HPLC-MS and MECC analysis of coumarins

Summary MECC separation of 11 coumarins has been achieved by use of a running electrolyte at pH 10.4 prepared from 50 mM boric acid, 10 mM sodium tetraborate and 100 mM sodium hydroxide. The buffer solution contained 50 mM SDS and, as organic modifier 1%n-propanol. The applied voltage was 25 kV and the temperature of the capillary was kept constant at 20°C. HPLC baseline separation of the coumarin mixture was obtained by use of a reversed-phase column and an acetonitrile-water solvent gradient. UV detection was performed at 205 nm. Peak assignment and purity control were achieved by HPLC-mass spectrometry with either an electrospray interface or an atmospheric-pressure chemical-ionization interface. Compounds were detected in either negative- or positive-ion modes. These MECC and HPLC-MS methods are suitable for ‘fingerprint’ analysis of a number of coumarin-containing plants, e.g. Fr.Ammi visnagae, Rd.Scopoliae and Rd.Imperatoriae.     

A capillary electrophoresis system with dual capacitively coupled contactless conductivity detection and electrospray ionization tandem mass spectrometry

A commercial system that is comprised of a CE coupled to an ESI triple quadrupole mass spectrometer was equipped with two capacitively coupled contactless conductivity detectors (C⁴Ds). The first C⁴D was positioned inside the original cartridge, and the second C⁴D was positioned as close as possible to the ESI probe entrance by using a 3D‐printed support. The C⁴Ds electropherograms were matched to the ESI‐MS electropherogram by correcting their timescales by the factor L_T/L_D, where L_T and L_D are the total capillary length and the length until the C⁴D, respectively. A general approach for method development supporting the simultaneous conductivity and MS detection is discussed, while application examples are introduced. These examples include the use of C⁴D as a simple device that dismiss the use of an EOF marker, a low‐selectivity detector that continuously provide information about unexpected features of the sample, and even a detector that can be more sensitive than ESI‐MS. The C⁴D used in this setup proved to have a smaller contribution to the peak broadening than ESI‐MS, which allowed that a C⁴D, positioned at 12 cm from the inlet of an 80‐cm‐long capillary, could be used to foresee position and shape of the peaks being formed 6.8 times slower at the ESI‐MS electropherogram.     

Capillary electrophoresis as an alternative to HPLC for determination of honey flavonoids

Summary The general objective is to provide an alternative methodology based on capillary electrophoresis (CE) to characterize flavonoids from honey and hence determine its botanical origin. The specific objective is to compare the separation of flavonoids by CE with those achieved by HPLC to assess CE as an alternative technique for the determination of honey flavonoids. Fourteen different flavonoids isolated from honey were analysed by MECC and compared to the HPLC separations. It was difficult to find specific experimental conditions to separate all the flavonoids from honey in a single MEKC run. Three chromatographic conditions are optimized and, depending on the flavonoid markers sought in honey, the appropriate detection method should be chosen. Compared to the HPLC results, it is clear that CE could be an alternative technique in honey flavonoids analysis and particularly in the study of its geographical and floral origin.     

Analysis of oligonucleotides using coupled high performance liquid chromatography-electrospray mass spectrometry

Summary Improved HPLC and ESMS conditions have been established, allowing the separation and analysis of oligodesoxyribonucleotides by coupled HPLC-ESMS.     

Analysis of human transferrin glycopeptides by capillary electrophoresis and capillary liquid chromatography-mass spectrometry. Application to diagnosis of alcohol dependence

In this study, capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry (CE-TOF-MS and μLC-TOF-MS) were used to detect and characterise human transferrin (Tf) glycopeptide glycoforms obtained by tryptic digestion. After selecting μLC-TOF-MS because of improved performance in analysis of N₄₁₃ and N₆₁₁ glycopeptide glycoforms, the proposed methodology was applied to serum samples. Two immunoaffinity columns were employed to isolate Tf from serum samples. Both columns were activated with the same anti-Tf antibody but using two different bonding chemistries. After immunoaffinity purification and digestion, serum samples from a teetotal individual (as control) and from individuals with low and high alcohol dependence were analysed by μLC-TOF-MS. Relative abundance of each glycoform was useful to estimate the degree of alcohol dependence of each individual. Finally, the established methodology was used to analyse serum samples from specific individuals with an unknown degree of alcohol dependence.     

