Microbore High-performance Liquid Chromatography Research Articles

Analysis and Pharmacokinetics of Apomorphine in Rat Brain by Microdialysis Coupled with Microbore HPLC Electrochemical Detection

The feasibility of an on-line microdialysis coupled with a sensitive microbore high performance liquid chromatography with electrochemical detection (HPLC-ED) system for the direct analysis of apomorphine was investigated. A microdialysis probe was inserted into the right striatum of male Sprague-Dawley rats, which had been administered apomorphine (10 mg/kg, i.v.).

Measurement and pharmacokinetic analysis of buspirone by means of brain microdialysis coupled to high-performance liquid chromatography with electrochemical detection

The feasibility of an electrochemical detection system with on-line microdialysis coupled with sensitive microbore high-performance liquid chromatography for the measurement and brain pharmacokinetic analysis of buspirone was investigated. A microdialysis probe was inserted into the right striatum of male Sprague-Dawley rats, which had been administered buspirone (10 mg/kg, i.v.). Dialysates were automatically injected through an on-line injector into a cyano microbore column coupled to an electrochemical detector. Samples were eluted with a mobile phase containing 0.1 M monosodium dihydrogenphosphate-acetonitrile-diethylamine (85:15:0.1, v/v/v, pH 3.0, adjusted with orthophosphoric acid) at a flow-rate of 0.06 ml/min. A biphasic phenomenon with a rapid distribution phase followed by a slower elimination phase was observed from the brain buspirone concentration-time curve. The results indicate that the brain pharmacokinetics of buspirone appear to conform to a two-compartment model.

Quantitative analysis of mevinolinic acid in human plasma by high-performance liquid chromatography coupled with negative-ion electrospray tandem mass spectrometry.

Mevinolinic acid (MVA), the major active metabolite of Lovastatin in human blood, is analysed by microbore high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Quantification is carried out by using methylmevinolinic acid (MMVA) as internal standard. Positive- and negative-ion mass spectra of these compounds are shown. Because of the higher sensitivity obtainable, the negative-ion mode is selected for the analysis. Solid-phase extraction cartridges are used off-line to prepurify and concentrate the sample and a microbore (1 x 100 mm) reversed-phase column is used for chromatography. Tandem mass spectrometry is carried out in the precursor-ion mode using a selected-reaction monitoring procedure. The [M-H]- precursor ions for MVA and MMVA are selected in the first quadrupole analyser and the second analyser is focused on the common product ion at m/z 319. Detection and quantification limits are ca. 50 pg/mL and ca. 200 pg/mL respectively. An example of the application of the method to the routine analysis of samples from a bioequivalence study is also shown.

Speciation of arsenic animal feed additives by microbore high-performance liquid chromatography with inductively coupled plasma mass spectrometry.

Phenylarsonic compounds have been used as poultry and swine feed additives for the purpose of growth promotion and disease prevention. Owing to the lack of suitable analytical methods, however, knowledge of their metabolism, environmental fate and impact remains incomplete. In order to compensate for this, analytical procedures were developed that allow the speciation of arsenic animal feed additives by using microbore high-performance liquid chromatography (microHPLC) coupled on-line with ICP-MS. More specifically, reversed-phase (RP) chromatographic methods were optimised to achieve the separation of various phenylarsonic acids from each other and from the more toxic inorganic arsenic compounds. This mode of chromatography, however, exhibits limitations, especially in the presence of naturally occurring organoarsenic compounds. The application of RP ion-pairing chromatography eliminates such shortcomings by minimising the co-elution of arsenic species. In general, the microHPLC-ICP-MS methods developed in this study provide high selectivity, extremely good sensitivity, low limits of detection (low-ppb or sub-pg amounts of As), require small sample volumes (< 1 microliter), minimise waste and operate most efficiently under low mobile-phase flow rates (15-40 microliters min-1), which are compatible for use with other types of mass spectrometers, e.g., electrospray. Reference materials containing naturally occurring arsenic compounds were spiked with phenylarsonic compounds and then analysed by using the procedures developed in this study.

