been successfully applied to analysis of small molecules and proteins. The unique characteristic of DESI is that it operates under ambient conditions.All other DI methods as described above normally require vacuum operation conditions, and sample manipulation during experiments is not feasible. DESI lifts the restriction on the vacuum constraints and can be very flexible in carrying out novel experiments. Potential applications include forensic analysis, explosive detection, and biological imaging experiments in tissues. 7.2.2 Historical View of Interfaces A critical component of the LC/MS system is the interface that connects an HPLC system to a mass spectrometer. The basic requirements for a success- ful interface include maintaining chromatographic performance (minimum additional peak broadening), high transfer efficiency from LC to MS, and no degradation in mass spectrometric performance. Historically, a main challenge in LC/MS interfaces was that high liquid flows from HPLC make it very dif- ficult to maintain the high vacuum required for the function of a mass spec- trometer. A number of different LC/MS interfaces have been developed over the years to address this issue and overcome the difficulty [3]. 7.2.2.1 Direct Liquid Introduction. One of the first attempted experiments to introduce liquids into a mass spectrometer was to minimize the amount of liquid into an MS, removing solvent by the vacuum system and ionizing the analyte in the gas phase. The pioneering work carried out by Tal’roze et al. [11] described the simplest direct liquid introduction interface. In their experiments, solvent was introduced into the mass spectrometer through a capillary at a flow rate below 1 µL/min. The ionization of analytes occurred by EI. The low flow rate used in the experiments was a limitation and would not give good sensitivities for analytes. In 1970s, McLafferty’s group employed a direct liquid introduction (DLI) interface to directly introduce a small fraction (<1%) of the liquid from HPLC into the ion chamber of a CI mass spectrometer [12]. The solvent acted as the ionizing reagent. The maintenance of the vacuum was assisted by using large pump systems and differential pumping. Micro-and nanobore chromatography (<1-mm-i.d. column) were suitable for DLI. A detection sensitivity of picogram level was achieved for full-scan analysis. 7.2.2.2 Moving Belt System. Initially developed by McFadden et al. [13], the moving belt system was based on the physical method of evaporation of the mobile phase through heat and vacuum that leave analytes as a thin coating on a continuously cycling polyimide belt. The analytes were trans- ported from atmospheric pressure region to the vacuum of the ion source through differentially pumped vacuum locks. Ionization methods used 286 APPLICATIONS TO SMALL MOLECULES included EI and CI for volatile analytes. The system has excellent enrichments and efficiencies, although it is often limited to the analysis of compounds which could have been analyzed by gas chromatography (GC)/MS. 7.2.2.3 Thermospray. The thermospray interface was introduced and devel- oped by Blakley and Vestal [14]. In their approach, a liquid flow from HPLC was directed through a resistively heated capillary connecting to the MS ion source. The heat and vacuum would evaporate the solvent from a supersonic beam of mobile phase produced in the spray, creating charged small micro- droplets. These small liquid droplets were further vaporized in the heated ion source. Ions present in the ion source were then transferred to the mass analyzer, and residual vapors were pumped away. Ionization process in thermospray involves ion desolvation/evaporation from charged liquid droplets and gas-phase ion/molecule reactions. Both pos- itively and negatively charged ions are formed in the process. Volatile buffers such as ammonium acetate are often used as part of HPLC effluent. These buffer ions act as CI reagent ions to form either protonated or deprotonated ions. The gas-phase proton affinity (for positive ion) or acidity (for negative ion) of the analyte relative to the buffers will determine whether the analyte will be ionized. If the proton affinity of the analyte is lower than that of the reagent ion, the analyte will not be ionized. This CI-like aspect of the ioniza- tion process results in thermospray mass spectra containing mostly molecular ions. When buffer is not used in thermospray experiments, an external ioniza- tion method is often applied, including EI filament and discharge ionization. These supplemental modes produce solvent-related CI mass spectra. A main advantage of thermospray is that it can handle commonly used HPLC eluents at higher flow rates (up to 2 mL/min) and generate good results for polar, nonvolatile, and thermolabile compounds. However, the sensitivity of the method is highly compound-dependent and not particularly attractive to high-molecular-weight compounds. 7.2.2.4 Continuous-Flow FAB. Continuous-flow FAB is a modified form of FAB method [15, 16]. In this modified method, the HPLC effluent with added FAB matrix (usually 5% aqueous glycerol) is continuously transported through a fused-silica capillary to the tip of a FAB probe residing inside of the ion source. The HPLC liquid with matrix deposited on the tip of the FAB probe is subjected to atom bombardment for ionization of analytes.The matrix addition can be done either pre-column or post-column, although post-column addition is preferred. The acceptable liquid flow rate in continuous-flow FAB is less than 10 µL/min. Flow splitting or the use of capillary chromatography is often required in the experiments. A major advantage in this method is the reduced chemical noise since much less matrix is used in continuous-flow FAB than in standard FAB experiments. This has led to improved detection limits to subpicomole range. Significantly, this