Drop-on-Demand Sample Introduction System Coupled with the

Oct 1, 2012 - J. Niklas Schaper,. †. Kevin P. Pfeuffer,. ‡. Jacob T. .... droplets were inserted close to the center of the cyclone, attaining the...
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Drop-on-Demand Sample Introduction System Coupled with the Flowing Atmospheric-Pressure Afterglow for Direct Molecular Analysis of Complex Liquid Microvolume Samples J. Niklas Schaper,† Kevin P. Pfeuffer,‡ Jacob T. Shelley,§ Nicolas H. Bings,† and Gary M. Hieftje*,‡ †

Institute of Inorganic and Analytical Chemistry, University of Mainz, 55128 Mainz, Germany Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States § Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States ‡

ABSTRACT: One of the fastest developing fields in analytical spectrochemistry in recent years is ambient desorption/ionization mass spectrometry (ADIMS). This burgeoning interest has been due to the demonstrated advantages of the method: simple mass spectra, little or no sample preparation, and applicability to samples in the solid, liquid, or gaseous state. One such ADI-MS source, the flowing atmospheric-pressure afterglow (FAPA), is capable of direct analysis of solids just by aiming the source at the solid surface and sampling the produced ions into a mass spectrometer. However, direct introduction of significant volumes of liquid samples into this source has not been possible, as solvent loads can quench the afterglow and, thus, the formation of reagent ions. As a result, the analysis of liquid samples is preferably carried out by analyzing dried residues or by desorbing small amounts of liquid samples directly from the liquid surface. In the former case, reproducibility of sample introduction is crucial if quantitative results are desired. In the present study, introduction of liquid samples as very small droplets helps overcome the issues of sample positioning and reduced levels of solvent intake. A recently developed “drop-on-demand” (DOD) aerosol generator is capable of reproducibly producing very small volumes of liquid (∼17 pL). In this paper, the coupling of FAPA-MS and DOD is reported and applications are suggested. Analytes representing different classes of substances were tested and limits of detections were determined. Matrix tolerance was investigated for drugs of abuse and their metabolites by analyzing raw urine samples and quantification without the use of internal standards. Limits of detection below 2 μg/mL, without sample pretreatment, were obtained.

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surfaces.7−10 The analysis of liquid samples has generally followed either of two strategies: (1) the sample is applied to a mesh or glass probe and allowed to dry, after which the mesh or probe is placed within the source7,11,12 where the analytes are desorbed and ionized, or (2) the analytes are desorbed directly from a liquid surface.4,13 The analysis procedure of the first strategy was similar to the atmospheric-pressure solids analysis probe (ASAP) technique described by McEwen, which allows the rapid analysis of volatile and semivolatile compounds by dipping a probe into the sample and placing this probe in the heated gas stream of a commercial electrospray or atmospheric pressure chemical ionization ion source.14,15 However, ASAP techniques do not use helium-based discharges as the ionization source and are therefore limited with respect to sensitivity, when compared to the FAPA source. Although qualitative analyses can easily be performed with ADI-MS, quantitation has often been significantly more problematic. This difficulty is due to many factors including irreproducible sample positioning and desorption/ionization matrix effects.16 Internal standardization with isotopically

he increasing security requirements of today’s society have led to a demand for simple-to-handle, fast, and reliable methods for the detection of weaponized chemicals such as explosives. Also, metabolites of drugs of abuse and illicit drugs themselves should be detected immediately, not after a long and costly laboratory analysis. Similarly, modern industrial processes demand on-line analysis of chemical reactions to optimize production and minimize consumption of raw materials and energy. Analytical chemists have put great effort into the development and enhancement of methods that are capable of meeting these often conflicting requirements. One field that is poised to meet this demand is ambient desorption/ionization mass spectrometry (ADI-MS). It offers excellent sensitivities for a variety of analytes of different kinds, no matter whether they be in the solid, liquid, or gaseous state.1,2 Although a wide variety of ADI-MS sources have been developed,2 only a few have gained significant attention in the literature; among these are direct analysis in real time (DART),3 the low temperature plasma probe (LTP),4 desorption electrospray ionization (DESI),5 and the flowing atmospheric-pressure afterglow (FAPA).6,7 Many of these sources (e.g., DART, LTP and FAPA) are based on electrical discharges maintained at atmospheric pressure. Gaseous samples can be introduced into these sources,6 although most studies have focused on direct desorption/ionization from solid © 2012 American Chemical Society

