Microbore Liquid Chromatography Research Articles

Determination of erythromycin in urine and plasma using microbore liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II) electrogenerated chemiluminescence detection.

Erythromycin is determined in both urine and plasma samples using microbore reversed-phase liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrogenerated chemiluminescence (ECL) detection. Ru(bpy)3(2+) is included in the mobile phase thus eliminating band broadening caused by post-column reagent addition. Extra column band broadening is an important concern in microbore liquid chromatography due to the small peak volumes. Erythromycin was studied in both water and biological samples. The detection limit for erythromycin in standards is 0.01 microM or 50 fmol injected with a S/N of 3 and a linear working range that extends four orders of magnitude. Human urine and blood plasma were also studied. Urine samples were diluted and filtered before injection. Ultrafiltration was used to remove protein from blood plasma samples prior to injection. Erythromycin was selectively detected in the body fluid samples without any further sample preparation. The detection limits obtained for erythromycin in urine and plasma are 0.05 and 0.1 microM, respectively, for 5 microl injected on a 150x1 mm I.D. C18 column.

New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection.

An automated microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin, dopamine and their metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Due to the chemical instability of the compounds, the addition of an antioxidant is required for automated analysis over a long period of time (e.g., 20 h). Therefore, the time stability of these substances was tested with different antioxidants. The stability for serotonin and 5-hydroxyindoleacetic acid was poor in acidic medium containing Na2EDTA but could greatly be improved by the addition of L-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.

Quantitative analysis of two opioid peptides in plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

Quantitative analysis of two opioid peptides, DSLET [( d-Ser 2)Leu-enkephalin-Thr 6] and Met-enkephalin-Arg-Gly-Leu, was performed using microbore liquid chromatography interfaced to electrospray ionization tandem mass spectrometry. Validation of the methodology was demonstrated for each peptide in plasma. Quantitative analyses were performed through the use of a deuterium labelled peptide analog as an internal standard. Linearity was observed for the analysis of DSLET (5–1000 ng/ml) and Met-enkephalin-Arg-Gly-Leu (1–1000 ng/ml) in plasma with a limit of detection of 0.25 ng/ml for Met-enkephalin-Arg-Gly-Leu and 1.0 ng/ml for DSLET. In general, the observed concentrations showed good reproducibility with coefficients of variation of within 15%. In the concentration range studied, only 0.5 ml of plasma was required for optimal detection of Met-enkephalin-Arg-Gly-Leu and 0.25 ml for DSLET. Application of this method was demonstrated by studying the disposition of DSLET in a rat. DSLET administered to a rat exhibited a short half-life and a high clearance value.

Endurance training effects on neurotransmitter release in rat striatum: an in vivo microdialysis study.

In the present study we use the in vivo microdialysis sampling technique to register extracellular levels of neurotransmitters in the striatum of trained and untrained rats. We further evaluate the influence of 1 h of exercise on the striatal release of dopamine (DA), noradrenaline (NA), glutamate (GLU) and gamma-aminobutyric acid (GABA) in trained and untrained rats. Male Wistars were randomly assigned to a training or control group. The exercise training consisted of running on a treadmill for 6 weeks, 5 days week-1, with running time and speed gradually increased from 30 min at 19 m min-1 during the first week to 80 min at 26 m min-1 during the final training week. The animals of the control group were placed on the treadmill twice a week, and received a total of four 'adaptation sessions', in which they exercised 15-45 min at 26 m min-1. Brain dialysates were analysed with microbore liquid chromatography (LC), with electrochemical detection (monoamines and GABA) and fluorescence detection (GLU). Soleus citrate synthase and basal striatal concentrations of DA, NA and GLU were significantly different between the trained and control animals. Sixty minutes of exercise significantly increased extracellular DA, NA and GLU levels in both groups, but there was no statistically significant difference in the exercise-induced increase between trained and control animals. There was no statistical difference in basal or exercise-induced GABA levels between trained and control animals. The results indicate that exercise training appears to result in diminished basal activity of striatal neurotransmitters, while maintaining the necessary sensitivity for responses to acute exercise.

Determination of Tetrahydrobiopterin and its Analogues in Biological Samples by Microbore Liquid Chromatography

Tetrahydrobiopterin and seven of its analogues (neopterin, xanthopterin, biopterin, pterin, tetrahydropterin, 6-methyltetrahydropterin, and 6-methylpterin) were separated on a 1 × 150 mm C18 microbore column. These analytes were detected by dual-electrode amperometry and UV absorption. Both a conventional glassy carbon electrode and an interdigitated array microelectrode were used. Low fmol amounts of xanthopterin, tetrahydropterin, tetrahydrobiopterin and 6-methyltetrahydropterin could be determined by electrochemical detection in the oxidative mode, but pmol amounts of the other analogues were determined by electrochemical detection in the reductive mode and with UV detection. Catecholamines and their metabolites do not interfere with the determination of tetrahydrobiopterin and its analogues in biological samples. The developed method was explored for the determination of tetrahydrobiopterin in samples of human urine and rat tissue (brain, liver, and kidney).

