Determination of ion and neutral loss compositions and deconvolution of product ion mass spectra using an orthogonal acceleration time-of-flight mass spectrometer and an ion correlation program

Exact masses of monoisotopic ions, and the relative isotopic abundances (RIAs) of ions greater in mass by 1 and 2 Da than the monoisotopic ion, are independent and complementary physical properties useful for distinguishing among elemental compositions of ions possible for a given nominal mass. Using these properties to determine elemental compositions of product ions and neutral losses increases the masses of precursor ions for which unique compositions can be determined. Compositions of the precursor ion, product ion, and neutral loss aid mass spectral interpretation and guide modest chemical literature searches for candidate standards to be obtained for confirmation of tentative compound identifications. This approach is essential for compound characterization or identification due to the absence of commercial libraries of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) product ion spectra. For a series of 34 exact mass measurements, an orthogonal acceleration time-of-flight mass spectrometer provided 34 and 29 values accurate to within 2 and 1 mDa, respectively, for ions from eight simulated unknowns with [M+H](+) ion masses between 166 and 319 Da. Of 36 RIA measurements for +1 Da or +2 Da ions, 35 were accurate to within 20% of their predicted values (or to within 0.4 RIA % when the RIA value was less than 1%) in the absence of obvious interferences, in cases where the monoisotopic ion peak areas were at least 1.7 x 10(5) counts and the ion masses exceeded 141 Da. An ion correlation program (ICP) provided the unique and correct compositions for all but three of the 34 ions studied. Manual inspection of the data eliminated the incorrect compositions. To test the utility of the ICP for deconvoluting composite product ion spectra, all 34 ions were tested for correlation. Six of eight precursor ions were identified as such, while two were compositional subsets of others and were not properly identified. The six precursor ion compositions were still found by the ICP even though ions with masses less than 158 Da were not considered since they could no longer be correlated with a single precursor ion. Finally, two unidentified analytes were characterized, based on data published by others and using the ICP together with mass spectral interpretation.     

Using a triple-quadrupole mass spectrometer in accurate mass mode and an ion correlation program to identify compounds

Atomic masses and isotopic abundances are independent and complementary properties for discriminating among ion compositions. The number of possible ion compositions is greatly reduced by accurately measuring exact masses of monoisotopic ions and the relative isotopic abundances (RIAs) of the ions greater in mass by +1 Da and +2 Da. When both properties are measured, a mass error limit of 6-10 mDa (< 31 ppm at 320 Da) and an RIA error limit of 10% are generally adequate for determining unique ion compositions for precursor and fragment ions produced from small molecules (less than 320 Da in this study). 'Inherent interferences', i.e., mass peaks seen in the product ion mass spectrum of the monoisotopic [M+H]+ ion of an analyte that are -2, -1, +1, or +2 Da different in mass from monoisotopic fragment ion masses, distort measured RIAs. This problem is overcome using an ion correlation program to compare the numbers of atoms of each element in a precursor ion to the sum of those in each fragment ion and its corresponding neutral loss. Synergy occurs when accurate measurement of only one pair of +1 Da and +2 Da RIAs for the precursor ion or a fragment ion rejects all but one possible ion composition for that ion, thereby indirectly rejecting all but one fragment ion-neutral loss combination for other exact masses. A triple-quadrupole mass spectrometer with accurate mass capability, using atmospheric pressure chemical ionization (APCI), was used to measure masses and RIAs of precursor and fragment ions. Nine chemicals were investigated as simulated unknowns. Mass accuracy and RIA accuracy were sufficient to determine unique compositions for all precursor ions and all but two of 40 fragment ions, and the two corresponding neutral losses. Interrogation of the chemical literature provided between one and three possible compounds for each of the nine analytes. This approach for identifying compounds compensates for the lack of commercial ESI and APCI mass spectral libraries, which precludes making tentative identifications based on spectral matches.     

