Ambient Mass Spectrometry
The forensic potential of DART, DESI and Paperspray MS
Literature thesis
Author
: Rudo Odijk
University
: University of Amsterdam
Student number : 10852484
Study track
: Analytical Chemistry
Supervisor
: Arian van Asten
Abstract
Since the development of mass spectrometry, this method is one of the most used techniques for the analysis of many kinds of molecules. In order to analyze samples with a mass spectrometer, the analyte needs to be in the gas phase and needs to be ionized. Ionization usually requires a high pressure, a vacuum or a way to transport the ionized molecules to the MS. This is a problem when this technique is used in the forensic field. The difference between normal and ambient MS is mainly the pressure. In ambient MS, ionization of analytes is realized under regular pressure. After the ionization the ions are transferred to a vacuum where the identification and quantification can take place. The advantage of AIMS is that shorter analysis times can be achieved because none or less sample treatment is needed. Also the analytes do not need to be exposed to high pressures and therefore AIMS is proved to be a non-invasive technique. This literature study discusses the potential of forensic applications of the ambient ionization techniques DART, DESI and PS MS for their use in the forensic field.
DART is a technique that can be applied in the field of forensic document and ink analysis where detailed results are obtained on the chemical composition of the paper and inks used. DART also provides information when drugs of abuse are being analyzed on a crime scene.
DESI is technique that can provide information on the composition of inks on paper documents but especially shows it worth when it comes to imaging. DESI can be helpful if the suspicion of forgery is present. Also DESI can detect cocaine residues in fingermarks by the use of imaging.
PS is a new and non-invasive technique that can be used for the determination of the chemical composition of all kinds of materials including inks on paper. The analysis of small amounts of dried blood spots provides information on the presence of pharmaceuticals or drugs of abuse and is one of the main advantages of PS.
Pitfalls for ambient ionization can be found in the fact that validation is not often present for each of these techniques due to the lack of research that is done. The biggest pitfall for DART is the lack of a second dimension like a separation technique so only identification is possible and the risk of overlapping patterns is present that only can be separated with another technique. DESI can be a risk because, in spite of the advantage of imaging, a small amount of a spray solvent is necessary in order to ionize the analyte. This can alter the matrix and can destroy part of the analyte. PS can analyze a great variety of analytes but has a decreased performance when compared to traditional methods. Nothing can be concluded about whether one of the three techniques is the best to use. All three ambient ionization techniques can provide good and detailed information on the matrices that have been explained and discussed in this report. When more research is done on the validation of these techniques, ambient ionization techniques will take part in the future of analytical and forensic chemistry.
Abbreviations
AccuTOF : Accurate Time-Of-FlightAIMS : Ambient Mass Spectrometry
AKD : Alkyl Ketene Dimer
DART : Direct Analysis in Real Time DESI : Desorption Electrospray Ionization ESI : Electrospray Ionization
GSR : Gun Shot Residue
MALDI : Matrix Assisted Laser Desorption Ionization
MS : Mass Spectrometry
MSI : Mass Spectrometry Imaging
PS : Paper Spray
PSI : Paper Spray Ionization
TLC : Thin Layer Chromatography
Table of contents
Abstract ... 2
Abbreviations ... 3
Table of contents ... 4
Introduction ... 5
1. Ambient MS - Background ... 7
1.1 Direct Analysis in Real Time (DART) ... 7 1.2 Desorption Electrospray Ionization (DESI) ... 7 1.3 Paperspray Ionization (PSI) ... 8
2. Applications of DART ... 9
2.1 Forensic document and ink analysis ... 9 2.1.1 Analysis of paper ... 9
2.1.2 Analysis of ink ... 10
2.2 Determination of drugs on a crime scene ... 12
3. Applications of DESI ... 14
3.1 Forensic ink analysis ... 14 3.2 Mass Spectrometry Imaging (MSI) ... 16
4. Applications of PS ... 17
4.1 Forensic ink analysis ... 17 4.1.1 Case sample of forgery ... 19
4.2 Blood analysis ... 20
Discussion ... 22
Ambient ionization ... 22 Direct Analysis in Real Time (DART) ... 22 Desorption Electrospray Ionization (DESI) ... 23 Paperspray Ionization (PSI) ... 23
Conclusion ... 25
References ... 26
Introduction
Since the development of mass spectrometry by J.J. Thomson in 1912, this method of detection has become one of the most frequently used techniques for the analysis of molecules ranging in size from small volatile alkanes to big proteins and hormones with a molecular mass larger then 100.000Da.1 Over the past century discoveries like Matrix Assisted Laser Desorption Ionization (MALDI), Time-of-Flight (TOF) and the Orbitrap, have resulted in an improvement of the specificity and selectivity of mass spectrometry (MS) and the detailed information that their spectra show.2 Due to its wide range of
compounds that can be analyzed and its ability to qualify and quantify these, it is a technique that is used in forensic science on a great scale to analyze e.g. pharmaceutical compounds, biological samples and possible fire starters.3
In order to analyze samples with a mass spectrometer, the analyte needs to be in the gas phase and needs to be ionized before it reaches the inlet of the MS. Ionization of analytes in a sample can be achieved by a great number of methods but they usually require a high pressure, a vacuum or a way to transport the ionized molecules to the MS.3, 4 These facts prevent the use of MS directly on a crime scene, because the apparatus is usually too big and the conditions are to extreme to achieve. Moreover, the sample treatment in normal MS can be destructive to the sample that needs to be analyzed so a less invasive way of sample treatment or ionization is needed.
