Development of sandwich HPLC microcolumns for analyte adsorption on the millisecond time scale

A new class of columns is reported that uses only microgram quantities of active support and that provides for the retention of biological compounds and other analytes on the millisecond time scale. This was accomplished by packing standard HPLC supports into layers as small as 60 microm in length and using only 90 microg of support material. This provided columns with effective residence times in the millisecond time range when routine HPLC flow rates and pressures were used. The retention of analytes by such columns was examined under both adsorption- and diffusion-limited conditions. The RPLC adsorption of hemoglobin (a system with diffusion-limited retention) was found to give 95% binding in as little as 4 ms. The adsorption of fluorescein by an anti-fluorescein antibody column (an adsorption-limited system) gave 95% retention in 100-120 ms. One application examined for these columns was their use in a chromatographic-based competitive binding immunoassay. This used bovine serum albumin (BSA) as the model analyte, and fluorescein-labeled BSA was used for detection. The resulting approach had a contact time of 180 ms between the sample and an anti-BSA immunoaffinity microcolumn and provided a signal within 5-25 s after sample injection. The columns developed in this work should also be useful in other situations that involve a small amount of a stationary phase or that require short column residence times.     

Assignment and quantification of 2-aminopyridine derivatized oligosaccharide isomers coeluted on reversed-phase HPLC/MS by MSn spectral library

2-Aminopyridine (PA)-derivatized oligosaccharides from IgG were analyzed by using reversed-phase HPLC/mass spectrometry (RP-HPLC/MS) and a MS(n) spectral library, in particular, focusing on two pairs of isomers incompletely separated or coeluted in chromatograms. We previously reported that MS(n) spectral matching considering both major fragment ions (m/z) and intensities is useful and applicable to the structural assignment of PA-oligosaccharide isomers. In this study, MS(n) spectral matching based on the MS(n) spectral library was applied to the assignment of these PA-oligosaccharide isomers in IgG. Its usefulness was investigated by comparing it to the conventional two-dimensional mapping method based on retention time indexes. Specifically, we focus on the assignment and quantification of the isomers, which are coeluted in chromatograms. From this, we propose a new method using MS(n) spectral matching and the working curve on which are plotted the relative intensities of selected fragment ions in their MS(2) spectra versus various mixtures of the isomers. This new method demonstrated that the obtained quantities coincide very well with those estimated after separating by a combination of lectin and reversed-phase columns. This means that separation by RP-HPLC/MS is greatly simplified because complete separation of the isomers is no longer required. Application of this new method was tested by using the two other pairs of fucosylated and nonfucosylated PA-oligosaccharides from IgG. The results showed that this method works for them as well.     

Simultaneous characterization of perfluoroalkyl carboxylate, sulfonate, and sulfonamide isomers by liquid chromatography-tandem mass spectrometry

A comprehensive method was developed to simultaneously separate and detect perfluorinated acid (PFA) and PFA-precursor isomers using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A linear perfluorooctyl stationary phase and acidified mobile phase increased separation efficiency, relative to alkyl stationary phases, for the many perfluoroalkyl carboxylate (PFCA), perfluoroalkyl sulfonate (PFSA), and perfluorooctyl sulfonamide (PFOSA) isomers and in combination with their distinct MS/MS transitions allowed full resolution of most isomers in standards. Utilizing the absence of the "9-series" and "0-series" product ions, several perfluorooctane sulfonate (C8F17SO3-, PFOS) isomers were structurally elucidated. In human serum, only perfluorooctane sulfonamide (C8F17SO2NH2, FOSA) and PFOS consisted of significant quantities of branched isomers, whereas PFCAs were predominantly linear. Interferences that coelute with the m/z 499 --> 80 transition of PFOS on alkyl stationary phases were simultaneously separated and identified as taurodeoxycholate isomers, removal of which permitted the use of the more sensitive m/z 80 product ion and a resulting 20-fold decrease in PFOS detection limits compared to the m/z 499 --> 99 transition (0.8 pg versus 20 pg using m/z 80 and 99, respectively). Interferences in human serum which caused a 10-20-fold over-reporting of perfluorohexane sulfonate (C6F13SO3-, PFHxS) concentrations on alkyl stationary phases were also simultaneously separated from linear PFHxS and identified as endogenous steroid sulfates. PFOSA isomers, generated with human microsomes, had different rates of metabolism, suggesting that the perfluoroalkyl branching pattern may affect the biological properties of individual isomers. This fact, and for reasons of improved accuracy and sensitivity, investigators are urged to utilize more efficient separation methods capable of isomer characterization in perfluoroalkyl research.     

