Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry

An experimental investigation and theoretical analysis are reported on charge competition in electrospray ionization (ESI) and its effects on the linear dynamic range of ESI mass spectrometric (MS) measurements. The experiments confirmed the expected increase of MS sensitivities as the ESI flow rate decreases. However, different compounds show somewhat different mass spectral peak intensities even at the lowest flow rates, at the same concentration and electrospray operating conditions. MS response for each compound solution shows good linearity at lower concentrations and levels off at high concentration, consistent with analyte "saturation" in the ESI process. The extent of charge competition leading to saturation in the ESI process is consistent with the relative magnitude of excess charge in the electrospray compared to the total number of analyte molecules in the solution. This ESI capacity model allows one to predict the sample concentration limits for charge competition and the on-set of ionization suppression effects, as well as the linear dynamic range for ESI-MS. The implications for quantitative MS analysis and possibilities for effectively extending the dynamic range of ESI measurements are discussed.     

Assessment of the various ionization methods in the analysis of metal salen complexes by mass spectrometry

Metal salen complexes are one of the most frequently used catalysts in enantioselective organic synthesis. In the present work, we compare a series of ionization methods that can be used for the mass spectral analysis of two types of metalosalens: ionic complexes (abbreviated as Com⁺X^?) and neutral complexes (NCom). These methods include electron ionization and field desorption (FD) which can be applied to pure samples and atmospheric pressure ionization techniques: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) which are suitable for solutions. We found that FD is a method of choice for recording molecular ions of the complexes containing even loosely bonded ligands. The results obtained using atmospheric pressure ionization methods show that the results depend mainly on the structure of metal salen complex and the ionization method. In ESI spectra, Com⁺ ions were observed, while in APCI and APPI spectra both Com⁺ and [Com + H]⁺ ions are observed in the ratio depending on the structure of the metal salen complex and the solvent used in the analysis. For complexes with tetrafluoroborate counterion, an elimination of BF₃ took place, and ions corresponding to complexes with fluoride counterion were observed. Experiments comparing the relative sensitivity of ESI, APCI and APPI (with and without a dopant) methods showed that for the majority of the studied complexes ESI is the most sensitive one; however, the sensitivity of APCI is usually less than two times lower and for some compounds is even higher than the sensitivity of ESI. Both methods show very high linearity of the calibration curve in a range of about 3 orders of magnitude of the sample concentration. Copyright © 2014 John Wiley & Sons, Ltd.     

A corona discharge initiated electrochemical electrospray ionization technique

We report here the development of a corona discharge (CD) initiated electrochemical (EC) electrospray ionization (ESI) technique using a standard electrospray ion source. This is a new ionization technique distinct from ESI, electrochemistry inherent to ESI, APCI, and techniques using hydroxyl radicals produced under atmospheric pressure conditions. By maximizing the observable CD at the tip of a stainless steel ESI capillary, efficient electrochemical oxidation of electrochemically active compounds is observed. For electrochemical oxidation to be observed, the ionization potential of the analyte must be lower than Fe. Ferrocene labeled compounds were chosen as the electrochemically active moiety. The electrochemical cell in the ESI source was robust, and generated ions with selectivity according to the ionization potential of the analytes and up to zeptomolar sensitivity. Our results indicate that CD initiated electrochemical ionization has the potential to become a powerful technique to increase the dynamic range, sensitivity, and selectivity of ESI experiments.     

Impact of APCI ionization source in liquid chromatography tandem mass spectrometry based tissue distribution studies

Measurement of test article concentration in tissue samples has been an important part of pharmacokinetic study and has helped to co‐relate pharmacokinetic/pharmacodynamic relationships since the 1950s. Bioanalysis of tissue samples using LC–MS/MS comes with unique challenges in terms of sample handling and inconsistent analyte response owing to nonvolatile matrix components. Matrix effect is a phenomenon where the target analyte response is either suppressed or enhanced in the presence of matrix components. Based on previous reports electrospray ionization (ESI) mode of ionization is believed to be more affected by matrix components than atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization. To explore the impact of ionization source with respect to bioanalysis of tissue samples, five structurally diverse compounds – atenolol, verapamil, diclofenac, propranolol and flufenamic acid – were selected. Quality control standards were spiked into 10 different biological matrices like whole blood, liver, heart, brain, spleen, kidney, skeletal muscle, eye and skin tissue and were quantified against calibration standards prepared in rat plasma. Quantitative bioanalysis was performed utilizing both APCI and ESI mode and results were compared. Quality control standards when analyzed with APCI mode were found to be more consistent in terms of accuracy and precision as compared with ESI mode. Additionally, for some instances, up to 20‐fold broader dynamic linearity range was observed with APCI mode as compared with ESI mode. As phospholid interferences have poor response in APCI mode, protein precipitation extraction technique can be used for multimatrix quantitation, which is more amenable to automation. The approach of multiple biological matrix quantitation against a single calibration curve helps bioanalysts to reduce turnaround time. Copyright © 2016 John Wiley & Sons, Ltd.     

