Enantioselective assay of chloroquine and its main metabolite deethyl chloroquine in human plasma by capillary electrophoresis

A sensitive assay for the determination of chloroquine (Clq) and its pharmacologically active metabolite deethyl chloroquine in plasma by capillary electrophoresis (CE) is developed. Plasma levels of drug and metabolite are measured using HeCd laser-induced fluorescence (LIF) detection over a range of three orders of magnitude from 2 to 1000 ng/mL after liquid-liquid extraction. A limit of detection of 0.5 ng/mL is achieved. Validation of the method yields intra- and interday precision data within the limits of 10% (20% at limit of quantitation) and intra- and interday accuracy data greater than 6% throughout the whole working range. The method is applied for the drug monitoring of patients treated with Clq. Based upon this assay, two enantioselective CE-LIF methods for Clq and its main metabolite are developed. Mixtures of substituted gamma-cyclodextrins are used as chiral selectors. A baseline separation of the enantiomers of both analytes in one run is achieved in less than 11 min (method A) and less than 9 min (method B), respectively. Hydroxychloroquine is used as the internal standard for both methods.     

Anthracycline analysis by capillary electrophoresis. Application to the analysis of daunorubicine in Kaposi sarcoma tumor.

Laser-induced fluorescence (LIF) detection is now a well-known sensitive and selective detection mode for capillary electrophoresis (CE) analysis. It has been shown to be 100- to 100,000-times more sensitive than UV detection and little work has been done using LIF in conjunction with high-performance liquid chromatography (HPLC). The need for greater resolution and higher sensitivity for the analysis of anthracyclines (fluorescent chemotherapic drugs), prompted us to compare CE-LIF and HPLC-LIF, for the detection of these substances. CE-LIF sensitivity based on quantity of anthracycline injected is 50-times greater than that obtained with HPLC-LIF, because of the injected sample volume. Analysis of daunorubicin in Kaposy sarcoma tumors and in plasma are presented. The decrease of the concentration of daunorubicin in the tumor and in the plasma following time show the same behavior, indicating identical concentrations of the anthracycline in both samples.     

A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection for the analysis of chelerythrine and sanguinarine in Chinese herbal medicines

Laser-induced fluorescence (LIF) is a highly sensitive detection method for capillary electrophoresis (CE). However, it usually requires analyte to be derivatized, unless the wavelength of native fluorescence of analyte matches the laser's. That limits its application in drug analysis. In this work, we introduced a rapid, simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence (NACE-LIF) detection for the analysis of chelerythrine and sanguinarine for the first time. As these two alkaloids have some native fluorescence, they were directly detected using a commercially available Ar⁺ laser without troublesome fluorescent derivatization. The fluorescence was enhanced by nonaqueous media. Compared with previously reported UV detection method, lower limit of detection (LOD) is achieved thanks to the high sensitivity of LIF detection (2.0 ng/mL for chelerythrine and 6.3 ng/mL for sanguinarine). Moreover, with NACE, the baseline separation of these alkaloids is finished within 3.5 min. This method is successfully applied to determine the contents of chelerythrine and sanguinarine in Macleaya cordata (Willd.) R. Br. and Chelidonium majus L.     

Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography-mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-beta-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo-N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE-LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE-LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-deethylzaleplon.     

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump‐free negative pressure sampling device was used for rapid bias‐free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in <1 min. The average migration times of the epinephrine (E), dopamine (DA), and norepinephrine (NE) were 34.26, 43.81, and 50.07 s, with an RSD of 1.05, 1.26, and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for E and 0.025～4.0 mg/L for DA and NE, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of E, DA, and NE were 2.5, 5.0, and 5.0 μg/L, respectively. The recoveries of E, DA, and NE in spiked urine samples were between 86 and 103%, with RSDs of 4.5～6.8% (n = 5). The proposed NAMCE with LIF detection combined with a pump‐free negative pressure sampling device is a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples.     

