Reduced matrix effects for anionic compounds with paired ion electrospray ionization mass spectrometry

It is well-known that matrix effects in high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) can seriously compromise quantitative analysis and affect method reproducibility. Paired ion electrospray ionization (PIESI) mass spectrometry is an approach for analyzing ultra-low levels of anions in the positive ion mode. This approach uses a structurally optimized ion pairing reagent to post-column associate with the anionic analyte, subsequently forming positively charged complexes. These newly formed complex ions are often more surface-active as compared to either the native anion or the ion pairing reagent. No studies have examined whether or not the PIESI approach mitigates matrix effects. Consequently, a controlled study was done using five analytes in highly controlled and reproducible synthetic groundwater and urine matrices. In addition, two different mass spectrometers (linear ion trap and triple quadrupole) were used. Compared to the negative ion mode, the PIESI-MS approach was less susceptible to matrix effects when performed on two different MS platforms. Using PIESI-MS, less dilution of the sample is needed to eliminate ionization suppression which, in turn, permits lower limits of detection and quantitation.     

纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用;单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。     

Acid‐based approach for separation of peptide epimers using IM‐MS

Chiral molecules frequently remain undistinguishable using ion mobility mass spectrometry (IM‐MS), due to insufficient differences of their collision cross sections at the available mobility resolution of the ion mobility drift tubes. The influence of the complexation with organic acids on the ion mobility separation of peptide epimers is evaluated using traveling‐wave ion mobility (TWIMS). The examined epimeric tripeptides containing Arg residue with the sequence: Ac‐Phe‐Arg‐Trp‐NH₂ formed stable complexes in the gas phase, and under the increased pressure in ion mobility drift tube, noncovalent associates formed with carboxylic or sulfonic monoacids and diacids with chiral variation of certain acids. Overall, the complexation with an acid leads to the improvement in stereodifferentiation among epimeric peptides, in comparison to the analysis of pure epimers. Detailed characterization of peptide epimer‐acid associates obtained for dibenzoyl‐D‐tartaric acid by theoretical calculations and collisional dissociation studies revealed that the presence of multiple hydrogen bonding interactions between carboxylate anions and hydrogens from N―H of both the guanidinium group of arginine and the indole of tryptophan, as well as the amide backbone hydrogens in the peptide, is responsible for stability of acid‐peptide complexes and for their differentiation in the ion mobility drift tube. The specificity of complex formation toward Arg was determined in terms of complex stability. Based on the reported results, we present general conclusions regarding the utility of the acid‐based complexation in the separation of peptide isomers.     

Investigations with O-linked protein glycosylations by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Posttranslational modifications such as glycosylation can play a fundamental role in signaling pathways that transform an ordinary cell into a malignant one. The development of a protocol to detect these changes in the preliminary stages of disease can lead to a sensitive and specific diagnostic for the early detection of malignancies such as ovarian cancer in which differential glycan patterns are linked to etiology and progression. Small variations in instrument parameters and sample preparation techniques are known to have significant influence on the outcome of an experiment. For an experiment to be effective and reproducible, these parameters must be optimized for the analyte(s) under study. We present a detailed examination of sample preparation and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) analysis of O-linked glycans globally cleaved from mucin glycoproteins. Experiments with stable isotope-labeled biomolecules allowed for the establishment of appropriate acquisition times and excitation voltages for MALDI-FT-ICR-MS of oligosaccharides. Quadrupole ion guide optimization studies with mucin glycans identified conditions for the comprehensive analysis of the entire mass range of O-linked carbohydrates in this glycoprotein. Separately optimized experimental parameters were integrated in a method that allowed for the effective study of O-linked glycans. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Calibration laws based on multiple linear regression applied to matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Operation of any mass spectrometer requires implementation of mass calibration laws to translate experimentally measured physical quantities into a m/z range. While internal calibration in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) offers several attractive features, including exposure of calibrant and analyte ions to identical experimental conditions (e.g. space charge), external calibration affords simpler pulse sequences and higher throughput. The automatic gain control method used in hybrid linear trap quadrupole (LTQ) FT-ICR-MS to consistently obtain the same ion population is not readily amenable to matrix-assisted laser desorption/ionization (MALDI) FT-ICR-MS, due to the heterogeneous nature and poor spot-to-spot reproducibility of MALDI. This can be compensated for by taking external calibration laws into account that consider magnetic and electric fields, as well as relative and total ion abundances. Herein, an evaluation of external mass calibration laws applied to MALDI-FT-ICR-MS is performed to achieve higher mass measurement accuracy (MMA).     

