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Recent advances in forensic drug analysis by DART-MS Mass spectrometry methods play a major role in many forensic applications. While gas chromatography–mass spectrometry methods are commonly used in crime laboratories and enforcement agencies, a variety of advanced techniques are now available that can improve upon standard methods and address emerging issues in forensic science. New mass spectrometry technologies include more versatile ionization sources, allowing the next generation of instrumentation to be more multipurpose and adaptable to the needs of the discipline. Direct analysis in real-time mass spectrometry is an ambient ionization method that allows direct testing of gas, liquid and solid samples without the need for any preparation or extraction, based on thermal desorption and ionization directly from the sample surface. This Review will provide an in-depth description of direct analysis in real-time time-of-flight mass spectrometry as applied to samples relevant to forensic science, with a focus on analysis and characterization related to forensic drug chemistry. MS techniques are now routinely employed in many aspects of forensic science, but advances in instrumentation are continually developed to provide improvements over current methods or even for new applications. Forensic science employs MS methods for any application that aims to identify a compound or components of a mixture based on their molecular mass, the molecular formula and the chemical structure. Accordingly, the broad applicability of MS analyses, along with the need to perform more rapid, efficient and sensitive measurements, has led to the development of more versatile and efficient MS formats. While these new MS formats have proved useful for an ever-growing list of forensic applications, they have also demonstrated their broad, general utility in chemistry, biochemistry, biology, medicine and pharmacology, among other disciplines, such that MS has become an essential tool for chemical identification, compositional profiling and structural analysis. Ideally, detection and analysis techniques should be robust, sensitive, informative, broad in terms of applicability and specific in terms of discrimination. While many different analytical techniques are available for forensic ana lysis, MS dominates certain applications due to its ability to realize most, if not all, of these attributes. Although traditional GC–MS serves as an essential instrument for forensic analyses, new MS technologies have been developed that provide some level of improvement across these various metrics. Improvements to the ionization process are arguably one of the most important
advancements, which include techniques such as ESI, MALDI, and ambient or atmospheric pressure ionization. Directly relevant to this Review is the application of ambient ionization methods, in particular the direct analysis in realtime (DART) ion source, although others will be mentioned briefly in relation to their forensic applications. The distinctive feature of ambient ionization methods as compared with conventional MS is the initial chemical manipulation of samples exterior to the instrumentation, as compounds are ionized in the open-air atmosphere in front of the mass spectrometer inlet, providing an instantaneous mass spectrum. It is important to note that ambient methods are employed without the use of chromatographic separation, such that all components of a mixture are subject to desorption and ionization, differentiated only by the resolution of the mass analyzer. A major benefit of ambient ionization is the broad utility in desorbing and ionizing samples in their native form, without the need for extraction, derivatization or other sample preprocessing prior to analysis. Improvements in mass analyzers are also extremely valuable, providing a relatively straightforward benefit, in that mass analyzers having higher resolution provide a greater identification potential. Accordingly, the DART ion source has been combined with a variety of different, more versatile, higher resolution mass analyzers, the most common being a time-of-flight (TOF) mass analyzer in the form of the DART-AccuTOF™ system
Ashton D Lesiak & Jason RE Shepard*
10.4155/BIO.14.31 © 2014 Future Science Ltd
Bioanalysis (2014) 6(6), 819–842
ISSN 1757-6180
Department of Chemistry, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA *Author for correspondence: Tel.: +1 518 442 4447 Fax: +1 518 442 3462 [email protected]
819
R eview |
Lesiak & Shepard
Key Terms Ambient ionization:
Mechanism to provide a positive or negative charge to an atom in the atmosphere outside of the mass spectrometer.
