Study of the effect of Wuzhi tablet (Schisandra sphenanthera extract) on tacrolimus tissue distribution in rat by liquid chromatography tandem mass spectrometry method

A liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining tacrolimus (FK506) in rat tissues to study the effect of Schisandra sphenanthera extract on FK506 tissue distribution. After a liquid–liquid extraction with ethyl acetate, FK506 and ascomycin (IS) were subjected to LC‐MS/MS analysis using positive electrospray ionization under multiple reactions monitoring mode. Chromatographic separation of FK506 and ascomycin was achieved on a Hypersil BDS C₁₈ column with a mobile phase consisting of methanol‐water (containing 2 mM ammonium acetate, 95 : 5, v/v). The intra‐ and inter‐batch precision of the method were less than 8.8 and 9.8%, respectively. The intra‐ and inter‐batch accuracies ranged from 97.5 to 104.0%. The lowest limit of quantification for FK506 was 0.5 ng/mL. The method was applied to a FK506 tissue distribution study with or without a dose of Wuzhi (WZ) tablet. Most of the FK506 tissue concentrations were slightly increased after a concomitant WZ tablet dose, but the whole blood concentration of FK506 was dramatically increased 3‐fold after a concomitant WZ tablet dose. These results indicated that the LC‐MS/MS method was rapid and sensitive enough to quantify FK506 in different rat tissues, and strict drug monitoring is recommended when co‐administering WZ tablet in clinical use. Copyright © 2009 John Wiley & Sons, Ltd.     

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract

A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.     

Ethyl acetate for the desorption of a liquid chromatographic precolumn on-line into a gas chromatograph

The feasibility of using ethyl acetate for the desorption of trace pollutants from a liquid chromatographic precolumn on-line into a diphenyltetramethyldisilazane-deactivated retention gap and, subsequently analysis by means of capillary gas chromatography has been demonstrated. First 5% of methanol are added to the water sample to prevent sorption of analytes onto parts of the preconcentration system. About 1 ml of this aqueous sample is injected onto a precolumn containing a polymeric stationary phase, using water–methanol (95:5, v/v) for transport and clean-up. The precolumn is desorbed with ethyl acetate and a fraction of 75 μl is injected on-line into the retention gap; separation is then achieved on a capillary CP Sil 19 column. No breakthrough of the test compounds was observed in the preconcentration step. The recovery was quantitative and the response obtained with flame ionization detection was linear in the range 0.1–100 ng/ml. The effect of varying the sorption flow rate on the recovery was studied. The system was applied to the analysis of river water.     

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.     

Determination of flunitrazepam and 7-aminoflunitrazepam in human urine by on-line solid phase extraction liquid chromatography-electrospray-tandem mass spectrometry

A method using an on-line solid phase extraction (SPE) and liquid chromatography with electrospray-tandem mass spectrometry (LC-ES-MS/MS) for the determination of flunitrazepam (FM2) and 7-aminoflunitrazepam (7-aminoFM2) in urine was developed. A mixed mode Oasis HLB SPE cartridge column was utilized for on-line extraction. A reversed phase C₁₈ LC column was employed for LC separation and MS/MS was used for detection. Sample extraction, clean-up and elution were performed automatically and controlled by a six-port valve. Recoveries ranging from 94.8 to 101.3% were measured. For both 7-aminoFM2 and FM2, dual linear ranges were determined from 20 to 200 and 200-2000 ng/ml, respectively. The detection limit for each analyte based on a signal-to-noise ratio of 3 ranged from 1 to 3 ng/ml. The intra-day and inter-day precision showed coefficients of variance (CV) ranging from 4.6 to 8.5 and 2.6-9.2%, respectively. The applicability of this newly developed method was examined by analyzing several urine samples.     

Determination of ampicillin in New Zealand white rabbit plasma using column switching technique and HPLC

Summary The purpose of this study was to develop a simple and fast analytical method for quantitation of ampicillin in rabbit plasma, suitable for analysis of large numbers of samples collected from experimental animals. The concentration of ampicillin in rabbit plasma was determined utilizing ion-pair reverse-phase high performance liquid chromatography (HPLC) with UV detection and a column switching technique. Plasma samples were treated with a perchloric acid solution to precipitate proteins and centrifuged to pellet the precipitated proteins. Cephalexin was used as an internal standard. The C₁₈ precolumn was placed in the injector loop of the Rheodyne injector. Samples were injected with the injector in the load position and the precolumn was washed free from interfering compounds. When the injector was switched to the inject position, the mobile phase was passed through the precolumn taking relatively pure compounds onto the analytical column. The limit of quantitation was established to be 400 ng mL^–1 of plasma. The standard curves were linear over the range of ampicillin concentrations assayed, 400 to 10,000 ng mL^–1 of rabbit plasma, and had a mean regression coefficient of 0.9962 (±0.0043). Intra-day variability was determined using six replicates of controls (low and high) analyzed on a single assay. The percent of relative accuracy for low and high controls were 5.67 and 1.12, respectively. Inter-day variability was determined over a four day period analyzing replicates of each control. The percent of relative accuracy for low and high controls were 4.33 and 1.63, respectively.     

