Sample preparation and nanoelectrospray ionization mass spectrometry

Abstract

Method for loading a sample of a target compound into a nanospray emitter tube for analysis by nanospray ionization mass spectrometry, wherein a cartridge having a fluid container, an inlet and an outlet is mounted onto a nanospray emitter tube on a nanospray emitter mount to form a nanospray emitter tube assembly, the assembly is mounted on a micro-centrifuge tube, a volume of the sample to be analyzed is loaded into the fluid container and the micro-centrifuge tube is spun on a centrifuge to transfer the sample into the nanospray emitter tube.

Claims

1. A method for isolation of a target compound from a solution thereof, and analysis of said target compound by nanospray ionization mass spectrometry, which comprises the steps of: a) providing: i) a cartridge having a fluid container with a cartridge inlet and a cartridge outlet, with a solid phase extraction bed of porous sorbent media covering or placed within the cartridge outlet, ii) a nanospray emitter tube having a distal end and a proximal end, iii) a nanospray emitter mount having a nanospray emitter mount inlet and a nanospray emitter mount outlet, said nanospray emitter mount inlet having a surrounding flange and said nanospray emitter mount outlet being adapted to receive and hold said nanospray emitter tube with the proximal end projecting through said nanospray emitter mount outlet and said distal end spaced away from the nanospray emitter mount inlet a distance that permits engagement of the cartridge outlet of said cartridge with the distal end of said nanospray emitter tube when said cartridge is mounted on said nanospray emitter mount, and iv) a micro-centrifuge tube having a tube inlet opening with a rim around the inlet opening said cartridge having a first flange, complimentary to the flange of said nanospray emitter mount, a second flange, complimentary to the rim of said micro-centrifuge tube, and said cartridge outlet of said cartridge being adapted to mate with the distal end of said nanospray emitter tube, with the inner surface of said cartridge outlet being tangential to the inner surface of said distal end of said nanospray emitter tube at at least one point, and provide non-turbulent flow of a solution of said target compound from said fluid container into said nanospray emitter tube when said cartridge is mounted on the nanospray emitter mount inlet of said nanospray emitter mount, with the cartridge outlet of said cartridge mating with said distal end of said nanospray emitter tube, to form a nanospray emitter assembly and said nanospray emitter assembly is mounted on a micro-centrifuge tube and spun in a centrifuge to force non-turbulent flow of said sample solution from said fluid container of said cartridge, through said cartridge outlet and into said nanospray emitter tube, b) installing said nanospray emitter tube in the outlet of said nanospray emitter mount with the proximal end of said nanospray emitter tube projecting through said outlet of said nanospray emitter mount and said distal end spaced away from the nanospray emitter mount inlet a distance that permits engagement of the cartridge outlet of said cartridge with the distal end of said nanospray emitter tube, c) mounting said cartridge on said micro-centrifuge tube, loading said solution of said target compound into the fluid container of said cartridge, placing said micro-centrifuge tube with the cartridge mounted thereon in a centrifuge and spinning said micro-centrifuge tube in said centrifuge to force said solution to flow through said porous media and into said micro-centrifuge tube, whereby said target compound is adsorbed by said porous media, d) removing said cartridge from said micro-centrifuge tube and installing it on the emitter tube mount inlet of said nanospray emitter tube mount with the nanospray emitter tube in place and engaging the cartridge outlet of said cartridge with the distal end of said nanospray emitter tube to form a nanospray emitter tube assembly, mounting said nanospray emitter tube assembly on an empty micro-centrifuge tube, loading a volume of an extraction solvent into said fluid container of said cartridge, said volume of extraction solvent being substantially less than the volume of said solution of said target compound that had been loaded into said fluid container in step c), and spinning said micro-centrifuge tube in said centrifuge to force a non-turbulent flow of said extraction solvent through said sorbent media to extract said target compound from said sorbent media and form a solution of said target compound in said extraction solvent and non-turbulent flow of said solution of said target compound in said extracton solvent into said nanospray emitter tube, e) engaging said nanospray emitter tube in a mass spectrometer applying sufficient voltage to said nanospray emitter tube to cause electrospray ionization to occur and analyzing said electrospray in said mass spectrometer.