Nebulization ionization and desorption ionization analysis of reactive organofunctionalized silanes in nanofilm products

Three different and recently developed desorption ionization techniques, transmission-mode desorption electrospray ionization (TM-DESI), low temperature plasma (LTP) ionization and nano-assisted laser desorption ionization (NALDI), are compared with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) for the analysis of two nanofilm products (NFPs) for surface coating, which contain hydrolysates and condensates of organo-functionalized silanes. The NFPs were characterized in different states from the liquid phase to the fully formed surface film. The LTP spectra were dominated by the silanes, while the corresponding di-, tri- and tetrasiloxanes were common in ESI, APCI and TM-DESI. This indicates readily condensation of the silanes during the ESI and APCI ionization processes leading to the observed siloxanes. NALDI showed larger siloxane structures than the other techniques, indicating film formation on the NALDI target. Real-time monitoring of the film formation on a glass surface by LTP showed a decreasing abundance of the silanes, while the abundances of the di-, tri and tetrasiloxanes increased significantly within the first 100 s. LTP was superior in showing the non-reacted content of the NFPs, while ESI, APCI and TM-DESI were characterized by artefact formation of siloxanes. NALDI was ideal for showing the siloxane structures of the formed film. The applicabilities of each of the ionization techniques were examined, showing the advantage of utilizing more than one ionization technique for the analysis of reactive species.     

Acetonitrile/water gas-phase reaction products observed by use of atmospheric pressure chemical ionization: adducts formed with sulfonamides

Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two most common mass spectrometric ionization methods used in the pharmaceutical industry. However, APCI analysis can sometimes lead to ambiguity in compound characterization and quantitation due to gas-phase reactions occurring between acetonitrile and water in the plasma, and between these plasma-generated compounds and the analyte. During the analysis of various sultams and sulfonamides we observed signals corresponding to m/z [M+44](+) and [M+60](+). Various solvent conditions and collisionally activated dissociation MS(n) experiments revealed that under the high-energy plasma conditions of APCI, the acetonitrile/water solvent mixture reacts undergoing acid-catalyzed hydrolysis producing acetamide, 59 Da. Further, the highly reactive 43 Da species ethanimine is also produced. These two compounds, normally not observed in APCI analysis, are stabilized by the sulfonamide and appear as adduct species in the mass spectra. The sulfone oxygens and the lone pair of electrons on the amide nitrogen play a role in stabilizing this adduct.     

Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry

The phenomena of ionization suppression in electrospray ionization (ESI) and enhancement in atmospheric pressure chemical ionization (APCI) were investigated in selected-ion monitoring and selected-reaction monitoring modes for nine drugs and their corresponding stable-isotope-labeled internal standards (IS). The results showed that all investigated target drugs and their co-eluting isotope-labeled IS suppress each other's ionization responses in ESI. The factors affecting the extent of suppression in ESI were investigated, including structures and concentrations of drugs, matrix effects, and flow rate. In contrast to the ESI results, APCI caused seven of the nine investigated target drugs and their co-eluting isotope-labeled IS to enhance each other's ionization responses. The mutual ionization suppression or enhancement between drugs and their isotope-labeled IS could possibly influence assay sensitivity, reproducibility, accuracy and linearity in quantitative liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, calibration curves were linear if an appropriate IS concentration was selected for a desired calibration range to keep the response factors constant.     

