Forensic Chemistry and Ambient Mass Spectrometry: A Perfect Couple

Jan 14, 2016 - Other countries, however, might only accept techniques in court when they are fully incorporated in forensic laboratories worldwide. ...
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Forensic Chemistry and Ambient Mass Spectrometry: A Perfect Couple Destined for a Happy Marriage? Ambient mass spectrometry has been demonstrated, via various proof-of-concept studies, to offer a powerful, rather universal, simple, fast, nondestructive, and robust tool in forensic chemistry, producing reliable evidence at the molecular level. Its nearly nondestructive nature also preserves the sample for further inquiries. This feature article demonstrates the applicability of ambient mass spectrometry in forensic chemistry and explains the challenges that need to be overcome for this technique to make the ultimate step from the academic world into forensic institutes worldwide. We anticipate that the many beneficial and matching figures of merit will bring forensic chemistry and ambient mass spectrometry to a long-term relationship, which is likely to get strongly consolidated over the years. Deleon N. Correa,†,‡ Jandyson M. Santos,† Livia S. Eberlin,§ Marcos N. Eberlin,*,† and Sebastiaan F. Teunissen*,† †

ThoMSon Mass Spectrometry Laboratory, University of CampinasUNICAMP, Campinas, São Paulo 13083-970, Brazil Technical-Scientific Police SuperintendenceIC-SPTC-SP, São Paulo, São Paulo 05507-06, Brazil § Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712 United States occupies a central position by focusing on the chemical characterization of crime evidence. Because of the numerous implications, the forensic chemist will focus on collecting highly informative and credible data, which will be used in writing a criminal report that he might need to defend in court. Such a report will likely contain crucial information from which an authority or judge will base their final “guilty−not guilty” decision. Evidence in forensic chemistry commonly consists of remains found at a crime scene or collected in a forgery or counterfeiting investigation. The chemical composition of the evidence should be disclosed in as much detail as possible since it might provide revealing clues related to the crime or contravention. Such samples are therefore highly diverse and might include hair, blood, semen, saliva, bones, nails, vitreous humor, questioned documents, banknotes, drugs, gunshot residue, explosives, fire residues, paints, inks, liquors, condoms, latent fingerprints, fuels, foods, agriculturals, pharmaceuticals, and many more. If the forensic chemist wants to provide prompt and confident answers, he or she must therefore have access to simple, robust, minimally destructive yet reliable analytical techniques. Rapid results are commonly requested in the early stages of forensic Nicolas Schwab investigations or when samples rapidly degrade or change over time. One could seek to apply specific techniques to particular samples and chemicals, but the ultimate goal of a forensic INTRODUCTION TO FORENSIC SCIENCES scientist would certainly be to have available as few and as Forensic science is dedicated to apply all the benefits of scientific universal as possible techniques, able to rapidly handle with knowledge to the legal system.1 Investigations in forensic science simplicity and robustness the large universe of highly diverse are therefore crucial to the establishment of an organized society samples faced daily in forensic investigations. since these investigations pursue the goal of solving crimes or detecting or demotivating illegal practices. Forensic science includes many subareas; and within them, forensic chemistry Published: January 14, 2016 ‡

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© 2016 American Chemical Society

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Figure 1. Schematics of the “top 5” ambient desorption/ionization MS techniques tested in forensic chemistry cases: (A) DESI, (B) PSI, (C) DART, (D) EASI, and (E) ELDI. Adapted with permission from ref 12. Copyright 2010 Springer.

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MS analysis and its “everything-everyone-everywhere” use. MS has become able to deal with highly complex chemical mixtures such as those found in biological samples and fluids common in forensic cases. The landmark breakthrough toward simplicity and generality was no doubt made by electrospray ionization (ESI), which was introduced in ∼1989 by Nobel prize laureate J. B. Fenn.7 ESI was so important for MS that it now divided it in the pre- and post-ESI eras8 establishing a bridge9 for the ions at atmospheric pressure to be moved from solution to the gas phase and vice versa. ESI brought about the ability to provide mass spectrometers with intact molecules that had been ionized in solution of polar solvents such as methanol and water from where they are directly “fished” to the gas phase. The gaseous ions produced via ESI are typically intact protonated [M + H]+ or deprotonated [M − H]− molecules (M) or ion/molecule adducts, such as [M + Na]+, [M + K]+, or [M + Cl]−. Another exceptional feature of ESI is that it forms ions with very low internal energies; hence in-source fragmentations are commonly suppressed. This gentleness results in a direct 1:1 molecule-to-

