Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells and cultured fibroblasts. OPA derivatives are separated on a reversed-phase HPLC column with an acetonitrile–sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) concentration is calculated by subtraction of the GSH level from the GSHt level. The assay shows high sensitivity (50 fmol per injection, the lowest reported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSSG close to 100%, and linearity (r>0.999). This HPLC technique is very simple and rapid. Its wide applicability and high sensitivity make it a convenient and reliable method for glutathione determination in various biological samples.     

Polymeric biocompatible iron oxide nanoparticles labeled with peptides for imaging in ovarian cancer

Compared with other nanomaterials, surface-modified iron oxide nanoparticles (IONPs) have gained attraction for cancer therapy applications due to its low toxicity, and long retention time. An innocuous targeting strategy was developed by generation of fluorescein isothiocyanate (FITC)-labeled peptide (growth factor domain (GFD) and somatomedin B domain (SMB)) functionalized, chitosan-coated IONPs (IONPs/C). It can be used to target urokinase plasminogen activator receptor (uPAR), which is a surface biomarker, in ovarian cancer. Binding affinity between uPAR and peptides (GFD and SMB) were revealed by in-silico docking studies. The biophysical characterizations of IONPs, IONPs/C, and IONPs/C/GFD-FITC or SMB-FITC nanoprobes were assessed via Vibrating Sample Magnetometer (VSM), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FT-IR). Prussian Blue staining, fluorescence spectroscopy, and fluorescence imaging were performed to confirm the targeting of nanoprobes with the surface receptor uPAR. The combination of IONPs/C/GFD+SMB showed efficient targeting of uPAR in the tumor microenvironment, and thus can be implemented as a molecular magnetic nanoprobe for cancer cell imaging and targeting.     

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.     

Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.     

Simultaneous determination of ampicillin and metampicillin in biological fluids using high-performance liquid chromatography with column switching

A new high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of metampicillin and its metabolite ampicillin in biological fluids. The plasma, urine and bile samples were injected onto a precolumn packed with LiChrosorb RP-8 (25–40 μm) after simple dilution with an internal standard solution in 0.05 M phosphate buffer (pH 7.0). The polar plasma components were washed out using 0.05 M phosphate buffer (pH 7.0). After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Ultracarb 5 ODS-30 column with a gradient system of acetonitrile-0.02 M phosphate buffer (pH 7.0) as the mobile phase. The method showed excellent precision, accuracy and speed with a detection limit of 0.1 μg/ml. The total analysis time per sample was less than 40 min and the coefficients of variation for intra- and inter-assay were less than 5.1%. This method has been successfully applied to plasma, urine and bile samples from rats after intravenous injection of metampicillin.     

A Low Picomole Fluorometric Detection System for Amino Acid Analysis

An amino acid detection system was developed for low picomole analysis of amino acids including proline, based on OPA-post-labeling with a non-switching NaCIO flow system. A computer-controlled HPLC equipped with a three-solvent gradient mixer was assembled with the detection system to analyze 17 kinds of amino acids in 85 min with a stable base line. The optimum conditions of the NaCIO and OPA solutions were determined as 0.002%–0.22ml/min and 0.16%–0.26ml/min, respectively. The use of NaCIO caused only approximately 30% decrease in fluorescence response of the usual amino acids, except for Pro and Cys, the latter even being enhanced by about 10 times. The detection limit for Pro and Cys was 500 and 1000 fmol, respectively, and that of the usual amino acids was 100 fmol. The calibration curves of all amino acids showed good linearity in a range between 5 and 500 pmol. This system was used as a detector for enantiomeric analysis of glutamic acid and cysteic acid with a reversed phase HPLC during stereochemical studies on the natural peptide derivative, oxycadystin.     

Monolithic column-based reversed-phase liquid chromatography separation for amino acid assay in microdialysates and cerebral spinal fluid

The development of a HPLC method using a monolithic C18 column is described using fluorescence detection for the assay of 21 amino acids and related substances with derivatisation using ortho-phthaldialdehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA). The method employs a tertiary gradient and has a run time of 24 min. Linearity (r2) for each amino acid was found to be greater than 0.99 up to a 10 microM concentration; reproducibility across all analyses (relative standard deviation (R.S.D.)) was between 0.97 and 6.7% and limit of detection (LOD) between 30 and 300 fmol on column. This method has been applied to the analysis of amino acids in both spinal microdialysis and cerebral spinal fluid samples.     

Development of a novel cytochrome p450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography

A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (beta-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (alpha-naphthoflavone, beta-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n=3; signal to noise [S/N]=3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n=1; S/N=3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.     

Amino acids analysis using a monolithic silica column after derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)

A fast HPLC method using a monolithic silica column was developed for the measurement of amino acids. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a monolithic silica column (MonoClad C18-HS, 250 mm × 3 mm I.D.). The separation of 19 NBD-amino acids was achieved within 18 min, which was only one-fifth of the time taken by the methods using a conventional particle-packed column, with the gradient elution of a mobile phase at the flow rate of 1.4 mL/min. The sensitivity was good with a limit of detection for the individual amino acids ranging from 2.94 to 53.4 fmol. The calibration curves for all the amino acids were found to be linear in the range of 200 fmol to 20 pmol with correlation coefficients of 0.997 or better. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.     

Direct plasma injection for high-performance liquid chromatographic–mass spectrometric quantitation of the anxiolytic agent CP-93 393

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC–ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC–ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10–1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 μl) were performed per column with unattended, automated analysis.     

