Competitive Ionization Research Articles

Effect of Sample Plates and Sample Matrix on the Quantification Capabilities of Surface-Assisted Flowing Atmospheric-Pressure Afterglow Mass Spectrometry (SA-FAPA-MS).

Ambient desorption/ionization mass spectrometry (ADI-MS) has been broadly applied to accomplish direct analysis without sample preparation or separation. However, quantification capabilities and analytical performance are sometimes limited. Here, we report signal enhancement effects and improved quantification capabilities in plasma-based ADI-MS, when a flowing atmospheric-pressure afterglow (FAPA) source is used to probe analytes on tailored thin-layer chromatography (TLC) plates. It was found that quantitative results could be achieved when the TLC plate merely served as a sampling plate without a preceding separation step. Specifically, the dynamic response of caffeine, nicotine, acetaminophen, and progesterone was investigated with FAPA-MS on a variety of different TLC surfaces (normal-phase silica, reversed-phase-modified silica, cyano [CN]-modified silica, and dimethyl [RP2]-modified silica). All analytes were studied as single-analyte standards and in a multianalyte mixture to evaluate the effect of sample plates and sample matrix on analytical performance and competitive ionization processes. Overall, dimethyl (RP2)- and CN-modified silica resulted in superior performance compared to other TLC materials. After careful optimization and without the use of internal standards, linear ranges of five orders of magnitude were accessible for caffeine and nicotine. Limits of detection down to femtomole amounts of analyte were achieved. Quantitation limits using RP2-TLC and FAPA-MS were 0.062, 0.062l, 0.31, and 14 pmol for caffeine, nicotine, progesterone, and acetaminophen, respectively. Interestingly, the presence of nicotine at relatively high amounts reduced the signal of the other analytes, an observation that was found to correlate with the differences in the enthalpy of vaporization (ΔHvap) and proton affinity. To prove the quantitative capabilities, nicotine quantification in a real matrix-heavy e-liquid sample was demonstrated using an isotopically labeled standard. The use of TLC-based surfaces with FAPA-MS can aid in the direct and quantitative mass spectrometric investigation of complex mixtures.

Evaluation of an internal standard for qualitative DART-MS analysis of seized drugs

Rapid and accurate screening tools for seized drug analysis continue to be needed due to the complexities associated with the emerging drug landscape. Direct analysis in real-time mass spectrometry (DART-MS) is one technique that has been used for this purpose and is seeing increased implementation due to its ability to provide high-fidelity information rapidly. As with any analytical technique, ensuring data integrity with DART-MS results is critical. To further enhance data integrity and eliminate some of the challenges associated with qualitative analysis of seized drugs using DART-MS, the incorporation of an internal standard was investigated. After evaluating a number of candidate compounds, tetracaine was chosen because of its desirable characteristics. An appropriate concentration of tetracaine was established that provided similar sensitivity to GC–MS for a panel of drugs. The presence of tetracaine in a drug extract was found to cause a reduction in signal for some common drugs due to competitive ionization, but did not cause complete suppression of signal at relevant concentrations. Evaluation of a set of 60 representative case samples with and without internal standard found that the presence of internal standard did not negatively impact the results and that its presence eliminated the false identification of noise peaks in negative samples. The use of an internal standard also provided within-sample mass calibration and analyte concentration checks. It also allowed for automated mass drift compensation, removing a time-consuming process for high resolution DART-MS data processing.

Validation Study on the Simultaneous Quantitation of Multiple Wine Aroma Compounds with Static Headspace-Gas Chromatography-Ion Mobility Spectrometry.

A new quantitative method based on static headspace-gas chromatography-ion mobility spectrometry (SHS-GC-IMS) is proposed, which enables the simultaneous quantitation of multiple aroma compounds in wine. The method was first evaluated for its stability and the necessity of using internal standards as a quality control measure. The two major hurdles in applying GC-IMS in quantitation studies, namely, nonlinearity and multiple ion species, were also investigated using the Boltzmann function and generalized additive model (GAM) as potential solutions. Metrics characterizing the model performance, including root mean squared error, bias, limit of detection, limit of quantitation, repeatability, reproducibility, and recovery, were investigated. Both nonlinear fitting methods, Boltzmann function and GAM, were able to return desirable analytical outcomes with an acceptable range of error. Potential pitfalls that would cause inaccurate quantitation, that is, effects of ethanol content and competitive ionization, were also discussed. The performance of the SHS-GC-IMS method was subsequently compared against that of a currently established method, namely, GC-MS, using commercial wine samples. These findings provide an initial validation of a GC-IMS-based quantitation method, as well as a starting point for further enhancing the analytical scope of GC-IMS.

