TITLE OF THE INVENTION METHOD OF FIELD INDUCED PHOTOIONIZATION OF MOLECULES USING LOW POWER POINTER LASER IN LASER ASSISTED PAPER SPRAY IONIZATION MASS SPECTROMETRY (LAPSI MS)

FIELD OF THE INVENTION

The present invention relates to a new form of ambient ionization mass spectrometric technique called laser assisted paper spray ionization mass spectrometry (LAPSI MS), which leads to signal amplification, high signal to noise ratio and ionization of poorly ionizable species.

BACKGROUND OF THE INVENTION

Ambient ionization methods such as electrospray ionization (ESI) (Yamashita & Fenn,

1984), desorption electrospray ionization (DESI) (Chen, Talaty, Takats, & Cooks, 2005; Cotte- Rodriguez, Takats, Talaty, Chen, & Cooks, 2005; Takats, Wiseman, Gologan, & Cooks, 2004), Low temperature plasma (LTP) (Harper et al, 2008) ionization, etc. have emerged as some of the most important directions in modern mass spectrometry. One of the important methods of ionization is paper spray ionization mass spectrometry (H. Wang, Liu, Cooks, & Buying, 2010), which has importance in several analytical situations such as blood clot analysis (Espy et al, 2014; Yannell, Kesely, Chien, Kissinger, & Cooks, 2017), forensic analysis (Domingos et al, 2017), food adulteration (Reeber, Gadi, Huang, & Glish, 2015; Q. Wang et al., 2015) and monitoring catalytic reactions (Banerjee, Basheer, & Zara, 2016), etc. Variation of the same such as leaf spray (LS) ionization has also been useful in a number of situations (Zhang et al., 2013). Despite the important advantages, the technique has serious limitations in analyzing specific category of analytes such as the analysis of alkanes, olefins, polycyclic aromatic hydrocarbons (PAH), etc. This stems from the fact that the electrospray ionization technique in general is inefficient in causing ionization for certain class of molecules. For such molecular systems, an approach to overcome this limitation is to use other stimuli, complementing the electric field available in paper spray.

The current invention shows that a simple laser pointer is adequate to assist the electric field and cause measurable ion intensity for a diverse variety of analytes. This modified paper spray referred to as laser assisted paper spray ionization mass spectrometry (LAPSI MS). The invention illustrates the application of LAPSI MS for several aromatic molecules which are not amenable for common paper spray ionization. Further, the invention shows that the utility of the methodology of paper spray in detecting laser induced transformations.

SUMMARY OF THE INVENTION

The present invention relates to a new form of ambient ionization mass spectrometric technique called laser assisted paper spray ionization mass spectrometry (LAPSI MS), which leads to signal amplification, high signal to noise ratio and ionization of poorly ionizable species.

In one embodiment, the invention shows a new ambient ionization technique named laser assisted paper spray ionization mass spectrometry (LAPSI MS), wherein, a 532 nm, <10 mW laser pointer was shone to a triangularly cut paper along with high voltage, to effect ionization. The analyte solution was continuously pushed through a fused silica capillary, using a syringe pump, at a preferred infusion rate. LAPSI MS showed enhanced ionization with high signal intensity of analytes, which provides field induced photoionization of molecules.

In other embodiment, the invention illustrates ionization of polycyclic aromatic hydrocarbons using LAPSI MS, which are normally not ionizable with similar ionization methods involving solvent sprays. LAPSI MS works both in positive and negative modes of ionization. A clear enhancement of signal intensity was visualized in the total ion chromatogram for an analyte in presence of the laser. The field-induced distortion of the potential well can be large in paperspray as the fibers constituting the paper are separated at tens of nanometers apart and consequently the analyte molecules are subjected to very large electric field of the order of 10 ⁷ Vcm ^"1 .

LAPSI MS can be used for monitoring in-situ photo-assisted reactions like, the decarboxylation of marcaptobenzoic acid in presence of gold and silver nanoparticles and dehydrogenation reaction of 2,3-dihydro-lH-isoindole. Impurities like mineral oils were detected in commercially available vegetable oil, pointing the way to application of social relevance. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Mass spectrum of benzene in presence (greytrace) and absence of laser (black trace). Inset shows schematic of the experimental setup of laser assisted paper spray ionization mass spectrometry (LAPSI MS). A green diode laser was pointed at the tip of the paper with a continuous supply of analyte through a fused silica capillary.

