High Resolution Separation of Urinary Organic Acids by High Performance Liquid Chromatography

Abstract

Reverse phase, anion exchange, and two-dimensional HPLC techniques were studied in order to increase resolution of organic urinary acids for eventual quantitative measurements. Reverse phase HPLC with a phosphate buffer/acetonitrile gradient yielded a separation of over 85 components in forty minutes and a peak area reproducibility of better than 5%. Connecting two reverse phase columns together resulted in the separation of 110 components. Anion exchange chromatography was determined to be of little use in resolving urinary acids in a resonable time except as the first stage in two-dimensional chromatography where fractions from the anion exchange column were injected into a reverse phase column. Over 139 components were separated by this two-dimensional method.     

β-Blocking Agents: Determination of Biological Levels Using High Performance Liquid Chromatography

Abstract

Nine β-blocking agents have been tested and dosed by high performance liquid chromatography. Six of them, acebutolol an acebutolol metabolite, atenolol, metoprolol, propranolol and sotalol are detected with a fluorometric detector. Oxprenolol, pindolol and timolol can be quantified by their UV absorption at variable wavelength. A method is developped to find the best conditions of extraction and detection for each blocking agent. Experimental trials have led to a simple procedure for all compounds. Only pindolol and timolol plasma levels are non suitable for high performance liquid chromatography and need mass fragmentography or gas chromatography with electron capture detection.

However, pharmacokinetic parameters can be reached, for timolol and pindolol, through urinary excretion since sensitivity of the procedure is within the range of urinary levels.

The method has been applied, as well, to pharmacokinetic studies on sotalol, acebutolol, acebutolol metabolite, atenolol, propranolol, pindolol and timolol.     

Urinary Uric Acid Determination by Reversed-Phase High Pressure Liquid Chromatography

Abstract

Reversed-phase high pressure liquid chromatography with UV detection was proven to be a powerful method for the separation and quantitation of urinary uric acid. We have compared three different treatments for urine samples previous chromatographic injection: alkaline methanol extraction, ethylacetate extraction and centrifugation. It was also studied storage conditions for urine samples.

Our findings show that the method has high specificity and reproducibility for urinary uric acid. Samples are stables and require only centrifugation previous injection to the chromatograph.     

Analysis of a Mixture of Polychlorinated Biphenyls,DDT and its Analogues by High-Performance Liquid Chromatography

Abstract

A simple and rapid method for separation and quantitative analysis of polychlorinated biphenyis (PCBs) and chlorinated pesticides (DDT and its analogues DDE and DDD in their o, o'-and p, p'-isomers) is described. The procedure consists of two steps: a) transformation of DDT and its analogues in o, p'-and p, p'-dichlorobenzophenone (DCBP); b)determination of the amount of PCBs and ∑DDT as DCBP by HPLC. Results obtained confirm that HPLC can be considered as an alternative or a supplementary methodology to conventional methods such as gas chromatography. The method is applied to marine organisms.     

Micromethod for Determination of Thiopental in Biological Fluids by High Performance Liquid Chromatography. Application to Therapeutic Follow-Up and Pharmacokinetic Study

Abstract

Thiopental is an ultra-short acting barbiturate, used either to induce anesthesia, or to manage intracranial hypertension associated with head injuries. A high performance liquid chromatography method vas proposed for the rapid and easy determination of thiopental in biological fluids. After precipitation of proteins with acetonitrile the chromatographic separation vas performed onto a reversed-phase column with an acetonitrile-perchlorate buffer mixture as mobile phase. Compounds were analysed at 300nm and the detection limit vas about 0.1 mg/1. The analytical procedure vas applied to the therapeutic follow-up in patients (8 childrens. 4 adults) treated with long-term infusion of thiopental after head injury, and to a pharmacokinetic study in patients receiving a single 1Y bolus of thiopental to induce anesthesia.     

Computer Enhanced Spectroscopic Separation of Coeluted Components in Supercritical Fluid Chromatography Coupled with an Ultraviolet Multichannel Detector

Abstract

Computer enhanced spectroscopic separation technique has been applied to identification of coeluted components in supercritical fluid chromatography by using an ultraviolet photodiode array multichannel detector. The powerful function such as peak deconvolution procedure has successfully offered thespectroscopic complete separation for even partially or completely coeluted components in super critical fluid chromatography.     

