Proposed High-performance Liquid Chromatography Method Research Articles

High‐performance Liquid Chromatographic Analysis of Docusate Sodium in Soft Gelatin Capsules

A rapid and simple high‐performance liquid Chromatographic (HPLC) method for the analysis of docusate sodium in soft gelatin capsules was developed with progesterone as the internal standard. The method requires a reversed‐phase column and a pairedion technique to separate docusate sodium from other components. A C22 column was used with a mobile phase of acetonitrile: water (70:30) containing 0.005M tetrabutylammonium phosphate. The flow rate was 1.8 mL/min, and the effluent was monitored at 214nm. Docusate sodium and progesterone had retention times of 4.5 and 6.8 min, respectively. The proposed HPLC method is linear, accurate, and precise. A mean assay value of 99.6% was obtained by the proposed method when five samples, each containing a composite of 10 capsules, were analyzed. The results obtained by the proposed HPLC, tetra‐n‐butylammonium iodide titration, and DSP XXII HPLC methods are compared.

Re-analysis of vitamin A values of selected Malaysian foods of animal origin by the AOAC and HPLC methods

Vitamin A values of 40 foods of animal origin from various food groups and several processed foods were studied using a newly developed, reverse-phase, high-performance liquid chromatography (HPLC) method. Carotenoids and retinol were separated isocratically on an octadecylsilane (C 18) column using a ternary mixture of acetonitrile, methanol and ethyl acetate. Two detectors connected in series were used to detect and quantify carotenoids simultaneously at 436 nm and retinol at 313 nm in a single chromatographic run. All samples were also simultaneously determined using the Association of Official Analytical Chemists (AOAC) open-column (alumina) chromatographic method. The AOAC method was found to give significantly higher retinol contents in the foods studied, due to the presence of other pigments that gave falsely elevated absorbance readings. Although there was no statistically significant difference in β-carotene contents given by the HPLC and AOAC methods, there were more foods with higher results given by the latter method. β-Carotene contents were generally low; only in seven foods did the carotene contribute more than 50% of the total vitamin A value. The contribution of other provitamin A carotenoids is probably insignificant. Thus, the total vitamin A activity of these foods was mainly contributed by retinol. The proposed HPLC method has been shown to be applicable to the determination of carotenoids in vegetables and fruits, as well as to the determination of carotenoids and retinol in foods of animal origin.

Liquid chromatographic method for the determination of calcium cyanamide using pre-column derivatization

A specific and stability-indicating high-performance liquid chromatographic (HPLC) method has been developed for the analysis of calcium cyanamide in bulk material and dosage form. Calcium cyanamide in samples was converted into dansyl cyanamide. A μBondapak C 18 column was employed for HPLC with 0.01 M sodium phosphate (pH 6.3)-acetonitrile (75:25, v/v) as the mobile phase. The proposed HPLC method was validated for linearity, specificity, accuracy and reproducibility.

Determination of malonaldehyde in oxidized biological materials by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was used to determine the level of malonaldehyde (MA) in materials containing unsaturated fatty acids and rat liver microsomes peroxidized in vitro. The detection limit was 8.3 pmol for fatty acid samples and 25 pmol for microsomal samples. The method was specific to MA and the relative standard deviation was 4.34–5.14%. The recovery of MA was about 100%. In general, the MA values in oxidized materials obtained by the proposed HPLC method were lower than those obtained by the thiobarbituric acid method, although similar results were obtained with both methods for microsomal samples oxidized by NADPH. The effect of temperature on the HPLC results was investigated and it was found that the MA values obtained by derivatization at 25°C, followed by separation using HPLC, reflected the situation of the peroxidation more accurately.

High-performance liquid chromatography of water-soluble vitamins. Part 3. Simultaneous determination of vitamins B1, B2, B6, B12 and C, nicotinamide and folic acid in capsule preparations by ion-pair reversed-phase high-performance liquid chromatography.

An ion-pair reversed-phase high-performance liquid chromatographic (HPLC) method for the separation and simultaneous determination of thiamine HCl (vitamin B1), riboflavin (vitamin B2), nicotinamide, pyridoxine HCl (vitamin B6), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C) and folic acid in capsule preparations is described. The above seven active substances were extracted from the preparations within 5 min using an electronically controlled extraction apparatus. Ion-pair reversed-phase HPLC was carried out on LiChrosorb RP-18 using methanol-water-concentrated phosphoric acid-octanesulphonic acid as the eluent. All seven active ingredients were separated in less than 4 min. The detection limits were 1–2 ng for all vitamins; however, amounts of 5–10 ng were required for the quantitative HPLC determination of the vitamins. The proposed HPLC method is suitable for the determination of these active ingredients in pharmaceutical preparations such as multivitamin capsules and has a coefficient of variation between 1.1 and 3.2%. The ease of extraction, accuracy and sensitivity of the method and the time required for analysis are emphasised.