Effects of salt concentration on analyte response using electrospray ionization mass spectrometry

The effect of salt concentration on analyte response using electrospray ionization mass spectrometry (ESI-MS) was measured and compared to that predicted by Enke's equilibrium partitioning model. The model predicts that analyte response will be proportional to concentration and that the response factor will decrease with increasing electrolyte concentration. The measured analyte response is proportional to concentration over four orders of magnitude when the electrolyte concentration is below 10(-3) M, as the model predicts. The concentration of excess charge ([Q]) generated by the ESI process increases significantly at 10(-3) M ionic concentration, but the response factor decreases at this concentration. Changes in shape of the spray that cause a loss of ion transmission efficiency may be the basis for the decrease in response. An increase in the analyte response factor with increasing electrolyte concentration is observed for electrolyte concentrations below 10(-3) M. An explanation for this based on the electrical double layer is proposed.     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of eluent on the ionization process in liquid chromatography-mass spectrometry

The most widely used ionization techniques in liquid chromatography-mass spectrometry (LC-MS) are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). All three provide user friendly coupling of LC to MS. Achieving optimal LC-MS conditions is not always easy, however, owing to the complexity of ionization processes and the many parameters affecting mass spectrometric sensitivity and chromatographic performance. The selection of eluent composition requires particular attention since a solvent that is optimal for analyte ionization often does not provide acceptable retention and resolution in LC. Compromises must then be made between ionization and chromatographic separation efficiencies. The review presents an overview of studies concerning the effect of eluent composition on the ionization efficiency of ESI, APCI and APPI in LC-MS. Solvent characteristics are discussed in the light of ionization theories, and selected analytical applications are described. The aim is to provide practical background information for the development and optimization of LC-MS methods.     

Towards the electrospray ionization mass spectrometry ionization efficiency scale of organic compounds

An approach that allows setting up under predefined ionization conditions a rugged self-consistent quantitative experimental scale of electrospray ionization (ESI) efficiencies of organic compounds is presented. By ESI ionization efficiency (IE) we mean the efficiency of generating gas-phase ions from analyte molecules or ions in the ESI source. The approach is based on measurement of relative ionization efficiency (RIE) of two compounds (B1 and B2) by infusing a solution containing both compounds at known concentrations (C1 and C2) and measuring the mass-spectrometric responses of the protonated forms of the compounds (R1 and R2). The RIE of B1 and B2 is expressed as logRIE(B1, B2) = log[(R1 . C2)/(C1 . R2)]. The relative way of measurement leads to cancellation of many of the factors affecting IE (ESI source design, voltages in the source and ion transport system, solvent composition, flow rates and temperatures of the nebulizing and drying gases). Using this approach an ESI IE scale containing ten compounds (esters and aromatic amines) and spanning over 4 logRIE units has been compiled. The consistency of the scale (the consistency standard deviation of the scale is s = 0.16 logRIE units) was assured by making measurements using different concentration ratios (at least 6-fold concentration ratio range) of the compounds and by making circular validation measurements (the logRIE of any two compounds was checked by measuring both against a third compound).     

Sonic spray ionization applied to liquid chromatography/mass spectrometry analysis of endocrine-disrupting chemicals in environmental water samples

A sonic spray ionization liquid chromatography/mass spectrometry (LC/SSI-MS) procedure combined with off-line solid-phase extraction was optimized for the analysis of 20 endocrine-disrupting chemicals (EDCs) in water samples. Method development included a comparison of the novel sonic spray ionization (SSI) with more traditional ion sources, i.e. pneumatically assisted electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). It was demonstrated that SSI and ESI spectra were very similar, but were more prone to the formation of solvent cluster ions as compared with APCI spectra. This phenomenon was most prominent for SSI and resulted in an increased chemical background in full-scan mass spectra. However, this chemical noise did not affect the overall sensitivity of SSI and ESI. After optimization of LC and MS parameters, the LC/SSI-MS method was validated. Recoveries ranged from 76.3 up to 113.4% for all compounds. Limits of detection (LOD) and quantitation (LOQ) were established between 3.0 and 11.5 ng/L and 9.9 and 38.0 ng/L, respectively. Within-day (n = 5) and between-day (n = 5) reproducibility were investigated at three levels and ranged from 3.3-16.5% and 7.6-19.2%, respectively. Eight-point calibration curves were established and showed linearity for all compounds (r(2) > 0.987) over a linear dynamic range of 10-10 000 ng/L.     

Atmospheric pressure photoionization. II. Dual source ionization

In this paper we describe results based on the combination of atmospheric pressure photoionization (APPI) with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The main purpose of combining more than one ionizer is to extend the range of compounds that can be simultaneously analyzed. Three modes of operation are presented; use of either ionizer, simultaneous use of two ionizers, and rapid switching between ionizers during a single chromatographic run. The dual ionizer configurations only minimally affect the performance of either ionizer relative to the standard single-ionizer sources. However, it is observed that the operation of both ionizers together does not typically give the sum signal from either source operating alone. For APCI/APPI the signal can range from less than that of either source alone to the sum of the two individual sources. For ESI/APPI, we observed large suppressions of the ESI multiply-charged signal of proteins when the APPI source was on. These behaviors are presumed to be due to the interaction of the initially formed ions by both sources and attests to the importance of ion-molecule reactions that occur during and after the primary ionization events. We give examples of compounds that are preferentially ionized by either APPI, APCI or ESI and present thermochemical arguments based on molecular structure and functionality to explain this behavior. The dual source is also shown to be able to operate in negative ion mode opening up the potential to conduct wide ranging chemical analyses.     