High-efficiency liquid chromatographic separation utilizing long monolithic silica capillary columns

Long monolithic silica-C18 capillary columns of 100 microm i.d. were prepared, and the efficiency was examined using reversed-phase HPLC under a pressure of up to 47 MPa. At linear velocities of 1-2 mm/s, 100,000-500,000 theoretical plates could be generated with a single column (90-440 cm in length) using an acetonitrile-water (80/20) mobile phase with a column dead time (t0) of 5-40 min. It was possible to prepare columns with a minimum plate height of 8.5 +/- 0.5 microm and permeability of (1.45 +/- 0.09) x 10(-13) m(2). The chromatographic performance of a long octadecylsilylated monolithic silica capillary column was demonstrated by the high-efficiency separations of aromatic hydrocarbons, benzene derivatives, and a protein digest. The efficiency for a peptide was maintained for an injection of up to 0.5-2 ng. When three 100 microm i.d. columns were connected to form a 1130-1240 cm column system, 1,000,000 theoretical plates were generated for aromatic hydrocarbons with retention factors of up to 2.4 with a t0 of 150 min. The fact that very high efficiencies were obtained for the retained solutes suggests the practical utility of these long monolithic silica capillary columns.     

多变量问题等值线形成的数学方法及物理意义

本文应用广义逆解法,将多变量散乱数据曲面(超曲面)拟合,把拟合后的曲面(超曲面)作为多变量问题等值线形成的数学模型,从而把超曲面、等值面、等值线这些几何概念,与等值线形成的物理意义结合起来,使人们能够从离散数据中揭示出所蕴含的物理规律。     

Advantages of the Use of Very Short and Ultra Short Hplc Columns for Drug Analysis in Dissolution Testing

The advantages of short (3–5cm long) or ultra short (4mm long) HPLC columns for drug analysis in dissolution testing are illustrated by reference to some example antihypertensive drug formulations.

Advantages include: selectivity, where interfering excipients or co-formulated drugs complicate UV spectrophotometric analysis: time savings, compared with conventional HPLC columns (although where UV spectrophotometry is applicable no time advantages may be obtained with short column HPLC): economy, as the columns are less expensive than conventional columns and there is reduced solvent consumption: increased sensitivity compared with conventional columns. Low dose potent. drugs with poor chromophores may be more readily quantitated: amenity to automation, including the use of laboratory robots.

These advantages would suggest the wide applicability to this mode of analysis in dissolution testing.     

Development of HPLC/ESI-MS and HPLC/1H NMR methods for the identification of photocatalytic degradation products of iodosulfuron

In the present study, HPLC/ESI-MS and stopped-flow HPLC/1H NMR methods were developed and applied to separate and characterize the byproducts arising from TiO2-catalyzed photodegradation of the herbicide iodosulfuron methyl ester (IOME) in aqueous solution under UV irradiation. Prior to identification, irradiated solutions of IOME (200 and 1000 mg.L(-1)) were concentrated by solid-phase extraction using two cartridges: Isolute C18 and Isolute ENV+. Analytical separation was achieved on a C18 reversed-phase column with ACN/H2O (HPLC/MS) or ACN/D2O (HPLC/NMR) as mobile phase and a linear gradient with a chromatographic run time of 35 min. The combination of UV and MS data allowed the structural elucidation of more than 20 degradation products, whereas 1H NMR data permitted an unequivocal confirmation of the identities of major products and the differentiation of several positional isomers, in particular, the hydroxylation isomers. The obtained results permitted us to propose a possible degradation scheme and to put in evidence the presence of privileged sites for the attack of OH radicals. This work shows, for the first time, the application of combined HPLC with UV, MS, and NMR detection for complete structural elucidation of photocatalytic degradation products, and it will be of particular value in studies on the elimination of pollutants in aqueous solutions by photocatalysis.     