Improved High Performance Liquid Chromatographic Method for the Quantification of 3-Methylhistidine in Serum and Urine

Abstract

We optimized an high-performance liquid chromatographic (HPLC) method for the determination of 3-methylhistidine (3-MeHis) in biological fluids. After pre-column derivatization with OPA, analytical separation was achieved on a reversed-phase C18 column by a simple gradient between sodium propionate buffer and acetonitrile. the method is accurate, reproducible and sensitive, and allows the determination of urinary 3-MeHis levels in about 55 min. Other additional 16 amino acids may be easily quantified while the 3-MeHis peak is well resolved from an unknown urinary compound potentially interfering.     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

Applicability of an HPLC System in the Analysis of Biodegraded Crude Oil Components

Abstract

High performance liquid chromatography has been used for the separation of biodegraded crude blend into fractions using back flushing procedure. The stationary phase was Energy analysis (3.9mm × 30cm) NH₂ column and the detection mode was UV absorptiometry. The method was compared with the liquid chromatography method.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

Evaluation of a new modified QuEChERS method for the monitoring of carbamate residues in high‐fat cheeses by using UHPLC–MS/MS

A simple and efficient method for the determination of 28 carbamates in high‐fat cheeses is proposed. The methodology is based on a modified quick, easy, cheap, effective, rugged, and safe procedure as sample treatment using a new sorbent (Z‐Sep⁺) followed by ultra‐high performance liquid chromatography with tandem mass spectrometry determination. The method has been validated in different kinds of cheese (Gorgonzola, Roquefort, and Camembert), achieving recoveries of 70–115%, relative standard deviations lower than 13% and limits of quantification lower than 5.4 μg/kg, below the maximum residue levels tolerated for these compounds by the European legislation. The matrix effect was lower than ±30% for all the studied pesticides. The combination of ultra‐high performance liquid chromatography and tandem mass spectrometry with this modified quick, easy, cheap, effective, rugged, and safe procedure using Z‐Sep⁺ allowed a high sample throughput and an efficient cleaning of extracts for the control of these residues in cheeses with a high fat content.     

Determination of Pentachlorophenol (PCP) in Samples of the Environmental Specimen Bank Using Isotope Dilution

Abstract

Within the program of the Environmental Specimen Bank a quick and efficient method for determination of pentachlorophenol (PCP) in various environmental matrices has been developed. The method includes alkaline hydrolysis of bound PCP, acidification, simultaneous steam distillation and extraction in one glass apparatus. After clean-up and derivatization with acetic anhydride the samples were analyzed by gas chromatography/mass spectrometry. Concentrations were calculated using ¹³C-labeled PCP as the internal standard. Validation was carried out with various environmental samples (soil, fish, conifer needles, kale).

The method can be used for various biological samples without any modification. The extracts are free of matrix components (lipids, chloropyll, terpenes, etc.) and other contaminants, which results in clear chromatograms with few peaks; therefore, correct integration is facilitated. Although the recoveries of PCP are in the range of 50–90%, due to losses during the several method steps, these losses can be corrected with the ¹³C-labeled internal standard, resulting in high precision (1.5–2.2% standard deviation).     

Analysis of Tamoxifen and Its N-Desmethyl and 4-Hydroxy Metabolites in Tissue

Abstract

A method is described for the analysis of the non-steroidal anti-estrogenic antineoplastic agent, tamoxifen and its 4-hydroxy and N-desmethyl metabolites in liver, uterine, fat and human breast tumor tissue, and whole blood, plasma and cerebral spinal fluid. The report focuses on separation of analytes from the biological matrix. After extraction, analytes are converted to fluorescent phenylphenanthrenes, which are separated by reverse phase paired ion chromatography with spectrofluorometric monitoring of column eluent. By control of column temperature, chromatographic analysis time could be significantly reduced and sensitivity increased.     