Improved intact peptide and protein quantitation by LC-MS: Battling the deleterious effects of analyte adsorption.

Peptide and protein quantitation by liquid chromatography-mass spectrometry relies on the assumption of linear signal response with concentration. At low concentrations, analyte adsorption to pipette tips, sample vials and equipment can have significant deleterious effects on signal response. Meanwhile at high concentrations, linearity breaks down due to competitive ionization, signal suppression, and the formation of peptide or protein multimers. These effects result in calibration curves that are more sigmoidal than linear. Linearity at low protein levels for identification and quantitation is of paramount importance in the discovery and validation of biomarker molecules. Herein, we demonstrate the benefits of using commercial low-bind microcentrifuge tubes and LC vials on the response of a 27-mer peptide, Vn96, and the intact proteins apomyoglobin and carbonic anhydrase. Linear curves were acquired for Vn96 while apomyoglobin required the addition of intact carbonic anhydrase as an adsorption competitor to achieve linearity. A linear calibration curve for carbonic anhydrase was also acquired by using the polypeptide ubiquitin as an adsorption competitor and internal standard. Linear response was recorded for approximately two orders of magnitude for apomyoglobin and carbonic anhydrase and three orders of magnitude for Vn96 with detection limits ranging from 0.33 to 19 fmol/µL. Finally, we used low-bind vials for the online enzymatic digestion of apomyoglobin where a high concentration of apomyoglobin acted as an adsorption blocker for the low level trypsin enzyme. Fortunately, the liberated tryptic peptides showed no affinity for the walls of the low-bind vials. In this study, we take a comprehensive approach to combat analyte adsorption by showing the significance of utilizing low-bind vials and adsorption competitors to greatly improve upon signal sensitivity at low concentrations of target molecules. The use of these methodologies should improve the low-level detection of molecules by mass spectrometry.

Research of n-type arsenic doped diamond: Theoretical analysis of electronic and mechanical properties

Substitutional defects of arsenic in diamond have been investigated in terms of the ionization energy, electronic property, and elastic constants for one and two arsenic doped configurations by density functional theory. Arsenic (As) particle doped diamond has been found to present attractive n-type doping properties. The ionization energy has been calculated based on different exchange-correlation functionals and competitive ionization energies are exhibited for one As atom doped and two As atoms doped (separated by one carbon atom) models. Besides, doping activities make the band gaps decrease for all configurations with the band structure of single As atom and separated site doped configurations remaining indirect, while the adjacent site of configuration with two As atoms has a direct band structure. Moreover, As doping makes the Young's module of diamond decrease and the elastic constants of adjacent site configuration present metastable state. Finally, one arsenic particle adsorption on diamond (001) surface has also been studied. It is identified that Bridge site is the most suitable adsorption site for arsenic doping during diamond growth process. As a conclusion, arsenic impurity is believed to be a competitive candidate of n-type doped diamond.

Identification of Suvorexant in Blood Using LC-MS-MS: Important Considerations for Matrix Effects and Quantitative Interferences in Targeted Assays.

Suvorexant (Belsomra®) is a novel dual orexin receptor antagonist used for the treatment of insomnia. The prevalence of suvorexant in forensic samples is relatively unknown, which demonstrates the need for robust analytical assays for the detection of this sedative hypnotic in forensic toxicology laboratories. In this study, suvorexant was isolated from whole blood using a simple acidic/neutral liquid-liquid extraction followed by analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Matrix effects were evaluated qualitatively and quantitatively using various extraction solvents, proprietary lipid clean-up devices and source conditions. The method was validated in terms of limit of detection, limit of quantitation, precision, bias, calibration model, carryover, matrix effects and drug interferences. Electrospray is a competitive ionization process whereby compounds in the droplet compete for a limited number of charged sites at the surface. As such, it is capacity-limited, and LC-MS-based techniques must be carefully evaluated to ensure that matrix effects or coeluting drugs do not impact quantitative assay performance. In this report, we describe efforts to ameliorate such effects in the absence of an isotopically labeled internal standard. Matrix effects are highly variable and heavily dependent on the physico-chemical properties of the substance. Although there is no universal solution to their resolution, conditions at the electrospray interface can mitigate these issues. Using this approach, the LC-MS/MS assay was fully validated and limits of detection and quantitation of 0.1 and 0.5ng/mL suvorexant were achieved in blood.