Received: July 17, 2012 Accepted: October 1, 2012 Published: October 1, 2012 9246

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labeled standards can enhance quantification in general,17 but the target analyte(s) must be known and labeled standards can be expensive and difficult to find or synthesize. Accordingly, a more general approach to overcome matrix effects would greatly benefit the field of ADI-MS. One potential method to reduce matrix effects is to limit the amount of the sample that is introduced into the desorption/ionization source; this approach has been successful with other analytical techniques such as inductively coupled plasma mass spectrometry (ICP-MS).18 Recently, a novel system has been reported for sample introduction in ICP-MS that is capable of handling liquid volumes of only a few picoliters.19 This drop-on-demand aerosol generator (DOD) is based on a modified thermal-inkjet printer cartridge and, in combination with a custom-built microcontroller, can reproducibly generate liquid droplets with a diameter between 32 and 39 μm. The droplet dispensing frequency and, thus, the resulting liquid flow rate is variable from single droplets [approximately 17 pL (32 μm diameter) for a single water droplet,19 for water−alcohol mixtures between 20 and 30 pL20 (39 μm diameter) per droplet] up to more than 2700 dosing events/s (2.74 μL/min). Theoretically, higher flow rates, of up to 2 mL/min, could be achieved by utilizing more than one nozzle simultaneously.19 In the present study, the DOD was used to introduce very small amounts of solution samples into the afterglow region of the FAPA to improve reproducibility of liquid sample analysis and to reduce matrix effects. Achievable limits of detection for several analytes and analysis of real samples having a high matrix load were also explored.

the atmosphere, while additional sample solution from the reservoir refills the nozzle chamber so the process can start over. A custom-built microcontroller offers access to all the important parameters of this droplet-generation process, namely the heating time of the thin-film resistor and the heating repetition rate. The whole cycle takes about 50 μs, so the generation of single droplets or repetition rates of several kHz can be achieved. The resulting velocity of the ejected droplets is approximately 8 m/s. In this study, a dropletintroduction frequency of 1 kHz was utilized, unless stated otherwise. Ionization Source. The FAPA design was similar to the one described by Andrade et al.21 and is outlined here only briefly. A direct-current, atmospheric-pressure glow discharge (APGD) was maintained in a Teflon body between a pin cathode and a plate anode. The pin was connected to the negative output of a high-voltage power supply (HewlettPackard Company, Palo Alto, CA, Model 6525A) through a 5 kΩ ballast resistor, while the plate was typically maintained between 60 and 100 V (GW Instek, Chino, CA, Model GPR 30H100) to produce a field-free region between the front plate of the FAPA source and the inlet of the mass spectrometer. A small orifice (1.3 mm) in this plate allowed ions, electrons, and excited species, formed in the discharge, to enter the atmosphere. The afterglow self-heats to around 200 °C, which aids in desorption of molecules from surfaces.13 Then, in the open atmosphere, charged and excited species from the discharge react with ambient constituents to form reagent ions, primarily protonated water clusters, which subsequently ionize analyte molecules. The FAPA was mounted on an xyz translation stage (Newport Corporation, Irvine, CA, 462 series) for precise alignment of the source relative to the inlet of the mass spectrometer. The APGD was operated in a currentcontrolled mode with currents ranging from 10 to 40 mA. Two helium flows, FAPA discharge gas and aerosol-transfer gas, were maintained with calibrated mass flow controllers (MKS Instruments, Andover, MA, type 1160B mass flow controller and type 247 4-channel readout) and were varied from 1.0 to 1.7 L/min and from 0.1 to 0.3 L/min, respectively. Gas flow rates, xyz position, and mass spectrometer potentials were optimized for each run, with only small day-to-day variations of these parameters. FAPA Solution Analysis Procedure. Previously, solution analysis with the FAPA has been carried out by applying a small amount of sample solution (2 μL) to a glass probe.13 After evaporation of the solvent, the glass probe was introduced into the FAPA afterglow and the analyte was desorbed and ionized from the glass surface. Three replicates of each sample, containing all analytes at the same time, were taken to ensure the sample was reproducibly introduced. The resulting peaks in the time traces of the analyte signal were integrated, resulting in an RSD for the probe analysis procedure of 35% for the peak area.

