WO2021242103A1 - Ionization source, functional combination with a mobile device and arrangement with an atmospheric inlet mass spectrometry system - Google Patents

Abstract

An improved ionization source (1) for use with any atmospheric pressure inlet (71) mass spectrometry system (7) is provided herein. The improved ionization source 1 comprises an electrode (2) having a deposition area (21) or a capillary channel (22) for introducing a sample, or an electrode (2) having a capillary channel (23) for introducing a solvent (or solvent mixture) while the sample is positioned in between (24) the ionization source (1) and the atmospheric pressure inlet (71) of the mass spectrometry system (7), and a power supply (3) having an output (32) coupled to the electrode and a USB-compatible input (31) and further comprising a controllable transformer (33) to transform an input supply voltage (Vs) provided at the USB-compatible input (31) into a high voltage (VH) at the power supply output, and a controller (34) for controlling the controllable transformer (33). The improved ionization source (1) is widely applicable as it can be directly coupled to any mobile device (5) having a USB-port. Moreover, control tasks can be performed by the mobile device (5) in response to user input, remote control and/or an autonomously operating application program functionalities.

Description

Title: Ionization source, functional combination with a mobile device and arrangement with an atmospheric inlet mass spectrometry system

BACKGROUND

The present invention pertains to an ionization source.

The present invention further pertains to a functional combination of the ionization source with a mobile device.

The present invention still further pertains to an arrangement comprising said functional combination and an atmospheric pressure inlet mass spectrometry system.

Mass spectrometry (MS) is due to its high versatility and high sensitivity one of the most used detection techniques in analytical chemistry and in life sciences (including but not limited to forensic, clinical, environmental, toxicological and food analysis). With recent (academic) developments, portable and transportable miniaturized mass spectrometers are becoming more prevalent and will eventually find their way from research laboratories to control laboratories to further on-site or at-line measurements. However, besides the (trans)portability of the mass analyzer, another prerequisite for on-site measurements is the simplification of sample preparation and ionization before introducing samples into the mass analyzer.

Recently, Blokland et al. demonstrated the potential of different ionization techniques suitable for on-site food safety analysis in “Potential of Recent Ambient Ionization Techniques for Future Food Contaminant Analysis Using (Trans)Portable Mass Spectrometry. Food Analytical Methods. (2019). “

Minimal sample preparation effort, absence of gas supply, direct analysis of trace amounts deposited on surfaces or dissolved in suitable solvents, ionization featuring low energy consumption, low weight, low-costs, and simplified user interfaces, and a wide range of available and inexpensive substrates are a prerequisite for the success of future on-site application. While the suitability of these techniques has been initially demonstrated for analysis in the field, they currently still require lab equipment and technician expertise. In contrast, future end-users of (trans)portable MS systems for on-site analysis will be most likely no longer lab technicians but, for example, on-site inspectors. Apart from training in basic skills, there is a need to provide a cost- efficient, low energy consuming, low weight and user-friendly ionization source for use in these systems.

SUMMARY

In order to address the above-mentioned need, an improved ionization source for use with an atmospheric pressure inlet mass spectrometry device is provided as specified in claim 1. The ionization source comprises:

- at least one deposition area or at least one capillary channel for introducing a sample (which may be in a solid, dissolved or gaseous state);

- at least one electrode for causing desorption and/or ionization and transfer of sample material from the deposition area or capillary channel towards an atmospheric pressure inlet of said mass spectrometry system;

- a power supply having a USB -compatible input, an output coupled to the electrode and a controllable transformer to transform an input supply voltage provided at the USB -compatible input into a high voltage at the power supply output, and;

- a controller for controlhng the controllable transformer.

The ionization source is cost-efficient, low energy consuming, low weight and user-friendly as it can be directly coupled with its USB -compatible input to a USB port of an ubiquitous mobile device such as a smartphone or tablet or even a battery with USB-output port, to therewith form a functional combination. Therewith a power source for the improved ionization source is always readily available.

In some embodiments the controllable transformer of the improved ionization source comprises a first transformer module to convert the input supply voltage received at the USB -compatible input into an intermediate voltage with a controllable voltage magnitude determined by the controller and a second transformer module to transform the intermediate voltage into an output DC- voltage having a voltage magnitude that is substantial proportional to the voltage magnitude of the intermediate voltage in accordance with a predetermined proportionality factor having a value of at least 100. Due to the fact that the second transformer module provides for the transformation of the intermediate voltage into the high voltage domain in a substantially proportional manner, the actual process of controlling the voltage can take place in a low voltage domain, so that the electronic components for this purpose can be provided at relatively low cost.

It is noted that various options are available. In one configuration the at least one deposition area is a surface area of the at least one electrode. In another configuration the at least one capillary channel contains the sample in electrical contact with the electrode. In some embodiments the capillary channel is mounted at the at least one electrode.

In another configuration, the at least one deposition area for introducing the sample is positioned in-between the at least one electrode with the at least one capillary channel and the atmospheric inlet of the mass spectrometer. In that configuration the at least one capillary channel only contains a solvent, or mixture thereof.

In some embodiments, the at least one deposition area is one of a plurality of deposition areas (including but not limited to microspots, nanospots and picospots) for introducing a plurality of samples.

In some embodiments, the at least one electrode is an electrode in an array of multiple electrodes, having each one or more deposition area(s), for introducing respective samples wherein the electrodes of the array of multiple electrodes either are subsequently powered by the power supply or are simultaneously powered by the power supply.

