A microfluidic chip platform with electrochemical carbon nanotube electrodes for pre-clinical evaluation of antibiotics nanocapsules

This paper presents a microfluidic chip platform with electrochemical carbon nanotube electrodes for preclinical evaluation of antibiotics nanocapsules. Currently, there has been an increasing interest in the development of nanocapsules for drug delivery applications for localized treatments of diseases. So far, the methods to detect antibiotics are liquid chromatography (LC), high performance liquid chromatography (HPLC), mass spectroscopy (MS). These conventional instruments are bulky, expensive, not ease of access, and talented operator required. In order to help the development of nanocapsules and understand drug release profile before planning the clinical experiments, it is important to set up a biosensing platform which could monitor and evaluate the real-time drug release profile of nanocapsules with high sensitivity and long-term measurement ability. In this work, a microfluidic chip platform with electrochemical carbon nanotube electrodes has been developed and characterized for rapid detection of antibiotics teicoplanin nanocapsules. Multi-walled carbon nanotubes are used to modify the gold electrode surfaces to enhance the performance of the electrochemical biosensors. Experimental results show that the limit of detection of the developed platform using carbon nanotubes electrodes is 0.1 μg/ml with a linear range from 1 μg/ml to 10 μg/ml. The sensitivity of the developed system is 0.023 mA ml/μg at 37°C. The drug release profile of teicoplanin nanocapsules in PBS shows that the antibiotics nanocapsules significantly increased the release of drug on the 4th day, measuring 0.4858 μg/(ml hr). The release of drug from the antibiotics nanocapsules reached 34.98 μg/ml on the 7th day. The results showed a similar trend compared with the measurement result using the HPLC instrument. Compared with the traditional HPLC measurements, the electrochemical sensing platform we developed measures results with increased flexibility in controlling experimental factors for long-term preclinical measurement of nanocapsules in real time and at low cost.     

Very small injected samples to study chloroquine and quinine in human serum using capillary-LC and native fluorescence

A comparison between HPLC with conventional fluorescence detection and capillary-LC (microHPLC) with native laser-induced fluorescence (LIF) detection was done to determine chloroquine (CQ) and quinine (Q) in human serum. HPLC experiments were run with parameters of the conventional fluorimeter set at the highest level of sensitivity. Results were compared with those obtained on microHPLC coupled to a ZETALIF (He-Cd 325 nm) detector which provided a 50-fold increase in sensitivity. In microHPLC-LIF injection volumes were 200 nL instead of 10 microL in conventional HPLC. The separation was completed within 3 min (6 min on HPLC). The limit of detection on microHPLC-LIF was 1.9 and 1.3 fmol for CQ and Q, respectively. Both experiments were validated on serum samples. The mean recovery was more than 95% for CQ and Q. The intra- and inter-day precision and accuracy were found to be within the acceptable limits (<10%).     

Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1, 2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.     

A comparison of thermospray and direct liquid introduction high-performance liquid chromatography/mass spectrometry for the analysis of candidate antimalarials

The analysis of antimalarials by high-performance liquid chromatography (HPLC)/mass spectrometry demonstrates a new dimension in specificity along with increased sensitivity compared to conventional HPLC detection methods. Both direct liquid introduction and thermospray HPLC/mass spectrometry interfaces provided molecular weight information as well as characteristic fragment ions for antimalarials not normally amenable to direct probe or gas chromatographic/mass spectrometric techniques. The direct liquid introduction interface, which incorporated a 1/100 split, showed a detection limit of 30 ng using selected ion monitoring. The thermospray technique showed less than 1 ng detection limits using selected ion monitoring.     