2. A method of loading a sample of a target compound into a nanospray emitter tube for analysis by nanospray ionization mass spectrometry, which comprises the steps of: a) providing: i) a cartridge having a fluid container, with a cartridge inlet and a cartridge outlet, ii) a nanospray emitter tube having a distal end and a proximal end, mounted in a nanospray emitter tube mount having a nanospray emitter tube mount inlet and a nanospray emitter tube mount outlet, said nanospray emitter tube mount inlet having a surrounding flange and said nanospray emitter tube mount outlet holding said nanospray emitter tube with the proximal end projecting through said nanospray emitter tube mount outlet and said distal end projecting towards and being spaced away from the nanospray emitter tube mount inlet a distance that permits engagement of the cartridge outlet of said cartridge with the distal end of said nanospray emitter tube, with the inner surface of said cartridge outlet being tangential to the inner surface of said distal end of said nanospray emitter tube at at least one point, when said cartridge is mounted on said nanospray emitter tube mount, and iii) a micro-centrifuge tube having a micro-centrifuge tube inlet opening with a rim around the micro-centrifuge tube inlet opening, said cartridge having a first flange, complimentary to the flange of said nanospray emitter tube mount, a second flange, complimentary to the rim of said micro-centrifuge tube, and said cartridge outlet of said cartridge being adapted to mate with the distal end of said nanospray emitter tube, b) mounting said cartridge on the inlet of said nanospray emitter tube mount and engaging the cartridge outlet of said cartridge with the distal end of said nanospray emitter tube to form a nanospray emitter tube assembly, mounting said nanospray emitter tube assembly on an empty micro-centrifuge tube, loading a volume of a sample to be analyzed into said fluid container of said cartridge, and spinning said micro-centrifuge tube, with said nanospray emitter tube assembly mounted thereon, in said centrifuge to transfer said sample into said nanospray emitter tube, said engagement of said cartridge outlet with said distal end of said nanospray emitter thereby being adapted to preserve non-turbulent flow from said cartridge into said emitter tube, c) engaging said nanospray emitter tube in a mass spectrometer applying sufficient voltage to said nanospray emitter tube to cause electrospray ionization to occur and analyzing said electrospray in said mass spectrometer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an expanded view of the components of a nanospray sample preparation system (100) illustrating nanospray emitter (2) in nanospray emitter mount (1), secondary fluidic element (3) with porous solid phase extraction bed (4) inserted therein, micro-centrifuge tube (5) and liquid tight cap (6) for the assembly.

(2) FIG. 2 is a detailed illustration of secondary fluidic element (3), showing sample reservoir (31), flange (32) for mating with the microcentrifuge tube, flange (33) for mating with the nanospray emitter mount, nozzle (34) for mating with the distil end of the emitter and porous solid phase extraction bed (4).

(3) FIG. 3 is a detailed illustration of nanospray emitter mount (1) with emitter (2) mounted therein, showing the distal end (22) and proximal end (23) of the emitter, bore (11) for the secondary fluidic element and flange (12) for mating with the micro-centrifuge tube.

(4) FIG. 4 is a detailed illustration of the nanospray emitter mount (1) with secondary fluidic element (3) mounted therein and nozzle (34) mating with distill end (22) of emitter (2).

(5) FIG. 5 is an isometric illustration of the elements of the nanospray sample preparation system shown in FIG. 1.

(6) FIG. 6 is an illustration of a nanospray source (7) for use with the nanospray sample preparation system (100), having a hole (71) to accept the nanospray assembly, a source housing (72), a high voltage source (73) with high voltage contact (74), slot (75), transfer arm (76) and release arm (77), shown supporting the nanospray preparation system by the flange on the micro-centrifuge tube. The high voltage contact (74) is adjacent to a mass spectrometer inlet (300).

(7) FIG. 7 illustrates transfer arm (76) being pushed to separate the nanospray emitter assembly of the nanospray emitter mount, the nanosopray emitter and the secondary fluidic element from the micro-centrifuge tube.

(8) FIG. 8 illustrates release arm (77) being pulled away from the micro-centrifuge tube, whereby the microcentrifuge tube is no longer supported and falls away.

(9) FIG. 9 illustrates transfer arm (76) being pushed to transport the nanospray emitter assembly through the slot to the end of the source (7) that is adjacent to the mass spectrometer inlet (300) and into contact with the high voltage contact (74).