Rapid analysis of anthracycline antibiotics doxorubicin and daunorubicin by microchip capillary electrophoresis

A simple and rapid method has been developed for the analysis of anthracycline antibiotics doxorubicin (DOX) and daunorubicin (DAU) in human serum using mirochip-based capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. In this study, method development included studies of the effect of buffer pH, buffer concentration, organic solvents and separation voltage on sensitivity and separation efficiencies for the CE separation of DOX and DAU. Acetonitrile was found to have significantly improved the sensitivity and separation efficiency. The method was validated with regard to reproducibilities, linearity and limit of detection (LOD). The optimum electrophoretic separation conditions were 10 mM sodium tetraborate buffer at pH 9.5 with 40% acetonitrile (V/V) and a separation voltage of 2.1 kV. DOX and DAU were separated in 60 s under the optimum separation conditions. Linear relationships were obtained between the concentration and peak area (or peak height) in the 1–75 µg mL^{− 1} range and with the detection limits of 0.3 and 0.2 μg mL^{− 1} for DOX and DAU, respectively. The stability of both migration time and peak height of the analytes showed relative standard deviations of less than 5% (n = 9). The potential of this method was verified by spiking a human serum sample with the two drugs and analyzing the recovery ratios.     

Rapid identification and high sensitive detection of cancer cells on the gold nanoparticle interface by combined contact angle and electrochemical measurements

In this study, we have proposed a novel strategy for the rapid identification and high sensitive detection of different kinds of cancer cells by means of electrochemical and contact angle measurements. A simple, unlabeled method based on the functionalized Au nanoparticles (GNPs) modified interface has been utilized to distinguish the different cancer cells, including lung cancer cells, liver cancer cells, drug sensitive leukemia K562/B.W cells and drug resistant leukemia K562/ADM cells. The relevant results indicate that under optimal conditions, this method can provide the quantitative determination of cancer cells, with a detection limit of ∼10³ cells mL⁻¹. Our observations demonstrate that the difference in the hydrophilic properties for target cellular surfaces and in the uptake efficiency of the anticancer drug daunorubicin for different cancer cells could be readily chosen as the elements of cancer identification and sensitive detection. This raises the possibility to advance the promising clinic diagnosis and monitoring of tumors with the aim of successful chemotherapy of human cancers.     

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.     

Kinetic Transport Analysis of Daunorubicin by LoVo and LoVo/DX Cells

We describe a fluorometric technique for the measurement of transport parameters of fluorescent drugs through cellular membranes. Unlike other procedures, this method gives an accurate measure of drug accumulated in the cells and measures the fraction of free and bound drug in the cell. The kinetic parameters of transport through cellular membranes are determined using a simple three-compartment model combined with fluorescence measurements performed on the extracellular medium and on Triton-permeabilized cells during daunorubicin incorporation. With this technique we found that LoVo cells have a greater daunorubicin uptake, a similar input rate constant and a lower output rate constant than the drug-resistant LoVo/DX cells.     

Simple and precise detection of lipid compounds present within liposomal formulations using a charged aerosol detector

In recent decades the use of liposomal preparations as drug delivery systems has become very attractive in pharmaceutical development. Therefore, thorough characterization and quantification of the lipids which form liposomes is wished from both investigators and regulatory authorities when the application in humans is being considered. In this study a new HPLC method for the detection of lipids in liposomal formulations was established using corona charged aerosol detection (CAD) which has the advantage to be independent of the chemical properties of the analytes. The superiority of this method over UV detection was demonstrated. Compared to UV detection no absorption effects of the organic solvent in the mobile phase interfering with the lipid signals were observed with CAD. CAD showed good linearity (R² > 0.990) for all liposomal compounds. The acceptance criteria for precision including repeatability were met. The average recovery for each of the excipients of the liposomal formulation was in the range of 90.0–110%.     

Novel nanocomposite of nano fe(3)o(4) and polylactide nanofibers for application in drug uptake and induction of cell death of leukemia cancer cells

Novel nanocomposites of polylactide (PLA) nanofibers and tetraheptylammonium-capped Fe3O4 magnetic nanoparticles have been prepared and utilized to realize the efficient accumulation of anticancer drug daunorubicin in target cancer cells. The observations of optical microscopy and confocal fluorescence microscopy indicate that the PLA nanofibers and Fe3O4 nanoparticles may contribute to their beneficial effects on intracellular drug uptake of leukemia K562 cell lines in which the efficiently enhanced accumulation of anticancer drug daunorubicin on the membrane of cancer cells could be observed. Meanwhile, the electrochemical detection and the microculture tetrazolium studies were also explored to probe the effect of the relevant nanomaterials on the drug uptake of cancer cells. The results illustrate that the nanocomposites could effectively facilitate the interaction of daunorubicin with leukemia cells and remarkably enhance the permeation and drug uptake of anticancer agents in the cancer cells, which could readily lead to the induction of the cell death of leukemia cells. This observation suggests a new perspective for the targeted therapeutic approaches of cancers.     

