Ionization source for mass spectrometry analysis

[0001] This invention relates to the field of mass spectrometry, and more particularly to improvements in the chemical ionization source to be applied to mass spectrometers.

[0002] A variety of ionization sources, for the analysis of molecules with medium-high molecular weight (like peptides and proteins) are essential components of modern mass spectrometric instruments. The ionization source transforms neutral molecules into ions which can be analyzed by mass spectrometry.

[0003] A mass spectrometer generally has the following components:

[0004] (1) a device, usually a Liquid Chromatograph, for the separation or de-salting of the molecules contained in a sample;

[0005] (2) an ionization source, contained in a chamber, to produce ions from the analyte;

[0006] (3) at least one analyzer or filter which separates the ions according to their mass-to-charge ratio;

[0007] (4) a detector that counts the number of the ions;

[0008] (5) a data processing system that calculates and plots a mass spectrum of the analyte.

[0009] The mass spectrometry techniques currently used for the analysis of macromolecules and, especially, proteins and peptides are based on the Electrospray Ionization (ESI) (U.S. Pat. No. 5,756,994; Cunsolo V, Foti S, La Rosa C, Saletti R, Canters G W, Verbeet M. Ph. Rapid Commun. Mass Spectrom. 2001; 15: 1817; Wall D B, Kachman M T, Gong S S, Parus S J, Long M W, Lubman D M. Rapid Commun. Mass Spectrom. 2001; 15: 1649; Fierens C, Stöckl D, Thienpont L M, De Leenheer A P. Rapid Commun. Mass Spectrom. 2001; 15: 1433; Li W, Hendrickson C L, Emmett M R, Marshall A G. Anal. Chem. 1999; 71: 4397; Fierens C, Stöckl D, Thienpont L M, De Leenheer A P. Rapid Commun. Mass Spectrom. 2001; 15: 451) and Matrix Assisted Laser Desorption Ionization (MALDI) (U.S. Pat. No. 5,965,884; Cozzolino R, Giorni S, Fisichella S, Garozzo D, La fiandra D, Palermo A. Rapid Commun. Mass Spectrom. 2001; 15: 1129; Madonna A J, Basile F, Furlong Ed, Voorhees K J. Rapid Commun. Mass Spectrom. 2001; 15: 1068; Basile A, Ferranti P, Pocsfalvi G, Mamone G, Miraglia N, Caira S, Ambrosi L, Soleo L, Cannolo N, Malorni A. Rapid Commun. Mass Spectrom. 2001; 15: 527; Galvani M, Hamdan M, Rigetti P G. Rapid Commun. Mass Spectrom. 2001; 15: 258; Ogorzalek Loo R R, Cavalcali J D, VanBogelen R A, Mitchell C, Loo J A, Moldover B, Andrews P C. Anal. Chem. 2001; 73: 4063).

[0010] Both techniques are highly effective for the production of ions of biomolecules in the gas phase, to be subsequently analyzed by Mass Spectrometry (MS).

[0011] In the case of ESI, multicharge ions of medium/high molecular weight compounds are produced. The mass of macromolecule compounds is then obtained using specific software algorithms.

[0012] Mass spectrometry represents an essential technology in the analytical field. It is usually coupled with other separative techniques, so as to identify chemical compounds and quantify complex biological mixtures. Proteins, for instance, are first separated, collected and then digested with Trypsin. The masses of the resulting peptides are determined by mass spectrometry (normal scan MS or tandem mass spectrometry MS/MS). In the case of the MS/MS approach, peptide ions of a single m/z ratio are fragmented by collision induced dissociation (CID) and then analyzed using various mass analyzers (triple quadrupole, ion trap, Fourier transform-ion cyclotron resonance). Each peptide gives origin to specific mass patterns for a given amino acid sequence. The peptide sequences can be obtained by computer analysis of the data using a dedicated software (database search and de novo sequence software). In order to obtain good MS/MS spectra doubly charge peptide ions are preferably fragmented (Cramer R, Corless S. Rapid Commun. Mass Spectrom. 2001; 15: 2058). The electrospray and MALDI techniques when are applied to the analysis of peptides with high molecular weight (2000-4000 Thompson (Th)) using the MS/MS approach have some limitations. For instance, when proteins or peptides with high molecular weight are analyzed, ESI multicharge ions are produced. These ions give rise to complex fragmentation spectra, difficult to interpret. For this reason only peptides with a maximum of 15 amino acidic residues can be analyzed by tandem mass spectrometry. In the case of MALDI only mono-charge ions are usually obtained. If the MALDI source is coupled with Time of Flight Mass Analyzer (TOF) the technique used to fragment the ions is the post source decay (PSD). This fragmentation technique give rise to some additional problems; in order to obtain good fragmentation spectra it is usually necessary to use peptide derivatization. A MALDI atmospheric pressure source has recently been coupled with an ion trap analyzer. This configuration makes possible the structural analysis of peptides by MS/MS and MS³. However, it must be emphasized that the MALDI source produces, mainly, mono-charge peptide ions that produce fragmentation spectra more complex and less specific than those obtained by fragmentation of the bi-charge ions.

