High-performance liquid chromatographic determination of busulfan in plasma

Summary Busulfan (Myleran; 1,4-bis-(methanesulfonyloxy) butane; BU) is a bifunctional alkylating agent used in clinical practice since 1959. It is currently included at high doses in conditioning regimens for bone marrow transplantation, usually in combination with cyclo-phosphamide. A high-performance liquid chromatographic method has been developed for the determination of BU in plasma. The basis of the assay is a derivatization with sodium diethyldithiocarbamate at 32°C in the presence of 1-bromo-1-deoxy-3,6-anhydrogalactitol as internal standard. Analysis is performed on a cyano column with heptane-isopropanol-glacial acetic acid as mobile phase and UV detection at 280 nm. The calibration graph was linear in the concentration range 0.18–46.40 μM BU in plasma. The limit of detection was 0.1 μM. The precision and accuracy were between the limits required by good laboratory practice. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, september 1–3, 1999     

High performance liquid chromatography method for the determination of meropenem in human plasma

Summary This paper describes an HPLC method for the determination of meropenem in human plasma. The method uses solid phase extraction (SPE) of the samples and has good sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.02 μg mL⁻¹. Calibration curves were linear over a large dynamic range, namely within 0.02–50 μg mL⁻¹. The method was applied to the determination of meropenem levels in patients receiving meropenem, as a single dose or at steady state.     

High-Performance liquid chromatographic determination of dibromomannitol in plasma

Summary Myelobromol 1,6-dibromo-1,6-dideoxy-D-mannitol (dibromomannitol, DBM) is a bifunctional alkylating agent that has been in clinical use since 1963. It is currently included at high dose in preconditioning regimen for bone marrow transplantation (BMT) and is a main-stay of treatment for polycythaemia vera. A high-performance liquid chromatographic method was developed for the determination of DBM in the plasma. The basis of the assay is a derivatization with sodium-diethyldithiocarbamate at 42°C in the presence of 1-bromo-1-deoxy-3,6-anhydro-galactitol as internal standard (IS). The analysis was carried out on a 250×4mm Hypersil 5 CPS column equipped with a 20×4 mm Hypersil 10 CPS precolumn. The eluent consisted of heptane:isopropyl-alcohol: glacial acetic acid=600:76:80 w/w. The flow rate was 1.2 mL min⁻¹. UV detection was performed at 254 nm. The calibration graph was linear in the concentration range of 2.5–260 μM of DBM in plasma. The limit of detection was 1.0 μM. The precision and accuracy of the method was between the good laboratory practice (GLP) required limits. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

High-performance liquid chromatographic determination of (−)-cathinone in plasma

Summary High-performance liquid chromatography (HPLC) for the determination of (–)-cathinone in rabbit and human plasma has been studied. The problem of dimerization during extraction from plasma was satisfactorily resolved. Detection was by UV at 257 nm. Concentration levels as low as 24 ng ml^–1 were satisfactorily determined. This level of sensitivity should be adequate for the detection of (–)-cathinone in the blood of khat users and also for the quantitative determination of (–)-cathinone in blood for pharmacokinetic purposes. The applicability of the assay procedure to pharmacokinetic studies is demonstrated.     

A high-performance liquid chromatographic method for determination of flecainide in human plasma using fluorescence detection

Summary A high performance liquid chromatographic method for the determination of flecainide in serum has been developed. The analysis is performed on a microparticulate silica column. The eluate is monitored by fluorescence detection at an excitation wavelength of 300nm and an emission wavelength of 370nm. No sources of interference were identified and a coefficient of variation of less than 8% was observed on repeated flecainide determinations. The method has a good reproducibility, specificity and accuracy, and can be applied in therapeutic drug monitoring of flecainide in patients.     

Determination of myristicin in rat serum samples using microbore high performance liquid chromatography with column-switching

Summary A microbore high-performance liquid chromatographic method with column-switching was developed for the analysis of myristicin from rat serum without prepurification. Deproteinization, fractionation, concentration and separation of analyte were carried out by appropriate switching of columns and using solvent mixtures. The method showed excellent precision, accuracy and speed with a detection limit of 10 ng mL⁻¹ from 25 μL of serum. The total analysis time per sample was 25 min and the coefficients of variation for intra- and inter-assay were less than 1.8%.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

High-performance liquid chromatographic analysis of azlocillin in serum

Summary A rapid and sensitive high performance liquid chromatographic method is described for the determination of azlocillin in serum. This method involves a short manual protein precipitation of the sample followed by an injection into a PR 18 column for separation and quantitation. The mobile phase was a 22% (V/V) solution acetonitrile in phosphate buffer pH 4.8 at a flow rate of 2,5 ml/min. The spectrophotometer detector was set at 220 nm with a sensitivity of 0.08 AUFS.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

