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 * Miniaturization of Mass Spectrometry and Development of Paper Spray Ionization**

Harrison Clark Drexel University Department of Chemistry 12/3/2011

The need for new types of chemical analysis increase as we learn more about our natural world and of the industrial world that we create. One of the most usefule quantitative and qualitative analysis methods is mass spectrometry. Mass spectrometry allows for analysis of both chemical and biological samples. There are a variety a mass spectrometry methods available as well as different kinds of mass spectrometers. The fundamental process of mass spectrometry is a sample is ionized by one of many processes, the ions flow into the mass spectrometer where they are then analyzed a compared by their mass-to-charge ratio (//m/////z//). The ions are collected and recored in a plot based on the intensity of the signal in comparison to the mass-to-charge, which is known as a mass spectrum(1). Interpretation and comparisons of the mass spectra will then provide identification of the compounds present as well as the concentration. Despite the advantages of high sensitivity and specificity, fast analysis times, and overall versatility of a mass spectrometer there are a few drawbacks. Sample preparation time can be long compared to the time for analysis. Mass spectrometer can be large and are a high cost pice of lab equipment.
 * Introduction**

In efforts to reduce costs as well as analysis time in field or in situ analysis has become necessary for mass spectral analysis. The key way to proceed is to eliminate the need to collect samples, prepare the samples, and then send the samples to laboratories for analysis. Instead the analysis could come to the area of collection by miniaturization of the mass spectrometer. The development of miniature mass spectrometers would come about by reducing the size of the key components of the mass spectrometer which are the mass analyzer, vacuum pumps and the sample interface(2).

The other half of the problem is to develop analysis methods that work well in many different situations. In the field there are many things that can not be planned for, but with a method that is robust enough to handle different samples would be efficient. One such technique that will be described is a new a emerging method of direct sample analysis called paper spray ionization. Paper spray (PS) makes use of the conductivity that paper has when it is wet to produce ions with an applied charge, but the main advantages to using the paper spray method is that it requires very little sample preparation, analysis time is fast, and it can reduce the high amount of training required to run a typical mass spectrometer allowing on site personnel to do the analysis.

What will follow is a description the steps involved in the miniaturization of the mass spectrometer from the early designs to what currently there is to offer. Then the development of the paper spray technique and the possibilities it has to offer.

In the early 1990's the first efforts were being made to produce smaller mass spectrometers. Though the size of the instrument was going to be reduced the effectiveness of the instrument did not want to be compromised too much. The limited mass range of a mass spectrometer was found to increase by reducing the size of the ion trap, but it also came with decreased resolution and produced artifact signals(3). A full scale reduction of a spectrograph was achieved in 1991 by reducing the size of the magnetic sector. The reduction of the magnet was done by making use of a new magnetic alloy Nd-B-Fe. The spectrograph weighed less than 10 kg which allwed for the instrument to be deployed in the field(4). It was found that simple geometry of the mass spectrometer could reduce the size of the instrument and still retain adequate performance. Precision machining allowed for more careful and precise manufactoring of the mass spectrometer as well which in turn allowed for geometries that were harder to impliment.
 * Mass Spectrometer Miniaturization**

The other major component of a mass spectrometer is the vacuum pumps used to lower the pressure in the mass spectrometer. The vacuum pumps play a major role in the cost and the size of the instrument. Early strategies for reducing the vacuum pumps was to pump down the instrument which was then removed from the rough pump. While in the field the mass spectrometer maintained the pressure with smaller ion pump during operation. Although it would allow the mass spectrometer to function at the desired pressure it was not sustainable and would eventually have to be reattached to the rough pump to maintain the vacuum. This limited the range and poratability of the mass spectrometer(2). Eventually manufactorers started to produce small cale rough pumps that were similar to their larger counterparts for the lab-scale mass spectrometers. Vacuum pumps can be reduced down to roughly three pounds and still maintain a vacuum of 10^-8 Torr, which far exceeds the pressure needed for a miniature mass spectrometer. Quadrupole ion trap mass spectrometer can maintain an optimal setting at 1 mTorr(5).