Rapid detection of fentanyl, fentanyl analogues, and opioids for on-site or laboratory based drug seizure screening using thermal desorption DART-MS and ion mobility spectrometry

Fentanyl and fentanyl analogues represent a current and emerging threat in the United States as pure illicit narcotics and in mixtures with heroin. Because of their extreme potency, methods to safely and rapidly detect these compounds are of high interest. This work investigates the use of thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS) and ion mobility spectrometry (IMS) as tools for the rapid and sensitive (nanogram to picograms) detection of fentanyl, 16 fentanyl analogues, and five additional opioids. Competitive ionization studies highlight that detection of these compounds in the presence of heroin is readily achievable, down to 0.1% fentanyl by mass with TD-DART-MS. With IMS, detection of nanogram levels of fentanyl in a binary fentanyl and heroin mixture is possible but can be complicated by decreased resolution in certain commercial instrument models. Modifications to the alarm windows can be used to ensure detection of fentanyl in binary mixtures. Additionally, three complex background matrices (fingerprint residue, dirt, and plasticizers) are shown to have a minimal effect of the detection of these compounds. Wipe sampling of the exterior of bags of questioned powders is shown to be a safe alternative method for field screening and identification, removing the need to handle potentially lethal amounts of material.

New urea-modified paper substrate for enhanced analytical performance of negative ion mode paper spray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) analysis in the negative ion mode is adversely affected by ionization suppression caused by electrical discharge and the presence of salts. Paper spray mass spectrometry (PS-MS) also suffers from the above phenomenon, being built on principles similar to those of ESI. Herein, we report the use of a paper substrate modified by 1-[3-(trimethoxysilyl)propyl]urea to improve the sensitivity of quantitative PS-MS analysis in the negative ion mode. The obtained results demonstrated that the urea modified paper substrate can effectively bind anions and highly polar compounds in the sample solution, decreasing competitive ionization in the negative ion mode of PS-MS and significantly reducing the signal intensity of Cl− adducts. In addition, the analyte responses were also significantly improved owing to the decreased electrical discharge observed for the less polar surface to decrease electrical discharge. Compared to non-modified PS-MS, urea-modified PS-MS exhibits a much higher sensitivity, showing 2−109 times improved signal-to-noise ratio (S/N). In real sample analysis, the limits of detection (LODs) of salicylic acid in urine and terpene lactones in Ginkgo biloba extract (EGb) were improved 10-fold and 2–40-fold, respectively, compared to that of non-modified PS-MS.

Microplasma-based flowing atmospheric-pressure afterglow (FAPA) source for ambient desorption-ionization mass spectrometry

A new direct-current microplasma-based flowing atmospheric pressure afterglow (FAPA) source was developed for use in ambient desorption-ionization mass spectrometry. The annular-shaped microplasma is formed in helium between two concentric stainless-steel capillaries that are separated by an alumina tube. Current-voltage characterization of the source shows that this version of the FAPA operates in the normal glow-discharge regime. A glass surface placed in the path of the helium afterglow reaches temperatures of up to approximately 400 °C; the temperature varies with distance from the source and helium flow rate through the source. Solid, liquid, and vapor samples were examined by means of a time-of-flight mass spectrometer. Results suggest that ionization occurs mainly through protonation, with only a small amount of fragmentation and adduct formation. The mass range of the source was shown to extend up to at least m/z 2722 for singly charged species. Limits of detection for several small organic molecules were in the sub-picomole range. Examination of competitive ionization revealed that signal suppression occurs only at high (mM) concentrations of competing substances.

Increased 14C AMS Efficiency from Reduced Competitive Ionization

AbstractObservation of 80 µA/mm2 C– current from a 0.5-mm-diameter sample compared to 20 µA/mm2 from a 1-mm-diameter sample contradicts the long-held surface ionization hypothesis of cesium sputter ion source operation. Resonant ionization occurs in neutral Cs plasma above a sample in a sputtered pit or well. A collision-radiation model of that plasma followed electronic excitation and radiation relaxation up to the Cs(7d) state. The Cs(5d) metastable state dominates plasma in a 1-mm-diameter well, but high electron densities in narrow wells drive a majority to Cs(7d) and higher. Competitive ionization by Al2 dimers from the sample holder reduces Cs(7s,p) states resonant in ionization energy with C–. Al anions from states above Cs(7p) in narrow wells also diminish radiation cascades to the Cs(7s,p), reducing C–. We tested sample wells of non-ionizing Zn to maintain high ionization efficiency for small samples in narrow wells.