Performance of monolithic silica capillary columns with increased phase ratios and small-sized domains

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecylsilylated monolithic silica column with the smallest domain size (through-pores of approximately 1.3 microm and silica skeletons of approximately 0.9 microm) showed a plate height of less than 5 microm at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of approximately 1, and approximately 7 microm at u = 8 mm/s. With a permeability similar to that of a column packed with 5-microm particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-microm particles, and showed performance in the "forbidden region" for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15,000-30,000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns.     

Determination of monosaccharide anhydrides in atmospheric aerosols by use of high-performance liquid chromatography combined with high-resolution mass spectrometry

A novel method for determination of the monosaccharide anhydrides (MAs) levoglucosan, mannosan, and galactosan in atmospheric aerosols has been developed. The method is based on solvent extraction of aerosol filter samples and chemical analysis performed with high-performance liquid chromatography (HPLC) combined with time-of-flight high-resolution mass spectrometry. The MAs were separated on two series connected 2.1 mm x150 mm reversed-phase HPLC columns and identified by negative electrospray ionization mass spectrometry using the m/z 113, 129, and 161 as monitoring ions. The limit of quantification (LOQ) at S/N 10 ranges between 20 and 40 pg for the three isomers. The LOQ can easily be improved. Samples from Elverum (urban background) and Oslo (urban) in Norway were used in validation experiments. Tetrahydrofuran was found to be the most efficient extraction solvent. The choice of solvent is of crucial importance to minimize adsorption to the glassware. Interactions between the MAs and active sites on the quartz filter surface are observed.     

Development of an Implantable,Biodegradable, Controlled Drug Delivery System for Local Antibiotic Therapy

Abstract

A novel drug delivery system was developed using a monoglyceride (Glycerol Monostearate) and a water-soluble release rate modifier as the matrix. Cefuroxime sodium (Zinacef®) was chosen as a model drug in this study. Formulations (cylindrical implants 6 × 6 mm) were prepared by a melt-dispersion method. Dissolution studies were performed using USP paddle method. The effect of glycerol, PEG 400 and their combination on drug release profiles was studied. Two assay methods (UV and HPLC) for cefuroxime analysis were compared. Percent recovery from four formulations (A-D) was higher with UV than HPLC assay. While both UV and HPLC assay methods were developed for cefuroxime, only HPLC assay is stability indicating. Glycerol showed higher accelerating effect than PEG 400 on the drug release. All formulations exhibited extended release of cefuroxime. Degradation of cefuroxime occurred mainly during dissolution suggesting drug stability in the formulations.     

General strategy for performing temperature programming in high performance liquid chromatography: prediction of linear temperature gradients

This paper describes how an empirical retention model is transferred from temperature-programmed gas chromatography (GC) to high temperature liquid chromatography (HT-HPLC). In order to evaluate the retention prediction, a temperature range from 50 to 180 °C was investigated using two test mixtures consisting of steroids and polycyclic aromatic hydrocarbons. In this temperature range, heating rates from 1.5 °C min(-1) up to 30 °C min(-1) were applied using four different high temperature stable HPLC columns with inner diameters of 1.0, 2.1, 3.0, and 4.6 mm. Temperature lag phenomena in the HPLC column as well as in the column oven are discussed, and it is shown that the linear elution strength (LES) model can be applied without any mathematical extension in order to take a temperature-dependent delay time into account. On the basis of this approximation, it is possible to perform a systematic method development using linear temperature gradients in liquid chromatography. Furthermore, it is shown that only two initial temperature gradient runs are necessary to predict the retention times of the analytes with a maximal relative error of less than 2%.     

