Ion source for lower limits of detection in spectrometric measurements

Technical field

The invention concerns the fields of mass spectrometry and ion mobility spectrometry employed for characterization of substances and their mixtures. These spectrometric measurements allow to obtain information about the structure of substances and their quantity in a sample. The use of these methods involves a wide range of applications from drug development to testing of food and environmental samples to analyses carried out on space probes.

Background art

The measurement of ion mobility ¹ ' ² and, in particular, mass spectrometry ³ are analytical techniques affording a wide range of significant information about both animate and inanimate systems (e.g. the structure and quantity of metabolites in an organism, presence of toxic compounds in the environment, distribution of constituents in minerals). Without mass spectrometry, many areas of research and even other more practical activities would not be conceivable today. It is used e.g. in drug development, testing of food, monitoring of the environment, medical diagnostics, toxicology, research on proteins and other biologically important compounds. The ion mobility spectrometry has some interesting applications too, e.g. at the airport security checks.

A basic prerequisite for the characterization of compounds by the methods of mass spectrometry and ion mobility spectrometry is conversion of the species into ions. The ionization takes place in the part of instrument referred to as ion source, yielding ions that are subsequently resolved based on their properties in another part of the instrument. Ion sources are based on various principles. The ionization can occur upon interaction of the species with an electron (electron ionization), interaction with another ion (chemical ionization), by means of electric field (field ionization, electrospray ionization), laser radiation (laser ionization) etc. The ionization process can run under reduced pressure (electron ionization, chemical ionization, field

1 Borsdorf H., Eiceman G.A.: Ion Mobility Spectrometry: Principles and Applications. Applied Spectroscopy Reviews 41, 323-375 (2006).

2 Eiceman GA. : Ion-mobility Spectrometry as a Fast Monitor of Chemical Composition. Trends in Analytical Chemistry 21, 259-275 (2002).

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ionization and other) but also at atmospheric pressure ³ ' ⁴ (atmospheric pressure chemical ionization (APCI) ³ ' ⁴ , electrospray ionization (ESI) ⁵ , desorption electrospray ionization (DESI) ⁶ , atmospheric pressure photoionization (APPI) ⁷ , atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) ⁸ , direct analysis in real time (DART) ⁹ , sonic spray ionization (SSI) ¹⁰ and other). The techniques operating at atmospheric pressure became widespread mostly during the last decade and contributed significantly to extending the scope of mass spectrometry and ion mobility spectrometry. Electrospray ionization allowed advancement in protein analysis and together with atmospheric pressure chemical ionization belongs to common ionization techniques used for coupling of liquid chromatography with mass spectrometry. DESI and DART are suitable for analysis of solid surfaces. Atmospheric pressure photoionization is a useful tool for ionization of nonpolar compounds and is today used for coupling of liquid chromatography with mass spectrometry too. In techniques operating at atmospheric pressure the region of ion source is connected to the ambient atmosphere. There is approximately atmospheric pressure in the ion source region, depending on the amount of sample (vapors of sample) introduced, amount of gases supplied and, possibly, on the amount of auxiliary liquids (amount of vapors of these liquids) supplied. A mild increase of pressure that may occur does not significantly influence the ionization as is shown below. The criterion of ionization efficiency is not only the number of ions formed in the ion source but also the number of ions transferred to the adjacent part of the instrument. The ionization efficiency is directly manifested in the measurement, determining the signal intensity. By comparing signal intensities achieved under various conditions, the ionization efficiency can be directly compared.

The atmospheric pressure ionization has, as any other analytical approach, certain limitations in terms of amount of substances necessary for their identification or determination. Under a certain concentration limit, the analysis cannot be successfully accomplished.

4 Bruins A.P.: Atmospheric Pressure Ionization Mass Spectrometry. I. Instrumentation and Ionization Techniques. Trends in Analytical Chemistry 13, 37-43 (1994).

5 Manisali L, Chen D. D.Y. , Schneider B. B.: Electrospray Ionization Source Geometry for Mass Spectrometry: Past, Present, and Future. Trends in Analytical Chemistry 25, 243-256 (2006).

6 Takats Z., Wiseman J.M., Gologan B., Cooks R.G.: Mass Spectrometry Sampling Under Ambient Conditions with Desorption Electrospray Ionization. Science 306, A11-4T3 (2004).