DART ionization: Ambient
ionization source that functions by Penning ionization, exposing the sample to a heated, metastable neutral gas stream that thermally desorbs and ionizes the sample in its native state before entering into the mass spectrometer
(Ionsense Inc., Saugus, MA, USA; JEOL USA, Inc., Peabody, MA, USA). Multiple reviews of DART-MS are available, as well as the seminal DART article by Cody et al., which describe in detail the chemistry and configurations of the process [1–4]. Herein, the focus will be on applications, in particular those related to forensic drug chemistry, including comparisons to standard forensic techniques, specifically GC–MS. As GC–MS is a mature, well-established technique, it is ubiquitous within the forensic community for the analysis of drugs. In contrast, ambient-ionization-MS methods, such as DART-MS, are less than a decade old and only beginning to find greater influence in forensic analyses, although the technique is currently being employed in multiple state and federal agencies. As the advantages that DART-MS provide are realized, including: rapid, instantaneous analyses; no requirement for sample preparation; soft ionization for analysis of easily fragmented drug molecules; and more informative, higher resolution data, the technique will continue to increase its usage within the discipline. DARTMS has been validated in forensic laboratories, accepted in the courtroom and has an extensive drug library in place that now positions the technique to influence the discipline for years to come. GC–MS In GC–MS, a sample must be in solution to be injected into the instrument, so is solubilized and/or extracted and possibly derivatized to increase the substances’ volatility. After chromatographic separation, ionization is performed through electron-impact (EI) ionization, where several tens of electron volts causes an electron to be ejected from the neutral species, creating a radical cation molecular ion with the mass of the compound and a single positive charge. The energy imparted to the molecular ion is enough to cause the molecule to fragment in a reproducible, chemically predictable manner. It is both the molecular ion and the reproducible fragmentation pattern that provide the chemical signature used for characterization. The EI process is generally suited for analysis of molecules that can be readily vaporized into the gas phase and will not overly fragment with the ionization energies associated with the process. For example, many classes of drugs, including amphetamines and cathinones, have been shown to extensively fragment under EI-GC-MS conditions, in some cases showing little to no parent
820
Bioanalysis (2014) 6(6)
peak, which is problematic for determination of a chemical formula. The chromatographic aspect not only allows separation of complex mixtures so that each component of the mixture is separately analyzed, but also produces a retention time that also can serve as a distinctive identifier. Together, both qualitative and quantitative analysis of the individual components can be determined by EI-GC–MS. DART-MS does not employ chromatography, but instead will ionize all components of the sample, with each component characterized by high mass accuracy measurements. The tradeoff between the information provided by retention time and rapid, high mass accuracy MS measurements is such that the two methods provide unique, but complementary data.However, despite its prevalent use, GC–MS does have some limitations, including the need for derivatization and the total ana lysis time, excessive fragmentation of fragile molecules, a limited mass range and the inability to vaporize larger, less volatile compounds, including some recent pharmaceuticals. Mass analyzers A variety of mass analyzers are employed in MS, including quadrupole, TOF, orbitrap and hybrid instruments, among others. Typical GC–MS mass analyzers employed in forensic laboratories include a linear quadrupole as they are compact, relatively low priced, efficient in terms of transmission of ions to the detector and serve the basic needs of the discipline, acting as a mass filter for chemical analysis. In comparison to the advanced methods described herein, quadrupole mass analyzers typically have unit resolution, which generally restricts its use to more straightforward forensic applications rather than more complicated needs, such as unknown determinations. However, more advanced mass analyzers are available with increased resolution, providing more informative content in MS analyses. For comparison related to this article, the AccuTOF mass analyzer commonly coupled to the DART ionization source has a significantly broader m/z range (up to 2000 Da), higher sensitivity (manufacturer specifications ~10 pg with S/N >10), increased resolution and more accurate mass measurements (>6,000 full width half maximum and 5 parts per million root mean square). The high mass accuracy is a key benefit, as it gives more detailed chemical information than a quadrupole, and in many cases, allows for positive identification without the need for chromatographic separations. future science group
