Low flow high-performance liquid chromatography solvent delivery system designed for tandem capillary liquid chromatography-mass spectrometry

A solvent delivery system is described that is designed to increase the efficiency of liquid chromatography-mass spectrometry (LC/MS) analyses. Gradients formed by using two low pressure syringe pumps are stored in a length of narrow bore tubing (gradient loop) mounted on a standard high pressure switching valve. The preformed gradient is pushed through the column by using a high pressure syringe pump. The system is fully automated and can be controlled with either a personal computer or the mass spectrometer data system. Advantages include gradient operation without the use of split flows, pressure programed flow control for rapid sample loading and recycling to initial conditions, and a flow rate range of 0.1–20 μL/min, which is suitable for packed capillary columns 50–500 μm in diameter. The system has been used extensively for rapid molecular weight determinations of intact protein samples, as well as LC/MS and liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures.     

Feasibility of using gravimetry to measure excess adsorption effects of a binary solvent system in a reversed-phase high-performance liquid chromatography column

A modified method for weighing HPLC columns filled with solvent is described. The method prevents the loss of traces of solvent from within the threads of the column. The method was tested by obtaining the weights of a C18 column filled with 10 different organic solvents, showing a standard deviation on the order of 0.1%. A plot of gross column weight versus solvent density showed excellent linearity. The method was then used to weigh a column filled with several acetonitrile–water mixtures. The gross column weights were lower than would have been predicted from the density of the acetonitrile–water mixtures. A likely explanation is the existence of an adsorbed acetonitrile-rich liquid on the surface of the C18 adsorbent, which caused the lower than expected weights due to the lower density of pure acetonitrile relative to the bulk mixtures. The volume of pure acetonitrile required for the observed weight discrepency was calculated. Based on the surface area of the column adsorbent, values of micromoles acetonitrile per square meter of surface area were determined. The values showed reasonable agreement with values obtained from published adsorption isotherm studies. This suggests that pycnometry may be a useful technique for adsorption studies. The limitations of the technique are discussed.     

Optimization of multicomponent solvent selection in high-performance liquid chromatography using a statistical method

A computer-assisted method is presented for optimization of multicomponent solvent mobile phase selection for separation of O-ethyl-N-isopropyl phosphoro (thioureido) thioates in reversed-phase HPLC and four geometric isomers of pesticides Decis in normal-phase HPLC. The method is based on Snyder's solvent selection triangle concept using a statistical method. The optimization of the separation over the experimental region is based on a special polynomial estimation from seven experimental runs, and resolution (R_s) is used as the selection criterion. Excellent agreement was obtained between predicted data and experimental results.     

Determination of pyrethroid pesticide residues in vegetables by solvent sublation followed by high-performance liquid chromatography

A novel method is developed for separating and enriching pyrethroid pesticides from vegetables by solvent sublation, and determination of the pyrethroids is performed by high-performance liquid chromatography (HPLC). The effects of organic solvent, pH of the solution, nitrogen flow rate, and sublation time on the sublation efficiency of pyrethroids are investigated in detail, and the optimal conditions of the solvent sublation are selected. The floated product of vegetables in the optimal conditions is determined by HPLC. The limit of detection values range from 1.4 microg/kg (for bifenthrin) to 4.2 microg/kg (for fenpropathin). The recoveries of spiked vegetable samples are from 85.7% to 110.4%, and relative standard deviation values are from 1.70% to 6.19%. The results are satisfactory.     

Determination of L-3,4-dihydroxyphenylalanine in blood by high-performance liquid chromatography after solvent extraction

The use of high-performance liquid chromatography combined with solvent extraction for sample purification is described for the determination of L-3,4-dihydroxyphenylalanine in blood plasma. It is extracted into n-hexanol via complexation of its catechol moiety with diphenyl borate and ion-pair formation of its carboxylic group with tetrapentylammonium ion in an alkaline buffer. Under optimal extraction conditions, L-3,4-dihydroxyphenylalanine and 3,4-dihydroxybenzylamine used as an internal standard are extracted from blood plasma by a simple procedure and in a short time and then separated by reversed-phase ion-pair chromatography. The analytical recovery (100.8%) and reproducibility (coefficient of variation = 2.3% for n = 6) from plasma samples are good enough for routine analysis. L-3,4-Dihydroxyphenylalanine levels in blood can be monitored by this method after oral intake of the substance.     

Use of high-performance liquid chromatography to quantitative thymine-containing pyrimidine dimers in DNA

We have developed two high-performance liquid chromatographic systems for the measurement of pyrimidine dimers in hydrolysates of DNA. Normal-phase chromatography on an NH₂ column in methanol—ethyl acetate (3:97) at an elution rate of 2.0 ml/min allowed quantitaion of thymine-containing (thymine-thymine plus thymine-uracil) pyrimidine dimers at levels as low as 0.1% of the total radioactivity as thymine in DNA. This system was unaffected by the presence of up to 1 mg of contaminating protein (bovine serum albumin) or 40 μg of DNA in hydrolysates prepared for chromatography. Reversed-phase chromatography on a μBondapak C₁₈ column allowed measurement of thymine-thymine dimers at concentrations as low as 0.02% of the total radioactivity. With 0.1% tetrahydrofuran in wateras the solvent at a flow-rate of up to 0.6 ml/min, thymine—thymine, thymine—uracil, and uracil—uracil dimers were completely resolved. We were not able to quantitate the latter two dimeric forms, however, owing to the presence of other radioactive components of undefined origin that eluted concomitantly with the uracil-containing dimers.     

