Narrow-bore Column Research Articles

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography–electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography–mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC 18 narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3–10 μg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of −6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC–ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography–electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography–mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC 18 narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3–10 μg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of −6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC–ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.

Gas chromatographic optimization studies on the side chain and ring regioisomers of methylenedioxymethamphetamine.

Gas chromatographic (GC) optimization studies are conducted for the 10 methylenedioxyphenethylamine regioisomeric substances related to the drug of abuse 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy). These 10 compounds, having the same molecular weight and equivalent major mass spectral fragments, are not completely resolved using typical GC-mass spectrometry screening methods for illicit drugs. MDMA coelutes with at least one nondrug regioisomer under standard drug screening conditions. Separation of the 10 regioisomers is studied using stationary phases of varying polarities. Resolution optimization shows that very slow program rates give the best separation for the nonpolar stationary phases, requiring analysis times of as much as 85 min. Narrow-bore columns containing the same nonpolar stationary phases improve the analysis time to approximately 29 min. The polar stationary phase DB-35MS allows high-temperature programming rates, yielding complete resolution of all 10 compounds in less than 7 min. Temperature program optimization studies on the DB-35MS phase allow the separation time to be reduced to approximately 4.5 min.

Instrumentation for advanced microseparations in pharmaceutical analysis and proteomics

Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.

Combining liquid chromatography with MALDI mass spectrometry using a heated droplet interface.

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 microL/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 microL/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrow-bore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 10(6) for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts.

Determination of Vitamin B 5 in a range of fortified food products by reversed-phase liquid chromatography–mass spectrometry with electrospray ionisation

Methods for Vitamin B 5 determination in food products remain limited by their low sensitivity and poor selectivity. Here, we have developed a liquid chromatography–mass spectrometry (LC–MS) method for Vitamin B 5 determination in wide range of fortified food products. Vitamin B 5 was extracted from food samples by heat treatment and analysed by LC–MS in the positive mode using electrospray ionisation (ESI). Vitamin B 5 was quantified using hopantenic acid (HOPA) as internal standard after their separation on a C 18 narrow-bore column with a gradient of mobile phase made of water/acetonitrile and trifluoroacetic acid (TFA) 0.025%. MS with single ion monitoring mode at mass m/z 220 was used for Vitamin B 5 quantification. Calibration curve between 0.5 and 10 μg/ml of Vitamin B 5 was linear ( r 2=0.9993) and the detection limit was determined to be 800 pg. The overall quantitative efficiency of the method was evaluated using Nestlé reference sample (infant formula). The intra-assay RSD was 4.8% ( n=8), the inter-assay RSD 6.4% ( n=4) and the recoveries of the spiked samples were above 95%. Application of the LC–MS method to Vitamin B 5 determination in wide range of fortified food products including three US National Institute of Standards and Technology (NIST) reference samples (RM 8435, RM 8415 and SRM 1546) shows consistent results with those obtained by microbiology and recoveries of Vitamin B 5 between 93 and 104% for the spiked samples.

Therapeutic drug monitoring for sirolimus in whole blood of organ transplants by high-performance liquid chromatography with ultraviolet detection

We developed and validated an accurate, sensitive, precise and rapid HPLC method with UV detection for the determination of sirolimus in blood samples from renal, cardiac and hepatic transplants. This method overcomes most of the problems related to previously published assays using a narrow-bore column with base deactivated C 18 reversed phase. Whole blood samples were purified by a combination of a precipitating blood matrix with zinc sulphate and a single step liquid–liquid extraction with acetone and 1-chlorobutane. Calibration curves (range 2.5–150 ng/ml), were linear with coefficients of correlation better than 0.996. The relative standard deviation was determined to be less than 8%. The present method has also been validated by a reference laboratory (St. George’s Hospital Medical School, London, UK). More of 300 clinical samples have been analysed with this method.