In some embodiments at least one capillary liquid channel has a shape and/or format different from that of a capillary, wherein optionally the liquid channel is a microfluidic or a nanofluidic channel. In some examples, using a technology known in microfLuidics, the capillary is provided by etching a rectangular channel in a substrate. In some examples thereof, the at least one capillary liquid channel is one of an array or bundle of multiple hquid channels in any shape or format, for introducing multiple samples, which multiple liquid channels are either subsequently powered or simultaneously powered by the power supply. In some embodiment the capillary channel comprises the sample in a sohd or vapor phase.

In a functional combination as specified above, the mobile device is particularly suitable to provide instructions and guidance to the user.

In some embodiments of this functional combination the controller is configured to operate according to an input control signal received from the mobile device via the USB -compatible input. Therewith, the controller in the improved ionization source can be provided very cost-efficiently as it only needs to perform basic control tasks, such as regulating the output voltage to a level prescribed by the input control signal. Ubiquitous mobile devices typically have a programmable processor capable to be programmed in order to provide the input control signal.

In accordance therewith a record carrier is provided comprising an apphcation program that when executed by the programmable processor of the mobile device in the functional combination causes the mobile device to provide said input control signal. In some examples, the record carrier is provided as an extension ROM that is plugged in the mobile device. In other examples, the record carrier is a general purpose rewritable memory proper to the mobile device, wherein the application program can be loaded prior to use of the functional combination, e.g. by a wired or a wireless connection.

In some embodiments of the functional combination the user is enabled to operate the ionization source with a user-interface on the mobile device. Therewith the programmable processor provides the input control signal in accordance with instructions provided by the user via the user-interface.

In some embodiments a remote operator is enabled to operate the ionization source. In that case the programmable processor provides the input control signal in accordance with instructions provided by the remote operator via the communication facilities of the mobile device. In some embodiments the programmable processor provides the input control signal both in accordance with instructions provided by the user via the user-interface and by the instructions provided by the remote operator via the communication facilities of the mobile device.

The present application further provides an improved arrangement comprising an atmospheric pressure inlet mass spectrometry device having an atmospheric pressure inlet and a functional combination according to embodiments as specified above. Therein the ionization source is arranged to transfer ionized material from the sample towards an atmospheric pressure inlet of said mass spectrometry system.

Whereas the improved ionization source is of particular advantage for use with a portable atmospheric pressure inlet mass spectrometry device it is also well applicable with atmospheric pressure inlet mass spectrometry devices that are not portable, e.g. MS-devices stationed in a laboratory or vehicle.

The concept of direct analysis by mass spectrometry has been demonstrated in numerous papers describing techniques such as, among others, direct electrospray probe (DEP), probe electrospray ionization (PESI), paper spray ionization (PSI), desorption electrospray ionization (DESI), direct analysis in real time (DART), atmospheric solids analysis probe (ASAP). While removal of sample clean-up steps and chromatographic separation could be achieved, apphcation to complex sample matrices is still limited due to severe ionization suppression (or enhancement) caused by matrix effects.

Recent developments of conductive blades coated with sohd-phase microextraction (SPME) material as a direct ionization technique, coated blade spray (CBS) is, in theory, an ideal compromise between sample clean-up and direct ionization. This technology enables both rapid extraction of target samples from complex matrices and provides a means of direct spray ionization. CBS- blades are geometrically sharp-tipped electrically conductive strips that are partially coated with a biocompatible SPME coating. The merging of SPME coating and metal carrier for direct spray ionization contributes to both the reduction of organic solvent used for extraction and the omission of gas supply, heaters or vulnerable laser to assist in desorption ionization. Furthermore, CBS allows for the extraction of compounds from liquids, which could be easily performed on-site, while adding crucial specificity for a wide range of samples by selecting blades with dedicated SPME stationary phase coatings. It was demonstrated that with a sufficiently large sample volume the amount extracted by the coating at equilibrium is directly proportional to the concentration of the sample and independent of the sample volume. Additionally, it is assumed that the metal structure of the blades also positively affects electrical conductivity and thus results in a more steady electric field and a more efficient and reproducible ion formation compared to less conductive substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects are described in more detail with reference to the drawing. Therein:

FIG. 1 schematically shows an embodiment of an improved ionization source;

FIG. 1A, B, C, D shows elements of a modified embodiment of an improved ionization source, respectively, containing a capillary channel, multiple electrodes, multiple capillary channels, concentric capillary;

FIG. 2 schematically shows another embodiment of an improved ionization source;

FIG. 3A, 3B, 4A and 4B show four further embodiments of an improved ionization source;

FIG. 5 and 6 show embodiments of a functional combination of an embodiment of the improved ionization source and a mobile device;

FIG. 7 shows an arrangement comprising such a functional combination and an atmospheric pressure inlet mass spectrometry system;

FIG. 8 shows measurement data obtained for various analytes;

FIG. 9a, b show output voltage curves for an embodiment of an improved ionization source and a known ionization source; FIG. 10 shows an experimental set-up and measurement results obtained therewith;

FIG. 11-14 show further measurement data obtained with an arrangement comprising an atmospheric pressure inlet mass spectrometry system and a functional combination of an embodiment of the improved ionization source and a mobile device.

