A miniature mass spectrometer for liquid chromatography applications

Research Article

Received: 6 July 2011 Revised: 16 August 2011 Accepted: 23 August 2011 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2011, 25, 3281–3288

A miniature mass spectrometer for liquid chromatographyapplications

Andrew Malcolm1, Steven Wright1*, Richard R. A. Syms2, Richard W. Moseley1,Shane O’Prey1, Neil Dash1, Albert Pegus1, Edward Crichton1, Guodong Hong1,Andrew S. Holmes2, Alan Finlay1, Peter Edwards1, Simon E. Hamilton3 andChristopher J. Welch4

1Microsaic Systems plc, Unit 2, GMS House, Boundary Rd, Woking, Surrey, GU21 5BX, UK2Department of Electrical and Electronic Engineering, Imperial College, Exhibition Rd, London, SW7 2AZ, UK3Merck Sharpe & Dohme, Hertford Road, Hoddesdon, EN11 9BU, UK4Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, NJ, USA

A miniature mass spectrometer capable of detecting analytes eluting from a high-performance liquid chromatogra-phy (HPLC) system is described and demonstrated for the first time. The entire instrument, including all pumpsand the computer, is contained within a single enclosure that may be conveniently accommodated at the base ofthe HPLC stack. The microspray ion source, vacuum interface, ion guide, and quadrupole ion filter are allmicroengineered. These components are fabricated in batches using microelectromechanical systems (MEMS)techniques and considered to be consumables. When coupled to a standard HPLC system using an integrated passivesplit, the limit of detection for reserpine while scanning the full mass range is 5 ng on-column (1 pg of which ispassed to the microspray). The mass range is m/z 100–800, and each spectrum is typically acquired at a rate of 1 scanper second. Copyright © 2011 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.5230

The miniaturization of analytical instrumentation has beenmostly driven by a desire to perform analyses in the field,particularly in challenging environments or when a rapidresponse is demanded. Techniques that were once confinedto the laboratory and practised by skilled operators are nowused for industrial process control, environmental monitor-ing, security applications, point-of-care medical diagnostics,and space exploration.[1,2] There is also a strong case for theuse of miniature and compact instrumentation within the tra-ditional laboratory. The growing amount of equipment beingused by chemists has forced greater consideration of factorssuch as the linear bench space occupied by instruments, noiseand vibration, heat generation, and workflow. In addition,cost-containment has become a priority within most sectors.Apart from the initial purchase price, long-term expensessuch as power consumption, downtime and servicing, sol-vent use, and consumables add significantly to the total costof ownership. These issues are of particular concern in thepharmaceutical industry, as the spiralling developmentexpenditure per approved new drug has forced a rationaliza-tion of costs and a drive towards more efficient and effectiveworking practices.[3]

The detection of analytes in solution by electrospray ioniza-tion mass spectrometry (ESI-MS) was first demonstrated byYamashita and Fenn more than 25 years ago.[4] Since then,

* Correspondence to: S. Wright, Microsaic Systems plc, Unit 2,GMS House, Boundary Rd, Woking, Surrey GU21 5BX, UK.E-mail: [email protected]

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liquid chromatography/mass spectrometry (LC/MS) hasbecome a pre-eminent technique in analytical chemistry,rivalled only by nuclear magnetic resonance (NMR), as theanalytical technique of choice for the confirmation and deter-mination of chemical identity. Unfortunately, despite manyyears of commercial development, ESI-MS systems remainexpensive, bulky, power hungry, and costly to maintain. Typi-cally, they are free-standing with attendant PC, monitor, andperipherals. Although generally hidden from the user, a largerotary pump is usually accommodated on the floor nearbyand connected to the instrument via a bulky vacuum hose.In many laboratories, an LC/MS system is often availableonly as a communal or centralized facility as a consequenceof the purchase price, high running costs, and limited avail-ability of bench space.