[0013] Another problem that affect both MALDI and ESI techniques is represented by the decrease in sensitivity when salts are present in the sample. In the case of ESI the problem may be solved by coupling the mass spectrometer with a pre-analytical separation step, such as by the use of an High Performance Liquid Chromatographer (HPLC) or other de-salting techniques. This obviously introduces another step in the whole procedure of analysis. The HPLC technique on the other hand cannot be used for the case of MALDI because in this case it is necessary to co-crystallize the analyte with a matrix molecule. Salts contained in the sample must, however, be eliminated before of the crystallization step by well known additional treatments of the sample.

[0014] The present invention is based on the introduction of a device for the ionization of neutral molecules in the gas phase. The device comprises an active surface carrying element that, according to this invention, is inserted in the ionization chamber. This technique has been named by us “Surface Activated Chemical Ionization” (SACI). SACI technique allows the ionization to be performed at atmospheric pressure.

[0015] Use of an atmospheric-pressure ionization has already been proposed and is known as the APCI technique. APCI instrument makes use of a needle-shaped corona discharge electrode inserted inside the ionization chamber. However, the high energy of the corona discharge electrode leads to the macromolecules fragmentation. The main problem of this method is the lower sensitivity with respect to ESI and MALDI techniques.

[0016] We have now surprisingly found that introducing into the ionization chamber a plate-like active-surface carrying element can bring to unexpected results in term of high sensitivity and possibility to detect molecules having a molecular weight in a broad range of values.

[0017] According to the invention, the solution containing the analyte is injected in the SACI source through an inlet aperture. The sample is nebulized by a gas flow and vaporized by heating. The ionization chamber contains an active surface carrying element onto which the vaporized molecules of the analyte bump, so that the analyte becomes ionized. This active surface can be made of various materials (steel, glass, quartz etc), both electrically conductive or not. Different molecules can also be bound or absorbed over the surface to improve the ionization process (H₂, D₂O and various acid and basic molecules). The analyte neutral molecules which are present in gas phase are ionized by various physical-chemical interactions which take place on the surface. Surface properties and function in catalyzing various kind of reactions is well known (U.S. Pat. No. 5,503,804; U.S. Pat. No. 5,525,308; U.S. Pat. No. 5,856,263; U.S. Pat. No. 5,980,843).

[0018] An interesting use of a surface in mass spectrometry is the Surface Enhanced Laser Desorption Ionization (SELDI) (U.S. Pat. No. 6,020,208; U.S. Pat. No. 6,124,137; U.S. Patent No. 20020060290; U.S. Pat. No. 5,719,060). In this case the probe of MALDI mass spectrometer carries an immobilized affinity reagent which binds the analyte on its surface. Furthermore an energy absorbing material is added to the dried sample and Laser Desorption Ionization mass spectrometry is used to analyze the sample. This technique however differs from the SACI because of the fact that the sample can be prepared in advance by deposition over the surface, so that this analysis is quite time consuming. Some ionization source make use of an electrical potential applied to a needle to ionize the sample, in gas phase, by using the corona discharge effect (U.S. Pat. No. 6,407,382; U.S. Pat. No. 5,684,300; U.S. Pat. No. 6,294,779; U.S. Pat. No. 5,750,988; U.S. Pat. No. 6,225,623; U.S. Pat. No. 5,756,994; U.S. Pat. No. 20020074491; U.S. Pat. No. 20020048818; U.S. Pat. No. 20020011560; U.S. Pat. No. 4,849,628).

