Semi-Quantitative Determination of Free Cyclic and Linear Polytetrahydrofurane in Coatings Using Critical Chromatography

Summary Liquid chromatography at critical conditions (LCCC) is being used more frequently, especially for the determination of cyclic structures in a matrix of the parent linear molecules, analyses that cannot be performed easily by other techniques. In practice, analyses are often more complex than separating the linear from the cyclic structures, e.g. in the determination of low concentrations of free (linear and cyclic) polyols like polytetrahydrofurane (pTHF) in functional polymers and coatings. To this end different chromatographic phase systems at the critical conditions for the polyols were investigated to obtain the maximum amount of information. The identification of different oligomeric series was performed by means of an on-line coupling of LCCC with atmospheric pressure chemical ionization – mass spectrometry (APCI-MS). Mass summated chromatograms were introduced to improve the signal to noise ratio of specific oligomeric series. MS cannot be used for quantitative purposes here; therefore the evaporative light scattering detector (ELSD) and the refractive index (RI) detectors were evaluated for routine analyses. Specific tailing problems observed with a drift tube in the ELSD are discussed. Two LCCC systems (reversed phase and normal phase) were compared for best selectivityPresented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

On-line coupling of liquid chromatography at critical conditions with electrospray ionization tandem mass spectrometry for the characterization of a nitroxide-mediated poly(ethylene oxide)/polystyrene block copolymer

Coupling of liquid chromatography at critical conditions (LCCC) with on-line mass spectrometry (MS) detection was implemented via an electrospray ionization (ESI) interface, using a mobile phase containing the cationizing agent. Critical conditions established for poly(ethylene oxide) were used to characterize a poly(ethylene oxide)/polystyrene block copolymer (PEO-b-PS) in both MS and MS/MS modes. As co-oligomer molecules were successfully separated according to the PS block size, structural information could be reached from simplified MS spectra. The microstructure of this copolymer, synthesized by nitroxide-mediated polymerization, could further be unambiguously characterized in LCCC/ESI-MS/MS experiments since the PS block size could be reached by both the co-oligomer chromatographic behavior and its MS/MS pattern.     

Characterization of poly(ethylene glycol)-b-poly(ε-caprolactone) by liquid chromatography under critical conditions: Influence of catalysts and reaction conditions on product composition

Amphiphilic block copolymers were synthesised by ring opening polymerization of ε-caprolactone with polyethylene glycol monomethyl ether (PEG-MME) using different catalysts (boron trifluoride, sodium hydride, and tin octoate) in a one pot procedure. The products obtained were characterized with respect to their molar mass distribution and content of homopolymers using size exclusion chromatography (SEC), Liquid chromatography under critical conditions (LCCC) and MALDI-TOF-MS. The homopolymers of caprolactone could be separated from the block copolymer by LCCC on a reversed phase column in tetrahydrofurane-water mobile phases with evaporative light scattering detection (ELSD). Residual monomer could be determined under the same conditions using density detection, a separation of the copolymer from residual initiator could be achieved on a normal phase column in acetone-water mobile phases.     

Endgroup-based separation and quantitation of polyamide-6,6 by means of critical chromatography

Polyamide-6,6 is a polycondensation product from the two monomers adipic acid and 1,6-hexamethylenediamine. Depending on the reacted amount of these monomers, different ratios of amine and carboxylic acid endgroups can be formed. Besides linear chains, cyclic polyamides will also be formed. Using critical chromatography, polyamide-6,6 can be separated independently of molar mass. Retention is based solely on endgroup functionality. It is demonstrated that high-molecular-mass polyamide-6,6 (Mw approximately 20,000-30,000) can be separated using this approach. The separation was optimized by using different parameters, such as percentage modifier, temperature and pressure. The concentration of phosphoric acid was used for selective retention of the different endgroup functionalities. Using this property, a new method called critical gradient chromatography was performed where the mobile phase changes from a weak to a strong solvent with respect to the endgroup functionality, while retaining the critical conditions of the backbone unit. Quantification using UV detection is discussed.     