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EXPERIMENTAL SECTION Reagents. All reagents were analytical-grade. High-purity helium (99.999% ultrahigh-purity helium; Airgas, Radnor, PA) was used as the discharge gas and droplet carrier gas in all cases. Pharmaceuticals were obtained as over-the-counter drugs from local supermarkets. Illicit drugs and metabolites were purchased as 1 mg/mL standard solutions in methanol or acetonitrile from Cerilliant (Round Rock, TX). The tebuthiuron pesticide standard was obtained from Sigma Aldrich (St. Louis, MO). Sample Treatment. Raw urine samples were obtained from a local individual and spiked with the drugs of abuse and their metabolites. DOD-FAPA-MS analysis was performed without any other treatment of the doped urine samples. Mass Spectrometric Analysis. Ions produced within the FAPA afterglow were detected with either a modified LECO Renaissance time-of-flight mass spectrometer or a LECO Unique molecular time-of-flight mass spectrometer operated in the positive-ion mode. Mass spectra were collected at a rate of 6.25 spectra/s. Time traces of each analyte ion as well as other ions of interest, primarily reagent ions, were collected with the LECO Renaissance spectrometer for diagnostic measurements, while all ions in a mass window from m/z 80 to m/z 400 were collected with the LECO Unique spectrometer. All reported limits of detection were obtained with the Unique system. Drop-on-Demand Generator. A detailed description of the DOD aerosol generator can be found elsewhere,19 so only key processes will be discussed here. The system is based on the thermal-inkjet-printing principle, where a passivated thinfilm resistor is in direct contact with the liquid sample in a small chamber. Droplet formation is a cyclic process, which starts with rapid heating of this thin-film resistor. The resulting solvent-vapor bubble presses the liquid through an orifice into

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RESULTS AND DISCUSSION Coupling DOD and FAPA. The droplets produced by the modified inkjet cartridge were introduced into the afterglow in two ways. Following the general concept of ADI-MS to keep everything as simple as possible, the direct introduction of droplets into the afterglow without any interface was first attempted. Unfortunately, the droplets traveled on the skin of the helium stream of the FAPA source, to produce a welldefined spot of dried particulate matter just above the sampling

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the droplets occurs. The mixing of the two gas flows by the tee provided a more intimate interaction between the reagent ions and the analyte molecules. It was important first to determine the response of the FAPA to the introduction of analyte-containing droplets. For this purpose, generation of droplets of a 1 mg/mL solution of acetaminophen was switched on and off (cf. Figure 2) while signals of the protonated molecular analyte ion (MH+) and the main reagent ions (NO+ and [H2O]2H+) responsible for analyte ionization were monitored. Before droplet introduction, reagent-ion signals were quite stable (2% RSD for NO+ and 4% RSD for [H2O]2H+). When the droplet generator was switched on, the analyte signal rapidly increased, with a rise time (background to 100%) of 2.7 s, while the signal for both reagent ions dropped at nearly the same speed. During the period of droplet introduction, the analyte signal was quite stable (4% RSD); however, an initial spike in the analyte signal occurred when droplets were first introduced. This finding was expected; it is known that the first few droplets after a long rest period have a different size than those formed under continuous operation.22 When the droplet generator was turned off, the analyte ion signal returned to its background level with a fall time (100% to background) of 2.1 s, while the signals for the reagent ions were similarly restored to their initial values. Two modes of operation of the DOD device can be considered. For greatest speed and flexibility, it would be desirable to employ an array of DOD nozzles, one for each standard solution and others for samples to be analyzed. However, this approach requires careful positioning of the various DOD units and likely an array of electronic drive systems. In a simpler approach, the one adopted here, only a single DOD assembly is utilized; this setup requires each

orifice of the mass spectrometer, and no analyte ion signal was detected. The second design involved a glass cone on which the DOD was mounted. The droplets were guided with an additional gas flow into a small tee, where they mixed with the afterglow of the FAPA (cf. Figure 1). The helium flow

