On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.     

Instrument dependence of electrospray ionization and tandem mass spectrometric fragmentation of the gingerols

The gingerols, including [6]-, [8]-, and [10]-gingerols, a series of chemical homologs differentiated by the length of their unbranched alkyl chains, have been identified as major active components in fresh ginger rhizome. The purpose of this study was to investigate the utility of ion trap liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an online tool to identify and quantify these compounds in raw or processed ginger rhizome samples. Negative mode electrospray ionization (ESI) was used in MS, MS/MS and MS(n) experiments in quadrupole ion trap instruments from two different manufacturers and in high-resolution and accurate mass MS and MS/MS experiments in a Fourier transform ion cyclotron resonance mass spectrometer to elucidate the ionization and fragmentation mechanisms of these compounds in these instruments. Positive mode ESI, which generated many more fragment ions in full scan MS even under gentle ionization conditions, was also used in LC/MS and MS/MS experiments and in direct infusion MS and MS/MS experiments. Consistent and predictable ionization and fragmentation behaviors were observed for all gingerols when analyzed in the same instrument. Instruments from different manufacturers, however, had different ionization mechanisms. The major difference between instruments was their ability to form covalent dimer adducts of the gingerols. Subsequent fragmentation patterns of the precursor ions were essentially identical. These results clearly demonstrate that LC/MS instruments produce data that cannot necessarily be replicated in other laboratories, especially if those laboratories do not have the same instrument model from the same manufacturer. This presents major problems for metabolite target analysis, metabolic profiling and metabolomics investigations, which would benefit from LC/MS mass spectrum libraries as they do from GC/MS mass spectrum libraries, because such libraries may not be valid across platforms.     

Metabonomics investigation of human urine after ingestion of green tea with gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and (1)H NMR spectroscopy

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose metabolism, citric acid cycle and amino acid metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants.     

Parallel high-throughput accurate mass measurement using a nine-channel multiplexed electrospray liquid chromatography ultraviolet time-of-flight mass spectrometry system

A nine-channel multiplexed electrospray (MUX) liquid chromatography ultraviolet time-of-flight mass spectrometry (LC/UV/TOFMS) system has been used to simultaneously measure accurate masses of eluting components from eight parallel gradient LC columns. Accuracies better than 5 and 10 ppm were achieved for 50 and 80% of samples, respectively, from a single batch analysis of ten plates (960 samples) of a Fmoc-Asp(OtBu)-OH and reserpine mixture. Combinatorial library compounds were analyzed using this parallel high-throughput system in both positive and negative modes to rigorously verify expected products and identify side products. A mass accuracy of 10 ppm root mean square (RMS) is routinely obtained for combinatorial library samples from this high-throughput accurate mass LC/MS system followed by automated data processing. This mass accuracy is critical in revealing combinatorial synthesis problems that would be missed by unit mass measurement.     

Characterization by mass spectrometry of an unknown polysiloxane sample used under uncontrolled medical conditions for cosmetic surgery

For a complete understanding of the raw material used for cosmetic surgery under uncontrolled medical conditions, an unknown sample of polydimethylsiloxanes has been investigated utilizing a combination of analytical techniques: pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS), electrospray ionization (ESI)-MS, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS, and liquid chromatography (LC)/MS. Among these techniques, the LC/APCI-MS coupling allowed the fastest and more effective analysis. In addition, the complexity of the mass spectra deduced from these LC/MS experiments was simplified compared to the mass spectra obtained by MALDI-TOF. In this work, we have demonstrated how the LC/APCI-MS coupling applied to polydimethylsiloxane samples permits the full characterization of samples where end groups of different nature can be present in very small quantities.     

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

LC coupled to single (LC–MS) and tandem (LC–MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC–MS/MS. Analyses were performed using a previously published LC–MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT–tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their “enhanced” product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.     

A strategy for a post-method-validation use of incurred biological samples for establishing the acceptability of a liquid chromatography/tandem mass-spectrometric method for quantitation of drugs in biological samples

