High Performance Liquid Chromatogrphy of Fat-Soluble Vitamins. III. Determination of Vitamin D3 in Pharmaceutical Preparations and in Blood

Abstract

A reversed phase high-performance liquid chromatographic method (HPLC) is described for separation and determination of colecalciferol (Vitamin D₃) in Vitamin preparations and in biological materials. Vitamin D₃ is extracted from the formulations and from the blood in a fully automated electronically controlled extraction apparatus. For HPLC a column of lichrosorb RP18 and methanol as eluent are used. The extraction, separation and determination of vitamin D₃ needs about 10–20 minutes. The described extraction and HPLC methods allow the detection of 1–2 ng per injection and are well reproduced with a maximum coefficient of variation of < 3,5%. Vitamin A-acetate is used as internal standard.     

High Performance Liquid Chromatography of Fat-Soluble Vitamins. II. Determination of Vitamin E in Pharmaceutical Preparations and Blood

Abstract

α-tocopherol acetate (Vitamin E-acetate) is extracted, separated and determined from pharmaceutical preparations and from biological materials in nanogram range in less than 25 minutes. The extraction of Vit. E-acetate is performed in a fully automated, electronically controlled extraction apparatus. A reversed phase high-performance liquid chromatography (HPLC) using a column of lichrosorb RP18 and methanol as eleuent has been developed for the separation and quantitative determination of Vit. E-acetate in formulations. The described extraction and determination methods are suitable for the analysis of Vit. E-acetat alone and in the presence of other water - and Fat-soluble Vitamins in pharmaceutical preparations and are reproducible with coefficient of variation of ～3%. Vitamin A-acetate can be used as internal standard.     

A Sensitive High Performance Liquid Chromatographic Method for U-80, 278A,A Substituted Aminotetralin,in Rat Plasma,Whole Blood and Brain Tissue

Abstract

A sensitive analytical method for U-80, 278A, a substituted aminotetralin analogue in rat plasma, whole blood and brain tissue has been developed. The method involves solid phase extraction, efficient reversed phase HPLC and fluorescence detection, and can measure 1 ng/ml from 50 μl samples. During method development, many analogues were investigated and a wide range of extraction and HPLC conditions were explored. These experiments enabled rapid modification and revalidation of the method to support animal experiments with novel analogues.     

High-Performance Liquid Chromatographic Analysis of Theophylline in the Presence of Caffeine in Blood Serum and Pharmaceutical Formulations

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid theophylline in the presence of caffeine -internal standard- in blood serum and in pharmaceutical preparations. The separation was performed on Spherisorb-5 RP-18 5μm reversed phase column using methanol: 0.038 M ammonium acetate: acetonitrile (38: 57:5) at a pH of about 7.20. The eluted alkaloids are detected at 272 nm. The retention time is 3.09 min for theophylline and 3.85 min for caffeine. The correlation of the integrated peak area with the concentration of theophylline showed a linear relationship between 0.05 to 5.0 theophylline in blood serum, tablets, sprinkels, syrups, suppositories and injectable solutions.     

HPLC Determination of Oxalic Acid in Cocoa

Abstract

An HPLC method is described for the determination of oxalic acid in cocoa and milk chocolate. Samples are extracted using 6N HCI; after extraction the pH of an aliquot is adjusted to 6.0 and interfering substances are eliminated through the use of a C₁₈ Sep-pak®. The final HPLC determination uses a monolayer reversed phase column with an ion-pairing mobile phase and electrochemical detection. The results indicate excellent accuracy and precision.     