After the early attempts at modifying mass spectrometers to produce a more portable and easy to use mass spectrometer system, all types of mass analyzers were considered for miniaturization from the basic quadrupole systems to time-of-flight (TOF)(6,7). A linear ion trap mass spectrometer was developed with power consumption of only 79 watts. It was able to produce uinit resolution with a mass range of 500 //m/////z//(8). Later development of a portable mass spectrometer with interchangeable ionizers and capability of tandem mass spectrometry were developed(9). The ion trap mass spectrometer proved very advantageous in miniaturization because it works at a higher pressure theh other ionization methods, it is capable of tandem mass spectrometry in a single instrument which gives a big reduction in size of the instrument and power consumption require(2).

Development in the area of the interface for sample analysis was also part of the miniaturization of mass spectrometers. The typical methods of sample introduction from full lab-scale mass spectrometers were adapted to use with the minature mass spectrometers. There was a portable version of the gas chromatograph-Mass spectrometer (GC-MS) (10). The ambient ionization techniques were better suited then some of the other typical sample introduction methods. The sampling would be done at atmospheric pressure most of the time when using a portable device, so sample introduction methods like electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure MALDI were appropriate introduction methods applied to the miniature mass spectrometers. Applications such as analyzing non-volatile compounds found in explosives and biomolecules from solution were able applications that a miniature mass spectrometer would be useful to analyze(2). There is still work to be done the miniature mass spectrometer has not yet been developed that contains a pumping system to obtain atmospheric pressure that will allow the flow of ions from the ambient region in to the mass analyzer. A miniature mass spectrometer with the right weight, size, pumping speed and efficiency of ion transfer is still the goal unattained(2). However, a few methods do seem to be coming closer to that goal. The development of the discontinous atmospheric pressure interface (DAPI) has been developed for a quadrupole ion trap instrument(11). The DAPI method along with other methods such as desorption electrospray ionization (DESI) are being implimented on miniature mass spectrometers and look promising for the advancement of the miniature mass spectrometer(12).

The traditional mass spectrometer is reaching it's limit of applications. The new applications see the need for specialized mass spectrometers and the miniature mass spectrometer a pushing into a new area for analysis. The specialized ionizaton techniques are also moving a head(13). Ionization methods like the DESI and DAPI lead to another method that allows for many of the qualities associated with the portable mass spectrometer. Paper spray looks to be the answer to ionization of biofluids, illicit drugs, foods, and pharmaceutical drugs with a common ionization soure of paper.

Direct sample ionizatoin has shown to come in a number different techniques. The desired abilities of a direct sampling technique is for it to be fast, reialbe, and easy to use. Portable mass spectrometry promotes the in situ approach to analysis. Allowing more analysis to be performed in the field greatly reduces the cost of laboratory expenses and drastically reduces the time for analysis. The miniaturized mass spectrometers also allw for less tehcnical training to be required to run the instruments. Paper spray (PS) ionization has become a powerful techniqure to emerge in the past few years. Coupled with the advances in portable mass spectrometers paper spray is able to take direct sample ionization further than of the other currently available techniques.
 * Paper Spray Ionization**

Paper spray mass spectrometer takes the porous material of paper, which is just interconnecting channels of cellulose and uses these channels to transfer analyte through the paper which is then ejected in a plume and into the mass spectrometer. A mascroscopically sharp point is wicked through the porous material for the analyte transfer. A high electric field applied across the paper ionizes the molecules as they travle through the paper. The paper is cut into a trianglular shape to force the accumulation of ions towards the tip of th paper. This creater the plume similar to that of elecrospray ionization. No pneumatic assistance is need for transport of the analyte through the paper, the high voltage supply applied to the wet paper is enough to cause the transport of the ions(14). Paper is readily available at a low cost which makes it the ideal as an ion source. Paper also has chromatographic properties. Paper is able to separate different substances with the right solvent added.