A novel, sensitive method for determining benzo[a]pyrene-diones using high-performance liquid chromatography with post-column zinc reduction

A novel and sensitive high-performance liquid chromatography (HPLC) method was developed to analyze dione metabolites of benzo[a]pyrene (BaP). Because BaP-diones do not fluoresce, detection of low concentrations is difficult to achieve when analyzing these chemicals with a simple HPLC system. We developed a method to increase the detection sensitivities for BaP-diones using reduction by zinc after the chromatographic separation. A post-column zinc reducer was used to convert BaP-diones, in-line, to their corresponding fluorescent BaP-hydroquinones, which can be measured by fluorescence detection with high sensitivity. With 20-muL injections, the limits of detection for the BaP-diones tested (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) were all below 1.0 nM. In addition to the high detection sensitivity, this HPLC method provides a wide linear dynamic range for BaP-dione detection (1.0-220 nM). We also studied the extraction recovery of BaP-diones from recombinant human cytochrome P450 and epoxide hydrolase. To demonstrate the application of this method, the kinetics of BaP-dione formation was studied by incubating BaP with these recombinant enzymes. The present method enhances the detection sensitivity for BaP-diones by more than two orders of magnitude compared with traditional ultraviolet detection. Moreover, the method avoids the time-consuming derivatization or reduction steps required by other methods.     

Analysis of 12 different pentacyclic triterpenic acids from frankincense in human plasma by high-performance liquid chromatography and photodiode array detection

For the determination of pentacyclic triterpenes of the boswellic acid family in human plasma a novel sensitive method was developed combining serial extraction on diatomaceous earth and graphitized carbon black followed by reversed phase high-performance liquid chromatography (HPLC) and photodiode array detection. The overall average extraction yield of 12 different pentacyclic triterpenic acids was approximately 66%. The calibration graphs were linear with coefficients of correlation for all compounds greater than 0.999. The overall within-day and between-day coefficients of variation (CV) for the 12 pentacyclic triterpenic acids were 5.6 and 6.8%, respectively. This HPLC procedure delivers the analytical sensitivity, precision and accuracy required for clinical pharmacokinetic and therapeutic studies.     

Determination of growth hormone-releasing hexapeptide by reversed-phase high-performance liquid chromatography with electrochemical detection

A novel HPLC method with electrochemical detection is described for the determination of a growth-hormone-releasing hexapeptide (GHRP-6). HPLC conditions, such as the column, mobile phase, and oxidation potential, were optimized for sensitivity and selectivity of analysis. GHRP-6 was separated on a reversed-phase CN column with 37% acetonitrile in 100 mM phosphate buffer (pH 7.0) as the mobile phase. The optimum electrochemical oxidation signal was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to two electroactive tryptophans and a histidine residue. Solid-phase extraction using octadecyl cartridges was optimized for sample cleanup of GHRP-6 from serum samples and the method was successfully applied over the concentration range of 5 to 100 ng/ml of analyte.     

Application of the dual-cell coulometric detector: a method for assaying monoamines and their metabolites

A new HPLC assay technique for monoamines and their metabolites, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite through which the samples flow, is described in comparison with a classical amperometric detector equipped with a glassy carbon electrode. Different potentials can be applied at each cell of the coulometric detector to improve sample resolution and detection sensitivity. The signal-to-noise ratio (s/n) calculated in similar conditions was 10 times lower for the coulometric detector than for the amperometric one. The dual-coulometric detector does not undergo daily decay or variation, and needs no particular care or preparation. It is therefore possible to achieve stable routine sensitivity in a range of 10 fmol. This new technique has been applied for assaying monoamines and their precursors and metabolites by direct injection of clear supernatant after centrifugation and for determination of catecholamine turnover in rat pineal gland and neuro- and adenohypophysis in samples purified by Al2O3 adsorption.     

Influence of extract purification on the recovery of citrinin in the HPLC analysis

Various analytical methods of citrinin determination are known from publications which cannot be applied without any difficulties. Low fluctuating recovery rates of standard additions, an insufficient extract purification for rye, and a rather low sensitivity based on improper HPLC conditions are being observed if the published methods are applied. An easily applicable, reliable and sensitive HPLC method in the lower ppb range on the basis of a solid phase extraction combined with a HPLC gradient which allows for a sensitive fluorescence detection, is under development. The results already achieved are described in the following.     

