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 trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

Determination of ambroxol in dog plasma by high-performance liquid chromatography and UV detection

Summary A new sensitive HPLC-UV method has been developed and validated for the determination of amboroxol in dog plasma enabling the investigation of a newly developed 75 mg ambroxol-containing retard capsule of EGIS Pharmaceuticals Ltd., Budapest, Hungary. A gradient method was used for removing the longer retained plasma components of no interest. The separation was performed on a BDS Hypersil C18 (5 μm, 250×2.1 mm) analytical column, supplied with a 10 mm guard column containing the same packing material. The detection was performed at 210 nm. The calibration curve was linear in the range 25–2000 ng·mL⁻¹. Nerisopam (EGIS-6775) was used as internal standard. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Sensitive determination of cinnarizine in human plasma by high performance liquid chromatography and fluorescence detection

Summary A sensitive high performance liquid chromatographic method has been developed for the determination of cinnarizine in human plasma. Cinnarizine and clocinizine (internal standard) were extracted from acidified plasma (pH 4.7) into carbon tetrachloride and the organic layer was evaporated. The products were separated on a Microspher C18 (3 m) column, using a mixture of 0.04 % triethylamine in 0.01 M ammonium dihydrogen phosphate (NH₄H₂PO₄), pH adjusted to 4.2 with orthophosphoric acid (H₃PO₄), and acetonitrile (2080, v/v) as mobile phase, at a flow rate of 1 ml/min at 40°C. Fluorescence detection (_ex = 245 nm, _em = 310 nm) was used; the detection limit was 0.5 ng/ml under the conditions used, and the calibration curve linear in the concentration range evaluated (1–60 ng/ml). The assay has been used to measure cinnarizine concentrations in plasma after oral administration to volunteers.     

Determination of thiopental in human serum and plasma by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic method for the analysis of thiopental in 100l of human serum or plasma is described. The procedure involves protein precipitation with acetonitrile. The supernatant is directly injected into a chromatograph containing a reversed-phase CLC-ODS (Shimadzu) column. A 5050 (v/v) mixture of water-acetonitrile, at a flow-rate of 1.0ml/min is used as the mobile phase. Detection is carried out ata wavelength of 280nm. Total analysis time per sample is 10min. The assay was found to be linear in the range of 0.1 to 120g/ml. Reproducibility was good, with intra-assay coefficients of variation from 1.780 to 3.208% and inter-assay coefficients of variation from 3.241 to 4.860%. The absolute recoveries were 97.4 to 101,4%. Other drugs were tested for potential interference with the assay, but none was found.     

Determination of risedronate in rat plasma samples by ion-pair high-performance liquid chromatography with UV detector

In the present work, an analytical method for determination of risedronate, a member of bisphosphonates, is described for the routine analysis in rat plasma. Sample pre-treatment involves protein precipitation, co-precipitation with calcium at alkaline pH, hydrolysis of possible derivatives of pyrophosphate and reprecipitation. A good separation was obtained by using a reversed-phase column (Hypersil ODS-2 C₁₈, 4.6 mm × 250 mm, 5 μm). The mobile phase was an aqueous solution of buffer (contained 1.5 mM EDTA-2Na, 1 mM sodium etidronate, 11 mM sodium phosphate and 5 mM tetrabutylammonium bromide as ion-pair reagent) - methanol (88:12, v/v) adjusted to pH 6.75 using 1 M NaOH. The flow rate was 1 ml min⁻¹. UV detection (λ = 262 nm) was used to quantitate risedronate in the concentration range of 10-500 ng ml⁻¹. The limit of detection and quantitation for risedronate were 7 and 10 ng ml⁻¹, respectively. The method was applied successfully to plasma samples from Wistar rats undergoing oral administration of risedronate mini-pills. Precision, extraction recoveries, as well as accuracy results, were satisfactory and no interference was found at the retention time of risedronate. Hence, the method is suitable for monitoring risedronate in rat plasma.     

Determination of picomole amounts of carbonyls as luminarin hydrazones by high-performance liquid chromatography with fluorescence detection

Summary Fluorogenic reagents (luminarin 3, luminarin 11 and luminarin 12), having a quinolizinocoumarin moiety as fluorophore and a carboxylic acid hydrazide function as reacting group, have been developed. These reagents were found to be highly sensitive fluorescence derivatization reagents for aldehydes and ketones in high-performance liquid chromatography. The reagents readily react with carbonyl compounds in aqueous sulphuric acid solution (0.1 M) at room temperature to produce the corresponding hydrazone derivatives, which can be separated on both reversed or normal-phase column. The structures of the derivatives were studied, together with their properties in reversed and normalphase chromatographic systems. UV absorbance, corrected fluorescence spectral data and quantum yields of luminarin 3, luminarin 11 and luminarin 12 are presented. The detection limits (signal to noise ratio=3) for aldehydes and ketones were in the sub-pmol range. Luminarin 3 was also applied to the determination of hydroxymethylfurfural (HMF) in orange juices and concentrates. The method for HMF involves the solid-liquid extraction of the juice by using a C-18 cartridge prior to derivatization and normal-phase separation of the derivative with fluorimetric detection at 387 nmex., 444 nm em. The calibration curve was linear for amounts of HMF ranging from 0.1 to 10 nmol. Intrarun relative standard deviation was 12.8% for 0.1 nmol and 2.6% for 1 nmol. Recovery studies indicated an average of 98.7±1.9% for juice concentrate and 99.8±3.2% for pasteurized juice.     

Determination of cisapride in human plasma by high-performance liquid chromatography with fluorescence detection

Summary A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL⁻¹. A demethoxy analogue of cisapride was used as internal standard.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography

Summary An HPLC method has been developed for the quantification of rantidine in plasma for pharmacokinetic studies. Metoclopramide was used as internal standard. The method uses a simple and rapid sample clean-up procedure involving single-step extraction with organic solvent to extract ranitidine from plasma. After evaporation and reconstitution the samples are chromatographed on a 250 mm×4 mm base-stable reversed-phase column with 0.05 M ammonium acetate-acetonitrile, 75∶25 (v/v) as mobile phase and UV detection at 313 nm. The calibration graph was linear for quantities of ranitidine between 10 and 2000 ng mL⁻¹. Intra- and inter-dayCV did not exceed 11.64%. The quantitation limit was 10 ng mL⁻¹ for human plasma. The applicability of this method for pharmacokinetic studies of ranitidine after oral administration are described. Approximately 90 samples can be processed in 24 h.     

Determination of phenazopyridine in human plasma by high performance liquid chromatography

Summary A simple, low-cost, sensitive and selective HPLC method was developed for the determination of phenazopyridine in human plasma. The method employs UV detection of phenazopyridine and of the internal Standard at 2 different wavelengths. Calibration curves were linear over a large dynamic range, i.e., within 0.05–10.0 μg mL⁻¹ with limit of quantification of 0.05 μg mL⁻¹, and a limit of detection of 0.01 μg mL⁻¹.     

An improved high-performance liquid chromatography method for the determination of homocysteine in human plasma

Summary Elevated plasma homocysteine is, a known risk factor in arteriosclerotic vascular disease. To measure homocysteine in a large number of samples, we have developed a rapid, simple, robust and inexpensive reversed-phase HPLC method for routine analysis. Mercaptopro-pionylglycine was used as the internal standard and an external calibration in plasma was performed. Improvement was achieved by the use of gradient elution (using a sodium acetate buffer and methanol) resulting in a higher number of samples analyzed per day. Plasma samples were reduced with tributylphosphine and the proteins were precipitated with perchloric acid before addition of internal standard. The analytes were derivatized by use of 7-fluorobenzofurazone-4-sulfonic acid ammonium salt. For calibration human plasma was spiked with nine different concentrations of homocysteine (range 2–50 μmol L⁻¹). The inter-assay precision of replicate (n=29) analysis of the concentration of homocysteine in a sample of pooled plasma was 3.0%. The limit of detection, defined as three times the signal-to-noise ratio, was 0.25 μmol L⁻¹. The linearity of the assay was confirmed for a plasma concentration range of 2–2000 μmol L⁻¹. The variation of duplicate analyses of 842 plasma samples was 2.6±1.7%.     

Analysis of dihydroartemisinin in plasma by liquid chromatography—Mass spectrometry

Summary Artemisinin (ART), or qinghaosu, a sesquiterpene lactone containing an endoperoxide bridge, is an efficient drug against chloroquine-resistant strains ofPlasmodium falciparum in the treatment of cerebral malaria. ART and its derivatives are rapidly metabolized into dihydroartemisinin (DHA), which is responsible for the antimalarial activity. After a simple liquid-liquid extraction (LLE), determination of DHA in plasma was conducted by liquid chromatography-mass spectrometry (LC-MS) with an electrospray ionization (ESI) interface. Addition of dodecylamine to the mobile phase and analysis in selected-ion monitoring (SIM) mode resulted in enhanced sensitivity and selectivity. Use of a primary amine, e.g. propylamine, pentylamine, hexylamine or dodecylamine, in the mobile phase can inhibit the formation of multimer clusters and ion adducts by forming one dominating species (analyte-primary amine adduct). Dodecylamine (DDA) was selected after several assays as a competitive agent for adduct formation. The procedure was validated and precision and accuracy were found to be good for the matrix studied. The method was applied to samples provided by patients receiving artesunate (ARS) for antimalarial treatment.     