Initial Validation of a New Procedure for Determining Aromatics in Petroleum Distillates

Abstract

The analytical data from an initial validation of a new method developed for determining aromatics in distillate fuels is reported. The method applies supercritical fluid chromatography (SFC) for the liquid chromatographic separation of total saturates and total aromatics, followed by flame ionization detection. A study was conducted to determine the validity of a SFC procedure after it was proposed to the Canadian national standard writing body for its adoption as a new standard method for determining aromatics. Inter-laboratory results suggest that SFC provides reproducible data among different laboratories and these SFC data correlate well with those obtained from other methods such as fluorescent indicator adsorption (FIA), nuclear magnetic resonance (NMR), and mass spectrometry (MS).     

Chromatographic Separation on Silica of Polar Aromatic Compounds II) Research of the Best Chromatographic System by Different Preloading in Thin Layer for a Transposition on Column

Abstract

A high performance thin layer chromatographic method for aromatic compounds separation with absorbant deactivation has been given in the precedent work. Our aim was to transpose the results obtain from HPTLC to HPLC. The two types of results were then related by a transposition coefficient, K_tR which interprets the geometrical variations of the two chromatographic methods. The values obtained for K_tR represent fairly well the linear relation between the retentions in thin layer chromatography and those in column chromatography. We have thus desmontrated the possibility of modifying adsorbent activity in the same manner in thin layer as in column chromatography by the fixation of chemical compounds, thus allowing the separation of relatively polar compounds.     

Simultaneous and rapid determination of deoxynivalenol and its acetylate derivatives in wheat flour and rice by ultra high performance liquid chromatography with photo diode array detection

A simple and reliable method of ultra high performance liquid chromatography coupled with photo‐diode array detection has been proposed for the simultaneous determination of deoxynivalenol and its acetylated derivatives in wheat flour and rice, especially focusing on the optimization of sample extraction, cleanup, and chromatographic separation conditions. Sample pretreatment consisted of a first step using a quick, easy, cheap, effective, rugged, and safe based extraction procedure and a subsequent cleanup step based on solid‐phase extraction. The method was extensively validated in wheat flour and rice, obtaining satisfactory analytical performance with good linearity (R² ≥ 0.999), acceptable recoveries (80.0–104.4%), and repeatability (RSDs 1.3–10.7%). The limits of detection (21.7–57.4 μg/kg) and quantitation (72.3–191.4 μg/kg) for deoxynivalenols were lower than those usually permitted by various countries’ legislation in these food matrices. The method was applied to 34 wheat and rice samples. The results were further compared with results of ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry.     

Determination of90Sr by thin layer radiochromatography

A simple method for the determination of⁹⁰Sr by using thin layer chromatography on silica gel or cellulose pretreated with calcium oxalate is proposed. In these conditions a complete separation between strontium and its daughter yttrium is obtained. Radioactivity of separated elements was measured by a linear multiscanner analyzer and the results were computer processed to obtain the activity of⁹⁰Sr. The method has been applied to samples of water and milk subjected to a very simple extraction procedure. Under the experimental conditions used, the detection limit is about 25 mBq of deposited radioactivity, which corresponds to about 6 Bq/l.     

Purification of Radio-Iodinated Cholecystokinin Peptides by Reverse Phase HPLC

Abstract

The purification of the iodinated tracers of CCK peptides using Sphadex G5O chromatography does not allow for a good separation between non-modified peptides and labelled peptides. We present, in this paper a simple and rapid purification method using reverse phase HPLC with a C-18 column for four of these tracers. The biological characteristics of the molecules obtained demonstrate their strong specific radioactivity and their high degree of purity.     

EcoFriendly Synthesis of Tetrahydropyrimidine Derivatives in Aqueous Medium Under Ultrasonic Irradiation

Abstract

A simple, efficient, and ecofriendly procedure was developed for the synthesis of 2-substituted 1,4,5,6-tetrahydropyrimidine derivatives catalyzed by N-bromosuccinimide using ultrasonic irradiation in aqueous medium. Prominent advantages of this new method are good yields, aqueous medium, short reaction times, and easy workup procedure. The compounds were characterized by infrared, NMR, liquid chromatography–mass spectrometry, and elemental analyses.