Recent advances in forensic drug analysis by DART-MS LC–MS While GC–MS is commonly employed for forensic analysis, LC–MS methods have established themselves in more niche applications. LC–MS employs ESI to generate gas-phase ions, in a more delicate manner useful for analysis of large, nonvolatile molecules that are difficult to get into the gas phase, such as biological materials [5,6]. With ESI, a solution is dispersed into a fine aerosol, to which a high voltage is applied to form small, highly charged liquid droplets. Desolvation of the droplets eventually leads to charge transfer from the droplet to the analyte molecules, allowing for a gentler ionization process. Quasi-molecular ions are produced, either molecular ions with a hydrogen cation, denoted as [M+H]+, salt adducts such as [M+Na]+, or multicharged species. The lower relative energy associated with ESI allows for a more intense molecular ion than in EI and substantially less fragmentation, if any at all. Accordingly, ESI provides information about the molecular weight of the compound, but limited information about the fragmentation unless tandem MS instrumentation in employed. Tandem MS, also known as MS/MS, MSn, QqQ, or triple quads, has provided the ability to induce fragmentation, essentially allowing for the selection of specific ions, which are then subjected to secondary fragmentation. The most straightforward application of the technology selects a precursor ion, such as the [M+H]+, and employs fragmentation to form product ions in order to gain added information on the precursor ion. Termed collision-induced dissociation (CID), the extent of fragmentation is controllable and inherently useful in determining structures, reaction mechanisms and elemental compositions, among others. LC–MS is also beneficial for complex samples that require separation, such as urine analysis. Regardless, although LC–MS instruments are less commonly found in crime laboratories and enforcement agencies, the combination of chromatography and MS provide separate, but complementary information that also allows the instruments to be used for quantitation, critical for drug and toxicology applications. Ambient ionization The past decade has experienced a renaissance in the development of different types of ionization methods. An inherent simplicity and finesse is associated with sampling a substance in its native state, and such developments have enabled analysis of compounds that are problematic future science group
| R eview
by conventional means, including nonvolatile compounds and solid materials, as well as tissue samples or those within complex backgrounds. While techniques such as MALDI are well established, a renewed push in ambient ionization technologies has demonstrated important new qualities previously unheard of in MS. MALDI is a highly sensitive technique, particularly for large molecules >100,000 Da and while such instrumentation is also less likely to be found in crime laboratories, it has demonstrated its power in protein analysis, peptide sequencing, proteomics and mapping biological tissues [6–8]. More recently developed, MALDI-MS imaging [9–14], has been employed in a forensic capacity. Francese and co-workers applied MALDI imaging to latent print development in tandem with chemical analysis of fingerprint residues [15–18]. High-resolution imaging of fingerprint features was possible, along with chemical ana lysis related to residues associated with sexual assault [15,16]. Continued refinement of the technique has led to characterization of the age of fingerprints based on decomposition and degradation products [15,18]. While DART-MS has been employed to identify endogenous compounds in fingerprints [19–21], in contrast with MALDI, DART-TOF targets small molecules (
Recent advances in forensic drug analysis by DART-MS Mass spectrometry methods play a major role in many forensic applications. While gas chromatography–mass spectrometry methods are commonly used in crime laboratories and enforcement agencies, a variety of advanced techniques are now available that can improve upon standard methods and address emerging issues in forensic science. New mass spectrometry technologies include more versatile ionization sources, allowing the next generation of instrumentation to be more multipurpose and adaptable to the needs of the discipline. Direct analysis in real-time mass spectrometry is an ambient ionization method that allows direct testing of gas, liquid and solid samples without the need for any preparation or extraction, based on thermal desorption and ionization directly from the sample surface. This Review will provide an in-depth description of direct analysis in real-time time-of-flight mass spectrometry as applied to samples relevant to forensic science, with a focus on analysis and characterization related to forensic drug chemistry. MS techniques are now routinely employed in many aspects of forensic science, but advances in instrumentation are continually developed to provide improvements over current methods or even for new applications. Forensic science employs MS methods for any application that aims to identify a compound or components of a mixture based on their molecular mass, the molecular formula and the chemical structure. Accordingly, the broad applicability of MS analyses, along with the need to perform more rapid, efficient and sensitive measurements, has led to the development of more versatile and efficient MS formats. While these new MS formats have proved useful for an ever-growing list of forensic applications, they have also demonstrated their broad, general utility in chemistry, biochemistry, biology, medicine and pharmacology, among other disciplines, such that MS has become an essential tool for chemical identification, compositional profiling and structural analysis. Ideally, detection and analysis techniques should be robust, sensitive, informative, broad in terms of applicability and specific in terms of discrimination. While many different analytical techniques are available for forensic ana lysis, MS dominates certain applications due to its ability to realize most, if not all, of these attributes. Although traditional GC–MS serves as an essential instrument for forensic analyses, new MS technologies have been developed that provide some level of improvement across these various metrics. Improvements to the ionization process are arguably one of the most important