Determination of B2 vitamers in the body fluid of plankton using high-performance liquid chromatography with fluorescence detection

A sensitive method, using high-performance liquid chromatography (HPLC) with fluorescence detection, has been developed for the separation and determination of B2 vitamers in the body fluid of plankton. B2 vitamers are liberated from the protein moiety in the body fluid by non-hydrolytic extraction with trichloroacetic acid and analysed by the proposed HPLC method. The major B2 vitamer in the body fluid of plankton is riboflavin, although there is also evidence to suggest the presence of endogenous enzymes that can decompose flavin mononucleotide and flavin adenine dinucleotide.

High-performance liquid chromatographic analysis of ammonium in human urine

A method for the quantitative determination of ammonium in human urine by high-performance liquid chromatography (HPLC) is described. After making fluorescent substances with fluorescamine, they were separated and quantified by their fluorometric intensity. The intensity (as measured by peak height) was linear between 0.5 and 5.0 μg, and coefficients of variation for elution time and peak height on replicate analysis of the standard were 0.15 and 4.2%, respectively. Recoveries of added ammonium were 96.5 and 97.3%, respectively, on 2.0 and 3.0 μg by this method. Detection limit of this method was 0.2 μg. There was good agreement between the proposed HPLC method ( X) and ion chromatographic method ( Y), giving the relationships as Y = 0.956 X + 0.012, r = 0.991.

Preparation and high-performance liquid chromatographic determination of guaiacol estrogen 17 beta-conjugates: the enzymatic O-methylation products of 2-hydroxyestradiol 17 beta-conjugates (clinical analysis on steroids. XXII).

For the direct assay of the enzymatic O-methylation products of 2-hydroxyestradiol 17β-sulfate (2) and 17β-glucuronide (3), the corresponding guaiacol estrogens have been prepared as authentic specimens and their high-performance liquid chromatography (HPLC) was investigated. The materials synthesized were : potassium 3-hydroxy-2-methoxyestra-1, 3, 5 (10)-trien-17β-yl sulfate (7), potassium 2-hydroxy-3-methoxyestra-1, 3, 5 (10)-trien-17β-yl sulfate (13), potassium [3-hydroxy-2-methoxyestra-1, 3, 5 (10)-trien-17β-yl-β-D-glucopyranosid] uronate (9), and potassium [2-hydroxy-3-methoxyestra-1, 3, 5 (10)-trien-17β-yl-β-D-glucopyranosid]-uronate (15). These sulfates and glucuronides were separated quantitatively by reversed-phase HPLC. The separation was performed with a mixture of acetate buffer (50 mM, pH 5.0) and methanol (50 : 50) as the mobile phase on a column of ODS SIL. The eluates were monitored in terms of the absorbance at 280 nm. Calibration curves between the amounts of conjugated guaiacols and the peak heights on chromatograms were all linear. The results obtained by proposed HPLC method for the quantification of O-methylated products obtained by the incubation of 2 and/or 3 with purified rat liver catechol O-methyltransferase in the presence of (3H3C)-S-adenosyl-L-methionine were in good agreement with the results obtained by a different procedure, the reverse isotope dilution method.

Clinical Evaluation of Acetylcysteine as a Mucolytic Agent in Cystic Fibrosis

A sensitive and rapid high-performance liquid chromatography (HPLC) method was developed to enantioseparation of N-acetyl-dl-cysteine after precolumn derivatization using o-phthaldialdehyde and primary aliphatic amines. Seven polysaccharide-based chiral columns were tested in a reversed phase mode. Under the optimal chromatographic conditions, N-acetyl-dl-cysteine derivatives were completely enantioseparated on Chiralcel OZ-3R column with the resolution more than 2.5. The impact of various primary aliphatic amine additives as co-reagents (ethyl-, 1-propyl-, 1-butyl-, 1-pentylamine, (R)-sec-butylamine, tert-butylamine, isobutylamine, cyclopropyl-, cyclobutyl-, cyclopentyl and cyclohexylamine) used in precolumn derivatization step on the retention behavior (retention factor, selectivity and column efficiency) of N-acetyl-dl-cysteine derivatives was investigated. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH, salt concentration in the mobile phase and column temperature on the retention and selectivity was investigated. The developed method was properly validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy, precision, intermediate precision and selectivity according to International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. Proposed HPLC method was successfully applied to the determination of optical purity in commercially available N-acetyl-L-cysteine samples.