Coupling of capillary electrophoresis with electrospray ionization multiplexing ion mobility spectrometry

In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two‐dimensional separation. To improve the performance of hyphenated CE‐IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5–250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE‐ESI‐IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra‐violet transparent compounds or none fluorescent compounds.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Twenty years of interface development for capillary electrophoresis-electrospray ionization-mass spectrometry

Capillary electrophoresis-electrospray ionization-mass spectrometry has the potential to become a preferred tool for the analysis of biological mixtures and other complex samples. The development of improved interfaces in the past twenty years has been critical in demonstrating the feasibility of this technique. However, a compromise still exists between interfaces that give optimal performance and those that are practical for commercial applications. The first section of this review focuses on the technological advances in CE-ESI-MS as they relate to the key interface features for both sheath-flow and sheathless systems: delivery of the sheath liquid, shaping of the emitter tip, formation of electrical contact, and practicality in terms of ease of use and lifetime. In the second section, we review the fundamental processes that affect interface performance. Because of the complex natures of both capillary electrophoresis and electrospray ionization, flow rate, arrangement of the electrical circuit, electrochemistry, tip geometry and location of electrical contact must all be carefully managed in the design of a successful interface.     

Impact of solvent conditions on separation and detection of basic drugs by micro liquid chromatography-mass spectrometry under overloading conditions

In this study the impact of solvent conditions on the performance of μLC/MS for the analysis of basic drugs was investigated. Our aim was to find experimental conditions that enable high-performance chromatographic separation particularly at overloading conditions paired with a minimal loss of mass spectrometric detection sensitivity. A focus was put on the evaluation of the usability of different kinds of acidic modifiers (acetic acid (HOAc), formic acid (FA), methansulfonic acid (CH₃SO₃H), trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), and heptafluorobutyric acid (HFBA)). The test mixture consisted of eleven compounds (bunitrolol, caffeine, cocaine, codeine, diazepam, doxepin, haloperidol, 3,4-methylendioxyamphetamine, morphine, nicotine, and zolpidem). Best chromatographic performance was obtained with the perfluorinated acids. Particularly, 0.010–0.050% HFBA (v/v) was found to represent a good compromise in terms of chromatographic performance and mass spectrometric detection sensitivity. Compared to HOAc, on average a 50% reduction of the peak widths was observed. The use of HFBA was particularly advantageous for polar compounds such as nicotine; only with such a hydrophobic ion-pairing reagent chromatographic retention of nicotine was observed. Best mass spectrometric performance was obtained with HOAc and FA. Loss of detection sensitivity induced by HFBA, however, was moderate and ranged from 0 to 40%, which clearly demonstrates that improved chromatographic performance is able to compensate to a large extent the negative effect of reduced ionization efficiency on detection sensitivity. Applications of μLC/MS for the qualitative and quantitative analysis of clinical and forensic toxicological samples are presented.     

Analysis of pyrimidine derivatives by capillary electrophoresis

Summary A capillary electrophoretic method has been developed for the determination of the main product as well as of by-products in technical samples of substituted pyrimidines. Both zone electrophoresis and micellar electrokinetic chromatography have been used for the separation employing electrolytes consisting of borate buffers (pH 9 to 9.4) with or without sodium dodecylsulfate. Optimization of separation selectivity could be achieved by addition of up to 20% 2-propanol or methanol to the carrier electrolyte. Quantification by internal standards resulted in relative standard deviations between 0.2 and 0.8%. By-products could be analyzed down to levels of 0.1% in technical samples.

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Analyse von Pyrimidinderivaten mitteles Kapillarelektrophorese

Zusammenfassung Für die Bestimmung von Haupt- und Nebensubstanzen in technischen Proben von substituierten Pyrimidinen wurde ein kapillarelektrophoretisches Analysenverfahren entwickelt. Sowohl Zonenelektrophorese als auch mizellare elektrokinetische Chromatographie mit Trägerelektrolyten bestehend aus Boratpuffern (pH 9 bis 9.4) mit oder ohne Natriumdodecylsulfat wurden für die Trennung eingesetzt. Eine Optimierung der Trennselektivität war durch die Zugabe von bis zu 20% 2-Propanol oder Methanol zum Trägerelektrolyten möglich. Quantifizierung mittels interner Standards ergab relative Standardabweichungen zwischen 0.2 und 0.8%. Nebenprodukte konnten in technischen Proben bis zu Gehalten von 0.1% analysiert werden.