Rapid and sensitive step gradient assays of glutamate, glycine, taurine and γ-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis samples

We developed a rapid step-gradient HPLC method for determination of glutamate, glycine and taurine, and a separate method for determination of γ-aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde–2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 μl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.     

Quantitative Analysis of Flavonoids in Chamomile Flowers (<Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.) by Microcolumn HPLC-UV

A reverse-phase microcolumn HPLC method with UV detection (330 nm) was developed for quantitative analysis of flavonoids of Matricaria chamomilla flowers using a ProntoSIL-120-5-C18 AQ column (60 mm × 1 mm × 5 μm), gradient elution system 0.2 M LiClO₄/0.006 M HClO₄–acetonitrile and cosmosiin as a reference compound. Optimal conditions for the hydrolytic process of flavonoids (KOH concentration 0.25%, extraction time 30 min) and the parameters of flavonoids extraction (particle size 0.25 mm, extraction temperature 60°C, a single extraction step lasting 30 min at a ratio 1: 100) were selected. Validation analysis showed that the proposed method is characterized by satisfactory metrological parameters. The limit of detection (LOD) and limit of quantification (LOQ) of cosmosiin were 84 and 255 ng/mL, respectively. The accuracy for cosmosiin content levels 80–120% was less than 101.93–103.00%. The method was used for the analysis of introduced and commercial samples of M. chamomilla flowers.     

Capillary chromatography-coupled mass spectrometry with column switching for rapid identification of proteins from 2-dimensional electrophoresis gels

An improved capillary liquid chromatography procedure, incorporating column switching in combination with mass spectrometry, is reported. The dual column system allows for rapid inject-to-inject cycle times to improve the speed of protein identification for proteomics applications. Full gradient elution of peptides from either of the two C18 columns can be achieved in less than 17 min while maintaining sufficient resolution for the peptides to be detected and fragmented by the mass spectrometer for protein identification. Importantly, the use of two columns for subsequent injections is reproducible and without carry-over. The limit of detection for the system is between 25 and 50 fmol per injection. This fully automated system is capable of analyzing and identifying proteins from an entire 96-well plate in about 27 h.     

Determination of metronidazole in vaginal tissue by high-performance liquid chromatography using solid-phase extraction

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of metronidazole in vaginal tissue is reported. The method uses a Zorbax SB phenyl column with a 0.01 M aqueous monobasic potassium phosphate buffer (pH 4.0)-absolute methanol (85:15, v/v) as mobile phase at a flow-rate of 1.0 ml/min and detection at 313 nm. Tinidazole was used as the internal standard. The method employed homogenization of tissue followed by solid-phase extraction. The quantitation was achieved within 30 min with sensitivity in the ng/g range. Metronidazole was linear in the 100–2000 ng/g range. The accuracy and precision were in the 1–4% range for the drug and the limit of detection was approximately 100 ng/g based on a signal-to-noise ratio of 3 and a 100-μl injection.     

Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography

The determination of quinine, (3S)-3-hydroxyquinine, 2′-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 μl cold methanol. Urine samples were 10–100× diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024–0.081 μM.     

Determination of aloesin in rat plasma using a column-switching high-performance liquid chromatographic assay

A column-switching high-performance liquid chromatography (HPLC) method for the determination of aloesin in rat plasma using column-switching and ultraviolet (UV) absorbance detection is described. Plasma was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The determination of aloesin was accurate and repeatable, with a limit of quantitation of 10 ng/ml in plasma. The standard calibration curve for aloesin was linear (r=0.998) over the concentration range of 10–1000 ng/ml in rat plasma. The intra- and inter-day assay variabilities of aloesin ranged from 1.0 to 4.7% and 1.1 to 8.8%, respectively. This highly sensitive and simple method has been successfully applied to a pharmacokinetic study after oral administration of aloesin to rats.     

Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol–chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15–20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15–20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC–CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.     

Serotonin,dopamine, noradrenaline and their metabolites: Levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine—a 5-HT2 receptor agonist

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection.2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it.3. [³H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a K_d of 5 nM and a concentration of B_max 180 fmol/mg protein.     

Dual microcolumn immunoassay applied to determination of insulin secretion from single islets of Langerhans and insulin in serum

A dual microcolumn immunoassay (DMIA) was developed and applied to determination of insulin in biological samples. The DMIA utilized a protein G capillary column (150 μm I.D.) with covalently attached anti-insulin to selectively capture and concentrate insulins in a sample. Insulins retained in the capillary immunoaffinity column were desorbed and injected onto a reversed-phase capillary column (150 μm I.D.) for further separation from interferences such as cross-reactive antigens and non-specifically adsorbed sample components. Bovine, porcine and rat insulin all cross-reacted with the antibody and could be determined simultaneously. Using a UV absorbance detector, the dual microcolumn system had a detection limit of 10 fmol or 20 pM for 500-μl sample volumes. The DMIA system was used to measure glucose-stimulated insulin secretion from single rat islets of Langerhans. Because of the separation in the second dimension, both rat I and rat II insulin could be independently determined. The system was also evaluated for determination of insulin in serum. Using microcolumns instead of conventional HPLC columns resulted in several advantages including use of less chromatographic material and improved mass detection limit.     

Quantitative high-performance liquid chromatography-based detection method for calphostin C,a naturally occurring perylenequinone with potent antileukemic activity

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca²⁺-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250×4 mm LiChrospher 100, RP-18 (5 μm) in conjunction with a 4×4 mm LiChrospher 100, RP-18 guard column (5 μm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile–water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05–40 μM for calphostin C in 50 μl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 μl plasma was 0.02 μM at a signal-to-noise ratio of ∼3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.