Rapid and simple determination of clenbuterol residues in retina by high-performance liquid chromatography with ultraviolet detection

A new method for the determination of clenbuterol by reversed-phase HPLC with UV detection has been developed. Clenbuterol was eluted on a C₈ column(250 × 4.6mm I.D.), using an isocratic eluent consisting of anacetonitrile-0.02 M phosphate buffer (25:75, v/v) adjusted to pH 2.8 with phosphoric acid. The method was linear from 2.5 to 50 ng injected. The detection limit was established to be 0.5 ng (signal/background ratio: 3), and the quantification limit was 2.5 ng. With the proposed method, we got a simple and rapid detection of clenbuterol in the retina, part of the animal where the biggest amount of clenbuterol is accumulated and where it remains for the longest time after any treatment.     

Rapid and simple determination of histamine-N-methyl transferase activity by high-performance liquid chromatography with UV detection.

A rapid, simple and low-cost assay method of histamine-N-methyltransferase activity was developed. Methylhistamine, which was separated from the enzymatic reaction system on reversed-phase high-performance liquid chromatography using an ion-paired chromatographic technique, was detected spectrophotometrically at 226 nm. The mobile phase used for the separation of methylhistamine was 0.05M NH4H2PO4 (pH 3.0) containing 2 mM of sodium octanesulfonate. The new assay technique could detect methylhistamine as an enzyme activity product of histamine-N-methyltransferase in the brain and kidney of rats. Chloropheniramine maleate, an antihistamine, activated the histamine-N-methyltransferase. Whether neurotransmitter or neuromodulator, the role of histamine in the brain has not yet been made clear. Therefore, the present method could be applicable for the enzymatic investigation of histamine metabolism in central nervous system or inflammatory reactions.     

Rapid determination of valaciclovir and acyclovir in human biological fluids by high-performance liquid chromatography using isocratic elution

A rapid high-performance liquid chromatographic assay with isocratic elution is developed for the simultaneous quantification of valaciclovir (VACV) prodrug and its active converted compound, acyclovir (ACV), in biological fluids of treated patients. For serum, the samples are deproteinized with perchloric acid in presence of 1-methylguanosine as the internal standard (IS). For urine and dialysis liquid, the samples are diluted with a mobile phase containing the IS, then filtered. VACV, ACV and the IS are separated on a SymmetryShield™ RP-8 column with acetonitrile–ammonium phosphate buffer as the mobile phase and detected at 254 nm. The chromatographic time is about 12 min. The relative standard deviations (RSD) of VACV and ACV standards are between 0.5 and 3.5%. Most endogenous nucleosides and their metabolites, psychotropic drugs and drugs of abuse are shown not to interfere with this technique. The method has been applied to study the pharmacokinetics of VACV and ACV in serum, dialysis liquid and urine of renal failure patients on continuous ambulatory peritoneal dialysis (CAPD) under oral treatment of VACV.     

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C₈ column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C₁₈ column (50×4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wavelength UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration—time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 μg/ml of succinylcholine. In a pilot study the plasma concentration—time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Determination of trimethoprim in biological fluids by high-performance liquid chromatography

A rapid, sensitive, and specific high-performance liquid chromatographic assay was developed for the determination of trimethoprim in blood, plasma, and urine using normal-phase (adsorption) chromatography on a microparticulate silica column and UV monitoring at 280 nm. Trimethoprim is selectively extracted from the biological sample matrix at alkaline pH with chloroform, providing nearly quantitative extraction (>95%) and a sensitivity limit of 0.01 to 0.02 μg/ml blood or plasma, without interference from sulfonamides.     

Simultaneous determination of moricizine and its sulphoxidation metabolites in biological fluids by high-performance liquid chromatography

A simultaneous assay for moricizine, its two sulphoxidation metabolites, morizine sulphoxide and moricizine sulphone, using high-performance liquid chromatography (HPLC) is described. The drug and metabolites and clozapine (internal standard) in biological fluids were extracted using pentanesulphonic acid into diethyl ether. The ethereal extract was evaporated to dryness and the residue was redissolved in the mobile phase (methanol-water-triethylamine, 65:35:0.5, v/v). The analyses were performed on a μBondapak reversed-phase C₁₈ column housed in a Waters Z-module, linked to a C₁₈ pre-column, with a run-time of 12 min. The retention times were 2.7, 3.5, 6.2 and 9.7 min for moricizine sulphone, moricizine sulphoxide, moricizine and clozapine, respectively. The recovery of the compounds from plasma ranged from 89.9% for the sulphoxide to 98.1% for clozapine. The limits of detection of the assay for moricizine, moricizine sulphoxide and moricizine sulphone were 20, 10 and 5 ng/ml, respectively.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Rapid and simple method for the determination of zolpidem in human plasma by high-performance liquid chromatography

A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.     

Measurement of catecholamines in biological fluids by high-performance liquid chromatography : A comparison of fluorimetric with electrochemical detection

An improved method for the determination of catecholamines in biological fluids, by reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection is presented. The pH titration previously employed in the alumina extraction was abandoned in favour of the use of a molar excess of pH 8.5 Tris—HCl buffer. A novel lyophilisation step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimised. The addition of 2 mM octanesulphonic acid to a citrate—phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity.That HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, has been demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the two techniques as applied to the measurement of urinary catecholamines. The HPLC—fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline or adrenaline at a signal-to-noise ratio of 2.0. Application of the method to the quantitative determination of catecholamines in human urine, plasma and rat brain homogenates is demonstrated.