Metabolic profiling of Escherichia coli by ion mobility-mass spectrometry with MALDI ion source

Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix-assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (<1500 Da) cause detection and/or identification of metabolites difficult by MS alone. However, ion mobility spectrometry (IMS) coupled with MS (IM-MS) provides a rapid analytical tool for measuring subtle structural differences in chemicals. IMS separates gas-phase ions based on their size-to-charge ratio. This study, for the first time, reports the application of MALDI to the measurement of small molecules in a biological matrix by ion mobility-time of flight mass spectrometry (IM-TOFMS) and demonstrates the advantage of ion-signal dispersion in the second dimension. Qualitative comparisons between metabolic profiling of the Escherichia coli metabolome by MALDI-TOFMS, MALDI-IM-TOFMS and electrospray ionization (ESI)-IM-TOFMS are reported. Results demonstrate that mobility separation prior to mass analysis increases peak-capacity through added dimensionality in measurement. Mobility separation also allows detection of metabolites in the matrix-ion dominated low-mass range (m/z < 1500 Da) by separating matrix signals from non-matrix signals in mobility space.     

Mass analysis of overlapping ion kinetic energy peaks arising from charge separation in dications

A triple-sector instrument of reversed geometry, BEQQ, has been employed to resolve overlapping ion kinetic energy peaks arising from charge separation of metastable dications. Separation was achieved through mass resolution of the dication in the magnetic sector and of the singly charged product ion of greater mass in the quadrupole mass filter accompanied by energy resolution with the electric sector; the electric sector was scanned to cover the complete range of each charge separation peak. Two overlapping ion kinetic energy peaks arising from charge separation of the diphenylenimine dication and up to four overlapping ion kinetic peaks arising from charge separations of dications arising from benzene-1,3,5-d₃ have been resolved. The kinetic energy releases have been calculated in each case. Enhanced z-discrimination is observed with the final stage of mass analysis.     

Identification of isobaric product ions in electrospray ionization mass spectra of fentanyl using multistage mass spectrometry and deuterium labeling

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N‐phenylpropanamide. 1‐(2‐Phenylethyl)‐1,2,3,6‐tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS³) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium‐labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Discrimination of isomers and isobars by varying the reduced-field across drift tube in proton-transfer-reaction mass spectrometry (PTR-MS)

Proton-transfer-reaction mass spectrometry (PTR-MS) is a powerful technique for the real time trace gas analysis of volatile organic compounds (VOCs). However, quadrupole mass spectrometer (MS) used in PTR-MS has a relatively low mass resolution and is therefore not suitable for differentiating isobars. Furthermore, because of the lack of chemical separation before analysis, isomers can not be identified, either. In the present study, by varying the reduced-field E/N in the reaction chamber with a range of 50–180 Td in PTR-MS, we studied the product ion distribution (PID) of three sets of isobars/isomers, i.e. n-propanol/iso-propanol/acetic acid, propanal/acetone and four structural isomers of butyl alcohol. The profiles of the reduced-field dependence (PFD) of the PID under the chosen E/N-values show obvious differences which can be used to discriminate between these isobars/isomers thus enabling the titled method. Noticeably, we have observed that even the isomers, in the case of four structural isomers of butyl alcohol, which show little difference with each other at high reduced-field, can be discriminated easily at low reduced-field. Finally, two examples for the application of this method are discussed: (1) cyclohexanone was identified to be a major compound in the headspace of medical infusion sets; and (2) the differentiation and quantification of propanal and acetone in three synthetic mixtures with different ratios. This study presents a potential method to distinguish and quantify isobars/isomers conveniently in practical applications of PTR-MS analysis without additional instrumental configurations.     