[0019] The use of the SACI ionization source which is disclosed in this invention, represents a key improvement for the production of ions with high molecular weight and low charge (bi-charge ions are usually much abundant). The innovative aspect of this invention over the previous known art can be so summarized:

[0020] a) Analytes with higher molecular mass can be studied since the technique is able to generate ions with high molecular weight and low charge, an essential feature useful for obtaining the mass of macromolecule compounds. Best results can be obtained if the source is coupled with a mass analyzer with high mass range like Fourier Transform—Ion Cyclotron Resonance (FT-ICR) or Time Of Flight (TOF).

[0021] b) A higher sensitivity can be obtained in the analysis of molecules with high mass and low charge (typically bi-charge ions). This is particularly useful for analyzing biological compounds, like proteins and peptides, which are frequently present at low concentration in biological samples (tissues, urine, etc).

[0022] c) The new technique makes it now possible to analyze molecules with medium/high mass and low charge (typically the bi-charge ions), by the MS/MS approach. This feature is useful to characterize proteins and high molecular weight peptides. In fact we have shown that peptides containing more than 15 amino acidic residues can be studied. This is particularly useful for the characterization of peptides with high mass, originated by missed cleavage during the enzymatic digestion reaction.

[0023] d) The SACI ionization source is much less affected by the presence of salts than the ESI and MALDI sources. The new invention makes it now possible to analyze liquid biological samples, which usually contain salts or buffers, by direct infusion into the mass spectrometer without using an HPLC systems or other desalting procedures. This is particularly useful for analyzing samples in high throughput applications. Samples containing a high concentration of salts are well known to give rise to serious problem when the ESI or MALDI techniques are used.

[0024] Table 1 summarize the critical improvements obtained by the application of SACI vs ESI technique.

[0025]

TABLE 1
A summary of the critical improvements obtained by
the application of SACI vs ESI techniques
SACI vs ESI
	Detect ions with high	Detect multicharge ions
	mass and low charge	with high mass
	High throughput	Pre-analytical steps limit
	“Tolerant” of salts	throughput
	Can sequence peptides with	Less tolerant of salts
	high molecular weight	Can not sequence peptides
	(more than 15 amino acid)	longer than 15 amino acid
	High sensitivity,	Higher chemical noise
	Low chemical noise	Lower sensitivity

[0049] The SACI source described in this invention and schematically represented in FIG. 1 produces ions that can be analyzed in a mass spectrometer. The spectrometer comprises the ionization source, the analyzer or filter for separating the ions by their mass-to-charge ratio, a detector for counting the ions and a data processing system. Since the structure of the spectrometer is conventional, it will not be described in more detail, but the ionization source device which is the subject of the present invention. The ionization source of the invention, on its turn, does not substantially differ, in its structure, from the known devices of this kind, so that a schematic representation thereof will be sufficient for the skilled man in this art to understand how it is constructed and works.

[0050] The ionization source device of the invention comprises an inlet assembly 11 which is in fluid communication with an ionization chamber 3.

[0051] The ionization chamber 3 comprises an outlet orifice, generally less than 1 mm in diameter, for communicating between the ionization chamber and the analyzer or filter. Generally, the angle between the axis of the inlet assembly 11 and the axis passing through said orifice is about 90°, but different relative positions can also be envisaged. Inside the ionization chamber 3 is positioned a plate 4. The plate 4 has at least one active surface 4′ which faces the internal aperture of the inlet assembly 11. Preferably, the plate 4 is inclined of an angle which allows the analyte to be reflected, once ionized, towards the outlet orifice bringing to the analyzer or filter, so that the highest number of ions can reach the analyzer (mirror effect). This will strongly improve the sensitivity of the method. The said inclination angle will depend of course on the relative position of the axes of both inlet assembly 11 and outlet orifice. For example, if such axes form an angle of 90°, the element 4 will be 45° inclined.

[0052] The plate 4 can have different geometries and shapes, such as squared, rectangular, hexagonal shape and so on, without departing for this from the scope of the present invention. It has been found that the sensitivity of the analysis increases when the active surface 4′ is increased. For this reason, the plate 4 surface will range preferably between 1 and 4 cm² and will be generally dictated, as the highest threshold, by the actual dimensions of the ionization chamber 3. While maintaining the dimension of the plate 4 fixed, the active surface 4′ area can be increased in various ways, for example by creating corrugations on the surface 4′. In particular cases, such as the case wherein low molecular weight molecules must be analyzed, high electrical field amplitude is required. In such cases, it may be advantageous to provide the active surface 4′ with a plurality of point-shaped corrugations, in order to increase in such points the electrical field amplitude.

