Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C₈ column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m² CP in a 1-h infusion demonstrated the applicability of the assay.     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Determination of a novel indolylpiperazine anti-migraine agent in rat, monkey, mouse and rabbit plasma by high-performance liquid chromatography with electrochemical detection

A specific, accurate, precise and reproducible assay for the quantitation of a novel indolylpiperazine anti-migraine agent (I) in plasma from various animal species is described. The method involves addition of internal standard (I.S.) and 1.0 M sodium carbonate to the plasma sample, vortex-mixing and extraction with ethylene dichloride. The organic layer is then back-extracted in a buffer consisting of 0.1 M tetramethylammonium hydroxide (TMAH), pH 3.0 and 0.1 M (NH₄)₂HPO₄, pH 3.0, in water. The aqueous layer is injected on to a Zorbax cyano analytical column with a mobile phase consisting of acetonitrile, methanol and water (15:5:80, v/v/v) with 0.01 M TMAH, pH 3.0 and 0.01 M (NH₄)₂HPO₄, pH 3.0. The eluate is monitored by electrochemical detection at 0.9 V (guard cell), 0.5 V (detector 1) and 0.8 V (detector 2). The retention times of I and I.S. were 7 and 10 min, respectively. In drug-free control plasma, there were no interfering peaks seen at the retention times of I or I.S. The standard curve was linear over the concentration range of 5–500 ng/ml in rat, monkey, mouse and rabbit plasma. The lower limit of quantitation in all four matrices was 5.0 ng/ml. Within- and between-assay variability of quality control samples was less than 9% relative standard deviation and the predicted concentration of the quality control samples deviated by less than 15% from the nominal concentration. The stability of I was established for up to 36 h in the autosampler tray, up to 10 months in plasma at −20°C and up to 2 h in plasma at room temperature. The assay is validated for determination of I in plasma.     

Application of cyanide-metabolizing enzymes to environmental control; enzyme thermistor assay of cyanide using immobilized rhodanese and injectase

The application of the enzyme thermistor in the analysis of cyanide in standard solutions as well as in blast furnace waste water is described. The heat signal is generated in the conversion of cyanide, catalyzed by the immobilized enzymes rhodanese (E.C. 2.8.1.1) and injectase (E.C. 4.4.19). Using the combination of cyanide-metabolizing enzymes and the enzyme thermistor unit, assays down to 20μM cyanide can be carried out. Linear relationships were obtained at 20–600μM cyanide for injectase and 20–1000μM for rhodanese. The stability at 27°C of the heat response was initially decreased, but soon stabilized at about 80% of the initial value and remained so for at least 200 hr. The technique was easily adapted to continuous analysis, applicable to environmental control (e.g., a “cyanide guard”) with a response time at present within 2–3 min after a sudden change in cyanide concentration has appeared.     

Liquid chromatographic determination of total celiprolol or (S)-celiprolol and (R)-celiprolol simultaneously in human plasma

A method has been developed for the determination of total celiprolol (sum of enantiomers) or the enantiomers (R)-celiprolol and (S)-celiprolol in plasma by high-performance liquid chromatography with UV and fluorescence detection. After extraction from alkalinized plasma with methyl-tert-butyl ether and back-extraction into 0.01 M HCl (for total celiprolol determination) or after evaporation of the organic phase and derivatisation with R(−)-1-(1-naphthyl)ethyl isocyanate (enantiomer determination), total celiprolol or its diastereomeric derivatives were chromatographed on a reversed-phase HPLC column with a mixture of acetonitrile and phosphate buffer pH 3.5 (+0.05% triethylamine). Acebutolol was used as internal standard. Linearity was obtained in the range of 5 to 2000 ng/ml for total and 2.5 to 500 ng/ml for enantiomer determination. Intra-day and inter-day variation was lower than 10%. The method can be applied for analysis of plasma samples obtained from patients treated with oral racemic celiprolol doses.     

Determination of lysergide in urine by high-performance liquid chromatography combined with electrospray ionisation mass spectrometry

A quantitative method which avoids derivatisation is described for the determination of lysergide (LSD) levels in urine. Sample preparation included addition of methysergide as an internal standard followed by solid-phase extraction. LSD was analysed on a system consisting of a C₁₈ stationary phase and a mobile phase of 0.1 M acetate buffer pH 8.0-acetonitrile-triethylamine (75:25:0.25, v/v). LSD was detected by electrospray ionisation mass spectrometry with selected ion monitoring. The quantification limit was 0.5 ng/ml and the method was linear up to 10 ng/ml of LSD in urine.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

An Evaluation of Irreversible Inhibition of Synaptosomal High-Affinity Choline Transport by Choline Mustard Aziridinium Ion

Abstract: Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC₅₀ value for high-affinity transport was 0.94 μM, compared to 29 μM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 × 10^?2 s^?1for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.     