Coupling Charge Reduction Mass Spectrometry to Liquid Chromatography for Complex Mixture Analysis.

Electrospray ionization (ESI) of solution mixtures often generates complex mass spectra, even following liquid chromatography (LC), due to analyte multiple charging. Multiple charge state distributions can lead to isobaric interferences, mass spectral congestion, and ambiguous ion identification. As a consequence, data interpretation increases in complexity. Several charge reduction mass spectrometry (MS) approaches have been previously developed to reduce the average charge state of gaseous ions; however, all of these techniques have been restricted to direct infusion MS. In this study, synthetic polyols and surfactants separated by liquid chromatography and ionized by positive mode ESI have been subjected to polonium-210 α-particle radiation to reduce the average charge state to singly charged cations prior to mass analysis. LC/MS analysis of 5000 molecular weight poly(ethylene glycol) (PEG5000) generated an average charge state of 5.88+; whereupon, liquid chromatography/electrospray ionization/charge reduction/mass spectrometry (LC/CR/MS) analysis of PEG 5000 generated an average charge state of 1.00+. The PEG5000 results demonstrated a decrease in spectral complexity and enabled facile interpretation. Other complex solution mixtures representing specific MS challenges (i.e., competitive ionization and isobaric ion overlap) were explored and analyzed with LC/CR/MS to demonstrate the benefits of coupling LC to CR/MS. For example, polyol information related to initiator, identity/relative amount of monomer, and estimated molecular weight was characterized in random and triblock ethylene oxide/propylene oxide polyols using LC/CR/MS. LC/CR/MS is a new analytical technique for the analysis of complex mixtures.

Tunable Ionization Modes of a Flowing Atmospheric-Pressure Afterglow (FAPA) Ambient Ionization Source.

Plasma-based ambient desorption/ionization sources are versatile in that they enable direct ionization of gaseous samples as well as desorption/ionization of analytes from liquid and solid samples. However, ionization matrix effects, caused by competitive ionization processes, can worsen sensitivity or even inhibit detection all together. The present study is focused on expanding the analytical capabilities of the flowing atmospheric-pressure afterglow (FAPA) source by exploring additional types of ionization chemistry. Specifically, it was found that the abundance and type of reagent ions produced by the FAPA source and, thus, the corresponding ionization pathways of analytes, can be altered by changing the source working conditions. High abundance of proton-transfer reagent ions was observed with relatively high gas flow rates and low discharge currents. Conversely, charge-transfer reagent species were most abundant at low gas flows and high discharge currents. A rather nonpolar model analyte, biphenyl, was found to significantly change ionization pathway based on source operating parameters. Different analyte ions (e.g., MH(+) via proton-transfer and M(+.) via charge-transfer) were formed under unique operating parameters demonstrating two different operating regimes. These tunable ionization modes of the FAPA were used to enable or enhance detection of analytes which traditionally exhibit low-sensitivity in plasma-based ADI-MS analyses. In one example, 2,2'-dichloroquaterphenyl was detected under charge-transfer FAPA conditions, which were difficult or impossible to detect with proton-transfer FAPA or direct analysis in real-time (DART). Overall, this unique mode of operation increases the number and range of detectable analytes and has the potential to lessen ionization matrix effects in ADI-MS analyses.

Direct analysis in real time mass spectrometry of potential by-products from homemade nitrate ester explosive synthesis