Systematic investigation of ion suppression and enhancement effects of fourteen stable‐isotope‐labeled internal standards by their native analogues using atmospheric‐pressure chemical ionization and electrospray ionization and the relevance for multi‐analyte liquid chromatographic/mass spectrometric procedures

In clinical and forensic toxicology, multi‐analyte procedures are very useful to quantify drugs and poisons of different classes in one run. For liquid chromatographic/tandem mass spectrometric (LC/MS/MS) multi‐analyte procedures, often only a limited number of stable‐isotope‐labeled internal standards (SIL‐ISs) are available. If an SIL‐IS is used for quantification of other analytes, it must be excluded that the co‐eluting native analyte influences its ionization. Therefore, the effect of ion suppression and enhancement of fourteen SIL‐ISs caused by their native analogues has been studied. It could be shown that the native analyte concentration influenced the extent of ion suppression and enhancement effects leading to more suppression with increasing analyte concentration especially when electrospray ionization (ESI) was used. Using atmospheric‐pressure chemical ionization (APCI), methanolic solution showed mainly enhancement effects, whereas no ion suppression and enhancement effect, with one exception, occurred when plasma extracts were used under these conditions. Such differences were not observed using ESI. With ESI, eleven SIL‐ISs showed relevant suppression effects, but only one analyte showed suppression effects when APCI was used. The presented study showed that ion suppression and enhancement tests using matrix‐based samples of different sources are essential for the selection of ISs, particularly if used for several analytes to avoid incorrect quantification. In conclusion, only SIL‐ISs should be selected for which no suppression and enhancement effects can be observed. If not enough ISs are free of ionization interferences, a different ionization technique should be considered. Copyright © 2010 John Wiley & Sons, Ltd.     

Atmospheric pressure ionization mass spectrometry techniques for the analysis of alkyl ethoxysulfate mixtures

This paper compares two liquid introduction atmospheric pressure ionization techniques for the analysis of alkyl ethoxysulfate (AES) anionic surfactant mixtures by mass spectrometry, i. e., electrospray ionization (ESI) in both positive and negative ion modes and atmospheric pressure chemical ionization (APCI) in positive ion mode, using a triple quadrupole mass spectrometer. Two ions are observed in ESI(+) for each individual AES component, [M + Na]⁺ and a “desulfated” ion [M − SO₃ + H]⁺, whereas only one ion, [M − Na]⁻ is observed for each AES component in ESI(−). APCI(+) produces a protonated, “desulfated” ion of the form [M − NaSO₃ + 2H]⁺ for each AES species in the mixture under low cone voltage (10 V) conditions. The mass spectral ion intensities of the individual AES components in either the series from ESI(+) or APCI(+) can be used to obtain an estimate of their relative concentrations in the mixture and of the average ethoxylate (EO) number of the sample. The precursor ions produced by either ESI(+) or ESI(−), when subjected to low-energy (50 eV) collision-induced dissociation, do not fragment to give ions that provide much structural information. The protonated, desulfated ions produced by APCI(+) form fragment ions which reveal structural information about the precursor ions, including alkyl chain length and EO number, under similar conditions. APCI(+) is less susceptible to matrix effects for quantitative work than ESI(+). Thus APCI(+) provides an additional tool for the analysis of anionic surfactants such as AES, especially in complex mixtures where tandem mass spectrometry is required for the identification of the individual components.     

LC‐MS analysis of estradiol in human serum and endometrial tissue: Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

Accurate measurement of estradiol (E2) is important in clinical diagnostics and research. High sensitivity methods are critical for specimens with E2 concentrations at low picomolar levels, such as serum of men, postmenopausal women and children. Achieving the required assay performance with LC–MS is challenging due to the non‐polar structure and low proton affinity of E2. Previous studies suggest that ionization has a major role for the performance of E2 measurement, but comparisons of different ionization techniques for the analysis of clinical samples are not available. In this study, female serum and endometrium tissue samples were used to compare electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) in both polarities. APPI was found to have the most potential for E2 analysis, with a quantification limit of 1 fmol on‐column. APCI and ESI could be employed in negative polarity, although being slightly less sensitive than APPI. In the presence of biological background, ESI was found to be highly susceptible to ion suppression, while APCI and APPI were largely unaffected by the sample matrix. Irrespective of the ionization technique, background interferences were observed when using the multiple reaction monitoring transitions commonly employed for E2 (m/z 271 > 159; m/z 255 > 145). These unidentified interferences were most severe in serum samples, varied in intensity between ionization techniques and required efficient chromatographic separation in order to achieve specificity for E2. Copyright © 2013 John Wiley & Sons, Ltd.     