(10) FIG. 10 illustrates the nanospray emitter assembly being withdrawn by the transfer arm to a position above the hole in the source body.

(11) FIG. 11 illustrates the transfer arm being separated from the nanospray emitter assembly, whereby the nanospray emitter assembly is no longer supported and falls away.

(12) FIG. 12 illustrates five minutes of data collection of both total ion current (top) and the ion current of protonated buspirone molecular ion (bottom) in the procedure of Example 1.

(13) FIG. 13 illustrates the results of an analysis of three samples using the system of Example 1.

(14) FIG. 14 illustrates the enhanced effect of mass spectrometer analysis with solid phase extraction (SPE) as compared to conventional (non SPE) analysis of triply charged Angiotensin I molecular ion at 433 m/z.

(15) FIG. 15 illustrates the enhanced effect of mass spectrometer analysis with solid phase extraction (SPE) as compared to conventional (non SPE) analysis of doubly charged Angiotensin II molecular ion at 524 m/z.

(16) FIG. 16 compares averaged full scan mass spectra for non SPE prepared samples (top) and SPE prepared samples (bottom).

(17) FIG. 17 shows three different embodiments (most least preferred, least preferred, and preferred) for the mating action between the nozzle of the secondary fluidic element (3) and the distal end of the nanospray emitter (22).

(18) FIG. 18 shows two additional embodiments (more preferred, most preferred) for the mating action between the nozzle of the secondary fluidic element (3) and the distal end of the nanospray emitter (22).

(19) FIG. 19 shows a detailed view of the most preferred embodiment for the nozzle of the secondary fluidic element (3) in relation to the distal end of the nanospray emitter (22).

(20) FIG. 20 shows the raw and normalized intensity distribution of a blue dye in relation to the proximal end of the nanospray emitter (23) as eluted from the SPE bed (4) as described in example 6.

EXAMPLES

Example 1

Efficient Loading of a Microliter Scale Sample

(21) An emitter assembly consisting of a nanospray emitter, nanospray emitter mount and secondary fluidic element, as shown in FIG. 4 was prepared. A nanospray assembly consisting of the emitter assembly and microcentrifuge tube was then assembled, as shown in FIG. 1 but without the cap. The mounted glass nanospray emitter was fabricated from 1.2 mm outside diameter, 0.69 mm inside diameter borosilicate glass tubing. The emitter had a 2 μm inside diameter orifice at it's tapered proximal end, and was coated with an electrically conductive platinum metal film. The nanospray emitter protruded approx. 20 mm from the emitter mount. Total emitter length was approximately 25 mm. The diameter of the though hole connecting the secondary fluidic element with the distal end of the nanospray emitter was 0.2 mm. The diameter of the sample reservoir in the secondary fluidic element was 3.1 mm. The secondary fluidic element was initially empty, and did not have a porous solid phase extraction bed in place.

(22) A sample of the chemical compound buspirone (CAS number 36505-84-7, formula weight=385.5 Da) was prepared at a concentration of 1 ug/mL in 50% acetonitrile, 0.1% formic acid. 5 uL of this sample was delivered into the sample reservoir of the secondary fluidic element using a conventional laboratory micro-pipettor (Eppendorf corp.). The nanospray assembly was capped and loaded into a fixed rotor centrifuge (Eppendorf model 5414C) and spun at a force of 326 g (2000 rpm) for approximately 30 sec.

(23) During centrifugation, the sample passed through the secondary fluidic element into the nanospray emitter. Greater than 85% of the total volume was transferred into the nanospray emitter. The nanospray assembly was then removed from the centrifuge.

(24) A nanospray source built to accept the nanospray assembly, and shown schematically in FIGS. 6 through 11, was mounted on a linear ion trap mass spectrometer (Thermo Fischer LTQ).

(25) The nanospray assembly was dropped into the hole on the top side of the nanospray source (FIG. 6). The assembly is prevented from falling through the source by a release arm that permits only partial penetration of the assembly though the source by interfering with the rim of the microcentrifuge tube. A transfer arm is then pushed in place by sliding it towards the nanospray assembly (FIG. 7). A tapered U-shaped element on the front of the transfer arm (A) physically separates the emitter assembly from the microcentrifuge tube by raising it approx. 2 mm above the rim of the microcentrifuge tube, and (B) captures the emitter assembly for positioning. As shown in FIG. 8, the release arm is pulled back, and the microcentrifuge tube falls away from the nanospray source due to gravity.