Measurement of Internal Acyl Migration Reaction Kinetics Using Directly Coupled HPLC-NMR: Application for the Positional Isomers of Synthetic (2-Fluorobenzoyl)-d-glucopyranuronic Acid

Ester glucuronides (1-O-acyl-β-d-glucopyranuronates) of many drugs may undergo internal acyl migration reactions, resulting in the formation of new positional isomers with both α- and β-anomers. We illustrate here a novel approach for the direct investigation of the acyl migration kinetics of ester glucuronides and show the application with respect to the isomers of synthetic (2-fluorobenzoyl)-d-glucopyranuronic acid. Individual isomers were separated from an equilibrium mixture containing the β-1-O-acyl, α- and β-2-O-acyl, α- and β-3-O-acyl, and α- and β-4-O-acyl isomers at pH 7.4 in 20 mM phosphate buffer. The interconverting isomers were separated using reversed-phase HPLC and pumped directly into a dedicated on-line NMR flow probe in a 600 MHz NMR spectrometer. The flow was stopped with each isomer in the NMR flow probe, and sequential NMR spectra were collected at 25 °C, allowing direct measurement of the production of positional isomers from each selectively isolated glucuronide isomer. All of the positional isomers and anomers were characterized, and relative quantities determined, and a kinetic model describing the rearrangement reactions was constructed. The acyl migration reaction kinetics were simulated using a theoretical approach using nine first-order rate constants determined for the acyl migration reactions and six first-order rate constants describing the mutarotation each of the 2-, 3-, and 4-positional isomers. The rate constants (in h(-)(1)) for the rearrangement reactions of the 2-fluorobenzoyl glucuronide isomers were as follows: β-1-O-acyl, 0.29 ± 0.01; α-2-O-acyl, 0.11 ± 0.01; β-2-O-acyl, 0.07 ± 0.01; α-3-O-acyl, 0.10 ± 0.01; β-3-O-acyl, 0.09 ± 0.01; α-4-O-acyl, 0.09 ± 0.01; and β-4-O-acyl, 0.06 ± 0.01. The α- and β-anomerization rates were estimated on the basis of the kinetics model; the anomerization rates of the 4-O-acyl isomers were additionally determined experimentally using directly coupled HPLC-NMR. The fitted anomerization rates for the 4-O-acyl isomer were 0.80 (α → β) and 0.50 h(-)(1) (β → α), whereas the experimentally estimated anomerization rates were 0.89 ± 0.1 and 0.52 ± 0.1 h(-)(1), respectively. The dynamic stop-flow HPLC-NMR approach allows unique kinetic information to be obtained relating to glucuronide reactivity, and this approach will be useful in future structure-reactivity studies on drug ester glucuronides and their properties.     

Separation and sequencing of isomeric oligonucleotide adducts using monolithic columns by ion-pair reversed-phase nano-HPLC coupled to ion trap mass spectrometry

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (+/-)-anti-7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to double-stranded (ds) 5'-PO4--ACCCGCGTCCGCGC-3'/5'-GCGCGGGCGCGGGT-3' oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter. The performance of three different stationary phases was also investigated. The chromatographic separation performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents C18 or PS-DVB. Different mobile phase organic solvents and ion-pairing reagents were also evaluated. An optimized mobile phase consisting of methanol and 25 mM triethylammonium bicarbonate resulted in the best chromatographic resolution and increased MS sensitivity of the oligonucleotides. By using a monolithic PS-DVB stationary phase fabricated in a nanocolumn, four positional isomeric (+/-)-BPDE-oligonucleotide adducts were separated and identified. In addition to four of the possible five positional isomers, three positional isomers were also resolved to several diastereoisomers, although their stereostructures could not be identified in the absence of reference standards.     

Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.     

