High-performance Liquid Chromatography Effluent Research Articles

Rapid detection and identification of angiotensin-converting enzyme inhibitors by on-line liquid chromatography–biochemical detection, coupled to electrospray mass spectrometry

An analytical method based on on-line liquid chromatography–biochemical detection (LC–BCD) coupled to electrospray mass spectrometry was developed for the detection and identification of angiotensin-converting enzyme (ACE) inhibitors in complex mixtures, such as hydrolyzed whey proteins. ACE inhibitory activity was detected by coupling a homogeneous, substrate conversion based bioassay on-line to high-performance liquid chromatography (HPLC). Chemical information was obtained by directing part of the HPLC effluent towards a mass spectrometer. After correlating the biochemical and chemical data, the accurate molecular masses of the bioactive peptides were used as search queries in protein databases. Combined with the recorded mass spectrometry (MS)–MS fingerprints, bioactive peptides were selected from the database search results. The results of LC–BCD–MS analyses were verified by establishing a bioactivity balance. Reference samples, containing several peptides at concentration levels similar to those observed in the hydrolyzed milk samples, were analyzed by LC–BCD–MS. High recoveries of biological activity were obtained, indicating that the correct ACE inhibitors were identified and that no co-elution of significantly bioactive molecules had occurred. Approximately, 30 ACE inhibitors were detected and identified. IC 50 values of ACE inhibitors, reported in literature, ranged between 43 and 580 μM.

On‐Line Detection for the HPLC Analysis of Water‐Soluble Vitamins in Multivitamin Tablets

Four water‐soluble vitamins, thiamine (B1), riboflavin (B2), pantothenic acid (B5), and pyridoxine (B6), frequently found in multivitamin tablets were analyzed by LC‐FTIR. The samples were run on a C18 column using gradient elution with a mobile phase of 0.01 M ammonium acetate and methanol. The separation was carried out on a LC‐FTIR system with a solvent elimination mode. The interface between high performance liquid chromatography (HPLC) and FTIR, which included an ultrasonic nebulizer and a heated drift tube removed the solvent from the HPLC effluent prior to FTIR detection. The analytes were deposited on a moving zinc selenide plate and FTIR spectra of the solid analytes were obtained. Spectral profiles of the vitamins of interest were constructed from the recorded standard spectra. The vitamins of interest in three multivitamin tablets were separated and identified, based on retention time and infrared spectral matching to those of standards.

High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.

A sensitive procedure for the study of β-carotene-d8 metabolism in humans using high performance liquid chromatography–mass spectrometry

This report describes the development of a robust method of high sensitivity for studying the metabolism of β-carotene-d8 in humans using a combination of liquid chromatography/particle beam–mass spectrometry (LC/PB-MS). The utility of the LC/PB-MS method was demonstrated in a pilot study. The carotenoids were extracted from plasma into hexane and separated by reverse phase high performance liquid chromatography (HPLC) using a C-18 column. The HPLC effluent was nebulized using helium and the solvent was removed under vacuum within the dual-stage particle beam interface. The de-solvated carotenoids were ionized in the negative-ion mode (electron capture) using methane chemical ionization and detected using selected ion monitoring. The limit of detection of the method was on the order of 0.3 ng (approximately 0.6 pmol) for β-carotene. β-Carotene-d8 was quantified in the plasma over a concentration range of two orders of magnitude using β-carotene-13C40 as an internal standard. The overall coefficient of variance (CV) for determining the concentration of the analytes from 30 μl of plasma was 3.9% for β-carotene and 2.4% for β-carotene-d8.Using the LC/PB-MS method, the concentration of β-carotene-d8 was determined in the plasma of a subject who had consumed a single 5-mg dose over a 30-day period. The sensitive semiautomated procedure is capable of high sample throughput and makes large comprehensive studies feasible.

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with fast liquid chromatography for quantitation and accurate mass measurement of five pharmaceutical drugs in human plasma.