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.     

High-performance liquid chromatography of phosphatidic acid

This paper reviews existing high-performance liquid chromatographic (HPLC) methods for the analysis of phosphatidic acid (PA) in various sample matrices. In addition to the introductory background discussion on important aspects of PA in lipid biochemistry, the review provides comprehensive coverage in the areas of derivatization techniques, detection methods, and HPLC separation techniques. Conversions of PA to suitable derivatives enhance the detection sensitivity and improve the chromatographic behavior of the analytes. Detection methods include the use of state-of-the-art detectors and are discussed in terms of sensitivity, specificity, and compatibility with analytical systems. Pertinent normal-phase and reversed-phase HPLC data for PA are compiled from published methods.     

Acetylcholine and Choline in Neuronal Tissue Measured by HPLC with Electrochemical Detection

Abstract: A simple, rapid method is presented for the determination of acetylcholine (ACh) and choline (Ch) in neuronal tissue using HPLC with electrochemical detection. The method is based on the separation of ACh and Ch by reverse-phase HPLC and mixing the effluent as it emerges from the column with acetylcholinesterase and Ch oxidase, which converts endogenous Ch and Ch produced by the hydrolysis of ACh to betaine and hydrogen peroxide. Production of hydrogen peroxide is continuously monitored electrochemically. The sensitivity of the procedure is 1 pmol for Ch and 2 pmol for ACh. Specificity of the method is based on HPLC, two specific enzymatic reactions, and the detection of hydrogen peroxide.     

Polarimetric detection in high-performance liquid chromatography

Chiroptical detection for HPLC is particularly useful as a selective detection method for chiral molecules, and in enantiomeric purity determination with partial chiral separation or without chiral separation. The recent development of laser-based polarimeters with microdegree sensitivity has increased the applicability of optical rotation detection in HPLC. The detection limit of these instruments is submicrogram on-column for many chiral compounds in analytical HPLC. A variety of applications of the selective detection of optically active molecules are reviewed. The use of polarimetric detection with partial chiral separation is considered, both as an aid to method development and for enantiomeric purity determination. Finally applications to enantiomeric purity determination without chiral separation are reviewed, with the dual use of nonchirally selective and chiroptical detectors to determine the total amount and optical purity of the analyte. Determinations of chiral purity for samples of high enantiomeric excess are described, which with laser-based instrumentation may give accuracies of better than +/- 1% with sample loadings of 50 micrograms on an achiral column. Applications to the study of enantioselective reactions are also considered, with determination of enantiomeric excess in near-racemates to better than +/- 0.1%.     

High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37°C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.     

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon alpha2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this way native conditions during SEC and structural changes of the protein during RP-HPLC were demonstrated. These results were confirmed by stand-alone fluorescence and CD measurements. With respect to HPLC tandem detection, the fluorescence detector compared favourably to the UV and CD detector regarding linearity, sensitivity and selectivity. SEC combined with intrinsic fluorescence scanning detection permits conformational analysis of small amounts of aggregates in the presence of excess native monomeric protein. In conclusion, HPLC with on-line UV and intrinsic fluorescence detection provides a promising concept for analysing the amount and conformational properties of a biopharmaceutical and its impurities.     

Sensitive column-switching high-performance liquid chromatography method for determination of propiverine in human plasma

A sensitive column-switching high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of propiverine in human plasma. Propiverine and internal standard, oxybutynin, were extracted from human plasma that had been made basic with 5N sodium hydroxide into methyl tert-butyl ether. The extracted plasma sample was injected onto the HPLC system consisting of a pretreatment column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The assay was linear in concentration ranges of 2-200 ng/ml for propiverine in human plasma. This method showed excellent sensitivity (a limit of detection of 0.5 ng/ml), good precision and accuracy. This method is suitable for bioequivalence studies following single dose in healthy volunteers.     