The introduction of Ambient Mass Spectrometry (AIMS) could provide a solution for the previous mentioned problems. The difference between normal and ambient MS is mainly the pressure. In ambient MS, ionization of analytes is realized under regular pressures instead of high vacuum.3 According to Huang et al. (2011), the ionization of analytes can be categorized in to either direct ionization, direct desorption or two
step ionization (Figure 1)4 Each of
the three ionization methods include a number of different techniques in which this type of ionization is used to gain information. After the ionization the ions are transferred to a vacuum where the identification and quantification can take place. The advantage of AIMS is that shorter analysis times can be achieved when compared to regular MS methods because none or less sample treatment is needed. Also the analytes do not need to be exposed to high pressures and therefore AIMS is proved to be a non-invasive technique and gives information about analytes in their native state.3, 4
Especially the non-invasiveness of ambient ionization techniques could be a major advantage for the forensic field in which it is important to maintain any type of evidence so different techniques can be used to contain information of the evidence and for the use of the evidence in court. But as it turns out, AIMS techniques are not yet used on a massive scale but remain mostly used on a research level. The gap between research and practice is too big due to the absence of proper results and validations, while validations in the forensic field are so important to increase the value of evidence. 5
Figure 1: Categorization of AIMS. Each of the three methods show their principle and the way that analyte ions are formed. 4
This literature study discusses the potential of forensic applications of DART, DESI and PS MS for their use in the forensic field. One technique is chosen for each of the three ionization methods in AIMS. An analysis was provided on these three ambient pressure techniques for forensic case work and contains an overview of their advantages and disadvantages as well as their robustness and validation. With each technique examples are given of the forensic fields in which these techniques could be implemented in to and in the case of DESI the value of imaging will be discussed. Finally the three techniques will be evaluated. Not to determine what is the best technique but to have an overview of each technique and their qualities.
1. Ambient MS - Background
In the introduction of this literature study, the different types of ionization in AIMS were discussed and in the chapters that follow, the applications of DART-MS, DESI-MS and PS-MS in the forensic field will be discussed. In order to fully understand how these different ambient techniques can be used to improve the value of forensic evidence or simplify the method of detection, a short background of the three AIMS techniques can be helpful.
1.1 Direct Analysis in Real Time (DART)
DART is a two step ionization technique and has first been described by Cody et al. in 2005 and allows the detection of gases, liquids and solids without the need of sample preparation.6 One of the most important advantages of DART is the state of the sample, which does not have to be altered due to the absence of a high voltage or pressure.7
In the source of DART, an inert gas like helium or nitrogen is introduced and collides with electrons by applying a high voltage between electrodes (Figure 2).8 Because of these collisions,
different kinds of electronic and vibronic excited molecules are produced, the so-called Penning ionization.4 Penning ionization
involves a transfer of energy from an excited gas to an analyte, which results in a radical cation of the molecule. This plasma moves through different grids of electrodes (Figure 2), to remove charged molecules. The neutral molecules are left to react with the sample. DART can be performed in both positive and negative ion mode and in both cases the ionization occurs mostly by Penning ionization. The result of a DART experiment is a mass spectra with respectively M.+ or [M+H]+ ions in the positive ion mode or [M-H]- in the negative ion mode but in both cases only single charged ions are formed.7
1.2 Desorption Electrospray Ionization (DESI)
DESI is a direct desorption ionization technique and has been developed by Cooks et al. in 2004 and is a method of mass spectrometry, which is based on electrospray ionization (ESI).9 An ion spray source or needle is spraying a solvent that consists of
charged micro-droplets towards a sample of interest.3 The micro-droplets desorb the analytes in the sample into the gas phase and induce ionization.3,9 The ions are then transported to the inlet of the mass spectrometer where the detection can take place (Figure 3). Before the ions reach the inlet, the solvent must be removed by heating it and making use of a vacuum.3
Figure 3: Schematic view of DESI. Under a flow of nitrogen, the needle produces charged droplets that ionize the analyte. The desorbed ions are transported to the MS inlet on the left.10 Figure 2: Schematic view of DART. 8
The desorption, ionization and transportation of the analytes of interest take place under ambient conditions. In contrast with DART, multiple charged ions can be formed when using DESI.9 This can
be of interest when analyzing peptides or proteins.