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-concentration band profiles and system peaks for a ternary solute system

High-concentration HPLC band profiles of single solutes and the individual band profiles of the components of binary and ternary mixtures are reported for benzyl alcohol, 2-phenylethanol, and 2-methylbenzyl alcohol. These solutes were eluted from a C18 column by a binary mobile phase (MeOH:H2O = 1:1, v/v). High-concentration system peaks were obtained using mixtures of benzyl alcohol and 2-phenylethanol at different relative concentrations as the feed and 2-methylbenzyl alcohol as the strong mobile phase additive. Band profiles and system peak profiles were calculated using the equilibrium-dispersive model of chromatography. The adsorption equilibrium in the multicomponent system was characterized by the competitive Langmuir model. Excellent quantitative agreement was found between the experimental and the calculated profiles. This work confirms that extremely unusual system peak profiles can be obtained even when the adsorption behavior is quite simple. Under certain circumstances, the use of a properly chosen additive could markedly increase the separation between bands and hence the production rate, the recovery yield, and/or the purity of the fractions.     

Determination of benzotriazole corrosion inhibitors from aqueous environmental samples by liquid chromatography-electrospray ionization-tandem mass spectrometry

The first method for the determination of commonly used corrosion inhibitors in environmental water samples by liquid chromatography-electrospray ionization-tandem mass spectrometry is presented. Benzotriazole (BTri) and the two isomers of tolyltriazole (5- and 4-TTri) are separated in an isocratic run. By gradient elution, BTri, 4-TTri, 5-TTri, and xylyltriazole can be determined simultaneously with three benzothiazoles, but here TTri isomers coelute. The instrumental detection limit of 2 pg allows the determination of the three most important benzotriazoles from municipal wastewater and most surface waters by direct injection into the HPLC system without previous enrichment. When solid-phase extraction is employed with mean recovery rates of 95-113%, the limit of quantification for benzotriazoles range from 10 ng/L in groundwater to 25 ng/L in untreated wastewater. BTri and TTri were determined in municipal wastewater in microgram per liter concentrations. Elimination in wastewater treatment appears to be poor, and BTri and TTri can be followed through a water cycle from treated municipal wastewater through surface water to bank filtrate used for drinking water production. The TTri isomers show markedly different biodegradation behavior with 4-TTri being more stable.     

Description and validation of a four-channel staggered LC-MS/MS system for high-throughput in vitro screens

A parallel four-channel LC-MS/MS system for quantitative high-throughput in vitro screens is described. The system comprises four pumps, a four-valve autosampler equipped with a nine-port stream selector valve, and a triple quadrupole mass spectrometer. Staggered injections across the sample list are performed by four injector valves onto separate columns in such a manner that the chromatographic window of interest is always eluting from one of the columns into the mass spectrometer. To demonstrate the applicability of the system, three different compounds (verapamil, propanolol, dextromethorphan) were analyzed. The results showed that the sample throughput could be increased more than three times in comparison with a conventional single-column LC-MS/MS system. Validation results for the three compounds showed an accuracy of 85.0-108%, a precision (given as relative standard deviation) of 2.60-10.7%, and a carryover of less than 0.1%.     

基于宽光谱光学遥感图像的细分光谱光学遥感图像的模拟

本文分析了现有遥感图像模拟方法的特点,介绍了地物波谱知识库的建立情况与应用意义,论述了光学传感器的成像特点及地面实测数据与遥感反演数据之间的转换关系.本文在对TM图像的光谱波段进行细划分的基础上,提出利用波谱知识库的数据支持来模拟获取具有更高光谱分辨率的细分光谱光学遥感图像的方法.本方法依据光谱数据库等先验知识的支持,通过遥感成像时辐射传输的模拟运算,在宽光谱光学遥感数据提供的图像纹理信息和地类信息的基础上,获取细分光谱波段的模拟光学遥感图像,同时避免了难以接受的计算量.细分光谱模拟图像的实验结果表明,本方法能够较为可靠的模拟出真实高光谱分辨率图像的信息,模拟方法可信.