New matrix of MALDI-TOF MS for analysis of small molecules

New matrix, metal-phthalocyanine (MPc), of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for analysis of small molecules (usually <500 Da). By using MPcs as matrices, small molecular samples were moved to high mass-to-charge region where there was no interference caused by the traditional matrices. The mass of the target analyte was obtained by simple calculation.     

Use of high‐resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose‐dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high‐resolution mass spectrometry led to unequivocal identification of the interfering peak as an N‐desmethyl metabolite of the parent analyte. High‐resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the ¹³C‐isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

Approaches for the analysis of low molecular weight compounds with laser desorption/ionization techniques and mass spectrometry

This review summarizes various approaches for the analysis of low molecular weight (LMW) compounds by different laser desorption/ionization mass spectrometry techniques (LDI-MS). It is common to use an agent to assist the ionization, and small molecules are normally difficult to analyze by, e.g., matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the common matrices available today, because the latter are generally small organic compounds themselves. This often results in severe suppression of analyte peaks, or interference of the matrix and analyte signals in the low mass region. However, intrinsic properties of several LDI techniques such as high sensitivity, low sample consumption, high tolerance towards salts and solid particles, and rapid analysis have stimulated scientists to develop methods to circumvent matrix-related issues in the analysis of LMW molecules. Recent developments within this field as well as historical considerations and future prospects are presented in this review.     

Discerning matrix-cluster peaks in matrix-assisted laser desorption/ionization time-of-flight mass spectra of dilute peptide mixtures

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is widely used for the analysis of peptide mixtures such as those resulting from protein digestion. Among several useful peptide matrices, alpha-cyano-4-hydroxycinnamic acid (4-HCCA) appears to be the most popular. This matrix does not generally give matrix-cluster peaks at the mass region covered by enzyme-digested peptides (i.e., m/z above approximately 500). However, when an analyte mixture is very dilute and/or the sample contains a large amount of salts, ion peaks from matrix clusters can be quite intense, compared to peptide peaks. This matrix-cluster interference becomes more pronounced as the amount of analyte decreases. In this paper, a simple scheme for matrix-cluster identification is reported. It is shown that matrix-cluster formation follows a systematic pattern, although the relative intensities of these cluster ions cannot be predicted. Discerning the matrix-cluster peaks from the peptide peaks is found to be critical in analyzing dilute peptide mixtures with both conventional and microspot MALDI-TOF techniques.     

Online Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with In situ Mixing

Online matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is reported with in situ matrix/analyte aerosol mixing. The analyte and matrix were dissolved in separate solvents and pneumatically atomized into particles. Continuous flows of analyte particles and matrix droplets in nitrogen were mixed in a tee. The resulting particles were sampled using a nozzle, focused into a beam with an aerodynamic lens system, and vertically deposited continuously on a movable target. The matrix/analyte mixture was desorbed/ionized using a 266 nanometers pulsed laser at an incident angle of 45°. The nascent ions were analyzed using reflectron TOF MS. The performance of online MALDI-TOF MS was evaluated by the analysis of palmityl palmitate with the lithium salt of 2, 4-dihydroxybenzoic acid as the matrix. Strong and stable MALDI signals of palmityl palmitate were obtained. The matrix solvent mixture and the analyte concentration were optimized and the results demonstrate the development of an alternative for online MALDI analysis.     

Determination of low levels of cadmium ions by the under potential deposition on a self-assembled monolayer on gold electrode

Despite the advantages of simplicity and high-throughput detection that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has over other methods, quantitative analysis of low-molecular-weight analyte is hampered by interference from matrix-derived background noise and signal fluctuation due to the inhomogeneous MALDI sample surface. Taking advantage of improved sample homogeneity through matrix-conjugated magnetic nanoparticles (matrix@MNP) and the seed-layer method, we report a new strategy for the rapid identification and quantification of drugs in urine samples, using morphine and 7-aminoflunitrazepam (7-aminoFM2) as model compounds. To our knowledge, this is the first attempt using the seed-layer method for small molecule analysis. By applying the proposed seed-layer method, which was specifically optimized for the 2,5-dihydroxybenzoic acid@MNP (DHB@MNP) matrix, homogeneous sample crystallization examined by microscopy analysis was obtained that generated reproducible MALDI signals (RSD<10.0%). For urine sample analysis, simple liquid-liquid extraction as a sample pretreatment step effectively reduced the ion suppression effect caused by the endogenous components in urine; good recoveries (82-90%) were obtained with a small ion suppression effect (<14% of signal decrease). This newly developed method demonstrated good quantitation linearity over a range of 50-2000 ng mL(-1) (R(2)>0.996) with reduced signal variation (RSD<10.0%). The detection limit is 30 ng mL(-1) with good precision (intra-day, 2.0-9.3%; inter-day, 5.0-10.0%) and accuracy (intra-day, 95.0-106.0%; inter-day, 103.0-115.5%). The nanoparticle-assisted MALDI-TOF MS combined with seed-layer surface preparation provides a rapid, efficient and accurate platform for the quantification of small molecules in urine samples.     