AMBIENT MASS SPECTROMETRY In the past, analyses applying solely mass spectrometry (MS) were rare in forensic investigations. The use of MS-only approaches was mostly limited by the restriction of the MS in handling only relatively small and volatile, ideally pure molecules. Modern MS has, however, eliminated this drawback and is currently able to handle most types of molecules in most types of matrixes and environments, even when the target analytes are present in highly complex chemical mixtures. MS is nowadays applicable nearly by everyone, to every sample and wherever it is needed. These universal characteristics of modern MS have become possible mostly due to the development of new and revolutionary ionization techniques, which are able to handle molecules with a large diversity of size and polarities: from atoms and small molecules such as benzene and pharmaceutical drugs such as acetyl salicylic acid and salts such as ionic liquids to large (bio)molecules as well as tissues,2 proteins,3 embryos,4 viruses,5 and intact bacteria.6 The emergence of these general and mild ionization techniques have also contributed to the simplicity of 2516

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solvent have been deposited.28 Variants of PSI have been developed and used in a variety of forensic applications. These variations were created by replacing the paper by similar surfaces or holding matrixes such as wooden toothpicks,29 swabs,30 or the sample itself such as in leaf spray.31 DART, together with DESI, forms the pair of the pioneering ambient MS techniques.13 DART is a solvent-free technique performed normally on a heated helium gas stream which combines several ionization mechanisms. Ionization is “promoted” by glow discharge that produces electronically excited He* atoms in a helium gas flow. Next, Penning ionization of atmospheric gases or water present on humid surfaces is promoted by He*, forming a series of ions such as H3O+ and O2−. Subsequently, via atmospheric pressure chemical ionization (APCI), the analyte ions are produced.32 EASI22 and its variants V-EASI,33 TI-EASI,34 and S-EASI35 are also spray-based techniques but operate via a distinct mechanism. No electrical potentials or heating are applied; hence, in EASI gaseous ions are not produced via the electrostatical removal of counterions but by a voltage-free “ion splitting” process, which produces a bipolar stream of charged droplets. Such droplets are formed due to a natural statistical unbalanced distribution of charge, of the anions and cations already present in solution, on the very minute droplets produced by sonic spray with low charge capacity. As these minute droplets evaporate, the excess of either cations or anions is ejected to the gas phase to form a bipolar stream of gaseous ions. Voltage-free EASI is therefore free from thermal degradation and electrochemical or discharge interferences. Being based on sonic spray ionization (SSI), which is the softest ionization technique, EASI also greatly favors the detection of intact molecular species.12 Spray desorption may, however, be quite inefficient. Ambient techniques based on laser desorption can therefore become highly desirable, particularly when more energetic desorption is required or for analytes more deeply located on the matrix. ELDI 24 and LAESI25,36 which use laser desorption in combination with ESI have therefore also been used in forensic chemistry. Other popular ambient MS techniques used in forensic applications have been secondary (extractive) electrospray ionization (SESI or EESI)37,38 and surface desorption atmospheric pressure chemical ionization (DAPCI).39 After the first review that covered the emerging uses of ambient MS in forensic science,15 numerous proof-of-principle studies have been reported in a diversity of cases. This Feature article will highlight some of the most illustrative cases we have selected in the hope to demonstrate that ambient MS and forensic science are indeed destined to enroll into a perfect, happy, and long lasting marriage. We also hope that this article will further motivate the search for new applications as well as proper validation and acceptance in court in order to establish ambient MS as a central, rather universal tool in the quest for simple, fast, and unquestionable forensic chemistry data.