7 Robb D.B., Covey T.R., Bruins A.P.: Atmospheric Pressure Photoionization: An Ionization Method for Liquid Chromatography-Mass Spectrometry. Analytical Chemistry 72, 3653-3659 (2000).

8 Moyer S. C, Cotter R.J.: Atmospheric Pressure MALDI. Analytical Chemistry 74, 469A-476A (2002).

9 Cody R.B., Laramee J.A., Dupont Durst H.: Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions. Analytical Chemistry 77, 2297-2302 ( 2005).

10 Hirabayashi A., Sakairi M., Koizumi H.: Sonic Spray Mass Spectrometry. Analytical Chemistry 67, 2878-2882 (1995).

Disclosure of the invention

The new design of the ion source allows to determine and identify compounds at concentration levels that are far below limit of quantitation or limit of detection for the contemporary atmospheric pressure design.

The new ion source design described here enables to generate more ions for subsequent measurement. Thus lower limits of quantitation, detection and possibly increased sensitivity of measurement (slope of the calibration line) are achieved. It is feasible then to analyze successfully samples with lower concentration of analytes which makes possible to get otherwise hardly accessible or inaccessible information about animate and inanimate systems. With this modification of contemporary ion sources operating at atmospheric pressure, the measured signal can be increased by order(s) of magnitude compared to current situation.

The design of the ion source (Figure 1) is proposed so that the working region where ionization takes place is isolated from ambient atmosphere to prevent pressure equalization, hi practice, the housing of the ion source is mounted to the evacuated part of the instrument with a seal between the two parts. The sample inlet, inlet of auxiliary media with controlled supply and outlet of the ion source to the ambient atmosphere are also sealed. The seals permit to maintain elevated pressure in the working region of the ion source which is achieved by curtailing or stopping the flow of gases and vapors through the outlet of the ion source to the ambient atmosphere. The pressure in the working region of the ion source is raised by supplying auxiliary media. The outlet of the ion source to the ambient atmosphere is coupled to a flow restricting device. This device is realized as a sealing plug or a plug with a small opening or an auxiliary pressure regulator. The auxiliary pressure regulator consists of unions, a T-piece through which the auxiliary medium is supplied, a flow restrictor equipped with a pressure gauge for monitoring of the pressure in the working region of the ion source and a needle valve which can be together with the controlled supply used to control the pressure in the working region of the ion source. The needle valve also drains liquid that can accumulate in the system upon introducing liquid samples or auxiliary fluids into the ion source. Such design of ion source allows to raise the pressure in the working region of the ion source to 120 IcPa and more.

The signal intensity as a function of increasing pressure in the ion source region is shown in Figures 2 to 5. Electrospray ionization was carried out on a commercial instrument equipped with an ion trap as the mass analyzer (the new design of ion source is applicable to other types of ionization operating at atmospheric pressure and other mass analyzers too). Small increase of pressure that may occur during common operation of ion sources working at atmospheric

pressure does not result in a significant increase of signal. By increasing the pressure to 120 IcPa and more, however, one-order-of-magnitude or higher increase of signal intensity can be achieved (see Figures 2 to 5). The fact that the cause of signal increase is not merely an increase of nitrogen flow rate is demonstrated in Figure 6. The signal intensity of protonated caffeine molecules at atmospheric pressure (101.3 kPa) was 4.43xlO ⁵ counts. Upon increasing the nitrogen flow rate from 0 L/min to 12 L/min while still maintaining atmospheric pressure (101.3 kPa) the intensity decreased to 1.02x10 ⁵ counts. At the same nitrogen flow rate (12 L/min) and at elevated pressure (141.3 IcPa) the signal intensity increased to 5.56xlO ⁶ counts, i.e. the intensity increased one order of magnitude. The pressure increase thus leads to a significant increase of ionization efficiency which is manifested as an increase of measured signal intensity. The pressure in the ion source can be gauged which is important for the control and reproducibility of results; however, the pressure monitoring is not a prerequisite for the efficiency of ionization process. The increase of pressure in the ion source region influences the processes of ionization and ion transmission to the evacuated part of instrument (e.g. the course of vaporization of liquids and sample solutions, respectively, in the ion source region, increased flow from the ion source to the evacuated part of instrument, more frequent collisions of particles in the ion source region).