DETAILED DESCRIPTION OF EMBODIMENTS

Like reference symbols in the various drawings indicate like elements unless otherwise indicated.

FIG. 1 schematically shows an embodiment of an improved ionization source 1 for use with an atmospheric pressure inlet mass spectrometry system.

As shown in FIG. 1, the improved ionization source 1 comprises an electrode 2 having a deposition area 21 for introducing a sample. Alternatively it may be provided with a capillary channel 22 for holding the sample solution, as shown in FIG. 1A. Alternatively, as shown in FIG. 3A, 3B, it may be provided with a capillary channel 23 for holding a suitable solvent (or solvent mixture) while the deposition area 24 with the sample is positioned in between the improved ionization source and the atmospheric pressure inlet 71 of the mass spectrometry system 7. Alternatively, the capillary channel 22 and 23 may be a set-up of concentric capillaries in order to allow gas-flow and/or liquid-flow assisted electrospray formation.

The improved ionization source 1 further comprises a power supply 3 having a USB -compatible input 31, an output 32 coupled to the electrode, and a reference output 32e to be coupled to the mass spectrometry system 7. The power supply 3 further comprises a controllable transformer 33 to transform an input supply voltage Vs provided at the USB -compatible input 31 into a high voltage V_H i.e. a voltage of 1 kV or more at the power supply output 32, and a controller 34 for controlling the controllable transformer 33. In this connection it is noted that a USB -compatible input 31 is understood to be an input that can be connected directly to a USB-port of a mobile device for example without requiring a voltage conversion between the USB-port voltage supply terminals and the USB -compatible input terminals. This implies that the improved ionization source is configured to operate on a supply voltage of approximately 1-lOV. In some embodiments the improved ionization source is configured to operate in a voltage range within the same order of magnitude, e.g. a voltage in the range of 1 to 10 V, e.g. 1.5 V. Though preferable, it is not necessary that the USB- compatible input is provided with a USB-plug that fits to a USB-port. As shown in dotted lines in FIG. 1, in use the improved ionization source 1 is positioned with the deposition area 21 or the capillary channel 22 in front of the atmospheric pressure inlet 71 of the mass spectrometry system 7 and the reference output 32e is electrically connected to the inlet 71.

FIG. 2 shows an alternative embodiment of the improved ionization source. In this embodiment the controllable transformer 33 comprises a first transformer module 331 and a second transformer module 332. The first transformer module 331 is configured to convert the input supply voltage Vs received at the USB- compatible input 31 into an intermediate voltage Vint with a controllable voltage magnitude determined by the controller 34.

The second transformer module 332 is configured to transform the intermediate voltage Vint in the range of 1-5 volt (0.5- 1.5 ampere) provided by the first transformer 331 into an output DC-voltage VH having a voltage magnitude that is substantially linearly dependent on the voltage magnitude of the intermediate voltage Vint in accordance with a predetermined proportionality factor having a value of at least 100. In the present embodiment the voltage range of 1-5 volt of the intermediate voltage corresponds to a voltage range of 0.5 to 5 kV at the output. Accordingly, the proportionality factor is 900. The second transformer 332 is composed of a cored transformer and multiple capacitors for high voltage generation. The total weight is less than 0.5 kg and the size is less than 10x10 cm.

For simplicity it is assumed in this embodiment and the subsequent embodiments that the analyte is provided onto a deposition area 21 of a blade like electrode 2 as shown in FIG. 1. However in each of these embodiments, the analyte may be alternatively provided within a capillary channel 22, as shown in FIG. 1A. Alternatively, in the embodiment shown in FIG. 3A and 3B, the at least one deposition area 24 for introducing the sample is positioned in-between the electrode 2 with the capillary channel 23 and the atmospheric inlet 71 of the mass spectrometer. The capillary channel 23 in this case only contains a suitable solvent (or solvent mixture).

Still further, as shown in the embodiments of FIG. 4A, 4B, it is possible that the electrode is provided as a needle 25 within a space holding a gaseous or aerosol phase of the sample. In these embodiments the high voltage VH supplied to the needle 25 ionizes the material of the sample by one or more reactions. This ionization approach is known as, for example, atmospheric pressure chemical ionization (APCI), or as atmospheric solids analysis probe (ASAP). FIG. 4A shows a modified version of FIG. 1, wherein the blade type electrode 21 is replaced by the needle 25. FIG. 4B shows the same modification apphed to the embodiment of FIG. 2.

FIG. 5 shows an arrangement wherein an embodiment of the improved ionization source 1, here the embodiment of FIG. 1, but alternatively the embodiment of FIG. 3A, 3B, 4A, 4B, or using the capillary tube 22 shown in FIG. 1A is functionally combined with a mobile device 5 having a USB-port 51. The ionization source 1 is coupled with its USB -compatible input 31 to the USB-port 51 of the mobile device 5 to receive the input voltage Vs. The mobile device 5 may be any portable device, even a portable USB-power supply. However, as will be apparent further below, a particular synergic effect is achieved in an arrangement wherein the mobile device 5 has data processing capabihties 52 and/or communication facilities 54. In the embodiment shown in FIG. 5, the controller 34 is configured to operate according to an input control signal Ci received from the mobile device 5 via the USB -compatible input 31.