ion relationship; hence, all the more polar constituents of chemical mixtures can normally and properly be accessed and instantaneously separated by ultrafast MS separation. Note that MS separation is fast and effective since it is based not on retention time but on mass differences of the resolved ions. Most isobars can therefore be readily separated whereas isomers will still overlap. Such set of highly suitable characteristics made “open air” ESI-MS very attractive in forensic chemistry.10 However, a second revolution, that of ambient MS,11−13 moved MS much further into simplicity.14 In ambient MS, sample preparation-free protocols were developed via desorption and ionization of analytes directly from their natural matrixes at ambient conditions. These ambient MS techniques, based on ESI and a variety of other related ionization strategies, are therefore pushing MS forward into an ideal and increasingly employed technique in forensic chemistry investigations. Ambient MS offers the direct analysis of the undisturbed precious forensic samples without the need of tedious chemical manipulation such as extraction or derivatization procedures. The risks of contamination or chemical alterations, which might bring doubts to the investigation in regard to sample integrity, are thereby lowered. The intact sample can now be handled via ambient MS with greater simplicity, speed and confidence, at open air, even by non-MS experts. Detailed chemical composition at the molecular level is obtained with nearly full sample preservation, allowing additional analysis at the same sample. When performed in miniaturized, portable, and user-friendly mass spectrometers, ambient MS becomes even more attractive as a rather universal tool in forensic science for immediate results that could be obtained even directly at a crime scene. The variety of ambient desorption/ionization MS techniques currently available to the forensic chemist15,16 in combination with miniaturized mass spectrometers offers, for instance, the opportunity to “swab” surfaces when collecting trace chemicals at crime scenes. This procedure can be used to screen for trace explosives, drugs, or other chemical markers, to rapidly have a direct on-site overview of the crime protocol. Such possibilities therefore facilitate the preselection of evidence and the speed of MS investigation.17,18 If a top five set of ambient MS techniques, applied to forensic science, were to be emphasized, such a set would vary according to the selection criteria but, based on papers demonstrating potential applications in forensic science, such a set would certainly include the pioneering and commercially available techniques of (a) desorption electrospray ionization (DESI)19 and its sister technique (b) paper spray ionization (PSI)20 and (c) direct analysis in real time (DART)21 as well as (d) easy ambient sonic spray ionization (EASI).22,23 Although several other techniques would closely compete, (e) electrosprayassisted laser desorption ionization (ELDI)24 certainly deserves a position in this set (Figure 1). DESI is based on ESI and is therefore a spray-based technique that combines all the beneficial features of electrospray with the additional benefits of direct surface desorption of target analytes.25 Typically in DESI, the spray of either positively or negatively charged droplets produced by ESI is used to bombard a surface, causing desorption (pick up) of the analyte and its ionization. When the analyte-containing droplets shrink due to desolvation, the analyte ions are ejected to the gas phase.26 DESI has been the major ambient MS technique used for MS imaging, which is also becoming a powerful tool in forensic chemistry.27 Paper spray ionization (PSI) is, similarly to DESI, based on ESI but in PSI a high voltage electrospray is produced on a porous triangular piece of paper in which both the sample and a proper

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EXPLOSIVES AND GUNSHOT RESIDUES Already in the very early days of ambient MS, detection of explosive traces directly from surfaces was immediately recognized as a very promising and suitable application in forensic chemistry. It was therefore one of the very first topics of attention in this area.40,41 Detection of explosive traces has always been a challenging task due to the very minute intact sample quantities left after an explosion, which requires sensitive and selective techniques. Although ion mobility has been, and still is, the method of choice for rapid, presumptive identification 2517

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Figure 2. (A) Picture from an explosion simulation, (B) ANFO residues remaining from a real crime scene. EASI-MS performed in negative ion mode from the surface of (C) an undisturbed Brazilian banknote; (D) banknote after ANFO explosion; and (E) an extract of ANFO residue remaining from a real crime scene. Adapted with permission from ref 45. Copyright 2015 Elsevier.

of explosives detection,42 the benefits of ambient MS for a much wider range of applications are evident and numerous. It offers great speed of analysis coupled to the ultrahigh sensitivity and selectivity for which MS is unmatched. A lot of representative explosives such as triacetone triperoxide (TATP), trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), octahydro-1,3,5,7tetranitro-1,3,5,7-tetrazocine (HMX), and trinitrohexahydro-