Determination of cyclosporin A in whole blood by high-performance liquid chromatography using automated column switching

A fully automated high-performance liquid chromatographic column-switching system is presented for the determination of cyclosporin A in whole blood. After blood proteins were precipitated with acetonitrile, the supernatant was automatically loaded on to a cyanopropyl column for initial separation, and then the fraction containing cyclosporin A was loaded on to a trimethylsilica column for final separation and quantitation. Cyclosporin A was detected by ultraviolet absorption at 205 nm. The minimum detectable concentration of cyclosporin A was 5 ng/ml in 100 microliter of blood. The coefficient of variation of the method was 1.755, 1.748 and 0.655% in whole blood when spiked at the 170, 425 and 850 ng/ml levels, respectively. One assay was completed in 15 min.     

LR12‐peptide quantitation in whole blood by RP‐HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study

A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells‐1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid‐phase extraction. Chromatographic separation was carried out in a gradient mode using a core–shell C₁₈ column (150 × 4.6 mm, 3.6 μm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50–500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre‐analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.     

Use of cotton as a sorbent for on-line precolumn enrichment of polycyclic aromatic hydrocarbons in waters prior to liquid chromatography determination

Using cotton as a solid-phase extraction sorbent of the precolumn, an on-line coupled precolumn preconcentration-liquid chromatography system with fluorescence detection was developed for the determination of PAHs in aqueous samples. Four PAHs including fluorene, phenanthrene, fluoranthene and benzo[k]fluoranthene were preconcentrated by a precolumn packed with 30 mg of absorbent cotton and then separated by C₁₈ analytical column. When 100 ml of sample was enriched, the proposed procedure provided detection limits in the range of 0.5-57 ng l⁻¹. Several water samples spiked with PAHs were analyzed with recoveries in the range of 92-119% at spiking level of 100 ng l⁻¹ for fluorene, phenanthrene and fluoranthene, and 10 ng l⁻¹ for benzo[k]fluoranthene, respectively.     

Analysis of free amino acids in Amur sturgeon by ultra‐performance liquid chromatography using pre‐column derivatization with 6‐aminoquinolyl‐carbamyl

A rapid, sensitive, and reliable ultra‐performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6‐aminoquinolyl‐carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C₁₈ column with Acetonitrile–acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5–1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.     

Characterization of the precolumn bio trap 500 C18 for direct injection of plasma samples in a column-switching system

Summary A new restricted access media (RAM) type of precolumn, Bio Trap 500 C₁₈, for direct injection of plasma samples in column-switching systems was evaluated with respect to the elution of plasma proteins in different mobile phases, the loading capacity of plasma samples, the chromatographic behavior during plasma injections and protein contamination of the packing and sealings. More than 95% of plasma proteins could be excluded from the precolumn within three minutes for all selected mobile phases. Quantitative analyte recoveries could be obtained by injecting plasma samples ranging from 5 to 500 μL with the analyte mass>150 ng onto a BioTrap 500 C₁₈ column (20×4 mm I.D.). One precolumn tolerated about 15 mL of plasma injection without out noticeable change in retention and pressure. Clogging of the precolumn was encountered (≥45 mL of plasma) due mainly to the adsorption of proteins on the packing. The performance of the analytical column (Kromasil C₁₈) was also examined. The column efficiency decreased by 60% after processing 45 mL plasma in total.     

Two-dimensional high-performance liquid chromatography at low ng/ml levels of the anti-proliferative agent B859-35 in serum with automated sample clean-up, solid-phase trapping and ultraviolet detection.

An automated non-chiral high-performance liquid chromatographic method is described for the determination of the new anti-proliferative agent B859-35 in serum. This method employs sample clean-up of 1 ml of biofluid by liquid-solid extraction with the AASP (Advanced Automatic Sample Preparation) system. First separation is achieved on a LiChrospher-60-RP-Select-B column. A fraction of this elute is then collected by solid-phase trapping. Thereafter, the final chromatogram is developed on a narrow-bore Hypers1-CPS column and quantified with ultraviolet detection at 230 nm. The limit of quantitation of the assay is 250 ng/ml. Linearity was proven in the range 0.25-100 ng/ml. Typical figures for precision at these concentrations are 7.4 and 3.3%, and for accuracy 8.0 and 1.3%, respectively. An application of this method to the study of pharmacokinetics of B859-35 in serum samples of cancer patients is given.     