Enhancements in travelling wave ion mobility resolution

The use of ion mobility separation to determine the collision cross-section of a gas-phase ion can provide valuable structural information. The introduction of travelling-wave ion mobility within a quadrupole/time-of-flight mass spectrometer has afforded routine collision cross-section measurements to be performed on a range of ionic species differing in gas-phase size/structure and molecular weight at physiologically relevant concentrations. Herein we discuss the technical advances in the second-generation travelling-wave ion mobility separator, which result in up to a four-fold increase in mobility resolution. This improvement is demonstrated using two reverse peptides (mw 490 Da), small ruthenium-containing anticancer drugs (mw 427 Da), a cisplatin-modified protein (mw 8776 Da) and the noncovalent tetradecameric chaperone complex GroEL (mw 802 kDa). What is also shown are that the collision cross-sections determined using the second-generation mobility separator correlate well with the previous generation and theoretically derived values.     

Utility of three types of mass spectrometers for determining elemental compositions of ions formed from chromatographically separated compounds

Concentration factors of 1000 and more reveal dozens of compounds in extracts of water supplies. Library mass spectra for most of these compounds are not available, and alternative means of identification are needed. Determination of the elemental compositions of the ions in mass spectra makes feasible searches of commercial and chemical literature that often lead to compound identification. Instrumental capabilities that constrain the utility of a mass spectrometer for determining ion compositions for compounds that elute from a chromatographic column are scan speed, mass accuracy, linear dynamic range, and resolving power. Mass peak profiling from selected ion recording data (MPPSIRD) performed with a double-focusing mass spectrometer provides the best combination of these capabilities. This technique provides unique ion compositions for ions of higher mass from compounds eluting from a gas chromatograph than can be obtained by orthogonal acceleration time-of-flight (oa-TOF) or Fourier transform ion cyclotron resonance mass spectrometry. Multiple compositions are usually possible for an ion with a mass exceeding 150 Da within the error limits of the mass measurement. The correct composition is selected based on measured exact masses of the mass peak profiles resulting from isotopic ions higher in mass by 1 and 2 Da and accurate measurement of the summed abundances of these isotopic ions relative to the monoisotopic ion. A profile generation model (PGM) automatically determines which compositions are consistent with measured exact masses and relative abundances. The utility of oa-TOF and double-focusing mass spectrometry using ion composition elucidation (MPPSIRD plus the PGM) are considered for determining ion compositions of two compounds found in drinking water extracts and a third compound from a monitoring well at a landfill. Published in 2002 by John Wiley & Sons, Ltd.     

Exact-mass library for pesticides using a molecular-feature database

An automated molecular-feature database (MFD) consisting of the exact monoisotopic mass of 100 compounds, at least one exact mass product ion for each compound, and chromatographic retention time were used to identify pesticides in food and water samples. The MFD software compiles a list of accurate mass ions, excludes noise, and compares them with the monoisotopic exact masses in the database. The screening criteria consisted of +/-5 ppm accurate mass window, +/-0.2 min retention time window, and a minimum 1000 counts (signal-to-noise (S/N) ratio of approximately 10:1). The limit of detection for 100 tested compounds varied from <0.01 mg/kg for 72% of the compounds to <0.1 mg/kg for 95% of the compounds. The MFD search was useful for rapid screening and identification of pesticides in food and water, as shown in actual samples. The combined use of accurate mass and chromatographic retention time eliminated false positives in the automated analysis. The major weakness of the MFD is matrix interferences and loss of mass accuracy. Strengths of the MFD include rapid screening of 100 compounds at sensitive levels compared with a manual approach and the ease of use of the library for any accurate mass spectrometer instrumentation capable of routine sub-5-ppm mass accuracy.     

Programmable gate delayed ion mobility spectrometry-mass spectrometry: a study with low concentrations of dipropylene-glycol-monomethyl-ether in air