Quantitative analysis of memantine in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry

A sensitive and specific method for the determination of memantine in human plasma is presented. Memantine was extracted from plasma and derivatized to the pentafluorobenzoyl derivative in a one-step procedure avoiding any sample concentration steps. Amantadine was used as an internal standard. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further processing. Using this detection mode, the fragment ions at m/z 353 and 325 were obtained at high relative abundance. Calibration graphs were linear over the range 0.117-30 ng ml(-1). At the limit of quantification (LOQ), the inter-assay precision was 2.00% and the intra-assay variability was 3.22%. The accuracy at the LOQ showed deviations of -1.42% (intra-assay) and -2.47% (inter-assay). The method is rugged, rapid and robust and was applied to the batch determination of memantine during pharmacokinetic profiling of the drug.     

Quantitative gas chromatographic/mass spectrometric analysis of morphine glucuronides in human plasma by negative ion chemical ionization mass spectrometry

A sensitive and specific method for the determination of morphine glucuronides in human plasma is presented. Morphine glucuronides, namely morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G), were extracted from plasma by solid-phase extraction on C(18) cartridges at pH 9.3 and derivatized to their pentafluorobenzyl ester trimethylsilyl ether derivatives. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 748 was obtained at high relative abundance for both target compounds. [(2)H(3)]-labeled morphine glucuronides were used as internal standards. Calibration graphs were calculated by polynomial fit within a range of 10-1280 and 15-1920 nmol l(-1) for the 6- and 3-glucuronide, respectively. At the limit of quantitation (LOQ), the inter-assay precision was 2.21% (M3G) and 2.23% (M6G) and the GC/MS assay variability was 1.8% (M3G) and 0.9% (M6G). The accuracy at the LOQ showed deviations of +4.92% (M3G) and +1.5% (M6G). The sample recovery after solid-phase extraction was 84.7% for both M3G and M6G. The method is rugged, rapid and robust and has been applied to the batch analysis of morphine glucuronides during pharmacokinetic profiling of the drugs.     

Analysis of impurities in pharmaceuticals by LC‐MS with cold electron ionization

Pharmaceuticals require careful and precise determination of their impurities that might harm the user upon consumption. Although today, the most common technique for impurities identification is liquid chromatography‐mass spectrometry (LC‐MS/MS), it has several downsides due to the nature of the ionization method. Also, the analyses in many cases are targeted thus despite being present, some of the compounds will not be revealed. In this paper, we propose and show a new method for untargeted analysis and identification of impurities in active pharmaceutical ingredients (APIs). The instrument used for these analyses is a novel electron ionization (EI) LC‐MS with supersonic molecular beams (SMB). The EI‐LC‐MS‐SMB was implemented for analyses of several drug samples spiked with an impurity. The instrument provides EI mass spectra with enhanced molecular ions, named Cold EI, which increases the identification probabilities when the compound is identified with the aid of an EI library like National Institute of Standards and Technology (NIST). We analyzed ibuprofen and its impurities, and both the API and the expected impurity were identified with names and structures by the NIST library. Moreover, other unexpected impurities were found and identified proving the ability of the EI‐LC‐MS‐SMB system for truly untargeted analysis. The results show a broad dynamic range of four orders of magnitude at the same run with a signal‐to‐noise ratio of over 10 000 for the API and almost uniform response.     

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrospray Lonization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Study on Sodium Azide Cluster Ions

Sodium azide has rarely been studied in gas phase or in the form of cluster ions and as a model of solid energetic substances and inorganic azide salt was ionized by electrospray ionization (ESI) and studied by high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) systematically. This paper highlights the effects of experimental conditions on the formation of salt cluster and the collision activation dissociation pathways of cluster ions to develop a microscopic understanding of inorganic azide salt clusters.     

