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.     

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.     

Application of HPLC to the Study of the Chloroplast Atpase Mg2+ Dependent Mechanism

Abstract

The determination by HPLC of released ADP from ATP by chloroplast ATPase (CF₁) is described.

The enzymatic rate measured by this method is well defined, over several minutes.

In the case of ? subunit-depleted CF₁ or activated CF₁, the rate is proportional to the enzyme concentration, the steady state theory is followed and the K_m and V_m constants have been calculated.

The enzymatic activity of CF₁ is inhibited by endogenous ? subunit and the inhibition constant has been measured.

The influences of ionic strength, pH, magnesium ion, phosphate and ADP concentrations have been studied and the results obtained by this method have been compared to previously reported data based on rate determination of released phosphate.     

HPLC Analysis of Adriamycine as N-2,4-Dinitrophenyladriamycine

Abstract

Derivatization of adriamycine (I) by reaction with 2,4-dinitrofluorobenzene, followed by HPLC analysis on reversed phase (RP-18 μ-Bondapak) or on normal phase (μ-porasil) and detection at 482 nm, provides a fast method (R_f=4.0 min) for determination of adriamycine [as N-2,4-dinitrophenyladriamycine (III)] in the 1–10 ppm range. Reaction with 2,4-dinitrofluoro[^?14C]benzene, followed by HPLC separation and detection on a γ counter, extends the detection limit to 0.04 ppm adriamycine.     

A Rapid Method For Cyclosporine A Determination By Hplc

Abstract

A rapid method is described for the determination of cyclo-sporine in whole blood by HPLC. The cyclosporine is extracted with an acetonitrile-isopropanol mixture, purified on a C₁₈ minicolumn, and finally injected on a CN column. Recovery of the drug is greater than 90%.     

HPLC Determination of α-Amino Acids in Phytoplanktonic Marine Cells

Abstract

A procedure for the quantitative determination of 17 amino acids in a marine matrix using HPLC is reported. Pre-column derivatization with o-phthalaldehyde, separation on C₁₈-bonded silica with phosphate buffer (pH 7.2)-acetonitrile as eluent and fluorescence detection have been used. The good variation coefficient (average 2% with working curves in real matrix) and the low detection limit (1-5 fmoles) make the procedure suitable for the determination of total or free amino acids in matrix cultures.     

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.     

The Determination of Sodium Monofluoroacetate (Compound 1080) in Formulation and Technical Samples by High Pressure Liquid Chromatography

Abstract

A high pressure liquid chromatographic (HPLC) procedure was developed for the determination of sodium monofluoroacetate (Compound 1080). The procedure utilized an amine (NH₂) bonded column for the reverse phase determination of sodium monofluoroacetate in formulation and technical samples.     

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.     

Serum Injection of the HPLC Column for Carbamazepine Assay

Abstract

Serum was injected directly on an HPLC column packed with C₁, 6.5 μm particle size, 300 Å pore-packing material for carbamazepine determination. The use of a wide-pore column with low hydrophobicity eliminated excessive pressure buildup in the column from protein precipitation which is usually caused by the high concentration of organic solvents in the mobile phase. This simple approach can be utilized for the determination of other drugs and endogenous compounds in serum.     

Aflatoxin Analysis by Reverse Phase HPLC Using Post-Column Derivatization for Enhancement of Fluorescence

Abstract

The application of a technique for the determination of aflatoxins by reverse phase HPLC and fluorescence detection incorporating post-column derivatization with iodine, is described. The procedure proved to be extremely sensitive and reproducible. Chromatograms of extracts from maize, peanut butter, sorghum malt and duckling mash are presented illustrating the value of the procedure for confirming the presence of aflatoxins B₁ and G₁.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

Measurement of Nicotine in Plasma by High-Performance Liquid Chromatography

Abstract

A new procedure has been developed to measure nicotine in blood plasma by high-performance liquid chromatography (HPLC). Nicotine is extracted from plasma by elution with cholorform. Final determination is achieved by isocratic HPLC with ultraviolet detection. Twenty microliters of plasma extract is deluted over a silica column at a flow rate of 1.0 ml/min with a dioxane:isopropanol:NH₄OH (80:3.0:0.4) mobile phase. The procedure is sensitive to 0.05 ug of nicotine per ml of plasma and is linear within the range of 0.05 to 10.0 ug/ml of plasma. When a known amount of nicotine was added to plasma, the concentration of nicotine measured averaged 99.9 + 3.9 (S.D.)% of the known concentration. The within-sample coefficient of variation was 3.9%     

