Unambiguous Peak Research Articles

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.

Temperature variations in caves induced by atmospheric pressure variations—Part 2: Unveiling hidden thermal signals

In underground cavities, temperature variations of the order of 10−3°C are permanently induced by the variations of atmospheric pressure, even at great depths, with couplings of the order of 0.2 to 20 × 10−3°C/hPa depending on frequency. In the first part of this study, we established the atmospheric pressure to temperature transfer function (TF) as a function of frequency from 8 × 10−7 to 8 × 10−4 Hz. Here, we use this TF to calculate the expected PIT variations, which, after being subtracted from the observed time-series, provide residual temperature time-series. We calculated such temperature residuals in four natural caves in France: Esparros, Aven d'Orgnac, Pech Merle and Chauvet-Pont d'Arc Caves, the last two containing unique prehistoric wall paintings. Temperature signals, as small as a few 10−3°C, due to human presence, are then conspicuous, with evidence of relaxation longer than several days and long-term cumulative effects. In addition, we observe temperature signals suggesting non-stationary states characterized by several processes which are not necessarily easy to separate, such as transient air currents, due to barometric winds or locally semi-confined convection cells, transient infiltration, or energy dissipation by evaporation-condensation at the rock surface. This background thermal agitation displays a scale-free amplitude spectrum, from 2 × 10−5 to 4 × 10−4 Hz, of the form f−α, with α varying from 0.1 to 0.6 depending on the site. Furthermore, at the Chauvet Cave, a weak but unambiguous peak emerges during some months at a period of about 82.2 ± 0.8 minutes, suggesting a Helmholtz-type resonance. Small but significant temperature signals are therefore detected in underground cavities once the effect of atmospheric pressure variations is corrected for. These signals reveal subtle coupled processes whose knowledge is essential to evaluate preservation strategies and to establish conditions for resilience of underground systems under artificial or natural influence including climate change.

A new approach to the determination of pKa and pKb of Brønsted–Lowry weak acid and weak base in aprotic solvent

• Developing a method of determining the dissociation constants of weak acid and base in acetonitrile. • Unambiguous voltammetric peak potentials were used to exactly measure pK a and pK b . • Compared with using potentiometry, the method is simple and rapid because of using cyclic voltammetry. The pK a and pK b of Bronsted–Lowry weak acids and weak bases in aprotic solvents are difficult to accurately determine. Herein, we found that in an aprotic solvent, the conjugate base of weak acid can produce a new, unambiguous anodic peak of hydroquinone, and the conjugate acid of weak base can produce a new cathodic peak of p-benzoquinone. These new peaks were due to the drastic change in pH near the electrode surface, and their potentials depended on the strength of conjugate acids and bases but not on the concentration of hydroquinone and quinone. Therefore, the conjugate acid–base dissociation constant can be measured by the new peak potential. Then, the weak acid–base dissociation constant can be obtained by the difference between the autoprotolysis constant of the aprotic solvent and the conjugate acid–base dissociation constant. The experimental results were consistent with those in literature.

A New Approach to the Determination of pK <sub>a</sub> and pK <sub>b</sub> of Brønsted-Lowry Weak Acid and Weak Base in Aprotic Solvent

It is difficult to accurately determine the pKa and pKb of Bronsted-Lowry weak acids and weak bases in aprotic solvents. We found that in aprotic solvent, the conjugated base of weak acid can cause a new unambiguous anodic peak of hydroquinone, and the conjugated acid of weak base can cause a new cathodic peak of p-benzoquinone. The new peaks are due to the drastic change of pH near the electrode surface, and their potentials are dependent on the strength of conjugated acids and bases, but independent of the concentration of hydroquinone and quinone. Therefore, the conjugated acid-base dissociation constant can be measured by the new peak potential. Then the weak acid-base dissociation constant can be obtained by the difference between the autoprotolysis constant of the aprotic solvent and the conjugated acid-base dissociation constant. The experimental results are consistent with those in the literatures.