High-performance liquid chromatographic measurement of 8-methoxypsoralen in plasma

Summary A simple high-performance liquid chromatographic method for the measurement of 8-Methoxypsoralen (8-MOP) in human plasma following a single 40mg dose has been described. After addition of phosphate-NaOH buffer, pH 12, and internal standard (trimethylpsoralen), the sample is vortex-mixed with diisopropylether. The resulting extract is analysed on a reverse phase column using phosphoric acid (0.05% v/v): acetonitrile (1:1) as mobile phase, and U.V. detection at 220nm. No interference from endogenous sources has been observed. The limit of sensitivity of the assay is 5ng/ml plasma. The measuring range is between 10–700ng 8-MOP/ml plasma, to be expected from oral doses of 0.6mg 8-MOP/kg body weight, and corresponds to the therapeutic plasma concentration. The relative standard deviation at 50ng/ml level of 8-MOP is 3.6%.     

Simultaneous determination of cefoxitin,cefuroxime, cephalexin and cephaloridine in plasma using HPLC and a column-switching technique

Summary A new high performance liquid chromatographic method was developed using a column-switching technique for the simultaneous determination of cephalexin, cefuroxime, cefoxitin and cephaloridine in plasma. The plasma samples were injected onto a precolumn packed with Corasil RP C₁₈ (37–50 m) after simple dilution with an internal standard solution in 0.01 M acetate buffer (pH 3.5). Polar plasma components were washed out using 0.01 M acetate buffer (pH 3.5). After valve switching, the concentrated drugs were desorbed in back-flush mode and separated on a Partisil ODS-3 column using acetonitrile in 0.02 M acetate buffer (pH 4.3) (1585, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed with a detection limit of 0.5 g/ml. The total analysis time per sample was less than 25 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9 %.This method has been successfully applied to plasma from rats after subcutaneous injection of cefuroxime.     

Isocratic high-performance liquid chromatographic determination of plasma adenosine

Summary Due to manifold physiological and cardioprotective actions of adenosine, the demand for a simple but accurate method to determine its concentration in plasma is increasing. The aim of this study was firstly to develop a simple isocratic method instead of the gradient elution or peak-shifting techniques used earlier and secondly to check conflicting data on the composition of stop-solution, added to the sample in order to prevent changes in adenosine concentration. Isocratic elution improved signal to noise ratio and concentrations of 100 mol L^–1 dipyridamole and 2.5 mol L^–1 erythro-9(2-hydroxy-3-nonyl)adenine in the blood sample effectively prevented both adenosine formation and degradation, even without the use of a 5-ecto-nucleotidase inhibitor. Lowering the concentration of dipyridamole to 25 mol L^–1 caused more than a tenfold increase of adenosine concentration in two out of five cases and even 100 mol L^–1 dipyridamole alone is not sufficient to inhibit adenosine deaminase in blood samples.     

High-performance liquid chromatographic determination of glaucine inGlaucium flavum crantz

Summary A method has been developed for the extraction and rapid analysis of D-glaucine inGlaucium flavum Crantz. Simple extraction of the drug with diethyl ether was followed by high-performance liquid chromatography on a μBondapak C₁₈ column using a mixture of acetonitrile, methanol and phosphate buffer as the mobile phase. Data on selectivity, sensitivity and precision demonstrate the reliability of this method.     

A new validated high-performance liquid chromatographic method for the determination of EGIS-9933 in rat plasma

Summary A new highly sensitive high-performance liquid chromatographic (HPLC) procedure for determination of EGIS-9933 (a newly developed anxiolytic compound) in rat plasma is described. A gradient, elution method with UV detection at 270 nm has been developed using a mobile phase of a mixture of A: methanol:acetonitrile 1:9 and B:0.5% triethilamine in water, the pH of B was adjusted to 3 with phosphoric acid. Solid phase extraction (SPE) was used for the sample preparation. The calibration was linear in the 10–10000 ng mL⁻¹ concentration range. The limit of quantification was 10 ng mL⁻¹. The bioanalytical method was validated according to internationally accepted criteria for biological samples. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Simultaneous determination of losartan and active metabolite EXP3174 in rat plasma by HPLC with column switching

Summary A new liquid chromatographic method with columnswitching has been developed for the simultaneous determination of losartan and its active metabolite, EXP3174 in rat plasma. The plasma samaple was injected onto a precolumn of Lichroprep RP-8 after dilution with 5% acetonitrile in 50 mM phosphoric acid. Polar plasma components were eluted using this diluent. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Inertsil ODS-2 column with acetonitrile-acetate buffer. The method showed excellent precision, accuracy and speed with detection limit 20 ng mL^–1. Total analysis time per sample was less than 40 min and the coefficients of variation for intra and inter-assay were 4.8%. This method has been successfully applied after oral administration of losartan to rat plasma samples.     

