Quantitation of antihistamines in pharmaceutical preparations by liquid chromatography with a micellar mobile phase of sodium dodecyl sulfate and pentanol.

A reversed-phase liquid chromatographic procedure with a micellar mobile phase of sodium dodecyl sulfate (SDS), containing a small amount of pentanol, was developed for the control of 7 antihistamines of diverse action in pharmaceutical preparations (tablets, capsules, powders, solutions, and syrups): azatadine, carbinoxamine, cyclizine, cyproheptadine, diphenhydramine, doxylamine, and tripelennamine. The retention times of the drugs were <9 min with a mobile phase of 0.15M SDS-6% (v/v) pentanol. The recoveries with respect to the declared compositions were in the range of 93-110%, and the intra- and interday repeatabilities and interday reproducibility were <1.2%. The results were similar to those obtained with a conventional 60 + 40 (v/v) methanol-water mixture, with the advantage of reduced toxicity, flammability, environmental impact, and cost of the micellar-pentanol solutions. The lower risk of evaporation of the organic solvent dissolved in the micellar solutions also increased the stability of the mobile phase.     

Determination of active ingredients in cough-cold preparations by micellar liquid chromatography

The chromatographic behaviour of some active ingredients in cough-cold pharmaceutical preparations, the antihistamine chlorpheniramine (or the dextro enantiomer dexchlorpheniramine), and the phenethylamines phenylephrine, phenylpropanolamine and pseudoephedrine, has been studied using a C(18) column, micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, and with UV detection. All possible combinations of chlorpheniramine/phenethylamine were resolved and determined using a mobile phase of 0.15 M SDS-6% (v/v) pentanol at pH 7, with analysis time below 7 min. Repeatabilities and within laboratory precisions were evaluated at four different drug concentrations in the range 0.5-25 mug ml(-1) (n=5), resulting RSDs below 1.6%. The drug amounts found in the analysis of 14 commercialised preparations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using an aqueous 60% (v/v) methanol reference mobile phase. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, caffeine, codeine phosphate, diphenhydramine, lactose, paracetamol, and prednisolone. The studied combinations required a rather high amount of methanol in conventional RPLC to be eluted from the column. In contrast, the proposed procedure used a much lower amount of organic solvent (pentanol), which is highly retained in the SDS solution, being also less toxic than methanol.     

Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

Chromatographic analysis of phenethylamine-antihistamine combinations using C8, C18 or cyano columns and micellar sodium dodecyl sulfate-pentanol mixtures

The chromatographic behaviour of binary and ternary mixtures of several phenethylamines (phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine and methoxyphenamine) and antihistamines (pheniramine, carbinoxamine, doxylamine, chlorpheniramine, dexchlorpheniramine, dexbrompheniramine, diphenhydramine, tripolidine, azatadine and phenyltoloxamine), found in cough-cold pharmaceutical preparations, was studied using C8, C18 and cyano columns, micellar mobile phases of sodium dodecyl sulfate (SDS) and pentanol and UV detection. Using a C8 column and mobile phases of 0.05 mol l-1 SDS-6% v/v pentanol or 0.15 mol l-1 SDS-2% v/v pentanol at pH 7, more than 30 different phenethylamine-antihistamine combinations can be resolved in < 15 min. Intra- and inter-day repeatabilities and reproducibilities evaluated at three different drug concentrations (0.5, 5 and 25 micrograms ml-1, n = 10) were below 1.6, 2.5 and 2.4%, respectively. The drug amounts found in 18 formulations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using a mobile phase of 55% v/v methanol at pH 7. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, bromhexine, caffeine, codeine, dextromethorphan, paracetamol, prednisolone, salicylamide and tartrazine. The proposed procedure has the advantage over the conventional aqueous-organic procedure of using a small amount of organic solvent, which is highly retained in the SDS solution. The efficiencies are also greater. On the other hand, in the micellar system, the retentions of phenethylamines and antihistamines are similar, although the compounds can be easily resolved. In contrast, using the methanol-water mobile phase, the phenethylamines are weakly retained, whereas the antihistamines usually show a high retention.     