Nanoscale separations combined with electrospray ionization mass spectrometry: Sulfonamide determination

Nanoscale capillary liquid chromatography (nCLC) and capillary zone electrophoresis (CZE) have been combined with quadrupole mass spectrometry via an electrospray ionization (ESI) interface. These methodologies have been applied to the separation and determination of a variety of sulfonamides. CZE/ESI/MS is the more rapid and sensitive technique, but nCLC/ESI/MS shows promise for the analysis of dilute samples. Ultimately, the two techniques provide complementary methods of analysis. The detection limits of these techniques in the full-scan mode are in the low picomole range. Dissociation of the sulfonamides can be induced by increasing the skimmer voltage. This provides a limited means of discriminating between compounds of identical molecular weight but, more important, provides fragments that could be used to confirm the presence of analyte within a sample.     

Drug impurity profiling by capillary electrophoresis/mass spectrometry using various ionization techniques

Capillary electrophoresis/mass spectrometry (CE/MS) is predominantly carried out using electrospray ionization (ESI). Recently, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) have become available for CE/MS. With the VUV lamp turned off, the APPI source may also be used for CE/MS by thermospray ionization (TSI). In the present study the suitability of ESI, APCI, APPI and TSI for drug impurity profiling by CE/MS in the positive ion mode is evaluated. The drugs carbachol, lidocaine and proguanil and their potential impurities were used as test compounds, representing different molecular polarities. A background electrolyte of 100 mM acetic acid (pH 4.5) provided baseline separation of nearly all impurities from the respective drugs. APPI yielded both even‐ and odd‐electron ions, whereas the other ionization techniques produced even‐electron ions only. In‐source fragmentation was more pronounced with APCI and APPI than with ESI and TSI, which was most obvious for proguanil and its impurities. In general, ESI and TSI appeared the most efficient ionization techniques for impurities that are charged in solution achieving detection limits of 100 ng/mL (full‐scan mode). APPI and APCI showed a lower efficiency, but allowed ionization of low and high polarity analytes, although quaternary ammonium compounds (e.g. carbachol) could not be detected. Largely neutral compounds, such as the lidocaine impurity 2,6‐dimethylaniline, could not be detected by TSI, and yielded similar detection limits (500 ng/mL) for ESI, APPI and APCI. In many cases, impurity detection at the 0.1% (w/w) level was possible when 1 mg/mL of parent drug was injected with at least one of the CE/MS systems. Overall, the tested CE/MS systems provide complementary information as illustrated by the detection and identification of an unknown impurity in carbachol. Copyright © 2009 John Wiley & Sons, Ltd.     

Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery

The performance of the atmospheric pressure photoionization (APPI) technique was evaluated against five sets of standards and drug-like compounds and compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI technique was first used to analyze a set of 86 drug standards with diverse structures and polarities with a 100% detection rate. More detailed studies were then performed for another three sets of both drug standards and proprietary drug candidates. All 60 test compounds in these three sets were detected by APPI with an overall higher ionization efficiency than either APCI or ESI. Most of the non-polar compounds in these three sets were not ionized by APCI or ESI. Analysis of a final set of 201 Wyeth proprietary drug candidates by APPI, APCI and ESI provided an additional comparison of the ionization techniques. The detection rates in positive ion mode were 94% for APPI, 84% for APCI, and 84% for ESI. Combining positive and negative ion mode detection, APPI detected 98% of the compounds, while APCI and ESI detected 91%, respectively. This analysis shows that APPI is a valuable tool for day-to-day usage in a pharmaceutical company setting because it is able to successfully ionize more compounds, with greater structural diversity, than the other two ionization techniques. Consequently, APPI could be considered a more universal ionization method, and therefore has great potential in high-throughput drug discovery especially for open access liquid chromatography/mass spectrometry (LC/MS) applications.     

Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities

Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r(2) = 0.9998) over two orders of magnitude of concentration of rac-1-phenylethylamine (PEA), which is converted to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (< or =5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes.