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.     

Chemische Vitamin D3-Bestimmung in Lebertran

The different aspects of Vitamin D₃ determination and the data reported in the literature are discussed. The spectrophotometric determination of Vitamin D₃ in cod-liver oil is carried out after the alkaline saponification, extraction of unsaponified parts, precipitation of accompanying sterols and the column- and thin-layer chromatographic purification and separation of vitamin D₃ on 40 cm plates from other vitamins. The dyestuff α-naphtholbenzein is suited well as standard substance for the better location and identification of vitamin D₃ zone on the thin-layer plate. The results obtained from the chemical method were checked through the simultaneous biological determination.     

High-Performance Liquid Chromatography of Fat-Soluble Vitamins. I. Determination of Vitamin A-Acetate in Pharmaceutical Preparations and Blood

Abstract

A reversed phase high-performance liquid chromatography (HPLC) has been developed for the quanititive determination of retinol acetate (vitamin A-acetate) in pharmaceutical formulations and biological materials. The extraction of vitamin A-acetate from capsules and dragees and from blood is performed in a fully automated, electronically controlled extraction apparatus within 3–10 minutes. For reversed phase HPLC a column of LiChrosorb RP18 and methanol as eluent were used. Vitamin A-acetate was separated in less than 1 minute. The detection limit was 1-2 ng. The described methods were proved useful for extraction and determination of vitamin A-acetate in pharmaceutical preparations such as capsules and dragees and in blood, and can be well reproduced with a maximum coefficient of variation of <4%.     

Chemische Vitamin D<Subscript>3</Subscript>-Bestimmung in Lebertran

The different aspects of Vitamin D₃ determination and the data reported in the literature are discussed. The spectrophotometric determination of Vitamin D₃ in cod-liver oil is carried out after the alkaline saponification, extraction of unsaponified parts, precipitation of accompanying sterols and the column- and thin-layer chromatographic purification and separation of vitamin D₃ on 40 cm plates from other vitamins. The dyestuff α-naphtholbenzein is suited well as standard substance for the better location and identification of vitamin D₃ zone on the thin-layer plate. The results obtained from the chemical method were checked through the simultaneous biological determination.     

Determination of Vitamins D2 and D3 in Feedingstuffs by High Performance Liquid Chromatography

Abstract

A sensitive and reliable HPLC method for complete separation and quantitation of vitamins D₂ and D₃ in complicated biological mixtures such as feedingstuffs has been developed and described. The method has been applied to the quantitative determination of D₂ and D₃ in feedingstuffs and related matrices at both high premix levels and low feed levels ranging down to a detection limit near 100 IU/1b or 0.22 IU/g sample. The procedures consist of the initial step of sample preparation by extraction; four sample cleanup stages: Sep/Pak/Silica Cartridge Cleanup, Millipore-Teflon Cleanup, Gel Permeation/Sephadex LH-20 Column Cleanup, and HPLC/Partisil-PAC Column Cleanup; and the final step of Reverse-Phase HPLC Separation, Identification, and Quantitation. The analytical Column used was Rainin Accupak 20 cm-3 um C-18 & Guard Columns. The Waters Associates Model 440 Fixed Wavelength UV Detector at 254 nm was used for all measurements. All separations and quantitations were carried out isocratically at room temperature and under subdued lighting. By these procedures, the sensitivity for both D₂ and D₃ is about the same (20 ng), and the resolution is excellent. Normally, the D₂ peak eluted at 30–31 min. and the D₃ peak followed at 2–3 min. later. By using the standard calibration and standard addition methods, the percent recovery ranges from 90.0%–104.8% with the mean value of 97.4%, whereas the accuracy is from 85.3% to 108.9% with the average of 97.8%. The standard deviation is ±5.2% and the coefficient of variation is 5.3%.     

The Spectrophotometric Determination of Vitamin a Using Iodine

Abstract

Methods are described for the spectrophotometric determination of vitamin A (0.15–12.5 μg) using iodine as the chromogenic reagent. The reaction is most sensitive in chloroform. Vitamin D₂ and ß-carotene do not interfere if the analysis is carried out in 1, 2 -dichloroethane.     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

Potential of High-Performance Liquid Chromatography for Distribution Coefficient Determination of 3-Hydroxyquinolin-4(1H)-one Derivatives