The paper spray method has undergone many experiments to find the ideal conditions for the paper spray technique and also the uses that paper spray has. Described below are a handful of experiments about the characterization and applications of paper spray ionization.

There are several choices of paper available for paper spray. A small sample of six typcial types of paper samples would be collected on were tested to see how they respond to the paper spray technique. Four different types of filter papers were chosen grade 1 (11 μm), grade 2 (8 μm), grade 595 (4-7 μm), and grade 6 (3 μm). The other two types of paper were glass microfiber filter paper and chromatography paper. The solver used on all papers as a mixture of methanol:water (1:1). The methanol:water mixture was added to th paper in 10 μL amounts containing 200 ng/mL of cocaine. All paper types were cut to the triangular form with the same dimensions. Each paper type produced a different background with peaks of different intensities. The glass microfiber paper produced peaks that were two orders of magnitude lower than the other types of paper. Also, the cociane peak at 304 //m/////z// ws unable to be identified with the microfiber paper and was deemed to be the poorest performing of the group. The chromatography paper with a thickness of 0.18 mm gave the highest signal-to-noise raito of the group. It was found that the paper could be rinsed and re-used to yield reproducible spectra(15)
 * Experimental - Types of Paper**

The paper is not in direct contact with the mass spectrometer inlet, but the spray produced by the high voltage ejects from the tip of the triangle towards the mass spectrometer, so it was necessary to optimize the relative position of the paper triangle to the inlet of the mass spectrometer. The paper was mounted so that is was moveable in the x and y directions. The paper was adjusted by 8 cm in the y direction and 3 cm in the x direction in 2 mm increments. A continuous spray of the cocaine spiked methanol:water mexiture was applied to the paper, the chromatography paper was used throughout the following experiments because it gave the best performance in the paper test. As the high voltage was applied the protonated cocaine peak was observed with the intensity recored in relation to the position of the tip of the triangle. The optimal x and y positions fell with in a 5 x 10 mm area(14).
 * Experimental - Position of Paper**

It is ideal that the paper accomadate as many types of molecules as possible giving acceptable results. The more versatile the paper the lower the cost of buying paper since you will be able to use one type of paper for multiple analyses. The positive and negative ion modes of aper spray were tested in this experiment as well. The modes are derentiated by the amount of voltage applied to the paper. Positive ion mode was set at 4.5 kV and the negative ion mode was set at -3 kV. A variety of molecules were chosen for the postive ion test which included serine, braydkinin 2-9, cyctochrom C, atrazine, and roxithromycin. Cytochrome C required the addition of 1% acetic acid to aid in the ionization process, but the rest of the molecules were treated with the methanol:water mixture. The ionization produced multiple protonated species typical of proteins and peptides. A good signal-to-noise ratio was obtained for most of the molecules at the 1 μg/mL level. The tandem MS produced good signal-to-noise for species at lower concentrations. A limit of detection was found to be 1 ng/mL. The negative ion mode produced very different results. Analytes were undetactable as well as the plume cloud from the tip of the paper. It is still unknown as to why it behaved this way, but further study and interest is expressed into understanding the cause of the lack of ions(15).
 * Experimental - Paper Versatility**

The real application of the paper spray method would be ta take raw untreated samples and perform in situ analysis. Blood samples are commonly collected as dried blood spots (DBS). They are capable of being stored for months without any special requirements. The dried blood spot papers are used for analysis of therapeutic drugs and other screen conditions(16). The paper spray analysis of dried bood spots applied a spked blood sample directly to the center of the paper which wasthen dried for 1 to 2 minutes. The methanol:water solution was then applied to the base of the paper triangle. 4.5 kV was applied to the paper for positive ion mode. The bovine blood samples were spiked with atenolol. The signal-to-noise ratio achieved was roughly 4 with MS at a concentration around 1 μg/mL. The tandem MS was able to produce higher signal-to-noise at lower concentrations. It was assumed that most of the analyte transferred through the paper into the mass spectrometer inlet leaving the majority of the blood in the paper. The methanol:water solvent was thought to carry the ions through the paper and leave the blood behind(15).
 * Experimental - Raw Samples**

