Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.     

High-throughput selected reaction monitoring liquid chromatography-mass spectrometry determination of methylphenidate and its major metabolite,ritalinic acid,in rat plasma employing monolithic columns

This work presents a high-throughput selected reaction monitoring (SRM) LC-MS method for the determination of methylphenidate (MPH), a central nervous stimulant, and its de-esterified metabolite, ritalinic acid (RA) in rat plasma samples. A separation of these two compounds was achieved in 15 s by employing a 3.5-ml/min flow-rate, a porous monolithic column and a TurboIonSpray source compatible with relatively high flow-rates. In addition, a relatively fast autosampler and a new data acquisition system resulted in a time lag of less than 17 s between consecutive injections. Overall, 768 protein-precipitated rat plasma samples (eight 96-well plates) containing both MPH and RA were analyzed within 3 h and 45 min. The partial method validation described in this report included an assessment of linearity, intra and inter-assay precision and accuracy, and method robustness. Deuterated internal standards for the target compounds, d(3)-MPH and d(5)-RA, were employed. The calibration curves ranged from 0.1 to 50 ng/ml for MPH and from 0.5 to 50 ng/ml for RA. The limit of quantification (LOQ) for MPH and RA was 0.1 and 0.5 ng/ml, respectively. For both analytes, the intra- and inter-assay precision (relative standard deviation, % C.V.) and accuracy (relative error) did not exceed 15% for the quality control samples (QCs) QC1, QC2 or QC3 (0.3, 1.5 and 40 ng/ml for MPH and 0.15, 15 and 40 ng/ml for RA) for either analyte and did not exceed 20% at the lower limit of quantitation (LOQ) level. No carry-over from the autosampler was detected. The retention times remained constant throughout the experiment. Baseline resolution of MPH and RA was consistently observed throughout the plates analyzed. The described method demonstrates the feasibility for employing monolithic HPLC columns to effect rapid bioanalytical SRM LC-MS analysis of representative biological samples.     

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column

Previously [Anal. Biochem., 232 (1995) 163–171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Determination of a new oral cephalosporin,S-1090, in human plasma and urine by direct injection high-performance liquid chromatography with ultraviolet detection and column switching

Direct injection high-performance liquid chromatographic (HPLC) methods with column switching and UV detection were developed for the rapid and accurate determination of S-1090 in human plasma and urine. An internal-surface reversed-phase pre-column and a C₁₈ analytical column were used for the plasma assay. Two pre-columns packed with cyano and phenyl materials and a C₁₈ analytical column were used for the urine assay. The calibration curves for plasma and urine assays were linear in the ranges 0.09–9 μg/ml and 0.5–100 μg/ml of S-1090, respectively. The relative standard deviations for plasma and urine assays were less than 6% with low relative errors. The established HPLC methods were demonstrated to be useful for clinical pharmacokinetic studies after oral administration of S-1090.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 microm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane-isopropanol with ethylene glycol, water or acetonitrile) are applied.     

Monolithic chromatography on conjoint liquid chromatography columns for speciation of platinum-based chemotherapeutics in serum of cancer patients

BackgroundMonolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run.MethodsHere, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. Both the CLC columns constructed from affinity Protein G and weak anion exchange diethylamine (DEAE) disks, were applied for the speciation of cisplatin, oxaliplatin and carboplatin in spiked standard serum proteins, spiked human serum and serum of cancer patients. The analytical performances of the CLC columns used were evaluated by comparing their robustness, selectivity, repeatability and reproducibility. The separated serum proteins were detected on-line by ultraviolet (UV) and eluted Pt species by inductively coupled plasma mass spectrometry (ICP-MS). For accurate quantification of the separated Pt species (unbound Pt-based chemotherapeutic from species associated to transferrin (Tf), human serum albumin (HSA) and Immunoglobulin G (IgG)), post column isotope dilution (ID)-ICP-MS was used.ResultsThe data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. It also enables more effective cleaning of monolithic disks and to analyse larger series of serum samples than the high-pressure CLC column. Analyses of serum samples of cancer patients treated with cisplatin or carboplatin showed that Pt-chemotherapeutics were bound preferentially to HSA (around 80%). The portion of unbound Pt in general did not exceed 2%, up to 5% of Pt was associated with Tf and approximately 20% with IgG. Column recoveries, calculated as a ratio between the sum of concentrations of Pt species eluted and concentration of total Pt in serum samples, were close to 100%.ConclusionsLow-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.     