Brain and plasma exposure profiling in early drug discovery using cassette administration and fast liquid chromatography-tandem mass spectrometry

A method using reverse phase liquid chromatography-tandem mass spectrometry and cassette administration was developed for in vivo brain and plasma exposure profiling to assist early CNS drug discovery programs. Three to four compounds were grouped in cassettes for dosing and analysis. Compounds in the cassettes were selected to minimize possible analytical interference from each other, as well as from their potential metabolites. In order to improve the confidence of cassette administration, an analogue of the study compounds, with well-established brain penetration data, was included in each cassette as a “biological internal standard”. Compounds were administered to rats by intraperitoneal injection and extracted from plasma or brain homogenate by simple protein precipitation. Fast chromatographic separation was achieved by using a short narrow-bore column at a flow rate of 1.0 ml/min with a fast gradient. The brain penetration of the compounds was evaluated by comparing their C max and AUC values in brain and plasma. This approach rapidly provided early brain penetration and plasma exposure information, thus making more of this data available to teams. Comparing the brain exposures to the EC 50 values (i.e. in vitro potency) of series compounds in the same discovery program provided another dimension of information to select lead compounds for future in vivo assessment. The method described here has been used for providing early brain penetration information in several CNS exploratory and discovery programs.

Targeted multidimensional gas chromatography using microswitching and cryogenic modulation.

A new method is described that allows fast target analysis in multidimensional gas chromatography by using a microswitching valve between two GC columns, with cryogenic trapping and rapid re-injection of trapped solutes in the second dimension. The essence of the procedure is that heart-cut fractions from the first column (1D) can be selectively transferred to column 2 (2D), where a moveable cryogenic trap first focuses the transferred solute(s) at the head of the second column and then permits their facile rapid analysis on 2D. Since 2D is a short narrow-bore column, which exhibits very fast analysis (on the order of a few seconds elution), peak responses (heights) are significantly enhanced (by up to 40-fold). Additionally, by using a 2D phase of a selectivity different from that used for 1D, it is possible to also separate components that are not resolved on the first column and to increase the resolution for other compounds. The heart-cut valve isolates the section(s) of solutes of interest from the first column separation, and this provides a considerable simplification to the chromatogram-in addition to the separation and sensitivity advantages. By using this method, multidimensional gas chromatography with multiple heart-cuts can be completed within the same time as the primary column separation. Since the described method permits non-heart-cut fractions to be transferred to a monitor detector, normal detection of these fractions is still permitted. By modulation of the cryotrap, it is also possible to achieve comprehensive two-dimensional gas chromatography for the heart-cut fractions; however, only those compounds passed to the second, separation column, which passes through the cryotrap, will be subjected to GC x GC analysis. The technique and the various modes of operation are described in this paper.

Direct HPLC analysis of quercetin and trans-resveratrol in red wine, grape, and winemaking byproducts.

A simple and fast reversed-phase HPLC method using diode array detection was developed and validated for the simultaneous determination of trans-resveratrol and quercetin in Sicilian red wine from the Nero d'Avola red grape variety. Investigation was also extended to the quantitative determination of resveratrol and quercetin in grape skins and winemaking byproducts obtained from the same cultivar. Samples were eluted using a C18 narrow-bore column under isocratic conditions in less than 20 min. Quantification of trans-resveratrol and quercetin in red wine was performed without any sample pretreatment, whereas the determination of these phenolic compounds in grape skins and wine pomage required a solvent extraction procedure. Linearity was demonstrated over the 0.39-12.5 and 0.45-57.6 microg/mL range for trans-resveratrol and quercetin, respectively. Detection limits in real samples were in the low ppm level (0.07 mg/L for trans-resveratrol and 0.12 mg/L for quercetin). The HPLC-UV/DAD method was applied for the routine analyses of red wine and grape skin and winemaking byproduct extracts to evaluate their trans-resveratrol and quercetin content. In particular, a very high content of quercetin was found in wine pomace, suggesting the use of this wine byproduct as a potential source of this health-promoting phenolic compound.

Application of High-Performance Liquid Chromatography on Short Narrow-bore Columns to the Determination of Priority Polycyclic Aromatic Hydrocarbons in Environmental Samples

Conditions were optimized for the separation of priority polycyclic aromatic hydrocarbons (PAHs) by high-performance liquid chromatography on short narrow-bore columns (2 × 75 mm). Twelve PAHs were determined by the direct injection of preconcentrated extracts from highly contaminated soil, solid phase of snow water, an aerosol, and surface water. PAH peaks were identified using retention times and spectral ratios (R). In the case of the detection of nonhomogeneous peaks (by the R value), the repeated separation of the sample was proposed with a change in the selectivity of the mobile phase, detection wavelength, and column temperature. The spread of the results of measurements of the PAH mass was estimated at different deviations of R from the standard value.