Figure 2 LAPSI MS spectra of PAHs, A) naphthalene, B) pyrene and C) benzanthracene in positive ion mode. Isotope distributions of the species.

Figure 3 shows A) Selected ion chromatogram for m/z 720 under the laser on/off condition. B), C) Mass spectrum of C ₆ o in negative mode in the absence and presence of laser, respectively. Inset in C shows the isotopic distribution of C ₆ o- Figure 4 Voltage dependence in the ion intensity of benzene (m/z 78), bromobenzene (m/z 157), naphthalene (m/z 128), pyrene (m/z 202) and benzanthracene (m/z 228).

Figure 5 Optimized structures for a) benzene, b) bromobenzene, c) naphthalene, d) pyrene and e) benzanthracene and their orientation for the applied electric fields.

Figure 6 IPs of A) benzene, B) bromobenzene, C) naphthalene, D) pyrene and E) benzanthracene in presence of electric fields at x, y and z directions.

Figure 7 Comparative mass spectrum of benzanthracene (m/z 228) A) ESI MS, B) PS MS and C) LAPSI MS, all in positive ion mode. Absence of the peak in ESI MS and PS MS indicates the non ionizability of the species. But presence of the peak in LASI MS spectrum shows the occurrence of ionization of benzanthracene.

Figure 8 Chromatogram of C ₆ o in negative mode and that of toluene in positive mode, merged together to illustrate the mechanism for the enhancement of the ion intensity in case of C ₆ o (in toluene). The increment in ion intensity of C ₆ o is 100 here (from 10 ² to 10 ⁴ ) and at the same time the intensity rise for toluene is from 0 to 10 ² .

Figure 9 Selected ion chromatogram of C ₆ o using A) chloroform, B) dichloromethane (DCM) and C) toluene at laser on/off conditions. The intensity enhancement of C ₆ o has not occur in case of chloroform and DCM due to non ionizability in LAPSI MS. But it occurs when C ₆ o was taken in toluene which implies that, toluene plays an important role to increase the ion density during the ionization.

Figure 10 In-situ monitoring of decarboxylation reaction of para-marcaptobenzoic acid (p- MBA) on AgNPs coated paper in A) presence and B) absence of laser. In absence of the laser the peak at m/z 153 corresponds to the deprotonated anion of the p-MBA [M-H] gets decarboxylate to the product ion peak (deprotonated benzene thiol [M-H]) at m/z 109.

Figure 11 Mass spectrum p-MBA on AuNPs coated paper in A) presence and B) absence of laser.

Figure 12 LAPSI MS spectrum collected for the dehydrogenation reaction of 3-dihydro-lH- isoindole in positive ion mode. Peak at m/z 196, shown in the spectrum, is for the molecular cation of 3-dihydro-lH-isoindole which gets transformed to m/z 196, corresponds to the molecular cation of the dehydrogenated product.

Figure 13 Mass spectrum of coconut oil, sold in the local market recorded in the A) absence and B) presence of the laser. Envelop of peaks due to hydrocarbon is marked.

Figure 14 Mass spectra of standard mixtures of coconut oil and paraffin oil in different ratios with laser off (left) and on (right) conditions.

Figure 15 Chromatogram and mass spectrum of pure paraffin oil in LAPSI MS in positive ion mode. Presence of the peaks during laser on condition indicates the ionization of the hydrocarbons in LAPSI MS.

Figure 16 Mass spectrum of pure coconut oil taken in LAPSI MS. The first envelop of spectra indicates the lipids present in coconut oil and the second envelop at higher m/z is for the dimers of the same.

Referring to the drawings, the embodiments of the present invention are further described. The figures are not necessarily drawn to scale, and in some instances the drawings have been exaggerated or simplified for illustrative purposes only. One of ordinary skill in the art may appreciate the many possible applications and variations of the present invention based on the following examples of possible embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

The following description is presented to enable any person skilled in the art to make and use the embodiments, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. Thus, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