advancements, which include techniques such as ESI, MALDI, and ambient or atmospheric pressure ionization. Directly relevant to this Review is the application of ambient ionization methods, in particular the direct analysis in realtime (DART) ion source, although others will be mentioned briefly in relation to their forensic applications. The distinctive feature of ambient ionization methods as compared with conventional MS is the initial chemical manipulation of samples exterior to the instrumentation, as compounds are ionized in the open-air atmosphere in front of the mass spectrometer inlet, providing an instantaneous mass spectrum. It is important to note that ambient methods are employed without the use of chromatographic separation, such that all components of a mixture are subject to desorption and ionization, differentiated only by the resolution of the mass analyzer. A major benefit of ambient ionization is the broad utility in desorbing and ionizing samples in their native form, without the need for extraction, derivatization or other sample preprocessing prior to analysis. Improvements in mass analyzers are also extremely valuable, providing a relatively straightforward benefit, in that mass analyzers having higher resolution provide a greater identification potential. Accordingly, the DART ion source has been combined with a variety of different, more versatile, higher resolution mass analyzers, the most common being a time-of-flight (TOF) mass analyzer in the form of the DART-AccuTOF™ system
Ashton D Lesiak & Jason RE Shepard*
10.4155/BIO.14.31 © 2014 Future Science Ltd
Bioanalysis (2014) 6(6), 819–842
ISSN 1757-6180
Department of Chemistry, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA *Author for correspondence: Tel.: +1 518 442 4447 Fax: +1 518 442 3462 [email protected]
819
R eview |
Lesiak & Shepard
Key Terms Ambient ionization:
Mechanism to provide a positive or negative charge to an atom in the atmosphere outside of the mass spectrometer.
DART ionization: Ambient
ionization source that functions by Penning ionization, exposing the sample to a heated, metastable neutral gas stream that thermally desorbs and ionizes the sample in its native state before entering into the mass spectrometer
(Ionsense Inc., Saugus, MA, USA; JEOL USA, Inc., Peabody, MA, USA). Multiple reviews of DART-MS are available, as well as the seminal DART article by Cody et al., which describe in detail the chemistry and configurations of the process [1–4]. Herein, the focus will be on applications, in particular those related to forensic drug chemistry, including comparisons to standard forensic techniques, specifically GC–MS. As GC–MS is a mature, well-established technique, it is ubiquitous within the forensic community for the analysis of drugs. In contrast, ambient-ionization-MS methods, such as DART-MS, are less than a decade old and only beginning to find greater influence in forensic analyses, although the technique is currently being employed in multiple state and federal agencies. As the advantages that DART-MS provide are realized, including: rapid, instantaneous analyses; no requirement for sample preparation; soft ionization for analysis of easily fragmented drug molecules; and more informative, higher resolution data, the technique will continue to increase its usage within the discipline. DARTMS has been validated in forensic laboratories, accepted in the courtroom and has an extensive drug library in place that now positions the technique to influence the discipline for years to come. GC–MS In GC–MS, a sample must be in solution to be injected into the instrument, so is solubilized and/or extracted and possibly derivatized to increase the substances’ volatility. After chromatographic separation, ionization is performed through electron-impact (EI) ionization, where several tens of electron volts causes an electron to be ejected from the neutral species, creating a radical cation molecular ion with the mass of the compound and a single positive charge. The energy imparted to the molecular ion is enough to cause the molecule to fragment in a reproducible, chemically predictable manner. It is both the molecular ion and the reproducible fragmentation pattern that provide the chemical signature used for characterization. The EI process is generally suited for analysis of molecules that can be readily vaporized into the gas phase and will not overly fragment with the ionization energies associated with the process. For example, many classes of drugs, including amphetamines and cathinones, have been shown to extensively fragment under EI-GC-MS conditions, in some cases showing little to no parent
820
Bioanalysis (2014) 6(6)
