Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Impact of halides on the simultaneous separation of aromatic amines and their acidic metabolites by capillary electrophoresis with laser-induced native fluorescence detection under acidic conditions

This paper describes a simple, sensitive, efficient, and rapid method for simultaneous analysis of biologically active amines and acids by capillary electrophoresis in conjunction with laser-induced native fluorescence detection (CE-LINF) using a diode pumped solid state nanolaser at 266 nm. In order to optimize resolution of the amines that were prepared in 10.0 mM formate-Tris (FT) solutions, 10.0 mM FT solutions with and without containing halides were used to fill the capillary and reservoirs, respectively. The electrophoretic mobilities of tryptamine (TA) and serotonin (5-HT) at pH 4.0 decrease with the increase in halide concentration (0-10.0 mM). Taken together with a great effect of iodide than other halides, we suggest that the formation of ion pairs is a main contributor for altering the migration of the amines. In order to simultaneously analyze the amines and their metabolites (acids) at low pH, a high bulk EOF is required. The analysis of 10 anlytes including amines and acids was completed within 12 min by CE-LINF using a capillary treated with 0.5M NaOH and then filled with 10.0 mM FT solutions (pH 4.0) containing 10.0 mM KCl prior to analysis. The limits of detection for TA and 5-hydroxyindole-3-acetic acid (5-HIAA) are 0.12 and 6.0 nM, respectively. The present method has been further validated by analyzing urine samples, with an RSD less than 3.1% (migration times) and 3.9% (concentration).     

Determination of amifloxacin and two of its principal metabolites in plasma and urine by high-performance liquid chromatography using automated column switching

The automated determination of amifloxacin and two of its principal metabolites in human plasma and urine by column-switching high-performance liquid chromatography is described. Plasma or urine samples, diluted 1:1 with 0.5 M sodium citrate buffer pH 2.5, were directly injected onto a cation-exchange pre-column. Following a 2.0-min wash of the pre-column with water at a flow-rate of 1.1 ml/min, the effluent from the pre-column was directed to the analytical column by a column-switching device. The precision of the plasma and urine methods ranged from a +/- 1.9 to +/- 3.6% for all compounds. The accuracies of the methods were within a range of -3.3% to 6.4% of the nominal values for all compounds. Linear responses were observed for all the standards in the range 0.10-5.0 micrograms/ml for plasma and 0.50-100 micrograms/ml for urine for all three compounds. The minimum quantifiable levels were 0.10 and 0.50 micrograms/ml for plasma and urine, respectively. The analytical methods may be used to quantify amifloxacin and the piperazinyl-N-desmethyl and piperazinyl-N-oxide metabolites in plasma and urine samples obtained from humans, monkeys, dogs and rats.     

Validated chiral high-performance liquid chromatography method for a novel anti-methicillin-resistant Staphylococcus aureus fluoroquinolone WCK 771

A sensitive, simple, specific, precise, accurate and rugged method for the assay and determination of enantiomeric purity of S-(-)-9-fluoro-6,7-dihydro-8-(4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid L-arginine salt tetrahydrate (WCK 771) in bulk drug has been developed. The method is RP-HPLC using endcapped C-18 stationary phase and chiral mobile phase. Chirality to the mobile phase was imparted with addition of beta-cyclodextrin. The UV-vis detector was operated at 290 nm. The flow rate of mobile phase was 2 ml/min. The method offers excellent separation of two enantiomers with resolution more than 2 and tailing factor less than 1.5. The method was validated for the assay of WCK 771 and quantification of R-(+)-enantiomer impurity in bulk drug. The calibration curves showed excellent linearity over the concentration range of 0.05-0.15 mg/ml for WCK 771 and 0.5-7.5 microg/ml for R-(+)-enantiomer. The precision (RSD) of the assay was 0.23%. The limit of detection and limit of quantitation of the method for WCK 771 were 0.015 and 0.06 microg/ml, respectively. The limit of detection and limit of quantitation for R-(+)-enantiomer were 0.025 and 0.09 microg/ml, respectively. The average recovery of the R-(+)-enantiomer was 100.5%. Same method was applied for the assay and determination of enantiomeric purity of WCK 771 in the intravenous formulation.     