Determination of Ten Organoarsenic Compounds Using Microbore High-performance Liquid Chromatography Coupled With Electrospray Mass Spectrometry–Mass Spectrometry

An analytical method based on reversed-phase microbore HPLC coupled on-line with electrospray mass spectrometry (ES-MS) is described. The method allows for the determination of up to ten organoarsenicals in a single chromatographic run. Excellent sensitivity and selectivity is achieved by operating the triple quadrupole mass spectrometer in the selected-reaction monitoring mode. This is possible because the ions produced by collision-induced dissociation of the protonated molecules or molecular ions of the ten organoarsenicals are characteristic of each compound. The best LODs, achieved in the positive-ion mode, were between 2 and 21 pg of arsenic and corresponded to arsenicals which exist as cations in acidic solutions. The selectivity achieved by using this method allows for successful determination of arsenicals co-eluting during HPLC. This is a major improvement over other hyphenated techniques already used for arsenic speciation, e.g. , HPLC–ICP-MS. Furthermore, the method was used for the analysis of an undiluted urine SRM in which arsenobetaine was determined to be present at the low µg l -1 level.

Quantitative determination of a nonpeptide antithrombotic in dog plasma by microbore high-performance liquid chromatography-tandem mass spectrometry utilizing pneumatically assisted electrospray ionization

A method has been developed and is described for the quantitative determination of a nonpeptide antithrombotic in dog plasma. The assay employs reversed phase microbore high-performance liquid chromatography in conjunction with tandem mass spectrometry utilizing pneumatically assisted electrospray ionization. The analyte and internal standard are isolated from the plasma matrix by solid-phase extraction. The mass spectrometer is operated in the positive ion multiple reaction monitoring mode and is set to detect the presence of a precursor-product ion pair for both the analyte and internal standard to generate product ion chromatograms for both species. The analyte is quantified by using weighted least-squares regression of the peak height ratio of drug:internal standard. The method provides linear response for plasma concentrations ranging from 5 ng/mL (25 pg on-column) to 2500 ng/mL. Statistical evaluation and examples of authentic sample assays are also presented.

The development of a data system for a combination of liquid chromatography or capillary electrophoresis with an ion trap storage/reflectron time-of-flight mass detector.

A data system based upon a 200 MHz transient recorder interface card in a Pentium PC computer is demonstrated for on-line analysis of microbore high-performance liquid chromatography (HPLC), capillary HPLC and capillary electrophoresis (CE) separations using a fast and sensitive ion-trap storage/reflectron time-of-flight mass spectrometric detector (IT-reTOFMS). Under the control of a user-written program, the system is capable of conducting the data acquisition and storage for a minimum of 30 min, at rates exceeding 10 Hz, of individual mass spectra containing 16,000 data points having 10 nsec resolution. The capability is mainly attributed to the use of a data reduction scheme in which only mass intensities higher than a preset threshold are saved as indexed flight-time/intensity pairs. This produces a typical reduction ratio of 30:1 in data set size, yielding faster storage with smaller file size, and permits the complete set of mass spectra to be held in the computer's memory. In addition, the data system is capable of displaying, for real-time evaluation of the analysis, each individual mass spectrum and the total-ion chromatogram. Further, the selected-ion chromatograms of given masses and a 3-dimensional topographic map describing a separation process can be rapidly generated from the collected data for the unambiguous and high fidelity identification of target analytes in a complex mixture.

Micropreparative Gel Electrophoresis of Low-Molecular-Weight Peptides: Purification of Highly Insoluble Amyloid Peptide Fragments

We have used the continuous-elution micropreparative gel electrophoresis device described by Baumann and Lauraeus (Anal. Biochem.214, 142–148, 1993) to purify low-molecular-weight peptide fragments from in-gel digested standard proteins as well as highly insoluble amyloid peptides. Alzheimer's amyloid β-peptide, gelsolin-derived amyloid peptide of the Finnish type, and a novel amyloid of the British type were purified from either homogenized brain or kidney tissue material to a high degree of purity in a single run. Using the high resolving capacity of the Tris–Tricine–SDS buffer system of Schaegger and von Jagow (Anal. Biochem.166, 368–379, 1978) we were able to isolate two synthetic peptides withMr4329 and 3284, differing only by 1045 in mass. The total peptide recovery, as determined by amino acid sequence analysis and scanning densitometry, ranged between 60 and 80%. In order to demonstrate the utility of this technique we subjected some of the purified peptides to direct N-terminal amino acid sequence analysis, mass spectrometry, microbore high-performance liquid chromatography, and immunochemical studies. Our results show that micropreparative gel electrophoresis is an effective tool for the isolation of not only larger polypeptides but also small peptide fragments in a form suitable for further biological use.