1,3,5-triazine (RDX) have been screened by ambient MS.43 The combination of ambient techniques with miniature and robust mass spectrometers has the potential to offer an efficient tool for rapid, selective, and sensitive on-site screening of explosives such as in airports and public buildings.44 EASI-MS has been shown to identify ammonium nitrate fuel oil (ANFO) either directly from the surface of banknotes or from samples of 2518

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Figure 3. (A) Schematics of the DEFFI-MS imaging setup. Spectra in the negative ion mode showing detection of (B) oleic acid and (C) HMX. DEFFIMS images of an HMX-laden artificial fingerprint deposited onto forensic lift tape for (D) oleic acid and (E) HMX as well as (F) a colocalization map of both oleic acid (blue) and HMX (red). Adapted with permission from ref 47. Copyright 2014 Royal Society of Chemistry.

distance which has shown the ability of trace level analysis for PETN and TNT.48 Identification of chemical warfare agents directly from glass49 and pepper spray products and tear gas has been demonstrated by DART-MS.50 Analysis of gunshot residues (GSR) is one of the most classical cases in forensic chemistry investigations. There are however some pitfalls in GSR analysis related to sampling and the high probability of false negatives. This inefficiency can be attributed to low deposition of particles after shooting and the ease with which gunshot residues might be cleaned from, e.g., hands.51 Ambient MS has therefore the potential to supply information in real time for the presence of GSR on surfaces such as skin, stubs, and clothes. Because of the ability of ambient MS to perform direct analysis, there are no special sampling processes required.

ANFO obtained in real forensic cases (Figure 2), even after the explosion, via the detection of the marker ion [(NO3)3Mg]−.45 The ability to link latent fingerprints to explosive traces may also be crucial in the combat of terrorism. As the pioneering work on the analysis of latent fingerprints by ambient MS has shown,46 ambient MS offers also a powerful technique for the chemical analysis of latent fingerprints. For instance, as Figure 3 illustrates, desorption electro-flow focusing ionization (DEFFI) MS imaging of fingerprints was able to detect endogenous components such as oleic acid but also traces of the HMX explosive in such fingerprints with proper spatial discrimination.47 Distant detection may also rely on techniques able to interrogate vapors transmitted over long distances such as demonstrated by the DESI-MS analysis of ambient vapors at 3 m 2519

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well as to detect invisible traces of colored antitheft markers.63 In a similar application, the use of DART-MS in the screening of antitheft device, 1-methylaminoanthraquinone (MAAQ), has been demonstrated.50

This ability favors the analysis of very low trace amounts since no GSR is lost during sampling. Although the composition of such residues is mostly inorganic, proper characterization of organic compounds such as methyl centralite and ethyl centralite has been demonstrated by DESI-MS.52,53 Diphenylamine is a common propellant stabilizer, and its degradation product has been detected by DESI.54

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COUNTERFEIT AND ILLEGAL DRUGS Ambient MS, due to its speed and direct application to the intact sample, has been shown to be applicable to the screening of drugs in general, particularly for counterfeiting screening and to characterize drugs of abuse. Direct analyses of seized cocaine by DESI-MS64 and DART-MS65 has demonstrated that, indeed, ambient MS offers a fast and robust methodology in this field and that mobile devices could be constructed to aid field investigations. For more complex formulations, simple TLC runs can be applied for fast separation followed by direct on-spot characterization. Drug analysis after TLC separation has been performed, for instance, via EASI-MS66 and DART-MS67 with the characterization of illicit drugs and their specific formulations. The elegant combination of TLC, the simplest chromatographic technique, widely used in forensic chemistry, with direct MS analysis68 facilitates the use by forensic scientists and reduces the risks of false positives or negatives that may occur when only the conventional TLC protocol is applied.69 DART-MS70 has also been applied to screen for carbinols61 whereas EASI-MS was used to screen for designer drugs such as meta-chlorophenylpiperazine (m-CPP).71 Figure 6 shows an interesting application for forensic investigations in which DESI-MS imaging was applied to latent fingerprints, drugs of abuse such as cocaine and Δ9-tetrahydrocannabinol, as well as explosives such as trinitrohexahydro-1,3,5-triazine (RDX, high-energy explosive) were imaged.46