Though, one of the most important advantages of DESI is the possibility of imaging. By using a small and concentrated beam of the reagent ion and combine this with moving the sample, two-dimensional information can be obtained.11, 12
1.3 Paperspray Ionization (PSI)
Paperspray ionization or paperspray (PS) is a direct ionization technique and was first described by Cooks and Oyang in 2010.4,13 In this type of AIMS the process of ionization and transfer takes place
on a piece of paper (or other porous material) with the sample and solvent applied on to. The piece of paper is cut into a point and running a high voltage through the paper results in the transportation and separation of molecules. At the tip a high field is responsible for the desorption of charged
micro-droplets containing the ionized molecules.14 The ionized analytes leave the paper tip in a spray with the shape of a Taylor cone towards the MS inlet.15 One of the advantages of PSI is that even at low voltages, analytes can be ionized and there is no carrier gas needed.16
Figure 4: Schematic view of Paper Spray (PS). The high voltage that runs towards the tip causes the analyte to move along the paper and ionize and flows finally into the MS inlet. 16
2. Applications of DART
As mentioned in the background of this study, one of the most important advantages of DART is the absence of a sample preparation step, that can increase the risk of contamination in your sample of interest due to side reactions or, even worse, to the degradation or destruction of forensic evidence. The use of DART for document examination and ink analysis will be discussed because the possible solvents used in the sample preparation step could have a great impact on the composition of the ink and the texture of the paper. In the analysis of explosives, DART could prove its value due to the fact that no high pressures are needed in order to contain spectral information as well as the absence of a sample treatment in which again side reactions can occur.
2.1 Forensic document and ink analysis
When documents, envelops, money or any other kind of paper material is obtained from a crime scene, the analysis of these types of evidence can provide information on compounds present on that evidence and on the type of paper that the evidence is made of. Also the analysis of ink or paint present on these carriers could tell something about the age of the document, whether it is forged or altered or about the origin of the ink.17
2.1.1 Analysis of paper
Because the chemical composition of printing and writing papers can be very different and even in the same brand or type of paper differences can occur, paper materials can provide detailed and unique information.17,18 To prove this, Adams (2010) created an experiment in which sixteen types of paper
were analyzed with DART-MS to obtain knowledge on the chemical components and their impact on long-term physical and optical stability. The types of paper varied from regular copy paper to different kinds of cardboard. An Accurate Time-Of-Flight (AccuTOF) mass spectrometer was used and combined with a DART ion source, as shown in Figure 2. The calibration was done by using polyethylene glycol (PEG-600) because it is a well-known calibrant that produces mostly single charged ions in both the positive and negative ion mode.19 The helium flow was set to 5.0 Lmin-1 and
the temperature of the helium flow was varied between 20 and 500°C. All the sixteen types of paper were introduced to the ion source by cutting a small piece of the paper and placing it with a distance of 1.0 mm from the outlet of the ion source.18 Figure 5 shows the mass spectra of modern copy paper at
different temperature ranges of the helium gas.
Figure 5: Mass spectra of modern copy paper obtained at a temperature range of 150-250°C (b), 250-350°C (c), >350°C (d) and an average specrum across all temperatures (e). [18]
The spectra in Figure 4 show different peaks arising at different temperatures of the helium flow. The analytes with a m/z value between 43 and 143, shown in spectra c and d, are mostly losses of compounds like the acylium ion (C2H3O+ m/z 43), furan (C4H5O+ m/z 69) and
3,5-dihydroxy-2-methyl-pyran-4-one (C6H7O4+ m/z 117). The analytes at the m/z values of 451, 479 and 507 (Figure 4b
and 4c) are the protonated ketones formed by the hydrolysis of alkene ketene dimers (AKD), which is a unique compound used in modern copy papers to make it more hydrophobic.18
As the temperature of the helium flow rises, more analytes show up with a lower m/z value. However, the analytes with a higher m/z value will show a lower relative isotope abundance (RIA) as the temperature comes above 350°C or eventually will even disappear. The lower RIA is mostly due to the temperature of the helium flow. At temperatures higher then 350°C, thermal decomposition is responsible for the disappearance of bigger compounds and these compounds fall apart into smaller and more volatile compounds.18,20,21 Whether analytes of a certain length are detected and shown in a
spectrum is mainly depending on the temperature of the helium flow. Therefore in the analysis of paper itself, it could be helpful to produce an average spectrum with all the temperatures combined to get the most information out of one spectrum or to optimize the temperature of the helium flow according to the nature of the sample that needs to be analyzed.18
2.1.2 Analysis of ink
The research of Adams (2010) has shown that the analysis of paper documents can provide unique information on the nature of the paper. However, what could be more important than the paper it self, is the information that is written on the paper. Different techniques have already been applied to the analysis of writing ink, but although an optical examination is non-destructive it only shows that two inks are similar.22-24 The use of Thin Layer Chromatography (TLC) has also been described, but
usually a match in TLC only means that the fluorescent components in two inks are the same. Therefore Jones and McClelland (2013) developed a method to analyze writing inks with DART-MS. In this research, three types of ink were analyzed on sixteen different kinds of paper and envelopes. All the spectra were obtained by using a AccuTOF mass spectrometer with a helium flow of 3.25 L/min. The heater between the electrodes was set to 225°C to help the analytes evaporate, but not discolor the paper. And again, higher temperatures could result in degradation of compounds.18,20,21 All
the spectra were obtained in the positive ion mode.