The quantitative determination and accurate mass measurement of five tricyclic amine pharmaceutical drugs (doxepin, desipramine, imipramine, amitriptyline and trimipramine) fortified in human plasma within a per sample run time of 18 s was accomplished by atmospheric pressure ionization (API) time-of-flight (TOF) mass spectrometry using a turboIonspray liquid chromatography/mass spectrometry (LC/MS) interface coupled with high-performance liquid chromatography (HPLC). The relatively short HPLC separation (18 s) was achieved using a short C18 column (15 x 2.1 mm i.d.) with a high aqueous mobile phase maintained at a flow-rate of 1.4 ml min(-1). An acquisition speed of 0.2 s per spectrum accommodates these fast separation conditions. This method employs a one-step liquid-liquid extraction procedure to isolate the five tricyclic amines from biological matrix components The overall extraction recovery was 75% for desipramine and >90% for the other four tricyclic amines. The lower level of quantitation was 1-2 ng ml(-1) for each compound. The calibration curve was linear from 2 to 100 ng ml(-1) for desipramine and from 1 to 50 ng ml(-1) for the other four tricyclic amines. A deuterated internal standard, imipramine-d3, was used for all five tricyclic amines. Acceptable intra- and inter-assay precision (1.0-17.7%) and accuracy (0.2-14.5%) were obtained. The linear dynamic range was extended to 200 based on a software upgrade for correcting ion current detection saturation. The accurate masses of the five tricyclic amines were determined by on-line LC/TOFMS analyses of biological extracts using two-point internal mass calibration. This was done by infusing a reference standard, Jeffamine D230, post-column into the HPLC effluent. All results showed a mass error not greater than 9 ppm for all the target compounds. These results were obtained from both synthetic mixtures when as little as 100 pg were injected or extracts of spiked human plasma samples with analytical concentration as low as 5 ng ml(-1). The factors influencing accurate mass measurements are discussed.

Enzyme amplification as detection tool in continuous-flow systems: II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compounds were biotinylated off-line with biotin aminocaproic hydrazide (BACH). On-column biotinylation was performed by preconcentration of anthracene aldehyde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affinity to biotin, was added post-column to the HPLC effluent. Excess of enzyme-labeled affinity protein was removed by means of an immobilized biotin column. After separation of free and bound fraction, substrate was added, which was converted to a fluorescent product by the enzyme label. Using alkaline phosphatase as an enzyme label, a mass detection limit after on-column preconcentration and biotinylation of 250 fmol was achieved.

The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples.

Two bioanalytical methods have been developed and validated utilizing high flow high performance liquid chromatography (HPLC) for on-line purification of plasma and serum samples and electrospray tandem mass spectrometry for detection and quantitation. Each plasma or serum sample, after mixing with an aqueous solution of the internal standard, was injected into a small diameter (1 x 50 mm) column packed with large particles of OASIS (30 microns), with a 100% aqueous mobile phase at a high flow rate (3-4 mL/min). The combination of the high linear speed (6-8 cm/s) of the aqueous mobile phase and the large particle size resulted in the rapid passage of the proteins and other large biomolecules through the column while the small-molecule analytes were retained on the column. During this purification period, the HPLC effluent was directed to waste. After the purification step, the HPLC mobile phase was rapidly changed from 100% aqueous to < or = 100% organic, the flow was reduced to 0.5-0.8 mL/min, and the column effluent was directed towards the mass spectrometer. The small molecule analytes were eluted during this period. In the method developed and validated for the quantitative determination of compound I in rat plasma (method A), the same OASIS column (1 x 50 mm, 30 microns) served as the purification and analytical (elution) column. In the method developed for the simultaneous determination of pravastatin and its positional isomer biotransformation product (SQ-31906) in human serum (method B), the purification column was connected to a conventional C18 analytical column (3.9 x 50 mm, 5 microns) to achieve the required chromatographic separation between the two isomers. For method A, where 50 microL of rat plasma mixed 1:1 with water containing the internal standard was injected, the standard curve range was 1 to 1,000 ng/mL. For method B, where 200 microL of a human serum sample mixed 4:1 with water containing the internal standard was injected, the standard curve range was 0.5 to 100 ng/mL. The total analysis time for each method was < or = 5 min per sample. The accuracy, inter-day precision and intra-day precision were within 10% for both methods.