Determination of disulfiram by micellar liquid chromatography in illicit preparations

Presently, disulfiram is used in aversion therapy for recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase, leading to high blood levels of acetaldehyde. A simple direct injection micellar liquid chromatographic procedure was developed to determine disulfiram in illicit preparations (ayurvedic, herbal, divine ash, and traditional medicine), as well as in pharmaceuticals and biological samples (urine). After application of a predictive optimization strategy, the proposed method was developed using a 0.1 M sodium dodecyl sulfate-butanol 4% (v/v) buffered to pH 7 as the mobile phase at a flow rate of 1 mL/min, an octyl silyl (C8) 150 mm column, and diode array detection at 248 nm. Under the above conditions, the analysis time was below 8 min. Validation studies were based on U.S. Food and Drug Administration guidelines. The LOD (3 x SD criterion) was 15 ng/mL and LOQ (10 x SD criterion) was 70 ng/mL for disulfiram. The intraday and interday precisions were below 3.5%, recoveries were in the range of 97-102%, and robustness was below 3%. The optimized and validated micellar liquid chromatographic method was successfully applied to the determination of disulfiram in ayurvedic, herbal, divine ash, and other samples. The procedure developed could also be used in the fields of QC, routine analysis, and pharmacokinetic studies.     

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CC(α)), detection capability (CC(β)), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples.     

Monitoring bronchodilators with direct injection

A procedure was developed for the determination of caffeine and theophylline using a C18 column (5 microm, 250 mm x 4.6 mm) and micellar liquid chromatography using hybrid mobile phases containing sodium dodecyl sulfate (SDS) and propanol, butanol or pentanol as modifiers. Detection was performed with a variable wavelength UV-vis detector at 272 nm. After the application of an interpretative strategy for the selection of the optimimum mobile phase, caffeine and theophylline can be resolved and determined in serum samples by direct injection, using a mobile phase made up of 50 mM SDS-2.5% (v/v) propanol-10 mM KH2PO4, pH 7, with an analysis time below 5 min. Calibration was linear in the range 0.05 to 50 microg mL(-1) with r > 0.999. The statistical evaluation of the method was examined by performing intra-day (n = 6) and inter-day calibration (n = 7) and was found to be satisfactory, with highly accurate and precise results. The proposed method was suitably validated and applied to the determination of caffeine and theophylline in serum samples of patients treated with bronchodilators.     

Comparison of the performance of butanol and pentanol as modifiers in the micellar chromatographic determination of some phenethylamines

A procedure was developed for the determination of several phenethylamines (amphetamine, arterenol, ephedrine, phenylephrine, phenylpropanolamine, mephentermine, methoxyphenamine, pseudoephedrine and tyramine), using micellar mobile phases of sodium dodecyl sulfate (SDS), a C18 column and UV detection. The drugs were eluted at short retention times with conventional acetonitrile-water or methanol-water mobile phases. In contrast, in the micellar system, they were strongly retained due to association with the surfactant adsorbed on the stationary phase, and needed the addition of butanol or pentanol to be eluted from the column. These modifiers allowed a simple way of controlling the retention. The chromatographic efficiencies obtained with the hybrid mobile phases of SDS-butanol and SDS-pentanol were also very high, mostly in the N=3000-7000 range, significantly greater than those achieved with a conventional acetonitrile-methanol-water mobile phase. Butanol and pentanol yielded similar selectivities, but the latter modifier permitted significantly shorter retention times than butanol, and was preferred to expedite the analysis of the pharmaceuticals. Most binary combinations of the nine phenethylamines can be resolved with these mobile phases. A mobile phase of 0.15 M SDS-5% pentanol was used to assay five of the phenethylamines (amphetamine, ephedrine, phenylephrine, phenylpropanolamine and pseudoephedrine) in 22 pharmaceutical preparations, which contained diverse accompanying compounds. The results agreed with the declared compositions and with those obtained with a mobile phase of methanol-acetonitrile-0.05 M phosphate buffer (pH 3) 10:5:85, with no interferences and relative errors usually below 2%. However, with the aqueous-organic mobile phase, the retention time for phenylephrine was too low and could not be usually evaluated.     