interface allows the LC/MS analysis of biomolecules that are traditionally analyzed by DI methods. IONIZATION METHODS AND LC/MS INTERFACES 287 7.2.3 Common Interfaces The early developed LC/MS interfaces as described above have played impor- tant roles in the evolution of LC/MS interfaces. However, their applicability, sensitivity, and robustness are very limited. The overwhelming popularity of LC/MS today is largely due to the development of atmospheric pressure ion- ization (API) interfaces, including ESI and APCI. 7.2.3.1 Electrospray. The first description of ESI was made by Zeleny in 1917 [17]. He described how a high electrical potential applied to a capillary caused the solvent to break into small droplets. In late 1960s and early 1970s, Dole and co-workers attempted to generate gas phase ions from macromole- cules in solution using an atmospheric pressure electrostatic sprayer by ion mobility spectrometry [18, 19]. In the late 1970s, Thomson and Iribarne suc- cessfully demonstrated the production of macro-ions from electrically charged droplets using MS [20, 21]. The very first applications of ESI were reported independently by Yamashita and Fenn [22] and Aleksandrov et al. [23] in the mid-1980s. Now ESI has become one of the most successful ionization methods / interfaces used in mass spectrometry [24]. The basic ESI apparatus consists of a spray needle at high electrical poten- tial (4–5 kV), a thermal/pneumatic desolvation chamber, and the vacuum inter- face (Figure 7-3). The ESI process is electrophoretic in nature. It may involve the generation of charged micro-droplets under a high electrical field and the subsequent evaporation of droplets using either a drying gas (N 2 ) or thermal desolvation. The first step of ion formation is the droplet formation at the needle tip when the high electrical field causes ions of the same polarity to 288 APPLICATIONS TO SMALL MOLECULES Figure 7-3. ESI source schematic diagram. The ion formation is illustrated in the pos- itive ion mode. form “Taylor cone” on the solution surface and emit charged droplets. The second step of ion formation is solvent removal, and its process is somewhat debatable. One theory is Dole/Fenn’s coulombic explosion. When the initially formed droplets become smaller droplets due to evaporation of solvents, the surface charge density increases and the coulombic forces exceed the surface tension (Rayleigh stability limit), with the droplets breaking into smaller droplets. Further evaporation process with Rayleigh droplet fragmentation produces analyte ions [22, 25]. One of the most important features in ESI is the formation of multiply charged ions for proteins/peptides [26]. Since a mass spectrometer measures mass-to-charge ratios of a compound, the multiply charged ions will appear in the mass spectrum at m/z values that are fractions of the mass (MW) of the ion. This allows the detection of high molecular weights of proteins/peptides using a standard quadrupole mass analyzer (3000-Da mass range). In addition, the detection of multiply charged ions provides precise measurements of molecular weights of proteins/peptides via the deconvolution method. A mass accuracy of better than 0.01% can be achieved for proteins with masses up to 100 kDa [27]. Another important characteristic of ESI is the softness of the ionization. It is a very mild process and can generate mainly molecular ions with little fragmentation. For small molecules, the singly charged molecular ions usually dominate the mass spectrum. The third characteristic of ESI is the simplicity of the source design and its operation at atmospheric pressure, allowing ESI to be readily coupled to HPLC. It is important to note that a low flow rate (~200 µL/min) of the sample solution is required in order to main- tain a stable spray in ESI. Thus, flow splitters are often utilized in ESI-LC/MS applications. This does not reduce the concentration sensitivity of ESI since ESI responses are directly related to the concentration of the analyte enter- ing the ion source. However, the mass sensitivity can be substantially increased with a lower flow rate if the same concentration sensitivity is maintained (c = m/v). This has led to the wide use of nano-spray (~nL/min) LC/MS for analysis of proteins and peptides, achieving femtomole sensitivity [24]. 7.2.3.2 Atmospheric Pressure Chemical Ionization. APCI is closely related to ESI. It was developed by Horning et al. [28] in the early 1970s. Figure 7-4 illustrates a typical APCI source. The sample solution is introduced into a nozzle spray device similar to that used in ESI, but without the high electrical potential applied to the nozzle. The nebulizing gas (usually N 2 ) is often added to assist the desolvation/ionization process. Although a heater at a tempera- ture of 400–500°C is used to vaporize solvents, minimal degradation of the sample occurs.A corona discharge needle at a high voltage (3–5 kV) is respon- sible for producing a discharge current and inducing solvent ionizations. The generated solvent reagent ions react with analyte molecules via gas-phase ion/molecule reactions and produce analyte ions. Clearly, the ion formation process is separated from solvent evaporation process in APCI (in contrast to ESI), allowing the use of solvents unfavorable for ion formation. For example, IONIZATION METHODS AND LC/MS INTERFACES 289 low-polarity solvents generally used in normal-phase chromatography can be evaporated for APCI ionization. Unlike ESI, APCI does not form multiple- charge ions for high mass compounds, and its response is more directly related to the absolute amount of analytes. APCI achieves optimal performance at high flow rates (1–2 mL/min), making it ideal as the LC/MS interface for con- necting to conventional HPLC without flow splitters. The other features of APCI include the appearance of CI-like mass spectra and suitability for analy- sis of volatile or semivolatile compounds. 