Determination of hexahydrophthalic acid and methylhexahydrophthalic acid in plasma after derivatisation with pentafluorobenzyl bromide using gas chromatography and mass spectrometric detection

A method for the simultaneous determination of hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) in human plasma was developed. The procedure was a rapid, single step extractive derivatisation with pentafluorobenzyl bromide as the derivatisation agent. The formed pentafluorobenzyl esters were analysed by gas chromatography-mass spectrometry in negative ion chemical ionisation mode with ammonia as the moderating gas. Deuterium-labeled HHP acid and MHHP acid were used as internal standards. The detection limit was 0.4 ng/ml for HHP acid (m/z 153) and 0.3 ng/ml for MHHP acid (m/z 365). The within-day precision of the method was between 2 and 3% and the between-day precision was between 3 and 12%. The overall recovery was between 65 and 83%. A comparison between HHP acid determinations with a previous and this method showed that the methods gave similar results. The method was applicable for analysis of plasma from occupationally exposed workers.     

Determination of sodium artesunate in plasma using ion-pairing high-performance liquid chromatography

A chromatographic method is described for the determination of sodium artesunate in plasma. This includes cetyltrimethylammonium bromide as a cationic pairing ion in a reversed-phase system using an octadecylsilica 100×4.6 mm I.D. 3 μm analytical column with a mobile phase of acetonitrile/acetate buffer at pH7. Column switching incorporating a 5 μm octadecylsilica 100×4.6 mm I.D. precolumn is used in addition to off-line solid-phase extraction for pretreatment of plasma samples in order to eliminate interference from endogenous components. Detection is by post-column derivatisation with 1.0 M methanolic KOH followed by UV detection at 289 nm. Calibration is linear over the range 100–1600 ng ml⁻¹ and the limit of detection is estimated as 20 ng ml⁻¹. Illustrative results are shown of the artesunate plasma levels determined by the proposed method following the administration of artesunate as tablets and as suppositories to healthy volunteers.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 mM phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogen phosphate (pH 3.4), 10 mM acetic acid and 36μM EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μM and the calibration line was linear up to 75 μM. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μM) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μM, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μM, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.     

Albino inflorescence proliferation of Dendrocalamus latiflorus

Summary Inflorescence proliferation is a plant tissue culture technique that, can be used to obtain in vitro inflorescences year-round without the intervening development of vegetative organs. In this study, we used albino mutant inflorescences of Dendrocalamus latiflorus as the original explant material to investigate, the effect of plant growth regulators on long-term inflorescence proliferation. The albino inflorescences proliferated on solidified Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), and the optimal concentration for successful long-term inflorescence proliferation was 0.45 μM TDZ. A combination of α-naphthaleneacetic acid (NAA) with 0.45 μM TDZ inhibited the inflorescence proliferation. Inflorescences cultured on a TDZ-free medium supplemented with 26.82 μM NAA rooted in 21 d, vegetative shoots formed by 42 d and, in one case, flowering occurred after 63 d. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D, 4.52 μM) and pieloram (4.14 μM) induced shoot formation. The protocol described can be used to produce large numbers of mutant inflorescences within a relatively short period of time.     

In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 μM (mean±S.D. 12.7±2.5 μM from the results of eight rats) and from 9.3 to 36.0 μM (mean±S.D., 24.3±8.9 μM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.     

High-performance liquid chromatographic determination of selenocysteine with the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid

The method described is based on derivatization of selenocysteine with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and responds linearly to selenocysteine spiked into plasma. Recovery is insensitive to inter-individual variation or use of serum versus plasma, but is decreased by hemolysis. The derivative is stable for at least three days. The total imprecision of determinations in plasma was 0.8–2.1% (coefficient of variation) over the range of 6–30 μM selenocysteine, with a detection limit of 0.4 μM (3 × S.D.). There was no significant interference from plasma thiols. This appears to be the first report of the selective reaction of free selenocysteine with a fluorescent reagent. This simple method works well in plasma and serum and may be adaptable to other types of samples.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.     

Determination of thiamphenicol in human plasma by micellar electrokinetic capillary chromatography

A micellar electrokinetic chromatographic method is described for the determination of thiamphenicol in human plasma. The plasma sample was basified by adding K₂HPO₄ and was then extracted with ethyl acetate. After the solvent was evaporated, the residue was reconstituted in water. Approximately 40 nl of the solution were injected hydrodynamically. The running buffer was 20 mM borate (pH 9.2) containing 40 mM sodium dodecyl sulfate and 10% acetonitrile. The applied voltage was 18 kV and the detector wavelength was set at 195 nm. On-column sample stacking was achieved during the analysis to enhance the sensitivity; the limit of quantitation was 0.1 μg/ml. Linearity was over the range of 0.2 to 10 μg/ml. Recovery was 93.7±3.3%, the intra-day precision and accuracy was 99.6±2.8%; the inter-day precision and accuracy was 98.4±3.4%. The concentration of thiamphenicol in human plasma from eight volunteers was measured after administering thiamphenicol capsules orally.     

Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron. A simple and rapid HPLC–UV method for the separate determination of ICL670 and Fe–[ICL670]₂ in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4°C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C₁₈ column using 0.05 M Na₂HPO₄ and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25–20 μg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.