Determination of phenoxyacid herbicides in water

Novel clean-up techniques for a polymeric precolumn (PLRP-S) for the subsequent determination of bentazone and eight phenoxy acid herbicides in surface water samples are described. After preconcentration of the components at pH 3 on a 10 x 2 mm I.D. precolumn, the technique consists of a clean-up with 1000 microliters of 0.1 mol/l sodium hydroxide solution (pH 12.5) and of a heartcut consisting of four precolumn bed volumes of eluent directed to waste followed by ten precolumn bed volumes of eluent directed to the analytical column. Analytical separation is performed with acetonitrile-water (30:70) containing 0.005 mol/l of tetrabutylammonium hydrogensulphate (pH 8.3) (which is also the desorption eluent during heartcutting) on a polymeric analytical column (PLRP-S). With 25 ml of surface water, spiked at 0.25 and 1 microgram/l, applied to the precolumn, recoveries for all components were over 85% with a relative standard deviation (n = 5) of ca. 9% at 0.25 microgram/l and ca. 2% at 1 microgram/l. Detection limits in surface water samples are 0.05-0.1 microgram/l. Owing to automation, the total analysis time is ca. 30 min.     

Quantitation of Verapamil and Norverapamil in Small Blood Samples From the Rat by High Performance Liquid Chromatography

Abstract

A simple, sensitive HPLC assay using flurescence detection was developed for quantitation of verapamil and its active metabolite, norverapamil in 100-200 μl blood samples from the rat. Baseline separation of verapamil, normverapamil and internal standard, propranolol, was attained within 14 minutes. Standard curves for verapamil and norverapamil were linear from 7 ng/ml to 1000 ng/ml with limit of detection of 4 ng/ml for both Compounds. the intraday and interday coefficients of variation in verapamil and norverapamil concentrations, determined from spiked whole blood samples, were less than 10%.     

Determination of polar aniline derivatives in aqueous environmental samples using on-line liquid chromatographic preconcentration techniques

Summary On-line precolumn sample handling is used to enrich polar aniline derivatives in order to preconcentrate them prior to their separation. Liquid-solid extraction is possible with a cation-exchanger precolumn after acidification of water samples at pH 3 and a clean-up in order to remove the high amounts of inorganic cations present in natural samples. Since inorganic removal cannot be total, overloading of the ion exchanger occurs rapidly. The volume which can be directly percolated through the cation-exchanger precolumn cannot exceed 30 ml and the amount preconcentrated is not sufficient for a determination at the 100 ppt level. A two-step preconcentration procedure is carried out in order to increase the sample volume: the direct percolation of samples through the cation-exchanger precolumn is avoided and the clean-up step is no longer necessary. Aniline derivatives are preconcentrated in their neutral form at pH 7 by a 9-cm long column packed with the copolymer-based PRP-1 sorbent; then, a small volume of water-methanol at pH 2 allows the cationic compounds alone to be desorbed from the PRP-1 column in their protonated form and to be transferred to a 1-cm long cation-exchanger precolumn. This precolumn is then coupled to an analytical C18 column and its content on-line analysed by an acetonitrile gradient. The PRP-1 column acts as a powerful filter to many neutral interferents and aniline derivatives can be thus determined from 150-ml drinking water samples with 10–50 ppt UV detection limits.Dedicated to Roland W. Frei     

Development,validation and application of a UHPLC–MS/MS method for quantification of the adiponectin-derived active peptide ADP355 in rat plasma

A UHPLC–MS/MS method for the quantification of ADP355, an adiponectin-derived active peptide, was developed and validated. The extraction method employed simple protein precipitation using methanol and chromatographic separation was achieved on anAccucore™ RP-MS C₁₈ column (100 × 2.1 mm, 2.6 μm, 80 Å), using 0.1% formic acid in both water and acetonitrile with gradient elution at the flow rate of 400 μl/min within 4.0 min. Detections were performed under positive ion mode with multiple reaction monitoring ion transitions m/z 1109.2 → 309.8 and 871.4 → 310.1 for ADP355 and Jt003 respectively at unit resolution. The linearity range of the calibration curve was 2–1,000 ng/ml with a lower limit detection of 0.5 ng/ml. The selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were validated, and all items met the requirement of US Food and Drug Administration guidance. This method was successfully applied to an intravenous pharmacokinetic study of ADP355 in rats and the in-vitro stability in rat serum, plasma and whole blood was also assessed.     

