Simultaneous determination of five 1,4-dihydropyridines in pharmaceutical formulations by high-performance liquid chromatography-amperometric detection

A high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of five 1,4-dihydropyridines: amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine. These drugs are widely used in the treatment of hypertension, angina pectoris and the therapy of cerebrovascular spasms of various origins. The chromatographic separation was performed on a Supelcosil LC ABZ + Plus C18 column with a mobile phase consisting of acetonitrile-10 mM acetate buffer (72:28, v/v) at a flow rate of 1 ml/min. The temperature was set at 30 +/- 0.2 degrees C. The amperometric detector, equipped with a glassy carbon electrode was operated at +1100 mV versus Ag/AgCl in the direct current mode. Under these chromatographic conditions, the drugs eluted in less than 12 min. The method showed to be linear over the range 4.5-15 microg/ml with a within-day and day-to-day repeatabilities in terms of R.S.D. lower than 15%, an accuracy greater than 98% and detection limits varying from 90 ng/ml (amlodipine) to 1.55 microg/ml (nitrendipine). The method was successfully applied to commercially available pharmaceuticals with relative errors lower than 5%. The validity of the method was examined comparing the results obtained with those of HPLC with photometric detection.     

LC-DAD Determination of Calcium Channel Blockers by Using an Experimental Design Approach

A high-performance liquid chromatographic method with diode array detection has been developed for the determination of five 1,4-dihydropyridines: amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine. A fractional design and a central composite design were used. The factors considered in the optimisation process were: percentage of organic modifier, pH of the aqueous buffer, buffer concentration and temperature. The chromatographic separation was performed using a Supelcosil LC-ABZ+Plus C18 column. An optimised mobile phase of acetonitrile-water (70:30, v/v), containing 10 mM CH₃COOH-CH₃COONa pH 5 at a flow rate of 1 mL min^?1 was used. The temperature was set at 30 ± 2 °C. The photometic detection was carried out at 237 nm. The method was applied to the determination of these compounds at μg mL^?1 concentration levels, obtaining intraday repeatabilities values lower than 5% in terms of relative standard deviations, accuracies higher than 98% and detection limits ranged from 0.03 (felodipine) to 0.35 μg mL^?1 (lacidipine). The chromatographic method allowed the analysis of the drugs in their pharmaceutical formulations with a total elution time of 12 min.     

Enantioselective determination of felodipine and other chiral dihydropyridine calcium entry blockers in human plasma

A sensitive method for the enantioselective determination of felodipine in human plasma is described. Following alkaline extraction with dichloromethane-pentane, racemic felodipine and its primary pyridine metabolite are simultaneously assayed using capillary gas chromatography on a DB-1 column, with electron-capture detection. The enantiomers of felodipine are quantitatively separated by high-performance liquid chromatography on a Chiralcel OJ column, containing tris(4-methylbenzoate)-modified cellulose coated on silica, and off-line detection using the same gas chromatographic system is applied. The limits of determination in plasma (and the inter-assay coefficient of variation (C.V.) at levels below 1 ng/ml) were 0.1 ng/ml (C.V. 13%) for felodipine, 0.1 ng/ml (C.V. 15%) for the enantiomers of felodipine and 0.3 ng/ml (C.V. 7%) for its pyridine metabolite. The method has proved to be applicable to several other chiral dihydropyridine calcium entry blockers, including nitrendipine, with comparable sensitivities.     

Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, -tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C₁₈ AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C₁₈ (150×3.9 mm, 4 μm) analytical column, using CH₃OH–0.05 M CH₃COONH₄ as mobile phase The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C₁₈ sorbent, on the above column. The mobile phase employed was MeOH–CH₃CN (95:5, v/v) working at a flow-rate of 2 ml min⁻¹ in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV–Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B₁₂) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.     

Determination of the carotenoid phytoene in blood by high-pressure liquid chromatography

A sensitive and specific high-pressure liquid chromatographic assay was developed for the determination of phytoene in blood with an overall recovery of 86 ± 6.0% and a limit of detection of 50–100 ng per ml of blood. This method provides for rapid and simple quantitation of phytoene using 1 ml or less of blood.