[0053] The plate 4 gas generally a thickness of between 0.05 and 1 mm, preferably of between 0.1 and 0.5 mm.

[0054] The active surface 4′ can be made of various materials, either of electrically conductive or non-conductive nature. Preferred materials can be a metal such as iron, steel, copper, gold or platinum, a silica or silicate material such as glass or quartz, a polymeric material such as PTFE (Teflon), and so on. When the active surface 4′ is comprised of a non-conductive material, the body of the plate 4 will be made of an electrically conductive material such as a metal, while at least a face thereof will be coated with the non-conductive material in form of a layer or film to create the active surface 4′. For example, a stainless steel plate 4 can be coated with a film of PTFE. It is in fact important that, even if of non-conductive nature, the active surface 4′ be subjected to a charge polarization. This will be achieved by applying an electric potential difference to the body plate, thus causing a polarization to be created by induction on the active surface 41 too. On the other: hand, if the surface 4′ is of electrical conductive nature, the plate 4 does not need to be coated. In this case, a good performance of the ionization source of the invention can be achieved even without applying a potential difference, i.e. by maintaining the surface 4′ at ground potential and allowing it to float.

[0055] The plate 4 is linked, through connecting means 5, to a handling means 6 that allows the movement of the plate 4 in all directions. The handling means 6 can be moved into the ionization chamber and also can be rotated. The connecting means 5 can be made of different electrically conductive materials and can take various geometries, shapes and dimensions. Preferably, it will be shaped and sized so as to facilitate the orientation of the plate 4 in an inclined position. In this case, the connecting means 5 will have a step-like shape (as shown in FIG. 1). The plate 4 is electrically connected to power supply means 20 in order to apply a potential difference to the active surface 4′.

[0056] Coming now to the description of the inlet assembly 11, the liquid sample containing the analyte is introduced into the chamber through the sample inlet hole 10. The inlet assembly 11 comprises an internal duct, open outwardly via the said inlet hole 10, which brings to a nebulization region 12. The said nebulization region is in fluid communication with at least one, typically two gas lines 14, 15 (typically, the gas is nitrogen) which intercepts the main flow of the sample with different angles, so that to perform the functions of both nebulizing the analyte solution (angle >45°) and carrying it towards the ionization chamber 3 (angle <45°). Downstream to the said nebulization region. 12, a heating region 13 is provided. The heating region 13 comprises heating means, such as a heating element connected to a power supply connector 16. The vaporized analyte is thus heated at temperatures ranging from 200° C. and 450° C., preferably of between 250° C. and 350° C. The internal duct of the inlet assembly 11 ends into the ionization chamber 3 in a position which allows the vaporized and heated analyte to impact the active surface 4′ of the plate 4, where the ionization of the neutral molecules of the analyte takes place. Without being bound to any particular theory, it is likely that a number of chemical reactions take place on the surface: proton transfer reactions, reaction with thermal electron, reaction with reactive molecules located on the surface, gas phase ion molecule reactions, molecules excitation by electrostatic induction. It is also possible that the dipolar solvent is attracted from the active surface 4′ by means of the charge polarization induced on it and so provide a source of protons that react with the analyte molecules to form ions. As said before, the plate 4 can be allowed to float—only if the active surface 4′ is electrically conductive, since in this case an electron exchange flow can be established between the solvent and the surface 4′—or a potential difference can be applied. Such a potential difference, as absolute value, will preferably be in the range of from 0 and 1000 V (in practice, can range between −1000 V and +1000 V, depending on the kind of polarization that is required on the active surface 4′), preferably of from 0 and 500 V, more preferably of from 0 and 200 V. High voltage, such as about 200 V, allows the ionization yield to be increased. The possibility given by the present invention device to work both with and without a voltage to be applied to the analyte is of pivotal importance. In fact, in some instances, there are molecules that do not suffer a strong electrical field, such as the macromolecules or even some small molecules like amphetamines, which degrade in such strong conditions. In general, it can be said that the absence of a voltage applied to the plate 4 avoids redox reactions to the analyte.

