Matrix‐enhanced secondary ion mass spectrometry: the influence of MALDI matrices on molecular ion yields of thin organic films

In this work the effect in secondary ion mass spectrometry (SIMS) of several frequently used matrix‐assisted laser desorption/ionisation (MALDI) matrices on the secondary ion intensities of low molecular weight (m/z 400–800) organic dyes and a pharmaceutical is tested. Matrix (10^?1 M) and analyte (10^?2 M) solutions were made in methanol. Mixtures with several concentration ratios were prepared from these solutions and spincoated on Si substrates prior to time‐of‐flight (TOF)‐SIMS analysis. In some cases the presence of the MALDI matrices caused a considerable increase in the positive secondary (protonated) molecular ion signals. Enhancements of a factor of 20 and more were recorded. Generally, of the matrices used, 2,5‐dihydroxybenzoic acid and 2,4,6‐trihydroxyacetophenone brought about the highest intensity increases. It was also shown that matrix‐enhanced (ME‐)SIMS is capable of lowering the detection limits for molecule ions. However, the enhancement effect is strongly influenced by the analyte/matrix combination and its concentration ratio. As a result, finding an optimal analyte/matrix mixture can be a very time‐consuming process. Mostly, the presence of the matrices causes changes in the relative ion intensities in the TOF‐S‐SIMS spectra. Compared to the spectra recorded from samples without matrices, only a few additional peaks, such as signals that originate directly from the applied matrix or adduct ions, are observed in the mass spectra. Sometimes molecule ions and some characteristic fragments at high m/z values, that cannot be recorded without matrix, do appear in the spectrum when a matrix is present. In the negative mode no enhancement effect is observed on applying the studied MALDI matrices. The results obtained from samples treated with MALDI matrices are also compared to SIMS results for the same samples after Ag and Au metallisation (MetA‐SIMS). For three of the four tested compounds Au MetA‐SIMS resulted in higher ion yields than ME‐SIMS. For both techniques possible mechanisms that can account for the enhancement effect are proposed. Copyright © 2005 John Wiley & Sons, Ltd.     

Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and <Superscript>18</Superscript>O-labeling

Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides (m/z 341) with 1–2, 1–4, and 1–6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1–3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1–3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. ¹⁸O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the “sidedness” of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing ¹⁸O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1–3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without ¹⁸O-labeling of the reducing sugar carbonyl group.     

Quantification of sofosbuvir in human serum by liquid chromatography with negative ionization mass spectrometry using the parent peak and its source‐induced fragment: Application to a bioequivalence study

In the mass spectrometry of sofosbuvir, a new orally administered antihepatitis C drug, a weak peak is detected at the m/z value of the parent ion (m/z 530) as a result of in‐source dissociation and current methods to its quantification, is based on monitoring of the parent peak using ultra high‐performance liquid chromatography with tandem mass spectrometry. With these methods serum concentration of the drug is quantifiable only up to 4–5 h postdose. However, the fragmentation of the molecule generates a more stable ion at m/z 287 (base peak) with a signal intensity of about tenfold compared to the parent ion. Our study was aimed to improve sensitivity of analysis by acquisition of the m/z value of the daughter ion from which it originated instead of the parent molecule. This novelty allows us to measure serum concentrations of the drug for a longer time postdose and provides more opportunity for pharmacokinetic studies of sofosbuvir. Our method was linear over the concentration range of 2–2560 ng/mL of sofosbuvir in human serum with a limit of quantification of 2 ng/mL compared to 10 ng/mL reported previously. The coefficient variation values of both inter and intraday analysis were less than 13.8%, and the percentage error was less than 6.3.     

Gas chromatographic mass spectrometric indication of double bond position in monounsaturated primary acetates and alcohols without derivatization

Electron impact mass spectra of straight chain, monounsaturated C₁₂ to C₁₈ acetates and alcohols have been shown to be useful in the indication of the double bond position on an empirical basis. The ratio of intensities of the m/z 55 and m/z 54 ions increases proportionately as the double bond is moved away from the acetate or alcohol functional group. The intensity of the m/z 61 ion also can be correlated with the double bond position. Results are generally accurate to ±1 carbon atom.     