The concept of using a short ionisation event, in this case a pulsed corona discharge, in conjunction with programmed gate delay is described. This technique is proposed for the selective study of different ionisation processes within the reaction region of an ion mobility spectrometer. The utility of such an approach was tested in a study of the ionisation of dipropylene-glycol-monomethyl-ether (DPM); a compound commonly used to test the operation of ion mobility spectrometers. Dipropylene-glycol-monomethyl-ether at a concentration of 113 microg m(-3) in air, with a water level of 75 mg m(-3) in air, was analysed using a switchable, high resolution ion mobility spectrometer, operating in the positive mode at 40 degrees C at ambient pressure. The ion mobility spectrometer was fitted with a pulsed corona discharge ionisation source, doped with ammonia at a concentration of 1.3 mg m(-3) in the reaction region, and interfaced to a mass spectrometer. Synchronisation of the ionisation event to the operation of the shutter grids for the drift region enabled different parts of the product ion population to be injected into the drift tube, and programming the gate delays produced a map of the gate delay verses drift time response surface. Ammonium bound dipropylene-glycol-monomethyl-ether was observed, [(DPM)NH4]+ (m/z 166) as well as the ammonium bound dimer [(DPM)2NH4]+ (m/z 314), the same as those observed with a 63Ni source. Two other species were also observed, but their molecular identity was not elucidated. One of them m/z 146, also observed with 63Ni, formed ammonium bound ions [(m/z 146)NH4]+ (K0= 1.49 cm2 V(-1) s(-1)), ammonium bound dimer ions [(m/z 146)2NH4]+(K0= 1.18 cm2 V(-1) s(-1)) and a mixed cluster ion with DPM [(m/z 146)(DPM)NH4]+(K0= 1.18 cm2 V(-1) s(-1)); while the other, m/z 88 a decomposition product, formed ammonium bound monomer [(m/z 88)NH4]+(K0= 1.68 cm2 V(-1) s(-1)), dimer ions [(m/z 88)2NH4]+(K0= 1.40 cm2 V(-1) s(-1)) and a mixed cluster ion containing DPM and ammonium, [(DPM)(m/z 88)2NH4]+(K0= 1.40 cm2 V(-1) s(-1)). The assignment of responses to these ions required the additional dimensionality in the data provided from the gate delay studies. The relationships evident in the programmable gate delay data enabled these ions to be differentiated from alternative assignments of possible nitrogen clusters, formed at the interface of the mass spectrometer.     

Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks

A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix-based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix-based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x - 0.005, 0.024 micro mol l(-1), and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated.     

Automated determination of precursor ion, product ion, and neutral loss compositions and deconvolution of composite mass spectra using ion correlation based on exact masses and relative isotopic abundances

After an accidental, deliberate, or weather-related dispersion of chemicals (dispersive event), rapid determination of elemental compositions of ions in mass spectra is essential for tentatively identifying compounds. A direct analysis in real time (DART)ion source interfaced to a JEOL AccuTOFmass spectrometer provided exact masses accurate to within 2 mDa for most ions in full scan mass spectra and relative isotopic abundances (RIAs) accurate to within 15-20% for abundant isotopic ions. To speed determination of the correct composition for precursor ions and most product ions and neutral losses, a three-part software suite was developed. Starting with text files of m/z ratios and their ion abundances from mass spectra acquired at low, moderate, and high collision energies, the ion extraction program (IEP) compiled lists for the most abundant monoisotopic ions of their exact masses and the RIAs of the +1 and +2 isotopic peaks when abundance thresholds were met; precursor ions; and higher-mass, precursor-related species. The ion correlation program (ICP) determined if a precursor ion composition could yield a product ion and corresponding neutral loss compositions for each product ion in turn. The input and output program (IOP) provided the ICP with each precursor ion:product ion pair for multiple sets of error limits and prepared correlation lists for single or multiple precursor ions. The software determined the correct precursor ion compositions for 21 individual standards and for three- and seven-component mixtures. Partial deconvolution of composite mass spectra was achieved based on exact masses and RIAs, rather than on chromatography.     

Determination of daughter ion elemental compositions in charge separation reactions

It is shown that the elemental composition of a product ion generated from a multiply charged ion in a charge separation reaction can be determined with a double focusing instrument under conditions of high mass and energy resolution. Results for a number of charge separation reactions in benzylamine are briefly discussed.     