Analysis of lamotrigine and lamotrigine 2-N-glucuronide in guinea pig blood and urine by reserved-phase ion-pairing liquid chromatography.

Lamotrigine is an investigational anticonsulvant drug undergoing clinical trials. A simultaneous assay was developed to quantitate lamotrigine and its major metabolite, lamotrigine 2-N-glucuronide, from guinea pig whole blood. The extraction procedure and reversed-phase high-performance liquid chromatographic (HPLC) assay employed sodium dodecylsulfate (SDS) as an ion-pairing reagent to selectively separate lamotrigine and lamotrigine 2-N-glucuronide from endogenous blood components, other anti-convulsant drugs, and their metabolites. The mobile phase was composed of acetonitrile-50 mM phosphoric acid (pH 2.2) containing 10 mM SDS (33:67, v/v), and components were detected at 277 nm. The total coefficients of variance (C.V.) for the blood assay were less than or equal to 9.4% for lamotrigine (0.25-20.0 micrograms/ml) and less than or equal to 13.4% for the glucuronide metabolite (0.25-10.0 micrograms/ml). Separate assays for lamotrigine and its glucuronide in urine were developed. In order to quantitate low levels of lamotrigine in guinea pig urine, lamotrigine was extracted with tert.-butyl methyl ether-ethyl acetate (1:1). The total C.V. for lamotrigine quantitation in urine was less than or equal to 7.5% (0.10-10.0 micrograms/ml). For the determination of lamotrigine 2-N-glucuronide, urine was diluted with an SDS-phosphoric acid buffer (1:4) and injected directly onto the HPLC system, total C.V. less than or equal to 4.2% (0.5-50 micrograms/ml).     

Validation of a high-performance liquid chromatographic method for the simultaneous determination of tramadol and its impurities in oral drops as a pharmaceutical formulation

The novel, rapid high performance liquid chromatographic method for the determination of tramadol hydrochloride and its three impurities was developed and validated. The method can simultaneously assay potassium sorbate, used as preservative, and saccharin sodium, used as sweetener in tramadol pharmaceutical formulation. The separation was carried out on a C(18) XTerra (150 mm x 4.6 mm, 5 mm) column using acetonitrile-0.015 M Na(2)HPO(4) buffer (2:8, v/v) as mobile phase (pH value 3.0 was adjusted with orthophosphoric acid) at a flow rate 1.0 ml min(-1), temperature of the column 20 degrees C and UV detection at 218 nm. The method was found to be linear (r > 0.999) in the range of 0.05-0.8 mg ml(-1) for tramadol hydrochloride, 0.1-1.2 mg ml(-1) for impurities B and C and for impurity A (r > 0.995) in the range 0.15-2.4 mg ml(-1). The low RSD values indicate good precision and high recovery values indicate excellent accuracy of the HPLC method. Developed method was successfully applied to the determination of tramadol hydrochloride, its investigated impurities and potassium sorbate in commercial formulation. The recovery of tramadol hydrochloride was 98.25% and RSD was 1.80%. The method is rapid and sensitive enough to be used to analyse Trodon oral drops.     

Determination of DNA with cetyltrimethylammonium bromide by the measurement of Resonance Light Scattering.

A simple assay of DNA was developed based on the measurements of enhanced signals of Resonance Light Scattering (RLS) of cetyltrimethylammonium bromide (CTMAB) by DNA. The enhanced RLS signals, measured by simultaneously scanning the excitation and emission monochromators of a common spectrofluorometer with lambda ex = lambda em, was optimized for the DNA assay with CTMAB. On the conditions of pH 2.21 and ionic strength 0.002, the enhanced RLS intensity at 470.0 nm, delta I, was found to be proportional to the concentration of DNA in the range 0-2.5 micrograms/ml if 1.5 x 10(-5) M CTMAB was used. Limits of determination for calf thymus DNA and fish sperm DNA were 4.9 ng/ml and 9.2 ng/ml, respectively. Synthetic samples were determined with the recovery ratio ranging from 93.2% to 105.1%, and the RSD is lower than 2.7%.     