peak, which is problematic for determination of a chemical formula. The chromatographic aspect not only allows separation of complex mixtures so that each component of the mixture is separately analyzed, but also produces a retention time that also can serve as a distinctive identifier. Together, both qualitative and quantitative analysis of the individual components can be determined by EI-GC–MS. DART-MS does not employ chromatography, but instead will ionize all components of the sample, with each component characterized by high mass accuracy measurements. The tradeoff between the information provided by retention time and rapid, high mass accuracy MS measurements is such that the two methods provide unique, but complementary data.However, despite its prevalent use, GC–MS does have some limitations, including the need for derivatization and the total ana lysis time, excessive fragmentation of fragile molecules, a limited mass range and the inability to vaporize larger, less volatile compounds, including some recent pharmaceuticals. Mass analyzers A variety of mass analyzers are employed in MS, including quadrupole, TOF, orbitrap and hybrid instruments, among others. Typical GC–MS mass analyzers employed in forensic laboratories include a linear quadrupole as they are compact, relatively low priced, efficient in terms of transmission of ions to the detector and serve the basic needs of the discipline, acting as a mass filter for chemical analysis. In comparison to the advanced methods described herein, quadrupole mass analyzers typically have unit resolution, which generally restricts its use to more straightforward forensic applications rather than more complicated needs, such as unknown determinations. However, more advanced mass analyzers are available with increased resolution, providing more informative content in MS analyses. For comparison related to this article, the AccuTOF mass analyzer commonly coupled to the DART ionization source has a significantly broader m/z range (up to 2000 Da), higher sensitivity (manufacturer specifications ~10 pg with S/N >10), increased resolution and more accurate mass measurements (>6,000 full width half maximum and 5 parts per million root mean square). The high mass accuracy is a key benefit, as it gives more detailed chemical information than a quadrupole, and in many cases, allows for positive identification without the need for chromatographic separations. future science group
Recent advances in forensic drug analysis by DART-MS LC–MS While GC–MS is commonly employed for forensic analysis, LC–MS methods have established themselves in more niche applications. LC–MS employs ESI to generate gas-phase ions, in a more delicate manner useful for analysis of large, nonvolatile molecules that are difficult to get into the gas phase, such as biological materials [5,6]. With ESI, a solution is dispersed into a fine aerosol, to which a high voltage is applied to form small, highly charged liquid droplets. Desolvation of the droplets eventually leads to charge transfer from the droplet to the analyte molecules, allowing for a gentler ionization process. Quasi-molecular ions are produced, either molecular ions with a hydrogen cation, denoted as [M+H]+, salt adducts such as [M+Na]+, or multicharged species. The lower relative energy associated with ESI allows for a more intense molecular ion than in EI and substantially less fragmentation, if any at all. Accordingly, ESI provides information about the molecular weight of the compound, but limited information about the fragmentation unless tandem MS instrumentation in employed. Tandem MS, also known as MS/MS, MSn, QqQ, or triple quads, has provided the ability to induce fragmentation, essentially allowing for the selection of specific ions, which are then subjected to secondary fragmentation. The most straightforward application of the technology selects a precursor ion, such as the [M+H]+, and employs fragmentation to form product ions in order to gain added information on the precursor ion. Termed collision-induced dissociation (CID), the extent of fragmentation is controllable and inherently useful in determining structures, reaction mechanisms and elemental compositions, among others. LC–MS is also beneficial for complex samples that require separation, such as urine analysis. Regardless, although LC–MS instruments are less commonly found in crime laboratories and enforcement agencies, the combination of chromatography and MS provide separate, but complementary information that also allows the instruments to be used for quantitation, critical for drug and toxicology applications. Ambient ionization The past decade has experienced a renaissance in the development of different types of ionization methods. An inherent simplicity and finesse is associated with sampling a substance in its native state, and such developments have enabled analysis of compounds that are problematic future science group
| R eview
by conventional means, including nonvolatile compounds and solid materials, as well as tissue samples or those within complex backgrounds. While techniques such as MALDI are well established, a renewed push in ambient ionization technologies has demonstrated important new qualities previously unheard of in MS. MALDI is a highly sensitive technique, particularly for large molecules >100,000 Da and while such instrumentation is also less likely to be found in crime laboratories, it has demonstrated its power in protein analysis, peptide sequencing, proteomics and mapping biological tissues [6–8]. More recently developed, MALDI-MS imaging [9–14], has been employed in a forensic capacity. Francese and co-workers applied MALDI imaging to latent print development in tandem with chemical analysis of fingerprint residues [15–18]. High-resolution imaging of fingerprint features was possible, along with chemical ana lysis related to residues associated with sexual assault [15,16]. Continued refinement of the technique has led to characterization of the age of fingerprints based on decomposition and degradation products [15,18]. While DART-MS has been employed to identify endogenous compounds in fingerprints [19–21], in contrast with MALDI, DART-TOF targets small molecules (