A General Unknown Screening Procedure for Drugs and Toxic Compounds in Serum using Liquid Chromatography-Electrospray-Single Quadrupole Mass Spectrometry

A complete general unknown screening procedure was developed using liquid chromatography-mass spectrometry (LC-MS), a coupling that can increase the range of compounds amenable to MS. Sample preparation was by solid-phase extraction on a mixed-mode support in parallel with serum deproteination in order to recover the most hydrophilic compounds. Chromatography employed a reversed-phase narrow-bore column (150 x 1-mm i.d.) and a 50-min gradient elution at low flow-rate (50 microL/min), compatible with the electrospray source used without splitting nor heating. The single quadrupole LC-MS instrument used was operated in the 100 to 1100 mu mass range in both the positive and negative modes, with two different, alternated collision-induced dissociation voltages in the source, in order to obtain the molecular or pseudo-molecular ions as well as fragments for the compounds analyzed. The addition of spectra obtained at low and high fragmentation voltages gave reconstructed spectra for each polarity, representing library entries. Finally, a program was created in order to detect the peaks of interest in the chromatographic noise using a very efficient signal processing algorithm, compute their relative retention time with respect to the internal standard (glafenine), draw their reconstructed spectra, search them in the libraries, and edit a report.

Factors influencing chromatographic data in fast gas chromatography with on‐column injection

AbstractThe use of larger volume injection with on‐column injection and fast GC commercial instrumentation was evaluated with the model mixture of n‐alkanes of a broad range of volatility (C10–C28). The presented configuration allows introduction of 40–80‐fold larger sample volumes without any distortion of peak shapes compared to “usual” fast GC set‐ups using narrow‐bore columns. A normal‐bore retention gap (1–5 m×0.32 mm ID) was coupled to a narrow‐bore (5 m×0.1 mm ID×0.4 μm film thickness) analytical column using a standard press‐fit connector. The connection was tight and reliable, and hence suitable for hydrogen as carrier gas. The effect of pre‐column and analytical column connector, injection volume, pre‐column length, column inlet pressure, and analyte volatility on peak shape, peak broadening, and focusing are discussed. The precision of chromatographic data measurements and peak capacity under optimised temperature programmed conditions for fast separations with large volume injection were found to be very good. The presented fast GC set‐up with on‐column injection extends the applicability of the technique to trace analysis.

Rapid Characterization of Hop Essential Oils Using Gas Chromatography–Time of Flight Mass Spectrometry

Hops (Humulus lupulus L.) are used by the brewing industry to add bitterness and flavor to beer. The analysis of hop essential oils helps in determining both hop quality and varietal discrimination. A new approach for the rapid analysis of essential oils is reported using capillary gas chromatography–time of flight mass spectrometry (GC-TOFMS), which is compared to conventional quadrupole gas chromatography-mass spectrometric analysis. Using narrow-bore capillary columns (0.18 mm i.d.) in combination with high spectral acquisition frequencies, inherent problems of peak coelutions were overcome using spectral deconvolution algorithms. High TOFMS acquisition rates afforded superior spectral purity that, in conjunction with greater high mass ion transmission than the quadrupole, gave excellent spectral data for species such as humulone and lupulone. Using a GC-TOFMS method, a reduction in total analysis time by a factor of 10 was achieved, successfully identifying 40 key common components. Modifying this method to incorporate quantitative analysis could enable a rapid method of quality control for hops.

Resistively heated gas chromatography coupled to quadrupole mass spectrometry

The potential of fast temperature-programmed gas chromatography (GC) was studied by combining a resistively heated gas chromatograph and a quadrupole mass spectrometer (MS). An EZ Flash GC was used, which generates temperature ramps up to 1200 °/min and cools down from 300 to 50°C in some 30 s; samples were injected via a conventional split/splitless injector in both injection modes. The combination of a short narrow-bore column (5 m×0.1 mm ID), a high gas flow rate, and fast temperature programs typically decreased conventional analysis times of 30 min to about 3 min. The scan speed of the quadrupole mass spectrometer (16 spectra/s in the range m/z 50–310) was found to be sufficient for a proper reconstruction of the chromatographic peaks, and good-quality mass spectra could be obtained. Six scans across a peak were sufficient for peak integration. The system was linear in the range of 0.01–3 ng injected mass and detection limits for pesticides were in the range of 1–25 pg in the full-scan and 0.1–5 pg in the selected ion monitoring mode. Series of apple and mole liver extracts were analyzed to demonstrate the high sample throughput and suitability of EZ Flash GC for fast screening.