Therewith, the controller 34 in the improved ionization source 1 can be provided very cost-efficiently as it only needs to perform basic control tasks, such as regulating the output voltage VH to a level prescribed by the input control signal Ci. Ubiquitous mobile devices typically have a programmable processor capable to be programmed in order to provide the input control signal so that the required control functionality can be achieved at modest costs. The mobile device 53 may be provided with a record carrier 57, for example as part of the data processing facilities 52. In that embodiment, the record carrier 57 comprises an application program that when executed by the data processing facilities 52, e.g. a programmable processor causes the mobile device 5 to provide the input control signal Ci at its USB-output 51. In some examples, the record carrier is provided as an extension ROM that is plugged in the mobile device. In other examples, the record carrier is a general purpose rewritable memory proper to the mobile device, wherein the application program can be loaded prior to use of the functional combination, e.g. by a wired or a wireless connection.

In an embodiment, a user is enabled to operate the ionization source 1 with a user-interface 53 on the mobile device 5. The application program in the record carrier 57 may support this functionality. In another embodiment, a remote operator is enabled to operate the ionization source 1 with the communication facilities 54 of the mobile device 5. In still further embodiments the programmable processor 52 provides the input control signal Ci both in accordance with instructions provided by the user via the user-interface 53 and by the instructions provided by the remote operator via the communication facilities 54 of the mobile device. Still further, a remote instructor may send instructions via the communication facilities 54 to the user interface 53 to instruct a user how to operate the improved ionization source 1 using the user interface 53. In the example shown in FIG. 5, the mobile device 5 is a mobile phone. In alternative arrangements the mobile device 5 is a laptop or a tablet.

FIG. 6 shows another arrangement wherein the embodiment of improved ionization source 1 of FIG. 2 is functionally combined with a mobile device 5.

FIG. 7 shows an arrangement comprising a mass spectrometry system 7 having an atmospheric pressure inlet 71 and a functional combination of an embodiment of an improved ionization source 1 and a mobile device 5. The mass spectrometry system 7 has its atmospheric pressure inlet 71 electrically connected to the reference output 32e. The improved ionization source 1 is positioned with the deposition area 21 of the electrode 2 in front of the inlet 71.

In case of the modification of FIG. 1A it is positioned with an opening of the capillary tube 22 towards the inlet 71. In case of an embodiment as shown in FIG. 4, it is positioned with the point of the needle 25 near the inlet 71. In case of the modification of FIG. 3A, 3B the deposition area 24 with the sample is positioned in between the electrode 2 with the capillary tube 23 and the inlet 71.

Various experiments were performed with an arrangement as shown in FIG. 7. These are described below.

In summary, therein the USB-powered ionization source 1 was used on two different mass spectrometry (MS) systems. The first one is a transportable single- quadrupole MS and the other one is a benchtop triple-quadrupole tandem MS. Various characteristics of the USB-powered ionization source 1, including high voltage generation and angular positioning, were studied. To demonstrate the apphcation potential of the newly developed ionization source 1, as an example, banned and regulated veterinary drugs such as b-agonists and sulfonamide antibiotics, covering a wide range of molecular weights and polarities, were analyzed showing a food analysis application.

Chemical components

Methanol (MeOH), acetonitrile (ACN), ethyl acetate (EtOAc), and Milli-Q water (all ULC/MS grade) were purchased from Actu-All (Oss, The Netherlands) and isopropanol (IPA) was purchased from Biosolve (Valkenswaard, The Netherlands). Analytical standards of clenbuterol, fenoterol, isoxsuprine, procaterol, salbutamol, salmeterol, terbutaline, ritodrine, metaproterenol, ractop amine, sulfadiazine, sulfamerazine, sulfamethizole, sulfamethoxazole, sulfamethoxypyridazine, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfamoxole, sulfacetamide, sulfaphenazole, lyophilized synthetic [D-Lys3]- GHRP-6 and the isotope-labelled brombuterol-d9 were purchased from Sigma- Aldrich (St. Louis, USA). Sulfadimidine, sulfachlorpyridazine, and sulfadoxine were provided by Riedel de Haen (Seelze, Germany). Ostarine and andarine were purchased from Selleck Chemicals (Houston, USA) and recombinant bovine somatotropin (rbST) in Tris buffer (300 pg/mL, at pH 9) was purchased from GenScript (Leiden, The Netherlands). Brombuterol, cimaterol, cimbuterol, clencyclohexerol, clenhexyl, clenpenterol, clenproperol, mabuterol, mapenterol, tulobuterol, chlorbrombuterol, hydroxymethylclenbuterol and the isotope-labelled sulfapyridine-13C6, sulfamerazine-13C6, sulfamethizole-13C6, sulfamethoxypyridazine-d3, sulfachlorpyridazine-13C6, sulfisoxazole-13C6, sulfadoxine-d3 and sulfaquinoxahne-13C6 were purchased from Witega (Berlin, Germany). Carbuterol, reproterol, zilpaterol and isotope-labelled clenbuterol-d6, salbutamol-d6, ractopamine-d5, mabuterol-d9, cimbuterol-d9, cimaterol-d7, clenproperol-d7, isoxsuprine-d5 and mapenterol-dll were kindly provided by BVL the EURL for B-agonist type substances (Berlin, Germany). Isotope-labelled zilpaterol-d7, terbutahne-d9, fenoterol-d6, reproterol-d4, clencyclohexerol-dlO and carbuterol-d9 were purchased from Toronto Research (Toronto, ON, Canada).