An improved method for the identification and quantitation of biological lipids by HPLC using laser light-scattering detection

We have developed a simplified and improved high performance liquid chromatography (HPLC) method for the detection and quantitation of tissue lipid using a new laser light-scattering detector (Varex model ELSD II). This detector has a limit of sensitivity of 50 ng for neutral lipid and 200 ng for most phospholipids with excellent reproducibility. By coupling the ELSD II with a ternary gradient normal phase HPLC system, we were able to separate and quantify the major lipid constituents of extracted tissue. This system was used to profile and quantitate the major lipids from rat brain, liver, and heart with greater sensitivity than other available techniques, with the exception of high performance thin-layer chromatography (HPTLC). However, the convenience of HPLC allows for a significant improvement in analysis time with only a threefold reduction in sensitivity when compared to HPTLC.     

Fast determination of low-level cytochrome P-450 1A1 activity by high-performance liquid chromatography with fluorescence or visible absorbance detection

A method to determine the activity of the cytochrome P-450 1A1 enzyme, by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity using high-performance liquid chromatography (HPLC) with fluorescence or with visible absorbance detection of resorufin, is described. The lowest quantifiable activity (0.2 pmol/mg min) is obtained by incubation of 0.3 mg of human duodenal microsomal proteins using HPLC fluorescence detection. Using HPLC with visible absorbance detection, sensitivity was ten times lower. However, the equipment for this last method is available in most laboratories. The use of both HPLC assays allows determination of the low EROD activity level in samples of small size, such as two or three human duodenal biopsies obtained by routine endoscopy. These methods will be a useful tool to study the role of drug intestinal metabolism by cytochrome P-450 1A1.     

Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography

Various analytical techniques have been developed to determine nitrite and nitrate, oxidation metabolites of nitric oxide (NO), in biological samples. HPLC is a widely used method to quantify these two anions in plasma, serum, urine, saliva, cerebrospinal fluid, tissue extracts, and fetal fluids, as well as meats and cell culture medium. The detection principles include UV and VIS absorbance, electrochemistry, chemiluminescence, and fluorescence. UV or VIS absorbance and electrochemistry allow simultaneous detection of nitrite and nitrate but are vulnerable to the severe interference from chloride present in biological samples. Chemiluminescence and fluorescence detection improve the assay sensitivity and are unaffected by chloride but cannot be applied to a simultaneous analysis of nitrite and nitrate. The choice of a detection method largely depends on sample type and facility availability. The recently developed fluorometric HPLC method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) and the enzymatic conversion of nitrate into nitrite, offers the advantages of easy sample preparation, simple derivatization, stable fluorescent derivatives, rapid analysis, high sensitivity and specificity, lack of interferences, and easy automation for determining nitrite and nitrate in all biological samples including cell culture medium. To ensure accurate analysis, care should be taken in sample collection, processing, and derivatization as well as preparation of reagent solutions and mobile phases, to prevent environmental contamination. HPLC methods provide a useful research tool for studying NO biochemistry, physiology and pharmacology.     

High-performance liquid chromatographic determination of PEG 600 in human urine

Polyethylene glycols (PEGs) are non-ionic, water-soluble synthetic polymers which have been widely used for many applications. Since they are of very low toxicity and are readily excreted in urine, PEGs in the molecular weight range 400–6000 have been used extensively in the study of intestinal physiology in man. A high-performance liquid chromatographic (HPLC) method has been developed for the determination of PEG 600 in human urine, which includes a pre-column derivatisation step. The dibenzoate derivatives of PEG 600 can be quantitatively prepared, and this, coupled with ultraviolet detection at 230 nm, has greatly improved the limit of detection for the determination of PEGs by HPLC. A suitable extraction procedure has also been developed which enabled PEG levels in urine to be monitored with much greater sensitivity than any previously reported method.