Optimization of the mass reflector parameters for direct ion extraction

The parameters of a mass reflector combined with direct ion extraction are optimized to reduce the time-of-flight spread caused by the initial velocity distribution of ions. Under the assumption that the initial ion velocity distribution is mass independent, one can find the mass range where the spread reaches its minimum by varying the electric field in the ion mirror. The mass dependence of this particular time-of-flight spread can be diminished when the electric field in the ion mirror changes with time. This was theoretically demonstrated for the mass region 5 x 10(3) < m < 10(5) Da, under conditions where the electric field in the mirror increases by approximately 20% during a time interval of several hundred microseconds. Copyright 2000 John Wiley & Sons, Ltd.     

Matrix-free high-resolution imaging mass spectrometry with high-energy ion projectiles

The importance of imaging mass spectrometry (MS) for visualizing the spatial distribution of molecular species in biological tissues and cells is growing. We have developed a new system for imaging MS using MeV ion beams, termed MeV-secondary ion mass spectrometry (MeV-SIMS) here, and demonstrated more than 1000-fold increase in molecular ion yield from a peptide sample (1154 Da), compared to keV ion irradiation. This significant enhancement of the molecular ion yield is attributed to electronic excitation induced in the near-surface region by the impact of high energy ions. In addition, the secondary ion efficiency for biologically important compounds (>1 kDa) increased to more than 10(10) cm(-2), demonstrating that the current technique could, in principle, achieve micrometer lateral resolution. In addition to MeV-SIMS, peptide compounds were also analyzed with cluster-SIMS and the results indicated that in the former method the molecular ion yields increased substantially compared to the latter. To assess the capability of MeV-SIMS to acquire heavy-ion images, we have prepared a micropatterned peptide surface and successfully obtained mass spectrometric imaging of the deprotonated peptides (m/z 1153) without any matrix enhancement. The results obtained in this study indicate that the MeV-SIMS technique can be a powerful tool for high-resolution imaging in the mass range from 100 to over 1000 Da.     

Isotope depletion of large biomolecules: Implications for molecular mass measurements

Isotope depletion (or enrichment) of large biomolecules is a procedure already used in high resolution Fourier transform ion cyclotron resonance mass spectrometry for improving the reliability and accuracy of biomolecular mass characterization. In this work, effects of isotope depletion on a number of mass spectrometric parameters are systematically studied. Implementation of the isotope depletion techniques in conjunction with lower resolution mass analyzers is discussed as well. We investigate theoretically the position of the centroid of the isotopic mass distributions (centroid mass) and the shift between the monoisotopic and the centroid masses of biopolymers as a function of the isotope abundance (e.g., ¹²C:¹³C ratio). The behaviour of other additive mass parameters, like the ratio between the monoisotopic and the first isotopic peak, is also discussed. We address by computer simulations the effects of different instrumental parameters like mass resolution and ion statistics as a function of isotope abundances and from there the achievable mass accuracy for high-mass biopolymers. We assess some of the practical issues of the isotope depletion technique, viz., to what degree and with what accuracy the depletion procedure should be performed for achieving the desired mass accuracy.     

Development of a novel mass spectrometer equipped with an electron cyclotron resonance ion source

The ionization efficiency of an electron cyclotron resonance ion source (ECRIS) is generally high, and all elements can be fundamentally ionized by the high-temperature plasma. We focused our attention on the high potentiality of ECRIS as an ion source for mass spectrometers and attempted to customize the mass spectrometer equipped with an ECRIS. Precise measurements were performed by using an ECRIS that was specialized and customized for elemental analysis. By using the charge-state distribution and the isotope ratio, the problem of overlap such as that observed in the spectra of isobars could be solved without any significant improvement in the mass resolution. When the isotope anomaly (or serious mass discrimination effect) was not observed in ECR plasma, the system was found to be very effective for isotope analysis. In this paper, based on the spectrum (ion current as a function of an analyzing magnet current) results of low charged state distributions (2+, 3+, 4+, ...) of noble gases, we discuss the feasibility of an elemental analysis system employing an ECRIS, particularly for isotopic analysis. The high-performance isotopic analysis obtained for ECRIS mass spectrometer in this study suggests that it can be widely applied to several fields of scientific study that require elemental or isotopic analyses with high sensitivity.     