A binary matrix for background suppression in MALDI-MS of small molecules

Application of matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) to small-molecule detection is often limited, because of high matrix background signals in the low-mass region. We report here an approach in which a mixture of two conventional MALDI matrices with different proton affinity was used to suppress the formation of matrix clusters and fragments. Specifically, when acidic α-cyano-4-hydroxycinnamic acid (CHCA) and basic 9-aminoacridine (9-AA) were used as the binary matrix, fewer background matrix peaks were observed in both positive and negative-mode detection of small molecules. In addition, the presence of CHCA substantially reduced the laser fluence needed for analyte desorption and ionization; thus better signal-to-background ratios were observed for negatively charged inositol phosphates in complex plant extracts. The mixing of MALDI matrices of different protonaffinities leads to suppression of matrix clusterformation and subsequently yields cleaner MS spectraof fewer background peaks in both positive andnegative detection of small molecules     

Unusual Analyte-Matrix Adduct Ions and Mechanism of Their Formation in MALDI TOF MS of Benzene-1,3,5-Tricarboxamide and Urea Compounds

Analyte-matrix adducts are normally absent under typical matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) conditions. Interestingly, though, in the analysis of several types of organic compounds synthesized in our laboratory, analyte-matrix adduct ion peaks were always recorded when common MALDI matrices such as 4-hydroxy-α-cyanocinnamic acid (CHCA) were used. These compounds are mainly those with a benzene-1,3,5-tricarboxamide (BTA) or urea moiety, which are important building blocks to make new functional supramolecular materials. The possible mechanism of the adduct formation was investigated. A shared feature of the compounds studied is that they can form intermolecular hydrogen bonding with matrices like CHCA. The intermolecular hydrogen bonding will make the association between analyte ions and matrix molecules stronger. As a result, the analyte ions and matrix molecules in MALDI clusters will become more difficult to be separated from each other. Furthermore, it was found that analyte ions were mainly adducted with matrix salts, which is probably due to the much lower volatility of the salts compared with that of their corresponding matrix acids. It seems that the analyte-matrix adduct formation for our compounds are caused by the incomplete evaporation of matrix molecules from the MALDI clusters because of the combined effects of enhanced intermolecular interaction between analyte-matrix and of the low volatility of matrix salts. Based on these findings, strategies to suppress the analyte-matrix adduction are briefly discussed. In return, the positive results of using these strategies support the proposed mechanism of the analyte-matrix adduct formation.

MoS2/Ag nanohybrid: A novel matrix with synergistic effect for small molecule drugs analysis by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS₂/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS₂. Moreover, both Ag nanoparticles and the edge of the MoS₂ layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS₂/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS₂/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.     

基质辅助激光解吸电离飞行时间质谱技术分析三七和海兔神经连索内的皂甙组分

基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱技术能高效解吸三七提取液(Panaxnotoginseng Extraction, PNE)中的混合皂甙分子为皂甙离子,并供质量分析器检测与分析.选用MALDI-TOF 质谱技术直接分析色谱纯皂甙样品的纯度,其检测灵敏度优于反相高效液相色谱法(RP-HPLC).优化提取中药三七(Panax notoginseng, PN)的混合皂甙, 选用MALDI-TOF质谱技术直接分析PNE中的皂甙种类和相对含量,发现PNE至少含有20种不同分子结构的皂甙组分,其中人参皂甙(Ginsenoside) Rg1和三七皂甙(Notoginsenoside)R1含量相对较高.选用薄板层析法(TLC)制备PNE中的R1皂甙.MALDI-TOF质谱技术研究蓝斑背肛海兔(Notarcus leachiicirrosus Stimpson, NLCS)神经连索内的超微量R1的组成与分布.建立PNE皂甙的指纹图谱,并适合于评价中药三七的品质和分析体内超微量皂甙的代谢过程与机理.     