[0057] For the reasons seen above, it is important that the solvent in which the analyte is dissolved be a dipolar solvent having acidic protons. Preferred solvents are H₂O, alcohols such methanol or ethanol, acetonitrile.

[0058] The impact angle of the analyte onto the active surface 4′ will be preferably 45° or less. Low impact angle values allow a better contact between the analyte and the active surface, thus improving the ionization performance.

[0059] In a preferred embodiment of the invention, the analyte solution also contains aminoacids such as glycine, lysine, istidine, aspartic acid and glutammic acid, which have the function of proton donors to promote the analyte ionization.

[0060] The ions so formed are reflected and directed to the analyzer 1 through the outlet orifice, as described above.

[0061] The essential feature of the invention consists in the introduction of a n active surface 4′ in the vaporization chamber 3, that enhances the ionization of the neutral analyte molecules present in gas phase. The SACI can be considered a soft ionization source, which can be of particular interest in several applications, such as in the field of drugs and anti-doping analysis.

[0062] It should be understood that the above description is intended to illustrate the principles of this invention and is not intended to limit any further modifications, which can be made following the disclosure of this patent application by people expert in the art.

[0063] The following, not limiting, examples are described to illustrate the novelty and usefulness of the invention.

[0064] A 10⁻⁷ M solution of Cytochrome C protein (MW: 12361) has been analyzed by direct infusion. FIG. 3a shows the protein signals obtained using the new SACI ionization source. The mono-charge, bi-charge and tri-charge ions were clearly detected using positive acquisition mode. This compares with results on the same solution achieved by the use of the ESI ionization source (FIG. 3b). In this latter case no multicharge distribution was detected in the 4000-14000 Th range. In fact signals obtained in this region of the spectrum by the use of the ESI ionization source are due to the chemical noise of the solvent. It is well known that the ESI ionization source cannot be used to analyze molecules with high molecular weight and low charge. Thus the ESI technique has serious limits for analyzing biological molecules with high molecular weight (like proteins). In order to overcome this limitation the MALDI ionization source is used since. The ionization source of MALDI is able to produce low charge ions in the range 1000-300000 Th. The application of MALDI technique, however, requires co-crystallization of the analyte with a matrix molecule. To ionize the sample a laser light that is mainly adsorbed by the matrix molecule is ordinary used. A micro explosion process (ablation) take place on the surface of the crystal and the excited matrix molecules ionize the sample molecules in gas phase (soft ionization reaction). For this reason a HPLC or similar on line separation methods cannot be used in the MALDI approach. It must be emphasized that the SACI ionization source is able, like the MALDI source, to generate ions with high molecular weight and low charge, but, in addition, it can be coupled in line with HPLC or other separatory methods.

[0065] Five high molecular weight standard peptides with molecular mass in the 2000-4000 Da range were analyzed. The results obtained using the SACI source are shown in FIG. 2a. As can be seen the mono and bi-charge peptide ions were clearly detected. The peptides were analyzed also by a mass spectrometer using the ESI ionization source (FIG. 2b). In this case the tri-charge peptide ions are the most abundant species. These species are located in a region of the spectrum (500-1100 Th) in which the chemical noise is high leading to decrease the S/N ratio.

[0066] The mass analyzer used to perform both experiments was an ion trap (LCQ^XP, ThermoFinnigan, USA) able to detect the signals in the 100-4000 Th and 1000-20000 Th range. The mass acquisition range can also be extended by coupling the SACI ion source with other kind of mass analyzer (for example TOF or FT-ICR) provided with a high mass acquisition range.

[0067] The SACI ionization source first described in the present invention is characterized by a higher sensitivity, as compared to the ESI technique, in the analysis of liquid samples of proteins and peptides. FIGS. 2a and 3a show the spectra obtained by direct infusion of solutions of five high molecular weight peptides (FIG. 2a) and Cytochrome C (FIG. 3a). A LCQ^XP (ThermoFinnigan, USA) provided with SACI ionization source was used. The solution concentration of each standard peptide and of the Cytochrome C was 10⁻⁷ M and the counts/s value was 10⁶ with a S/N ratio of the most abundant peak of 500 for the high molecular weight peptides and 300 for the Cytochrome C protein. The comparison of these results with those obtained, for the same solutions, using the ESI ionization source (FIGS. 2b and 3b) shows that the SACI ionization source increases the sensitivity. As can be seen for the case of the ESI spectra of the same high molecular weight peptides (FIG. 2b) the most abundant signals (tri-charge ions) are detected in the 500-1100 Th range, due to the multicharge phenomenon. Furthermore, the chemical noise is higher (S/N ratio of the most abundant peak=100) using the ESI technique than that obtained by the SACI ionization source (S/N ratio of the most abundant peak=500).