Micellar electrokinetic capillary chromatography method for direct determination of glucuronides of entacapone and its (Z)-isomer in human urine

This paper describes the validation of a micellar electrokinetic capillary chromatography method for the direct determination of the 3-O-glucuronides of entacapone and its (Z)-isomer, the main urinary metabolites of entacapone in humans. Entacapone is a novel drug which, as a potent inhibitor of catechol-O-methyltransferase (COMT), is used as an adjunct in the standard therapy of Parkinson's disease. The 3-O-glucuronide of another COMT inhibitor, nitecapone, was used as internal standard (I.S.). The validation experiments were performed by using spiked urine samples that were extracted with Sep-Pak C18 cartridges before analysis. Determinations were carried out in a buffer of pH 7.0 containing 25 mM of phosphate, 50 mM of borate and 20 mM of sodium dodecyl sulfate, and by applying 15 kV over a 67 cm (60 cm to the detector) x 75 microns fused-silica capillary. UV detection was at 335 nm. The validity of the method was assessed by investigating the identity of the analytes, selectivity, limit of quantitation, linearity, within-day precision, extraction recovery, between-day precision and accuracy, electroosmotic flow stability and analyte stability. The method proved to be reproducible, sufficiently selective and accurate. Extraction recoveries of the analytes were > 94%. The limit of quantitation (LOQ) was 2 micrograms/ml and the assay was linear in the range 2-150 micrograms/ml with correlation coefficients better than 0.999 for both glucuronides. The repeatability of the method, expressed as the ratio of corrected peak area of the analytes to that of I.S., gave RSD values of < 5% even at the LOQ. Between-day precision (RSD) was < 7.5% for both glucuronides at 7.5 micrograms/ml. Determination of the glucuronide concentrations in urine samples of 34 patients treated with entacapone either orally (200 mg) or intravenously (25 mg) showed the method to be suitable for monitoring the concentrations of the glucuronide of entacapone after both oral and intravenous administration and those of the glucuronide of its (Z)-isomer after oral administration. The limited long term stability of the system requires, however, frequent recalibration in applications involving long sample series.     

Simultaneous separation and determination (in serum) of phenytoin and carbamazepine and their deuterated analogues by high-performance liquid chromatography--ultraviolet detection for tracer studies

The use of stable isotope-labeled tracer compounds is the safest and most effective method to perform many steady state pharmacokinetic and drug interaction studies. We describe a method by which the heavily deuterated 2H10 analogues of carbamazepine (2H10 CBZ) and phenytoin (2H10 PHT) can be chromatographically separated by high-performance liquid chromatography from unlabeled CBZ and PHT. All compounds are quantitated against an internal standard (IS) (10,11-dihydrocarbamazepine) and measured using conventional UV detection rather than mass spectrometry. Baseline resolution of extracted serum containing 2H10 CBZ, CBZ, 2H10 PHT, PHT and IS is achieved on a heated (55 degrees C) 25 cm x 4.6 mm BioAnalytical Systems Phase II 5 microns ODS column with an isocratic mobile phase consisting of water-acetonitrile-tetrahydrofuran (80:16:4, v/v/v) at 1.2 ml/min. Eluting compounds were monitored at a UV wavelength of 214 nm. Calculated resolution of 2H10 CBZ from CBZ and of 2H10 PHT from PHT were 1.3. Serum standard curves were linear (R greater than or equal to 0.999) over a range of 0.5-14 micrograms/ml for 2H10 CBZ, 0.5-20 micrograms/ml for CBZ, 0.5-20 micrograms/ml for 2H10 PHT, and 0.5-30 micrograms/ml for PHT. Within-day percent relative standard deviations (precision) were less than 6% in all cases.     