Identification of stress proteins in lysates of human cell lines separated by two-dimensional electrophoresis and electroblotted simultaneously onto two different membranes.

A protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two-dimensional electrophoresis (2-DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N-terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on-line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2-DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N-terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).

AC corona-discharge aerosol-neutralization device adapted to liquid chromatography/particle beam/mass spectrometry.

An AC corona-discharge device was inserted upstream of a thermospray vaporizer tip in a liquid chromatography/particle beam mass spectrometer to neutralize static aerosol charging. Response of a test analyte was measured with or without discharge initiation. If the solvent contained no ammonium acetate buffer, increased analyte signal was associated with the discharge. However, in the presence of ammonium acetate the benefit of AC discharge neutralization was either not observed or was more subtle. This led to the conclusion that the previously observed ammonium acetate "carrier" effect is attributable, at least in part, to neutralization of static electric charges produced spontaneously during the solvent nebulization process. In a second experiment, the pattern of particles issuing from the system momentum separator was examined by aiming the particle beam at a cold target located within a mass spectrometer ion source. Variations in particle density were observed depending on (i) whether or not the aerosol had been neutralized and (ii) the proximity of electron-beam-collimating magnets to the particle beam trajectory. These results are consistent with a hypothesis that electrostatic charging occurs spontaneously during the nebulization process in which an aerosol is formed from the high performance liquid chromatography effluent. Such electrostatic charging introduces a factor likely to degrade system performance by at least two modes: through interactions of the charged aerosol particles (i) with the walls of the aerosol transmission pathway, and, after they are accelerated into a particle beam and introduced into the mass spectrometer, (ii) with the magnets used for electron beam collimation in many mass spectrometer ion sources.

Thermally assisted electrospray interface for liquid chromatography/mass spectrometry

AbstractAn electrospray interface for liquid chromatography/mass spectrometry (LC/MS) is described that combines the direct heating of High‐performance liquid chromatography (HPLC) effluent that is characteristic of thermoooospray‐type interfaces. This interface is called the thermally assisted electrospray interface for LC/MS. The device can accept the total effluent from a liquid chromatograph at flow rates ranging up to 500 μL/min. The HPLC effluent is nebulized into charged droplets by a combination of heat and an applied electric field. Ions emitted from charged droplets are mass‐analyzed by a triple quadraupole mass spectrometer equipped with an atmospheric pressure ion sampling orifice. Singly or multiply charged ions, indicating the molecular weight of a sample, are observed with minimal fragmentation. The extent of multiple charging observed in electrospray and ion spray mass spectrometry is decreased somewhat in thermally assisted electrospray. Labile compounds which undergo thermal decomposition by conventional thermospray show no decomposition with thermally assisted electrospray.

Assessment of a thermospray interface for the coupling of high-performance liquid chromatography and Fourier transform infrared spectrometry

A thermospray interface has been developed to couple high-performance liquid chromatography (HPLC) with Fourier transform infrared (FT-IR) spectrometry. The system has been constructed for operation in normal- and reversed-phase HPLC. The HPLC effluent is thermally desolvated and deposited on to a moving metal substrate, which continually transfers the solutes into the diffuse reflectance (DRIFT) accessory of an FT-IR spectrometer, enabling identification by measurement of the IR reflectance spectrum. Various aspects of the interface design have been investigated and optimised, and the interface has been applied to the analysis of several types of compound, with molecular identification being indicated for the antioxidant Irganox 565 at concentrations lower than 50 μg ml −1