Microbore liquid chromatography analysis of monoamine transmitters.

Ion-pair reversed-phase liquid chromatography (LC), as introduced by Johansson et al. in 1978 (1) is still the most widely used separation technique for analysis of the biogenic amines. It is usually coupled with electrochemical detection (ECD). The use of microbore LC was introduced by Scott and Kucera in 1976 (2). These systems use columns with an internal diameter (id) of 1 mm or less. Microbore LC provides a significant advantage over conventional LC: The sample injected on to a microbore column is diluted by about 20-fold less than when injected on to a “normal” bore (e.g., 4.6 mm id) column. In this way, the compounds of interest become easier to detect. Microbore LC offers several other advantages:

Nanoliter volume, high-resolution NMR microspectroscopy using a 60-micron planar microcoil.

Previous studies demonstrated the feasibility of using 100-microns inner diameter planar spiral inductors (microcoils) as detectors in 1H nuclear magnetic resonance (NMR) microspectroscopy. However, high-resolution NMR applications were not possible due to poor spectral resolution and low signal-to-noise ratio (SNR). These limitations in performance have now been largely overcome by using a nonconductive liquid fluorocarbon (FC-43) to minimize the effects of susceptibility mismatch between materials, and by carefully optimizing the microcoil geometry for maximum SNR. In this study, liquid samples were loaded into a fused silica capillary (75-microns inner diameter, 147-microns outer diameter). The capillary was positioned 50 microns above a 3.5-turn microcoil so that approximately 1 nL of the sample was present in the sensitive region of the microcoil. The microcoil was fabricated on a gallium arsenide substrate with an inner diameter of 60 microns, an outer diameter of 200 microns, trace width of 10 microns, trace spacing of 10 microns, and trace height of 3 microns. At 5.9 T (250 MHz) in 1H-NMR microspectroscopy experiments using a spectral width of 1 kHz, 4096 sampled data points, and a recovery delay of 1 s, a SNR of 25 (per acquisition) and a spectral linewidth of less than 2 Hz were obtained from a sample of water. These results demonstrate that planar microcoils can be used for high-resolution NMR microspectroscopy. Such coils may also be suitable for localized NMR studies at the cellular level and as detectors in capillary electrophoresis or microbore liquid chromatography.

Assessment of injection volume limits when using on-column focusing with microbore liquid chromatography

It had been ascertained that in the use of microbore liquid chromatography (LC) and on-column focusing (peak compression), there were advantages to be had in using the strongest eluting injection solvent that would still bring about on-column focusing. In such cases, it was found that the scope for the use of on-column focusing with microbore LC was greater than previously imagined. It has been possible to explain these results by considering on-column focusing to take place in two distinct phases, i.e. focusing of the sample band due to the injection solvent, followed by further focusing of the sample band by the sample solvent/mobile phase step gradient. In this content, an equation has been developed which provides a much more accurate estimate of the maximum allowable injection volume for a given injection solvent and mobile phase than has been possible until now.

Microbore liquid chromatography analysis of amino acid transmitters.

Microbore liquid chromatography analysis of amino acid transmitters.

New dual electrochemical detector for microbore liquid chromatography determination of dopamine and serotonin in rat striatum dialysates

A new type of liquid chromatographic (LC) dual thin-layer amperometric detector for the simultaneous measurement of trace levels of dopamine and serotonin in microdialysates is described. The concentrations of these analytes in rat dialysates are usually in the sub-nanomolar concentration range (typically, 0.10–5.00 pg in 5-μl dialysates). With this dual electrode, a glass-lined microbore column provides excellent sensitivity, selectivity, and separation. In addition, a three- to five-fold improvement in anodic current or cathodic responses over conventional dual electrodes in microbore LC can be achieved. Due to the irreversible electrochemical properties of some interference peaks, this dual electrode provides reliable measurement of dopamine based on the cathodic signal. The detection limit (signal-to-noise ratio=3) of this assay is 0.02 pg per injection for dopamine or serotonin. This new dual electrode allows the simultaneous measurements of basal dopamine and serotonin in rat striatum dialysates without the use of re-uptake inhibitors in perfusion medium.