A technical limitation accompanying use of the new ion source can be insufficient power of the vacuum pumps. In such case, the pressure increase in the ion source may be limited since the pressure in the evacuated part of instrument could increase too much and the instrument could be e.g. shut down automatically. Then it would be necessary to employ more powerful vacuum pumps or more vacuum pumps for the instrument. Another difficulty that can arise when a sealing plug is used at the outlet of ion source to the ambient atmosphere is that the liquids introduced to the ion source region do not drain. These liquids are converted to vapors but partly can remain in the liquid phase. This problem can be simply solved by using e.g. the auxiliary pressure regulator or the plug with a small opening. Both solutions allow to achieve sufficient pressure resistance to prevent a pressure decrees in the ion source but at the same time the liquid is pushed out by the overpressure in the ion source through the auxiliary pressure regulator or the plug with a small opening.

Examples

Example 1

A representative of smaller organic molecules for the realization of the invention is caffeine. The ionization was carried out by electrospray in the positive mode. An ion trap mass analyzer was used. The outlet of ion source into the ambient atmosphere JO was sealed with a pressure gauge. The pressure in the working region of ion source 3 was increased by means of higher flow rate of nitrogen as the auxiliary medium. The nitrogen was supplied through the inlet for auxiliary media with controlled supply 8. Solution of caffeine at a concentration of 5 μg/mL (dissolved in water : methanol mixture = 1 : 1, v/v) was analyzed by flow injection analysis where 1 μL of the solution was injected into the flow of methanol (at a flow rate of 0.2 mL/min). The parameters of the mass spectrometer were tuned to caffeine at atmospheric pressure; the basic parameters of the measurement were: spray voltage 5.6 IcV, nebulizing gas flow rate 1.2 L/min, heated capillary temperature 200 ⁰ C, heated capillary voltage 9.51 V (the capillary - a narrow aperture 6 connecting the evacuated part of instrument I with the working region of ion source 3). For the same injected amount (5 ng), the response of caffeine was 4.43x10 ⁵ counts at atmospheric pressure (101.3 IdPa) and at elevated pressure (141.3 kPa) the response of caffeine increased to 5.56x10 ⁶ counts (Figure 6). The spectra in Figure 6 also prove that to increase the signal a mere increase of nitrogen flow rate is not sufficient but the pressure in the working region of ion source 3 must be increased as well (compare the spectrum measured at the pressure of 101.3 IcPa and the nitrogen flow rate of 12 L/min with the one measured at the pressure of 141.3 IcPa and the nitrogen flow rate of 12 L/min).

Example 2

A mass spectrometric analysis of reserpine using atmospheric pressure chemical ionization in the positive mode and an ion trap mass analyzer was performed. The outlet of ion source into the ambient atmosphere JjO was coupled to the auxiliary pressure regulator J/7. The pressure in the working region of ion source 3_ was increased by means of higher flow rate of nitrogen as the auxiliary medium. The nitrogen was supplied through the inlet for auxiliary media with controlled supply 8. Reserpine is a substance often used in the area of mass spectrometry to characterize the sensitivity of instruments or for demonstration of the limit of quantitation or detection. Solution of reserpine at a concentration of 5 μg/mL (dissolved in water

: methanol mixture = 1 : 1, v/v) was analyzed by flow injection analysis where 1 μL of the solution was injected into the flow of methanol (at a flow rate of 0.2 mL/min). The parameters of the mass spectrometer were tuned to reserpine at atmospheric pressure; the basic parameters of the measurement were: vaporizer temperature 300°C, corona discharge current 5.7 μA, nebulizing gas flow rate 0.6 L/min, heated capillary temperature 175°C, heated capillary voltage 9.51 V (the capillary - a narrow aperture 6 connecting the evacuated part of instrument I with the working region of ion source 3_). For the same injected amount (5 ng), the response of reserpine was 2.3xlO ⁴ counts at atmospheric pressure (101.3 kPa) and at elevated pressure (141.3 kPa) the response of reserpine increased to 1.0x10 counts (Figure 7).