Figures 6 and 7 show the spectra of the analysis of ballpoint ink, gel ink and fluid ink after a couple of days and after a eleven months to show the effect of the age of ink and the difference in volatility in the components of ink.
Figure 6: DART spectra of ballpoint ink after four and 322 days [17].
All three inks gave similar results in the first week when the spectra were obtained. In the lower mass region the more volatile compounds can be found. This is also a major contribution to the fact that after eleven months only the bigger components were still detected with the same relative intensity. After eleven months the smaller and more volatile components will show a lower relative intensity or will even disappear from the spectra. Because of their small mass and high volatility this was to be expected. Figure 6a is a good example of where the bigger components remain unchanged over time. The peak with a m/z value of 367 is due to the presence of protonated dibutylglycol and after eleven months the intensity of this peak did not change and therefore seems stable over time. The small peak with a m/z value of 374 is probably due to contaminants. The expectations were to see a peak at a m/z value of 372 which is crystal violet, a major component in ballpoint ink. Instead there was a peak detected with a m/z value of 374. The detection of this peak specifically, could be related to the presence of leucocrystal violet.
The spectra of the gel ink (Figure 7 - left) showed less peaks than the spectra of the ballpoint ink and after eleven months even fewer peaks could be observed. To make the gel ink dry fast enough, it contains more volatile components then the ballpoint ink and therefore show fewer peaks in the spectrum, even when the spectra were obtained in the first week. Triethanolamine with a mass of 150 seems to be the most stable compound in gel ink. This in contrast with a relative high peak with a mass of 301 that disappears completely, but could not be identified. The fluid ink in figure 7 - right shows a broad range of polyethyleneglycols with a very stable and high peak with a mass of 239, which is caused by the presence of pentaethylene glycol. Over time all the polyethylene derivates reduce in intensity and ultimately disappear from the spectrum.17
Figure 7: DART spectra of gel ink (left) and fluid ink (right) after a week and eleven months [17].
2.2 Determination of drugs on a crime scene
The analysis of drugs with DART is, along with forensic document and ink analysis, an important field of interest. The fast, easy and non-invasive analysis and detection of several drugs of abuse or medicine that can be found on a crime scene proves to be of a great value for forensic purposes. This way it could be easier and faster to determine the type of drugs that is found on a crime scene and when other matrices can be analyzed in the same way it can give information on the traces of drugs present in a victim or suspect and if the pure substance and the traces have the same origin.
Brown et al. (2016) developed a method of DART combined with a portable mass spectrometer for the identification of common drugs on a crime scene.25 They used a DART source with nitrogen or
compressed air that was generated by a common air compressor. Helium could not be used, as it requires a compresses cylinder that is too big for normal vehicles. Samples were directly placed between the DART source and the inlet for the MS. For the mass spectrometer part a MT Explorer 50 was used with a 180µm capillary at 120°C. The instrument size was 30x43x50cm with a total weight of 35kg. In addition to the use of nitrogen or compressed air, metal hydride cartridges were used that supply amounts of hydrogen and this addition eliminates the need for helium. For the library spectra pure standards of 1mg/ml were dissolved in methanol and placed on the closed end of a glass capillary. When dried, the samples were placed between the DART source and the MS.25
Prior to the analysis of pure standard solutions or mixtures of drugs a background signal and mass spectrum of ambient air were obtained. These spectra are shown in Figure 8. When the pure standard solution of cocaine was introduced to the DART source and MS, spectra were obtained as shown in Figure 9. The spectra B3 and B2 show cocaine in its pure form. After this the ion trap was switched to MS/MS mode to detect a daughter ion of cocaine with a m/z value of 182. Brown mentioned that the signal of the sample could only be seen when fully introduced to the inlet of the MS and decreased rapidly when removed from the source. To avoid contamination the samples of interest were placed on a metal mesh because only traces of the analyte are required for analysis.
The standard solutions that were tested all could be distinguished from one another except for isobaric and isomeric compounds which is a well known problem in mass spectrometry.25
Figure 8: Background signal (above) and mass spectrum (below) of ambient air. [25]
Next to solutions of pure compounds, complex mixtures of drugs were analyzed in order to see if certain types of drugs could be identified within the mixture. Brown used a tablet of Percocet, which contains oxycodone and acetaminophen. The tablet was placed in to the DART source without any extraction and the obtained spectra is shown in Figure 10. As can be seen in this figure both acetaminophen and oxycodone could be identified separately and furthermore oxycodone was identified by obtaining a MS/MS spectrum of oxycodone at a m/z value of 316.