Quantification of gangliosides by microbore high performance liquid chromatography.

A highly sensitive analytical method was developed that allows the separation of ganglioside mixtures and quantification of individual non-derivatized gangliosides in the concentration range between 2 pmol and 1 nmol. Gangliosides were separated with a gradient of acetonitrile/phosphate buffer on a 1 mm diameter microbore HPLC column packed with Spherisorb-NH2. They eluted according to their number of sialic acid residues with increasing phosphate and decreasing acetonitrile concentrations. The separation of different gangliosides with equal sialic acid content is also described. The column effluent was monitored at the maximum of absorption at 197 nm. The sensitivity is higher than resorcinol staining of fractionated gangliosides by thin layer chromatography, previously the standard method for ganglioside analysis. The separated gangliosides can be analyzed by further methods. The HPLC method described here has been applied to the analysis of serum and oligodendroglioma specimens.

Microscale Flow Injection and Microbore High-Performance Liquid Chromatography Coupled with Inductively Coupled Plasma Mass Spectrometry via a High-Efficiency Nebulizer

A high-efficiency nebulizer has been used for coupling microscale flow injection and microbore high-performance liquid chromatography with inductively coupled plasma mass spectrometry (ICPMS). The microscale flow injection system was configured to minimize band broadening between the injection valve and the nebulizer, and it was evaluated for flow rates between 20 and 120 μL/min. Various materials, including certified reference materials, were analyzed for their arsenic and lead contents. Separation of arsenic species was carried out on microbore liquid chromatography columns. The absolute detection limits (femtogram range) for various arsenic compounds were excellent compared with those obtained previously by using conventional column (1 mL/min eluent flow) high-performance liquid chromatography/inductively coupled plasma mass spectrometry. Other advantages of this system include use of small sample volumes (≤1 μL), the resulting minimization of sample matrix introduction into the plasma, and reduced waste generation compared with conventional ICPMS sampling systems.

Aspirin inhibits collagen-induced platelet serotonin release, as measured by microbore high-performance liquid chromatography with electrochemical detection.

A sensitive microbore high-performance liquid chromatographic method with electrochemical detection has been developed for the measurement of small quantities of serotonin (5-hydroxytryptamine; 5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in rabbit platelets. The limit of detection of 5-HT is 0.1 ng/ml. To evaluate the 5-HT release from platelet suspension, the aggregating agent collagen was added at concentrations of 10, 30 and 100 micrograms/ml and the 5-HT concentrations rose from the base level (11.80 +/- 0.42 ng/ml) to 123.97 +/- 11.02, 361.96 +/- 17.90 and 470.45 +/- 35.46 ng/ml, respectively. Further results demonstrated that aspirin inhibits collagen-induced 5-HT release from platelet suspension significantly.

Microbore liquid chromatography of tertiary amine anticholinergic pharmaceuticals with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection.

The post-column chemiluminescent reaction of six anticholinergic alkaloid compounds with tris(2,2'-bipyridine)ruthenium(III) (Ru(bpy)3(3+)) is applied to microbore high-performance liquid chromatography (HPLC). At flow rates less than 200 microL/min, the capillary mixing cell in which Ru(bpy)3(3+) and the analyte are mixed directly allows for good light detection. In contrast, a diminished signal occurs at these low flow rates with conventional post-column mixing in a tee. Optimal chemiluminescent pH conditions for atropine, scopolamine, dicyclomine, cyclopentolate, cyclobenzaprine, and procyclidine are determined at moderately basic conditions (pH 7 to 9). 2-Butanone is found to be compatible with the chemiluminescent reaction, whereas tetrahydrofuran and propionitrile cause an increase in background noise and a chemiluminescent signal loss. As 2-butanone is more nonpolar than acetonitrile, it assists in the elution of these hydrophobic anticholinergic compounds. Five anticholinergic compounds are resolved successfully with a PRP-1 polymeric column and a slightly basic mobile phase, but a C8 silica column is better suited for the more hydrophobic compounds (cyclobenzaprine, procyclidine, and dicyclomine).