(26) The transfer arm is then pushed fully forward (FIG. 9). This carries the remaining elements of the nanospray assembly, i.e., the emitter assembly, through a slot in the bottom of the nanospray source housing to the forward position. In this position, the conductive coating on the nanospray emitter is in contact with a spring loaded electrical contact within the body of the nanospray source housing. This contact is in turn connected to a 1.5 kilovolt high voltage power supply coming from the mass spectrometer.

(27) This voltage is sufficient to cause electrospray ionization to occur. As the proximal end of the nanospray emitter is now in close proximity to the mass spectrometer inlet (within 3 mm), ionization signal is obtained. FIG. 12 shows five minutes of data collection of both total ion current (top) and the ion current of the protonated buspirone molecular ion (bottom) at a mass-to-charge ratio of 386 m/z from the mass spectrometer.

(28) After data collection, the transfer arm is pulled away until the nanospray emitter assembly is positioned over the hole (FIG. 10). Further pulling of the transfer arm causes the nanospray emitter assembly to make contact with the side of the housing wall. This releases (FIG. 11) the emitter assembly from the transfer arm and it falls away from the source housing due to gravity.

Example 2

Rapid Analysis of Multiple Samples

(29) The system of example 1 was used to evaluate the performance for the analysis of multiple samples. Samples A, B, and C of commercially available peptides (Sigma-Aldrich Corporation) were prepared individually at a concentration of 1 ug/mL in 50% acetonitirle and 0.1% formic acid for a total volume of 100 uL each. Sample A was Angiotensin I (Sigma catalog number A9650-1 MG, formula weight=1296 Da). Sample B was valine substituted Angiotensin I (Sigma catalog number A9402-1 MG, formula weight=1282 Da). Sample C was Angiotensin II (Sigma catalog number A9525-1MG, formula weight=1045 Da). As in example 1, 5 uL of samples A, B, and C were individually loaded into the secondary fluidic element of three nanospray assemblies, respectively. The three assemblies' were placed in the rotor of the micro-centrifuge used in example 1 and simultaneously spun using the same conditions.

(30) The mass spectrometer was placed into data collection mode and a data file was acquired during the entire nanospray source loading process. After removal of the three micro-centrifuge tubes from the centrifuge, the first sample, sample A, was placed in the nanospray source and the transfer and release arms were manipulated as in example 1 (FIG. 9), positioning sample A in the signal collection position. Signal was collected for approximately 20 seconds. The transfer arm was pulled back to eject sample A from the source. The release arm was then pushed forward and sample B was loaded into the source. The release arm and transfer arm were again manipulated as in example 1 (FIG. 9) to position sample B for signal collection. Signal was collected for approximately 20 seconds. The transfer arm was pulled back to eject sample B from the source. The release arm was then pushed forward and sample C was loaded into the source. The release arm and transfer arm were again manipulated as in example 1 (FIG. 9) to position sample C for signal collection. Signal was collected for approximately 20 seconds. The transfer arm was pulled back to eject sample C from the source. The data collection of the mass spectrometer was then stopped.

(31) FIG. 13 shows the output of the resulting data collection file. The total ion current, and molecular ion current for samples A, B, and C are shown. Sample A shows the triply protonated molecular ion at a 433 mass-to-charge ratio. Sample B shows the triply protonated molecular ion at a 428 mass-to-charge ratio. Sample C shows the doubly protonated molecular ion at a 524.5 mass-to-charge ratio. This device demonstrates fast sample throughput with zero experimental carry-over from one sample to the next due to the non-redundant fluid path in which a single assembly (100) is used only once for the processing and analysis of an individual sample. Even with manual loading, the analysis of three samples occurred within a 1.5 minute time frame.

Example 3

Analysis Combined with Solid Phase Extraction for Sample Preparation

(32) The system of example 1 was modified so that the secondary fluidic element contained a porous sorbent media, suitable for sample preparation and concentration by solid phase extraction. The porous sorbent media was contained within the narrow portion of the fluidic element's inner through bore, just prior to the proximal end of the fluidic element that mates with the distal end of the nanospray emitter.