To make sure that the combination of the DART source with the MS would detect and qualify the drugs of interest, the samples were analyzed with a GC-MS for correct identification. 25
Figure 9: Mass spectra of a 1mg/ml solution of cocaïne in methanol. The spectra B2 and B3 show the detection of the parent ion of cocaine at a m/z value of 304. The spectra C2 and C3 show one of the daughter ions of cocaine at a m/z value of 182. [25] Figure 10: Mass spectra of a Percocet tablet that contains acetaminophen and oxycodone. The spectrum below. [25]
3. Applications of DESI
The other ambient ionization technique, which can be of great value for the analysis of evidence in forensic casework, is DESI. The disadvantage of DESI when compared to DART is that the analytes in a sample are being exposed to a micro-droplet spray of solvent that is responsible for the ionization. This or every other method of ionization that require a solvent in order to cause ionization of an analyte can cause degradation of the evidence of some kind or side reactions with compounds in the evidence, which prevents the use of other detection techniques and decreases the value of the evidence. However, the combination of DESI with an imaging technique can provide additional information that makes this type of AIMS beneficial over a technique like DART. For this chapter the forensic applications of DESI will be discussed for the use in Gun Shot Residue analysis (GSR) and in forensic document and ink analysis.
3.1 Forensic ink analysis
In the field of forensic document and ink analysis Ifa et al. (2007) analyzed different types of ink with DESI-MS to see if this type of ambient ionization could provide the same information as techniques like MALDI but with a lower risk of sample destruction and the possibility of imaging samples that provide additional information. For this experiment Ifa used glossy photographic paper on which he wrote with a red (asterisk sign) and blue (the letter m) permanent marker. These markers contain rhodamine 6G and Basic Blue 7. The DESI ion source consists of an inner capillary for the spray solvcnt and an outer capillary for the nebulizing gas. The MS spectra were obtained with a Thermo Finnigan LTQ spectrometer. In both cases 100% methanol was used as the spray solvent and the MS spectra were obtained in positive ion mode. For the sample imaging every pixel of the written letter was measured twice and were performed by continuously scanning the surface of the sample. Figure 11 shows a schematic set up for this experiment.26
Charged droplets that are generated in the ion source are delivered to the surface. The inlet for the mass spectrometer is able to move over the sample surface in order to gain imaging information. The analytical spot size of DESI depends on the size of the capillary and the flow rate of the spray solvent. For the imaging experiment an area of 5.4*107 µm2 was scanned in both the x and y direction to end up with an array of 1260 pixels. Figure 12 shows the results of this experiment. 26
Figure 12: Results of DESI-MS for permanent markers on photographic paper. A: Scanned optical image of the sample. B: and C: Mass spectra of Rhodamine 6G and Basic Blue 7 as analyzed with DESI-MS. D: Molecular ion image recorded by DESI-MS. [26]
The mass spectra in figure 12 show that the intact cations of Rhodamine 6G (m/z: 443.5) and Basic Blue 7 (m/z 478.4) can be recorded from the glossy photographic surface with DESI-MS. When the imaging technique was applied, the structure of the letter and asterisk sign were revealed by plotting the intensity of the signals to the distribution of the two inks. This structure reveals the features of the original image in 4A.
Another combination of DESI-MS and imaging is reported by Hamid et al. (2015) for the detection of forged documents and to distinguish between isobaric components like rhodamine B and rhodamine 6G. Hamid used a red ink to write the letter F, and used a different red ink to convert that F into the number 8. Both compounds have a mass of 443 but rhodamine B produces an ion with m/z 399 due to the loss of CO2 and rhodamine 6G produces an ion with m/z 415 due to the loss of ethylene. Figure 13
shows the results of this experiment were A is the original picture, B is a DESI image of both rhodamine 6G and rhodamine B, C is the DESI image of m/z 399 and D is the DESI image of m/z 415.27
3.2 Mass Spectrometry Imaging (MSI)
One of the advantages of DESI-MS combined with imaging is reported by Bailey et al. (2015). Bailey collected fingerprints in order to determine the presence of cocaine and its metabolites in fingermark residues. To make sure that a good correlation was found between the different metabolites, Bailey analyzed oral fluid tests as well. Figure 14 shows a DESI image of a fingerprint that was coated with cocaine and deposited on a glass surface. The image on the right shows that multiple important details are collected to identify this fingerprint and therefore proves the use of DESI-MS imaging.
When this technique is compared to other methods that try to detect drugs in fingermarks, DESI has the advantage. Kaplan-Sandquist et al. (2014) tried to determine drugs in fingermarks with MALDI-TOF-MS but were not able to detect the targeted drug ions.28
Figure 14: DESI image of distribution of cocaine on a glass surface. [28]
4. Applications of PS
The last AIMS technique that will be discussed in this study is PS. While paperspray can not be combined with an imaging technique like described for DESI, it is a technique that can provide rapid analysis, no sample preparation and highly precise quantitation for a great variety of samples like blood, raw tissues and even food additives and samples on dirty surfaces.29 In this chapter the
applications for paperspray will be described based on its use in the detection of drugs in blood spots and in forensic ink analysis.