Determination of purine bases and nucleosides by conventional microbore high-performance liquid chromatography and gas chromatography with an ion-trap detector

A reversed-phase high-performance liquid chromatographic method has been developed for the analysis of purine and pyrimidine bases, uric acid and nucleosides largely relating to the purine synthetic and degradation metabolic pathways, with particular attention to the separation of hypoxanthine, xanthine and guanine. Complete separation and quantitation of the purines has been accomplished in the nanogram-microgram scale on conventional 4.6 mm I.D. columns with a standard gradient HPLC instrumentation as well as on 1 mm I.D. microbore columns with a dedicated isocratic micro-HPLC system using a dioxane-sodium acetate buffer. For the definite identification of components in excreta of ticks a GC-MS method has been described involving formation and GC of the trimethysilyl derivatives on a 25-m DB-5 column directly coupled with an ion trap detector. The methods are demonstrated on the analysis of the purine metabolites having an assembly pheromone effect on argasid ticks.

Rapid measurement of the monoamine content in small volumes of rat plasma

A method for the simultaneous measurement of serotonin, catecholamines, and their metabolites, 5-hydroxy-indoleacetic acid, homovanillic acid, and 3, 4-dihydroxyphenylacetic acid, by ultrafiltration and microbore high-performance liquid chromatography with dual electrochemical detection in small plasma volumes was established. Unlike the traditional assays which require at least 1–2 ml of plasma for each measurement, the present method uses only a 20-μl sample volume. Since blood loss is minimal, repeated blood sampling from a single animal as a rat becomes practicable. The present microassay provides low detection limits (signal-to-noise ratio = 3) for all analytes (0.2–0.5 pg per 5-μl injection or 50–120 pg/ml plasma). Isocratic separation of these analytes on a microbore column is achieved within 15 min. This rapid and sensitive method can be used as a routine research tool in various physiological or pharmacokinetic studies especially in small animals.

Reutilizing 32P-Labeled Probes to Reduce Radioactive Waste

For the determination of the cytotoxic drug 5-fluorouracil in tissue, a sensitive and selective assay has been developed based on microbore high-performance liquid chromatography and fluorescence detection after precolumn derivatization with 4-bromomethyl-7-methoxycoumarin. 5-Chlorouracil was found to be an appropriate internal standard. A column switching protocol has been devised to separate the analyte from late eluting matrix components and the natural uracil. The drug can be determined at concentrations in the low ng/g wet tissue range with a detection limit of 3 ng/g; furthermore, the same protocol can be applied to analyze serum and plasma samples. The procedure was employed to determine the drug in samples taken from human liver tumors and metastases after an intra-arterial bolus administration and from epithelial carcinomas after systemic continuous application.

Enantioselective assay for the determination of nisoldipine in dog, rat and mouse plasma by chiral microbore high-performance liquid chromatography combined with gas chromatography-mass spectrometry

A sensitive, selective and validated method for the enantioselective determination of (+)- and (−)-nisoldipine in rat, mouse and dog plasma following administration of nisoldipine racemate is described. The alkalized plasma samples containing [ 13C 4]nisoldipine racemate as internal standard (ISTD) were extracted once with toluene. The enantiomers of nisoldipine were quantitatively separated by high-performance liquid chromatography on a 250 × 2 mm I. D. column containing tris(4-methylben-zoate)-modified cellulose on silica. The fractions containing either the (+) or (−)-enantiomer of the analyte and [ 13C 4]ISTD were analysed by gas chromatography with mass-selective detection in the single-ion monitoring mode. The limits of determination and detection were 0.5 and 0.2 ng/ml, respectively, the total precision was better than 7% (R.S.D. at 5 and 50 ng/ml, n = 35) and the accuracy was better than 10% (0.5–100 ng/ml, n = 23). The sum of the concentrations of the enantiomers determined with this assay corresponds to the concentration of the racemate determined independently by capillary gas chromatography with electron-capture detection (accuracy better than 15%, 1–80 ng/ml). The method was used for the analysis of more than 500 plasma samples obtained from toxicokinetic studies.