(33) In this specific example, the porous sorbent media consisted of a plug of Empore™ C18 extraction disk media (3M corporation, part number 2315). The plug of Empore was approx. 0.43 mm in diameter by 0.5 mm thick, representing a total volume of approx. 0.073 μL. The Empore disk is a fibrous network of PTFE (Teflon®) with adsorbent particles (90% by weight) embedded and bonded to the PTFE (10% of the disk by weight). This porous disk allows the passage of liquid through the disk pass but traps semi- or non-volatile organic compounds that are adsorbed by the embedded sorbent particles. Other types and chemical formulations of sorbent media would also prove suitable, such as conventional packed particle beds or polymerized monolithic structures.

(34) A sample containing a mixture of known peptide standards was prepared in 0.1% formic acid at a concentration of 1 ug of protein, per millileter for each peptide. Peptides in the mixture included those used in example 2. The mixture contained: Angiotensin I (Sigma catalog number A9650-1MG, formula weight=1296 Da), valine substituted Angiotensin I (Sigma catalog number A9402-1 MG, formula weight=1282 Da), and Angiotensin II (Sigma catalog number A9525-1MG, formula weight=1045 Da).

(35) A secondary fluidic element containing the Empore extraction media was placed on top of an empty micro-centrifuge tube. The Empore extraction media was then chemically conditioned prior to use, according to the manufacturer's recommendations. A 40 uL aliquot of methanol was transferred by pipette into the secondary fluidic element reservoir. This assembly was placed in a micro-centrifuge and briefly spun at a force of 326×g (2000 rpm for less than 1 minute) forcing the methanol through the media. 40 uL of 0.1% formic acid was then added to the reservoir of the secondary fluidic element and again spun in the centrifuge to condition the sorbent just prior to sample loading.

(36) A 40 μL aliquot of the sample mixture was then loaded into the secondary fluidic element reservoir by pipette and it was again spun in the centrifuge under identical conditions. At this point, any chemical compounds present in the mixture, having sufficient hydrophobic character, will be chemically adsorbed to the surface of the sorbent media particles.

(37) The loaded secondary fluidic element is then transferred to an assembly consisting of a second micro-centrifuge tube and a nanospray emitter assembly as previously described in example 1.

(38) A 5 μL aliquot of extraction solvent consisting of 0.1% formic acid and 80% acetonitrile (by volume) was added to the secondary fluidic element reservoir.

(39) The assembly was then transferred to the centrifuge and again spun at a force of 326×g (2,000 rpm) for less than one minute.

(40) The assembly was then placed into the nanospray source apparatus operated and identically as described in example 1.

Example 4

(41) The same sample peptide mixture of example 3 was then analyzed in a manner identical to that of example 1, to generate baseline data for a non-SPE prepped sample. This comparison allows one to characterize the efficiency and benefit of the SPE sample preparation step of example 3.

(42) Data from each acquisition (non-SPE and SPE preparation) of this sample is shown in FIGS. 14, 15, and 16.

(43) FIGS. 14 and 15 show the surprisingly enhanced effect of this implementation of solid phase extraction compared with analysis of the conventional (non SPE) ion intensities, shown as black crosses. The obtained intensity with SPE sample preparation is shown as open circles for the triply charged Angiotensin I molecular ion at 433 m/z (FIG. 14) and the doubly charged Angiotensin II at 524 m/z (FIG. 15). The expected signal intensity assuming 100% trapping and extraction efficiency is shown by the dashed line in each figure.

(44) In each case, the obtained peak ion intensity is nearly an order of magnitude higher in intensity that that predicted by the volumetric ratio of the sample volume to extraction solvent volume (8:1). This means that the effective extraction volume (the volume that the analyte is contained in) must be much smaller than the actual applied extraction volume, thus providing a surprising and advantageous analytical outcome. A non-homogenous distribution of analyte within the nanospray emitter tube explains this favorable outcome. The inventive device and method enables the results one would obtain with the use of the smallest volume necessary for the extraction of analyte from the sorbent bed. Because the invention is able to use a larger volume than necessary to complete the extraction process while preserving the results of the smallest necessary volume, there is no additional requirement for exceptional expertise, apparatus, or equipment for effective use of smaller volumes and little analytical penalty for the use of higher-than-necessary extraction volumes.