Chromatography of human prothrombin from Nitschmann fraction III on Q Sepharose Fast Flow using axial and radial flow column

An axial column (Hitrap Q 5 ml, 2.5 2 1.6 cm) and a radial flow column (3.5 2 5 cm) packed with Q Sepharose Fast Flow media had been evaluated for the separation of human prothrombin. Nitschmann fraction III dissolved in buffered saline (0.10 M sodium chloride buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Effects of sample flow rate of the two columns were screened. Under radial flow conditions using the radial column, sample flow rate up to 15 ml/min (i.e. 18 bed volumes/h) was achieved and the operating pressure was below 0.2 MPa eventhough the elution velocity was 30 ml/min. Breakthrough capacity was determined by analyzing the total protein and prothrombin activity of the target protein-containing fraction under subsaturating conditions and both columns had almost the same breakthrough capacity per ml media, indicating that the sample loading was independent of radial column geometry. It was concluded that the radial column is an attractive alternate to traditional axial packed bed column, exhibiting very good potential for use in the separation of human prothrombin.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 μm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane–isopropanol with ethylene glycol, water or acetonitrile) are applied.     

High-yield high-performance liquid chromatographic analysis of steroid hormone receptors on glass columns

The HPLC characteristics of extensively purified chicken oviduct progesterone receptors were compared on TSK 3000 SW size-exclusion and DEAE-5-PW media packed in either glass or stainless-steel columns. Recoveries of [3H]progesterone-labeled receptor from size exclusion were 75-95% in glass columns and less than 10% in stainless-steel columns. Similarly, recoveries from DEAE were greater than 90% in glass columns but only approximately 45% in stainless-steel columns. Recoveries in glass columns were similar on several HPLC systems. Thus, the requisite component for high yields from extensively purified receptor preparations was the glass column itself. While receptor B exhibited ionic strength-dependent mobility similar to several standard proteins on size-exclusion glass column HPLC, receptor A was very peculiar. Resolution of receptors A and B was superior to previous reports using size exclusion open-end chromatography. We also resolved functionally active proteolytic fragments. Finally, the generality of glass column size-exclusion HPLC was demonstrated by high-yield analysis of different steroid hormone receptors from different tissues and species.     

Practice of solid-phase extraction and protein precipitation in the 96-well format combined with high-performance liquid chromatography–ultraviolet detection for the analysis of drugs in plasma and brain

C₁₈ Empore 96-well extraction disc plates have been employed for the analysis of three drugs with different polarities in plasma in conjunction with HPLC–UV, rufinamide, ICL670 and an anticonvulsant agent (AA1) in an early stage of development. With the most polar compound (AA1), ion-pair extraction at pH 12 was applied. The method developed for the assay of AA1 in plasma was applied to its determination in brain using an Oasis HLB plate following homogenisation in a pH 7.4 buffer and protein precipitation with NaOH–ZnSO₄, thereby saving time for method development. Protein precipitation in the 96-well format with filtration of the precipitate was applied to the determination of ICL670, a highly protein-bound compound (>99.5%), with a good recovery (78%). Reversed-phase chromatography was applied using a short 5 cm column packed with 3 μm particles for the determination of ICL670 and AA1 and two parallel columns (15 cm long) for the determination of rufinamide. The methods were used routinely, one plate per analysis day being processed, resulting in increase in sample throughput and saving in solvents.     

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.     