Achiral-Chiral Two-Dimensional Liquid Chromatography Platform to Support Automated High-Throughput Experimentation in the Field of Drug Development.

Automated high-throughput experimentation (HTE) is a powerful tool for scientists to explore and optimize chemical transformations by simultaneously screening yield, stereoselectivity, and impurity profiles. To analyze the HTE samples, high-throughput analysis (HTA) platforms must be fast, accurate, generic, and specific at the same time. A large amount of high-quality data is critical for the success of machine learning models in the era of big data. Conventional chiral liquid chromatography-mass spectrometry (LC/MS) HTE methods are hampered by compound co-eluting, possible ion suppression, and limited chiral column lifetime in the presence of crude reaction mixtures or complex sample matrices. To overcome these limitations, a generic and fast achiral-chiral heart-cutting two-dimensional (2D)-LC method has been developed to determine both the yield and stereoselectivity of chemical transformations within a 10 min run time. Successful implementation of the 2D-LC HTA platform in a routine drug development environment was achieved for real-world project support, with the analysis so far of over 2000 reaction mixtures prepared in the 96-well plate format. Excellent performance of the method was demonstrated by relative standard deviation (RSD) lower than 0.83% for the 1D and 2D retention times, and determination coefficients higher than 0.99. The presented HTA 2D-LC platform has had a significant impact on drug development by analyzing the HTE samples rapidly with unambiguous peak tracking and providing a robust approach for accurately generating a large amount of high-quality data in a short time.

Unambiguous Peak Identification of a Silicon Fabry‐Perot Temperature Sensor Assisted With an In-Line Fiber Bragg Grating

Wavelength interrogation has been widely used for fiber-optic sensors based on Fabry‐Perot interferometers (FPIs) because of its high measurement resolution. However, phase ambiguity often limits the measurement range to be less than one free-spectral range (FSR) of the FPI. Here, we investigate a simple but effective method that can eliminate the phase ambiguity of a silicon FPI temperature sensor using a fiber Bragg grating (FBG) fabricated on the lead-in fiber close to the silicon FPI. The reflection spectrum of the sensor shows sinusoidal fringes from the FPI and a single peak from the FBG. Assuming the FBG and the silicon FPI have similar temperatures, the FBG peak can be used merely as a marker to identify an FPI fringe peak without ambiguity even when it shifts by more than an FSR. Although the temperature resolution of the FBG is lower than that of the silicon FPI, the measurement range of the silicon FPI can be greatly improved without sacrificing the measurement resolution. Using a temperature sensor based on a 75- μ m-thick silicon FPI, we demonstrated temperature measurement over a range of −60 to 140 °C (corresponding to a spectral shift of about five FSRs) with a resolution of 0.0033 °C. The measurement range with unambiguous peak recognition is expected to be larger than demonstrated in the experiment and only limited by the spectral bandwidth of the sensor system and the capability of materials used for the sensor construction.

The use of high-resolution mass spectrometry (HRMS) for the analysis of DNA and other macromolecules: A how-to guide for forensic chemistry

High-resolution mass spectrometry (HRMS) is a highly effective technique for the analysis of DNA and other biological macromolecules and has the potential to be a valuable tool for forensic chemists. In this work, we introduce concepts of HRMS, discuss benefits of the technique, and outline the steps involved in developing an application utilizing this platform. We examine the importance of optimizing instrumental conditions, selecting an appropriate solvent, and interpreting mass spectra with considerations to determining charge state, mass accuracy and resolution. Specifically, we present a method for the analysis of short single stranded oligonucleotides (26- and 28-base) using Fourier-transform ion cyclotron mass spectrometry (FT-ICR MS), while exploring the importance of sample matrix. Results of this work indicate that analysis of small oligonucleotides with 50:50 water:acteonitrile + 20 mM imidazole (C3H4N2) produced a mass spectrum with the −5 charge state as the base peak. Charge states of −4 up to −15 were also observed, although at much lower ion intensities. Analysis of the oligonucleotides using water and 20 mM imidazole produced a charge state distribution from −5 to −11 over the mass range m/z 200–2500. These two solvents, paired with the optimized HRMS method, produced multiple charge states, high resolution mass spectra, and high ion signal intensity that allowed for unambiguous peak identification. This work serves as a “how-to guide” for forensics scientists who require efficient and accurate HRMS protocols. Our research supports using nucleic acids for diagnostics to analyze oligonucleotides, short DNA fragments and other biological macromolecules using HRMS.