(45) FIG. 16 compares averaged full scan mass spectra (17 scans taken from the 0.5 minute after the start of acquisition) for no-prep (top) and SPE prep sample (bottom). Note the far superior signal-to-noise ratio for the SPE prepped sample. Again the volumetric ratio of the SPE process would predict ion intensity approx. 8× that of that obtained of the unprocessed sample. Actual ion intensities for the prepped sample are significantly higher. For example the 524 m/z ion shows an intensity between 6×10.sup.5 to 1×10.sup.6 counts in the no-prep sample. Assuming a 100% sample trap and elute efficiency for SPE, the volumetric benefit of SPE should yield a signal intensity of approx. 8× this level, or 8×10.sup.6 counts. In actuality, the signal obtained for the SPE prep was approx. 3.6×10.sup.7, a realized gain of 35×. This is a signal intensity that is 437% greater than the maximum expected for a fully efficient trap and extraction procedure. A similar result, based on the use of smaller extraction volumes with traditional methods, would require a reduction of the actual eluent volume from 5 μL to 1/35 that value (0.14 μL).

Example 5

Use of an Alternate Sorbent Media

(46) The apparatus used in examples 3 and 4 were modified to demonstrate the use of the invention with an alternate solid phase sorbent media, substituting for Empore media, inside the secondary fluidic element.

(47) The Empore extraction membrane was replaced with a layered-sorbent bed that consisted of a glass fiber filter disk frit (Whatman Corporation Filter paper type GF/A) and a packed bed of 5 μm spherical and porous (30 nm pore) octa-decylsilyl (C18) bonded silica particles (W.R. GRACE corporation). The overall dimensions of the glass filter and packed particle bed inside the secondary fluidic element were roughly the same as the Empore membrane disk, having a diameter of 0.43 mm and a thickness of between 0.5 to 0.6 mm. This type of layered construction for an SPE device and method is well known and described in the prior art.

(48) An identically prepared sample to that used in example 3 was processed to establish the relative performance for SPE enrichment of the packed bed secondary fluidic element.

(49) The bed of the secondary fluidic element was treated prior to sample loading by conditioning the bed with 40 uL of methanol and spinning in the centrifuge at 2,000×g (5,000 rpm) for 15 seconds. This was followed by the addition of 10 uL of pure water and spinning again at 2,000×g (5,000 rpm) for 15 seconds. Two 40 μL aliquots of the sample mixture were loaded into the secondary fluidic element reservoir by pipette and spun in the centrifuge at 2,000×g (5,000 rpm) for 30 seconds. At this point, any chemical compounds present in the mixture, having sufficient hydrophobic character, will be chemically adsorbed to the surface of the packed bed C18 media.

(50) The loaded secondary fluidic is then transferred to an assembly consisting of a second micro-centrifuge tube and a nanospray emitter assembly as previously described in example 1.

(51) A 5 μL aliquot of extraction solvent consisting of 0.1% formic acid and 80% acetonitrile (by volume) was added to the secondary fluidic element reservoir. The assembly was then transferred to the centrifuge and again spun at a force of 2,000×g (5,000 rpm) for 15 seconds.

(52) The assembly was then placed into the nanospray source apparatus operated identically as described in example 1. Full-scan mass spectrometric data was acquired for 5 minutes.

(53) A reference sample of peptides at 1 pmol/uL in 80% acetonitrile and 0.1% formic acid, prepared identically as that of example 4, was subsequently loaded into the apparatus as described in example 1 to provide a reference signal for a non-SPE prepared sample. Full-scan mass spectrometric data was acquired for 5 minutes. Data analysis of the triply charged Angiotensin I molecular ion at 433 m/z was analyzed post acquisition to compare performance with the SPE and non-SPE processed samples. For the SPE processed sample a peak intensity and average intensity (30 second average) of 1.72×10.sup.7 and 6.19×10.sup.6 were observed. For the non-SPE processed sample, a peak intensity and average intensity (30 second average) of 8.6×10.sup.5 and 2.86×10.sup.5 were observed. This represents observed ratios of 19.9 and 21.6 fold for peak and average ion intensities respectively.