Bovine urine samples were obtained from multiple bovine animals of different ages and gender from routine control programs in the Netherlands. Bovine milk samples were present at Wageningen Food Safety Researchand predetermined blank for sulfonamide antibiotics.

Instruments and consumables

The electrode blades 2 used in the improved ionization source 1 were coated with either Bondelut Certify (Agilent, Amstelveen, the Netherlands) or Oasis HLB (Waters, Milford, MA, USA) stationary phase material at Waterloo University (Waterloo, ON, Canada). The USB powered CBS ionization source was designed and constructed by coupling an HV generator to the adjustable output transformer board. The very low USB-provided input voltage was maintained at 1.5 V for all positive ionization experiments and 2.0 V for all negative ionization experiments. Coated blades were mounted via a (toothless crocodile) clamp connected to the HV output wire. The ground wire was connected to a grounded part of the mass spectrometer.

The mass spectrometers used were a benchtop model Quattro Premier XE triple quadrupole tandem MS system (Waters) and a model Acquity QDa (Waters) transportable single quadrupole MS detector. To perform CBS analysis, both the Premier XE and QDa were slightly modified. At the Premier XE, the ESI source housing and probe were removed and interlocks were bypassed by a dummy plug (MS Vision, Almere, The Netherlands). The QDa was slightly modified by removing the ESI source housing and changing the instrument settings, according to Trim pin et al. in Matrix-Assisted Ionization on a Portable Mass Spectrometer: Analysis Directly from Biological and Synthetic Materials. Analytical Chemistry. 88, 10831-10836 (2016). These modifications allowed the positioning of the USB-CBS in front of the MS sample cone inlet.

CBS methodology

Coated blades 2 were preconditioned before sampling using MeOH: Water (50:50 v/v) and vortex mixing for 30 seconds (s) at 650 revolutions per minute (rpm). After preconditioning, sampling of blades 2 was performed by placing the blade 2 into an aliquot of 300 pL of standards, blanks, or fortified urine samples and vortex at 650 rpm for 60 s. Some of the sample matrix contaminants were removed by a twofold wash with 300 pL of water, discarding the liquid in between the washing steps, and vortexing during 10 s. Blades 2 were dried in the open-air for approximately 5 minutes. Desorption and ionization were performed using 10 pL of MeOH, followed by the application of the selected HV for approximately 20 s.

Benchton MS/MS measurements

Characterization of the developed USB-CBS ion source 1 was performed on the Quattro Premier XE triple quadrupole MS. The electric potential on the blade was set to 1.6 or 3.2 kilovolts (kV) in positive ionization mode; source and desolvation temperatures were set at 100 °C, and 20 °C respectively. Desolvation and cone gas flows were both set to 0 L/hr, and collision-induced dissociation (CID) was performed utihzing a 0.18 mL/min argon (purity > 99,998%) gas flow.

Targeted USB-CBS-MS/MS analysis of sulfonamide antibiotics was performed in the multiple reaction monitoring (MRM) mode. For each sulfonamide antibiotic two product -ions MRM channels were recorded. The cone voltage, collision energy, m/z values of the precursor and product-ions and the corresponding internal standards are given in the table reproduced in FIG. 8. Instrument control and analysis of MS data were carried out using MassLynx v4.1 software (Waters).

Transportable single quadrunole MS measurements

Initial QDa MS data were obtained in full scan positive mode at a cone voltage of 15 V and source temperature of 150 °C. Further target analyses were performed in the selected ion recording (SIR) mode, whereby the theoretical monoisotopic mass of each precursor ion was measured in positive ionization mode. Instrument control and analysis of MS data were performed using MassLynx v4.1 software.

Data processing

For the quantitative determination of the analytes of interest, the area under the specific ion signal in the smoothed reconstructed ion chromatogram (RIC) was used. Smoothing of the RIC was done by applying the Savitzky-Golay algorithm using a window size of 2 Da. The ratios between the area of the RICs of the analyte and its internal standard were expressed as a response factor (RF). Calibration curves corresponding to individual analytes were constructed by plotting the RF versus the analyte concentration. Linearities were determined using correlation coefficients based on least-squares linear regression.

Single-day initial validation of the USB-CBS-MS concept

Method linearity was determined using the correlation coefficients of the constructed calibration curves. Calibration curves were constructed in triplicate by multianalyte fortified matrices in the concentration range 0-200 pg/kg (n=5). The limit of detection (LOD) of individual analytes was defined as the first concentration in the calibration curve with an RF higher than the previous lower calibration concentration used. Results and Discussion

Design and characterization of the USB generated high voltage

For creating an universal CBS ionization source, a positive direct current to direct current (DC/DC) HV generator module was composed, characterized and applied. To test the components, a laboratory benchtop test set-up was built to mimic the ionization set-up in the mass spectrometer. The set-up allowed testing in a controlled environment, thus avoiding possible damage to the mass spectrometer by electric arc discharge. By testing various input voltages and distances of the CBS needle tip to the inlet of the MS, the optimal settings were obtained. Even with a low input voltage (1.5 V) arc discharge was still occurring. Increasing the distance between the blade and MS inlet ceased the discharge process. Still, it enabled spray formation as was also seen on the laboratory benchtop experiments. These initial tests clearly show Taylor cone formation and the associated spray jet. However, the spray onset of the USB-powered device also showed a transient aerosol outburst when the HV was activated. To determine the origin of the observed aerosol outburst, an oscilloscope high- voltage probe was used. A blade was fixed in the clamp and placed in front of the MS inlet. The oscilloscope high-voltage probe enabled the recording of the HV build-up curve of the created USB-CBS device. The recorded electric potential build-up curve is shown in Figure 9a. As the recorded HV build-up curve in Figure 9a shows, the maximum electrical potential output of the created USB- CBS device was approximately 2700 V, i.e. in an appropriate range for electrospray-like coated blade spray.