7.2.4 Special Interfaces There are other LC/MS interfaces that are less commonly used than ESI and APCI, but are often employed by researchers for analysis of nonpolar or neutral compounds, including particle beam and atmospheric pressure pho- toionization (APPI). 7.2.4.1 Particle Beam. Particle beam interface, also known as MAGIC (monodisperse aerosol generation interface for chromatography), was devel- oped by Browner and co-workers in the early 1980s [29]. It uses a momentum separator to eliminate volatile solvents and to transport analyte in the form of micro-aggregates particles to the EI/CI source of a mass spectrometer.Typ- ically, the LC effluent is forced into a small nebulizer using a helium gas flow to form aerosol droplets. These uniform droplets go through a desolvation chamber and evaporate into particles that are further separated from solvent vapors by a multistage momentum separator. The flow rate of liquid samples in particle beam interface ranges from 0.1 mL/min to 0.5 mL/min. The inter- face is limited to relatively volatile compounds. One of advantages in using a particle beam interface is the database searching capability with the EI-MS library for structural identifications. 7.2.4.2 Atmospheric Pressure Photoionization. Photoionization has been used in mass spectrometry to ionize a variety of compounds [3]. The forma- 290 APPLICATIONS TO SMALL MOLECULES Figure 7-4. APCI source schematic diagram. [...]... works best for most polar molecules, while APCI performs better with low to moderately polar molecules.Alternating between ESI and APCI modes is suitable for detection of unknown compounds so that the best ionization method can be selected for signal optimization As in the case of polarity mode selection, HPLC conditions also contribute to the performance of ESI/APCI (see later sections for discussions)... higher solvent flow rate is used For example, a typical flow rate of 400 L/hr is maintained for a solvent flow rate at 50 µL/min The desolvation temperature varies from 100°C for less than 10 µL/min of solvent flow to about 400°C for over 50 µL/min of solvent flow For LC/APCI-MS, the APCI probe temperature is normally set at 400°C, which can be higher for involatile samples or lower for volatile samples It is... necessary to perform both positive ion and negative ion LC/MS experiments to obtain structural information It is common to perform alternating positive ion and negative ion LC/MS experiments for initial assessment of unknown compounds Modern instruments have the capability to switch rapidly between positive ion and negative ion modes Its limitation is the reduced analysis time on a specific mode for a compound,... Thus TOF is best coupled with ion sources producing ions in pulses Naturally, MALDI is ideal for the combination with TOF In fact, MALDI-TOF is one of the most widely used systems for analysis of large biomolecules For a continuous ion source, the ions can be stored for a short period of time and pulsed out for analysis In the case of ESI, orthogonal injection provides efficient injection of ions from... ions It can provide valuable information for interpretation of fragmentation patterns Modern MS/MS experiments often rely on the scanning of the second mass analyzer to record the product ions This type of scan mode is a product ion scan that is widely used to obtain structural information of the parent ion (and its neutral form of the parent molecule) The other two forms of MS/MS scan modes include... separation and the performance of a mass spectrometer Depending on the column used in separation, different flow rates are applied For example, optimum flow rate of 1.0 mL/min may be employed for a 4.6-mm-i.d column A 2.1-mm-i.d column has optimum flow rate of 0.2 mL/min For LC/ESI-MS, ESI-MS is compatible with LC capillary columns and conventional analytical columns The optimum performance of ESI may require... ion formation by ESI-MS operated in the negative ion mode They performed extensive ESI-MS experiments designed to determine the underlying principles in the formation of proton-bound dimer ions and sodium-bridged dimer ions from the halide-substituted benzoic acid derivatives [60] and six acidic pharmaceuticals [59] The relative gas-phase basicity and proton affinity had significant effects on the formation... are generally formed by the analyte– adduct interaction in the solution system that is preserved as a result of the soft ionization of the ESI/APCI process These ions are also formed by analyte-adduct gas-phase collisions in the spray chamber [49] The exact mechanisms of how the analyte adducts are formed in ESI/APCI still remain unresolved at this point More often than not, the adduct ion formation is... ion mode can be used to form a protonated or cationized molecule For acidic compounds, a deprotonated molecule is formed in the negative ion mode The negative ion MS offers selectivity and sensitivity since only limited 300 APPLICATIONS TO SMALL MOLECULES Figure 7-9 Schematic diagram of a typical LC/MS system compounds can be ionized in this mode If a compound exists in the form of salts (i.e., quaternary... [60] For those compounds lacking highly acidic sites and less prone to undergo deprotonation, chloride ion attachment in the presence of chlorinated solvents such as chloroform can promote the formation of [M + Cl−] in the negative ion mode [24] This is very useful when compounds do not respond well in the negative ion ESI mode Another complicating factor in molecular ion determination is the formation . of mass-to-charge ratios, 281 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons,. in the form of high-performance liquid chromatography (HPLC) / MS (LC/MS), mass spectrometry has become the principal method of mixture analysis in pharmaceutical