Screening and sequencing of complex sialylated and sulfated glycosphingolipid mixtures by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

A protocol for negative ion nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (-)nanoESI-FTICR MS, investigation of complex biological mixtures consisting of sialylated or sulfated glycosphingolipids (GSL) expressing high heterogeneity in the ceramide portion is described. Different instrumental and solvent conditions were explored and optimized to promote efficient ionization, reduce the in-source fragmentation and consequently enhance the detection of intact molecular species from complex mixtures. Using the novel optimized (-)nanoESI-FTICR MS protocol, a reliable and detailed compositional fingerprint of the polysialylated ganglioside mixture isolated from human brain was obtained. Sustained off-resonance irradiation collision-induced dissociation mass spectrometry (SORI-CID MS2) was introduced for the first time for structural elucidation of polysialylated gangliosides. Under well-defined conditions, an informative fragmentation pattern of the trisialylated ganglioside GT1 was obtained. The compositional mapping of a complex mixture of sulfated glucuronic acid containing neolacto-series GSLs extracted from bovine Cauda equina provided hard evidence upon previously described components and new structures not identified before by any other analytical method. Negative ion nanoESI-FTICR MS at 9.4 T is shown here to represent a valuable method in glycolipidomics, allowing a high resolution and mass accuracy detection of major and minor GSL glycoforms and identification of known and novel biologically relevant structures.     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

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.     

Preparative isolation and purification of chemical constituents from the root of Adenophora tetraphlla by high-speed counter-current chromatography with evaporative light scattering detection

Preparative high-speed counter-current chromatography (HSCCC), as a continuous liquid-liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of non-chromophoric chemical components from Chinese medicinal herb Adenophora tetraphlla (Thunb.), Fisch. Nine compounds, including alpha-spinasterol, beta-sitosterol, nonacosan-10-ol, 24-methylene cycloartanol, lupenone, 3-O-palmitoyl-beta-sitosterol, 3-O-beta-d-glucose-beta-sitosterol, eicosanoic acid and an unknown compound, were obtained. The compounds were all above 95% determined by high-performance liquid chromatography (HPLC)-ELSD, and their structures were identified by (1)H NMR and chemical ionization mass spectroscopy (CI-MS). The results demonstrate that HSCCC coupled with ELSD is a feasible and efficient technique for systematic isolation of non-chromophoric components from traditional medicinal herbs.     

Characterization of linear and cyclic polylactic acids and their solvolysis products by electrospray ionization mass spectrometry

Linear and cyclic polylactic acids (PLAs) were characterized using electrospray ionization mass spectrometry (ESI-MS) as part of our ongoing investigation of the hydrolysis mechanism of biodegradable polymers. The condensation oligomers of linear polylactic acid (LPLA) were synthesized by thermal dehydration of L-lactic acid. The trimer and tetramer base polymers of cyclic polylactic acid (CPLA) were obtained by cyclization reactions of lactic acid trimers and tetramers, respectively. In the ESI-MS/MS measurement, LPLA yielded three types of product ion series, while CPLA yielded only one type, from which the repeated units of CPLA were removed. The MS/MS spectrum of the NH4+ adduct ion for both cyclic and linear PLA showed loss of one ammonia molecule. The postsource decay (PSD) spectrum of CPLA by matrix-assisted laser desorption ionization (MALDI) mass spectrometry was similar to the ESI-MS/MS spectrum, while that of LPLA was different. In addition, the degradation of cyclic and linear PLAs by solvolysis was investigated. Solvolysis with anhydrous MeOH was quite feasible, but did not readily occur in the presence of even a small amount of water in the MeOH solvent.     

Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry

Techniques in mass spectrometry (MS) combined with chemical cross-linking have proven to be efficient tools for the rapid determination of low-resolution three-dimensional (3-D) structures of proteins. The general procedure involves chemical cross-linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. We present a method for mapping low-resolution 3-D protein structures by combining chemical cross-linking with high-resolution FTICR (Fourier transform ion-cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo-bifunctional, amine-reactive cross-linking reagents that bridge distances from 6 to 16 A. The non-digested cross-linking reaction mixtures were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to determine the extent of cross-linking. Enzymatically digested reaction mixtures were separated by nano-high-performance liquid chromatography (nano-HPLC) on reverse-phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-ESI (electrospray ionization) source. Cross-linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross-linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross-linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme.     

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

高效液相色谱/电喷雾飞行时间质谱分析太子参中环肽类化合物

采用高效液相色谱/电喷雾飞行时间质谱联用方法(HPLC/ESI-TOFMS)分析太子参中的环肽类化合物。实验采用反相C18色谱柱,二元线性梯度洗脱,分离并检测了太子参中6种环肽类化合物;通过与电喷雾飞行时间质谱联用获得了这几种化合物的准确分子量信息,由于ESI-TOFMS具有高分辨率,能够测定化合物精确的分子质量而不降低灵敏度,对6种环肽类化合物成分进行了定性鉴定。该方法简便、快速、准确。     

Rapid and simple liquid chromatography tandem mass spectrometry method for the quantification of zidovudine in rat plasma

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of zidovudine in rat plasma. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 268/127 for zidovudine and m/z 230/112 for the internal standard. The method exhibited a linear dynamic range of 5-500 ng/mL for zidovudine in rat plasma. The lower limit of quantification was 5 ng/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 plasma samples per day. The validated method was applied for pharmacokinetic studies of the novel drug delivery systems of zidovudine in rats.     