The voltage curve of the MS powered high voltage set at 1600 V was likewise oscilloscopic recorded and is shown in Figure 9b. A comparison of both oscilloscopic derived HV curves demonstrates remarkable differences in voltage build-up. At the configured USB-powered device 1 the voltage rapidly increases prior to stabilization, while the MS powered set-up yields a much slower HV build-up followed by slope deflection before stabilizing at 1600 V. It is conjectured that the observed instantaneous increase may be beneficial to overcome the impact of any less conductive local imperfections in the CBS material. This is subject of further research.

In some cases the rapid increase of the output voltage may have as a result that the obtained measurement signal is less stable compared to that obtained with a slower voltage build up as shown in Figure 9b. However, using an internal standard can correct for these differences in signal as demonstrated by analyzing clenbuterol, spiked in an aqueous solution at three concentrations (5,

50 and 100 pg/L) and a corresponding internal standard clenbuterol- d6 (5 pg/L). The two different methods of HV generation, USB- and MS-powered, demonstrated similar linearities (R2=0.996 and R2=0.997) after correcting with internal standard.

Angular blade positioning to the mass analyzer

Due to limited access and space for positioning of the CBS device in front of the inlet of different brands of mass spectrometers, angular positioning of the USB-CBS device to MS inlet would be essential for the ease of placement. While the significance of angular positioning of substrate-spray techniques relative to the MS inlet has not been reported in literature, the characterization of tolerance to horizontal positioning of CBS for ionization in front of the MS inlet has been described. In previous research stable high signal intensities were achieved for 5 mm in all directions from the center of an ion-transfer capillary. Similar tolerances to positioning are described for PSI-MS analysis. It is believed that the central position of the spray ionization device does not embody the highest spray efficiency due to the shape of Taylor cones. Since minor offsets in relative angular positioning of the USB-CBS device could have similar effects as small offsets in horizontal placement, the effect to total ion current (TIC) and the RIC of 50 pg/L clenbuterol and 5 pg/L clenbuterol-d6 were investigated. Blades were kept at a similar distance of 8 mm to the MS inlet and directly pointed at the center of the MS inlet. The straight central positioning of the CBS device in front of the MS inlet is considered as an angular placement of 180°. The four tested offsets are 10°, 30°, 45° and 60° resulting in angles relative to the MS inlet of 190°, 210°, 225° and 240° respectively, as depicted in Figure 10. The upper portion of FIG. 10 shows the representation of the conventional 180° and the four tested angular offsets utilized for characterization of tolerance to angular positioning relative to the MS inlet. The lower portion of FIG. 10 shows area RICs and calculated RF of clenbuterol (diagonal stripes) and clenbuterol-d6 (dots) at the different angular offsets, including standard deviation (n=3). A minor effect of lower RICs of clenbuterol was observed at higher angular offsets. As shown in Figure 10, an increase in standard deviation (% real.SD) is observed by offsets beyond 10° when not corrected for the added internal standard. In contrast, the use of isotope- labelled internal standard results yields RF values of 0.96; 1.00; 1.05; 1.10 and 0.99, respectively. Consequently, the tested angles could all be applied without compromising linearity. To reduce errors caused by manual blade fixation, it would be recommended to assure consistent fixation by using dedicated holder cartridges, as previously commercially developed for paper spray.

Proof of principle of USB-CBS-transnortable-MS for on-site analysis

For proof of principle of USB-CBS-transportable-MS for on-site analysis, the improved ionization source 1 was combined with a transportable single- quadrupole mass spectrometer system (QDa). This commercial MS system has a small footprint, can be easily transported and is up and running in only 10 minutes. The applicability of USB-CBS-transportable-MS for b-agonist analysis was demonstrated using b-agonists. 6-agonists are illegal growth-promoting agents in livestock, due to the anabolic like side-effects, i.e. the dramatic increase in muscle mass and partially preventing or restoring muscle loss.

FIG. 11 shows a full scan mass spectrum of 20 signals of an aqueous solution composed out of 22 b-agonist at different concentrations, showing 20 m/z signals corresponding to theoretical m/z values of: 40 pg/L of clenbuterol (9), mabuterol (16), mapenterol (19), tulobuterol (3), chlorbrombuterol (18), hydroxymethylclenbuterol (12). 100 pg/L of clenpenterol (11), clenproperol (7), cimaterol (2), cimbuterol (4), isoxsuprine (13) en ritodrine, 200 pg/L of ractopamine (13), clencyclohexerol (17), salbutamol (5), salmeterol (2), zilpaterol (6), fenoterol (15), clenhexyl (14), procaterol (11) en carbuterol (8), 2000 pg/L of metaproterenol (1).