The major product ion of S‐adenosyl‐L‐methionine arises from a neighbouring group reaction

Previous studies have shown that low‐energy collision‐induced dissociation (CID) of the important sulfonium ion metabolite S‐adenosyl‐L‐methionine (AdoMet, m/z 399) yields five main product ions: an ion at m/z 250 arising from methionine loss; ions at m/z 102 and 298, which arise via cleavage of the γ C? S bond of methionine; and ions at m/z 136 and 264, which arise via loss of protonated and neutral adenine, respectively. These metabolomics studies have, however, either totally ignored the mechanisms that govern the formation of the major product ion at m/z 250 (Gellekink H, van Oppenraaij‐Emmerzaal D, van Rooij A, Struys EA, den Heijer M, Blom HJ. Clin. Chem. 2005; 51: 1487), or have proposed an oxonium ion structure that must arise via a rearrangement involving a 1,2 hydride shift (Cataldi TRI, Bianco G, Abate S, Mattia D. Rapid Commun. Mass Spectrom. 2009; 23: 3465). Here DFT calculations on a model system are used to examine potential mechanisms for the formation of the major product ion of AdoMet. These calculations suggest that a neighbouring group mechanism is preferred over a 1,2 hydride shift mechanism. Copyright © 2010 John Wiley & Sons, Ltd.     

Ion-Neutral complexes from protonated propyl phenyl ethers. Gas-phase solvolysis versus elimination-readdition

Ion-neutral complexes, well attested as intermediates in the expulsion of alkenes from M⁺? and MH⁺ ions from primary alkyl phenyl ethers, are shown to intervene in the decomposition of the MH⁺ ion of a secondary alkyl phenyl ether, (CD₃)₂CHOPh. Chemical ionization (CI) (methane reagent gas)-mass-analysed ion kinetic energy spectroscopy (MIKES) shows ions of both m/z 96 and 97, indicating that the proton deposited by the CI reagent exchanges with the methyl deuterium atoms. The ratio of daughter ion intensities, as well as the proportions of ions of m/z 95, 96 and 97 from the MH⁺ of CD₃CH₂CD₂OPh, agree with predictions based on the gas-phase solvolysis mechanism, in which [i-Pr⁺ PhOH] complexes form from the protonated parent via simple bond heterolysis. An alternative mechanism, elimination-readdition, would proceed via [propene PhOHD⁺] complexes. This latter mechanism predicts a ratio of daughter ion intensities that is very different from gas-phase solvolysis and which disagrees with experiment. The elimination-readdition pathway is effectively ruled out, while the gas-phase solvolysis mechanism is reinforced.     

Lipid analysis of flat-mounted eye tissue by imaging mass spectrometry with identification of contaminants in preservation

Matrix-assisted laser desorption/ionization imaging mass spectrometry was used to analyze donor eye tissue specimens for phospholipid content to evaluate lipid distribution. Phosphatidylcholines and sphingomyelins were detected in the positive ion mode using 2,5-dihydroxybenzoic acid as the matrix. During this study, unknown ion signals in the lower m/z region (less than m/z 400) were detected, mainly in the far periphery of human flat-mounted tissue but not in age-matched rhesus monkey tissue prepared in a similar manner. The unknown ion signals occurred at m/z 304, 332, 360, and 388. These ions were subjected to tandem mass spectrometry directly from the tissue sample, and exact mass measurements of extracts were prepared for further identification. These ions were identified as alkyl dimethylbenzylammonium surfactants (benzalkonium chlorides (BACs)). The classification of these species was verified by comparing an eye tissue extract to an over-the-counter eye-care product containing BACs.     

Thermal degradation behavior of a carboxylic acid-terminated amphiphilic block copolymer poly(ethylene)-b-poly(ethylene oxide)

The thermal degradation of an amphiphilic block copolymer poly(ethylene)-b-poly(ethylene oxide)-carboxylic acid terminated (PE-b-80%PEO–CH₂COOH) and its salt obtained as intermediary product from chemical oxidation of the end group of poly(ethylene)-b-poly(ethylene oxide) (PE-b-80%PEO) has been studied using a thermogravimetric mass spectrometry (TG/MS) coupled system. The isothermal fragmentation of PE-b-80%PEO–CH₂COOH showed a more complex fragmentation pattern than PE-b-80%PEO owing to the simultaneous occurrence of the polyether block and the carboxylic end group fragmentations. This led to the appearance of four overlapping ion current peaks of fragments with m/z 44 and two peaks relative to m/z 18 at different times by acid-terminated copolymer. For the PE-b-80%PEO copolymer, two ion current peaks associated to m/z 44 and one large peak relative to m/z 18 fragments were detected. The intermediary product (PE-b-80%PEO–CH₂COO⁻ K⁺) showed differences related to the fragmentation behavior. It has more defined ion current signals and presented characteristic peaks attributed to m/z 43 fragment at the very beginning of the thermal degradation process, which it not detected in the acid copolymer.     