Direct observation of cross-polarized excitons in aligned single-chirality single-wall carbon nanotubes

Optical properties of single-wall carbon nanotubes (SWCNTs) for light polarized parallel to the nanotube axis have been extensively studied, whereas their response to light polarized perpendicular to the nanotube axis has not been well explored. Here, by using a macroscopic film of highly aligned single-chirality (6,5) SWCNTs, we performed a systematic polarization-dependent optical absorption spectroscopy study. In addition to the commonly observed angular-momentum-conserving interband absorption of parallel-polarized light, which generates $E_{11}$ and $E_{22}$ excitons, we observed a small but unambiguous absorption peak whose intensity is maximum for perpendicular-polarized light. We attribute this feature to the lowest-energy cross-polarized interband absorption processes that change the angular momentum along the nanotube axis by $\pm 1$, generating $E_{12}$ and $E_{21}$ excitons. The energy difference between the $E_{12}$ and $E_{21}$ exciton peaks, expected from asymmetry between the conduction and valence bands, was smaller than the observed linewidth. Unlike previous observations of cross-polarized excitons in polarization-dependent photoluminescence and circular dichroism spectroscopy experiments, our direct observation using absorption spectroscopy allowed us to quantitatively analyze this resonance. Specifically, we determined the energy and oscillator strength of this resonance to be 1.54 and 0.05, respectively, compared with the values for the $E_{11}$ exciton peak. These values, in combination with comparison with theoretical calculations, in turn led to an assessment of the environmental effect on the strength of Coulomb interactions in this aligned single-chirality SWCNT film.

Excited states of neutral donor bound excitons in GaN

We investigate the excited states of a neutral donor bound exciton (D0X) in bulk GaN by means of high-resolution, polychromatic photoluminescence excitation (PLE) spectroscopy. The optically most prominent donor in our sample is silicon accompanied by only a minor contribution of oxygen—the key for an unambiguous assignment of excited states. Consequently, we can observe a multitude of Si0X-related excitation channels with linewidths down to 200 μeV. Two groups of excitation channels are identified, belonging either to rotational-vibrational or electronic excited states of the hole in the Si0X complex. Such identification is achieved by modeling the excited states based on the equations of motion for a Kratzer potential, taking into account the particularly large anisotropy of effective hole masses in GaN. Furthermore, several ground- and excited states of the exciton-polaritons and the dominant bound exciton are observed in the photoluminescence (PL) and PLE spectra, facilitating an estimate of the associated complex binding energies. Our data clearly show that great care must be taken if only PL spectra of D0X centers in GaN are analyzed. Every PL feature we observe at higher emission energies with regard to the Si0X ground state corresponds to an excited state. Hence, any unambiguous peak identification renders PLE spectra highly valuable, as important spectral features are obscured in common PL spectra. Here, GaN represents a particular case among the wide-bandgap, wurtzite semiconductors, as comparably low localization energies for common D0X centers are usually paired with large emission linewidths and the prominent optical signature of exciton-polaritons, making the sole analysis of PL spectra a challenging task.