A New Method for the HPLC Analysis of Pt(II) in Urine

Abstract

An analytical method has been developed to measure Pt(II) in urine via derivatization and UV or HPLC analysis. A measured quantity of urine is heated briefly with diethyl ammonium diethyl-dithiocarbamate, and the resulting Pt(Et₂NCS₂)₂ is extracted into a measured volume of chloroform. Concentrations of Pt(II) are determined by UV absorption at 346 nm or by reverse phase HPLC analysis. The detection limit for Pt(II) as its dithiocarbamate is ～ 1 ng by HPLC; the concentration limit for HPLC analysis by direct extraction was ～ 25 ng/ml. Chromatographic response was linearly related to Pt(II) concentration over the range 100-4, 000 ng/ml; dilution of more concentrated samples has extended this range to at least 30, 000 ng/ml. This method has been applied to the analysis of Pt(II) in the urine of patients who have received cis-dichlorodiamniineplatinum(II) (CDDP) chemotherapy.     

Determination of Bicascarosides in Cascara Fluid Extract by High Pressure Liquid Chromatography

Abstract

A quantitative determination of bicascarosides by HPLC in pharmaceutical preparations is described. These compounds are transformed by oxidative hydrolysis into their aglycones which can be separated on a RP-18 column. Concentrations as low as 0.05% can be determined without interference from monomeric aglycones.     

An Accurate,Specific HPLC Method for the Analysis of a Decapeptide in a Lactose Matrix

Abstract

A high-performance liquid chromatographic (HPLC) method is described for the determination of an analog of the hormone LH-RH in lyophilized vials at the low parts-per-million level. The peptide (Schally analog) is quantitatively recovered from the glass lyophilization vials after reconstitution with mobile phase. The peptide solution is eluted on a reversed-phase, C₁₈ column and monitored with ultraviolet (UV) detection at 220 nm. The chromatography resolves Schally analog from a number of synthetic impurities and decomposition products.     

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.     

High Performance Liquid Chromatographic Separation of Bacitracin Methylenedisalicylate

Abstract

Our previously described isocratic RP HPLC procedure was convenient for monitoring and quality control of bacitracin production and Zn - bacitracin feed grade preparations. Separation and quantitative determination of the main active bacitracin components (A₁ B₁ and B₂) are possible and those elution is not interrupted by other ingredients in this type of samples. But when the methylenedisalicylic salt of bacitracin was tested some modification of method were necessary for the correct separation of bacitracin components. Mobile phase had to be modified and polystyrene based packing was an alternative and useful complement to octadecylated silica gel packings.     

Preparation,Isolation and Characterization of all of the Regioisomeric 61, 6N-Bis-O-(Monomethoxytrityl) and-(Dimethoxytrityl) Derivatives of Cyclomalto-Olgosaccharides

Abstract

Regioisomeric 6¹, 6ⁿ-bis-O-(monomethoxytrityl) or 6¹, 6ⁿ-bis-O-(dimethoxytrityl) cyclomaltohexaose, -cyclomaltoheptaose (n = 2-4), and -cyclomaltooctaose derivatives (n = 2-5) were prepared by the reaction of cyclomaltohexaose (1, cG₆, αCD), cyclomaltoheptaose (11, cG₇, βCD) or cyclomaltooctaose (21, cG₈, γCD) and 4-monomethoxytrityl chloride or 4,4′-dimethoxytrityl chloride in pyridine. Products were isolated by HPLC. The regiochemical determination of these positional isomers was done by converting these compounds to the respective 6¹, 6ⁿ-bis-O-(tert-butyldimethylsilyl) derivatives¹ whose structures have been already established.     

High Pressure Liquid Chromatographic Determination of Isoniazid and Acetylisoniazid in Human Plasma

Abstract

A quantitative high pressure liquid chromatographic (HPLC) assay has been developed for the determination of isoniazid (INH) and acetylisoniazid (ACINH) in human plasma. Plasma samples were taken from a patient after oral administration of INH (with proven tuberculosis infection). A C₁₈ reversed phase radial compression column was used to separate INH and ACINH from other plasma components. The analysis takes 10 minutes per sample and the lower limit of detection for each compound is 0.10 ug/ml plasma.