The potential of reversed-phase HPLC for the determination of distribution coefficient D _7.4 of selected 3-hydroxyquinolin-4(1H)-ones (3HQs) as compounds with significant biological activity was studied. Various stationary phases with C18 as well as hexyl-phenyl modification reflect current trends in RP-HPLC development such as higher sorbent silanophilicity, core–shell technology, hybrid and/or charged surface particles. Because of significant peak tailing of 3HQs at physiological pH on reversed-phase sorbents the separations at pH 3 were performed as well. Surprisingly, the pH change did not affect significantly the partition coefficients of 3HQs. Very affordable and common standards such as anisole, acetophenone, benzyl alcohol, brombenzene, ethylbenzoate and trichlorethylene were applied in the described methodology. The best linearity (R ² 0.9895) of the correlation between log P and log k _w for standards was obtained for hexyl-phenyl sorbent, but this stationary phase was shown to be unsuitable for HPLC separation of 3HQs. The highest linearity (R ² 0.9499) of the relationship between log D _7.4 determined by the classic shake-flask method and log D determined by means of HPLC for 3HQs was attained with Cortecs C18+ column at pH 7.4. The described methodology with Cortecs C18+ as stationary phase offers fast and accurate estimation of log D _7.4 of the tested 3HQs. In an effort to increase the throughput of the HPLC method for log D _7.4 determination, we evaluated almost aqueous mobile phase that contained only 3 % of acetonitrile. Although a worse correlation between log D _7.4 determined by shake-flask method and HPLC with almost aqueous mobile phase was observed, the described procedure offers a very simple and high-throughput alternative for the estimation of log D _7.4.

     

A Study On Air Pollution by Asphalt Fumes

Abstract

A TLC/HPLC procedure for the determination of polycyclic aromatic hydrocarbons (PAH), occuring in asphalt fumes (adsorbed on particular matter), is described. The method is based on extraction of asphalt fume particles, collected on glass fibre filters, using CCK₄. Following a clean up step by the aid of a TLC procedure on Al₂ O₃ thinlayer plates, using a mixture of cyclohexane/acetone/ether as the mobile phase. Under UV-light, occuring PAH are indicated as fluorescent spots. A separation of the collected PAH into individual components and their identification is performed by the aid of a HPLC procedure. Futher-more, an approach was made to verify the separated PAH by their fluorescence spectra and their mass spectra.     

Quantitation of Creatinine in Urine and Plasma Samples by Reversed Phase HPLC

Abstract

Creatinine determination in urine and plasma affords an index of the renal function. Reversed-phase high pressure liquid chromatography was used for the separation and quantitation of creatinine in normal and arsenic exposed human urine samples. Acetonitrile/water (1:1) was the mobile phase. The method was compared with the Jaffé alkaline picrate reaction. Results show that the HPLC procedure has high reproducibility and samples are stable at the storage conditions. Plasma samples required depro-teinization and extraction with CH₃CN prior to HPLC analysis, while urine samples required only centrifugation.     

Sensitive Spectrophotometric Determination of Vanadium in Environmental Samples at PPB Levels

Abstract

A new, sensitive and selective method for separation, and extraction - spectrophotometric determination of V (V) with pyrogallol {Ar (OH)₃} and hydroxyamidines (HOA) is described. The molar absorptivities of the colored species with six substituted HOA lie in the range of (0.52–1.5)x 10⁶ L mole^?1 cm^?1 at λ_max 430–440 nm. Of these, the simplest compound, N-hydroxy-N, N- diphenylbenz-amidine (HDPBA) gives the most sensitive color reaction and hence, it is used for detailed studies. No interference of almost all diverse ions tested is noticed. The method offers the determination of vanadium in different environmental samples.     

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.     

Determination of Bitrex,Quassia Powder and Sucrose Octaacetate Next to Diethyl Phthalate and Camphor in Specially Denatured Alcohols by Liquid Chromatografhy

Abstract

A HPLC method for determination of the bitter principles, bitrex, quassia and sucrose octaacetate, next to other ingredients in Specially Denatured Alcohol formulations is described. The method is based on evaporation of a sample, extraction of the residue with hexane and analysis of the extracted residue on a Cyano-type column with acetonitrile-water used as the eluent. Baseline separation of compounds and satisfactory quantitation has been achieved. Samples without pretreatment can occasionally be analysed with slight modifications of the procedure.     

High Performance Liquid Chromatographic Determination of Trimethoprim and Sulphadiazine in Medicated Fish Feedstock

Abstract

A high performance liquid chromatographic (HPLC) method for the determination of sulphadiazine and trimethoprim in medicated fish feedstock is described. It is based on the extraction of the drugs into methanol followed by separation on a μ-Bondapak column using an aqueous acetonitrile mobile phase of pH 3.0. The method was validated by determining intra- and inter-assay precision and the coefficient of variation was found to be less than 8% for both drugs. Callbration curves were linear over the range 160–4000 pg drug per g of feedstock and the method was applied to the determination of a commercial feedstock formulation.     

Quantification of physiological levels of vitamin D3 and 25‐hydroxyvitamin D3 in porcine fat and liver in subgram sample sizes

Most methods for the quantification of physiological levels of vitamin D₃ and 25‐hydroxyvitamin D₃ are developed for food analysis where the sample size is not usually a critical parameter. In contrast, in life science studies sample sizes are often limited. A very sensitive liquid chromatography with tandem mass spectrometry method was developed to quantify vitamin D₃ and 25‐hydroxyvitamin D₃ simultaneously in porcine tissues. A sample of 0.2–1 g was saponified followed by liquid–liquid extraction and normal‐phase solid‐phase extraction. The analytes were derivatized with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione to improve the ionization efficiency by electrospray ionization. The method was validated in porcine liver and adipose tissue, and the accuracy was determined to be 72–97% for vitamin D₃ and 91–124% for 25‐hydroxyvitamin D₃. The limit of quantification was <0.1 ng/g, and the precision varied between 1.4 and 16% depending on the level of spiking. The small sample size required for the described method enables quantification of vitamin D₃ and 25‐hydroxyvitamin D₃ in tissues from studies where sample sizes are limited.     