Some LC/MS applications, particularly those involvinganalysis of biomolecules, demand the sensitivity afforded byhigh-end mass spectrometers. However, there are many otherapplications, currently served by conventional entry-levelsingle quadrupole mass spectrometers, that require much lesssensitivity. For example, in small-molecule synthetic chemis-try, the monoisotopic molecular mass of intermediates is usedas confirmation of molecular identity. Generally, solutions ofthese intermediates, which are typically available in relativelyabundant quantities, are prepared to a convenient concentra-tion prior to analysis.[5] Given the undoubted desirability ofLC/MS as an analytical tool, there is a clear need for a moreaffordable, economical, miniature mass spectrometer capableof a level of performance sufficient for use in routine analysisby synthetic chemists at the bench.

Copyright © 2011 John Wiley & Sons, Ltd.

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Although a number of miniature mass spectrometersystems have now been reported,[6–17] they have beendesigned primarily for monitoring and security applications.Some of these systems employ electron ionization inside thevacuum chamber, and accept gaseous analytes drawn inthrough a membrane or narrow capillary,[7,11,12] absorbed ona fibre,[6] or from a gas chromatography (GC) column.[8,10]

They are mostly compact and light by virtue of the smallpumps needed to displace the very low gas loads involved.Such instruments are clearly unsuitable for directly analyzingliquid samples eluting from an LC system. Several miniaturemass spectrometers,[13–17] which are described in more detailbelow, have been coupled to atmospheric pressure ionsources, but none have been demonstrated in combinationwith an LC system.An instrument used to detect gaseous analytes is an early

example.[13] It has an atmospheric pressure chemical ioniza-tion (APCI) source, two vacuum stages, and weighs less than20 kg. A skimmer in the first stage operates at the relativelyhigh pressure of 10 Torr and passes a small fraction of theflow through the 80 mm inlet nozzle to a monopole mass filterin the second vacuum stage, which tolerates a pressure of upto 10–3 Torr.Researchers at Purdue University have developed a minia-

ture instrument that admits ions generated by various atmo-spheric pressure ionization techniques into a single vacuumstage through a capillary.[14,15] The operating pressure is1.5� 10–2 Torr, which is close to the upper limit that can betolerated by the miniature rectilinear ion trap used for massanalysis. As a result of the high operating pressure, the reso-lution achieved is relatively poor, and difficulties associatedwith arcing have been reported.[15] Better performance isobserved when a pinch valve, pulsed at a rate of 1 Hz, is usedto control the flow of gas through the capillary and into thevacuum system. This normally closed valve is opened for20 ms at the start of each cycle, with the mass scan timed tostart 500 ms later, once the gas load has been evacuated.Although suited to the periodic nature of ion trap operation,this pulsed mode of ion introduction is likely to be inefficientwhen using an analyzer that is scanned continuously.The same group also demonstrated a more conventional

three-stage system in which ions sampled by a skimmer inthe first stage are transferred by an ion guide in the secondstage to a miniature cylindrical ion trap in the final stage.[16]

Although the vacuum chambers and components thereinare considered miniature, a large rotary pump is used topump the first stage. However, Griffin Analytical Technolo-gies (West Lafayette, IN, USA) have developed a prototypeminiature system based on a very similar design.[17] To avoidthe need for a floor-standing rotary pump, a small drag pumpbacked by a diaphragm pump is used to pump the first stage.The instrument weighs 45 kg and is contained within a 0.1 m3

enclosure (approximately a cube of side 45 cm).Using the techniques and materials that have emerged

from the field of microelectromechanical systems (MEMS),Microsaic Systems (Woking, UK) has developed a numberof miniature mass spectrometer components.[6,18–22] Theintention has been to batch produce accurately engineeredbut inexpensive devices to replace parts that have tradition-ally been high value. MEMS technology is a genericdescription of integrated devices incorporating componentswith a characteristic dimension of less than 1 mm. These are

wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wile

typically fabricated by repetitive use of planar processessuch as photolithography, material deposition, and etch-ing.[23] MEMS technology has been extensively employedin the fabrication of micro total analysis and lab-on-chipsystems,[24] and is now being increasingly applied to massspectrometry.[25,26]

Several years ago, we developed a simple quadrupole massfilter based on a multilayer silicon frame.[18,19] This filter wasintegrated into a miniature system[6] intended for field analy-sis of samples collected by solid-phase microextraction(SPME). It remains the only portable mass spectrometersystem to incorporate a MEMS mass spectrometer. The massfilter was mounted together with an electron ionizationsource and a multiplying detector in a small, interchangeablecartridge that was plugged into a mating socket in thevacuum chamber.