A Procedure for the Determination of 5-Fluorouracil in Tissue Using Microbore HPLC and Fluorescence Detection

For the determination of the cytotoxic drug 5-fluorouracil in tissue, a sensitive and selective assay has been developed based on microbore high-performance liquid chromatography and fluorescence detection after precolumn derivatization with 4-bromomethyl-7-methoxycoumarin. 5-Chlorouracil was found to be an appropriate internal standard. A column switching protocol has been devised to separate the analyte from late eluting matrix components and the natural uracil. The drug can be determined at concentrations in the low ng/g wet tissue range with a detection limit of 3 ng/g; furthermore, the same protocol can be applied to analyze serum and plasma samples. The procedure was employed to determine the drug in samples taken from human liver tumors and metastases after an intra-arterial bolus administration and from epithelial carcinomas after systemic continuous application.

Probing high order structure of proteins by fast-atom bombardment mass spectrometry

During the past decade, numerous investigations have demonstrated that the rate at which amide hydrogens located at peptide linkages undergo isotopic exchange is a sensitive probe of the high order structure and dynamics of proteins. The present investigation demonstrates that microbore high-performance liquid chromatography (HPLC) continuous-flow fast-atom bombardment mass spectrometry (FABMS) can be used to accurately quantify deuterium located at peptide linkages in short segments of large proteins. This result is important because it demonstrates the feasibility of using mass spectrometry as a tool for studying the high order structure and dynamics of large proteins. Following a period of deuterium exchange-in, a protein was placed into slow-exchange conditions and fragmented into peptides with pepsin. The digest was analyzed by continuous-flow HPLC FABMS to determine the molecular weights of the peptides, from which the number of deuterons located at the peptide linkages could be deduced. The HPLC step was used both to fractionate the peptides according to their hydrophobicities and to remove through back-exchange all deuterium except that located at peptide amide linkages. This approach has been applied to α-crystallin, a lens protein composed of two gene products with monomer molecular weights of 20 kDa and an aggregate molecular weight approaching 1000 kDa. Results from this study show that some of the peptide amide hydrogens in α A-crystallin exchange very rapidly (k > 10 h −1) while others exchange very slowly (k < 10 −3 h −1). The ability not only to detect that a conformational change has occurred, but also to identify the specific regions within the protein where the change occurred, was demonstrated by measuring changes in the exchange rates within these regions as the deuterium exchange-in temperature was increased from 10 to 80 °C.

Confirmation of danofloxacin residues in chicken and cattle liver by microbore high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

A specific assay is described for the confirmatory identification of danofloxacin residues in edible tissues of cattle and chicken. The assay utilizes on-line microbore high-performance liquid chromatography and pneumatically assisted electrospray tandem mass spectrometry (MS/MS). Collision-induced dissociation of the danofloxacin protonated molecule results in two significant daughter ions. Monitoring both ions provides the specificity required for this confirmatory assay. Optimum electrospray and MS/MS operating conditions permitted the specific monitoring of danofloxacin and the confirmation of its residues in chicken and cattle liver extracts down to 50 ppb. The analysis of control liver or the commercially available antibacterial quinolones enrofloxacin and its metabolite ciprofloxacin gave no response under the assay conditions. The ratios of the two daughter ions were similar for danofloxacin standard solutions, fortified tissues and incurred tissues.

An Improved Assay for Adenosine in Rat Brain Microdialysates Using Microbore High Performance Liquid Chromatography

Abstract A high-performance liquid chromatographic method using a microbore system consisting of a microbore column (100 mm – 1mm I. D.- 3 μm), microbore pump (60 μl/min) and UV absorbance detector has been developed for the determination of adenosine in rat brain microdialysis samples. UV absorbance of adenosine at 259 nm was used to measure adenosine. The limit of detection (LOD) was 50 pg and the limit of quantitation (LOQ) was 10 ng/ml. The methodology is applicable for the quantitation of adenosine in small volumes (3 – 10 (μl) of microdialysate and does not require sample preparation.