4.1 Forensic ink analysis
Da Silva Ferreira et al. (2015) described the use of paperspray for forensic ink analysis and they proved that several ballpoint inks could be distinguished by the use of PS-MS with minimal sample preparation and no degradation of the analyzed samples.30
For their research, ordinary white office paper was cut in to triangular pieces. Twelve different ballpoint inks were used to draw straight lines on the triangular piece of paper. The samples were then put on a copper clip and placed in front if the MS inlet. For this experiment a Thermo LCQ Fleet mass spectrometer was used in the positive ion mode with a voltage of 4.5kV, a capillary temperature of 275°C and a capillary voltage of 35V. Because literature has shown that the most common dyes and additives in ballpoint inks have basic and cationic characteristics, the positive mode was chosen. The mass spectra were obtained within a range of 100-1000m/z. To obtain a MS spectrum, 7µl of 90% methanol was added to the paper.30
Figure 10: Mass spectra of four types of ballpoint ink based on the composition of dyes and additives that can be found in these types of ink. The most abundant peaks in the spectra are originated from a m/z value of 372 (Basic Violet 3), a m/z value of 478 (Basic Blue 7) and a m/z value of 470 (Basic Blue 26). [30]
Figure 10 shows the spectra of four different types of ballpoint ink analyzed with paperspray MS. All analyzed inks show the presence of Basic Violet 3, although in varying abundances. Besides Basic Violet 3, three other dyes were detected that are frequently used in ballpoint inks. The ions with a m/z value of 212 and 268 can be explained by the presence of so called aryl guanidines. These compounds are used as an additive to raise the pH of the ink.
Eight of the twelve types of ballpoint ink have an identical composition of Basic Violet 3 and Basic Blue 26 so based on a visual inspection of the obtained mass spectra, these types of ballpoint ink could not be distinguished from one another. Da Silva Ferreira proposed an experiment in which the relative ion intensity of these dyes was measured to determine the ratio of the ion of interest to other compounds. The relative ion intensity was calculated by characterization of the demethylated amine groups in Basic Violet 3 and Basic Blue 26. Figure 11 shows the mass spectra of eight different ballpoint inks that contain BV3 and BB26. Each spectrum contains information on the relative ion intensity of these compounds.30
Figure 11: Mass spectra in positive ion mode for eight different ballpoint inks. The RII value within each spectra represents the ratio of the demethylated amine groups to the unmethylated dye and gives information on the quantitation of these compouynds within the dye. [30]
4.1.1 Case sample of forgery
A hotel receipt that was originally signed in 2005, was altered by changing the year of signing to 2006 amongst other changes. Several pieces of paper were cut in triangular shapes and used to obtain their mass spectra. The results are shown in Figure 12.30
Figure 12: Mass spectra obtained from several segments of a hotel receipt. Mass spectra (a) is originated from an original segment of the receipt. Mass spectra (b-e) are forged regions of the receipt. Every forged region was made with another type of ball point ink. [30]
The mass spectra in figure 12 show that there is a big difference in relative ion intensities of BV3 and BB26 within the forged regions. In all cases the RII of both BV3 and BB26 increased compared to these values of the original sample of the receipt. Further analysis of these results indicates that, when documents are stored under fluorescent light, natural light or even in the absence of light, the way in which these dyes degrade is significant for the detection of these compounds. This means that even after several years, documents can still be analyzed and differentiation of dyes is still possible.30
4.2 Blood analysis
Another field of interest where paperspray MS can prove its worth is when blood samples need to be analyzed in order to find drugs of abuse, therapeutic drugs or other pharmaceuticals. Regular analysis of blood requires time-consuming sample preparation steps. The matrix can cause severe contamination and storage of blood does have its own risks. Because paperspray is a non invasive and fast technique it can improve the quality of the analysis and can be implemented in a high throughput experiment.29
Espy et al. (2014) set up an experiment for the quantification of eight different drugs of abuse in whole blood. For this experiment 10µl eight different drugs were spiked in to 190µl whole blood. The whole blood was incubated to 37°C prior to use. The paperspray procedure was performed using an automated ion source with disposable cartridges that contained teardrop shaped pieces of paper. 12µl of blood was spiked onto the paper and dried for 20 minutes in a 37°C oven to simulate forensic lab settings. The mass spectrometry was performed using a TSQ Quantum Access MAX mass spectrometer with a capillary temperature of 300°C and a voltage of 35V.31
In order to achieve a good sensitivity of the method a mixture of organic solvents is added to the blood spot with a delay between the injections. The reason that the solvent is added in steps is to produce gradual solvent introduction and to maximize analyte extraction. The voltage of the spray was set to 3500V because at values lower than 2500V the paperspray method became unstable. Figure 13 shows the mass spectra obtained with paperspray for one of the eight drugs analyzed, THC.