The affinity of B-agonists for Bondelut Certify coated material has been previously described and was expected the same for all the B-agonists given the similarities in basicity and amine moieties. The compatibihty of the Bondelut blades was confirmed by a full scan mass spectrum of an aqueous solution containing 25 B-agonists on the transportable MS. However as depicted in Figure 11, clear signals could be obtained for only 22 B-agonists. The recorded mass spectrum included two overlapping signals for clenpenterol (m/z 291) and procaterol (m/z 291), ractopamine (m/z 302) and isoxsurpine (m/z 302), due to similar monoisotopic masses being inseparable by the single-stage MS. Signals of reproterol (m/z 390), brombuterol (m/z 367), and zinterol (m/z 379) could not be detected. Reproterol, brombuterol and zinterol are also less sensitive in the LC- MS/MS method used in our institute for routine analysis. To determine the LOD a cahbration series of B-agonists spiked in bovine urine in the range of 2 to 2000 pg/L was analyzed in duplicate in SIR mode using m/z values of the selected B- agonists. For clenbuterol, mabuterol, mapenterol, tulobuterol, chlorbrombuterol, hydroxymethylclenbuterol, clenproperol, cimaterol, cimbuterol, ritodrine, salmeterol, clenhexyl and procaterol the LOD was in the rage of 20 to 50 pg/L. The LOD was for clenpenterol, isoxsuprine, ractopamine, clencyclohexerol, salbutamol, zilpaterol, fenoterol, carbuterol and metaproterenol in the range of 100 to 2000 pg/L, all above the concentration recommended by the EURL. While signals of 22 B-agonists at high concentrations in an aqueous solution and urine could be observed by low-resolution single-quadrupole MS, this MS system currently provides insufficient specificity and sensitivity at the low regulatory limits for B-agonists in bovine urine.

USB-CBS-MS(7MS) analysis of SARMs. GHRPs and rBST

To gain specificity, required to compensate for the lack of chromatographic separation prior to ionization as demonstrated by the B-agonist experiment, a tandem mass analyzer is needed. Further experiments were performed on an outdated benchtop triple-quadrupole MS/MS system, assuming to match the (limited) sensitivity of future truly portable triple-quadrupole mass spectrometers. Model analytes with different physicochemical properties were selected to demonstrate the capability of USB-CBS for different molecular weights: the selective androgen receptor modulators (SARMs) ostarine and andarine (380 and 441 Da), a growth hormone-releasing peptide (GHRP, 930 Da) and recombinant growth hormone somatotropin protein (rbST, 21.8 kDa). Moreover, by using the SARMs example, negative USB-CBS ionization was shown as well. Less background interference and thus higher selectivity associated with negative ionization could yield lower LODs and overall quantitative performance. Aqueous solutions of ostarine (50 pg/L), andarine (50 pg/L), [D-Lys3]-GHRP-6 (1 pg/mL) and rbST (30 pg/mL) were used and were sampled with Oasis HLB coated blades. Mass spectra were recorded in negative product-ion scan mode for ostarine and andarine (Figure 12a and 12b), positive ion scan and product-ion scan modes for [D-Lys3]-GHRP-6 (Figure 12c), and rbST (Figure 12d). Full scan spectra of [D-Lys3]-GHRP-6 and rbST demonstrated (multiple) protonated molecular ions. While [D-Lys3]-GHRP-6 was recorded as [M+2H]2+ at m/z 465, rbST displayed a charge state distribution typical of proteins.

Analysis of sulfonamide antibiotics in milk USB-CBS-MS/MS

A method was developed and an initial single day validation was performed for sulfonamides in milk. For CBS-MS analysis of sulfonamide antibiotics in bovine milk using Oasis HLB coated blades 2 for thirteen sulfonamide antibiotics two individual MRM transitions were selected. Sulfonamide antibiotics were spiked at maximum residue limit (MRL) concentration (100 pg/kg) in blank bovine milk. Oasis HLB coated blades were preconditioned and placed in an aliquot of 300 pL of bovine milk sample. Simplified rinsing with water was used to wash the sampled blades, before desorption and ionization at the MS inlet. This sampling approach was developed to be used in the future on-site by inspectors. FIG. 13 shows MRM chronograms of the most intense fragment of 13 sulfonamide antibiotics in bovine milk. The solid hnes represent chronograms of bovine milk spiked at MRL (100 pg/kg) and the dotted lines represent chronograms of blank bovine milk.

Clear MRM transitions were recorded for 13 sulfonamide antibiotics spiked at MRL in bovine milk and shown in Figure 13. The MRM transitions showed low negligible noise signals in blank matrix due to the selective extraction capabihties of the coating on the coated blades and additional selectivity of a tandem mass analyzer. Calibration curves with three replicates of 13 sulfonamide antibiotics and 9 corresponding internal standards were prepared in bovine milk. The constructed matrix-matched calibration curves demonstrated quantitative performance by using internal standards to correct for variances and relative matrix effects in milk. The matrix-matched calibration curves were used to determine the linearity and the LOD. The determined hnearity and LODs of individual sulfonamide antibiotics are given in the table reproduced in FIG. 14.