Example 3

Electrospray ionization is a suitable and widely used technique in peptide analysis. The outlet of ion source into the ambient atmosphere K) was sealed with a pressure gauge. The pressure in the working region of ion source 3 was increased by means of higher flow rate of nitrogen as the auxiliary medium. The nitrogen was supplied through the inlet for auxiliary media with controlled supply 8. Using an ion trap mass analyzer, the MRFA peptide (L- methionine-arginine-phenylalamne-alanine acetate monohydrate) was analyzed in the positive mode of ionization. Its solution at a concentration of 5 μg/mL (dissolved in water : methanol mixture = 1 : 1, v/v) was analyzed by flow injection analysis where 1 μL of the solution was injected into the flow of methanol (at a flow rate of 0.2 mL/min). The parameters of the mass spectrometer were tuned to the MRFA peptide at atmospheric pressure; the basic parameters of the measurement were: spray voltage 5.6 kV, nebulizing gas flow rate 1.2 L/min, heated capillary temperature 200°C, heated capillary voltage 29.8 V (the capillary - a narrow aperture 6 connecting the evacuated part of instrument I with the working region of ion source 3). For the same injected amount (5 ng), the response of MRFA was 2.05xl0 ⁶ counts at atmospheric pressure (101.3 kPa) and at elevated pressure (141.3 IcPa) the response of MRFA increased to 1.97xlO ⁷ counts (Figure 8).

Example 4

An example of negative mode measurement is the analysis of glutamic acid using electrospray ionization and an ion trap mass analyzer. The outlet of ion source into the ambient atmosphere 10 was coupled to the auxiliary pressure regulator 17. The pressure in the working

region of ion source 3 was increased by means of higher flow rate of nitrogen as the auxiliary medium. The nitrogen was supplied through the inlet for auxiliary media with controlled supply 8. Solution of glutamic acid at a concentration of 5 μg/mL (dissolved in water : methanol mixture = 1 : 1, v/v) was analyzed by flow injection analysis where 1 μL of the solution was injected into the flow of methanol (at a flow rate of 0,2 mL/min). The parameters of the mass spectrometer were tuned to glutamic acid at atmospheric pressure; the basic parameters of the measurement were: spray voltage -4.53 kV, nebulizing gas flow rate 1.2 L/min, heated capillary temperature 200°C, heated capillary voltage -10.8 V (the capillary - a narrow aperture 6 connecting the evacuated part of instrument 1 with the working region of ion source 3_). For the same injected amount (5 ng), the response of glutamic acid was 7.56xlO ³ counts at atmospheric pressure (104.7 IdPa) and at elevated pressure (144.7 kPa) the response of glutamic acid increased to 1.05x10 ⁵ counts (Figure 9).

Example 5

Interesting application fields are analysis of complexes and analysis of chiral compounds. Both these fields are represented in the analysis of mixture of copper(II) ions, phenylalanine (Phe) and tryptophane (Trp). For the discrimination of optical isomers, it is necessary to obtain a diastereomeric complex of the formula [Cu(Trp) ₂ Phe-H] ⁺ during the ionization. The measurement was carried out using electrospray ionization in the positive mode with an ion trap mass analyzer. The outlet of ion source into the ambient atmosphere JX) was sealed with a pressure gauge. The pressure in the working region of ion source 3 was increased by means of higher flow rate of nitrogen as the auxiliary medium. The nitrogen was supplied through the inlet for auxiliary media with controlled supply 18. Working solution in mixture of water : methanol (1 : I ₅ v/v) with the following concentration of individual components - 50 μmol/L of Cu ²⁺ , 50 μmol/L of tryptophane and 10 μmol/L of phenylalanine - was directly infused into the ion source at a flow rate of 5 μL/min. The parameters of the mass spectrometer were tuned to the complex of interest at atmospheric pressure; the basic parameters of the measurement were: spray voltage 5.6 IcV, nebulizing gas flow rate 1.2 L/min, heated capillary temperature 200 ⁰ C, heated capillary voltage 9.5 V (the capillary - a narrow aperture 6 connecting the evacuated part of instrument 1 with the working region of ion source 2). During the working solution infusion, the response of the complex was 1.18x10 ³ counts at atmospheric pressure (104.7 kPa) and at elevated pressure (144.7 kPa) the response of the complex increased to 8.49x10 ⁵ counts (Figure 10). The figure also shows increases of intensity of the phenylalanine ion (m/z= 166.05) from

5.62xlO ⁴ counts to 3.83xlO ⁶ counts and of the tryptophane ion (m/z=205.23) from 2.03xl0 ⁵ counts to 8.86xlO ⁶ counts when the pressure is increased from 104.7 kPa to 144.7 kPa (Fig. 10).