This work demonstrates the coupling of direct analysis in real time (DART) ionization with time-of-flight mass spectrometry (MS) in an off-axis configuration for the trace detection and analysis of potential partially nitrated and dimerized by-products of homemade nitrate ester explosive synthesis. Five compounds relating to the synthesis of nitroglycerin (NG) and pentaerythritol tetranitrate (PETN) were examined. Deprotonated ions and adducts with molecular oxygen, nitrite, and nitrate were observed in the mass spectral responses of these compounds. A global optimum temperature of 350°C for the by-products investigated here enabled single nanogram to sub nanogram trace detection. Matrix effects were examined through a series of mixtures containing one or more compounds (sugar alcohol precursors, by-products, and/or explosives) across a range of mass loadings. The explosives MS responses experienced competitive ionization in the presence of all by-products. The magnitude of this influence corresponded to both the degree of by-product nitration and the relative mass loading of the by-product to the explosive. This work provides a characterization of potential by-products from homemade nitrate ester synthesis, including matrix effects and potential challenges that might arise from the trace detection of homemade explosives (HMEs) containing impurities. Detection and understanding of HME impurities and complex mixtures may provide valuable information for the screening and sourcing of homemade nitrate ester explosives.

Trace detection and competitive ionization of erythritol tetranitrate in mixtures using direct analysis in real time mass spectrometry

Trace detection and competitive ionization of a homemade nitrate ester explosive and its sugar alcohol precursor using DART-MS.

Comparing MALDI‐MS, RP‐LC‐MALDI‐MS and RP‐LC‐ESI‐MS glycomic profiles of permethylated N‐glycans derived from model glycoproteins and human blood serum

The glycomic profiling of purified glycoproteins and biological specimen is routinely achieved through different analytical methods, but mainly through MS and LC-MS. The enhanced ionization efficiency and improved tandem MS interpretation of permethylated glycans have prompted the popularity of this approach. This study focuses on comparing the glycomic profiling of permethylated N-glycans derived from model glycoproteins and human blood serum using MALDI-MS as well as RP-LC-MALDI-MS and RP-LC-ESI-MS. In the case of model glycoproteins, the glycomic profiles acquired using the three methods were very comparable. However, this was not completely true in the case of glycans derived from blood serum. RP-LC-ESI-MS analysis of reduced and permethylated N-glycans derived from 250 nl of blood serum allowed the confident detection of 73 glycans (the structures of which were confirmed by mass accuracy and tandem MS), while 53 and 43 structures were identified in the case of RP-LC-MALDI-MS and MALDI-MS analyses of the same sample, respectively. RP-LC-ESI-MS analysis facilitates automated and sensitive tandem MS acquisitions. The glycan structures that were detected only in the RP-LC-ESI-MS analysis were glycans existing at low abundances. This is suggesting the higher detection sensitivity of RP-LC-ESI-MS analysis, originating from both reduced competitive ionization and saturation of detectors, facilitated by the chromatographic separation. The latter also permitted the separation of several structural isomers; however, isomeric separations pertaining to linkages were not detected.

Ion Mobility Spectrometry: A Comprehensive and Versatile Tool for Occupational Pharmaceutical Exposure Assessment

The qualitative and quantitative capabilities of ion mobility spectrometry (IMS) as a comprehensive and powerful tool in workplace air monitoring have been demonstrated on the example of a Spanish pharmaceutical company. The developed IMS based procedure is capable of detecting and determining in air samples the active pharmaceutical ingredients (APIs) manipulated and/or produced in this pharmaceutical industry. Sensitivity, in the ng-pg range, selectivity, possibly to provide results in near real time, and reduction of analysis costs are the most important properties that ratify IMS as a serious alternative in occupational exposure assessment. The possibility of false positives by drift time interferences and false negatives by competitive ionization and also desorption process interferences has been deeply evaluated. Moreover, chemometric strategies based on self-modeling curve resolution (SMCR) have been applied to obtain qualitative and quantitative individual component information from overlapped peaks. The IMS procedure has been successfully applied to evaluate the concentration of APIs (nimesulide, dexketoprofen, deflazacort) handled by the pharmaceutical company employees in the making of tablets and granulates, and control measures have been suggested in accordance.

Production and fragmentation of negative ions from neutral N‐linked carbohydrates ionized by matrix‐assisted laser desorption/ionization