Unambiguous Peak Recognition for a Silicon Fabry–Pérot Interferometric Temperature Sensor

Wavelength tracking method is widely used for fiber-optic sensors based on Fabry–Perot interferometers (FPIs). Although wavelength tracking provides high resolution, it encounters the well-known problem of direction ambiguity when the spectrum shifts by more than half of the free-spectral range. In this paper, we propose a new signal processing method that can unambiguously recognize the spectral peaks of a silicon FPI by obtaining their unique order values to overcome the direction ambiguity and achieve measurement over extended range. New elements of the proposed method include: 1) an average peak order calculated from multiple peak pairs is used to reduce variation of the calculated order number, and 2) a modified dispersion model of silicon is involved in the calculation of the peak order to suppress drift of the calculated peak order. The averaging method generally decreases the data processing rate, but does not affect the response time of the sensor. We experimentally demonstrate the feasibility of the proposed method using our recently developed fiber-optic temperature sensor using a 200-μm-thick silicon FPI. Results show that the measurement of temperature over 0 to 100 °C is realized, leading to an increase of more than five times in dynamic range.

Use of High Energy Collision Induced Dissociation (HE-CID) in TOF-SIMS for Unambiguous Peak Identification and Imaging

Use of High Energy Collision Induced Dissociation (HE-CID) in TOF-SIMS for Unambiguous Peak Identification and Imaging

Extension of the two-dimensional mass channel cluster plot method to fast separations utilizing low thermal mass gas chromatography with time-of-flight mass spectrometry

Implementation of a data reduction and visualization method for use with high-speed gas chromatography and time-of-flight mass spectrometry (GC-TOFMS) is reported. The method, called the “2D m/z cluster method” facilitates analyte detection, deconvolution, and identification, by accurately measuring peak widths and retention times using a fast TOFMS sampling frequency (500 Hz). Characteristics and requirements for high speed GC are taken into consideration: fast separations with narrow peak widths and high peak capacity, rapid data collection rate, and effective peak deconvolution. Transitioning from standard GC (10–60+ minute separations) to fast GC (1–10 min separations) required consideration of how to properly analyze the data. This report validates use of the 2D m/z cluster method with newly developed GC technology that produces ultra-fast separations (∼1 min) with narrow analyte peak widths. Low thermal mass gas chromatography (LTM-GC) operated at a heating rate of 250 °C/min coupled to a LECO Pegasus III TOFMS analyzed a 115 component test mixture in 120 s with peak widths-at-base, wb (4σ), of 350 ms (average) to produce a separation with a high peak capacity, nc ∼ 340 (at unit resolution Rs = 1). The 2D m/z cluster method is shown to separate overlapped analytes to a limiting Rs ∼ 0.03, so the effective peak capacity was increased nearly 30-fold to nc ∼10,000 in the 120 s separation. The method, when coupled with LTM-GC-TOFMS, is demonstrated to provide unambiguous peak rank (i.e. the number of analytes per overlapped peak in the total ion current (TIC)), by visualizing locations of pure and chromatographically overlapped m/z. Hence, peak deconvolution and identification using MCR-ALS (multivariate curve resolution – alternating least squares) is demonstrated.

Modifications in the glycerophospholipid composition between the Coxiella burnetii phase Iand phase II cells suggest an association with phase variation of the bacterium.

Glycerophospholipids (GP) extracted from the Coxiella burnetii strain Nine Mile in virulent phase I(NM I) and low virulent phase II (NM II) were analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) that gave asuperior mass resolution and mass accuracy allowing unambiguous peak recognition and precise assignment of ions. We showed that GP present in the pathogen'souter membrane underwent considerable modifications during the phase variation that might be related to impact of various environmental factors. It was found that GP from phase Icells were much more complex than those from phase II cells. While glycerophosphoethanolamines (PE), glycerophosphocholines (PC) and glycerophosphoglycerols (PG) were present in both phases of C. burnetii, major differences were observed in the presence of glycerophosphates (PA) and glycerophosphoserines (PS). Thus, PA but no PS were detected in NM Ivariant in contrast with NM II cells where PS but no PA were identified. It is suggested that enzymes for PA head group modifications to form PS, PE, and PG become active during the phase variation of the bacterium.