High-performance liquid chromatographic-tandem mass spectrometric evaluation and determination of stable isotope labeled analogs of rofecoxib in human plasma samples from oral bioavailability studies

A method for the simultaneous determination of a cyclooxygenase-2 inhibitor, 4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib, I) and [13C7]rofecoxib, (II), in human plasma has been developed to support the clinical oral bioavailability (BA) study of I. The method is based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in the negative ionization mode using a heated nebulizer interface. Two different stable isotope labeled analogs of I were initially evaluated for their use as intravenous (i.v.) markers in the BA study. [13CD3]Rofecoxib was shown to be isotopically unstable in plasma and water containing solvent and an efficient deuterium exchange prevented its use in the study. On the other hand, the isotopic integrity of the subsequently synthesized [13C7]rofecoxib (II) was maintained, as expected, in plasma and other solvent systems. The results of these experiments clearly demonstrated the need for the careful evaluation of the isotopic integrity of the stable isotope labeled compound for the successful utilization of these compounds in BA studies and also as internal standards in the quantitative analysis of drugs in biological fluids. After liquid-liquid extraction of I, II, and internal standard (III) from plasma, the analytes were chromatographed on a narrow bore (100 mm x 3.0 mm) C18 analytical column, with mobile phase consisting of acetonitrile-water (1:1, v/v) at a flow-rate of 0.5 ml/min. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in the selected reaction monitoring mode. The precursor-->product ion combinations of m/z 313-->257, 320-->292, and 327-->271 were used to quantify I, II, and III, respectively. The assay was validated in the concentration range of 0.1 to 100 ng/ml of plasma for both I and II. The precision of the assay (expressed as relative standard deviation) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. The assay was utilized to support the clinical BA study in which oral doses of I were administered together with an i.v. dose of II to determine the oral BA of rofecoxib at 12.5- and 25-mg doses.     

On-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma with a weak ion exchange monolithic column

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u = 4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u = 8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol–chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15–20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15–20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC–CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.     

High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.     

High-speed liquid chromatographic method for the monitoring of carbamazepine and its active metabolite, carbamazepine-10,11-epoxide, in human plasma

The assays of antiepileptic drugs, which are performed by central laboratories in Phase II and III clinical trials, require both a very fast turn-around time and a suitable specificity. In order to decrease the run time and to keep the powerful specificity of the liquid chromatography (HPLC), the use of a reversed-phase 1.5 μm monosized non-porous silicon dioxide microspheres column instead of regular columns containing spherical porous C₁₈ material was studied. The determination of carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZ-E), in human plasma or serum was chosen to demonstrate the utility of these columns. As a prerequisite of this work, no modification of a regular HPLC system was allowed. The samples were prepared in autosampler vials by protein precipitation with acetonitrile, followed by a quick centrifugation. Without any change to a conventional HPLC system, CBZ and CBZ-E are well separated in less than 2.5 min using a Kovasil MS C₁₄ column. No interference was observed with endogenous compounds and with nine antiepileptic drugs commonly prescribed as co-medication, and their metabolites. Due to the very low specific surface area of the packing, the required organic modifier volume per chromatographic run was decreased by a factor of 25. The method was validated. The developed method is well suited for the determination of CBZ and CBZ-E in clinical trials. It can be easily adapted to the monitoring of other antiepileptic drugs. No modification of a regular HPLC system was required.     

Fast analysis using monolithic columns coupled with high-flow on-line extraction and electrospray mass spectrometric detection for the direct and simultaneous quantitation of multiple components in plasma

In this work, monolithic columns were used as a fast separation tool for multiple-component quantitative liquid chromatography-tandem mass spectrometry (LC-MS-MS) assays of drug candidates in biological fluids. A considerably reduced runtime was achieved while maintaining good chromatographic separations. This significantly improved separation speed demanded higher throughput on sample extraction. To this end, monolithic separations were coupled on-line with high-flow extraction, which allowed for the fast extraction and separation of samples containing multiple analytes. An evaluation of this system was performed using a mixture of fenfluramine, temazepam, oxazepam, and tamoxifen in plasma. A total cycle time of 1.2 min was achieved which included both sample extraction and subsequent monolithic column separation via column switching. A total of over 400 plasma samples were analyzed in less than 10 h. The sensitivity and responses were reproducible throughout the run. The system has been routinely used in the authors' laboratory for high-throughput quantitation of compounds in biological fluids in support of drug discovery programs. The assay for samples from a 9-in-1 dog pharmacokinetic study is shown as an example to demonstrate the capability of this system.