More recently, an advanced quadrupole mass analyzer, avacuum interface, and a microspray ionization source haveall been developed using MEMS technology. The designs,operating principles, methods of fabrication, and perfor-mance characteristics of these individual components havebeen reported in detail elsewhere.[20–22] In the present paper,we demonstrate for the first time, a complete miniature ESI-MS system (Microsaic 3500 MiD) in which all the componentsused for ion generation, transmission, and mass analysis aremicroengineered. The intention has been to develop a minia-ture laboratory instrument that may be interfaced to conven-tional LC systems, rather than a portable instrument for fieldapplications. Below, we first give an overview of the systemarchitecture and its component parts, then present data illus-trating the mass range, resolution, and sensitivity of theinstrument.

EXPERIMENTAL

Miniature mass spectrometer system

The miniature mass spectrometer system, including all thepumps, weighs 27 kg and is mostly contained within a steelbox measuring 35� 18� 62 cm. A molded cover attached tothe front fascia panel protects the microspray assembly,which is mounted outside the main enclosure for ease ofaccess. In addition, a keyboard, mouse, and monitor arerequired to operate the system. The instrument is showncoupled to a conventional high-performance liquid chroma-tography (HPLC) system (1100 Series, Hewlett-Packard,Santa Clara, CA, USA) in Fig. 1. It is possible to position theinstrument next to the HPLC system, either laid flat or onits side, rather than at the base of the stack as shown. At therear of the enclosure, there are connections for the nitrogensupply, pump exhaust, master/slave control, monitor, andwireless transceiver for the mouse and keyboard. An Ethernetconnection and a USB port are also provided for data transfer,and an RS-232 port is available for communication with asyringe pump.

An on-board computer hosts the graphical user interfaceand handles data acquisition, post-processing, calibrationroutines, and all low-level control tasks. Data is stored on a160 GB internal hard drive. During operation, the graphicaluser interface displays two panes. One shows the total ionchromatogram (TIC), an extracted ion chromatogram (EIC),

y & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 3281–3288

Figure 1. Photograph of a Microsaic 3500 MiD miniature mass spectrometercoupled to a standard HPLC stack. The instrument can also be placed on its sidenext to the stack. The inset shows the microspray assembly, which is normallyconcealed by a protective cover.

Miniature mass spectrometer for LC applications

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a selected ion monitoring (SIM) chromatogram, or the basepeak chromatogram (BPC), while the other displays theindividual mass spectra as they are acquired. Followingacquisition, the data may be examined in a separate analysiswindow. Dragging the cursor across any peak in the chro-matogram yields a summed mass spectrum for that periodof the data acquisition.The power requirement is approximately 250 W during

initial pump down, 300 W during operation with the RFpower supply at its maximum output, and just 220 W whenthe system is pumped down but idle. This compares veryfavourably with conventional systems, which typically havea power consumption of 1–2 kW. The noise level, measuredat a distance of 1 m using an ST 8820meter (Reed Instruments,Ste-Anne-De-Bellevue, Quebec, Canada), is 58 dB(A) relativeto the standard 20 mPa (RMS) reference level.A three-stage vacuum system is employed, which has a