For eleven out of thirteen sulfonamide antibiotics, LODs below the MRL of 100 pg/kg milk were determined. Nine sulfonamide antibiotics had linearities of R2>0.91 close to the acceptance criterium of R2>0.99. Poorer quantitative performance was achieved for sulfacetamide, sulfamethizole, sulfachloropyridazine and sulfaphenazole. Increased background signals from the blank bovine milk matrix were also present for these compounds, as shown in Figure 13, suggesting less specificity for the coating on the blades during the extraction procedure. Also, no isotopic labeled analogs of these four sulfonamide antibiotics were used to correct for analyte recovery and variances in the bovine milk matrix sulfamerazine-13C6 was used. This could, therefore, limit the correction of specific differences in analyte recovery or ionization performance for these sulfonamide antibiotics. As for use of this method a full validation is needed, including precision, selectivity and specificity, and ruggedness, the preliminary results for the linearities and LODs demonstrate the proof-of-concept of USB-CBS-MS/MS as a semi-quantitative analysis tool for multiple sulfonamide antibiotics in bovine milk. There is great potential for the future when this technique can be used on-site if suitable MS/MS (trans)portable mass spectrometers would come available.

Summary

The present invention provides an improved ionization source. A simplified inexpensive universal applicable USB-powered ionization source has been developed for direct MS analysis that can be manufactured with low-cost components. As an example, the arrangement of the USB-powered ionization source 1 and a triple-quadrupole tandem MS showed to be fit-for-purpose as a semi- quantitative screening tool for most regulated sulfonamide antibiotics at their Maximum Residue Limit (MRL) (limits of detection between 25 and 200 pg/kg) in bovine milk.

Claims

1. Ionization source (1) for use with an atmospheric pressure inlet mass spectrometry system, comprising:

- at least one deposition area (21, 24) and/or at least one capillary channel for introducing a sample (22) or a solvent (23);

- at least one electrode (2) for causing desorption and/or ionization and transfer of sample material from the deposition area (21, 24) or the capillary channel (22) towards an atmospheric pressure inlet (71) of said mass spectrometry system;

- a power supply (3) having:

- an output (32) coupled to the electrode and;

- a controllable transformer (33) to transform an input supply voltage (V_s) into a high voltage (V_H) at the power supply output, and;

- a controller (34) for controlling the controllable transformer (33), characterized in that the ionization source comprises a USB -compatible input (31) configured to be coupled to a USB-port (51) of a mobile device to receive the input supply voltage (Vs).

2. The ionization source (1) according to claim 1, wherein the at least one deposition area (21) is a surface area of said at least one electrode (2) and/or wherein a content in the at least one capillary channel (22) is in electrical contact with the at least one electrode (2).

3. The ionization source (1) according to claim 2, wherein the at least one deposition area (24) for introducing the sample is positioned in-between the at least one electrode (2) with the at least one capillary channel (23) and the atmospheric inlet (71) of the mass spectrometer, wherein the at least one capillary channel (23) contains a solvent or solvent mixture.

4. The ionization source (1) according to one of the preceding claims, wherein the at least one deposition area is one of a plurality of deposition areas (including but not limited to microspots, nanospots and picospots) for introducing a plurality of samples.

5. The ionization source (1) according to claim 2 or 4, wherein the at least one electrode is an electrode in an array of multiple electrodes, having each one or more deposition area(s), for introducing respective samples wherein the electrodes of the array of multiple electrodes either are subsequently powered by the power supply or are simultaneously powered by the power supply.

6. The ionization source (1) according to claims 2 and 3, wherein the at least one capillary channel (22) and (23) has a shape and/or format different from that of a capillary, wherein optionally the channel is a microfluidic or a nanofluidic channel.

7. The ionization source (1) according to claims 2 and 3, wherein the at least one capillary channel (22) and (23) has the format of concentric capillaries.

8. The ionization source (1) according to claim 2 or 6, wherein the at least one capillary channel is one of an array or bundle of multiple capillary channels in any shape or format, for introducing multiple samples, which multiple capillary channels are either subsequently powered or simultaneously powered by the power supply.

9. The ionization source (1) according to one of the preceding claims, wherein the controllable transformer (33) comprises a first transformer module (331) to convert the input supply voltage (V_s) received at the USB -compatible input (31) into an intermediate voltage (Vi_nt) with a controllable voltage magnitude determined by the controller (34) and a second transformer module (332) to transform the intermediate voltage (Vi_nt) into an output DC-voltage (VH) having a voltage magnitude that is substantial proportional to the voltage magnitude of the intermediate voltage (Vi_nt) in accordance with a predetermined proportionality factor having a value of at least 100.

10. System comprising the ionization source (1) according to one of the preceding claims and a mobile device (5), wherein the ionization source is coupled with its USB -compatible input (31) to a USB-port (51) of the mobile device to receive the input voltage from the mobile device.

11. The system according to claim 10, wherein the mobile device has data processing facilities (52) and/or communication facihties (54), wherein the controller (34) is configured to operate according to an input control signal (Ci) received from the mobile device (5) via the USB -compatible input (31).

12. The system according to claim 11, wherein a user is enabled to operate the ionization source (1) with a user-interface (53) on the mobile device (5).

13. The system according to claim 11 or 12, wherein a remote operator is enabled to operate the ionization source (1) with the communication facilities (54) of the mobile device (5).

14. The system according to one or more of claims 10-13, wherein the mobile device is one of a mobile phone, a laptop or a tablet.

15. An arrangement comprising an mass spectrometry system (7) having an atmospheric pressure inlet (71) and a system according to either one of claims 10- 14, wherein the ionization source (1) is arranged to transfer ionized material from the sample towards the atmospheric pressure inlet (71) of the mass spectrometry system (7).

16. Record carrier comprising a program that when executed by a programmable processor of a mobile device in the system of either of the claims 10-14, or in the arrangement of claim 15 causes the mobile device to provide said input control signal (Ci).