On-line racemization by high-performance liquid chromatography

An on-column stopped-flow bidimensional recycling HPLC procedure was developed to obtain an enantiomeric enrichment starting from a racemic mixture. The method developed was applied to two chiral compounds of pharmaceutical interest, (±)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide (1) and (±)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((±)IDRA21, (2)), since the pharmacological activity of the two benzothiadiazine derivatives investigated has been ascribed to only one enantiomer. Starting from a racemic mixture it was possible to obtain about 95% of pure enantiomer. The procedure was applied both in reverse-phase mode and in normal-phase mode. The scaled up and automatization of the novel analytical HPLC procedure represents a powerful tool to obtain pure enantiomer starting from racemic compounds without cumbersome stereoselective synthesis or expensive enantiopurification processes.     

Determination of thiamine by high-performance liquid chromatography

High-performance liquid chromatographic methods for the determination of thiamine (vitamin B1) in foodstuffs or biological tissues and fluids are outlined and discussed. The methods are often similar and interchangeable, sample extraction and clean up procedures being the major difference. Most of the methods use either ultraviolet or fluorescence detection. Fluorescence detection requires either precolumn or postcolumn oxidation of thiamine to thiochrome. A number of methods are recommended and problems with standardization are emphasized.     

Use of high-performance liquid chromatography to assess airborne mycotoxins. Aflatoxins and ochratoxin A

An HPLC analytical method combining methanol-deionised water (80:20, v/v) extraction, methanol-acetonitrile (50:50, v/v) extraction and fluorescence detection was implanted to analyse ochratoxin A and aflatoxins B1, B2, G1 and G2 of air samples collected during the usual production process in a number of workplaces of a coffee factory to assess the occupational exposure of the engaged workers. The average levels of airborne ochratoxin A and aflatoxins were less than 1.2 and 0.4 ng/m3, respectively, using 50 L air samples. When 150 L air samples were used, levels lower than 0.04 ng/m3 ochratoxin A and 0.013 ng/m3 for aflatoxins B1, B2, G1 and G2, could be detected.     

Determination of aprotinin by high-performance liquid chromatography

Summary A highly accurate and reproducible method for the determination of aprotinin (bovine pancreatic trypsin inhibitor) by HPLC is described. In the experiments, the relative standard deviation was 1.2% and detection limit 1 FIP-U cm^–3. Also, the method is quick and selective and active ingredients from difference source correlate well with enzymatic method. Analyses at different laboratories can be compared directly.     

Use of optimization software to determine rugged analysis conditions in high-performance liquid chromatography

The ruggedness evaluation of an analytical method is now generally required for further validation. By considering ruggedness at an early stage of method development, major disappointments and amount of work could be avoided. This work shows that the optimization software OSIRIS can be helpful for the chromatographer during a method development, as it takes into account the method ruggedness. The ruggedness of the analysis conditions is then evaluated all along the selectivity optimization procedure. This optimization software belongs to the interpretive methods that consist of predicting the optimum conditions by modeling first the solute retention over the parameter space using a minimum number of preliminary runs. The choice of a response function is studied. This response function must be able to take into account several individual criteria: analysis time, minimal resolution and ruggedness of each parameter. Some optimum separations, determined using a ruggedness criteria or not, are given and compared in terms of long term repeatability.     

Determination of beta-carbolines in foodstuffs by high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method combined with fluorimetric detection is described for the determination of beta-carboline (norharman) and 1-methyl-beta-carboline (harman). The analysis of foodstuffs for the identification of beta-carbolines is facilitated by clean-up samples using Bond Elut PRS cartridges. Recoveries were excellent. Further, a high-performance liquid chromatographic-mass spectrometric method was also developed for their identification. The concentration of beta-carboline among the foodstuffs and alcoholic beverages varied greatly. Also, norharman and harman were observed in uncooked foodstuffs, whereas acetaldehyde was found in most fermented food. The toxicological implication of beta-carbolines in foodstuffs is discussed.     

Alkanol-based supramolecular solvent microextraction of organophosphorus pesticides and their determination using high-performance liquid chromatography

A sensitive method for extraction and determination of three organophosphorus pesticides (chlorpyrifos, diazinon, phosalone) using a supramolecular solvent (SUPRAS) made of inverted hexagonal aggregates of alkanol and high-performance liquid chromatography with ultraviolet detection (HPLC–UV) was developed. The studied factors were alkanol amount, THF percentage (v/v), pH and vortex time. According to the full factorial design results, the effective parameters were alkanol amount, THF percentage (v/v) and pH. Then, a CCF was applied to obtain optimal conditions. The optimized conditions were obtained at 100 mg of alkanol, 5% of THF and pH 3.9. The limits of detection of pesticides were 0.5–1.3 ng/mL. The linearity was 1.6–500.0 ng/mL for different pesticides. Relative standard deviations for intra- and inter-day extraction of pesticides were 3.3–5.0 and 5.1–6.3, respectively, for five measurements. The method was also successfully applied for the determination of the pesticides in fruit juice and tap water samples.     

Use of a displacement model for solvent sorption to study non-specific selectivity in reversed-phase liquid chromatography

Summary A thermodynamic equation is derived for the non-specific selectivity of alkyl bonded phases as a function of the mobile phase composition using a displacement mechanism to model the sorption of solvents into the bonded phase. The equation is used to calculate the thermodynamic parameters which characterize the incremental behavior of a hydrophobic group in ethyl alkanoate and methyl perfluoroalkanoate ester solutes chromatographed with water-methanol and water-acetonitrile mobile phases on both octyl and octadecyl bonded phases.