Simultaneous determination of ethamsylate, tramadol and lidocaine in human urine by capillary electrophoresis with electrochemiluminescence detection

Ethamsylate, tramadol and lidocaine, partly excreted by the kidney, are generally used as hemostatic, analgesic and local anesthetic in surgery. We developed a simple and sensitive method for their simultaneous monitoring in human urine based on CE coupled with electrochemiluminescence detection by end-column mode. Under optimized conditions the proposed method yielded linear ranges from 5.0 x 10(-8) to 5.0 x 10(-5), 1.0 x 10(-7) to 1.0 x 10(-4) and 1.0 x 10(-7) to 1.0 x 10(-4) M with LODs of 8.0 x 10(-9) M (36 amol), 1.6 x 10(-8) M (72 amol) and 1.0 x 10(-8) M (45 amol) (S/N = 3) for ethamsylate, tramadol and lidocaine, respectively. The RSD for their simultaneous detection at 1.0 x 10(-6) M was 2.1, 2.8 and 3.2% (n = 7), respectively. For practical application an extraction step with ethyl acetate at pH 11 was performed to eliminate the influence of the sample ionic strength. The recoveries of ethamsylate, tramadol and lidocaine at different levels in human urine were between 87 and 95%. This method was used for simultaneous detection of ethamsylate, tramadol and lidocaine in clinic urine samples from two medicated patients. It was valuable in clinical and biochemical laboratories for monitoring these drugs for various purposes.     

Fast high-performance liquid chromatographic analysis of mianserin and its metabolites in human plasma using monolithic silica column and solid phase extraction

A fast high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of mianserin (MIAN) and its metabolites desmethylmianserin (DMM), 8-hydroxymianserin (HM) and mianserin-N-oxide (MNO) in human plasma. Each compound, together with internal standard (propranolol) was extracted from the plasma matrix using solid phase extraction. Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column ( mm i.d.) under isocratic conditions using a mobile phase of 74:26 (v/v) 25 mM phosphate buffer (pH 5.3 adjusted with phosphoric acid): acetonitrile. The elution of the analytes were monitored at 292 mm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 3.5 ml/min. The total run time of the assay was 5 min. The method was validated over the range of 10-200 ng/ml for MIAN, 10-150 ng/ml for DMM, 20-300 ng/ml for HM and 25-500 ng/ml for MNO. The method proved to be precise (within-run precision ranging from 1.6 to 6.9% R.S.D. and between-run precision ranging from 1.3 to 7.2% R.S.D.) and accurate (within-run accuracies ranged from 1.4 to 6.4% and between-run accuracies ranging from 1.5 to 4.5%). The mean absolute recoveries were 95.7, 94.8, 99.6, and 102.6% for MIAN, DMM, HM and MNO, respectively. The limit of quantitation (LOQ) for MIAN and DMM was 10 ng/ml and for HM and MNO were 20 and 25 ng/ml in human plasma, respectively. The limit of detection (LOD) for MIAN, DMM, HM and MNO was 5, 2.5, 10 and 15 ng/ml, respectively. The described method demonstrates the feasibility for employing monolithic columns to effect rapid bioanalytical HPLC analysis for the quantitative determination of MIAN and its major metabolites in human plasma.     

High-performance liquid chromatographic procedures for the determination of temafloxacin in biological matrices.

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination of temafloxacin and its trace level metabolites in biological matrices. Plasma samples are ultrafiltered after addition of an internal standard in a displacing reagent containing sodium dodecyl sulfate and acetonitrile. Plasma ultrafiltrates or diluted urines are chromatographed on a reversed-phase analytical column, using an ion-pair chromatographic mobile phase and fluorescence detection. The chromatographic system allows resolution and quantitation of temafloxacin's oxidative metabolites, which collectively account for less than 2% of the administered dose. The mean intra-assay coefficient of variation for determination of temafloxacin concentrations in plasma ranging from 0.05 to 10.0 micrograms/ml was 0.7%. The procedure was implemented at four laboratories for the analysis of over 12,000 samples from clinical studies. Inter-assay coefficients of variation estimated from routine analyses of quality control samples in these studies averaged 4% or lower for concentrations in the 0.15-10 micrograms/ml range. The limit of quantitation of the procedure is approximately 10 ng/ml; inter-assay coefficients of variation at 15 ng/ml averaged under 9%. Calibration curves were reproducible and highly linear, with correlation coefficients typically averaging over 0.9995. An alternative, more complex procedure, involving methylene chloride extraction, which extends the detection limits to below 1 ng/ml, is also described.     

