Variable UV Detector Research Articles

High-performance liquid chromatography for determination of α-tocopherol in vegetables

A simple method for the determination of α-tocopherol in vegetables is described. The procedure consists of the following steps: saponification, extraction, silica-column clean-up, and high-performance liquid chromatography. Elution time for D, L-α-tocopherol was 9.0 min using a Zorbax Sil (250 x 4.6 mm) column and an isocratic mobile phase of hexane-methanol (99.3 + 0.7), with a flow rate of 1 ml/min, and detection at 292 nm using a variable UV detector. The average recovery of α-tocopherol was 91.2%, and the minimum detectable amount was 0.1 mg/100 g of fresh vegetable tissue. This method is comparable to gas-chromatographic determination of α-tocopherol, but has fewer analytical steps and gives more reproducible results.

Quantitative determination of diazepam, nitrazepam and flunitrazepam in tablets using thin-layer chromatography–densitometry technique

A simple and reliable assay for diazepam, nitrazepam and flunitrazepam in tablets by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)–densitometry is described. A quantity of a ground tablet mass, equal to the average weight of one tablet was sonicated in MeOH, mixed with appropriate internal standard, filtered and either injected directly into the liquid chromatograph, or after evaporation and reconstitution of an aliquot of the extract, was spotted on a silica gel thin-layer plate. A variable UV detector, operated at 254 nm was employed in both procedures. A C18, reversed phase 7 μm column was used for HPLC analysis; the mobile phase was a 1:1 (v/v) mixture of MeOH (40 °C) and 0.01 M phosphate buffer (pH 7, 80 °C). The TLC plate was developed in an unsaturated chromatographic chamber containing 100 ml chloroform–acetone (9:1); at room temperature, the mobile phase was allowed to travel 15 cm. The percentage of the active ingredient content of each tablet obtained by both procedures, was in the range of the stated amount except for one brand of diazepam tablets which contained ∼23% less active ingredient than the minimum prescribed amount. The TLC densitometry, although yields slightly higher values than the HPLC method, is preferred due to its simplicity, ease and low cost.

Determination of the urinary metabolites of styrene: estimation of the method evaluation function and evaluation of reference values in Danish subjects.

A European study on styrene exposure was initiated in 1989 to evaluate the health effects of environmental and occupational exposure. A part of this study included the development of an analytical method for use in a biological monitoring program. The urinary metabolites of styrene, mandelic acid (MA) and phenylglyoxylic acid (PGA) were quantitated by a direct and convenient high-performance liquid chromatography method. Urine samples were diluted with eluent and analysed by HPLC with a C8 reversed-phase column and a buffer to acetonitrile (9:1) eluent with a counterion added. The detector used was a variable UV detector and the wavelength was lambda = 210 nm. The method was statistically evaluated by a method evaluation demonstrating no systematic error. The uncertainty was 23.8 mumol/l and 11.5 mumol/l for MA and PGA, respectively. The limit of detection (LOD) of MA is 71.4 mumol/l and the LOD of PGA is 34.5 mumol/l, sufficiently low for the measurement of styrene exposure at a low exposure level. The present study indicates that reference values for MA and PGA are low. The fraction of reference values below LOD was 0.80 for MA and 0.66 for PGA; consequently, the reference values were described by a non-parametric one-sided tolerance interval. The 95% one-sided upper tolerance limits calculated for MA and PGA were 31.0 mumol/mmol creatinine and 20.1 mumol/mmol creatinine, respectively, with the coverage 0.95 +/- 0.045 for both metabolites. The method has been used for biological monitoring in several studies of environmentally and occupationally exposed subjects in concentrations up to 200 mumol/mmol creatinine for MA and 150 mumol/mmol creatinine for PGA.

Determination of urinary tryptophan and its metabolites along the nicotinic acid pathway by high performance liquid chromatography with ultraviolet detection

A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites on the nicotinic acid pathway. Kynurenine, tryptophan, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid and xanthurenic acid were isocratically eluted and completely resolved with a mobile phase of acetonitrile + sodium acetate buffer, pH 4.76 (4:96, v/v). The flow rate was 0.8 mL/min at the beginning and was then linearly increased to 1.2 after 6 min; after 14 min the flow was augmented from 1.2 to 2 mL/min. The effluent was monitored with a variable UV detector set at 254 nm for the first five peaks and at 280 and 325 nm for the penultimate peak and final peak. Analytical recoveries of the compounds after deproteinization varied between 64% and 98%. The reported method should enable one to examine easily, extensively, quantitatively and routinely urinary tryptophan and the most important metabolites of the nicotinic acid pathway.

A simple reverse-phase high pressure liquid chromatographic determination of conjugated bile acids in serum and bile using a novel radial compression separation system

A new HPLC method for analyzing conjugated bile acids in bile and especially in serum is presented. For separation a novel radial compression system with a 10-cm flexible-walled reverse phase column (RCM-100 Module, Waters Ass.) packed with μ-Bondapak C 18 was used. Without any laborious hydrolysis and derivatization steps, 10 conjugated bile acids are separated easily with a mobile phase of methanol/0.01 mol/l KH 2PO 4 (150:50, v/v; pH 6.0) in about 30 min, determined with a variable UV detector at 200 nm and a flow rate of only 0.5 ml/min. Dexamethasone was used as internal standard. Conjugated bile acids are extracted from serum with Sep-pak C 18 cartridges after a 1:8 dilution with a mixture of the mobile phase and 0.2 mol/l NaOH (6:8, v/v). Extraction from bile was also performed using Sep-pak cartridges after a 1:20 dilution with 0.5 mol/l phosphate buffer (pH 7.0). The complete analysis time of the method, with Sep-pak extraction, is less than one hour. Recoveries for the different conjugated bile acids varied from 89.4% to 96.7% in serum. The HPLC method for serum and bile was validated for the primary bile acids cholate and chenodeoxycholate against the previously described GLC method. Agreement between the two methods was excellent. Duplicate analyses in serum and bile samples also showed that the reproducibility was good.

High-performance liquid-chromatographic analysis of trimethoprim and sulphamethoxazole in plasma

4 combination preparations of Trimethoprim (TMP) and Sulphamethoxazole (SMZ) were administered to 8 healthy male subjects and plasma samples were analysed after extraction of TMP and SMZ by high-performance liquid chromatography with a variable UV-detector at 230 and 280 nm. The Chromatographic system consisted of a Waters reversed-phase C18 column and a mobile phase containing phosphate buffer, pH 5.0, and ethanol (80 + 20). A dose of 80 mg TMP × 400 mg SMZ results in a plasma concentration of 0.05–1.0 μg/ml TMP and 1–20 μg/ml SMZ during 26 h after drug administration. The relative standard deviation for TMP was found to be ±5–7% and for SMZ ±2–3%.

High Speed Liquid Chromatographic Separations of Phthalic Esters, Carbohydrates, TCA Organic Acids and Organic Mercury Compounds

The high speed liquid chromatographic separations of phthalic esters, carbohydrates, TCA cycle organic acids and organic mercury compounds are demonstrated by using several detectors (RI detector, FID, variable UV detector and atomic absorption spectrophotometer) and new solvent systems.