A more recent experiment for DBS was perfomred with a more quantiative approach by the additon of an internal standard. Making use of the paper spray ionization method a single set analysis approach to the DBS samples for monitoring therapeutic drugs was tested. There are many benefits for DBS sampling with a method like paper spray over the traditional HPLC-MS methods. If DBS are found useful for analysis with a fast and reliable method this allows for greater convenience for the collection of samples. Instead of blood draws finger pricks could be used to get enough for a full analysis. Results can be made available to the patient immediately. Sampling time can vary at convenience. The reduction of biohazard material with the small amount of sample required(17). The blood cars were used as the paper material and cut into triangles of 6.5 mm x 9 mm. The internal standard was first added to the paper in 2 μL aliquots. THe therapeutic drugs for monitoring were amitriptyline and sunitinib. The internal standards for these molecules were the deuterated species, [ 2 H 6 ]amitriptyline and [ 2 H 10 ]sunitinib. The spiked bovine blood was applied on top of the internal standard. The paper was then allowed to dry. Before analysis each samples was treated with 25 μL of 95%methanol:5%water solvent. The internal standard for anitriptyline produced acceptable results in the normal therapeutic range of 50-200 ng/mL. Subitinib also performed with acceptable accuracy and precision from 1-500 ng/mL with a normal therapeutic range of 50-100 ng/mL. The experment proved that a one step sample preperation technique can perform quantitative analysis. The dried blood spot analysis proved to be low cost, fast, and gave results that were precise and accurate on a routinley basis(18).

Urine analysis is also an application for paper spray mass spectrometry. The analysis of illicit drugs in urine was another test for the use of paper spray. Dried urine spots were spiked with 0.4 μL raw urine. The MS and MS/MS analysis of the urine samples were almost instantaneous with the paper spray method. The only sample preparation required was the addition of the methanol:water solution. Heroin in the urin was detectable at 125 ng/mL and was easily identified from the fragmentation in the MS/MS which also produced good peak intensity. Other ambient methos such as atmospheric pressure chemical ionization-MS and Direct ESI do not provide good results with raw or diluted urine. The urine analysis shows again that the paper spray method is a good application ofr biofluids even with the limited sample preparation.(15).

The use of paper allows for samples to be collected by wiping off a surface. An experiment was designed to place a known amount of heroin on desktop, which was then wiped with the triangular cut paper pre-wetted with a mixture of methanol:water. The positive ion analysis indicated that 50 ng/mL of heroin was present on the desktop, which also contained dust and other chemical residues with little interferance. Heroin identification was found to be as low as 100 pg with the wiping method and tandem MS fragmentation. An investigatoin into the wiping method on other surfaces was performed with analysis of a lemon peel.(15). Thiabendazole and imazalil were two fungicides identified from the analysis. Other agrochemical analysis have been perfomred by methods such as DESI(19). The wiping is able to collect more analyte by analyzing a larger area as opposed to the direct analysis of the DESI coupled instrument. The pak intensity for the thiabendazole was larger for the paper spray method.
 * Experimental - Sample Wiping**

Direct sample analysis by the paper spray technique has proved that it is capable of functioning in many areas with many different sample matrices. It shows that limited sample preparation can still obtain acceptable results. The combination of paper spray with mass spectrometry should allow for in situ analysis by the appropriate personnel with proper training. The clinical use for paper spray for therapeutic drug monitoring and dried blood spot screening are promising for the future of the method.The impact of paper spray is also seen in more environmentally friendly ionization with that of leaf ionization, which uses the leaf similar to the paper as the substrate for the ionization to take place(20).The mechanisms involved in the sample transport and ionization of paper spray are not yet completely understood, but are being pursued for further investigation. The method of analysis for complex mixtures is available with simple and rapid analsyis.
 * Conclusion**

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