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QUESTIONED DOCUMENTS Chemical characterization of documents also occupies a prominent position in forensic chemistry. The use of powerful techniques able to provide chemical fingerprinting and markers has been of high demand due to the increasingly advanced technologies nowadays used for document forgery. Implementation of high resolution, rapid, and non- or minimally destructive techniques for questioned documents screening at the molecular level seems therefore inevitable if forensic chemistry is to keep up with modern day sophisticated forgery technologies. Such characterization has been traditionally performed using optical techniques such as UV−vis, Raman, infrared, and fluorescence spectroscopy,55 but complementary or more powerful techniques have been increasingly required. These morphological techniques are relatively simple and nondestructive but lack detailed molecular information. Ambient MS has been shown to provide a vital complementary technique in such area of forensic investigations. Different inquiries have been addressed such as the identification of the paper itself, the ink, the age of writings and printings and the use of different pens and printers, as well as the order of ink deposition of writings/printing crossings. The pioneering work on ink writing fingerprinting was performed by DESI-MS imaging (Figure 4).56 A further

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BIOLOGICAL SAMPLES Because of their utmost complexity, and great variety, the classical protocols normally applied by the forensic chemist to the analysis of target analytes in biological material are often laborious and time-consuming. Ambient MS offers therefore an attractive alternative due to its simplicity, speed, and sample preparation free protocols. Especially in forensic toxicology, this might expedite the work of a forensic medical examiner, revealing for instance, the presence of drugs, pesticides, or poisons in whole blood72 or urine.73 Such ability could facilitate fast verdicts of whether a person died under suspicious circumstances or in emergency rooms for the immediate application of the right treatment. Innumerous ambient MS techniques have been applied to biological samples. An illustrative example is the application of a modified V-EASI-MS system in the form of an “intelligent knife” to screen for cancer biomarkers in real time during surgery.74,75 The recently developed touch spray ionization (TSI) technique has used a stainless steel needle probe to sample the material. A proper solvent and electrical potential is subsequently applied to cause desorption and ionization. TSI-MS has been applied to whole blood, tissues, as well as illicit drugs (Figure 7).76 PSI-MS has also become popular for the analysis of biological samples77 and has been applied, for instance, to the analysis of drugs of abuse and prescribed drugs in whole blood (Figure 8).78,79

Figure 4. DESI-MS images from a forged number on paper. (A) Ion image of Basic Violet 3 of m/z 372; (B) ion image of Sovent Blue 2 of m/ z 484; (C) overlay of parts A and B combining Basic Violet 3 and Basic Blue 2 ion images; and (D) optical image of the surface. Adapted with permission from ref 56. Copyright 2007 Royal Society of Chemistry.

comprehensive study on ink writings by EASI-MS included the demonstration of a linear correlation of ink markers with age and the ability to investigate the ink lines crossing (Figure 5).57 That is, by constructing a 3D-image of the crossing ink lines, the order in which the inks had been deposited on the document might be determined. MS imaging could therefore provide key information on whether, e.g., a signature was written before or after the construction of a document. To screen for counterfeit money bills, DART,58 DESI,59 EASI,59,60 and ELDI61 have been applied with the characterization of, for instance, typical fingerprints of euro, dollar, and Brazilian Real bills. Recently, EASI-MS has also been used to characterize authentic forms of Brazilian vehicle documents,62 as

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FOOD, COSMETICS, AND CONTRACEPTIVE PRODUCTS Counterfeiting of food and cosmetic products is also a common but illegal activity of great concern worldwide and a major issue 2520

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Figure 5. (A) Schematics of an EASI(+)-MS setup used to inspect the intersection of two blue ink lines (B). Parts C, D, and E represent the mass spectra obtained from line 1, line 2, and the crossing point, respectively. Adapted with permission from ref 57. Copyright 2010 Royal Society of Chemistry.