Figure 1. Diagram of FAPA-DOD combination. The DOD is mounted on top of a glass cone with an additional gas inlet to aid in transport of the droplets. The glass cone is inserted into the vertical segment of a tee, while the horizontal connection is adjacent to the front plate of the pin-to-plate FAPA.

inside the cone is thought to form a cyclonic profile and the droplets were inserted close to the center of the cyclone, attaining the velocity of the gas. The small lower end of the cone was positioned very close to the gas flow exiting the FAPA source. Because of the use of dry helium, partial evaporation of

Figure 2. Time traces of selected ions produced within the FAPA afterglow while droplet generation was switched on and off. During droplet introduction, the analyte signal RSD is approximately 4%, neglecting the initial spike. 9248

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successive solution to be added to the DOD reservoir and necessitates a washout period. To assess this washout time, the DOD was cleaned thoroughly in an ultrasonic bath and mounted on top of the glass cone. The reservoir was filled with 170 μL of a blank solution and mass-spectrometric signal acquisition was started. Aerosol production was switched on 10 s later and 10 μL of a 0.1 mg/mL lidocaine solution was added to the blank solution 10 s after aerosol production was initiated. The resulting time trace of the protonated analyte ion (MH+ = 235) is shown in Figure 3. The background at the target analyte

calibration strategy similar to the “Null Point Technique“23 of Bastiaans and Hieftje can be employed. This so-called “dosing frequency-based calibration” (DFC)24 enables a calibration plot to be created with just a single standard solution by changing the number of droplets that are introduced per unit time. A representative analyte, lidocaine, was dissolved in water and also in a 1:1 (v/v) mixture of water and methanol to investigate the influence of solvent on the analyte signal (cf. Figure 4). The net signal of the analyte in water/methanol was

Figure 3. Time trace of analyte ion signal after introduction of a new sample solution. At time = 0, the DOD was activated with a blank solution in it, and after 20 s, 10 μL of a 0.1 mg/mL analyte solution was added to the liquid reservoir. An acceptably stable signal level is reached after 15 s.

Figure 4. Averaged signal for analyte ion and main proton donor species obtained at different frequencies of droplet introduction and in two different solvents. The same concentration of analyte was dissolved in two selected solvents and the average analyte signal was found to rise with the introduction frequency. Also, the corresponding decline in reagent ion signal is shown.

mass climbed slightly when the aerosol generator was switched on, because droplet introduction into the afterglow region changes the conditions in the afterglow. Once this trace had stabilized, the analyte solution was added with a pipet as close as possible to the entrance of the nozzle. The analyte signal appeared 15 s afterward. After the initial peak, the signal steadily declined over the course of the experiment as the amount of analyte steadily decreased due to imperfect mixing. However, the rapid migration of the analyte inside the DOD reservoir shows that samples can be changed with little carryover. Rinsing the reservoir two times with 200 μL of a blank solution was enough to ensure that no sample was left; this rinsing procedure was followed throughout all experiments. With the knowledge that an added solution migrates to the orifice within 15 s, the signal level was seen to decrease to background level with the following rinsing procedure: the sample was removed with a pipet from the reservoir and the reservoir was filled with blank solution, while the DOD continued producing droplets. The blank solution was removed after about 30 s and fresh blank solution was added again. This blank solution was then replaced by the next sample. These rinsing steps were sufficient to completely remove from the reservoir sample solutions with a concentration of up to 100 mg/L and to allow the signal to return to a level consistent with blank. Dosing Frequency-Based Calibration (DFC). The flux of solvent and analyte introduced into the FAPA source is directly proportional to the droplet-introduction frequency, which is tunable from tens of mHz to several kHz. Consequently, a