In figure 13 the mass spectrum is shown for THC. In the upper spectrum THC is detected with the same solvent mixture as the other drugs, a mixture of methanol, water and acetic acid. In the lower spectrum sulphuric acid was added because the THC in the blood needs to react with the sulphuric acid in order to gain an improved sensitivity of 50 times due to opening of the cyclohexyl ether. The addition leads to protonated THC. As can be seen in figure 13 THC could be detected, after the addition of sulphuric acid, with a high relative intensity. 31
Figure 2: MS spectrum of THC with paperspray MS. [31]
The same parameters were used to quantitate pharmaceuticals in blood. With the use of the same solvents and mass spectrometer as described by Espy et al (2014), Manicke et al. (2010) quantified spiked pharmaceuticals like propranolol in bovine blood. Manicke found a good detection and quantitation of this pharmaceutical. Its mass spectrum is shown in Figure 14. The protonated propranolol is found at a m/z value of 260. Selecting this ion as the parent ion results in a spectrum where the most abundant daughter ion is present at a m/z value of 183. 32,33
Because propranolol is a pharmaceutical that can bind to proteins in blood, Manicke analyzed if the detection of propranolol would be affected by the drug protein binding. They found out that the ratio of propranolol in blood and a pure solution was not significantly different and therefore concluded that the protein binding does not affect the analyte detection.32,33
Figure 14: Mass spectrum of propranolol. The upper spectrum shows the protonated propranolol at m/z: 260. The lower spectrum shows its daughter ion at m/z: 183. [33]
Discussion
Although ambient ionization mass spectrometry can prove its worth in the forensic field, there are still some pitfalls that have to be taken in to account. In the discussion the advantages and disadvantages of DART, DESI and PSI will be discussed as well as an overview of the advantages that ambient ionization techniques can have in contrast with the traditional methods.
Ambient ionization
Ambient ionization mass spectrometer techniques are the future in the forensic field of analysis. In general the advantage of AIMS can be found in the fact that these techniques offer rapid and sensitive analysis with high specificity. The analysis times are significantly shorter than common analytical techniques. Besides this a great advantage is that samples require little to no sample preparation.21 Samples are not exposed to a high vacuum and therefore the analysis of the sample in its native state is possible. In addition to the analysis in the native state, using an ambient ionization technique results in a non-invasive way of analysis, which allows samples to remain in their original state so that multiple identical or other techniques can be applied.34
One example of the advantage of ambient ionization can be found in the analysis of trace explosives, which include a high level of reproducibility and accuracy, even in complex matrices.20
If we apply the use of ambient ionization on the overlapping technique described in this report, the analysis of inks and papers, the biggest advantage is the minimal sample destruction and the possibility for direct analysis on-site in their native state. Analysis in the open environment without prior preparation turns ambient ionization in a technique that is very suitable in the forensic field.30 The only disadvantage that is reported for the use of ambient ionization techniques is the lack of validation. Multiple techniques describe a good identification of analytes but quantitation is not so much reported. Also the combination of the ambient ionization with a mass spectrometer is a challenge, because the machinery is often heavy and big which prevents the use on a crime scene.35
Direct Analysis in Real Time (DART)
Direct analysis in real time is an ambient ionization technique, which takes advantage of the production of metastable gas atoms generated from a glow discharge plasma in a heated gas stream. What makes DART a unique technique is the fact that DART allows samples to be introduced directly into the gas stream between the source and mass spectrometer. This technique provides a way to analyze samples under atmospheric conditions with high temperatures and voltages but without a high potential. DART can be used for the quick identification of volatile and non-volatile polar organic compounds in different matrices without extractions, derivatizations and chromatographic separations and can be easily combined with traditional chromatographic techniques like HPLC-MS and GC-MS. Three samples per second are reported for DART.18,20
A variation of studies has shown that applications of DART can be found in the fast analysis of drugs of abuse. Also a difficult matrix like hair is easily analyzed with a technique like DART, because every segment of the hair can be analyzed separately when held in front of the source. Even in the field of Gun Shot Residues (GSR) and trace explosives DART can provide good results.28,36
When the analysis of inks and paper is studied, DART can provide detailed information on inks, without altering the appearance of the document. This is because no sample needs to be cut out and it only removes a little fragment of the dyes and pigments that constitute the visible portion of the ink.17 In contrast with the positive quality of DART, which allows the rapid and simultaneous detection of hair, THC was the only analyte that could be detected. There was no proof that other analytes or metabolites were present in the matrix and even the concentration of THC that was reported, was not in line with the recommended cut-off ranges.36
In spite of the fact that DART is a useful ambient ionization technique, the lack of a second dimension, like a separation technique, makes it difficult to confirm the identification of trace explosives for instance due to similar fragmentation patterns of explosions in the same class. And as last, due to the risk of shared ions and a mass overlap of similar fragmentation patterns, false positives are easily accounted for.20
Desorption Electrospray Ionization (DESI)
Desorption Electrospray ionization is a versatile and high throughput ambient ionization technique that requires minimal sample preparation and is a sensitive and specific technique that offer a high discriminating power. DESI is able to analyze samples in different forms like tablets, gels and powders. It can be applied to the analysis of trace levels of peptides, proteins, nucleotides, drugs and nitro-aromatic substances on or in solids, liquids and mixtures, which is important in forensic applications. DESI proves its worth because only a small fraction of the specimen is exposed to the spray solvent and therefore destroyed. It is a technique that can easily be combined with an ion mobility TOF mass spectrometer for instance.
DESI offers an analysis time of seconds, which is significantly better than the time needed to separate and analyze compounds using traditional techniques. Three samples per minute are reported for DESI.34,37 In comparison with a technique like MALDI, DESI is complementary including the fact that
MALDI is applied to proteins and peptides where DESI works better with lipids. MALDI gives a higher resolution but requires more sample preparation.