Electrochemical detection in bioanalysis

This review article presents an overview of current research on the use of amperometric electrochemical detectors in bioanalytical chemistry. Topics covered include microdialysis and ultrafiltration membranes for in-vivo sampling; microbore liquid chromatography; capillary electrophoresis; enzyme, photochemical, and chemical post-column reactions; electrochemiluminescence; and thin-film electrode materials. Selected references are cited.

Carbon film based ring‐disk and split‐disk dual electrodes as detectors for microbore liquid chromatography

AbstractThe electrochemical properties of carbon film based ring‐disk and split‐disk electrodes in small active volume (0.24–2μL) radial flow cells were studied in order to employ them in conjunction with microbore liquid chromatography (LC) for determining catecholamines. The high conversion of dopamine (DA) at the disk of a ring‐disk electrode makes the DA limiting current lower than the theoretical value at a low flow rate. The collection efficiency (CE) increases with decreasing flow rate and cell thickness, which is different from previously reported results. A high CE of 0.96 was achieved because of the narrow gap of 5 μm.On the other hand, a higher limiting current was observed with the split‐disk than with a commercially available dual‐disk electrode because of its efficient geometry for analyte collection. The cross talk of the DA after reacting at one of the electrode is only 2%. The ring‐disk and split‐disk electrodes were suitable as series and parallel electrochemical detectors for small bore LC. A detection limit of 103 fg was obtained for DA at the disk of the ring‐disk because of the high signal in the thin‐layer radial flow cell and the low noise level of the carbon film. In contrast, very slight interference was observed from neighboring electrodes when determining catecholamines at the split‐disk.

Carbon Film-Based Interdigitated Ring Array Electrodes as Detectors in Radial Flow Cells

We have fabricated the first carbon film-based interdigitated ring array (IDRA) electrodes for application as detectors in thin-layer radial flow cells. The limiting current of dopamine (DA) at the electrode is proportional to the one-third power of the volume flow rate (v1/3) when two electrodes in the IDRA are held at 750 and 50 mV, respectively. In contrast, it is not proportional to v1/3 when only one electrode is held at 750 mV with the other electrode disconnected, due to the high conversion of DA in the flow cell at a low flow rate. The collection efficiency (CE) and redox cycles (Rc) increase with decreasing flow rate, since the redox cycling of the IDRA suffers less influence from the flow. The IDRA electrode was used in combination with microbore liquid chromatography (LC) for catecholamine detection. The CE and Rc are particularly high in the usual flow rate range for microbore LC, which is from 0.02 to 0.1 mL/min. Rc values of 4.3 and 3.2 were achieved for DA and epinephrine, respectively, at ...

Determination of acetylcholine and choline with platinum-black ultramicroarray electrodes using liquid chromatography with a post-column enzyme reactor

A method for the highly sensitive determination of acetylcholine (ACh) and choline (Ch) was developed using platinum (Pt) black microarray electrodes as detectors in a microbore liquid chromatography (LC) with a post column enzyme reactor. The electrodes were prepared by plating a gold (Au) film electrode with sub-μm Pt-black particles. Since hydrogen peroxide generated by the enzymatic reaction of ACh and Ch was oxidized only at the Pt-black microarray electrode, each Pt-black particle (typically 0.1–0.2 μm in size) operated as an ultramicroelectrode. A high signal-to-noise ratio was achieved because of the high current density at the Pt-black microarray electrodes and because the Au film has a much lower baseline noise than the Pt. Detection limits of 5.7 (ACh) and 6.0 (Ch) fmol were obtained, with a wide linear range. The ACh and Ch signals with an Au film electrode modified with Pt-black particles retained more than 70% of their initial value after 5 days with continuous potential application. This is better stability than for a bare platinum electrode which retained only 40% of its initial response under comparable conditions.

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.

Dual-electrode amperometric detection for the determination of SR4233 and its metabolites with microbore liquid chromatography