total pumped volume of approximately 800 cm3. The firststage is a microengineered vacuum interface. A smalldiaphragm pump is used to differentially pump this compo-nent. The second and third vacuum stages are pumped byturbomolecular pump technology backed by a second dia-phragm pump. Ions generated by the microspray ionsource are first drawn through the front orifice of themicroengineered interface. A fraction of the gas and entrainedions is transmitted through the rear orifice and into the sec-ond vacuum stage, where a short microengineered quadru-pole ion guide directs the ions towards an aperture in thebulkhead between the second and third vacuum stages. Afterpassing through this aperture, ions are filtered according totheir mass-to-charge ratio (m/z) by a microengineered quad-rupole mass filter, and then accelerated towards a conversiondynode. The resulting secondary electrons are detected by amultiplying detector operating in pulse counting mode.The microspray ion source, vacuum interface, ion guide,

quadrupole mass filter, and ion detection components are allmounted on a piston that is accommodated by the circular

Copyright © 2011Rapid Commun. Mass Spectrom. 2011, 25, 3281–3288

bore of the vacuum manifold. This piston may be entirelyremoved from the system as all electrical connections aremade with push-fit connectors, and O-ring seals are used toseparate the various stages of differential pumping. Each ofthe microengineered components, which are considered tobe consumables, can be readily removed from the piston.

Microspray ion source

Users of microspray and nanospray ion sources oftenencounter difficulties associated with making multiplemicrofluidic connections, replacing emitter tips, and optimi-zation of the emitter tip position relative to the vacuuminterface. These tasks are generally time-consuming andrequire more than a casual familiarity with the equipment.As described in previous reports,[21,22] a microengineeredmicrospray source has been developed that incorporatesfeatures to accurately align a standard capillary emitter tiprelative to an integrated counter electrode, and a means ofsupplying a coaxial flow of nebulizing gas. The completeassembly is essentially an ion gun that may be positionedrelatively freely with respect to the vacuum interface. Bothsilica and metallic capillary emitter tips with internaldiameters in the range of 15–30 mm have been used success-fully. Many examples of chip-based nanospray ion sourceshave been reported.[25] However, the emitter tip is invari-ably formed as part of the microfabrication process. A keyadvantage of integrating a standard emitter tip with amicroengineered alignment bench, as described here, is thata number of vendors supply emitter tips of generally goodand repeatable quality. As a result, parameters such as oper-ating voltage and spray current are consistent andreproducible.

In Fig. 2, a microspray source can be seen mounted on theright-hand side of a polymer sub-unit. To the left there is afluidic connector and an electrical contact to the electrodes.A hole located on the underside (not visible) provides a path

wileyonlinelibrary.com/journal/rcmJohn Wiley & Sons, Ltd.

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Figure 2. Photograph of the miniature components used to generate, transmitand filter ions. A 150 mm ruler is shown to establish the scale. The microen-gineered component of the microspray ion source is seen mounted on theright-hand side of a polymer sub-unit. A stainless steel collar supports thedifferentially pumped vacuum interface, which consists of two etched circularsilicon dies that have been placed back-to-back and bonded together. Both themicroengineered quadrupole mass filter and the quadrupole ion guide areconstructed using cylindrical rods mounted in pairs on two glass platesseparated by spacers. A short Brubaker prefilter can be seen to the left ofthe main mass filter rods.

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for the nebulizing gas. This sub-unit is screwed into an elbowreceiver that, in addition to providing mating electrical, flu-idic, and gas connectors, also incorporates a passive flow splitand a high-voltage connection to a microfluidic conductiveunion. Typically, nebulizing gas is supplied at a rate of2.5 L/min, and the split is set such that the fluid flow rateto the emitter tip is 0.3–0.8 mL/min. The inset in Fig. 1 showsthe receiver elbow in situ. A short length of standard 1/16" o.d.PEEK tubing connects the HPLC system to the elbow, while asecond length of tubing allows the majority of the flow todrain from the elbow to a waste bottle. To replace the micro-spray sub-assembly, the elbow is pulled against the resis-tance of two seals in order to disengage it from the vacuuminterface flange.