always found to be higher than in pure water, suggesting that the evaporation rate of the solvent has an impact on the evaporation/desorption process; also, the dominant reagent ion in the mass spectrum changes from protonated water clusters to protonated methanol when the solvent is switched to the water/methanol mixture. Simultaneous monitoring of reagent ions along with protonated analyte ions illustrates that the concentration of reagent ions in the afterglow declines with increasing droplet-introduction frequency. This behavior indicates that reagent ions might be depleted at very high droplet generation frequencies. The simultaneous monitoring of protonated water clusters and solvent ions (cf. Figure 4) shows that solvent ions are more abundant than analyte ions. Of course, the number of solvent molecules in the solution exceeds the number of analyte molecules, so this finding is expected. However, an ionized solvent molecule might still act as a proton donor, if the difference in gas-phase acidity is large enough. Therefore, the main depletion source might be the solvent, but a detailed study should be made to identify the source of depletion. Indeed, this depletion is evident in Figure 5, which shows calibration plots obtained with the DFC method for acetaminophen under different FAPA operating conditions. The standard deviations of the averaged signal at each dropletintroduction frequency are very small; at 25 mA FAPA operating current, they range from 7% at 100 Hz to 1% at 1.6 kHz for a time window of 15 s. 9249

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Figure 5. Calibration similar to the “Null-Point” method,20 based on different droplet-introduction frequencies. The transferred mass is calculated as the product of the droplet volume (25 pL), the respective droplet-introduction frequency and the concentration. At lower discharge currents, the calibration rolls off toward higher transferred masses because of the limited concentration of reagent ions in the afterglow region and possibly because of reduced droplet desolvation efficiency.

sections, each 10 s long, and the signal from each section was averaged. These sections were averaged again and standard deviations calculated. Representative analytes from several substance families were explored, including pharmaceuticals, chemical-warfare agent simulants, pesticides, and drugs of abuse and their metabolites. Analytes that were not soluble in a water/methanol mixture were dissolved in a water/acetonitrile mixture as indicated in Table 1, but the solvent system had no

Of course, the signal must be directly proportional to the droplet introduction rate as a prerequisite for use of the DFC method. The results for each calibration plot have been tested for outliers according to DIN 32645 at a 95% confidence level. The data for 1 ng/s at a discharge current of 40 mA as well as 10 ng/s at a discharge current of 25 mA have been identified as outliers. The calibrations obtained at discharge currents of 25 and 40 mA are linear, and the slopes are very comparable. However, the overall background is increased at a discharge current of 40 mA, so a current 25 mA was chosen for operation. The only nonlinear calibration plot is at low discharge currents; it rolls off at higher droplet introduction frequencies. This roll off could be due to depletion of reagent ions discussed above or to incomplete droplet desolvation at higher introduction frequencies due to a lower discharge temperature. Increasing the discharge current provides not only more reagent ions, but also a hotter afterglow and, consequently, more efficient desolvation. However, the assignment of a concentration to a certain frequency is not easy, because the mean droplet volume has to be known. For inorganic analytes and water as solvent, an ejected volume of 17 pL has been determined.24 For a water/ methanol mixture (1:1 v/v), a volume of 25 pL was found.20 The transferred mass is the product of this volume with the respective dosing frequency and the concentration utilized. For the example in Figure 5, the chosen frequency range of 0.1−1.6 kHz corresponds to a transferred mass range of 1.25−20 ng/s. Limits of Detection (LODs). Conventional calibration can be performed by introducing droplets of solutions containing a range of known analyte concentrations, and by integrating and averaging the observed signal. LODs for DOD-FAPA-MS were determined by introducing droplets of standard solutions for 2 min, in which the first 10 s were allowed for stabilization and the last 10 s were used as a buffer time, which was not taken into evaluation. The resulting time trace was divided into 10

Table 1. LODs for Selected Analytes in Pure Solvents or Solvent Mixtures of Methanol (MeOH), Acetonitrile (AcN), and Water (H2O) analyte

MH+

LOD/ μg/ mL

Acetaminophen Lidocaine Tebuthiuron Methyl 2hydroxybenzoate Triethylphosphate Benzoylecgonine Cocaethylene MDMA/Ecstasy Methadone Cocaine

152 235 229 153

0.9 0.1 0.1 0.8

MeOH/H2O MeOH/H2O MeOH/H2O MeOH/H2O

183 290 318 194 310 304

0.2 0.05 0.06 0.04 0.08 0.07

MeOH/H2O MeOH AcN MeOH MeOH AcN

solvent

remark Drugs Pesticide Simulants of chemical warfare agents Illicit Drugs or Metabolites

influence on the general trend of results. LODs for a set of analytes in pure solvents or solvent mixtures, compiled in Table 1, range from 0.04 to 0.9 μg/mL. These LODs were achieved without MS/MS experiments to confirm the analyte, and thus represent an improvement in solution analysis for ambient mass spectrometry. Reported LODs for the LTP utilized tandem mass spectrometry and are of the same order of magnitude as those obtained here.11 9250