3-Amino-1,2-benzotriazine-2-di- N-oxide (SR4233) is a promising new antineoplastic agent based on reductive activation. SR4233 and its major metabolites (SR4317 and SR4330) are all easily reduced at a carbon electrode. Reductive amperometric detection can therefore provide high selectivity and low detection limits with chromatographic analysis and is an ideal approach to detection of SR4233 in microdialysis samples. However, in order to use amperometric detection in the reductive mode, sample deoxygenation is necessary. This is typically done by purging the sample with either argon or nitrogen prior to injection. This approach is not feasible for microdialysis samples because only 5–10 μl is usually available. In this report, a microbore liquid chromatographic method with dual-electrode amperometric detection is described for the determination of SR4233 and its metabolites without predeoxygenation. A dual-electrode amperometric detector was used in the series configuratioin with an upstream potential of −450 mV to reduce SR4233 and its metabolites to a common product and a downstream potential of +400 mV to oxidize this product. Oxygen is only electroactive at the upstream electrode because of its irreversible behavior. This method is compatible with the small sample volumes provided by microdialysis sampling. Linear calibration graphs were obtained up to 55 μM for SR4233, and 140 μM for both SR4317 and SR4330. The detection limits were 70 nM for SR4233, and 50 nM for SR4317 and SR4330. The average intra-day variation over 5 days was 1.8% (SR4233), 1.4% (SR4330), and 1.8% (SR4317), whereas the inter-day variation over 5 days was 14.1% (SR4233), 8.6% (SR4317), and 2.6% (SR4330).

On-line NMR detection of amino acids and peptides in microbore LC.

The combination of liquid chromatography (LC) and nuclear magnetic resonance (NMR) offers the potential of unparalleled chemical information from analytes separated from complex mixtures. However, the application of LC-NMR has been hindered by poor detection sensitivity. We develop a theoretical model for predicting signal-to-noise ratio (SNR) performance while scaling NMR detection cells for flowing experiments. The model includes the effects of separation parameters, coil geometry, and NMR acquisition parameters on SNR performance. Although the detector cell should be as large as possible to ensure adequate efficiency for a given separation, reducing the detector cell volume does not significantly degrade SNR. For example, our model predicts a 2-fold reduction in SNR for a 400-fold reduction in cell volume. The results of static NMR measurements of amino acids and peptides in a 50-nL-volume cell (approximately 1 microgram of each) demonstrate the performance of such a small-volume NMR microcell. Using this 50-nL detector cell with microbore LC, two-dimensional LC-NMR chromatograms are shown for amino acid and peptide separations.

Liquid chromatography-time-of-flight mass spectrometry with continuous-flow matrix-assisted laser desorption ionization

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been combined with liquid chromatography (LC) by using a continuous-flow sample inlet system. The on-line interface consists of two major components: a three-port mixing tee for post-column matrix addition and a flow probe for introducing the solution directly into a time-of-flight mass spectrometer. In the experiment, the LC effluent and the matrix solution flow into the mixing tee through two separate ports. The resulting mixture is directed to the flow probe for MALDI. Both conventional LC and microbore LC have been successfully interfaced to MALDI for on-line detection of proteins. It is demonstrated that the interface does not degrade the chromatographic performance significantly. With microbore LC, LC-MALDI can be performed with total-sample injection in the low-picomole region. An example is also given to illustrate the application of on-line LC-MALDI for peak identification in a protein mixture separation.

Detection of basal acetylcholine in rat brain microdialysate

A liquid chromatography-electrochemistry (LC-EC) method is described for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. This method is based on the separation of ACh and choline (Ch) by microbore liquid chromatography followed by passage of the effluent through a post-column immobilized enzyme reactor (IMER), containing acetylcholinesterase (AChE) and choline oxidase (ChO), and then the electrochemical detection of the hydrogen peroxide produced. Instead of the conventional platinum electrode used for the anodic detection of hydrogen peroxide, a peroxidase-redox polymer modified glassy carbon electrode operated at + 100 mV vs. Ag/AgCl has been used to detect the reduction of hydrogen peroxide. With this method, a detection limit of 10 fmol (injected) for ACh ( S/ N = 3:1) was obtained and the basal ACh concentration in striatal microdialysate was determined without using esterase inhibitors.

Improved detection limit for catecholamines using liquid chromatography-electrochemistry with a carbon interdigitated array microelectrode.

The detection limit of catecholamines can be lowered by using a carbon-based interdigitated array (IDA) microelectrode as a detector for liquid chromatography (LC). The IDA electrode is more sensitive than conventional glassy carbon electrodes due to the high current density caused by radial diffusion at each microband, and redox cycling between two microband arrays. Since the number of redox cycles increases at lower flow-rates, the carbon IDA is particularly useful for microbore LC. In an LC system with a 1-mm microbore column and a carbon IDA electrode, the peak height of dopamine (DA) and DOPAC did not decrease with decreasing flow-rate because of this redox cycling. A low detection limit of 5 fg (32 amol) and 9.6 fg (57 amol) was obtained for DA and DOPAC due to the high current density and low background noise level (0.1 pA) at the carbon IDA electrode. The total charge generated by oxidizing DA at the anodic array was more than the value calculated by assuming that all the DA molecules were oxidized.