Vacuum interface

Traditionally, the first vacuum stage of an ESI-MS instrumentis pumped to a pressure of approximately 1–2 Torr using arotary pump, and a fraction of the gas and entrained ionsflowing through the inlet is transmitted to the next vacuumstage through a sampling cone or skimmer. Typically, the inletflow is 500–1000 standard cubic centimeters per minute(sccm), which in turn dictates that the rotary pump has aspeed of approximately 30 m3/h at the required pressure.Such pumps weigh in excess of 25 kg, have high power con-sumption, and generate high levels of both heat and noise.Unfortunately, the size and weight of the rotary pumpneeded to pump the interface do not scale favourably as theinlet flow is decreased. For example, a rotary pump capableof displacing 50 sccm at 1 Torr typically weighs 10 kg.As discussed in detail elsewhere,[21] a microengineered

vacuum interface operating at a much higher pressure andpumped by a small diaphragm pump is a more appealingprospect for a miniaturized system. The interface shown in


Fig. 2 is conceptually similar to the structure describedpreviously,[21] which was the first example of a differen-tially pumped MEMS vacuum interface. The gold-plated,microengineered component is seen mounted centrally on astainless steel collar that engages with alignment features ona flange. It consists of two silicon dies that have been placedback-to-back and bonded together. The circular dies havebeen patterned and etched so as to define inlet and outletcapillaries, an internal cavity, and channels that allow gas tobe pumped out from the internal cavity. These features cannotbe seen in Fig. 2, as the capillaries are only visible on amagnified scale,[21] while the internal cavity and pumpingchannels are concealed within the assembled structure. Thisinterface provides a simple means of partitioning the gasflow. A portion of the flow through the inlet capillary canbe pumped away via the internal cavity and the pumpingchannels, thereby reducing the gas load entering the secondvacuum stage through the exit capillary. In some conven-tional vacuum interfaces, a skimmer is used to pierce theinitial free jet expansion. The required skimmer profile iscomplex, particularly when the operating pressure is raisedabove 1 Torr. The difficulties associated with fabrication ofsuch profiles using MEMS techniques are formidable. Conse-quently, no such structure has been incorporated into themicroengineered vacuum interface described here. Neverthe-less, this simple and inexpensive miniature interface transfersa flux of ions to the second vacuum stage that is sufficient forthe present application.

Quadrupole filter and ion guide

The primary difficulty encountered when miniaturizing aquadrupole mass filter is that the geometry is critical andthe fractional alignment accuracy needed to achieve unit reso-lution must be maintained as the diameter of the rods is


Figure 3. Total ion chromatogram (TIC) and base peakchromatogram (BPC) acquired in positive ion mode follow-ing injection of a mixture of melamine, reserpine, warfarin,and simvastatin. The concentration of each analyte in themixture was 50 mg/mL, except for melamine, which waspresent at a concentration of 100 mg/mL. The TIC has beenscaled and vertically offset to aid presentation.


reduced.[27] Consequently, to date, the majority of miniaturemass spectrometer systems have been based on ion traps, asthese appear to be less sensitive to assembly accuracy, andmay be constructed using highly simplified approximationsto the ideal electrode geometry.[28]

We have pursued the alternative strategy of attemptingto fabricate high-performance, miniature quadrupole filtersusing MEMS techniques to achieve the required accuracy.In an early design, 0.5 mm diameter rods were held in asilicon frame using leaf springs.[18,19] The mass range wasm/z 0–400, and the maximum resolution was m/Δm= 80at m/z 219, measured at 10% peak height. Although theperformance compared favourably with that of other massspectrometers of a similar size, it became clear that themass range, resolution, and transmission were inadequatefor LC/MS applications. As a result, this first-generationdevice was abandoned in favour of a more advancedmicroengineered quadrupole filter[20] based on a silicon-on-glass substrate and slightly larger, 0.65 mm diameterrods. The structural support and electrical isolation offeredby the glass layer allowed a more orthodox, less intrusiverod mounting arrangement to be adopted. Consequently,complex integrated optics to couple ions into and out ofthe structure were not needed, and a Brubaker prefiltercould be positioned in close proximity to the main rodsto further improve the performance. A prefilter is a shortrf-only quadrupole that prevents scattering of ions by thefringing fields at the entrance of the main filter. In addi-tion to an increased signal level, an improved resolutionof m/Δm= 150 at m/z 219, again measured at 10% peakheight, was demonstrated. Moreover, the excellent electri-cal isolation offered by the substrate allowed the massrange to be increased to m/z 1250.For the application described in the present paper, this