Migration behaviour and separation of tramadol metabolites and diastereomeric separation of tramadol glucuronides by capillary electrophoresis

Capillary electrophoresis with UV detection was used to separate tramadol (TR), a centrally acting analgesic, and its five phase I (M1, M2, M3, M4, M5) and three phase II metabolites (glucuronides of M1, M4 and M5). Several factors were evaluated in optimisation of the separation: pH and composition of the background electrolyte and the influence of a micellar modifier, sodium dodecyl sulfate. Baseline separation of TR and all the analytes was obtained with use of 65 mM tetraborate electrolyte solution at pH 10.65. The lowest concentrations of the analytes that could be detected were below 1 microM for the O-methylated, below 2 microM for the phenolic and ca. 7 microM for the glucuronide metabolites. The suitability of the method for screening of real samples was tested with an authentic urine sample collected after a single oral dose (50 mg) of TR. After purification and five-fold concentration of the sample (solid-phase extraction with Oasis MCX cartridges), the parent drug TR and its metabolites M1, M1G, M5 and M5G were easily detected, in comparison with standards, in an interference-free area of the electropherogram. Diastereomeric separation of TR glucuronides in in vitro samples was achieved with 10 mM ammonium acetate-100 mM formic acid electrolyte solution at pH 2.75 and with basic micellar 25 mM tetraborate-70 mM SDS electrolyte solution at pH 10.45. Both separations showed that glucuronidation in vitro produces glucuronide diastereomers in different amounts. The authentic TR urine sample was also analysed by micellar method, but unambiguous identification of the glucuronide diastereomers was not achieved owing to many interferences.     

Analysis of amino acids and biogenic amines in breast cancer cells by capillary electrophoresis using polymer solutions containing sodium dodecyl sulfate

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and NDA-biogenic amine derivatives by CE in conjunction with light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO) solutions containing sodium dodecyl sulfate (SDS). After sample injection, via EOF 0.1% PEO prepared in 100 mM TB solution (pH 9.0) containing 30 mM SDS entered a capillary filled with 0.5 M TB solution (pH 10.2) containing 40 mM SDS. Under this condition, 14 NDA-amino acid and NDA-amine derivatives were separated within 16 min, with high efficiency ((1.0–3.2) × 10⁵ theoretical plates) and sensitivity (LODs at S/N = 3 ranging from 2.06 to 19.17 nM). In the presence of SDS and PEO, analytes adsorption on the capillary wall was suppressed, leading to high efficiency and reproducibility. The intraday analysis RSD values (n = 3) of the mobilities for the analytes are less than 0.52%. We have validated the practicality of this approach by quantitative determination of 9 amino acids in breast cancer cells (MCF-7) and 10 amino acids in normal epithelial cells (H184B5F5/M10). The concentrations of Tau and Gln in the MCF-7 cells were different than those in the H184B5F5/M10 cells, respectively. Our results show the potential of this approach for cancer study.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography.

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C18 column with isocratic elution with acetate buffer (25 mM, pH 4.5)-methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35 degrees C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 microgram/ml for BA and HA and 5.0 micrograms/ml for PAA and PA with a 20-microliters injection.     