[0068] In the spectrum of the Cytochrome C, obtained by the ESI ionization source. (FIG. 3b), no protein signal has been detected in the 4000-14000 Th range. This is due to the extensive multicharge phenomenon that takes place in the ESI ionization source. For this reason the multicharge distribution is usually compressed in the 100-2000 Th range (FIG. 3c) where the chemical noise is higher.

[0069] The tandem mass spectrometry (MS/MS) of bi-charge ions, that are abundantly produced by the SACI source, can be further characterized. In FIG. 4a the SACI-MS/MS spectrum of the bi-charge ion of Vasoactive Intestinal Peptide Fragment 6-28 is shown. The bi-charge ion was isolated into the ion trap analyzer and fragmented by Collision Induced Dissociation (CID). The results of the peptide identification and its relative statistical correlation score, by the use of the SEQUEST database search program, were as follows:

[0070]

	Peptide	Xcorr	DeltCn
	Vasoactive Intestinal Peptide	3.5382	0.204
	Fragment 6-28

[0071] Xcorr is a spectra correlation score and DeltCn is the 1.0—normalized correlation score. A correctly identified peptide has a value of Xcorr score higher than 3. The peptide was also analyzed using the ESI ionization source (FIG. 4b). In this case the bi-charge peak at m/z 1409 had a too weak intensity to obtain an MS/MS spectrum. Thus, the tri-charge ion at m/z 940 was fragmented. The statistical correlation score and the DeltCn in this case were as follows:

[0072]

	Peptide	Xcorr	DeltCn
	Vasoactive Intestinal Peptide	1.2280	0.608
	Fragment 6-28

[0073] As can be seen by the Xcorr and DeltCn scores so calculated, the peptide characterization is statistically more accurate using the SACI-MS/MS spectrum obtained fragmenting the bi-charge ions at m/z 1409.

[0074] FIGS. 5a and 6a show the mass spectra of a solution of five standard peptides and of peptides obtained by Cytochrome C tryptic digestion all in 50 mmol/L NH₄HCO₃ buffer. The SACI ionization source was used. In both cases the solution concentration was 10⁻⁷ M. The counts/s value was 10⁶ and the S/N ratio was 500 in the case of the high molecular weight peptides and 450 in the case of Cytochrome C peptides. The results obtained using the ESI ionization source is shown in FIGS. 5b and 6b. As can be seen in these latter cases the mass spectra show a high chemical noise, due to the presence of the buffer. This leads to a decrease in sensitivity as compared to that obtained by the use of SACI ionization source. In fact the counts/s value was an order of magnitude lower (10⁵) and the S/N ratio of the most abundant peak (100) is 5 times lower.

[0075] In order to show that the S/N ratio is not affected by salts, FIG. 7 reports the mass spectra of five high molecular weight peptides acquired without (FIG. 7a) and with (FIG. 7b) salts in the sample solutions. The SACI ionization source was used in both cases. As can be seen salts do not lead to a decrease of the spectrum quality. This fact is very important when biological mixtures are analyzed. In fact these mixtures almost always contain salts or buffers (as for example NH₄HCO₃ used for the tryptic digestion) that give rise to well known effect on the ESI mass spectra.

[0076] FIG. 8 shows the spectra obtained by analyzing the high molecular weight peptide solutions in absence (FIG. 8a) and in presence. (FIG. 8b) of salts by the standard ESI technique. In both cases the spectra show a higher chemical noise than in those obtained using the SACI ionization source (respectively shown in FIGS. 7a and 7b). The addition of the NH₄HCO₃ buffer to the solution analyzed by the ESI technique decrease the peptide signals at m/z 1068, 1006, 778 and 954. For this very reason an HPLC or other separation steps system is coupled with the ESI ionization source. A chromatographic analysis, however, takes time and increases the number of manipulation of the sample before analysis. This is a limit especially when many samples must be analyzed.