architecture has been scaled to accommodate a rod diameterof approximately 2 mm, and this has yielded a substantialincrease in transmission and a much improved resolution.In Fig. 2, a quadrupole filter is shown mounted on a supportplate. Two of the main rods, each held at their ends by siliconsupports, can be seen through the top glass plate. To the leftof the main rods, a third set of supports holds the shortprefilter rods. Also shown in Fig. 2 is a miniature quadrupoleion guide, which has been constructed according to a verysimilar design.

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RESULTS AND DISCUSSION

The mass range, sensitivity, and resolution of the Microsaic3500 MiD miniature mass spectrometer are the primary perfor-mance characteristics of interest. Each of these characteristicsis demonstrated below using data obtained either with theinstrument coupled to a conventional HPLC system, or bydirect infusion of analyte solutions using a syringe pump.ATIC and a BPC obtained in positive ion mode following a

10 mL injection of a mixture of reserpine, warfarin, simva-statin, and melamine (Sigma-Aldrich, Poole, UK) is shownin Fig. 3. A mass range of m/z 100–800 was scanned at a rateof 1 scan/s to generate each data point. The solvent gradientof acetonitrile and water, both with 0.1% formic acid, was setsuch that the aqueous component was 95% at 0 min, 95% at2 min, 5% at 8 min, 5% at 10 min, and 95% at 12 min. An


Ascentis Express C18 LC column (10 cm� 0.46 cm, Sigma-Aldrich) was used. Four prominent peaks, each approximately7 s wide, can be seen in both the TIC and the BPC. The concen-tration of each analyte in the mixture was 50 mg/mL, except formelamine, which was present at a concentration of 100 mg/mL.The flow rate through the columnwas 1.8 mL/min, which wassplit such that the flow to the microspray was approximately0.35 mL/min. It follows that the quantity of reserpine, warfarin,and simvastin passed to the mass spectrometer was 10 ng ineach case.

Cumulative mass spectra obtained by summing the 6–8separate mass spectra corresponding to each of the peaksin the TIC are shown in Fig. 4. The inset plots show, onan expanded scale, a region of each spectrum in the vici-nity of the [M+H]+ ion. The interval between data pointsis 0.2 m/z units. None of the mass spectra presented in thispaper have been smoothed or subjected to backgroundsubtraction. The first isotope peaks are distinct and wellresolved, as is the second isotope peak in the case of reser-pine. In combination with the nominal mass of the [M+H]+ ion, the relative intensities of the isotope peaks canhelp to uniquely identify an unknown analyte. It shouldbe noted that the resolution has been set during the auto-mated tuning procedure such that the full width at halfmaximum (FWHM) is approximately 0.7 m/z units acrossthe full mass range, as this is the usual criterion for rou-tine analysis. However, the ultimate resolution achievablewith the quadrupole mass filter, albeit at lower sensitivity,is significantly better. For example, at m/z 609, the ultimateresolution measured at FWHM is m/Δm= 3050. OtherMEMS quadrupole mass filters have not achieved thislevel of performance. A resolution of m/Δm= 28 at m/z219 has been demonstrated using a novel square rod fil-ter,[29] while a resolution of m/Δm= 99 at m/z 219 has beenreported for a filter constructed using microengineeredout-of-plane support structures.[30]


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Figure 4. (a)–(d) Cumulative mass spectra obtained by summing the 6–8 separate mass spectracorresponding to each of the peaks in the TIC shown in Fig. 3. The inset plots show, on an expandedscale, a region of each spectrum in the vicinity of the [M+H]+ ion.