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Table 2. DOD-FAPA and FPA LODs of Several Drugs of Abuse in Doped Urine without Sample Pretreatment and without Internal Standards Compared to Other Methods Reported in the Literaturea LOD/(μg/mL)

a

analyte

MH+

DOD-FAPA

FAPA

LTP-MS/MS11

DART/w. prec.30

ELISA31

Benzoylecgonine Cocaethylene MDMA/Ecstasy Methadone Cocaine

290 318 194 310 304

1.3 0.05 1 0.04 0.1

7 1 nq nq 4

1 nqb nq 0.1 0.01

0.024 0.01 0.2532 nq 0.004

0.3 nq 1 0.3 nq

For FAPA and DOD-FAPA analysis, all five substances were present in the sample at the same time. bnq = not quantifiable.

Direct Urine Analysis. To assess the performance of the DOD-FAPA combination with complex samples, samples of unfiltered and undiluted raw urine were spiked with five different drugs and metabolites spanning a concentration range of 1−15 μg/mL each. The total sample volume in these measurements, consumed over a time interval of 6 s, was only about 150 nL. Compared to the established probe analysis procedure, where each replicate consumes the volume that can be reproducibly and conveniently handled with a pipet, 2 μL, the sample volume is reduced dramatically by use of the droplet-introduction strategy. The dominant ion in the mass spectra is a protonated dimer of urea at m/z 121, but analyte ions could still be detected even at the lowest concentration of 1 μg/mL, suggesting that the method could be used for fast and easy screening in the search for drugs of abuse and their metabolites, and without any sample pretreatment. Lack of need for any internal standard, sophisticated mass spectrometric equipment, or MS/MS capability makes the new method especially attractive. Limits of detection were determined also by means of an earlier FAPA solution-analysis procedure, in which a 2 μL droplet is placed on a glass probe and the dried residue analyzed. Peak-area variations were about 35% for three replicates. In contrast, the RSD for a 10-fold, 60-s long DOD-FAPA analysis was usually around 8%. This improvement in precision is due to a more stable sample introduction position within the FAPA source than in the probe technique. All five analytes present at the same time in the spiked urine samples could be detected with both FAPA methods. However, not all analytes could be quantified with the probe-FAPA approach because of nonlinear calibration plots for MDMA and methadone, probably caused by competitive ionization. The two analytes, MDMA and methadone, are not quantifiable by probe-FAPA when in a sample with the other three analytes, due to strong signal suppression. In DOD-FAPA analysis, MDMA and methadone are quantifiable, but the signal is suppressed compared to analysis of neat samples. Other molecules, cocaine, benzocylgocline and cocaethylene, are enhanced compared to MDMA and methadone when all 5 analytes are present in the sample. Finally, suppression does not take place when only MDMA or methadone is present in the sample. This competitive ionization in DOD-FAPA will be the subject of future studies. LODs achieved with the two FAPA methods are compiled in Table 2 and compared to reported values for LTP-MS/MS and preconcentration-DART.11,30 The LODs for the LTP-MS/MS and preconcentration-DART methods are quite similar for most but not all of the compounds of interest determined for DOD-FAPA. This finding is unexpected, as methods that use MS/MS or preconcentration in combination with isotopically