Fast gas chromatography-time-of-flight mass spectrometry of polychlorinated biphenyls and other environmental contaminants.

Practical applications of fast gas chromatography (GC) with time-of-flight mass spectrometry (TOFMS) are presented. A narrow-bore column (0.10-mm i.d.) is used to analyze over 100 specific polychlorinated biphenyl congeners in an Aroclor mix and a sediment sample in 10.5 min. Sample preparation is minimized for the sediment to more closely match the speed advantage gained by using fast GC-TOFMS. The possibility of using a 0.53-mm-i.d. column operated under vacuum-outlet conditions for fast GC-TOFMS is established for Aroclors and a suite of environmental contaminants. Fast acquisition rates and automated peak-find and spectral deconvolution capabilities are demonstrated for TOFMS.

A Study of the Critical Criteria for Analyte Stability in High-Temperature Liquid Chromatography

There are three major impediments to the use of high-temperature ultrafast liquid chromatography. First, the stationary phase must be thermally stable. Over the past decade, a series of thermally stable, highly efficient stationary phases have been developed that can withstand temperatures exceeding 200 degrees C. Second, the temperature mismatch between the incoming eluent and the column must be minimized (<5 degrees C), because such a mismatch is a very serious cause of peak broadening, especially in ultrafast separations. The thermal mismatch problem can be significantly ameliorated at high column linear velocities by using narrow-bore columns (2.1-mm i.d.). Third, analytes that are exposed to high temperatures must be thermally stable on the time scale of the chromatographic run. We report here a study of the ability of a number of pharmaceuticals to withstand superambient temperatures on the time scale of fast separations. We propose criteria by which a particular analyte may be rejected as a candidate for high-temperature analysis, and we demonstrate that complex molecules are amenable to quantitation, even at temperatures in excess of 100 degrees C in the aqueous media. We also show that as the time an analyte spends on hot column decreases, the extent of on-column reaction decreases for those analytes that do react. Although the seminal work of Antia and Horvath addresses these issues from a theoretical perspective, we hope to further alleviate fear of the use of high temperatures in liquid chromatography through the empirical approach used here.

Temperature programming in liquid chromatography

The present state of temperature programming in liquid chromatography, including electrochromatography, is reviewed. A relatively limited number of papers making use of temperature programming as a technique in LC has been published so far, while the various effects of temperature are frequently discussed. As a part of the ongoing trend of miniaturization in chromatography, the active use of temperature as a variable in LC is expected to increase significantly. This paper includes an overview of the effects of temperature on column efficiency, retention, and selectivity, particularly in relation to narrow-bore columns, instrumental features, and some selected applications.

Temperature programming in liquid chromatography

The present state of temperature programming in liquid chromatography, including electrochromatography, is reviewed. A relatively limited number of papers making use of temperature programming as a technique in LC has been published so far, while the various effects of temperature are frequently discussed. As a part of the ongoing trend of miniaturization in chromatography, the active use of temperature as a variable in LC is expected to increase significantly. This paper includes an overview of the effects of temperature on column efficiency, retention, and selectivity, particularly in relation to narrow-bore columns, instrumental features, and some selected applications.

Fast generic-gradient reversed-phase high-performance liquid chromatography using short narrow-bore columns packed with small nonporous silica particles for the analysis of combinatorial libraries.

The extremely large number of samples generated for the quality control analysis of combinatorial libraries that are developed in the pharmaceutical research for drug discovery requires fast generic methods such as rapid-gradient reversed-phase high-performance liquid chromatography (HPLC). These methods are necessary as standard procedures to produce up to several hundreds of analytical results per day and should be optimized in order to be applied to library products of widely differing polarities. This work presents an optimized generic method using a narrow-bore column packed with 1.5-microm nonporous particles and a completely automated HPLC workstation configured for the best efficiency, throughput, and robustness with this column. A test mix of 12 compounds with a wide polarity range is separated within 1.5 min with a cycle time of 3.5 min. The throughput is further enhanced using a Gilson 233XL dual-injection sampler to feed two parallel HPLC systems in order to perform 34 analyses per hour.