(54) This result is greater than that expected for a system demonstrating 100% extraction and elution efficiency, which means that the invention yields a surprising effect whereby the effective elution volume is smaller than the total applied elution volume loaded into the device. The expected signal intensity, assuming 100% trapping and extraction efficiency, would yield an approximate 16-fold increase in ion intensity, representing the ratio of sample-to-elution volumes (80/5=16). The results are similar to that obtained if one were to reduce the actual applied volume of eluent by 1/20, from 5 μL to 0.25 uL. This is particularly advantageous since handling sub-microliter volumes with normal laboratory apparatus is considered either impractical or highly time-consuming and requiring expert practice. For extraction of miniaturized volumes (less than 10 uL) it is particularly advantageous to extract in the smallest practical volume.

Example 6

Alternate Validation of the Concentration Distribution

(55) Example 3 was repeated as described with two substitutions: (A) the substitution of a colored dye solution replaced the peptide sample and (B) a nanospray emitter without a conductive coating was used to permit visual observation of the emitter's contents. Using a colored dye allows for the direct visual observation and photo-documentation of analyte concentration. The colored dye consisted of a 40 uL aliquot of FD&C BLUE 1 food coloring (McCormick & Co. Inc.) that had been previously diluted by 1000 fold in distilled water. The sample loading and elution operation was as described in example 3.

(56) Immediately subsequent from elution of the sample into the nanospray emitter (2), the nanospray emitter (2) and nanospray emitter mount (1) were manually removed from the assembly (100) and placed under a conventional stereomicroscope (50× magnification) and digitally photographed using reflected light illumination. The digital photo was then analyzed with a quantitative image processing program (Image J from the National Institutes of Health; http://www.imagej.org). The program was used to measure the relative absorbance and the distribution of dye inside the nanospray emitter.

(57) The results of this analysis are shown in FIG. 20. The light line shows the raw pixel intensity representing the concentration of dye within the emitter. It was clearly observed that the distribution was non-uniform, and the concentration of dye increased closer to the proximal end (23) of the emitter (2). The actual concentration increase was then normalized (shown as the heavy line in FIG. 20) to the diameter of the emitter (2). Because the proximal end (23) was tapered with a cone angle of approximately 12 degrees, there was less of an optical path length for dye absorption the closer one is to the proximal end (23). Normalizing the raw intensity with the measured diameter of the emitter shows a response that is well correlated to the dye concentration. The peak normalized intensity, near the proximal end of the emitter, was 28 fold higher than the mean level of dye at a distance 5 mm away from the proximal end.

(58) Note that the normalized peak intensity of dye is very similar to the distribution of peptide ion current obtained from example 3. Therefore a rational explanation of the increase in observed ion intensity is that the analyte has a non-uniform distribution within the nanospray emitter.

(59) It is important that the desirable concentration distribution is preserved inside the nanospray emitter tube. The dimensions of the interior volume of the nanospray emitter with respect to the total elution volume is critical for preservation of the concentration gradient. As the emitter sits prior to analysis, diffusion will drive the contents of the emitter to a homogeneous distribution. In the examples presented here the inside diameter of the nanospray emitter was 1.2 mm at the distal end (22). At the proximal end (23) of the emitter the inside diameter tapered to a 2-4 μm orifice over a total length of approx. 4.5 mm with a cone angle typically between 12-14 degrees. Using a nanospray emitter that was both longer, and of a narrower inside diameter would improve the preservation of the concentration gradient over time. The effective use of a smaller elution volume would require a smaller ID nanospray tube, a longer taper region at the proximal end (23), or both, for effective use. A smaller diameter, and/or longer taper would also relax the need for immediate analysis by mass spectrometry since longitudinal diffusion inside the emitter tube would be reduced. A larger elution volume would not benefit from a larger ID nanospray tube however, since axial and longitudinal diffusion would be enhanced with inside diameters much greater than or equal to 2 mm.

(60) The examples presented within all use a centrifuge to generate the forces for the transfer of liquid sample and eluent from the secondary fluidic element into the nanospray emitter. As is known to those skilled in the prior art of solid-phase extraction, other physical means of effecting the fluidic transfer are viable. These other methods include the use of a pressure differential (either high-pressure gas or vacuum or both) across the secondary fluidic element and/or nanospray emitter to induce flow.