Rapid high-performance liquid chromatographic assay for the simultaneous analysis of non-steroidal anti-inflammatory drugs in plasma

A high-performance liquid chromatographic assay has been developed for the determination of a number of non-steroidal anti-inflammatory drugs in plasma. The samples were prepared by adding acetonitrile and perchloric acid to 200 microliter of plasma. Diclofenac, fenoprofen, ketoprofen, naproxen, phenylbutazone, piroxicam and sulindac were quantified in the supernatant produced using a mobile phase of phosphoric acid 0.03% (pH 2.5)-acetonitrile and a detecting wavelength of 254 nm. The reproducibility, linearity, precision and specificity of the assay were determined and found to be satisfactory. Alteration of the detection wavelength to 229 nm also permitted accurate determination of ibuprofen concentration in plasma. While reduction of the organic solvent content of the mobile phase and alteration of wavelength to 313 nm produced a system capable of quantifying salicylate and its metabolites in plasma and by further reducing the detecting wavelength to 237 nm, aspirin also was quantifiable. These methods have been applied in a cross-sectional study of medication compliance among rheumatoid arthritis patients treated with non-steroidal anti-inflammatory drugs.     

Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum

A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiolspecific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory.     

Stereoselective high-performance liquid chromatographic assay for pirmenol enantiomers in dog plasma.

Pirmenol enantiomers in dog plasma were quantified using a stereospecific high-performance liquid chromatographic method with ultraviolet detection at 262 nm. Racemic pirmenol and internal standard, (+)-propranolol, were isolated from dog plasma by a three-step extraction procedure using toluene, 0.1 M hydrochloric acid and hexane, respectively. A chiral analytical column (Chiralcel OJ) was used with a mobile phase consisting of hexane-isopropanol-diethylamine (98.9:1.0:0.1). Linear calibration curves were obtained in the concentration range 0.0200-5.00 micrograms/ml for each enantiomer. Precision of the method, expressed as coefficient of variation for nine quality control samples, was 7.1% for (+)-pirmenol and 6.4% for (-)-pirmenol. Bias was +/- 2.2% for (+)-pirmenol and +/- 1.5% for (-)-pirmenol in quality control samples.     

Rapid high-performance liquid chromatographic assay of dorzolamide in rabbit aqueous humor

A rapid high-performance liquid chromatographic method using a C(18) reversed-phase column (Hypersil ODS) with UV detection at 254 nm and a simple pre-treatment of samples is presented for the analysis of dorzolamide, a carbonic anhydrase inhibitor, in rabbit aqueous humor. A water solution containing 2% ZnSO(4) small middle dot7H(2)O was used to deproteinize aqueous humor samples. The mobile phase consisted of 7% CH(3)CN and 93% of a solution containing 1% TEA adjusted to pH = 3.5 with H(3)PO(4). Paracetamol was found to be a suitable internal standard. The standard curves were linear in the detection range. The precision and the accuracy were <5% for both intra- and inter-day assays.     

Stereospecific high-performance liquid chromatographic assay of acebutolol in human plasma and urine

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.     

Automated high-performance liquid chromatographic assay for the determination of 7-ethoxycoumarin and umbelliferone.

An improved high-performance liquid chromatographic assay is presented for the determination of 7-ethoxycoumarin O-deethylase activity. Following a 30-min microsomal incubation, 7-ethoxycoumarin, 4-methylumbelliferone (internal standard), and the metabolite umbelliferone were extracted with chloroform. Separation was achieved with an isocratic mobile phase using a microBondapak phenyl (300 mm x 3.9 mm I.D.) analytical column. The effluent was monitored by fluorescence detection with an excitation wavelength of 360 nm and an emission wavelength of 470 nm. The intra- and inter-assay coefficients of variation were 10 and 6%, respectively. A detection limit of 0.07 micrograms/ml was achieved, making this method suitable for characterizing P-450 activity of human livers.     

A validated high-performance liquid chromatographic assay for determination of lumiracoxib in human plasma

Lumiracoxib {2-[(2-fluoro-6-chlorophenyl)amino]-5-methyl-benzeneacetic acid} is a highly selective and potent cyclooxygenase-2 (COX-2) inhibitor, which is chemically distinct from other coxibs in that it contains a carboxylic group and is weakly acidic. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of lumiracoxib in human plasma. The analyte was separated on a reversed-phase column with acetonitrile and 0.05% trichloracetic acid in water (35:65, v/v) as mobile phase at a flow rate of 1 mL/min, and UV detection at 270 nm. The retention times for lumiracoxib and niflumic acid (internal standard) were 16.9 and 10.4 min, respectively. The validated quantitation range for lumiracoxib was 10-10,000 ng/mL. The developed procedure was applied to assess the pharmacokinetics of lumiracoxib following administration of a single oral 200 mg dose to a healthy male volunteer.     

Selective and sensitive high-performance liquid chromatographic assay for the metabolites of nomifensine in human plasma

A selective high-performance liquid chromatographic method for the determination of the three metabolites of nomifensine in human plasma is described. All metabolites and the internal standard, mexiletine, are extracted with diethyl ether and then back-extracted into an acidic aqueous phase. After subsequent extraction into diethyl ether the metabolites are analysed by high-performance liquid chromatography. A reversed-phase C18 column is used with a mobile phase of dioxane-methanol-potassium phosphate buffer (pH 2.25). The sensitivity of the method is 0.007 micromol/l for all metabolites. Extraction efficiencies are 84.6%, 75.8%, and 78.2% for 4'-hydroxynomifensine, 4'-hydroxy-3'-methoxynomifensine and 3'-hydroxy-4'-methoxynomifensine, respectively. The reproducibility of the method is good, the coefficients of variation (%) varying between 2.1% and 9.9% in the concentration range 0.05-1.00 micromol/l. The procedure was applied to human plasma samples from a volunteer who had received a single oral dose of nomifensine. The method is accurate and sensitive for pharmacokinetic studies on the metabolites of nomifensine.     

Sensitive high-performance liquid chromatographic assay using 9-fluorenylmethylchloroformate for monitoring controlled-release lidocaine in plasma

A sensitive high-performance liquid chromatographic (HPLC) assay using fluorescence detection for quantifying lidocaine levels in plasma (in the ng/ml range) was developed. This novel HPLC assay has made possible the simultaneous monitoring of lidocaine levels in coronary and peripheral plasma obtained after myocardial controlled-release matrix administration (0.92 mg/kg during 4 h) in the arrhythmic dog. The method employed extracts the drug from plasma using 1-chlorobutane and a subsequent derivatization with 9-fluorenylmethylchloroformate in acetonitrile at 110 degrees C. The derivative was chromatographed on a C18 reversed-phase column and measured with fluorescence detection (excitation 254 nm, emission 313 nm). N-Methylephedrine was found to be suitable as an internal standard, post-derivatization. The derivatization product of lidocaine was identified and characterized by mass spectral analysis. It was found to have a unique and reproducible dicarbamate structure, which was stable for at least three days at room temperature. The method was tested with human plasma as well as on dog plasma. Analytical recoveries were 88.6 +/- 3.6 and 77.4 +/- 3.0% (mean +/- S.E.), respectively, at levels ranging from 25 to 200 ng/ml. The lower detection limit was 1 ng/ml lidocaine. In conclusion, this rapid and convenient analysis was found to be suitable for the bioavailability pharmacokinetic assessment of lidocaine following low-dose regional drug administration.