Figure 7. Schematics of the sampling of (A) solids and (B) liquids by TSI using an angled teasing needle. (C) Application of high voltage and solvent causes an ESI-like plume of analyte-containing charged droplets, from which gaseous analyte ions are produced. Adapted with permission from ref 76. Copyright 2014 Royal Society of Chemistry. Figure 6. (A) Δ9-THC and/or cannabidiol on paper as identified by the MS/MS transition m/z 313 > 245; (B) Δ9-THC, distinguished from cannabidiol by the unique MS/MS transition m/z 313 > 191; MS/MS of protonated cannabidiol (C), and Δ9-THC (D). Adapted with permission from ref 46. Copyright 2008 American Association for the Advancement of Science.

analysis of flavor and fragrance raw materials in complex matrixes such as fabric and hair.80 In a recent study, an application of ambient MS in the investigation of sexual assaults was demonstrated using both DESI-MS and EASI-MS, which were used to characterize different brands of condoms.81 EASI-MS and its variants have also been investigated in their ability to screen for counterfeiting in food and cosmetic products. These techniques have been used to screen adulteration and counterfeiting of valuable vegetal oils such as olive oil,82 exotic

in forensic chemistry investigations. Ambient MS has therefore been tested and demonstrated to offer a powerful tool in this area. DART-MS has been shown, for instance, to allow for the 2521

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Figure 8. (A) Schematics of PSI-MS analysis using an autosampler cartridge in which eight drugs of abuse are simultaneously quantified. (B) PSI-MS of a single dried blood spot on paper. Adapted with permission from ref 28. Copyright 2010 Wiley. Adapted from ref 79. Copyright 2014 American Chemical Society.

Figure 9. (A) Workflow of TI-EASI analysis: a piece (∼1 cm2) of meat or fat is manually sliced into ∼10 mm thick sections; it is then placed on a brown Kraft paper surface and a few drops (∼3) of a MeOH/CHCl3 solution (2:1, v/v) are dripped on the sample surface; the sample surface is heated for 20 s (for fats) or 90 s (for meat); the TAG content imprinted on the paper surface is submitted to EASI-MS. Typical mass spectra obtained for trout and salmon are presented. Adapted with permission from ref 34. Copyright 2012 Royal Society of Chemistry.

oils from the Amazon forest,83 as well as to detect propolis,84 softeners,85 and perfume86 forgery. Food mislabeling is also a common illegal practice worldwide, such as the sale of cheaper salmon trout as if it was salmon. For such cases, TI-EASI-MS could be applied since it has been demonstrated to offer a fast and direct screening method for meat and fish34 (Figure 9) as well as for caviar87 and ham.88 DESI-MS89 and DART-MS90 have also been applied to characterize edible oils as a demonstration of their ability to screen for adulteration and forgery.

combat this illegal practice, either in laboratories as well as for onsite monitoring. The use of fuels as accelerants in arsons is another area of forensic investigations that benefits from the application of ambient MS, as exemplified by a DART-MS investigation of household materials.91 Screening of kerosene, diesel, gasoline,92 and biodiesel93 adulteration has also been demonstrated using EASI-MS. Figure 10 shows the variation of composition based on fatty acid methyl esters (FAME), m/z 317 ([FAME + Na]+), for soybean biodiesel in different concentrations of biodiesel blends in petroleum diesel.

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PETRO AND BIOFUELS Because of the high consumption and enormous financial revenues, fuels are a highly attractive and common target for fraud and adulteration worldwide. Again for fuels, due to attractive figures of merits, ambient MS techniques have been demonstrated to offer a suitable tool for the forensic chemist to

DISCUSSION

At this point it would be important to discuss why it is that other techniques such as gas chromatography (GC) or liquid chromatography (LC) coupled to MS are widely encountered in forensic laboratories, while ambient MS has not yet fully found 2522

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Figure 10. (A) Schematics of EASI-MS analysis of biodiesel samples. EASI-MS fingerprints with characteristic profiles of [FAME + Na]+ ions are instantaneously obtained and used for biodiesel typification and quality control. (B) EASI(+)-MS of representative Bn blends (n = amount of biodiesel in petrodiesel). Note that the ion of m/z 317 is the major [FAME + Na]+ ion for soybean biodiesel, whereas the ion of m/z 335 is from the internal standard. Adapted with permission from ref 94. Copyright 2010 Elsevier. Reprinted from ref 95. Copyright 2012 American Chemical Society.