LAPSI MS: The present invention shows that using a green laser pointer (532 nm, <10 mW) along with standard paper spray mass spectrometryresults in a new ambient ionization technique called laser assisted paper spray ionization mass spectrometry (LAPSI MS). Figure 1 shows the experimental setup whereas Thermo scientific LTQ XL mass spectrometer was used for all the mass spectrometric measurements. A Whattman 42 filter paper was cut into a triangular shape and was connected to a high voltage power supply through a copper clip in such a way that one of the vertices was pointed towards the inlet of the mass spectrometer. The average area of the paper taken was 3.2 cm ² for all the experiments. The distance between the tip of the paper and the mass spectrometer inlet was set to be 10 mm in all the cases. Laser was placed vertically at a distance of 10 cm from the paper. The laser spot was pointed at the tip of the paper and the visible spot diameter was approximately 3 mm. Although the laser runs on a battery, it was connected to a switch mode power supply (SMPS) to give constant input power, enabling constant laser intensity throughout the experiments. The spray voltage was varied from 1 kV to 5 kV depending on the analyte. Capillary and tube lens voltages were set to ±45 V and ±100 V for positive and negative modes, respectively. Capillary temperature was 250° C and the sheath gas pressure was set to zero. The solvent or the analyte solution was continuously supplied to the paper through a fused silica capillary (300 μηι outer diameter, 100 μηι inner diameter), connected to a 500 μL Hamilton gas tight syringe, using a syringe pump. Solution infusion rate was optimized by trial and error method to be 8μL/min, for all our experiments. Methanol, toluene and mixture of these two solvents at different proportions were used as solvents for the present experiments.

Synthesis of Au NPs: Citrate protected Au NPs were synthesized by reduction of HAuCl ₄ .3H ₂ 0 with trisodium citrate using Turkevich method (Kimling et al, 2006). About 5 mM 10 mL aqueous solution of HAuCl ₄ .3H ₂ 0 was taken and diluted with 180 mL of distilled water followed by heating until it started boiling. An aqueous solution of trisodium citrate (10 mL, 0.5%) was added to it and then heated again till the solution become wine red in color. The solution was cooled and used directly as catalyst for in-situ monitoring of laser induced transformations. Synthesis of Ag NPs: The Turkevich method was used for the synthesis of citrate protected Ag NPs (Turkevich, Stevenson, & Hillier, 1951). About 40 mg trisodium citrate was added to ImM aqueous solution of silver nitrate at 100° C and the solution was heated until a pale yellow color was observed. The final solution was cooled and used in LAPSI MS. Monitoring in-situ chemical reactions: Citrate protected Au NPs and Ag NPs were drop cast over the filter paper and kept for drying under laboratory conditions. It was cut into triangular shape for LAPSI MS measurements. Spray voltage was set to be 3 kV during the experiment. In the case of decarboxylation reaction (Chakraborty et al, 2016) an ethanolic solution of MBA (50 pM) was used as reactant. For the dehydrogenation reaction (Tsai, Chung, Chou, & Hou, 2008), 50 pM solution of 2,3-dihydro-lH-isoindole in methanol was pushed directly to the paper source and the mass spectrum was collected in presence and absence of the laser.

Detection of adulteration in coconut oil: 1 : 1 (V/V) ratio of methanol and toluene was used to make the solution of both paraffin oil and coconut oil. Pure coconut oil was used as standard sample. Standard mixtures were made by mixing paraffin oil (1, 5, 10, 20 and 30%) in a solution of coconut oil. Experiments were conducted in two different ways. In the first case, spotted the oil on the paper source and then eluted with a solvent mixture to collect the mass spectrum. In the second case, the solution of oil mixture was fed to the paper source continuously. Characterization of LAPSI MS: In standard paper spray, molecules like benzene cannot be ionized. However, LAPSI MS gives a clean spectrum for benzene with isotopic distribution (Figure 1). The peak corresponding benzene disappeared completely in the absence of laser. This shows that LAPSI MS can be a potent method to ionize hydrocarbons which cannot be ionized easily in standard paper spray condition. The same methodology can be applied to other aromatic hydrocarbons (PAH). Figure 2

A, B and C show the mass spectra collected for naphthalene, pyrene and benzanthracene, respectively in positive mode, where the peaks at m/z 128, 202 and 228 represent the molecular cations of the respective analyte. Inset shows their isotopic distributions in all the cases. It clearly shows that LAPSI MS has the capability to ionize a certain type of molecules in their molecular ion form and polycyclic aromatic hydrocarbons are one such category.