Determination of ephedra alkaloids by high-performance liquid chromatography

Summary Two simple methods were developed for the simultaneous determination of six alkaloids (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine and methyl-pseudoephedrine) inEphedrae Herba by high-performance liquid chromatography. The first method was carried out by using a Cosmosil 5C₁₈-MS column with a gradient solvent system consisting of a phosphate buffer and acetonitrile, and detection at 210 nm. The contents of alkaloids in non-pretreated ephedra herb extracts could be determined easily in 50 min. Alternatively, the alkaloids could be determined within 35 minutes by using a Cosmosil 5C₁₈-MS column with an isocratic solvent system of a sodium dodecyl sulfate-acetonitrile solution. The two methods are compared and discussed.     

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

Turbidimetric titration and gradient high-performance liquid chromatography of polystyrene samples

Summary Polystyrene samples of narrow molecular-weight distribution have been eluted according to their molecular weight from columns packed with bare silica Si50, phenyl, or C18 bonded phase by gradients of methanol and tetrahydrofuran (THF) or ofiso-octane and THF. Among the six combinations investigated,iso-octane/THF with a silica column formed a proper normal-phase system whereas methanol/THF with a C18 column formed a proper reversed-phase system. The combinations of C18 column andiso-octane/THF or of Si50 column and methanol/THF gradient did not correspond to the approved polarity rules in high-performance liquid chromatography but were nevertheless effective in separating polystyrene mixtures by molecular weight. Methanol andiso-octane are nonsolvents for polystyrene whereas THF is a solvent. The solubility of polystyrene as a function of molecular weight and concentration was determined by means of turbidimetric titration of solutions in THF with the nonsolvents used in the gradients. The solubility and elution characteristics were almost identical on C18 columns or in methanol/THF combinations. The elution from phenyl bonded phase and Si50 columns usingiso-octane/THF gradients required more THF than the solubility experiments. Information is also given on the occurrence of multimodal elution patterns.     

Direct electrogenerated chemiluminescence detection in high-performance liquid chromatography for determination of ofloxacin

Ofloxacin (OFLX) exhibited strong electrogenerated chemiluminescence (ECL) in NaNO₃ solution with a dual-electrode system when constant current was exerted. Based on this observation, a sensitive direct ECL method coupled with high-performance liquid chromatography (HPLC) separation was developed for determination of OFLX in human serum. Factors affected the ECL emission were investigated. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of OFLX in the range of 1.0 × 10⁻⁸ to 4.0 × 10⁻⁶ g mL⁻¹ and the detection limit was 4 × 10⁻⁹ g mL⁻¹ (S/N = 3). The proposed method was sensitive, simple and convenient to operate.     

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.     

Separation of L-DOPA and four metabolites in plasma using a porous graphitic carbon column in capillary liquid chromatography

Summary A sensitive HPLC method with marbofloxacin (MAR) as internal standard and fluorescence detection is described for the analysis of ofloxacin (OFL) enantiomers in plasma samples. Plasma samples were prepared by adding phosphate buffer (pH 7.4, 0.1m), then extracted with trichloromethane.S-OFL,R-OFL, and the internal standard were separated on a reversed-phase column with water-methanol, 85.5∶14.5, as mobile phase. The concentrations ofS-OFL andR-OFL eluting from the column (retention times 7.5 and 8.7 min, respectively) were monitored by fluorescence detection withλ _ex = 331 andλ _em = 488 nm. The detection and quantitation limits were 10 and 20 ng mL⁻¹, respectively, forS-OFL and 11 and 21 ng mL⁻¹ forR-OFL. Response was linearly related to concentration in the range 10 to 2500 ng mL⁻¹. Recovery was close to 93% for both compounds. The method was applied to determination of the enantiomers of OFL in plasma samples collected during pharmacokinetic studies.     

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     

Determination of chloride in biological fluids as pentafluorobenzylchloride by reversed-phase high-performance liquid chromatography and UV detection

Summary A reversed-phase high-performance liquid chromatographic method for the determination of chloride in plasma, urine, saliva, sweat and aqueous solution is described. Chloride, in solution in aqueous acetone, is converted by means of pentafluorobenzyl bromide into pentafluorobenzyl chloride. This derivative is separated on a ODS-5 m reversed-phase column using isocratic elution with acctonitrile/water, 50/50, v/v, at a flow rate of 2.0 ml/min, and detected by a UV detector at 264 nm. The method is rapid, accurate and sufficiently sensitive for the determination of chloride in less than 10 l sample volume of a biological fluid.