Synthesis, characterization and nonlinear absorption of novel octakis-POSS substituted metallophthalocyanines and strong optical limiting property of CuPc

In this study, the preparation of some novel metallophthalocyanine (MPcs) complexes substituted with octakis(mercaptopropylisobutyl-POSS) functional group was achieved. By the reaction of [1-(3-mercapto)propyl-3,5,7,9,11,13,15-isobutylpentacyclo[9.5.1.1(3,9).1(5,15).1(7,13)]octasiloxane 1 with 4,5-dichloro-1,2-dicyanobenzene 2 in THF as the solvent in the presence of K2CO3 as the base, the phthalonitrile derivative 3 was synthesized. Compound 3 reacted with CoCl2 x 6H2O in ethylene glycol to furnish a novel cobalt(II) phthalocyanine . The tetramerization of 3 with urea and CuCl in the absence of solvent gave the novel Cu(II) phthalocyanine 4; while with Zn(OAc)2 x 2H2O in dry DMF gave the novel zinc(ii) phthalocyanine 6. The structures of the target compounds were confirmed by elemental analysis, UV/VIS, IR, MALDI-TOF MS and 1H NMR spectra. Nonlinear absorptions of MPcs in chloroform solution were investigated by using Z-scan measurement technique with 4 ns pulses at 532 nm wavelength. While CuPc 5 showed very high nonlinear absorption, MPcs 4 and 6 did not show considerable nonlinear absorption. Investigations of optical limiting properties of 5 revealed that this material is a very good candidate for optical limiting applications.     

Characterization of unknown compounds from stainless steel plates in matrix-assisted laser desorption/ionization mass spectrometry

Peaks originating from unknown compounds on stainless steel plates used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are observed around m/z 304.3, 332.3, 360.4, and 388.4 regardless of the matrix and/or solvent, and are even observed with bare plates. These peaks were characterized using three different types of MALDI-MS instrumentation: MALDI-TOF MS, MALDI-TOF/TOF MS, and MALDI-FTMS. The fragmentation data from MALDI-TOF/TOF MS and accurate mass determination by MALDI-FTMS enabled identification of the chemical formulae and structures. The unknown compounds are, in fact, likely benzylalkylmethylammonium salts, as confirmed by closely matching fragmentation patterns with a commercially available benzalkonium chloride.     

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectra of poly(butylene adipate)

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) was employed to analyze four poly(butylene adipate) (PBAd) oligomers and to investigate their fragmentation pathways as a continuation of our work on the MALDI-TOF/TOF-MS/MS study of synthetic polymers. MALDI-TOF/TOF-MS/MS analysis was performed on oligomers terminated by carboxyl and hydroxyl groups, methyl adipate and hydroxyl groups, dihydroxyl groups, and dicarboxyl groups. The sodium adducts of these oligomers were selected as precursor ions. Different end groups do not influence the fragmentation of sodiated polyester oligomers and similar series of product ions were observed in all the MALDI-TOF/TOF-MS/MS spectra. According to the structures of the most abundant product ions identified in the present work, three fragmentation pathways have been proposed to occur most frequently in PBAd: beta-hydrogen-transfer rearrangement, leading to the selective cleavage of the --O--CH(2)-- bonds; --CH(2)--CH(2)-- (beta--beta) bond cleavage in the adipate moiety; and ester bond scission.     

Controlling matrix suppression for matrix‐assisted laser desorption/ionization analysis of small molecules

The need for high‐throughput methodologies providing both qualitative and quantitative information has grown substantially in the pharmaceutical laboratory in recent years. Currently, tandem mass spectrometry (MS/MS) using quadrupole technology offers analysis in the minutes time scale. The use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) offers the advantage of speed and automation and enables analysis in the seconds time scale with accurate mass capabilities that are not typically found in quadrupole MS/MS. However, one of the limitations of MALDI for the analysis of small molecules is the abundance of interfering matrix peaks in the low molecular weight region of the mass spectrum. Described herein is an evaluation of a pre‐prepared MALDI target plate that has been coated with a thin layer of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and nitrocellulose. This modified plate has been shown to suppress or eliminate CHCA matrix signals without any significant loss of analyte sensitivity when compared with analysis of the same sample using an unmodified target plate. Copyright © 2004 John Wiley & Sons, Ltd.     

Studies of pesticides by collision-induced dissociation, postsource-decay, matrix-assisted laser desorption/ionization time of flight mass spectrometry

The use of collision-induced dissociation, postsource decay (CID-PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of small organic molecules is demonstrated. Three pesticides: paraquat, diquat, and difenzoquat were chosen for this study. The matrices 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA), and sinapinic acid (SA) were selected to investigate the effect of the matrix on the CID-PSD MALDI spectra of these molecules. Alpha-CHCA and DHB were found to be appropriate matrices for the pesticides studied. Spectra for a given pesticide obtained from different matrices were compared with each other, and the differences between them are discussed. A comparison of CID-PSD MALDI with fast-atom bombardment MS/MS spectra is presented; the agreement of pesticide fragmentation patterns between the two methods indicates that CID-PSD MALDI MS is a reliable and efficient technique for structural elucidation of small molecules.     