The assay was used in the determination of phytoene blood levels in the dog following intravenous and oral administration of 10-mg/kg doses.     

Determination of lasalocid with sensitized terbium(III) luminescence detection

The luminescence of the lasalocid-terbium(III) system in the presence of Triton X-100 and trioctylphosphine oxide has been studied by obtaining kinetic and equilibrium measurements and using the stopped-flow mixing technique. The initial rate and luminescence signal of this system are directly proportional to the lasalocid concentration, which allows one to develop very simple, fast, automatic methods for the determination of this analyte. Kinetic and equilibrium data can be obtained in only 0.1 and 10 s, respectively. The calibration graphs were linear over the range 0.004-5.0 mug ml(-1) (kinetic method) and 0.01-5.0 mug ml(-1) (equilibrium method) and the detection limits achieved were 1 and 3 ng ml(-1), respectively, equivalent to 2 and 6 ng g(-1) lasalocid in a chicken liver sample, which are similar to those afforded by the chromatographic methods described for this determination. The relative standard deviation of both methods was close to 2%. The analytical recoveries obtained by applying the kinetic and equilibrium methods to drinking water, poultry feed and chicken liver samples ranged from 95.6 to 102.1% and from 95.9 to 104.9%, respectively.     

Development of a liquid-liquid extraction procedure for five 1,4-dihydropyridines calcium channel antagonists from human plasma using experimental design

A liquid–liquid extraction method using diethyl ether as organic solvent was optimized simultaneously for five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine) belonging to the group of calcium channel blockers. Some experimental tools such as a full factorial design, a central composite design and the Multisimplex program were used to optimise the concentration of NaOH, volume of organic solvent and shaking time as main factors that influence the liquid–liquid extraction procedure. Following the extraction, the quantitation of the 1,4-dihydropyridines concentrations were performed by high-performance liquid chromatography with diode-array detector. Therefore, the studied compounds were separated quantitatively on a Supelcosil ABZ+Plus, 25 cm × 4.6 mm i.d., 5 μm column which was set at 30 °C, using as mobile phase, a mixture of acetonitrile–water (70:30, v/v) containing 10 mM acetate buffer (pH 5) and setting the detector at a wavelength value of 360 nm. It was concluded that the main factors that influence in the extraction process were the volume of organic solvent and the shaking time. The Multisimplex program suggested as optimal conditions an average of 6 ml of organic solvent and 23 min of shaking time. For these values, the optimised liquid–liquid extraction method showed good values of recoveries (80% for amlodipine and higher than 90% for the rest of the compounds) and low values of R.S.D. (<10%) in the reproducibility of the extraction what makes it reliable for the quantification of all the studied compounds in human plasma.     

纺织品中残留氯酚的毛细管气相色谱测定法

采用稀硫酸浸湿样品,正己烷提取,乙酸酐衍生后以毛细管气相色谱分离测定的方法对纺织品中五氯酚、三氯酚残留量进行了同时测定,探讨了提取、净化及色谱分析条件。方法回收率范围三氯酚84.8％～98.1％,五氯酚88.0％～100.2％。相对标准偏差三氯酚1.54％～2.33％,五氯酚3.48％。方法的检出限（质量分数）分别为2,4,6-三氯酚1.0×10     

Optimizing recoveries of two chlorotriazine herbicide metabolites and 11 pesticides from aqueous samples using solid-phase extraction and gas chromatography–mass spectrometry

A method was developed for solid-phase extraction of two chlorotriazine herbicide metabolites, deethylatrazine (DEA) and deisopropylatrazine (DIA), from aqueous samples. Two C₁₈ phases in cartridge format were compared and recoveries were found to be highly sensitive to sorbent amount, sample volume and presence of parent compounds. Recoveries were significantly improved using a partially non-endcapped C₁₈ phase compared to the normal C₁₈ phase, particularly for DIA, apparently due to polar interactions. Combinations of sample volume and sorbent amount were tested using deionized water to determine an optimal combination of 200 ml and 1.0 g, respectively. Recoveries from a variety of river, stream, runoff and ground waters averaged 105–116% and 109–117% at concentrations of 0.5–1.0 ng/ml for DIA and DEA, respectively, with minimum detection limits of 0.05 ng/ml. Other pesticides tested also have acceptable recoveries using this method.     