Toxaphene analysis in Great Lakes fish: a comparison of GC-EI/MS/MS and GC-ECNI-MS, individual congener standard and technical mixture for quantification of toxaphene

Toxaphene is considered to be a problematic organochlorine pollutant because of its bioaccumulation potential and persistence in aquatic environments. In this study, whole lake trout and walleye composites were used to evaluate two analytical techniques for total toxaphene and selected congener analysis. The efficacy of using gas chromatography electron ionization tandem mass spectrometry (GC-EI/MS/MS) and electron capture negative ionization mass spectrometry (GC-ECNI-MS) were compared. Although the sensitivity using GC-ECNI-MS was approximately five times greater than GC-EI/MS/MS, the latter provided more consistent inter-Parlar relative response factors (RRF). When using technical calibration mixtures, these results suggest a more accurate total toxaphene measurement was obtained using the GC-EI/MS/MS method. Total toxaphene concentrations in lake trout composites from both methods were highly correlated (R ² = 0.985) with the MS/MS concentrations approximately half of those determined by ECNI, suggesting systematic high bias in toxaphene concentrations when measured using GC-ECNI.     

微波辅助萃取-气相色谱-负化学电离/电子轰击电离-飞行时间质谱法测定烟草中农药残留

建立了微波辅助萃取结合气相色谱-飞行时间质谱(GC-TOF MS)技术在负化学电离(NCI)源和电子轰击电离(EI)源两种模式下测定烟草中24种农药残留的分析方法。烟叶样品于100℃下用二氯甲烷-正己烷(3:1, v/v)混合溶剂微波萃取10 min,萃取液经弗罗里硅土固相萃取柱净化后进行检测分析。在NCI源和EI源质谱模式下,24种农药的线性关系均良好(r²>0.99),相对标准偏差分别小于8.6%和9.1%,定量限分别为0.3~6.9 μg/kg和10.2~44.9 μg/kg,加标回收率分别为75.2%~94.8%和75.0%~95.1%。比较两种离子源模式下的色谱图和质谱图,NCI源模式较EI源模式的选择性好、灵敏度高、基质干扰小、图谱简单易于解析,检出限低一个数量级以上,在分析低含量、复杂基质的样品时更具优势。     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

气相色谱-负化学源质谱快速测定母乳中的多溴联苯醚

建立了母乳中8种多溴联苯醚(PBDEs：BDE28,BDE47,BDE99,BDE100,BDE153,BDE154,BDE183,BDE209)的气相色谱-负化学源质谱测定方法(GC-NCI/MS)。样品经索氏提取、酸化硅胶除脂、硅胶氧化铝色谱柱净化后,在7 m长的毛细管气相色谱柱上快速分离,NCI/MS以选择离子监测模式测定目标化合物。其中,三溴～七溴联苯醚采用内标法定量,十溴联苯醚(BDE209)采用同位素稀释法定量。8种PBDEs的检出限为1.74～6.35 pg/g(以脂肪计)。加标回收试验的回收率为61.5%～108%,相对标准偏差为2.06%～10.1%(n=6)。并采用母乳参考物质进一步证实了该方法的准确可靠。该方法提高了BDE209的分析灵敏度,而且分析成本相对较低,分析时间短,适于推广。     

Determination of misoprostol free acid in human breast milk and serum by gas chromatography/negative ion chemical ionization tandem mass spectrometry

To study an expected transition of misoprostol from human blood into breast milk, a novel method for the determination of its active metabolite misoprostol acid (MPA) was developed. MPA was determined in serum and breast milk samples by an isotope dilution assay using gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS). After addition of (15S)-15-methylprostaglandin E(2) (15-methyl-PGE(2)) as an internal standard, MPA was extracted from both matrices using a reversed-phase cartridge. The prostanoids were derivatized with O-2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride (PFBHA) and 2,3,4,5,6-pentafluorobenzyl bromide (PFBB) to the pentafluorobenzyl oxime (PFBO)-pentafluorobenzyl ester (PFB) derivatives. The sample was subjected to thin-layer chromatography with ethyl acetate-hexane (1 : 1 (v/v)) as the developing solvent. The corresponding zone was extracted. After derivatization to the trimethylsilyl ether, MPA was determined by GC/NICI-MS/MS using the [molecule (M) - pentafluorobenzyl (PFB)](-) ([P](-)) ions as precursor in the negative ion chemical ionization mode. The product ions used for quantification were [P - 2TMSOH - C(6)F(5)CH(2)OH](-) (MPA) and [P - 2TMSOH - C(6)F(5)CH(2)OH - CO(2)](-)(15-methyl-PGE(2)), respectively. The limit of quantification for MPA was approximately 1 pg ml(-1) in breast milk and serum samples. The correlation coefficients of the calibration curves for MPA were r > 0.997 in the 0.5-2000 pg ml(-1) range for both tested matrices.     