Applications of ambient MS to the analysis of forensic evidence could be subdivided based on their complexity. On-site preselection is likely to require the least intensive validation and hence might be the easiest to implement. Both sample comparison by use of fingerprinting and in-lab product identification and quantitation are considered to be significantly more difficult to implement due to their intensive validation requirement upon implementation. On-site preselection and inlab product identification and quantitation seem to be very similar applications but they differ significantly based on their degree of identification certainty. The amount of certainty in the on-site (e.g., on crime scenes) identification of evidence needs only to be enough to have sufficient support to select it as “evidence” or “nonrelevant”. When, subsequently, this evidence is analyzed more thoroughly in a forensic laboratory, the identification and potentially even quantitation need to have a degree of certainty sufficient enough to be used in a forensic report and to withstand in court. More importantly, the degree of certainty of identification needs to be accurately defined. It is within these latter two aspects that the controversy of the application of ambient MS in forensic science might be the highest, primarily due to selectivity concerns related to the use of chromatographic separation-free protocols.99 However, several studies in ambient MS have shown that such previous separation is not as indispensable as generally thought. The selectivity in an LC−MS/MS setup is defined as the probability of the occurrence of a compound showing the same LC−MS/MS characteristics as the compound of interest.100 Whereas this probability can be quantitatively defined in an LC−MS/MS setup, it can also be quantitatively defined in an ambient-MS setup in which chromatography is not used. In summary, although implementation of ambient MS in forensics will likely encounter some resistance, this resistance should not withstand once methods are properly validated and all points from the Daubert standard

its way from the academic environment into routinely used practice in forensic laboratories worldwide. Traditional GC−MS and LC−MS are still dominant because these are fully consolidated MS techniques which have already been around for many decades and are therefore widely accepted and available by the community of analytical chemists and forensic scientists. Validated protocols for GC−MS and LC−MS are therefore also extensively described and generally accepted.96−98 Hence, although the advantages and applicability of ambient MS has been clearly demonstrated in numerous proof-of-concept studies, naturally it will take time before this advantageous but new approach is fully incorporated into forensic laboratories. Fully validated and implemented LC−MS or GC−MS methods are associated with years and years of experience, and once it is decided to replace these methods by new (ambient) techniques, new methods will also need to be developed and fully validated and sometimes new instrumentation will have to be acquired. Such a replacement is time-consuming and the strict quality criteria required for case work investigations hamper the introduction of new science and technology in forensic laboratories. Second, because of the controversy related to ion suppression effects and the potential failure to properly provide a comprehensive description of a complex mixture or to quantitate each of its individual components without chromatographic separation or the inherent inability of MS-only analysis to separate isomers, a general acceptance by the scientific community might require time and effort. The degree of acceptance of such new techniques in court might also vary strongly per country. In some countries, new techniques might be accepted in court when they are properly validated, error rate is known and the technique is published in a peer-reviewed scientific journal. Other countries, however, might only accept techniques in court when they are fully incorporated in forensic laboratories worldwide. 2523

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Analytical Chemistry definition101 are met, and once the advantages of the methodology, particularly related to its unsurpassed speed, simplicity and sample preparation free figures of merit, are considered.

UNICAMP, Brazil. His research interests are mainly focused on the application of MS in forensic sciences, working at the interface between academic method development and its applications in actual forensic cases.

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Jandyson Machado Santos is a Ph.D. student at the University of Campinas, UNICAMP, Brazil. He has received a B.Sc. in Chemistry and M.Sc. in Analytical Chemistry from the Federal University of Sergipe, Brazil. His research plans are to apply MS with emphasis in forensic science and petroleomic studies, developing selective tools for the analysis of vegetable oils and crude oils using ambient MS techniques and ultra-high-resolution and accuracy mass spectrometry.