The effect of the laser induced enhancement in ionization is dramatic in the case of C ₆ o. Figure 3 illustrates 10 fold enhancements in the molecular ion intensity of C ₆ o in presence of laser. The laser was switched on and off in a periodic fashion approximately in the same time interval. The chromatogram in Figure 3A is a selected ion chromatogram for the peak at m/z 720 (molecular anion of C ₆₀ ). It clearly reflects the intensification of the C ₆ o signal intensity in presence of laser. Figure 3B and 3C allow us to compare the mass spectrum taken for C ₆ o in toluene (1 mg/ml or 1.39 mM) in laser-off and laser-on conditions where we clearly see the signal intensification. The intensity of the molecular ion is so high that we cannot see any background in this case. These results indicate that the LAPSI MS method works well both in positive and negative ion modes.

LAPSI MS is an ambient ionization technique involving low power laser along with high voltage of the standard paper spray method. The mechanism of ionization is different from the usual laser based ionization techniques where a high energy laser is used to produce ions such as matrix assisted laser desorption ionization (MALDI) (Hillenkamp, Karas, Beavis, & Chait, 1991), laser desorption/ionization (LDI) (Vidova et al, 2010), or techniques where laser and electrospray are in combination such as, electrospray assisted laser desorption ionization (ELDI) (Huang, Hsu, Lee, Jeng, & Shiea, 2006), and laser ablation electrospray ionization (LAESI) (Nemes & Vertes, 2007). It has also been shown that UV pulsed laser produces soft ionization during DESI imaging due to the solvent cage effect (Lee, Jansson, Nam, & Zare, 2016). In order to understand the mechanism of the observed ionization event, ion yield was measured from the selected ion chromatogram of various analytes with increasing electric field. Plot of normalized intensity vs applied potential shown in Figure 4 proves the direct correlation between ionization potential (IP) and applied voltage.

The field assisted photoionization can be explained with the example of benzene. At 532 nm, the available photon energy is 2.33 eV. Therefore, without the assistance of the electric field, the photon energy is inadequate to cause ionization as the IP of benzene is 9.17 eV. For an applied potential of 4.8 kV, which can be termed as threshold potential, the benzene molecules

7 1

feel an electric field of more than 10 Vcm ^" at the sharp tip of the triangular paper (Espy, Muliadi, Ouyang, & Cooks, 2012). This is due to the fact that the molecules are trapped in- between the fibers of the paper. This electric field causes a destabilization of the ground state to a large extent, which brings down the IP to 2.33 eV. Depending on the n system of the molecule, the threshold voltage needed for ionization varies. From Figure 4, it shows that the threshold voltage is decreasing upon increasing the n electron density in the molecule. In the case of benzene, the threshold is around 4.8 kV and at the same time, benzanthracene needs only around 2.5 kV to start ionization.

Density functional theory (DFT) study was useful in understanding electronic properties such as IP of the analytes. Field dependent IP was calculated using different methods(Davari, Aastrand, Unge, Lundgaard, & Linhjell, 2014; Deleuze, Claes, Kryachko, & Francois, 2003). In the present invention, the field dependent IP was calculated at B3LYP/ cc-pVDZ level of theory. Figure 5 (A to E) show the energy optimized structures for benzene, bromobenzene, naphthalene, pyrene and benzanthracene and their orientations with respect to the applied electric fields.

Figure 6 shows the IPs for all the analytes in presence of electric field ranging from 0 to 500 MV/cm along all the three axes. Due to the high IP of benzene (9.17 eV, calculated in zero field), it required a larger electric field (up to 450 MV/cm) for ionization. In general, for all the molecules, the influence of electric field on the IP along the z axis is less in comparison to other axes. This may be because the molecules are planer and the field is normal to that plane.

Although IP at low electric fields (up to 100 MV/cm) only differ slightly along the x and y axes for benzene, the calculated IP is invariant at higher electric fields as shown in Figure 6A and it is attributed to the high molecular symmetry (D _6h ) of benzene. When the field reaches 408 MV/cm, the IP is reduced to 2.3 eV in both the axes. Hence, benzene exhibits ionization when laser is applied at this electric field. In support to the speculated mechanism, other analytes are studied follow a similar fashion. Figure 6 (B to E) show a clear decrease in the threshold ionization voltage with increasing electron density in the system.