Simultaneous determination of three opiates in serum by micellar liquid chromatography using direct injection

A simple and reliable micellar liquid chromatographic method was developed for the simultaneous determination of 3 opiates (codeine, morphine, and thebaine) in serum, using direct injection and ultraviolet detection. The separation of the drugs was optimized on a C18 column, thermostatically controlled at 25 degrees C, by evaluating mobile phases containing sodium dodecyl sulfate (SDS) and various modifiers (propanol, butanol, or pentanol). Adequate resolution of the opiates was obtained with a chemometrics approach, in which retention was modeled as a first step by using the retention factors for several mobile phases. Next, an optimization criterion that takes into account the position and shape of the chromatographic peaks was applied. The 3 opiates were totally resolved and determined in 12 min with the mobile phase 0.15M SDS-7% (v/v) butanol buffered at pH 7. The limits of detection for codeine and morphine were greatly improved by using fluorimetric detection. Repeatability and intermediate precision were tested for 3 different concentrations of the drugs, and the relative standard deviations were <0.8% for most of the assays. Finally, the method was successfully applied to the determination of morphine and codeine in serum samples.     

Comparison of C18 silica bonded phases selectivity in micellar liquid chromatography

The paper describes a new test designed in micellar LC (MLC) to compare the commercial C18 stationary phase properties. This test provides the total hydrophobicity, hydrophilicity, steric selectivity, hydrogen bonding, and ion‐exchange capacity properties calculation of the ODS stationary phases. Both the test compounds and chromatographic separation conditions choice for column characterization in MLC are detailed. The chromatographic performance of several stationary phases that are used in MLC was evaluated with specific chromatographic test comprising nine test compounds, possessing different physico‐chemical properties, which were injected on different supports with two micellar mobile phases: one at pH 7.0 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol), and other at pH 2.7 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol adjusted to pH by TFA). Fundamental column chromatographic properties were obtained under these conditions and were treated by hierarchical cluster analysis. From the results of cluster analysis, two closely related groups of columns are distinguished, and it was shown that the chosen column characteristic parameters allow characterizing both sorbent and micellar chromatographic system properties. Eleven columns were analyzed by this test, which allows a comparison of columns with the aim of the selection of suitable and analogous column for the analysis with MLC.     

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%.     

Anserine and carnosine determination in meat samples by pure micellar liquid chromatography

Anserine and carnosine, which are both imidazole dipeptides, are natural antioxidants that are present in some types of meat. A pure micellar liquid chromatographic procedure was developed using a micellar mobile phase of 0.10 M sodium dodecyl sulphate buffered at pH 7, an amino column and UV detection. Three types of stationary phases (C18, phenyl and amino columns) were examined and the procedure was used to determine the two compounds in meat samples. They were completely resolved without any interference from the protein band. Total analysis time was 12 min. The limits of detection (ng/mL) were 71 and 53 for anserine and carnosine, respectively. Calibration curves were constructed on three different days (r>0.998). Repeatability and intermediate precision were evaluated at three different concentrations in meat matrices, the residual standard deviations being below 2.1%. Meat samples of poultry, pork and beef were injected directly into the chromatographic system after extraction in a sodium dodecyl sulphate solution and filtration. The possibility of direct injection using micellar liquid chromatography reduces the cost and time of analyses, and decreases error sources owing to minimised risks of losses and chemical changes in the analytes. Moreover, the selection of a pure mobile phase of sodium dodecyl sulphate allows this procedure to offer a number of advantages, such as non-toxicity, non-flammability, biodegradability and low cost, in comparison with aqueous-organic solvents. Its simplicity, then, makes it a good candidate for application in routine analysis in the area of food control and quality.     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma.

A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.     

Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.     