11] Fourier transform infrared spectroscopy of retinoids

This chapter discusses the fourier transform infrared (FTIR) spectroscopy of retinoids, and presents the FTIR spectra of 15 retinoids along with band positions which correlate with the retinoid structures. FTIR spectroscopy offers sufficient instrumental sensitivity, accuracy, and speed of analysis to be the method of choice. The wavelength multiplexing advantage and high energy throughput allow FTIR to be used in conjunction with low signal applications as might be the case where small quantities of material are to be analyzed or where the analysis method itself lacks efficient spectral throughput (e.g., diffuse reflectance). There are several versatile new methods for analysis which may prove to be of immense value on retinoid mixtures. In particular, attenuated reflectance cylinders allow measurement of flowing aqueous streams, such as the effluent of high-performance liquid chromatography (HPLC) systems. The ordinate performance of better quality FTIR spectrometers permits adequate blanking of the solvent concentration. Diffuse reflectance permits examination of HPLC effluent by allowing the carrier solvent to evaporate, leaving the analyte physisorbed on KCI powder. Photoacoustic spectroscopy allows examination of powders, films, or liquids without the need for sample preparation or correction for Kristiansen dispersion. All of these promise potent new alternatives for accurate and quantitative study of small quantities of retinoids. The chapter also discusses summary of absorption bands, and the purification procedures and infrared analysis.

High-sensitivity radioassay in chromatographic effluents

We have devised convenient systems for fractionating the effluent of high-performance liquid chromatography (HPLC) columns and concentrating the fractions for radioassay. A succession of aliquots of the HPLC effluent is deposited in wells formed in non-wetting fluorocarbon film and evaporated to near-dryness where the aliquots form droplets of uniform size. The droplets are then quantitatively transferred to filter paper impregnated with scintillator, where they form uniform circles 2–3 mm in diameter. Papers containing samples and standards are then exposed to photographic film for a time dependent on the radioactivity of the sample, the film is developed and teh autoradiographs are scanned with a thin-layer chromatography scanner used as a densitometer. The density of the spots was proportional to the radioactivity and could be quantified by comparison with the density produced by the standards. Scans of successions of spots from samples collected during elution of amino acids from HPLC columns reproduced the shapes of the peaks recorded by the UV-absorbance detector flow-cell, demonstrating that the resolution of the analysis was preserved. Film blackening sufficient for quantification was obtained with samples containing 14C in the 100–1000 dpm range in as little as 6 h of exposure with further increases in sensitivity in proportion to the increase in time of exposure. Hundreds of fractions from HPLC effluent and samples from several effluents could be assayed simultaneously, thus offering a large workload capability along with high sensitivity. We postulate that the same technique will produce similarly high sensitivity with little loss of resolution when used in conjunction with methods previously described for stripping radioactive compounds from gas—liquid chromatographic effluent into flowing liquid streams.

Solid Phase Reactions for Derivatization in Hplc (HPLC-SPR)

Abstract As in all other forms of chromatography, high performance liquid chromatography (HPLC) has come to rely quite heavily on derivatization of individual analytes for improved qualitative and quantitative identifications (1–13). Virtually all of this work has involved the use of homogeneous type derivatizations, wherein the sample solution to be injected, in the pre-column approach, or the HPLC effluent, in the post-column approach, are fully mixed with the derivatization reagents in solution. Clearly, homogeneous type derivatizations can be done off-line or on-line, in either the pre-or post-column modes, but in general, pre-column methods have been done offline, and post-column approaches have been done both on-line and off-line. Online type derivatizations, in either the pre-or post-column modes, appear to offer some very significant advantages. Major among these is the ability to perform derivatization-separation-detection or separation-derivatization-detection following injection of the sample m...

Nitrogen-selective detector for high-performance liquid chromatography

A nitrogen-selective detector for high-performance liquid chromatography (HPLC) is described. The HPLC effluent is introduced into a furnace using a concentric atomizer. Organic nitrogen is converted to ammonia by reaction in hydrogen over a nickel catalyst. The ammonia is collected into water-alcohol and the ammonium ion is measured with a Tracor Hall electrolytic conductivity detector. The limit of detection is 1 ng nitrogen per sec injected onto the chromatographic column. The carbon to nitrogen selectivity ratio is 10,000. Precision is 3.4% relative standard deviation when 0.56 μg nitrogen are injected. The detector is linear over a narrow range. Deviation from linearity is most likely due to incomplete partitioning and ionization of ammonia in the conductivity solution. Applications using both normal and reversed-phase conditions are discussed.