Endogenous high‐performance liquid chromatography/tandem mass spectrometry interferences and the case of perfluorohexane sulfonate (PFHxS) in human serum; are we overestimating exposure?

Perfluorinated acids have received increasing scientific attention due to their widespread global distribution, environmental persistence and bioaccumulation in wildlife and humans. For perfluorohexane sulfonate (PFHxS, C₆F₁₃SO, m/z 399), all existing human data have been generated using high‐performance liquid chromatography (HPLC) and its most sensitive tandem mass spectrometric (MS/MS) transitions (m/z 399/80 [SO₃]^? or m/z 399/99 [SO₃F]^?), but this may be problematic because of co‐eluting endogenous steroid sulfates that share common fragmentation pathways. We examined the magnitude of over‐reporting for PFHxS in pregnant women (n = 29), and in pooled serum of males, non‐pregnant and pregnant females (n = 3, 100 samples per pool), by comparing m/z 399/80 and 399/99 data with an interference‐free transition, m/z 399/119. PFHxS concentrations in pregnant women determined using m/z 399/80 and 399/99 (p < 0.05), but not m/z 399/119, were positively correlated to the response of the steroid sulfates. This led to an average overestimation of PFHxS by 1.5‐ and 4.7‐fold, using m/z 399/80 and 399/99, respectively, and validated the use of m/z 399/119 for the first time. The interferences were a problem in all human serum samples, and analysis of pooled serum revealed statistically significant over‐reporting by m/z 399/80 and 399/99 for pregnant women > non‐pregnant women > men. The magnitude of over‐reporting here represents a worst‐case scenario, but the extent to which the published literature values are biased is unknown due to limited details of methods in existing reports. Instead of using the less sensitive m/z 399/119 transition, we showed that an alternative selection of column and mobile phase can allow for sufficient chromatographic separation of the interferences. In conclusion, it was shown that routine analytical methods are prone to systematically overestimating PFHxS concentrations in serum of men or women, but that this can be avoided by alternative chromatographic steps or MS/MS transitions. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of isomeric 5‐hydroxytryptophan‐ and oxindolylalanine‐containing peptides by mass spectrometry

Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5‐hydroxytryptophan (5‐HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2‐nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5‐HTP residues. The mass shift of 152.98 m/z units allowed identifying 5‐HTP‐ besides Trp‐containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia‐ and 5‐HTP‐derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y₁ ions at m/z 175.12 derived from peptides with C‐terminal arginine residues. The pseudo MS³ spectra acquired on a quadrupole time‐of‐flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia‐containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5‐HTP‐cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification. Copyright © 2012 John Wiley & Sons, Ltd.     

The role of self-protonation under direct GC-MS determination of amphetamine hydrochloride

Summary We studied the possibility of direct GC-MS determination of amphetamine hydrochloride. In order to avoid the double peak we determined the optimal injector temperature (260°C) and heating rate (40°C min⁻¹). In the direct analysis of amphetamine hydrochloride containing samples, calibration for fragmentm/z=59 (originating from amphetaminium ion fragmentation; the calibrating agent is amphetamine hydrochloride) gave better results than calibration form/z=44 (derived from both free amphetamine and amphetaminium ion; calibration agents were amphetamine or amphetamine hydrochloride). We also compared the mass spectra of amphetamine and amphetamine hydrochloride taken by ion-trap GC-MS. The fragmentation path was cleavage of the C-C bond at benzyl position for the free base or between the phenyl group and the aliphatic moiety, in the case of free base and amphetaminium ion, respectively.     

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of fenofibric acid in rat plasma

A rapid and sensitive LC‐MS/MS method for the quantification of fenofibric acid in rat plasma was developed and validated. Plasma samples were prepared by liquid–liquid extraction with a mixture of N‐hexane–dichloromethane–isopropanol (100:50:5, v/v/v). Isocratic chromatographic separation was performed on a reversed‐phase Discovery C₁₈ column (2.1 × 50 mm, 5 µm). The mobile phase was methanol–water–formic (75:25:0.25, v/v/v). Detection of fenofibric acid and the internal standard (IS) diclofenac acid was achieved by ESI MS/MS in the negative ion mode using m/z 317 → m/z 213 and m/z 294 → m/z 250 transitions, respectively. The method was linear from 0.005 to 1.250 µg/mL when 100 μL plasma was analyzed. The lower limit of quantification was 0.005 µg/mL. The intra‐ and inter‐day precision values were below 8.2%, and accuracy ranged from ?0.9 to 2.1% in all quality control samples. The recovery was 90.3–94.7% and 83.3% for fenofibric acid and IS, respectively. Total run time for each sample analysis was 2.5 min. The validated method was successfully applied to a pharmacokinetic study in six rats after oral administration of fenofibrate, the ester prodrug of fenofibric acid (equivalent to fenofibric acid 5 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid fenofibric acid determination. Copyright © 2011 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of febuxostat in human plasma