labeled standards should yield LODs that are some orders of magnitude better than methods without preconcentration. Strong advantages of the new DOD-FAPA method are the lack of any need for preconcentration or internal standardization and the much lower susceptibility to matrix interferents than the DART method, where it was shown that a direct analysis could “produce unacceptable results with a poor response for all the analytes”.30 In addition, the DODFAPA method is convenient to use, as sample introduction is simple and sample consumption is very low. The method also offers a fast and thus time-saving analysis with no costly equipment, compared to high-resolution mass spectrometers. LODs for a popular method for screening drug and drug metabolites based on the specific binding of a ligand reagent to the target molecule, the so-called enzyme-linked immunosorbent assays (ELISA) are also compared in Table 2 to those obtained with the DOD-FAPA approach.25,26,31 LODs of the DOD-FAPA and probe-FAPA techniques are different from those obtained with just methanol or acetonitrile as solvents, but are still in an acceptable range between 0.1 and 20 μg/mL. Comparable studies for some of the analytes in Table 2 were performed with the LTP and tandem mass spectrometry; they confirmed the analyte mass and yielded LODs of the same order of magnitude. A similar study was conducted also with DART and a preconcentration−microextraction step and internal standardization; reported LODs for illicit drugs and metabolites were again comparable.30 A different approach involving preconcentration and detection by DART-TOF was reported for MDMA determination, and yielded an LOD of 0.25 μg/mL.32 Screening experiments might be easier with the probe method, but quantification is better by DOD-FAPA due to reliable sample introduction. Also, methods involving MS/MS or preconcentration of analytes are more complex. Immunoassay tests, which also require very little sample pretreatment, offer comparable LODs for some target analytes,31 but they can suffer from cross contamination.27 Other detection methods for drugs of abuse described in the literature are mostly used for confirmation of first results based on a screening. These methods use LC/MSn or GC/MS to detect the target molecules, and involve extensive sample preparation such as centrifugation for protein precipitation, filtration drying and dissolving28 or incubation with reagents for 1 h, pH adjusting, liquid−liquid extraction, centrifugation, evaporation, dissolving and finally derivatization.29

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CONCLUSIONS A drop-on-demand generation system has been coupled successfully to the flowing atmospheric-pressure afterglow (FAPA) ambient desorption/ionization mass spectrometry 9251

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source. Rapid and repeatable sample introduction is possible with the DOD-FAPA combination, with rise times less than 2.7 s and good reproducibility (4% RSD). Also, changing the droplet-introduction frequency enables calibration curves to be generated rapidly (less than 5 min) while the reservoir of the DOD needs to be loaded only once with a minimum of 100 μL standard solution. Moreover, the actual sample consumption rate is only 1.2 μL/min so the volume needed to generate a complete calibration is lower. In an ideal DOD system, the volume of the sample reservoir would be minimized. The dynamic range of this dosing-frequency based calibration was so far determined to extend to 2 orders of magnitude. Competitive ionization that can be present in all ADI-MS methods would affect the accuracy of a calibration, but the ease of generating calibration plots and standard additions with the DOD-FAPA method can compensate somewhat for this handicap. Also, standard-addition methods can be easily implemented by adding standard solution to the DOD reservoir to compensate for competitive ionization. Calibration plots can also be generated in the conventional way, with a range of standard solution concentrations. Both calibration strategies resulted in the construction of accurate linear calibration plots (R2: 0.99) for all investigated analytes. However, different sensitivities were observed for the two calibration methods and will be addressed in further studies. LODs between 0.05 and 0.1 μg/mL were determined for several classes of analytes in pure solvents or solvent mixtures, using the conventional calibration strategy. Finally, doped urine was analyzed for drugs of abuse and their metabolites with LODs in the 0.1 μg/mL range with no sample preparation, no internal standard, and no use of tandem mass spectrometry. The results here are based on the analysis of relatively small molecules. Thermal mechanisms are dominant in the FAPA desorption process; as a consequence, the range of compounds that can be detected with the FAPA source is limited to molecules that can be driven into the gasphase by heat. Additionally, differences in gas-phase proton affinity determine whether a proton exchange can occur. Thus, the potential for the determination of molecules of higher molecular weight, for example, proteins, nucleic acids, or complex sugars, will depend on the vapor pressure and gasphase proton affinity of the compound of interest. Also, fragmentation of molecules has to be assessed on a case-by-case basis and will be addressed in future studies.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.N.S. would like to thank the German Academic Exchange Service (DAAD) for financial support of his research stay at the Department of Chemistry at Indiana University. The authors would like to acknowledge funding from Prosolia, through a National Institutes of Health (NIH) STTR grant and Leco Corporation for the generous donation of equipment. Partial salary support was provided by the US Department of Energy through grant DE-FG02-98ER14890. 9252

dx.doi.org/10.1021/ac3020164 | Anal. Chem. 2012, 84, 9246−9252