     

啤酒中单糖的衍生化HPLC-ESI-MS测定方法研究

单糖类样品在溶液中非常稳定,难于离子化,不适合于进行ESI-MS检测。采用1-苯基-3-甲基-5-吡唑啉酮(PMP)将糖类物质衍生化,HPLC-ESI-MS在线联用,选择性离子扫描方式对几种啤酒样品中的5种单糖进行了分离检测。检出限可达到80pg。     

Determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry

The determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) was investigated. Both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes of operation were studied. The optimal separation of four cobalamin species (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5′-deoxyadenosylcobalamin) and a potentially harmful corrinoid analogue (cobinamide dicyanide) was obtained using CZE at a pH of 2.5. Both 20 mM phosphate and 20 mM formate buffers were used with success, although the formate buffer provided improved resolution. The CZE-ICP-MS method was used to quantify cyanocobalamin in a vitamin supplement and the analytical results were in good agreement (±5%) with values obtained by ICP-MS for total Co levels. The solution detection limits for cobalamins using CZE-ICP-MS were approximately 50 ng/ml. MEKC was found to be useful for the screening of vitamin preparations because it provided a rapid means of distinguishing cyanocobalamin (the form most commonly used in vitamin preparations) from free cobalt. The separation of free cobalt and cyanocobalamin using MEKC was achieved in less than 10 min.     

Separation and identification of higher fullerenes in soot extract by liquid chromatography-mass spectrometry

Summary Two-step liquid chromatographic separation (LC) has been applied to soot extract and the identification of higher fullerenes has been accomplished by LC-MS measurements using an ESI interface. The first separation step is preparative-scale LC using a 50 mm i.d. column packed with monomeric octadecylisilica (ODS) because elution is mainly controlled by relative molecular mass. 39 batches of five fractions each were collected and then as the second separation step each fraction was analysed by analytical-scale LC using a conventional column of a polymeric ODS phase which can elute fullerenes according to shape and structure. This stationary phase can also separate many isomers of higher fullerenes, consequently the existence of several higher fullerenes larger than C₈₆ has been confirmed and their UV-Vis spectra were obtained by the photodiode array detection system coupled to the analytical LC.     

Comprehensive ultra-high pressure capillary liquid chromatography/ion mobility spectrometry

Summary An ultra-high pressure liquid chromatograph was interfaced to a moderately high-resolution nanoelectrospray ionization ion mobility spectrometer operating at ambient temperature and pressure to achieve fast multidimensional separations. The potential of using ion mobility spectrometry as the second dimension in a comprehensive arrangement with liquid chromatography to offer improved qualitative information for compounds under specific operating conditions, is discussed. Separation and detection of selected benzodiazepines and triazine herbicides are demonstrated. Composite peak capacities of 39 and 33 for benzodiazepine and triazine herbicide mixtures, respectively, were achieved in less than 75 s using a 16.5 cm×50 μm internal diameter fused silica capillary liquid chromatographic column packed with 1.5 μm diameter ODS-bonded silica particles.     

Separation techniques hyphenated to electrospray-tandem mass spectrometry in proteomics: capillary electrophoresis versus nanoliquid chromatography