Maybe the most advantageous part of DESI is the fact that it allows imaging of a sample. One field in which this is used is the detection of cocaine residues in fingermarks and the analysis of forged documents based on the analysis of inks. DESI good for cocaine residues in fingermarks.28,37
DESI suffers from ion suppression or enhancement though. The amount of charged ions in the gas phase that reaches the detector can be affected due to the presence of volatile compounds that can change the efficiency of droplet formation in the DESI source. This parameter is of importance because in the forensic field, samples are rarely clean and often involve complex matrices.
Automatic sampling is until now not present for DESI and because parameters have to be set up manually, it still is a time consuming technique and the results are not as reproducible as desired. The signal intensity is surface dependent so if no sample preparation is required, the surface might result in a poor sensitivity. Complex mixtures can be hard to analyze because chromatographic steps are missing which can only be resolved by a combination with MS/MS for instance.34
The use of DESI can be less advantageous due to the fact that a fine spray of solvent is used, which can alter the matrix, for instance the document or the ink that needs to be analyzed.17 The use of DESI on-site can also be challenging because there is a limited use of miniature mass spectrometers due to the high gas and solvent flows that are necessary for DESI.38
Paperspray Ionization (PSI)
Paperspray mass spectrometry is preferred due to the fact that it does not require pneumatic assistance in order to transport the analyte to the mass spectrometer. The simple set up of the method allows a much easier operation and instrumentation and therefore makes it much more compatible with miniature mass spectrometers when compared to DESI and DART. PS can be easily transformed in to a high throughput method because only multiple pieces of paper or similar materials are required and therefore multiple analyses are possible. This has a positive effect on the speed in which a paperspray analysis can take place and numbers up to seven samples per second are reported.
PS requires minimal sample preparation and it does not destroy or alter samples in any way. That makes it possible to perform multiple identical analyses of the same sample or even chromatographic analyses when requested.29,30
PS-MS has been used for the analysis of pharmaceuticals in different matrices, therapeutic drugs, protein complexes and the analysis of inorganic ions. It even can be used in the direct analysis of drugs of abuse in blood spots and raw urine and cocaine residues in various material surfaces.
The small amount of blood as mentioned in this report offers a quick and flexible sample collection and analysis and it can also be easily coupled to portable miniature mass spectrometers for on-site analysis and roadside testing.31
Although paperspray MS offer good, reliable, quick and a great number of results in a short amount of time the choice of the mass spectrometer can be difficult. A mass spectrometer is a somewhat unsuited choice because of its high costs and the complexity of the construction, especially when the vacuum requirement has to be taken into account. This is a great contrast with the low costs of the paperspray method itself.37
Also the use of paperspray MS results in a decreased performance when this method is compared to the more traditional HPLC-MS methods in spite of the fact that paperspray MS proves to shorten the workflow, allows the analysis of more samples pin the same time and decreases the complexity of the MS quantitation.33
Conclusion
In this report a literature study has been done on the forensic potential of ambient ionization techniques like DART, DESI and PS. For each of the three ambient ionization technique a couple of applications were studied and explained in this report.
For the forensic analysis of documents and inks each of the techniques has proven it self to provide detailed information on the chemical composition of the ink or paper it was written on and on the differentiation between two types of ink within one document.
DART can provide detailed information when an analysis is required of drugs of abuse found on a crime scene but it seems to be limited to the identification of analytes. Due to the lack of a second dimension quantitation remains difficult.
DESI has an advantage when it comes to imaging. In the analysis of forged documents DESI can provide good results. The use of a solvent spray though must be taken into account as it can destroy a small part of the sample.
Paperspray has a very broad range of samples that can be analyzed due to the fact that no sample preparation is required but one of the biggest advantages can be found in dried blood spots that still provide good information on the chemical composition of analytes within the blood. The performance of paperspray is lower than traditional methods though in spite of its advantages.
In Table 1 an overview is given on the characteristics of DART, DESI and paperspray.
Technique DART DESI PS
Introduction 2005 2004 2010
Ionization Plasma Spray Spray
State of sample Solid, Liquid, Gas Solid, Liquid Solid, Liquid
Imaging No Yes No
Speed of analayis 3 samples/sec 3 samples/min 7 samples/sec
Non-Destructive Minimally No Yes
Sample preparation No No No
Miniature MS-coupling Difficult Difficult Yes
Quantitation No No Yes
Overview of ambient ionization characteristics
Table 1: Overview of ambient ionization characteristics.
Nothing can be concluded about whether one of the three techniques is the best to use, because it depends on the combination of spectrometry that is added to the ambient ionization, the type of sample and matrix that are needed to be analyzed and what the hypothesis is of the sample. All three ambient ionization techniques can provide good and detailed information on the matrices that have been explained and discussed in this report. When more studies are performed on the quantitation of analytes with ambient ionization and when more research is done on the validation of these techniques, especially in combination with their use within the forensic field, ambient ionization techniques definitely will take part in the future of analytical and forensic chemistry.
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