Improved efficiency in micellar liquid chromatography using triethylamine and 1-butanol as mobile phase additives to reduce surfactant adsorption

The effect of triethylamine as a mobile phase modifier on chromatographic efficiency in micellar liquid chromatography (MLC) is reported for nine different columns with various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, and perfluorinated. Reduced plate height (h) versus reduced velocity (nu) plots were constructed for each column and the A' and C' terms calculated using a simplified Van Deemter equation introduced in our previous work. To further explore the practicality of using triethylamine in the micellar mobile phase, the efficiency of nine polar and non-polar substituted benzenes was studied on seven columns. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with the addition of 2% (v/v) triethylamine to a 1-butanol modified aqueous micellar mobile phase. This finding is supported by the lower amount of surfactant adsorbed onto the stationary phase when TEA is present in the mobile phase compared to an SDS only or a 1-butanol modified SDS mobile phase.     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

Determination of furosemide in urine samples by direct injection in a micellar liquid chromatographic system

A sensitive, selective and efficient micellar liquid chromatographic (MLC) procedure was developed for the determination of furosemide (4-chloro-N-furfuryl-5-sulfamoylanthranilic acid) in urine samples by direct injection and UV detection. The procedure makes use of a C18 reversed-phase column and a micellar mobile phase of 0.05 mol l(-1) sodium dodecyl sulfate-6% v/v propanol and phosphate buffer at pH 3 to resolve furosemide from its photochemical degradation products. The importance of protecting the standards and urine samples to be analysed from light in the assay of furosemide, avoiding its degradation, was verified. The limit of quantification was 0.15 microg ml(-1) and the relative standard deviation of the inter-day assay was 0.8-0.04% in the 6-82 microg ml(-1) range. Detection of urinary excretion of furosemide was followed up to 12 h after ingestion of the drug by a healthy volunteer. No potential interference from the major metabolite (furosemide acylglucuronide) and its hydrolytic product (4-chloro-5-sulfamoylanthranilic acid) was observed. Commonly administered drugs also did not interfere. The proposed MLC procedure permits the rapid and reproducible measurement of low levels of furosemide in a small amount of urine.     

A comparative study of the separation of oleanolic acid and ursolic acid in Prunella vulgaris by high-performance liquid chromatography and cyclodextrin-modified micellar electrokinetic chromatography

A high-performance liquid chromatographic (HPLC) method and a cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method were developed to separate and determine oleanolic acid (OA) and ursolic acid (UA) in Prunella vulgaris. HPLC separations were carried out on a Hedera ODS C18 column with methanol -H₂O- acetic acid (85:15:0.3, v/v/v) as mobile phase at a flow-rate of 0.8 ml min^?1. CD-MEKC analysis was performed on a CL1030 capillary electrophoresis system with a 6% (v/v) methanol solution (pH = 9.0) containing 10 mM disodium tetraborate, 10 mM sodium dihydrogen phosphate, 50 mM sodium dodecylsulfate (SDS), 15 mM 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) as background electrolyte. The analytical results of HPLC and CD-MEKC were compared with each other. CD-MEKC has better analytical efficiency for two components, and the analytical time (15 min) was shorter than that of HPLC (35 min).     

Rapid determination of propyphenazone in plasma by high-performance liquid chromatography.

A rapid and simple high-performance liquid chromatographic assay for the determination of propyphenazone in plasma is described. Phenylbutazone was used as the internal standard. Plasma proteins were precipitated with acetonitrile before injection onto a 3-microns Supelcosil LC-18 column. The mobile phase, ethanol containing 0.2% (v/v) heptylamine-0.005 M potassium dihydrogenphosphate (30:70, v/v), was used at a flow-rate of 1.3 ml/min. The quantitation was performed by ultraviolet detection at a wavelength of 270 nm. The chromatographic time was 7 min. The within- and between-day coefficients of variation were less than 6% and the recoveries close to 100% for concentrations between 0.4 and 22 mumol/l. The limit of quantitation was 0.4 mumol/l (ca. 100 ng/ml).