High-performance liquid chromatographic determination of urocanic acid isomers in cosmetic products

Summary A method has been developed for the determination of trans and cis urocanic acid in oil-in-water cosmetic emulsions. It involves an extraction of the sample in 1:3 methanol-aqueous NaOH (10⁻³ M), by ultrasonication, which leads to quantitative recoveries, and a reversed-phase HPLC isocratic elution for the analysis of the extract. Chromatography is performed on a C18 column using 0.1 M sodium perchlorate (pH 3.0)-acetonitrile 98:2 (v/v), as the mobile phase, and UV detection at 263 nm. The separation of the isomers is good. Linearity and precision of the method have been assessed.     

An improved high-performance liquid chromatographic determination of chlorpropamide in human plasma

Summary Samples were extracted with dichloromethane and the organic layer evaporated to dryness. The residue was dissolved in methanol, and 10 μl aliquot injected onto the column. Tolbutamide was used as the internal standard for chlorpropamide. The UV detector response was linear over the range 0–200 μg ml⁻¹ with a correlation coefficient of 0.999; detection limit: 0.002 μg ml⁻¹. Within-day and between-day assay variation was generally ≤7%. No interference from endogenous constituents was observed. The utility of the method was demonstrated by determining chlorpropamide in samples from six healthy volunteers following a single oral dose of 250 mg. The procedure is simple and requires small volumes of plasma.     

High-performance liquid chromatographic analysis of dinitrobenzene isomers

Summary An efficient, reproducible and rapid high-performance liquid chromatographic method, in normal phase mode, for the analysis of the three dinitrobenzene isomers is described. The method affords good linearity for each isomer in the range 10–160 g ml^–1. The total analysis time is only 10 minutes, and the method shows an accuracy of ±1.25% with a coefficient of variation from 0.30% to 2.85% for different levels of the dinitrobenzene isomers.     

High-performance liquid chromatographic assay for simultaneous determination of tramadol and its active metabolite in human plasma. Application to pharmacokinetic studies

Summary A sensitive liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its active metabolite is described. Fluconazole was used as internal standard. The assay involved a singletert-butyl methyl ether extraction and LC analysis with fluorescence detection. Chromatography was at 30°C pumping an isocratic mobile phase of acetonitrile-water (19∶81, v/v) containing 0.06M NaH₂PO₄ and 0.05M triethylamine, adjusted to pH 7.90, at 1 mL min⁻¹ through a reversed-phase, 250×4 mm base-stable column. The limit of quantitation of tramadol and its active metabolite was 1 ng mL⁻¹, only 0.5 mL plasma sample was required for the determination. The calibration curve was linear from 1–1000 ng mL⁻¹. Intra and inter-day precision (C.V.) did not exceed 10%. Mean recoveries of 96.38% for tramadol and 96.62% forO-demethyltramadol with CVs of 0.43% and 1.46% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study on normal volunteers who received 100 mg tramadol intravenously.     

Full automation of serotonin determination by column-switching and HPLC

Summary A simple, fast, fully automated method for plasma serotonin determination is described. Full automation is obtained by coupling two devices: a sample processing station and a solid-phase autosampler. The sample processing station dilutes the plasma sample and is then connected, on-stream, with the solid-phase autosampler. It firstly fills a loop with all the solvents necessary for the sample clean-up, then, inverting the flow, pumps these solvents through the silica-bonded cation-exchange disposable extraction cartridge positioned on the autosampler. For the elution, the cartridge is switched on-stream with the HPLC analytical column and serotonin is eluted by the HPLC mobile-phase. The HPLC separation is performed by ion-pairing reversed-phase liquid chromatography. The column effluent is completely reduced by an electrochemical reactor and serotonin is detected in an oxidation-mode by a dual-cell electrochemical detector. The plasma sample is 50 l, the plasma sensitivity is 40 ng/l, the retention time is 6 min and the recovery is 95%. The repeatibility, the normal ranges for platelet-poor and for platelet-rich plasma have been established and correlation with manual HPLC calculated.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.