Determination of piperazine in working atmosphere and in human urine using derivatization and capillary gas chromatography with nitrogen- and mass-selective detection

A reliable routine method is presented for the determination of piperazine down to the sub-ppm level in aqueous solutions and in urine. The method includes a two-phase derivatization procedure with ethyl- or isobutyl chloroformate as the reagent, followed by a capillary gas chromatographic determination using nitrogen- or mass selective detection. The addition of ammonia ensured a quantitative recovery. Detection limits for piperazine in urine were ca. 20 ng/ml using nitrogen-selective and ca. 1 ng/ml with mass-selective detection. The calibration plots were linear in the investigated range, 100-10,000 ng/ml with nitrogen-selective and 30-3000 ng/ml with mass-selective detection. The precision was ca. 6% at a concentration of 300 ng/ml. Acid anhydrides were investigated as alternative reagents in the two-phase derivatization procedure, and heptafluorobutyric acid anhydride in aqueous solutions gave approximately 100% recovery. However, in urine the recoveries of the investigated acid anhydride derivatives were unsatisfactory.     

Simultaneous determination of clozapine, clozapine N-oxide, N-desmethylclozapine, risperidone, and 9-hydroxyrisperidone in plasma by high performance liquid chromatography with ultraviolet detection

A simple high performance liquid chromatography techniques with ultraviolet detection (HPLC–UV) method is described for the simultaneous determination of clozapine (CZP), clozapine N-oxide (CNO), N-desmethylclozapine (NCZ), risperidone (RSP) and 9-hydroxyrisperidone (9-OHRSP) in human plasma. After extraction process, the analytes were separated on a C₁₈ column (150 mm×3.9 mm i.d.) by the mobile phase (methanol–water–dimethylamine, 60:40:0.04 (v:v:v)). Relative recoveries of five analytes were quantitative. The precision and accuracy of intra- and interday assays were all below 8.2% for R.S.D. and 5.6% for RE, respectively. Based on 1 ml of plasma, the limits of detection were 2.0 ng/ml for CZP, 0.2 ng/ml for CZP N-oxide, 1.0 ng/ml for NCZ, 1.0 ng/ml for RSP, and 0.5 ng/ml for 9-OHRSP (S/N=3). The calibration curves were linear (r≥0.988). This method was applied to therapeutic drug monitoring of schizophrenia patients receiving CZP or RSP therapy.     

Simple and sensitive method for determination of glycoalkaloids in potato tubers by high-performance liquid chromatography with chemiluminescence detection

A novel, simple and sensitive high-performance liquid chromatographic method for the determination of the potato glycoalkaloids, alpha-solanine and alpha-chaconine, based on the chemiluminescent reaction of tris(2,2'-bipyridine)ruthenium(III) has been developed. The calibration graph was linear in the range of 5 ng/ml-10 microg/ml for both alpha-solanine and alpha-chaconine. The detection limits of alpha-solanine and alpha-chaconine were 1.2 and 1.3 ng/ml, respectively. This method was successfully applied to a potato tuber sample without cleanup, pre-concentration, and derivatization steps. The recoveries (mean +/- standard deviation, %) of alpha-solanine and alpha-chaconine spiked in tuber pith at 10 microg/g (n = 6) were 101.0 +/- 4.4% and 103.6 +/- 7.1%, respectively.     

Microanalysis of dialkylphosphorus metabolites of organophosphorus pesticides in human blood by capillary gas chromatography and by phosphorus-selective and ion trap detection