Appearance of Antiferromagnetic Dipole Order in Ce0.5La0.5B6 with Pr Ion Doping

We have performed a neutron diffraction experiment on Pr-doped Ce0.5Pr0.1La0.4B6, in which an antiferromagnetic octupole order with \(\boldsymbol{{q}} = (1/2,1/2,1/2)\) could be anticipated by analogy with Ce0.7La0.3B6. Contrary to this natural expectation, we detected an unambiguous magnetic peak at \(\boldsymbol{{q}} = (1/4,1/4,1/2)\), which is the same q-vector frequently realized in the magnetic ordered phases of RB6 (R = rare earth) compounds. No significant signal was observed at \(\boldsymbol{{q}} = (1/2,1/2,1/2)\) at zero magnetic field. This result shows that the normal antiferromagnetic dipole moment is also one of the competing multipole order parameters in the CexLa1−xB6 system. The relevant order parameters are close in energy and can be tuned by a weak perturbation.

Raman scattering of neutron irradiated 6H-SiC

Confocal micro-Raman spectroscopy was preformed to investigate the structural damage of SiC neutron irradiated with the fluences of 1.72×1019 and 1.67×1020 n/cm2. In addition to characteristic peaks, several additional signals related to Si–Si, Si–C, and C–C vibration modes were monitored. The vibration mode associated with CSiV C complexes was identified to be the unambiguous peak at 575 cm−1 which appears initially in the sample post-irradiation annealed at 800 °C. The defect-induced phonon confinement effect results in an asymmetric broadening with a low-frequency tail of the optical phonon peaks. The sigmoidal thermal recovery behavior of the optical phonon frequencies indicates that the reduction of FLO0/6–FTO2/6 splitting originates mainly from the isolated vacancies and interstitials.

Linear Ion Trap MSn of Enzymatically Synthesized 13C-Labeled Fructans Revealing Differentiating Fragmentation Patterns of β (1-2) and β (1-6) Fructans and Providing a Tool for Oligosaccharide Identification in Complex Mixtures

Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of β-d-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as Lolium perenne. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MS(n) method for the separation and detection of fructan isomers and present here evidence for specific MS(n) fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MS(n) analysis of (13)C labeled fructan oligomers produced by L. perenne fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards.

Analysis of high-molecular-weight fructan polymers in crude plant extracts by high-resolution LC-MS.

The main water-soluble carbohydrates in temperate forage grasses are polymeric fructans. Fructans consist of fructose chains of various chain lengths attached to sucrose as a core molecule. In grasses, fructans are a complex mixture of a large number of isomeric oligomers with a degree of polymerisation ranging from 3 to >100. Accurate monitoring and unambiguous peak identification requires chromatographic separation coupled to mass spectrometry. The mass range of ion trap mass spectrometers is limited, and we show here how monitoring selected multiply charged ions can be used for the detection and quantification of individual isomers and oligomers of high mass, particularly those of high degree of polymerization (DP > 20) in complex plant extracts. Previously reported methods using linear ion traps with low mass resolution have been shown to be useful for the detection of fructans with a DP up to 49. Here, we report a method using high-resolution mass spectrometry (MS) using an Exactive Orbitrap MS which greatly improves the signal-to-noise ratio and allows the detection of fructans up to DP = 100. High-sugar (HS) Lolium perenne cultivars with high concentrations of these fructans have been shown to be of benefit to the pastoral agricultural industry because they improve rumen nitrogen use efficiency and reduce nitrous oxide emissions from pastures. We demonstrate with our method that these HS grasses not only contain increased amounts of fructans in leaf blades but also accumulate fructans with much higher DP compared to cultivars with normal sugar levels.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-5374-8) contains supplementary material, which is available to authorized users.