Figure 5. Plot of peak height at m/z 609 against concentra-tion of reserpine in the injected sample. A mass range ofm/z 100–800 was scanned at a rate of 1 scan/s, and thepeak height was extracted from the summed spectra corre-sponding to reserpine elution. The straight line is a linearleast mean squares fit to the data. The limit of detectionis determined to be 500 ng/mL, which corresponds to5 ng on-column.

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In each of the cumulative mass spectra there is a cluster ofsmall peaks between m/z 100 and 220 as well as other minorpeaks elsewhere. This background is mostly due to low-levelcontaminants. For example, on close inspection, peaks at m/z149, 205, 279 and 391 can be identified. These can be attributedto phthalate esters,[31] which are widely used as plasticizers.Some contaminants are present in the as-purchased mobilephase solvents, while others are introduced during storage,preparation, and handling, or as the solvent passes throughthe LC system. Chemical noise of this type is ubiquitous inLC/MS and generally difficult to eradicate. The drifting base-line in the TIC can be attributed to variation in the chemicalnoise composition and intensity as the mobile phase gradientchanges,[31] while the broad feature at 12 min corresponds toelution of accumulated contaminants when the gradient changesabruptly to a high aqueous composition. Summation of all thechemical noise intensity results in a significant background con-tribution to the TIC. However, the background is reduced inthe BPC, since the peaks due to the four analytes are much moreintense than any of the individual chemical noise peaks.Declustering and fragmentation are controlled by the voltage

applied to the vacuum interface relative to the following ionoptical element. Typically, a voltage difference of 20–50 Vyieldsthe best compromise between declustering, fragmentation, andsignal level. In Fig. 4, there is little evidence of clusters, wateradducts, or significant fragmentation. In the simvastatin spec-trum, a small peak due to loss of the ester side chain[32] can beidentified at m/z 303, while in the warfarin spectrum a smallpeak at m/z 617 can be attributed to a dimer ion.The limit of detection (LOD) has been determined for

reserpine with the miniature mass spectrometer coupled toa standard HPLC system, as described above, and the massrange set to m/z 100–800. Solutions of reserpine in a 50:50


mixture of acetonitrile and water, each with 0.1% formic acid,were prepared by sequential dilution of a stock solution. Foreach injection, the height of the m/z 609 peak has beenextracted from the cumulative mass spectrum correspondingto reserpine elution. A plot of peak height against concentra-tion is shown in Fig. 5. One of the injections was repeated fivetimes in order to give an indication of the error limits. TheLOD (3:1 signal-to-noise (S/N) ratio, peak-to-peak definition)



in this mode of operation is determined to be 500 ng/mL. Atthis concentration, the peak height is approximately 10–20counts per bin (a bin is an array element that stores the cumu-lative ion count at a discrete m/z value). Given that the injec-tion volume was 10 mL, it follows that the LOD correspondsto 5 ng of reserpine on-column. However, due to the approxi-mately 5000:1 post-column split, only 1 pg of reserpine isactually passed to the mass spectrometer. Clearly, the use ofa high split ratio results in inefficient use of the analyte. Ifonly trace quantities of analyte are available it would beadvantageous to use a micro-flow LC system, which could becoupled directly to the mass spectrometer without a split, orwith a low split ratio. Although the full mass range wasscanned in order to demonstrate the LOD for an unknownanalyte, it is often sufficient (when quantifying a knownanalyte, for example) to monitor only one ion signal (selectedion monitoring (SIM) mode). Clearly, a higher sensitivity(1–2 orders of magnitude) is possible when this is the case,as the signal of interest can be integrated for a much longerperiod of time.Direct comparison with other ESI-MS systems is not