Validated LC Method, with a Chiral Mobile Phase, for Separation of the Isomers of Fexofenadine Hydrochloride

A rapid, simple and sensitive high-performance liquid chromatographic method (HPLC) has been developed to assay atomoxetine HCl in capsules. The HPLC analysis used a reversed phase C18 (150 × 4.6 mm i.d. 5 μm particle size) analytical column and a mobile phase consisting of monobasic potassium dihydrogen orthophosphate and acetonitrile (95:5 v/v), with UV detection at 269 nm. The validation data showed that the assay is sensitive, specific and reproducible for determination of atomoxetine HCl in this dosage form. Calibration curves were linear from 1 to 10 μg mL⁻¹ (R ² > 0.997). The accuracy of the method ranged from 98.13 to 101.5%. Mean inter- and intra-assay relative standard deviations (RSD) were less than 1.0%. The proposed method provided an accurate and precise analysis of atomoxetine HCl in its pharmaceutical dosage form.     

Simultaneous Determination of Diazepam and its metabolites in Plasma by High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic method (HPLC) for the simultaneous determination of diazepam and its three active metabolites, nordazepam, oxazepam and temazepam, in plasma was proposed. The compounds were isolated by solid-phase extraction. The chromatographic mobile phase was metanol-water (55:45, v/v) at a flow rate of 1 mL/min. UV detection was performed concurrently at 240 and 254 nm.     

Rapid Analysis for Codeine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid codeine in pharmaceutical preparations and in body fluids. The minimum detectable concentrations for body fluids were 5ppb and 7ppb respectively for urine and whole blood with an analysis time of under 5 min. A RP-8 Spheri-5 guard column and a RP-8 Lichrosorb-10 column were used and codeine was detected at its absorption maximum wavelength of 212 nm using an eluting system of methanol: 0.5% w/v aqueous ammonium acetate (70:30) at a pH of about 7.0.     

HPLC Determination of Cyclosporine in Whole Blood

Abstract

A rapid sensitive method for measuring cyclosporine concentration in whole blood by HPLC has been developed. The pre-chromatography isolation steps are convenient and rapid and are based on salting out acetonitrile with simultaneous extraction of cyclosporine from the blood. A reversed phase column is used with detection by absorbance at 200 nm.     

Differential Measurement of Cortisol and Cortisone in Human Saliva by HPLC with Uv Detection

Abstract

Both cortisol and its dehydro metabolite cortisone are present in normal human saliva. A method for differential Measurement of both compounds in 1 ml samples of saliva by HPLC/UV is described. the method uses an extraction column having a cyclodextrin bonded phase to retain the compounds of interest while allowing elution of interfering compounds. A steroid-bearing fraction is eluted from the cyclodextrin column, dried, reconstituted in a weak mobile phase, and injected on a reversed phase HPLC/UV system provided with an injector-mounted reversed phase extraction column. Samples containing corticosteroid concentrations as low as 0.5 ng/ml can be effectively analyzed by this method.     

Comparative Spectrophotometric,Spectrofluorometric, and High‐performance Liquid Chromatographic Study for the Quantitative Determination of the Binary Mixture Felodipine and Ramipril in Pharmaceutical Formulations

Abstract

Two‐component mixtures of felodipine (FLD) and ramipril (RMP) were assayed by derivative UV spectrophotometry, spectrofluorometry, and high performance liquid chromatography (HPLC). The spectrophotometric methods included a zero‐crossing first‐ and second‐order derivative procedure and a derivative compensation technique for the determination of binary mixtures with overlapping spectra. The spectrofluorometric method was based on first‐ and second‐order derivatives of the emission spectra (zero‐crossing point). Results from these methods were compared with those obtained by an exclusively developed isocratic reversed phase HPLC method. A reversed‐phase Adsorbosil DS analytical column, with methanol‐acetonitrile‐water (50∶30∶20, v/v) mobile phase at a flow rate of 1.5 ml/min, was used with a UV detector. The temperature was set at 25±0.2°C. Results obtained by the spectrophotometric and spectrofluorometric methods were comparable to those obtained by the HPLC method, as far as analysis of variance (ANOVA) test results were concerned. It is concluded that the developed methods are equally accurate, sensitive, and precise; with direct and simple application to pharmaceutical formulations of felodipine and ramipril combination, without interference from common pharmaceutical adjuvants.     