Although negative ion fragmentation mass spectra of neutral N-linked carbohydrates (those attached to Asn in glycoproteins) provide much more structural information than spectra recorded in positive ion mode, neutral carbohydrates are reluctant to form negative ions by matrix-assisted laser desorption/ionization (MALDI) unless ionized from specific matrices such as nor-harmane or adducted with anions such as chloride. This paper reports the results of experiments to optimize negative ion formation from adducts of N-linked glycans with respect to ion abundance and fragment ion production. The best results were obtained with 2,4,6-trihydroxyacetophenone (THAP) as the matrix with added ammonium nitrate as the salt providing the anion. This approach is demonstrated to be applicable for a wide range of N-linked glycan structures. Phosphate adducts, analogous to those that are usually encountered in electrospray spectra from N-glycans released by protein N-glycosidase F, were produced by addition of ammonium phosphate to the matrix but in relatively low yield allowing competitive ionization of endogenous anionic compounds leading to complex spectra. Fragmentation of the nitrate adducts, which were formed in higher yield, generally paralleled that seen by collision-induced dissociation following ionization by electrospray, with the first stage of the dissociation being the elimination of the nitrate with a proton from one of the hydroxyl groups of the sugar. The spectra of the resulting [M-H](-) species displayed very specific fragment ions, mainly cross-ring and C-type glycosidic cleavage products, that revealed more structural (linkage and branching) information of the compounds than the mainly glycosidic cleavage products that dominated the positive ion spectra.

Optimized thermal desorption for improved sensitivity in trace explosives detection by ion mobility spectrometry

In this work we evaluate the influence of thermal desorber temperature on the analytical response of a swipe-based thermal desorption ion mobility spectrometer (IMS) for detection of trace explosives. IMS response for several common high explosives ranging from 0.1 ng to 100 ng was measured over a thermal desorber temperature range from 60 °C to 280 °C. Most of the explosives examined demonstrated a well-defined maximum IMS signal response at a temperature slightly below the melting point. Optimal temperatures, giving the highest IMS peak intensity, were 80 °C for trinitrotoluene (TNT), 100 °C for pentaerythritol tetranitrate (PETN), 160 °C for cyclotrimethylenetrinitramine (RDX) and 200 °C for cyclotetramethylenetetranitramine (HMX). By modifying the desorber temperature, we were able to increase cumulative IMS signal by a factor of 5 for TNT and HMX, and by a factor of 10 for RDX and PETN. Similar signal enhancements were observed for the same compounds formulated as plastic-bonded explosives (Composition 4 (C-4), Detasheet, and Semtex). In addition, mixtures of the explosives exhibited similar enhancements in analyte peak intensities. The increases in sensitivity were obtained at the expense of increased analysis times of up to 20 seconds. A slow sample heating rate as well as slower vapor-phase analyte introduction rate caused by low-temperature desorption enhanced the analytical sensitivity of individual explosives, plastic-bonded explosives, and explosives mixtures by IMS. Several possible mechanisms that can affect IMS signal response were investigated such as thermal degradation of the analytes, ionization efficiency, competitive ionization from background, and aerosol emission.

Competitive ionization processes of anthracene excited with a femtosecond pulse in the multi-photon ionization regime

To clarify the ionization mechanism of large molecules under multi-photon ionization conditions, photo-electron spectroscopic studies on anthracene have been performed with electron imaging technique. Electron kinetic energy distributions below a few eV reveal that three kinds of ionization channels coexist, viz., vertical ionization, ionization from Rydberg states, and thermionic hot electron emission. Their relative yield is determined by the characteristic of the laser pulse. The duration in particular influences the ratio between the first two processes, while for higher intensities the last process dominates. Our results provide strong evidence that internal conversion plays an important role for the ionization of the molecule.

Pros and cons of benzodiazepines screening in human saliva by ion mobility spectrometry

The usefulness of ion mobility spectrometry as a screening methodology for the on-site benzodiazepine analysis in saliva samples has been critically evaluated. The procedure involved the injection of clear supernatant extracts after centrifugation and provided limit of detection values ranging from 2.0 to 18 μg L(-1), and a precision, expressed as relative standard deviation, from 2.9% to 16%, depending on the different benzodiazepines studied. Those values are appropriate for their positive identification in saliva samples in which benzodiazepine concentration, after a chronic or acute dose, is in the range of 2-30 μg L(-1). Problems related with overlapped benzodiazepine signals have been successfully overcome by application of multivariate curve resolution, which is a helpful tool to improve the resolution of the technique, without sacrificing the method simplicity and frequency of analysis. The possibility of false positives caused by the presence of interferents with the same drift time as the benzodiazepines and the possibility of false negatives due to the presence of interferents by competitive ionization have been critically evaluated. The satisfactory results obtained for the analysis of real saliva samples after an acute dose of diazepam through sublingual and oral intakes confirm the capability of the technique to be used as a screening methodology in the analysis of benzodiazepines in oral fluids.