Example 6

An important subject area of analytical chemistry is protein analysis. Electrospray ionization and an ion trap mass analyzer were used to analyze a protein (bovine cytochrome c) in the positive mode. The outlet of ion source into the ambient atmosphere H) was coupled to the auxiliary pressure regulator 17. The pressure in the working region of ion source 3_ was increased by means of higher flow rate of nitrogen as the auxiliary medium. The nitrogen was supplied through the inlet for auxiliary media with controlled supply 8. Solution of the protein at a concentration of 10 μg/mL (dissolved in water : methanol mixture = 1 : 1, v/v) was directly infused into the ion source at a flow rate of 5 μL/min. The parameters of the mass spectrometer were tuned to the protein of interest at atmospheric pressure; the basic parameters of the measurement were: spray voltage 5.6 kV, nebulizing gas flow rate 1.2 L/min, heated capillary temperature 200°C, heated capillary voltage 25 V (the capillary - a narrow aperture 6 connecting the evacuated part of instrument 1 with the working region of ion source 3). During infusion of the protein solution, the response of the most intense ion of the protein was 4.02x10 ⁴ counts at atmospheric pressure (105.4 kPa) and at elevated pressure (145.4 IcPa) the response increased to 2.05xl0 ⁵ counts (Figure 11).

Figure 1

A scheme of ion source for lower limits of detection in mass spectrometric and ion mobility measurements. A- side view of the mounting of ion source 2 to the evacuated part of instrument I; B - frontal cross-section of ion source 2 with auxiliary pressure regulator 17- 1 evacuated part of instrument, 2 ion source, 3 working region of ion source, 4 housing of ion source, 5 seals, 6 narrow aperture for transmission of ions into evacuated part of instrument, 7 sample inlet, 8 inlet of auxiliary media with controlled suply, 9 mount of ion source to evacuated part of instrument, H) outlet of ion source into the ambient atmosphere, JJ, union, 12 T-piece, 13 _. controlled supply of auxiliary medium, J_4 flow restrictor, 15 needle valve, 16 pressure gauge, 17 auxiliary pressure regulator.

Figure 2

Signal intensity of protonated molecule of caffeine (m/z 195.20) as a function of pressure in the working region of ion source 3_.

Figure 3

Signal intensity of protonated molecule of tryptophane (m/z 205.23) as a function of pressure in the working region of ion source 3.

Figure 4

Signal intensity of complex ion of [Cu(Trp) ₂ Phe-H] ⁺ (m/z 635.18) as a function of pressure in the working region of ion source 3_.

Figure 5

Signal intensity of protonated molecule of MRFA peptide (L-methionine-arginine-phenylalanine- alanine acetate monohydrate; m/z 524.70) as a function of pressure in the working region of ion source 3.

Figure 6

Mass spectra of caffeine showing increase of signal intensity with increased pressure in the working region of ion source 3. Relevant parameters are given in the spectra.

Figure 7

Mass spectra of reserpine showing increase of signal intensity with increased pressure in the working region of ion source 3_. Relevant parameters are given in the spectra.

Figure 8

Mass spectra of peptide (MRFA, L-methionme-arginine-phenylalanme-alanine acetate

monohydrate) showing increase of signal intensity with increased pressure in the working region of ion source 3. Relevant parameters are given in the spectra.

Figure 9

Mass spectra of glutamic acid showing increase of signal intensity with increased pressure in the working region of ion source 3. Relevant parameters are given in the spectra.

Figure 10

Mass spectra of [Cu(Trp) ₂ Phe-H] ⁺ complex showing increase of signal intensity with increased pressure in the working region of ion source 3. Relevant parameters are given in the spectra.

Figure 11

Mass spectra of bovine cytochrome c showing increase of signal intensity with increased pressure in the working region of ion source 3_. Relevant parameters are given in the spectra.

Industrial applicability

The invention can be utilized in the area of instrumental technology for manufacturing ion sources of mass spectrometers and ion mobility spectrometers. These instruments are used in pharmaceutical industry, food industry, environmental monitoring, in research of the processes in living organisms and in other disciplines where mass spectrometry or ion mobility spectrometry are applied.