Efficient enrichment and identification of phosphopeptides by cerium oxide using on-plate matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

An efficient and simple method for enrichment and identification of phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using cerium oxide is presented. After pretreatment of tryptic digests of phosphoproteins with CeO(2), nonphosphopeptides are discarded and phosphopeptides are enriched. By applying the separated CeO(2) on a target plate and analysis using MALDI-TOF MS, peaks of phosphopeptides and their correspondingly series of dephosphorylated peptides are observed in the mass spectra. Thus, the phosphopeptides are very easy to identify with the mass difference, which are all 80 Da between adjacent peaks in the same series, and clear background in the spectra owing to elimination of signal suppression from large amounts of nonphosphopeptides. Furthermore, the phosphopeptides can be dephosphorylated completely after a further NH(4)OH elution. Tryptic digest products from several standard proteins are pretreated using CeO(2) to demonstrate the efficiency of this method. Phosphopeptides from a very small quantity of human serum are enriched and analyzed, and proteins also identified by searching against a database using Mascot on MALDI-TOF/TOF fragments, which indicates that this method may be employed in complex samples for further application.     

Matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry of Pt(II) and Pd(II) complexes

In this work, we have analysed matrix-assisted laser desorption and ionisation time-of-flight (MALDI-TOF) mass spectra of [PtCl₂(en)], [PtCl₂(dach)] and [PdCl(dien)]Cl acquired either with 2,5-dihydroxybenzoic acid (DHB) or α-cyano-hydroxycinnamic acid (CHCA) as matrices. For certain experiments, small amounts of trifluoro acetic acid (TFA) or higher concentration of inorganic salts (NaCl or KCl) was added to the matrix solution. The majority of peaks arising from the Pt(II) and Pd(II) complexes could be identified, but certain ions detectable in the spectra were generated upon ligand loss. Additionally, the analysis of Pt(II) complexes was also possible in the presence of a higher salt content, which is a commonly used analysis condition for the samples of biological origin. While DHB appears to be the best suited for MALDI-TOF mass spectrometric analysis of Pt(II) complexes, CHCA seems to be a better matrix for Pd(II) complex used in this study. On the other hand, small amounts of TFA improve the spectra quality of Pt(II) complexes, but lead most probably to the degradation of Pd(II) complex. Taken together, we have demonstrated that the analysis of metallo-drugs using MALDI-TOF MS, though accompanied with certain identification problems, is easy and reliable. On the other hand, having in mind that some complexes (i.e. a combination of a particular transition metal/ligand) cannot be analysed under conditions usually applied for others, we deem it necessary to find out the best conditions for MALDI-TOF MS analysis of each metal complex.     

A second‐generation ionic liquid matrix‐assisted laser desorption/ionization matrix for effective mass spectrometric analysis of biodegradable polymers

A second generation ionic liquid matrix (ILM), N,N‐diisopropylethylammonium α‐cyano‐4‐hydroxycinnamate (DEA‐CHCA), was developed for the characterization of polar biodegradable polymers. It is compared with five solid matrices typically used for the characterization of these polymers and one other new ILM. It is shown that use of the ILM, DEA‐CHCA, allows maximum signal with minimum laser intensity which minimizes polymer degradation. In these conditions, the DEA‐CHCA ILM is able to assist in the ionization of analytes in an efficient but soft manner. These qualities produce spectra that allow an accurate and sensitive determination of the number average molecular weights, weight average m.w., and polydispersity index of labile polar polymers. With such polymers, many solid matrices produce spectra showing extensive polymer degradation leading to the underestimation of molecular weights. The distribution of intact analyte peaks obtained with the ILM DEA‐CHCA allows for identification of the fine structure of complex copolymers. ILMs were much less susceptible to effects of extraction delay times on molecular weight determination than were solid matrices. The liquid nature of the matrix is an important reason for the outstanding results obtained for labile analyte polymers. No comparable results could be obtained with any known solid matrices or other ILMs. In many cases, the manufacturers' listed molecular weights and polydispersity measurements for biodegradable polymers are determined by size‐exclusion chromatography and the data obtained by that method may differ considerably from the high‐precision matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) results presented here. Copyright © 2009 John Wiley & Sons, Ltd.     

Measuring salty samples without adducts with MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used for the discovery of hundreds of novel cell to cell signaling peptides. Beyond its advantages of sensitivity and minimal sample preparation requirements, MALDI MS is attractive for biological analyses as high quality mass spectra may be obtained directly from specific locations within prepared tissue sections. However, due to the large quantity of salts present in physiological tissues, these mass spectra often contain many adducts of cationic salts such as sodium and potassium, in addition to the molecular ion [M + H]⁺. To reduce the presence of cation adducts in MALDI mass spectra obtained directly from tissues, we present a methodology that uses a slow condensation procedure to enable the formation of distinct regions of matrix/analyte crystals and cation (salt) crystals. Secondary ion mass spectrometric imaging suggests that the salts and MALDI matrix undergo a mutually exclusive crystallization process that results in the separation of the salts and matrix in the sample.