Micellar electrokinetic chromatography as a fast screening method for the determination of 1,4-dihydropyridine calcium antagonists

A micellar electrokinetic chromatographic method (MEKC) was optimized for the separation of six calcium antagonists. The effects of the buffer (concentration and pH), concentration of sodium dodecyl sulphate (SDS), the organic modifier, the injection time, and the voltage applied were studied. A final appropriate electrolyte of 50 mM borate buffer, pH 8.2, containing 20 mM SDS and 15% (v/v) acetonitrile was found to provide the optimum separation with respect to resolution and migration time. The samples were introduced hydrostatically for 4 s at 50 mbar injection pressure and the applied voltage was +25 kV. The screening of the six compounds was achieved in less than 15 min: nifedipine (migration time, tm = 6.9 min), nimodipine (tm = 10.1 min), felodipine (tm = 12.2 min), nicardipine hydrochloride (tm = 12.7 min), lacidipine (tm = 13.5 min) and amlodipine besylate (tm = 14.1 min, tm = 8 min). The method developed showed to be linear at least up to 70 micrograms/ml with a detection limit of about 5 micrograms/ml for each compound. The within-day and inter-day area reproducibility (R.S.D.) were, respectively, lower than 4.8 and 8.6% for six replicate samples.     

Two high-performance liquid chromatographic methods for quantitation of theophylline in plasma

Two high-performance liquid chromatographic methods are described for the assay of theophylline in plasma. Both allowed the separation of theophylline from the caffeine metabolites, theobromine and 1,7-dimethylxanthine. Method A, using 8-chlorotheophylline as internal standard, involved back extraction of theophylline from organic extract with 0.1 M sodium hydroxide. Method B used generally accepted solvent extraction followed by evaporation and beta-hydroxyethyltheophylline as internal standard. High-performance liquid chromatographic analyses were performed on reversed-phase phenyl columns (25 X 0.46 and 25 X 0.41 cm) using 20% methanol in 20 mM phosphate buffer at pH 5.6 for Method A and 2% acetonitrile and 8% methanol in 20 mM phosphate buffer for Method B. The column effluent was monitored at UV 273 nm. Standard curves for both Methods A and B were fitted by linear regression (r greater than 0.999) in the concentration range of 0.05-50 micrograms/ml. Either method was selective, accurate and reproducible over the concentration range 0.08-26 micrograms/ml. However, compared with Method B, Method A provided significant advantages in terms of simplicity, speed and efficiency.     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Enantiomeric resolution of bupivacaine by high-performance liquid chromatography and chiroptical detection

This work reports a new analytical procedure for the separation and determination of the enantiomers of bupivacaine and the determination of the enantiomeric purity. The isomers were separated using a Chirex 3020 (250 mm x 4.6 mm) with a mobile phase of n-hexane:dichloroethane:ethanol (82:9:9, v/v/v) at a flow-rate of 1 ml min(-1) and UV, polarimetric and circular dichroism (CD) detection. Obtained retention times were 5.93 and 7.53 min (R and S) with a resolution of Rs=2.36. Precisions (RSD) were 1.83 and 2.02% (CD detection) and 3.07 and 1.26% (UV detection) for R- and S-enantiomers, respectively (at 10 microg level). Detection limits were 0.5 and 0.5 microg (R and S) with CD detection, and 0.9 and 0.3 microg with UV detection. Polarimetric detection was inadequate to perform a quantitative method at similar concentration ranges as UV and CD because of poor sensitivity. A procedure for determination of enantiomeric purity using a conventional chromatographic column (RP18, Luna) coupled to a CD detector and anisotropy factor (CD/UV) as analytical signal was also developed. Obtained results show that RSDs of 6.7-1.6% were obtained in the range of 0-100% enantiomeric purity.     

Capillary electrophoretic determination of inorganic anions in the drainage and surface water samples.

Capillary electrophoresis was used for separation and quantitation of several inorganic anions in the drainage and surface water samples from the region with extensive use of fertilisers. Baseline separation of 13 small anions including nitrite and nitrate up to the concentrations of 100 mg/l was achieved in less than 5 min. The electrolyte consisted of 3 mM K2CrO4, 30 microM cetyltrimethylammonium bromide and 3 mM boric acid at pH 8. The method yielded precisions of 1.8-7.2% (RSD, n = 10) and detection limits from 4 micrograms/l (Cl-) up to 500 micrograms/l (citrate). The results of the CE method were compared to ion chromatography using water-acetonitrile (86:14) at pH 8.6 adjusted with NaOH as the mobile phase and consistent results were obtained.