Mass and charge assignment for electrospray ions by cation adduction

The assignment of the mass (m) value from the m/z value for ions with a multiple number of charges (z) in electrospray mass spectra usually utilizes multiple peaks of the same m but different z values, or unit-mass—separated isotopic peaks of the same z value from high resolution spectra. The latter approach is also feasible with much less resolving power using adduct ions of much higher mass separation. The application of this to mixture spectra containing many masses, such as spectra from tandem mass spectrometry (MS/MS) ion dissociation, does not appear to have been pointed out previously. Thus, replacing two protons by one Cu²⁺ ion increases the mass by 61.5 Da, with this shift providing a mass scale for assignment of m and z from this pair of m/z values. The more common Na⁺ adduct peaks provide a 22.0 Da separation, of utility for 1000 resolving power only below approximately 10 kDa. Further, collisional dissociation lowers the degree of Cu²⁺ adduction in the resulting sequence-specific fragment ions much less than that of the corresponding Na⁺ adducts, making the Cu²⁺ adducts far more useful for m and z determination in MS/MS studies.     

Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning

An API 3000 triple-quadrupole instrument and a QSTAR Pulsar quadrupole time-of-flight (TOF) mass spectrometer were compared for the determination of phosphopeptides by precursor ion scanning in both the positive and negative nanoelectrospray ionization modes. The limits of detection for synthetic phosphopeptides were similar (500 amol microl(-1)) for both types of instruments when monitoring precursors of -79 Da (PO(3)(-)). However, the quadrupole TOF system was approximately fivefold more sensitive (1 fmol microl(-1)) than the triple-quadrupole instrument (5 fmol microl(-1)) when monitoring precursors of 216 Da (immonium ion of phosphotyrosine). The recently introduced Q(2)-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF part, improved the sensitivity of precursor ion scans on a quadrupole TOF instrument. The selectivity of precursor ion scans is much better on quadrupole TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection at no expense of sensitivity. This minimizes interferences from other peptide fragment ions (a-, b-, and y- type) of the same nominal mass but with sufficient differences in their exact masses. As a result, the characteristic immonium ion of phosphotyrosine at m/z 216.043 can be utilized for the selective detection of tyrosine phosphorylated peptides. Our data suggest that, in addition to their superior performance for peptide sequencing, quadrupole TOF instruments also offer a very viable alternative to triple quadrupoles for precursor ion scanning, thus combining high sensitivity and selectivity for both MS and MS/MS experiments in one instrument.     

Rapid screening method for intact glucosinolates in Chinese medicinal herbs by using liquid chromatography coupled with electrospray ionization ion trap mass spectrometry in negative ion mode

An optimized method using liquid chromatography coupled with electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) in negative ion mode has been developed for screening different structural classes of intact glucosinolates in six Chinese medicinal herbs. The glucosinolates were extracted with hot methanol/water (70:30 v/v) and separation of the individual glucosinolates was achieved using a reversed-phase C18 column with an aqueous ammonium acetate/methanol gradient. Identification of the intact glucosinolates was based on the detection of compounds with a constant neutral loss of 242 Da corresponding to the combined loss of anhydroglucose (162 Da) and sulfur trioxide (80 Da) in collision-induced dissociation. The structures of the identified glucosinolates were confirmed with the use of group-specific product ions at m/z 195, 241, 259, 275 in their corresponding MS/MS product ion spectra. Differentiation of intact glucosinolates was achieved through their respective retention times and molecular masses as well as the characteristic product ions. The limits of detection were at the low nanogram level per injection, based on constant neutral loss scans. Significant variation in the compositions of intact glucosinolates was identified in the cruciferous herbs. This method was applied in the differentiation and quality control of two pairs of easily confused herbs. Copyright (c) 2008 John Wiley & Sons, Ltd.