Electrospray ionization mass spectrometric study of end‐groups in peroxydicarbonate‐initiated radical polymerization

Initiation by diethyl peroxydicarbonate (E‐PDC), di‐n‐tetradecyl peroxydicarbonate (nTD‐PDC), di‐n‐hexadecyl peroxydicarbonate (nHD‐PDC), and di‐2‐ethylhexyl peroxydicarbonate (2EH‐PDC) of free‐radical polymerizations of methyl methacrylate in benzene solution was studied by end‐group analysis via electrospray ionization mass spectrometry (ESI‐MS). Unambiguous assignment of ESI‐MS peaks allows for identification of the type of radical that starts chain growth. In case of initiation by dialkyl peroxydicarbonates with linear alkyl groups, almost exclusively alkoxy carbonyloxyl species, which are the primary fragments from initiator decomposition, occur as end‐groups. With 2EH‐PDC, however, both the primary 2‐ethylhexoxy carbonyloxyl fragment and a second moiety, which is formed by decarboxylation of the 2‐ethylhexoxy carbonyloxyl radical, are clearly observed as end‐groups. The decarboxylation process is described by a concerted mechanism which involves a 1,5‐hydrogen shift reaction. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6071–6081, 2008     

Degradation of Poly(methyl methacrylate) Model Compounds at Constant Elevated Temperature Studied via High Resolution Electrospray Ionization Mass Spectrometry (ESI‐MS)

The products of the thermal degradation at 95 °C over 10 months of ω‐saturated and non‐saturated poly(methyl methacrylate) (pMMA) model compounds were identified with high accuracy via quadrupole ion trap and quadrupole ion trap‐time of flight (Q‐ToF) mass spectrometry. Analysis of the samples taken via these techniques indicated that degradation of vinyl terminated pMMA proceeds via the incorporation of oxygen via the formation of ethylene oxide type end groups, which subsequently rearrange under the expulsion of formaldehyde and 2‐oxo‐propionic acid methyl ester. The corresponding saturated model compounds were demonstrated to be stable over the same time period. The present findings highlight for the first time that poly(methyl methacrylate) degradation does not necessarily and exclusively proceed via radical intermediates.

     

High‐resolution MALDI‐TOF MS study on analysis of low‐molecular‐weight products from photo‐oxidation of poly(3‐hexylthiophene)

High‐resolution matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometry (TOF MS) was used for the analysis of the low‐molecular‐weight products from the photo‐oxidation of poly(3‐hexylthiophene) (P3HT) in solution and thin film. Eight new peak series were observed in the low‐mass range of the mass spectra of the products degraded in solution, and the formulas of the eight components were determined from the accurate mass. From SEC/MALDI‐TOF MS, two components were identified as the degraded products, and the other six components were derived from the fragmentation of the degraded products during the MALDI process. A mechanism for the formation of these components was proposed on the basis of the results of MALDI‐TOF MS. For the thin film degradation, a part of products in the solution degradation were observed, which supports that the oxidation of P3HT in solution and thin film proceeded in the same mechanism. This study shows that high‐resolution MALDI‐TOF MS is effective for the analysis of the low‐molecular‐weight products from P3HT photo‐oxidation and expected to be feasible for the degradation analyses of other polymers. Copyright © 2015 John Wiley & Sons, Ltd.     

Uncharged Water‐Soluble Metal‐Bis‐Porphyrins Like Molecular Tweezers for Amino Acids

Some new water‐soluble bis‐porphyrins, constituted of two porphyrin units spaced by means of aliphatic bridges of different lengths, were synthesized and characterized by MALDI‐TOF mass spectrometry, ¹H NMR and UV‐vis spectroscopy. The hydrosolubility of these uncharged compounds was guaranteed from the presence of six long PEG chains bound on the peripheral positions of the two porphyrins. Cobalt and zinc derivatives were also prepared. In the case of Co‐bis‐porphyrin, the appearance of induced circular dichroism (ICD) signals in water solution confirmed the formation of stable complexes with some amino acids, in which the bis‐porphyrin behaves like molecular tweezers.