The Analysis of Sulphonamide Drug Residues in Pork Muscle using Automated Dialysis

ABSTRACT

The aim of this study was to assess the suitability of automated dialysis, using a commercial system, for the analysis of sulphonamides in porcine tissue. The system involves automated dialysis, followed by trace enrichment of the dialysate prior to HPLC determination. The procedure was applied to the analysis of nine sulphonamide drug residues using reversed phase HPLC as the method of determination. Muscle samples were blended in saline, centrifuged and the supernatant was filtered before dialysis for an optimised time of 11 min. The resulting dialysate was concentrated on a reversed phase trace enrichment cartridge prior to HPLC analysis with UV detection at 280 nm. The developed method was evaluated by carrying out intra- and inter-assays on fortified porcine muscle. Mean recoveries, evaluated from the inter-assay study, were 80% or higher for the nine sulphonamides studied and the limit of determination for the method was 40 ng g^?1.     

Rapid High Performance Liquid Chromatographic Determination of Ampicillin in Human Urine

Abstract

A rapid, sensitive and specific HPLC assay for the determination of ampicillin in human urine is developed.

Ampicillin was directly measured in human urine at 225 nm using a reversed phase column (Synchropack RP-P) and a mobile phase composed of (1:9 methanol-sodium acetate solution, 0.01 M, pH 4). The analysis required no longer than 10 min. Linear correlation between the peak height ratio of ampicillin to cefoxitin sodium (internal standard) and ampicillin concentration in urine over the range 10–100 μg ml^?1 was obtained. The developed method proved to be advantageous as it monitors ampicillin level in urine. Moreover, the urinary excretion of ampicillin in human subjects after an oral administration of 500 mg ampicillin capsules was established using the proposed method.     

Determination of Cinnarizine in Whole Blood and Plasma by Reversed Phase HFLC and its Application to a Pharmacokinetic Study

Abstract

Cinnarizine is determined in whole blood and plasma by reversed phase HPLC on a RP-18 stationary phase. The one-step extraction is performed with a chloroform/hexane (2/3) mixture. A high recovery of 91% and a detection limit of 2 ng/ml are obtained as well as a good precision. The internal standard is meclozine. Pharmacokinetic parameters found are in accordance with data cited in literature.     

Determination of Trimethoprim and Sulphamethoxazole in Serum by Reversed-Phase and Ion Pair HPLC

Abstract

HPLC serum assay methods for trimethoprim and sulpha-methoxazole are described. Octadecylsilane coated silica is used as the stationary phase in both analyses. The determination of trimethoprim involves extraction and chromatography employing reversed phase ion-pair HPLC with u.v. detection at 229nm. Using lml of serum, trimethoprim at levels of 10ng/ml can be measured. Sample preparation of sulphamethoxazole consists of protein precipitation only. Chromatography is by ion-supression and monitoring at 254nm. The sulphamethoxazole can be detected in serum at concentrations in the region of 0.1μg/ml. Results are presented to show the variation of trimethoprim and sulphamethoxazole in serum of eight healthy subjects over a 72 hour period following ingestion of 20ml co-trimoxazole suspensions from two manufacturers.     

The Use of HPLC/Thermospray Ms for the Confirmation of Aflatoxins in Peanuts

Abstract

An HPLC/Thermospray MS method is described for the determination of aflatoxins B₁, B₂, G₁ and G₂ in peanut extracts. Samples are extracted and prepared for analysis using an SPE method. The final determination utilizes reversed phase HPLC with a thermospray MS detector. The use of the MS allowed for unequivocal identification of aflatoxins in the extracts.     