Determination of methotrexate and its major metabolite, 7-hydroxymethotrexate, using capillary zone electrophoresis and laser-induced fluorescence detection

High-dosage methotrexate therapy requires careful monitoring of the drug in serum to ensure minimal toxic effects. A simple, rapid and sensitive method for the separation and quantitation of methotrexate and its major metabolite, 7-hydroxymethotrexate, using high-voltage capillary zone electrophoresis combined with laser-induced fluorescence detection is described. The detection limit for methotrexate is as low as 5.10(-10) M (signal-to-noise ratio = 3), while that for 7-hydroxymethotrexate is 2.10(-9) M. The linearity of the system extends over nearly four orders of magnitude for both methotrexate and 7-hydroxymethotrexate. The extraction efficiency for the drug and its metabolite from serum is 80-85% using a Sep-Pak C18 cartridge. Quantitation of methotrexate in serum was possible in the 10(-10) M range, nearly two orders of magnitude lower than that currently obtainable by existing methods. Good correlation (r = 0.99) for serum methotrexate concentrations was obtained with an enzyme-multiplied immunoassay technique. Comparison with an enzyme inhibition assay also provided similar results.     

Detection of specific antibodies using immunosubtraction and capillary electrophoresis instrumentation

Solution-phase immunoassays based on capillary electrophoresis (CE) separations have been shown to be rapid and simple to perform. The potential for sample matrix interference and incompatibility with multiplexing conditions for antibody detection when dealing with real samples, however, has prompted the development of an assay that utilizes an immunosubtraction methodology. A model assay for the detection of specific antibodies that relies on solid-phase extraction, CE and laser-induced fluorescence (LIF) detection is described. The method, called immunocapture-immunosubtraction (ICIS), incorporates an antibody capture/purification protocol using magnetic particles. The detection of specific antibodies is achieved by CE-LIF analysis of a probe solution following incubation with the captured antibodies. As an example of the ICIS assay's capabilities, the relative quantification of anti-fluorescein in serum is presented.     

Determination of sertraline and N-desmethylsertraline in human plasma by CE with LIF detection

A method has been developed for the analysis of the antidepressant drug sertraline together with its main metabolite N-desmethylsertraline (DMS) in human plasma. It is based on CE with LIF detection (lambda = 488 nm). A SPE procedure is employed for biological sample pretreatment, followed by a derivatization step with FITC; reboxetine was the internal standard. The effect of CD, acetone and N-methyl-D-glucamine (GLC) as constituents of the BGE for analyte separation was investigated. The final BGE consisted of 20 mM carbonate buffer, pH 9.0, with 2.5 mM heptakis(2,6-di-O-methyl)-beta-CD, 50 mM GLC and 20% v/v acetone. With 30 kV applied voltage, the electrophoretic run is completed in 7.5 min. Linearity was observed in the plasma concentration range from 3.0 to 500 ng/mL for sertraline and 4.0 to 500 ng/mL for DMS. Extraction yield was >97.1%, precision - expressed as RSD% - was <3.7, accuracy (recovery) was >95.6%. Due to its sensitivity and selectivity, the method was suited for the analysis of plasma samples from patients undergoing therapy with sertraline.     