Validated liquid chromatography/tandem mass spectrometric (LC/MS/MS) methods are now widely used for quantitation of drugs in post-dose (incurred) biological samples for the assessment of pharmacokinetic parameters, bioavailability and bioequivalence. In accordance with the practice currently accepted within the pharmaceutical industry and the regulatory bodies, validation of a bioanalytical LC/MS/MS method is performed using standards and quality control (QC) samples prepared by spiking the drug (the analyte) into the appropriate blank biological matrix (e.g. human plasma). The method is then declared to be adequately validated for analyzing incurred biological samples. However, unlike QC samples, incurred samples may contain an epimer or another type of isomer of the drug, such as a Z or E isomer. Such a metabolite will obviously interfere with the selected reaction monitoring (SRM) transition used for the quantitation of the drug. The incurred sample may also contain a non-isomeric metabolite having a molecular mass different from that of the drug (such an acylglucuronide metabolite) that can still contribute to (and hence interfere with) the SRM transition used for the quantitation of the drug. The potential for the SRM interference increases with the use of LC/MS/MS bioanalytical methods with very short run times (e.g. 0.5 min). In addition, a metabolite can potentially undergo degradation or conversion to revert back to the drug during the multiple steps of sample preparation that precede the introduction of the processed sample into the LC/MS/MS system. In this paper, we recommend a set of procedures to undertake with incurred samples, as soon as such samples are available, in order to establish the validity of an LC/MS/MS method for analyzing real-life samples. First, it is recommended that the stability of incurred samples be investigated 'as is' and after sample preparation. Second, it is recommended that potential SRM interference be investigated by analyzing the incurred samples using the same LC/MS/MS method but with the additional incorporation of the SRM transitions attributable to putative metabolites (multi-SRM method). The metabolites monitored will depend on the expected metabolic products of the drug, which are predictable based on the functional groups present in the chemical structure of the drug. Third, it is recommended that potential SRM interference be further investigated by analyzing the incurred samples using the multi-SRM LC/MS/MS method following the modification of chromatographic conditions to enhance chromatographic separation of the drug from any putative metabolites. We will demonstrate the application of the proposed strategy by using a carboxylic acid containing drug candidate and its acylglucuronide as a putative metabolite. Plasma samples from the first-in-man (FIM) study of the drug candidate were used as the incurred samples.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Automated liquid chromatographic/tandem mass spectrometric method for screening beta-blocking drugs in urine

An automated liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method is presented for the screening and confirmation of 16 beta-blocking drugs in clinical and autopsy urine samples. The described method involved C(18) solid phase extraction, LC separation and MS analysis on a triple-stage quadrupole mass analyser. Samples were initially pre-screened for the presence of any beta-blocking drugs using LC/MS with selected ion monitoring. Any compounds tentatively identified as beta-blocking drugs on the basis of their LC retention time and protonated molecular ion were then automatedly subjected to a second analysis in which the relevant MS/MS product ion mass spectra were acquired. These product ion mass spectra were then automatically searched against a 400-substance mass spectral library containing previously acquired beta-blocking drugs. The results demonstrated that library search of beta-blocking drugs in urine with MS/MS product ion mass spectra was more reliable and produced fewer false negatives than library searching with mass spectra derived from single-stage quadrupole MS. The limits of identification in the MS/MS product ion scan ranged from 0.02 mg l(-1) for carvedilol to 1.2 mg l(-1) for pindolol, the majority of the values being below 0.2 mg l(-1).     

Qualitative and quantitative evaluation of Oroxylum indicum (L.) Kurz by HPLC and LC‐qTOF‐MS/MS

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples.     

Detection and identification of drugs and toxicants in human body fluids by liquid chromatography–tandem mass spectrometry under data-dependent acquisition control and automated database search

Systematic toxicological analysis (STA) is aimed at detecting and identifying all substances of toxicological relevance (i.e. drugs, drugs of abuse, poisons and/or their metabolites) in biological material. Particularly, gas chromatography–mass spectrometry (GC/MS) represents a competent and commonly applied screening and confirmation tool. Herein, we present an untargeted liquid chromatography–tandem mass spectrometry (LC/MS/MS) assay aimed to complement existing GC/MS screening for the detection and identification of drugs in blood, plasma and urine samples. Solid-phase extraction was accomplished on mixed-mode cartridges. LC was based on gradient elution in a miniaturized C18 column. High resolution electrospray ionization-MS/MS in positive ion mode with data-dependent acquisition control was used to generate tandem mass spectral information that enabled compound identification via automated library search in the “Wiley Registry of Tandem Mass Spectral Data, MSforID”. Fitness of the developed LC/MS/MS method for application in STA in terms of selectivity, detection capability and reliability of identification (sensitivity/specificity) was demonstrated with blank samples, certified reference materials, proficiency test samples, and authentic casework samples.     

UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation

A new approach to obtain fragmentation information in liquid chromatography/mass spectrometry (LC/MS) studies of small molecules in complex mixtures is presented using simultaneous acquisition of exact mass at high and low collision energy, MS(E). LC/MS-TOF and LC/MS/MS-TOF are powerful tools for the analysis of complex mixtures, especially those for biological fluids allowing the elucidation of elemental composition and fragmentation information. In this example the composition of rat urine was studied using this new approach, allowing the structures of several endogenous components to be confirmed in one analytical run by the simultaneous acquisition of exact mass precursor and fragment ion data. The spectral data obtained using this new approach are comparable to those obtained by conventional LC/MS/MS as exemplified by the identification of endogenous metabolites present in rat urine.     

Investigation of unknown related substances in commercial erythromycin samples with liquid chromatography/mass spectrometry

A selective reversed phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of erythromycin impurities and related substances in commercial erythromycin samples. Mass spectral data are acquired on a LCQ ion trap mass spectrometer equipped with an electrospray interface operated in positive ion mode. The LCQ is ideally suited for identification of impurities and related substances because it provides on-line LC/MS(n) capability. Compared with UV detection, this hyphenated LC/MS(n) technique provides as a main advantage efficient identification of novel substances without time-consuming isolation and purification procedures. Using this method four novel related substances were identified in commercial samples.     

Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3)

The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step. In this paper, we explored the advantages of a combination of FFE and liquid chromatography/mass spectrometry to extend the dynamic range of a proteomic analysis of a complex cell lysate. In this study, the whole cell lysate of a chronic myelogeneous leukemia cell line, K562/CR3, was prefractionated by FFE into 96 fractions spanning pH 3-12. Of these, 35 fractions were digested with trypsin and then analyzed by LC/MS. Depending on the algorithm used for peptide assignment from MS/MS data, at least 319 proteins were identified through database searches. The results also suggested that pI could serve as an additional criterion besides peptide fragmentation pattern for protein identification, although in some cases, a pI shift might indicate post-translational modification. In summary, this study demonstrated that free flow electrophoresis provided a useful prefractionation step for proteomic analysis and when combined with LC/MS allowed the identification of significant number of low level proteins in complex samples.     

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/mass spectrometry libraries

Liquid chromatography/ultraviolet (LC/UV) and mass spectrometry/mass spectrometry (MS/MS) libraries containing 39 phenolic compounds were established by coupling a LC and an ion trap MS with an electrospray ionization (ESI) source, operated in negative ion mode. As a result, the deprotonated [M-H]- molecule was observed for all the analyzed compounds. Using MS/MS hydroxybenzoic acid and hydroxycinnamic acids showed a loss of CO2 and production of a [M-H-44]- fragment and as expected, the UV spectra of these two compounds were affected by their chemical structures. For flavonol and flavonol glycosides, the spectra of their glycosides and aglycones produced deprotonated [M-H]- and [A-H]- species, respectively, and their UV spectra each presented two major absorption peaks. The UV spectra and MS/MS data of flavan-3-ols and stilbenes were also investigated. Using the optimized LC/MS/MS analytical conditions, the phenolic extracts from six representative wine samples were analyzed and 31 phenolic compounds were detected, 26 of which were identified by searching the LC/UV and MS/MS libraries. Finally, the presence of phenolic compounds was confirmed in different wine samples using the LC/UV and LC/MS/MS libraries.     

Improving peptide identification using an empirical peptide retention time database

Peptide retention time (RT) is independent of tandem mass spectrometry (MS/MS) parameters and can be combined with MS/MS information to enhance peptide identification. In this paper, we utilized peptide empirical RT and MS/MS for peptide identification. This new approach resulted in the construction of an Empirical Peptide Retention Time Database (EPRTD) based on peptides showing a false‐positive rate (FPR) ≤1%, detected in several liquid chromatography (LC)/MS/MS analyses. In subsequent experiments, the RT of peptides with FPR >1% was compared with empirical data derived from the EPRTD. If the experimental RT was within a specified time range of the empirical value, the corresponding MS/MS spectra were accepted as positive. Application of the EPRTD approach to simple samples (known protein mixtures) and complex samples (human urinary proteome) revealed that this method could significantly enhance peptide identification without compromising the associated confidence levels. Further analysis indicated that the EPRTD approach could improve low‐abundance peptides and with the expansion of the EPRTD the number of peptide identifications will be increased. This approach is suitable for large‐scale clinical proteomics research, in which tens of LC/MS/MS analyses are run for different samples with similar components. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

Investigation of the identification point system adaptation in cocaine,benzoylecgonine and ecgonine methyl ester using a single quadrupole mass spectrometer

At present, no official criteria exist for drug identification using single quadrupole mass spectrometers although the European Union (EU) criteria for compound identification have been adopted. These criteria are evaluated with respect to the confirmation of cocaine and its metabolites by single quadrupole liquid chromatography/mass spectrometry (LC/MS) and problems are highlighted. Spiked samples, proficiency testing samples, certified reference materials and samples from real cases that had screened positive for cocaine derivatives by immunoassay were subjected to confirmation by LC/MS using single ion monitoring with in‐source fragmentation. The EU criteria for compound identification were applied for the confirmation of cocaine, benzoylecgonine and ecgonine methyl ester. The use of the identification point (IP) system in spiked, proficiency testing samples and certified reference materials provided acceptable results in all cases while in some cases real positive samples did not provide acceptable results. Failure to meet the EU criteria was attributed to low fragmentation at the lower concentrations and the ion suppression effect while both factors affected compliance with the IP system. The identification of cocaine and its metabolites was considerably improved by using a combination of ammonium formate and formic acid as the LC mobile phase. It appears that poor in‐source fragmentation in single quadrupole LC/MS and ion suppression may constitute a problem with drug identification when implementing the IP system in real samples, resulting in false negative results. Further investigation is needed for the use of such IP systems to be suitable for use in LC/MS methods. Copyright © 2009 John Wiley & Sons, Ltd.