A procedure for the determination of dialkyphosphorus metabolites of organophosphorus pesticides in human blood has been worked out. Dimethyl and diethyl phosphates, phosphorothioates and phosphorodithioates, extracted with diethyl ether from plasma acidified with hydrochloric acid, were methylated with diazomethane and analysed by capillary gas chromatography with an alkali flame ionization detector and an ion trap detector. The extraction of metabolites was preceded by n-hexane extraction of parent organophosphorus pesticides without a negative effect on the efficiency of metabolite extraction. If plasma samples, containing 2 μ/ml of each metabolite, were not saturated with sodium chloride before extraction, only dialkyl phosphorothioates were recovered by more than 80%. The recoveries of other metabolites were less than 25%. The extraction of plasma samples saturated with sodium chloride resulted in higher recoveries of all metabolites. At concentrations ranging from 0.2 to 2.8 μg/ml the accumulation effeciencies (%±S.D.) of dimethyl and diethyl phosphorothioates were 92±20 and 97±11, and those of corresponding phosphorodithiotes 79±7 and 71±4. A significantly lower recovery (36±12%) was determined for dimethyl phosphate at concentrations in plasma below 2 μg/ml. The recovery of diethyl phosphate was dependent on the initial metabolite concentration in plasma being 31±5% at concentrations lower than 0.5 μg/ml, 51±12% at concentrations ranging from 0.7 to 1.7 μg/ml and 97±3% at concentrations at or above 2 μg/ml. Detection limits of metabolites in plasma using the phosphorus selective detector were 150 ng/ml for dimethyl phosphate and 50 ng/ml for other metabolites. Those values were for dialkyl phosphates and phophorothioates three to five times lower and for diakyl phosphorodithiotes even 30 times lower than detection limits achieved by the use of ion trap detector. The procedure was applied for the evidence and confirmation of human poisoning with organophosphorus pesticides.     

Determination of cilnidipine, a new calcium antagonist, in human plasma using high performance liquid chromatography with tandem mass spectrometric detection

A rapid, sensitive and reliable high performance liquid chromatographic method coupled with tandem mass spectrometry (HPLC–MS/MS) has been developed and validated for the determination of cilnidipine, a relatively new calcium antagonist, in human plasma. The reversed-phase chromatographic system was interfaced with a TurboIonSpray (TIS) source. Nimodipine was employed as the internal standard (IS). Sample extracts following protein precipitation were injected into the HPLC–MS/MS system. The analyte and IS were eluted isocratically on a C18 column, with a mobile phase consisting of CH₃OH and NH₄Ac (96:4, v/v). The ions were detected by a triple quadrupole mass spectrometric detector in the negative mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 for cilnidipine and for the IS, respectively. The analysis time for each run was 3.0 min. The calibration curve fitted well over the concentration range of 0.1–10 ng mL⁻¹, with the regression equation Y = (0.103 ± 0.002)X + (0.014 ± 0.003) (n = 5), r = 0.9994. The intra-day and inter-day R.S.D.% were less than 12.51% at all concentration levels within the calibration range. The recoveries were between 92.71% and 97.64%. The long-term stability and freeze-thaw stability were satisfying at each level. The present method provides a modern, rapid and robust tool for pharmacokinetic studies of cilnidipine.     

Determination of etretinate and its main metabolite in human plasma using normal-phase high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of astemizole and its primary metabolite in plasma and animal tissues. Both compounds and the internal standard were extracted from alkalinized plasma with heptane--isoamyl alcohol and analyzed using a reversed-phase column and UV monitoring at 254 nm. The detection limits for both compounds were 1 ng/ml of plasma and 5 ng/g of tissue and extraction recoveries were sufficiently high (71-84%). The method was applied to plasma and tissue samples from dogs after repeated oral administration, and to plasma samples from a volunteer taking a 300-mg oral dose of the drug. The results were compared with those obtained by a formerly developed radioimmunoassay.     

High-performance liquid chromatographic determination of nafimidone and its major metabolite nafimidone alcohol in human plasma and urine

A rapid, sensitive and selective method for the determination in plasma and urine of nafimidone, a new antiepileptic drug, and its major metabolite, nafimidone alcohol, has been developed which uses a high-performance liquid chromatographic system and a fluorescence detector for nafimidone or ultraviolet detector for nafimidone alcohol. The detection limits for nafimidone and nafimidone alcohol are 5.0 and 12.5 ng/ml, respectively.     