Quantification of pegylated liposomal doxorubicin and doxorubicinol in rat plasma by liquid chromatography/electrospray tandem mass spectroscopy: Application to preclinical pharmacokinetic studies

A high-performance liquid chromatography-tandem mass spectrometric method (LC/MS/MS) has been developed and validated for the determination of pegylated liposomal doxorubicin and its metabolite doxorubicinol in rat plasma. One hundred microliters plasma samples were treated with Triton X-100 to immediately disperse the liposome. Then the samples were extracted by a single methanol:acetone protein precipitation step in the presence of additional 50microL of 70% (w/v) zinc sulfate, and subsequently analyzed by LC/MS/MS using positive turbo-ion spray ionization mode operating the instrument in the multiple-reaction-monitoring (MRM) mode. The related compound daunorubicin was used as internal standard. The validated concentration ranges were from 20 to 8000ng/mL for doxorubicin and from 0.05 to 20.0ng/mL for doxorubicinol. An effective LC-MS/MS method was developed to quantify trace amount of doxorubicinol with little interference from doxorubicin. The autosampler carryover was minimized from 285 to 10.5% by increasing the washing times of the valves when the used pentafluorophenylpropyl HPLC column had no contribution to the carryover. The relative matrix effect from six unique lots was absent for both compounds. Results obtained from the GLP validation study demonstrated good accuracy (85-110%) and precision (CV less than 14%) across the calibration ranges for both compounds. This method was applied to study the pharmacokinetic profiles of doxorubicin and doxorubicinol in rats after a single dose administration of Stealth-49 liposomal doxorubicin HCl. The mean AUC value for doxorubicinol was found to be only 0.011% of that of doxorubicin.     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.     

Rapid Determination of Free Amino Acids in Milk by Microchip Electrophoresis Coupled with Laser‐induced Fluorescence Detection

A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis (MCE) coupled with laser‐induced fluorescence (LIF) detection was developed. Seven kinds of standard amino acids were derivated with sulfoindocyanine succinimidyl ester (Cy5) and then perfectly measured by MCE‐LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions: 100 mmol·L^?1 sodium borate solution (pH 10.0) as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 µmol·L^?1 to 1.0 µmol·L^?1 and the detection limit was as low as about 1.0 nmol·L^?1. This MCE‐LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.     

Nonaqueous capillary electrophoresis coupled with laser-induced native fluorescence detection for the analysis of berberine, palmatine, and jatrorrhizine in Chinese herbal medicines

LIF detection is one of the most sensitive detection methods for CE. However, its application is limited because the analyte is usually required to be derivatized with a fluorescent label. As a result, LIF is seldom used to analyze active ingredients in plants. In this work, we introduce a rapid, simple, and sensitive method of nonaqueous CE (NACE) coupled with laser-induced native fluorescence detection for the simultaneous analysis of berberine, palmatine, and jatrorrhizine. This method skillfully utilizes the native fluorescence of these alkaloids and requires no troublesome fluorescent derivatization. As these alkaloids can fluoresce to some degree, they were simply detected by a commercially available 488 nm Ar+ laser. The native fluorescence of the analytes was greatly enhanced by nonaqueous media. Compared with the reported UV detection method, much lower LOD was achieved (6.0 ng/mL for berberine, 7.5 ng/mL for palmatine, and 380 ng/mL for jatrorrhizine). This method was successfully applied to analyze berberine, palmatine, and jatrorrhizine in two Chinese herbal medicines, Rhizoma coptidis and Caulis mahoniae.     

Uptake of amitriptyline and nortriptyline with liposomes, proteins, and serum: implications for drug detoxification

Liposomes composed of DOPG and DMPC were studied for their ability to sequester amitriptyline and nortriptyline under physiological conditions. The liposomes reduced the free drug concentration in protein mixtures and in human serum, but the drug uptake efficiency of liposomes was reduced in the presence of plasma proteins, perhaps due to adsorption of proteins on the liposomes. The reduction was significantly more for the pure DOPG liposomes. The 50:50 DMPC:DOPG liposomes (0.72 mg lipid/mL) reduced the free amitriptyline concentration by 50-60% in the presence of 7% proteins (4% albumin (w/w), 2% fibrinogen (w/w), 1% globulins (w/w)). In human serum, the free drug reduction was 35-70% with the same 50:50 liposomes (0.72 mg lipid/mL). The liposomal systems were equally efficient at sequestering nortriptyline, which is a major metabolite of amitriptyline. The drug binding to liposomes in the presence of serum proteins is also quick and reversible and the likely mechanism of drug sequestration is adsorption of drug on the surface of liposomes. Accordingly, the drug uptake increases with increased charge and lipid loading. Even though the serum proteins reduced the effectiveness of the liposomes at sequestering the drug, the 50:50 DMPC:DOPG liposomes may be effective at treating amitriptyline overdose patients.