Control experiments were performed to check the importance of laser in LAPSI MS. Figure 7 shows that there is no ionization in the absence of laser even up to 5 kV of spray voltage. But ionization occurs in presence of laser. Figure 7 (A to C) also shows a comparison of LAPSI with ESI and PSI using benzanthracene as the analyte (all the parameters were exactly the same in all the cases).

ESI as well as standard PSI do not show a peak at m/z 228 due to benzanthracene while LAPSI shows an intense signal. Hence, both ESI and PSI are blind to the molecule whereas our method can see it. This result clearly indicates that the ionization mechanism of LAPSI MS is significantly different from ESI and also from field enhanced ionization of PS.

The enhanced ionization of C ₆ o in the negative mode follows a one electron capture mechanism. The ionization involves the solvent ionization first followed by capture of the released electron by the unionized C ₆ o molecule. Hence the ion number density in the spray gets increased. Figure 8 show that the enhancement in signal intensity in the case of C ₆ o in the negative mode equals approximately the intensity enhancement for toluene in positive mode, in presence of laser. These results support the speculated mechanism of electron capture by C ₆ o. For further confirmation of the mechanism, C ₆ o was taken in two different solvents, dichloromethane (DCM) and chloroform which are unresponsive to LAPSI MS in the studied potential range. Figure 9A and 9B present the results where one can see from the selected ion chromatogram that the intensity of m/z 720 remains constant during the laser on off condition in both the solutions of C ₆ o _, but was increased in toluene (Figure 9C). This confirms the mechanism.

Monitoring in-situ photochemical reaction by LAPSI MS: LAPSI MS provides additional advantage as it involves irradiation of a laser along with paper spray which allows one to monitor photochemical reactions. Figure 10 presents dearboxylation of p-M A over Ag nanoparticles-coated paper. During the laser off condition, the detected mass peak at m/z 153 corresponds to p-MBA [M-H] in the negative ion mode over Ag NPs coated paper (Figure 10B). But when the laser was turned on, a peak at m/z 109 which corresponds to the decarboxylated product of p-MBA appeared (Figure 10A). Similar result was seen in the case of Au NPs-coated paper (Figure 11).

Dehydrogenation reaction was performed on 2,3-dihydro-lH-isoindole in presence of the laser. Figure 12 shows the mass spectrum of the reaction during laser on and off conditions. The peak at m/z 196 is the molecular ion of 2,3-dihydro-lH-isoindole in the positive ion mode during the laser off condition and when laser was turned on, the molecule loses two hydrogens to get aromatized and the product peak at m/z 194 was formed.

Detection of hydrocarbons as adulterants in coconut oil by LAPSI MS: In order to use this methodology in an analytical context, we chose to study the adulteration of vegetable oils by mineral oils, which is a common problem in many countries. As shown in Figure 13, LAPSI MS was able to detect the presence of paraffin oil in commonly sold coconut oil, purchased from the market. It is important to note that paraffin oil is undetectable in conventional paper spray or by electrospray ionization. By preparing standard sample and correlating the intensity of the selected ion at m/z 429 (Figure 14) which corresponds to one of the peaks present in the mass spectrum of paraffin oil, its contamination in the real sample was estimated to be about 10%. Figure 15 provides the chromatogram and the mass spectrum of pure paraffin oil by LAPSI MS. Envelop mass peaks in the spectrum corresponds to the hydrocarbons present in the paraffin oil. Figure 16 represents the mass spectrum taken for the pure coconut oil, where the first branch of mass peaks corresponds to the lipids present in the coconut oil. The next brunch of lipids is the dimers of the same.

Thus the present invention provides a new form of paper spray mass spectrometry called laser assisted paper spray ionization mass spectrometry (LAPSI MS) which is useful for the detection of saturated and unsaturated hydrocarbons, by including a simple 532 nm, <10 mW green pointer laser to it. This method extends the scope of ambient ionization. This invention also shows that the photoionization is field assisted from various control experiments. Solvent induced ionization enhancement of C ₆ o has also been demonstrated. The present invention shows the capability of the system to monitor some of the photoinduced transformations in-situ which makes LAPS MS more useful. This technique can be expanded to routine analytical situations. This analytical methodology will be useful for the easy detection of analytes in diverse situations. As ionization is photo-assisted, use of other light sources is possible by varying the threshold potential used for ionization.

It may be appreciated by those skilled in the art that the drawings, examples and detailed description herein are to be regarded in an illustrative rather than a restrictive manner.