straightforward, as the sensitivity depends on the analyte,how the analyte is introduced, the dwell time per data point,the data analysis method, and the extent of any smoothing.Nevertheless, it is useful to consider the sensitivity specifica-tion of a conventional mass spectrometer, such as the Agilent6130 (Agilent Technologies Inc., Santa Clara, USA), which is atypical research grade single quadrupole ESI-MS instrument.When the scan rate, scan range, and flow to the ESI source areset at 2500 u/s, m/z 100–650, and 400 mL/min, respectively,the extracted ion response due to 50 pg of reserpine has aS/N ratio of 20:1 (peak-to-peak), according to the manufac-turer’s specifications. It follows that the LOD (3:1 S/N ratio)is expected to be approximately 30 pg on-column if thisinstrument is coupled via a 4.5:1 flow split to an HPLCsystem operating at 1.8 mL/min.Figure 6 shows a mass spectrum of erythromycin at a

concentration of 25 mg/mL. The solution was preparedusing a 50:50 mixture of acetonitrile and water with 0.1%

Figure 6. Positive ion mass spectrum of erythromycin at aconcentration of 25 mg/mL. A syringe pump was used todeliver the solution directly to the microspray ion source.A resolution of m/Δm=1245 is demonstrated in the insetplot, which shows the [M+H]+ ion and its isotope peaks.Loss of water results in a prominent peak at m/z 716.7.


formic acid. A syringe pump was used to deliver the solu-tion to the microspray at a rate of 0.4 mL/min. The spectrumwas acquired in positive ion mode over a period of 20 s,and data points were recorded at a reduced interval of0.1 m/z units in order to better define the peak shapes. Thesmall [M+H]+ ion peak at m/z 734.8, which is shown onan expanded scale in the inset plot, demonstrates a resolu-tion of m/Δm= 1245 at FWHM. Spectra of erythromycinrecorded using a conventional instrument have been reportedelsewhere.[33] Under normal conditions, the [M+H]+ peakdominates the spectrum, whereas a number of product ionsbecome prominent when the instrument parameters arechanged so as to induce fragmentation. On first inspection,the additional peaks at m/z 716.7 and 558.5 in Fig. 6 appearto suggest some degree of fragmentation. Indeed, thesepeaks are consistent with an initial loss of water from the[M+H]+ ion followed, to a lesser extent, by loss of thesugar moiety. However, it is also known that erythromycinreadily loses water in acidic solutions.[34] Given thelimited fragmentation seen in Fig. 4 and the addition of0.1% formic acid to the solvent, the high intensity of thepeak at m/z 716.7 is probably mostly due to this reactionin solution, rather than to processes occurring after thevacuum interface.

CONCLUSIONS

There has been much progress in the development ofminiaturized mass spectrometers for use in applicationsrequiring a portable instrument. However, to date, minia-ture mass spectrometers have generally not made a signif-icant impact on mainstream applications and markets,as the benefits of miniaturization are often outweighedby the compromises associated with restrictive methodsof sample introduction and comparatively low levels ofperformance. In this paper, a miniature mass spectrometerthat may be coupled to a conventional HPLC system hasbeen demonstrated. The sensitivity is such that columnloadings typically used in small-molecule syntheticchemistry can be readily detected. In contrast to otherminiature mass spectrometers, the resolution achievedis sufficient to resolve isotope clusters over the entiremass range.

The microspray ion source, vacuum interface, ion guide,and quadrupole mass filter are all batch produced on wafersusing MEMS techniques, and packaged as easily removedsub-units. In a shift from current working practices, it isenvisaged that these inexpensive components will beregarded as consumables, to be replaced by users rather thanservice technicians when faulty or contaminated.

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AcknowledgementsThe authors are indebted to M.-A. Schwab, R. Thomas, C.Wright, D. Rotherham, C. Sopellsa, T. Wallace, W. Boxford,and E. Yeatman for their help and encouragement. Fundingfrom Merck & Co. Inc., the European Commission throughthe Seventh Framework Programme, and the European Unionthrough the Eurostars Programme is also acknowledged.


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A miniature mass spectrometer for liquid chromatography applications

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