Determination of Vitamin B12 in Multivitamin Tablets by High Performance Liquid Chromatography

Abstract

Two procedures for separation and determination of vitamin B₁₂ in multivitamin tablets by reversed phase high performance liquid chromatography are proposed. Sample preparation is very simple: tablets are dissolved in distilled water, centrifuged and filtered. The sample solution is directly applied in the sample loop injector and chromatograms are obtained with gradient elution using water-methanol and water-acetonitrile as solvents. The peak of vitamin B₁₂ from samples of B-complex tablets is well separated with the two procedures. For multivitamin tablets, however, only the procedure with water and methanol as solvents was good for separation and quantification of vitamin B₁₂. Both procedures were verified by the standard addition method and also compared to a previously developed method using electrothermal atomic absorption spectrometry for vitamin B₁₂ determination.     

Determination of Ketoconazole in Plasma and Dosage Forms by High-Performance Liquid Chromatography and a Microbiological Method

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of ketoconazole in plasma and in tablets was developed. the method employs benzafibrate as internal standard and is sufficiently rapid and sensitive for use in pharmacokinetic studies. Separation of the drug from plasma was achieved by extraction with acetonitrile followed by a reversed phase chromatography on a μ Bondapak column using the isocratic mobile phase of methanol-water-glacial acetic acid (67.5:32:0.5). With this eluting solvent ketoconazole and the internal standard. were well separated from the components of plasma. A linear relationship was obtained between the ratio of the area under the peak of drug to that of the internal standard versus the concentration of the drug. Data comparing the microbiological assay with the HPLC procedure, which was developed, are shown. In the microbiological assay, Candida albicans, was the test organism, using the agar diffusion technique. Both methods were applied to the assay of ketoconazole in plasma and in tablets. Excellent agreement was observed between the results from the two methods.     

Determination of butamyrate citrate in cough preparations by derivative UV spectrophotometry and high performance liquid chromatography.

Derivative spectrophotometric procedures and an isocratic high performance liquid chromatographic method for the determination of butamyrate citrate (Sinecod, Safarol) in cough syrups have been developed. In the spectrophotometric method, direct measurement of the drug at its absorption maxima is impossible because of interference from different absorbing excipients. Extraction of butamyrate citrate was performed with n-pentane/isopropyl alcohol. Quantification was carried out through the use of 1D derivative at a trough depth of 253.6 nm where interferences from other coextracted compounds are negligible. The extraction efficiency expressed as a % recovery and precision were assessed by fortifying placebo syrup(s) with known amounts of the compound. Also, a reversed phase high performance liquid chromatographic method was used with a mobile phase containing 0.015 M aqueous tetraethylammonium hydrogen sulfate, methanol and acetonitrile 40:30:30 adjusted to pH 3.50 with ammonium hydroxide. The retention behavior of butamyrate citrate as a function of both pH and salt concentration in the aqueous portion of the mobile phase was investigated. Quantification was achieved with UV detection at 258 nm based on peak area. The HPLC method clearly separates the analyte from its degradation products derived after storage of samples under different stress conditions such as acid, alkaline, temperature, oxygen and light. The described methods were successfully applied to the determination of butamyrate citrate in commercial pharmaceutical products and in placebo syrups prepared in the laboratory with good accuracy and precision. The results of the present study show that the use of the derivatives and the HPLC procedure provide precise and sensitive methods for the determination of the compound in pharmaceutical formulations.     

Analysis of Bunaprolast,An Esterase Unstable Drug,with An Active Metabolite Subject to Oxidative Degradation,in Blood Plasma

Abstract

This report describes a sensitive, selective and robust assay for bunaprolast and an active metabolite in canine and human plasma. Method development was complicated in that bunaprolast is quickly hydrolysed by esterases even in vitro, and the metabolite is rapidly oxidised in dilute aqueous solutions. Effective measures to stabilize analytes in biological matrices and during sample extraction are described.

The method involves the selective solid phase extraction of analytes with a close analogue as internal standard followed by reversed phase HPLC and fluorescence detection. The limit of quantification was typically less than 1 ng/ml, and the assay was linear over the range 1 - 1,000 ng/ml.