Micro-columns packed with Chlorella vulgaris immobilised on silica gel for mercury speciation

A method has been developed for mercury speciation in water by using columns packed with Chlorella vulgaris immobilised on silica gel. The method involves the retention of CH₃Hg⁺ and Hg²⁺ in micro-columns prepared by packing immobilised algae in polypropylene tubes, followed by selective and sequential elution with 0.03 and 1.5 M HCl for CH₃Hg⁺ and Hg²⁺, respectively. The adsorption capacity of the micro-algae for Hg²⁺ and CH₃Hg⁺ has been evaluated using free and immobilised C. vulgaris. The efficiency uptake for both species at pH 3 was higher than 97%. Studies were carried out on the effect of retention and elution conditions for both species. Furthermore, the stability of mercury species retained on algae-silica gel micro-columns and lifetime of the columns were also investigated. Hg²⁺ showed a higher stability than CH₃Hg⁺ at 0 °C (21 and 3 days, respectively) and a better lifetime than for the organic species.

The developed method was applied to the analysis of spiked tap, sea and wastewater samples. Recovery studies on tap and filtered seawater provided results between 96 ± 3 and 106 ± 2 for Hg²⁺ and from 98 ± 5 to 107 ± 5 for CH₃Hg⁺, for samples spiked with single species. For samples spiked with both CH₃Hg⁺ and Hg²⁺, the average recoveries varied from 96 ± 5 to 99 ± 3 and from 103 ± 6 to 115 ± 5 for Hg²⁺ and CH₃Hg⁺, respectively. However, the percentages of retention and elution on wastewater and unfiltered seawater were only adequate for the inorganic species.     

Determination of perfluorooctanesulfonate and perfluorooctanoic acid in sewage sludge samples using liquid chromatography/quadrupole time-of-flight mass spectrometry

A new method is developed for the determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) in sewage sludge samples. The analytes in sewage sludge samples are extracted by methanol and formic acid, cleaned by C18 solid-phase extraction, then separated, identified and quantitated by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC–QTOF-MS). A C18 column (150 mm × 2.1 mm, 3.5 μm) with gradient elution of MeOH–H₂O (60:40) containing 5 mmol/L ammonium acetate and MeOH–H₂O (80:20) is used for the chromatographic separation. [M−K]⁻ ions at m/z 498.93 for PFOS and [M−COOH]⁻ ion at m/z 368.97 for PFOA are selected for QTOF-MS in the negative electrospray ionization mode. The detection limits for PFOS and PFOA in sewage sludge samples are 0.5 and 0.8 ng/g, respectively. The spiked recoveries are in the range of 85–114 and 71–98% for PFOS and PFOA, respectively. The proposed method is successfully applied to the analysis of PFOS and PFOA in 16 sewage sludge samples from China. PFOS and PFOA are detected in most sewage sludge samples and the concentrations of PFOS and PFOA are up to 5383 and 4780 ng/g (oven dry weight), respectively.     

Simultaneous determination of vanadium and molybdenum as N-benzoyl-N-phenylhydroxamate complexes by combining solvent extraction and liquid chromatography

A normal-phase high-performance liquid chromatographic method for the selective simultaneous determination of vanadium and molybdenum with N-benzoyl-N-phenylhydroxylamine (BPHA) is described. The V(V)-BPHA and Mo(Vl)-BPHA complexes were preconcentrated by solvent extraction into chloroform and injected on to a nitrile-bonded column for chromatography. The mobile phase was a 5.9· 10⁻⁴ M solution of BPHA in chloroform (stabilized with amylene). The detection limits for vanadium and molybdenum were 2.1 and 3.3 ng ml⁻¹, respectively, for an aqueous to organic phase-volume ratio of 20:1. The procedure, applied to the analysis of a synthetic water, showed satisfactory accuracy and precision.     

Sensitive simultaneous determination of benzo(a)pyrene, perylene and chrysene by synchronous spectrofluorometry in nonionic micellar media

A synchronous spectrofluorometric method was developed for the simultaneous determination of benzo(a)pyrene, perylene and chrysene in a POLE micellar medium, with detection limits of 0.05 ng/ml, 0.28 ng/ml and 0.64 ng/ml, respectively. Good recoveries were obtained for sea water samples spiked with each hydrocarbon.