Liquid chromatography (LC) nanoelectrospray-tandem mass spectrometry (MS/MS) is a key technology for the study of proteomics, with the main benefit to the characterization of sensitive peptides from complex mixtures. Capillary electrophoresis coupled to mass spectrometry (MS) has been taken into consideration sporadically due to the highly efficient separation and ability to handle low sample amount, yet classified as being less sensitive with respect to analyte concentration. The limitation in capillary zone electrophoresis (CZE) injection volumes can be overcome by on-line solid-phase extraction (SPE). Such an on-line SPE-CZE system was explored in combination with an ion trap (IT) mass spectrometer. Thus, it was possible to inject more than 100 microL sample solution on to the CZE capillary. Concentration limits of detection as low as 100 amol/microL were demonstrated for a peptide standard. This SPE-CZE-microelectrospray ionization (ESI)-MS/MS setup was compared directly to nanoLC/nanoESI using the same sample of a tryptic digest of bovine serum albumin (BSA) as a reference standard. Measurements were made on one IT mass spectrometer with identical acquisition parameters. Both chromatography systems enabled the separation and detection of low levels of peptides from a mixture of moderate complexity, with most peptides identified using both techniques; however, specific differences were obvious. The nanoLC-MS is about five times more sensitive than the CZE-MS, yet the difference was less pronounced than expected. The CZE-MS technique showed reduced loss of peptides, especially for larger peptides (missed cleavages) and is about four times faster than the nanoLC-MS approach.     

Separation by capillary zone electrophoresis of the active anthraquinone components of the Chinese herbPolygonum multiflorum Thunb

Summary An optimization strategy was applied to explore the capability of hybrid micellar eluents of sodium dodecyl sulphate (SDS), using acetonitrile or pentanol as modifiers, to resolve mixtures of eleven steroids showing a wide range of hydrophobicity (clostebol acetate, dehydrotestosterone, dydrogesterone, medroxyprogesterone, medroxyprogesterone acetate, methandienone, methyltestosterone, progesterone, testosterone, testosterone enanthate and testosterone propionate). The accurate prediction of the retention behaviour of the steroids, with relative errors in the 0.8–1.7% and 0.4–2.9% ranges for SDS-acetonitrile and SDS-pentanol eluents, respectively, demonstrated the reliability of the methodology. Acetonitrile and pentanol had a complementary effect in these analyses. The elution strength of acetonitrile was weaker, but allowed higher efficiencies. A 0.094 M SDS-16% acetonitrile eluent separated seven steroids (dehydrotestosterone, dydrogesterone, medroxyprogesterone, medroxyprogesterone acetate, methandienone, methyltestosterone and testosterone) in 27 min, while the most hydrophobic steroids were strongly retained. In contrast, a 0.125 M SDS-5.8% pentanol eluent permitted the elution of a mixture of eight steroids (dehydrotestosterone, dydrogesterone, medroxyprogesterone acetate, methandienone, methyltestosterone, progesterone, testosterone enanthate and testosterone propionate) of diverse hydrophobicity in 14 min. With this eluent, however, the peaks of dehydrotestosterone-medroxyprogesterone and methandienone-testosterone were highly overlapping.     

Analysis of diastereoisomer impurities in chiral pharmaceutical compounds by capillary electrophoresis

Summary Micellar electrokinetic capillary chromatography (MECC) has been applied to the separation and analysis of diastereoisomer impurities in chiral pharmaceutical compounds. Differences in separation mechanism and selectivity make MECC useful as an alternative method to HPLC for analysis of these synthetic inpurities. Advantages of MECC include high efficiency separations and low consumption of sample and solvents. Water soluble and insoluble pharmaceutical compounds are used to illustrate the separation characteristics and quantitative capabilities of this versatile new analytical technique.     

Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.     

Micellar electrokinetic capillary chromatography as an alternative method for determination of hydrocortisone and its most important associated compounds in local pharmaceutical preparations

Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone, hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when compared with reference spectrophotometric and HPLC methods.     

Analysis of lignin degradation products by capillary electrophoresis

Summary A method for the quantitative analysis of phenolic lignin degradation products by capillary electrophoresis (CE) with on-column UV detection has been developed. The liquid biomass solutions contain low molecular hemicellulosic sugars and phenolic lignin degradation products with various degrees of polymerization. Special attention has been paid to the monomeric phenolic components of lignin degradation fragments, e.g. derivatives of phenolic acids, aldehydes, and alcohols. Uncoated fused silica capillaries and borate-phosphate buffer systems at moderate pH conditions were used in order to separate the compounds of interest. To provide validation of the method, the same samples were analyzed independently by HPLC using gradient elution on a RP-C18 column. As sugar degradation fragment, furan-2-carboxylic acid was detected.