Identification and quantitation of trace impurities in fatty alcohol ethoxylates using HPLC and MALDI-TOF mass spectrometry

A two-step HPLC separation that combines isocratic and gradient elution has been developed for the analysis of trace impurities in fatty alcohol ethoxylates (FAEs). Quantitative estimates of the early eluting impurities were obtained from a calibration curve based on the responses of an evaporative light scattering detector (ELSD). Isocratic conditions were used to elute the impurities and gradient conditions for the main FAE components. In this manner, an acceptable compromise was obtained between resolution and analysis time. Fractions were collected and the impurities identified using MALDI-TOF mass spectrometry.     

基于诊断离子策略的超高压液相色谱-线性离子阱-轨道离子阱质谱联用技术解析中药虎杖的化学成分

建立了基于超高压液相色谱-电喷雾-高分辨质谱联用技术(UHPLC-ESI-HRMS)的中药虎杖化学成分的快速定性分析方法.采用Waters UPLC C₁₈色谱柱,乙腈-甲酸水系统梯度洗脱,采用ESI-LTQ-Orbitrap高分辨杂交质谱技术负离子采集,线性离子阱质谱进行多级质谱碎裂,轨道离子阱高分辨检测,获取多维质谱数据.利用代表性成分的诊断离子进行搜寻,结合质谱偶电子规律、氮规律、不饱和度和同位素峰等信息,参阅文献,总结主要成分的裂解规律,对主要峰进行指认与鉴定.结果显示,整个分析过程在10 min内完成,该方法分离良好,灵敏度高.对虎杖根中的34种成分进行了结构分析,研究从中药虎杖中发现了多种酚酸取代二苯乙烯苷、酚酸取代蒽醌苷和蒽醌硫酸盐衍生物等成分.研究结果表明本方法为虎杖的后续化学成分研究、相关产品的质量控制和药效物质基础的研究提供了一种高效、可靠的检测手段.     

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.     

Polyketide analysis using mass spectrometry,evaporative light scattering,and charged aerosol detector systems

In this study, a mass spectrometer (MS), an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD) were used to analyze an erythromycin precursor (termed 6-deoxyerythronolide B). The work highlights the capabilities of each detector to analyze a representative polyketide compound that does not possess a natural chromophore, and presents the first comparison to include a charged aerosol system. Each detector was evaluated based upon limit of detection (LOD), dynamic range, and precision in the context of polyketide analysis. Due to its low LOD, wide dynamic range, and ability to provide molecular weight information, the MS was deemed the best detection option for the analysis of low-concentration, poorly identified polyketide compounds. Alternatively, both the CAD and ELSD systems studied showed better precision and accuracy. The ELSD demonstrated the best precision at 3%, but its LOD was limited to concentrations primarily greater than or equal to 1 mg/L. The Corona CAD demonstrated a LOD (0.012 mg/L) and dynamic range comparable to mass spectroscopy and therefore serves as a more cost-efficient alternative for polyketide production schemes with low titers.     

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.     

Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia

An approach for the separation and identification of components in a traditional Chinese medicine Psoralea corylifolia was developed. Ion-exchange chromatography (IEC) was applied for the fractionation of P. corylifolia extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further separated on an ODS column with detection of UV absorbance and atmospheric pressure chemical ionization mass spectrometer (APCI/MS), respectively, and also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes. Totally more than 188 components in P. corylifolia extract were detected with this integrated approach, and 12 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS. The obtained analytical results not only demonstrated the powerful resolution of integration IEC fractionation with reversed-phase liquid chromatography (RPLC)-APCI/MS and MALDI-TOF/MS for analysis of compounds in a complex sample, but also exhibited the superiority of APCI/MS and MALDI-TOF/MS for identification of low-mass compounds, such as for study of traditional Chinese medicines (TCMs) and metabolome.     

Determination by MALDI-TOF MS of the structures obtained from polytransesterification of diethyl 2,6-pyridinedicarboxylate and poly(ethylene glycol)

Polyesters from reaction of diethyl 2,6-pyridinedicarboxylate with poly(ethylene glycol) 1000 were synthesized through a polytransesterification process using titanium(IV) alkoxide as catalyst. The resulting polyesters were characterized by size-exclusion chromatography (SEC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The structures of the polyesters were established and revealed the formation of several cyclic and linear populations of varying size. Detailed analysis of the MALDI-TOF mass spectra of the obtained polyesters has revealed the presence of linear polycondensates bearing various end groups among which those attributed to an exchange reaction between alkyl groups of the catalyst and ester groups of the polycondensates.