A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with positive ion electrospray ionization was developed for the determination of febuxostat in human plasma using d₇‐febuxostat as the internal standard (IS). A simple protein precipitation was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on a Capcell PAK C₁₈ column (4.6 × 100 mm, 5 µm) using acetonitrile–5 mm ammonium acetate–formic acid (85:15:0.015, v/v/v) as the mobile phase at a flow rate of 0.6 mL/min. An Agilent 6460 electrospray tandem mass spectrometer was operated in the multiple reaction monitoring mode. The precursor‐to‐product ion transitions m/z 317 → m/z 261 (febuxsotat) and m/z 324 → m/z (261 + 262) (d₇‐febuxostat, IS) were used for quantitation. The results were linear over the studied range (10.0–5000 ng/mL), and the total analysis time for each chromatograph was 3 min. The intra‐ and inter‐day precisions were less than 7.9 and 7.2%, respectively, and the accuracy was within ±4.2%. No evidence of analyte instability in human plasma was observed storage at ?20°C for 31 days. This method was successfully applied in the determination of febuxostat concentrations in plasma samples from healthy Chinese volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Losses of ions during forward and reverse scans in a quadrupole ion trap mass spectrometer and how to reduce them

The higher order fields present in the quadrupole ion trap may have beneficial effects such as increases in mass resolution in the mass-selective instability or resonance ejection modes of operation, but may also result in losses of ions due to nonlinear resonances. In this work, the reduction in ion intensities observed in the mass spectra of polyethylene glycol (PEG 1000) has been utilized to monitor the ion losses resulting from these higher order fields during the rf voltage scans in both the forward and reverse directions. Extensive ion losses were observed in reverse rf voltage scans at q _z=0.64 (a _z=0), which corresponds to octopole resonance at β _z=1/2. The losses depended upon rf voltage scan rate and ion mass being greater for lower scan rates and lower masses. For ions of m/z 877, losses of up to 60% of the stored ions were observed at low scan rates (<1×10⁴ Da/s), but were minimal at higher scan rates. Thus, it is possible to avoid such losses during reverse scans by scanning the region q _z=0.64 at rates in excess of 4×10⁴ Da/s. In forward rf voltage scans, ion storage was considerably more reliable, with significant losses observed only at very high scan rates near the region q _z=0.78 (hexapole resonance at β _z=2/3).     

Reactivity of gaseous sodiated ions derived from benzene dicarboxylate salts toward residual water in the collision gas

The sodium adduct of disodium salts of benzene dicarboxylic acids (m/z 233), when subjected to collision‐induced dissociation (CID), undergoes a facile loss of CO₂ to produce an ion of m/z 189, which retains all the three sodium atoms of the precursor. The CID spectrum of this unusual m/z 189 ion shows significant peaks at m/z 167, 63 and 85. The enigmatic m/z 167 ion, which appeared to represent a loss of a 22‐Da neutral fragment from the precursor ion is in fact a fragment produced by the interaction of the m/z 189 ion with traces of water present in the collision gas. The change of the m/z 167 peak to 168, when D₂O vapor was introduced to the collision gas of a Q‐ToF instrument, proved that such an intervention of water could occur even in collision cells of tandem‐in‐space mass spectrometers. The m/z 189 ion has such high affinity for water; it forms an ion/molecule complex even during the brief residence time of ions in collision cells of triple quadrupole instruments. The complex formed in this way then eliminates elements of NaOH to produce the ion observed at m/z 167. In an ion trap, the relative intensity of the m/z 167 peak increases with longer activation time even at the lowest possible collision energy setting. Similarly, the m/z 145 ion (which represents the sodium adduct of phenelenedisodium, formed by two consecutive losses of CO₂ from the m/z 233 ion of meta‐ and para‐isomers) interacts with water to produce a fragment ion at m/z 123 for the sodium adduct of phenylsodium. Other uncommon ions that originate also from water/ion interactions are observed at m/z 85 and 63 for [Na₃O]⁺ and [Na₂OH]⁺, respectively. Tandem mass spectrometric experiments conducted with appropriately deuterium‐labeled compounds confirmed that the proton required for the formation of the [Na₂OH]⁺ ion originates from traces of water present in the collision gas and not from the ring protons of the aromatic moiety. Copyright © 2010 John Wiley & Sons, Ltd.     