CONCLUSION AND PERSPECTIVES Ambient MS offers to the forensic scientist simple, robust, and fast tools for direct analysis of forensic samples. With a single instrument and the selection of a few or even a single desorption/ ionization technique, ambient MS can be used as a quite universal, nearly nondestructive protocol, applicable to many forensic investigations. The destructive sample preparation step, which was indispensable in conventional MS, is no longer necessary in modern day ambient MS. This is a huge step forward, finally removing “destructive” as a “feature-of-drawback” for MS in the field of forensic chemistry where the availability of a single sample generally hampers the use of destructive techniques. Forensic samples applicable to ambient MS can vary dramatically from explosives, drugs, fuels, printed and hand written documents, stamps, seals to food products, fuels, and biological samples. Whereas the amount of ambient MS sources that are becoming commercially available is increasing, homemade assembling is also possible using inexpensive and common laboratory parts which should make these techniques available to everyone and everywhere. Even entirely disposable sources can be constructed, as the S-EASI source has illustrated. Miniature and portable mass spectrometers are being developed and becoming commercially available. They are nowadays operated at very modest vacuum making even the dream of atmospheric pressure MS analyses not so “unrealistic” as it was thought of before. The use of such miniature MS system in combination with ambient desorption/ ionization techniques will certainly facilitate real time on-site analysis even for crime scene investigators. For instance, a portable MS device could be taken to a crime scene to look for traces of explosives, to gas stations to check for the authenticity of fuels, to a body on a crime scene to check for drugs in urine or blood spots, and to banks to monitor for money authenticity. Onsite preselection of evidence by ambient MS is a great benefit for forensic science and would not only effectively lower the burden on forensic laboratories, but it could also provide crucial guiding information in the early stages of investigations. We expect the major developments of recent years to proceed in terms of new applications of ambient ionization techniques as well as improvements in miniaturization and portability of mass spectrometers. Indeed as the normal inertia for the application of new protocols is surpassed, a perfect couple has a great potential to be formed, as ambient MS and forensic sciences perfectly complement each other. This happy marriage, once consolidated, is likely to endure and get much stronger over the years.

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Livia Schiavinato Eberlin is an Assistant Professor at the Chemistry Department of The University of Texas at Austin. She received her B.Sc. in chemistry from the University of Campinas, UNICAMP, Brazil. She completed her Ph.D. in Analytical Chemistry at Purdue University, under the supervision of Prof. R. Graham Cooks. Her research focused on developments and applications of desorption electrospray ionization MS imaging. She pursued her postdoctoral research at Stanford University in the laboratory of Prof. Richard N. Zare. At the University of Texas at Austin, she continues to develop and to use novel ambient ionization methods for MS to address critical problems in cancer research. Marcos N. Eberlin received his B.Sc. and Ph.D. in Chemistry from the University of Campinas, UNICAMP and was a visiting scientist at the Aston laboratory at Purdue University directed by Prof. R. Graham Cooks. He is currently a full professor at UNICAMP where he coordinates the ThoMSon Mass Spectrometry Laboratory. He is former president of the International MS society and a founder and current executive president of the Brazilian MS society. His research group is focused on the development of new MS techniques and the search of applications of MS techniques in different fields of science. Sebastiaan Frans Teunissen is a visiting forensic scientist at the University of Campinas, UNICAMP, Brazil. He has received a B.Sc. and a M.Sc. in Analytical Chemistry at the Free University of Amsterdam, The Netherlands, and a Ph.D. in Bioanalytical Chemistry at the University of Utrecht, The Netherlands. After working several years as a forensic scientist at the Toxicology Laboratory of The Netherlands Forensic Institute, he now focuses on the development and application of analytical methods using ambient MS in forensic science.

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ACKNOWLEDGMENTS ́ We thank Coordenaçaõ de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) Process No. 23038.006844/2014-46 and Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo, Brasil (FAPESP) Process No. 2013/19161-4 for financial support.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Deleon N. Correa is a forensic expert at the Technical-Scientific Police Superintendence, Criminalistic Institute from São Paulo State, Brazil. He has received a B.Sc. in Chemistry, M.Sc. in Inorganic Chemistry, and a Ph.D. in Analytical Chemistry from the University of Campinas, 2524

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