Determination of Coptis Chinensis Franch and Phellodendron Amurense Rupr in Qingwei-Huanglian Wan by Pls-HPLC Method

Abstract

Coptis chinensis Franch contains berberine(1), palmatine(2), jatrorrhizine(3) and etc. And phellodendron amurense Rupr also contains above components. Qingwei-Huanglian Wan is made from Coptis chinensis Franch, phellodendron amurense Rupr and etc. Berberine, palmatine and jatrorrhizine in Qingwei-Huanglian Wan were determined by HPLC. The optimal composition of mobile phase CH₃COOEt-HCOOH-EtOH (15:3:2) for HPLC separation of berberine, palmatine and jatrorrhizine was successfully determine by using window diagram technique. Detection wavelength was 345nm. Flow rate: 1.5ml/min. Calibration graphs for (1), (2) and (3) were rectilinear for 0.06–0.39μg, 0.06–0.61μg and 0.01–0.12μg respectively. The basic principle and method of partial least squares method (PLS) is presented in this paper. The data from HPLC were treated with PLS program to obtain the contents of Coptis chinensis corresponding with the requirement. All these indicate that PLS-HPLC method is new and feasible for the determination of crude drugs in Chinese Patent Medicine.

2. PLS is a new multivariate statistical method, and its performance is better than other traditional methods such as ordinary multivariate regression and principal components regression. This is because the calibration technique, which makes good use of the information in concentration matrix Y and content matrix X, is used in PLS method.

3. The method is applicable to the quanlity control of the products. The contents of IV and V in VI can be predicted accurately fast, if the method described here is used by factories making VI.     

Use of Derivative Spectrophotometry for the in Vitro determination of Phenylbutazone and Oxyphenbutazone in Human Plasma

Abstract

Fourth derivative-difference (ΔD₄.) spectrophoto metry is presented for the simultaneous assay of phenylbutazone and oxyphenbutazone as metabolites of bumadizone either in laboratory made mixtures or in human plasma. Phenylbutazone and oxyphenbutazone are determined through the measurement of ΔD₄ signals at 293 and 282 nm, respectively. The linear relationship of these values versus concentration of both compoundsin the range 1–5 μg/ml for phenylbutazone and 3–8 μg/ml for oxyphenbutazone permits their determination with high accuracy and good reproducibility. The relative standard deviation is less than 1.35% The proposed method could be used for monitoring bumadizone metabolites.     

A High-Performance Liquid Chromatographic Method for the Determination of Doxefazepam in Human Plasma Using a Solid-Phase Extraction Column

Abstract

A procedure for the rapid, quantitative isolation of doxefazepam from plasma with Supelclean LC-18 cartridges is described together with a sensitive HPLC assay for the quantitative determination of the drug. The recovery of doxefazepam was greater than 80 % over an investigated range of 0.1–2.0 μg/ml of plasma. The column extraction of doxefazepam coupled with the versatility of HPLC make this procedure well suited for detailed pharmacokinetic studies and as well as routine plasma analysis of doxefazepam.     

A New Spectrophotometric Method for Determination of Vitamin B12 as Cobalts

Abstract

A spectrophotometric method for determination of Vitamin B₁₂ (Cyanocobalamin) as cobalt in pharmaceutical preparations is described. The sample is first decomposed by concentrated sulfuric and nitric acids and the dry residue is solubilized in distilled water. The cobalt present is then spectrophotometrically determined at Λ= 317 nm as Co-HMPA-SCN complex. The complex is stable for a long time in the range of pH 3–10. The presence of many other metal cations does not interfere.     

Spectrophotometric determination of vitamins D2 and K1 in presence of rutin

Summary Methods are given for determination of vitamin D₂ and K₁ in presence of rutin added as stabilizer, and applied to assessment of the rate of breakdown of these vitamins when exposed to ultraviolet light. Because the absorption maxima of these vitamins, rutin and some of the decomposition products are very near to each other a preliminary separation is necessary. Thin layer chromatography with chloroform on silica gel H is suitable.

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Spektropbotometrische Bestimmung von Vitamin D₂ und K₁ in Gegenwart von Rutin

Zusammenfassung Für die Bestimmung von Vitamin D₂ und Vitamin K₁ in Gegenwart von Rutin, das als Stabilisator zugegeben wird, wurden Methoden angegeben und zur Bestimmung der bei UV-Bestrahlung eintretenden Zersetzung dieser Vitamine angewendet. Da deren Absorptionsmaxima alle sehr nahe beieinander liegen, ist eine vorherige Trennung nötig. Dazu hat sich die Dünnschichtchromatographie mit Chloroform auf Silicagel H als geeignet erwiesen.

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Presented at the 8th International Microchemical Symposiums Graz, August 25–30, 1980.