Solid-Phase Extraction of Carbamazepine and Two Major Metabolites from Plasma for Analysis by HPLC

Abstract

A rapid, sensitive and simple to operate HPLC method for the simultaneous determination of carbamazepine, carbamazepine 10,11-epoxide and 10,11-dihydro-10,11-trans-dihydroxycarbamazepine in plasma is described. The drug and its metabolites are extracted from plasma using commercially available reversed-phase octadecylsilane bonded-silica columns (Bond Elut C₁₈, 2.8 ml capacity). Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile - methanol - water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Waters Assoc. Nova-Pak C₁₈ column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Waters Assoc. Z-module RCSS and protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak μBondapak C₁₈ insert. Using ultraviolet detection at 214 nm, levels in the region of 50–100 ng/ml for CBZ and its metabolites can be measured with only 250 μl of plasma. The method has been used to determine steady-state concentrations of the drug and its metabolites in paediatric patients.     

Sensitive and selective bioanalytical assay for the determination of dobutamine in rat plasma samples

Summary Dobutamine is one of the synthetic catecholamines acting directly onβ ₁-receptors. For the analysis of dobutamine in rat plasma samples, a selective and sensitive liquid chromatographic method is described. After a simple liquid-liquid extraction, separation of the analyte was performed using a reversed-phase ion-pair system with an octyl modified silica column. The solute was detected by fluorescence detection, applying an excitation wavelength of 285nm and an emission wavelength of 313nm. The (im)possibilities of the application of the normally used assays for the isolation, concentration and quantitation of catecholamines are discussed. By the addition of a minimum amount of modifier to the mobile phase, the selectivity of the system was increased significantly. With this method the detection limit is 9ng/ml in 0.2ml plasma samples. The application of the method is shown in rat plasma samples by measuring the concentration-time curves to establish plasma level-effect relationships for this drug.     

Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

A column-switching system for the direct injection of plasma or serum samples, followed by isocratic high-performance liquid chromatography and ultraviolet detection, is described for the simultaneous quantitation of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline and the E- and Z-isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline. The method included adsorption of amitriptyline and metabolites on a reversed-phase C8 clean-up column (10 microns; 20 mm x 4.6 mm I.D.), washing of unwanted material to waste and, after on-line column-switching, separation on a cyanopropyl analytical column (5 microns; 250 mm x 4.6 mm I.D.). The compounds of interest were separated and eluted using acetonitrile-methanol-0.01 M phosphate buffer (pH 6.8) (578:188:235, v/v) within less than 20 min. Various drugs frequently co-administered with amitriptyline or other antidepressants did not interfere with the determinations. In plasma samples spiked with 25-300 ng/ml, the recoveries were between 84 and 112% and the inter-assay coefficients of variation were 3-11%. After a minor modification, as little as 5 ng/ml could be quantitated. There were linear correlations (r greater than 0.99) between drug concentrations of 5-500 ng/ml and the detector signal. The method allows routine measurements of amitriptyline, nortriptyline and hydroxylated metabolites in blood plasma or serum of patients treated with amitriptyline or nortriptyline, and enables the results to be reported within 1 h.     

A sensitive column-switching HPLC method for aripiprazole and dehydroaripiprazole and its application to human pharmacokinetic studies

A simple and sensitive column‐switching HPLC‐UV method was developed for the simultaneous determination of aripiprazole, a novel atypical antipsychotic drug, and its active metabolite, dehydroaripiprazole in human plasma. Aripiprazole, its active metabolite and 7‐[5‐[4‐(3‐chloro‐2‐methylphenyl)‐1‐piperazinyl]pentyloxy]‐3,4‐dihydro‐2(1H)‐quinolinone (OPC‐14558) as an internal standard were extracted from 1 mL of plasma using a mixture of chloroform/n‐heptane (3:7, v/v), and the extract was injected into a column I (TSK BSA‐ODS/S precolumn, 5 μm) for cleanup and column II (C₁₈ STR ODS‐II analytical column, 5 μm) for separation. Peaks were detected with an UV detector set at a wavelength of 254 nm, and the total time for chromatographic separation was ～20 min. Mean absolute recoveries were 74.0 and 74.7% for aripiprazole and dehydroaripiprazole, respectively. Intra‐ and inter‐day CVs were less than 7.5 and 7.1% for aripiprazole concentrations ranging from 2 to 600 ng/mL, and 9.2 and 4.5% for dehydroaripiprazole concentrations ranging from 2 to 160 ng/mL. The validated concentration ranges for this method were 1–500 ng/mL and the limits of detection were 0.5 ng/mL for both aripiprazole and dehydroaripiprazole. This method was applied to pharmacokinetic study in human volunteers and patients taking aripiprazole.