Authentication of Cereal Flours by Multivariate Analysis of GC–MS Data

In this work flour samples of various cereal species, a group of different cultivars of small grain species (wheat, Triticum spp.; barley, Hordeum vulgare; oat, Avena sativa; rye, Secale cereale; triticale, Triticosecale) and cultivars of corn species, Zea Mays, growing in the same period and in the same geographical area, were selected to establish differences between each other, using a new rapid method: a comparison of derivatized hexane extracts by GC–MS and multivariate analysis, using the characteristic fragmentation ion m/z 74, without performing qualitative and quantitative analysis of eluting components. Obtained results were compared with the results obtained using a common electron microscopy method. Flour samples made of every corn and oat cultivar showed complete differences compared to flour samples of each wheat, barley, rye and triticale cultivar investigated in this study. The GC–MS approach combined with multivariate analysis outperforms the standard electron microscopy method in a faster and easier way, and may be used to verify flour types in the market.

     

Quantitative determination of major platelet activating factors from human plasma

Platelet activating factor (PAF) is a potent lipid mediator that is involved in many important biological functions, including platelet aggregation and neuronal differentiation. Although an ELISA assay has been used to measure PAF levels, it cannot distinguish between its isoforms. To achieve this, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used instead. However, isobaric lysophosphatidylcholine (lyso PC), which is often present in large amounts in complex biological samples and has similar retention times in many LC conditions, can affect the accurate measurement of PAF. The present study examined the fragmentation behavior of major PAF and lyso PC during various MS/MS conditions. Fragment ions at m/z 184 and at m/z 104 were abundantly observed from MS/MS of lyso PCs. PAF provided a dominant fragment ion at m/z 184, but a fragment ion at m/z 104 was almost never produced, regardless of the collision energy. Thus, the two fragment ions at m/z 184 and m/z 104 were used to accurately measure PAF levels. First, the fragment ion at m/z 184 and the retention time of PAF in LC-MS/MS were used to identify and quantitate PAF. However, if there were small retention time shifts, which are common in multiple sample runs, and lipid composition in a sample is very complicated, the fragment ion at m/z 104 was used to confirm whether the fragment ion at m/z 184 belonged to PAF. This novel method accurately determined the major PAF (C16:0 PAF, C18:0 PAF, and C18:1 PAF) levels in human plasma.     

Identification of Proteins and Phosphoproteins Using Pulsed Q Collision Induced Dissociation (PQD)

Pulsed Q collision induced dissociation (PQD) was developed to facilitate detection of low-mass reporter ions from labeling reagents (e.g., iTRΑQ) in peptide quantification using an LTQ mass spectrometer (MS). Despite the large number of linear ion traps worldwide, the use and optimization of PQD for protein identification have been limited, in part due to less effective ion fragmentation relative to the collision induced dissociation (CID). PQD expands the m/z coverage of fragment ions to the lower m/z range by circumventing the typical low mass cut-off of an ion trap MS. Since database searching relies on the matching between theoretical and observed spectra, it is not clear how ion intensity and peak number might affect the outcomes of a database search. In this report, we systematically evaluated the attributes of PQD mass spectra, performed intensity optimization, and assessed the benefits of using PQD on the identification of peptides and phosphopeptides from an LTQ. Based on head-to-head comparisons between CID (higher intensity) and PQD (better m/z coverage), peptides identified using PQD generally have Xcorr scores lower than those using CID. Such score differences were considerably diminished by the use of 0.1% m-nitrobenzyl alcohol (m-NBA) in mobile phases. The ion intensities of both CID and PQD were adversely affected by increasing m/z of the precursor, with PQD more sensitive than CID. In addition to negating the 1/3 rule, PQD enhances direct bond cleavage and generates patterns of fragment ions different from those of CID, particularly for peptides with a labile functional group (e.g., phosphopeptides). The higher energy fragmentation pathway of PQD on peptide fragmentation was further compared to those of CID and the quadrupole-type activation in parallel experiments.     

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

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