Combining advanced NMR techniques with ultrahigh resolution mass spectrometry: A new strategy for molecular scale characterization of macromolecular components of soil and sedimentary organic matter

Molecular level characterization of complex biopolymers in nature is a key element to understanding the composition of natural organic matter (NOM) and fossil organic matter formation, such as kerogen and coal. Characterization of such geopolymers is difficult due to their heterogeneity and insolubility in common aqueous or organic solvents. Here, a strategy for extracting a representative sample is presented using the combined techniques of nuclear magnetic resonance (NMR) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for chemical analysis and characterization of NOM. A variety of NOM samples (wood, kerogen, bitumen, whole sediments) are shown as examples for implementing the strategy, which include solvent extractions using pyridine. For most samples, the extracts are confirmed to be chemically representative of the insoluble solid, by comparing the liquids NMR spectrum of the extract to that of the whole, unfractionated NOM utilizing high resolution magic angle spinning (HRMAS) NMR. To assist in unambiguous peak assignment, a technique for post acquisition spectral denoising, using wavelet transformation (WT), is also employed on the 1D and 2D NMR spectra. The findings from the NMR data lay a foundation for the subsequent electrospray ionization FTICR-MS analysis of the pyridine extracts, since this instrument has the limitation of only analyzing liquid samples. The FTICR-MS analyses can, therefore, be used to molecularly represent the structural components of the NOM. Various compositional insights have been obtained on these samples, indicating the efficacy of the analytical techniques used.

Overestimation of the importance of phytate in NaOH–EDTA soil extracts as assessed by 31P NMR analyses

Solution 31P NMR analysis of NaOH–EDTA extracts is the most widely used method for determining the speciation of organic P in soils. In this paper, we bring together results from a number of our recent studies that highlight some of the difficulties in obtaining accurate quantitative results and propose that these difficulties may have resulted in the systematic overestimation of soil phytate concentrations. The first problem is peak identification, which is complicated by variations in chemical shift with pH and ionic strength, and the impracticality of the primary 31P chemical shift reference (85% H 3PO 4). Unambiguous peak assignment can be achieved by spiking suspected compounds directly into soil extracts prepared for NMR analysis; this ensures native and added compounds experience exactly the same chemical environment. The second problem is quantifying NMR signal for individual species in the crowded and overlapping phosphate monoester region. Spectral deconvolution has been employed for this purpose, but very different results are obtained depending on whether or not the fitting procedure includes a broad signal that probably comes from P in large, “humic” compounds. In particular, failing to consider this broad peak in the deconvolution procedure results in overestimation of small organic P compounds, including phytate. The ready decomposition of phytate is demonstrated with an incubation experiment in which phytate added at various concentrations to a calcareous soil is shown to be decomposed to produce orthophosphate. The decomposition follows a first order exponential decay with a half-life of 4–8 weeks.

Nonaqueous capillary electrophoresis‐electrospray ionization‐ion trap‐mass spectrometry analysis of pyrrolo‐ and pyrido[1,2‐a]azepine alkaloids in Stemona

Stemona alkaloids represent an outstanding class of natural compounds due to their pharmacological profile and their complex and unusual molecular structures. The aim of this study was the development of the first CE method for the separation, identification and quantification of these pyrrolo- and pyrido[1,2-a]azepine derivatives in three Stemona species. The best results were obtained with a NACE-ESI-IT-MS method, utilizing an electrolyte of 50 mM ammonium acetate, 1 M acetic acid and 10% methanol in ACN and a separation voltage of 30 kV. Samples were injected voltage-assisted with 20 kV for 1 s. Isopropanol:water (1:1) was used as ESI sheath liquid at a flow rate of 3 microL/min. The assay was applied for the qualitative profiling of Stemona alkaloids in S. curtisii, S. collinsae and S. tuberosa. For unambiguous peak assignment of more than forty unidentified alkaloids MS/MS experiments were performed and fragmentation patterns studied. Subsequently the method was validated for the quantitative determination of four selected derivatives (RSD inter- and intraday <6%, LODs <7.5 microg/mL, LOQs <25.0 microg/mL, for all analytes, recovery rates >98.9%) in several Stemona sp. extracts.