Determination of oxytetracycline in blood serum by high-performance liquid chromatography with direct injection.

A direct injection analysis by high-performance liquid chromatography has been developed for oxytetracycline in serum of animals and fish. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of methanol-0.2 M oxalic acid (10:90, v/v, pH 7.0) with ultraviolet detection at 360 nm were used. The standard calibration curves in serum of chicken, hog, cattle and rainbow trout were linear over the range 0.1-20 micrograms/ml. The recoveries of oxytetracycline from all serum samples determined at two different concentrations (0.5 and 2.0 micrograms/ml) were 88-103%. The detection limit was 0.05 micrograms/ml for every serum sample.     

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

Determination of creatinine in serum by high-performance liquid chromatography: a comparison of three ion-exchange methods

Three ion-exchange high-performance liquid chromatographic methods for the determination of creatinine in serum have been compared. In method 1 a strong cation exchanger was used. In method 2 a reversed-phase column was given strong cation-exchange properties by the addition of N-methyloleoyl taurate to the mobile phase. In method 3 a weak cation exchanger was used. Elution was with a pH gradient in methods 1 and 2, and isocratic elution was used in method 3. The imprecision was similar for the three methods and varied between 0.9 and 2.5% as studied within-day and between 1.4 and 3.2% from day-to-day. The lowest coefficient of variation was obtained around the upper reference limit. Analytical recoveries were quantitative for the three methods. The method with N-methyloleoyl taurate showed no advantages over the conventional strong cation exchanger. With the weak cation exchanger no interferences were detected from compounds investigated, but with the strong cation exchanger a slight interference was obtained with uric acid. We prefer the weak cation-exchange method because of its simplicity, higher throughput and absence of interference from hitherto tested compounds.     

Towards a definitive assay of creatinine in serum and in urine: separation by high-performance liquid chromatography

A fast and sensitive method for the separation of serum and urinary creatinine is described. For the preliminary purification of serum and urine, a cation-exchange column is used to remove protein, anions and neutral compounds prior to isolation of creatinine by high-performance liquid chromatography. A reversed-phase system with 0.01 M ammonium acetate solution as the mobile phase can separate creatinine in 7.5 min at a flow-rate of 1 ml/min. The purity of the separated creatinine is proved by derivatization using trifluoracetic anhydride, followed by gas chromatography and mass spectrometry. Although this method of purification was designed for incorporation into a definitive assay, the ease and speed of analysis makes is very attractive for routine clinical use.     

On-line coupling of sol-gel-generated immunoaffinity columns with high-performance liquid chromatography.

The paper demonstrates the possibility to use sol-gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol-gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol-gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol-gel immunoaffinity columns. The system consists of a sol-gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.     

Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography

This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 x 4.6 mm I.D., 10 microm particle size and 120 A pore size) to an octadecyl Hypersil column (150 x 4.6 mm I.D., 5 microm particle size and 120 A pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 microl) of the appropriated standard solutions were added to each culture tube containing an 180 microl of plasma and a solution of 0.30 mM tetrabuthylammonium phosphate (60 microl). They were vortexed for 15 s and then 290 microl were transferred to autosampler vials. Aliquots (250 microl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 microl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).     

Characterization of an avidin-bonded column for direct injection in reversed-phase high-performance liquid chromatography

An automatic method for the determination of metabolites of Ropivacaine in urine was set up. It utilizes supported liquid membrane extraction for sample clean-up and enrichment, followed by ion-pair chromatography determination using UV detection. The extraction was very selective with no observed interfering compounds from the urine matrix, permitting simple isocratic chromatographic analysis. The detection limits for spiked urine samples were 2–18 nM for the different compounds. The repeatability was 1–3% (RSD) with an internal standard that was also extracted, and about twice without this standard. A throughput of 3.3 samples per hour was achieved and the liquid membrane was stable for more than a week.     

Analysis of sulthiame in serum by narrow-bore high-performance liquid chromatography. Comparison of direct sample injection with pre-column switching and extrelut extraction.

Two high-performance liquid chromatographic methods for the analysis of sulthiame in serum are described. In the first method direct injection of serum samples onto a 4 x 4 mm I.D. (C18, 25 microns) precolumn in a column-switching device was used. After a purge step, the adsorbed analytes were eluted onto a 250 x 3 mm I.D. (C18, 5 microns) narrow-bore column for chromatographic separation. In the second method the sample pretreatment was an Extrelut extraction with dichloromethane-propanol-2 (95:5). After evaporation of the solvents, the residue was dissolved in methanol. The chromatographic separation was carried out on the same analytical column as used in the column-switching method. Both sample pretreatment methods were compared with respect to their suitability of routine analysis of sera from patients also receiving other antiepileptic drugs.     

Quantitation of insulin injection by high-performance liquid chromatography and high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was evaluated as a potential technique for the regulatory analysis of commercial dosage forms of insulin. A comparison was made to a liquid chromatographic analysis presently being proposed as an official monograph in the United States Pharmacopeia. The salient points of this comparison were accuracy, precision and ease of use. Both authentic (i.e. single blind, spiked) samples and commercial pharmaceutical formulations (injections) were examined. Chromatographic analyses of both commercial formulations and authentic samples were characterized by good precision, with accuracy being supported by results from authentic (spiked) samples. Conventional HPCE (by which is meant a non-micellar electrolyte used with an uncoated, unmodified fused-silica capillary) achieved reasonable accuracy, but less than impressive precision, when applied to authentic samples. When used for commercial formulations, this type of HPCE did not produce a level of accuracy suitable for regulatory purposes, even with the use of an internal standard.     

Avidin protein-conjugated column for direct injection analysis of drug enantiomers in plasma by high-performance liquid chromatography.

A new concept in high-performance liquid chromatography supports is proposed for the direct injection analysis of drug enantiomers in plasma. The new supports are designed with disuccinimidyl suberate as a hydrophobic internal region, and avidin protein as a hydrophilic and bulky surface region. Plasma proteins are excluded by the avidin phase and are eluted immediately from the column, whereas low-molecular-mass analytes can penetrate the surface region and interact with disuccinimidyl suberate. Enantiomers interact differentially with avidin, and are thereby separated. This column was used in reversed-phase high-performance liquid chromatographic analysis to determine ketoprofen enantiomers in plasma by direct injection. The recovery of racemic drug from plasma was almost 100%.     

Fluorimetric detection of serum corticosterone using high-performance liquid chromatography.

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.     

Determination of creatinine and purine derivatives in ruminants' urine by reversed-phase high-performance liquid chromatography.

A procedure is described for the rapid and simultaneous determination of allantoin, creatinine, uric acid, hypoxanthine and xanthine in sheep urine. Separation was achieved on a Novapak C18 column under isocratic conditions. The mobile phase was potassium phosphate buffer (10 mM, pH 4.0). A flow-rate of 0.5 ml/min, detection at 218 nm and a column temperature of 25 degrees C were employed with a total analysis time of less than 15 min. Detection limits for allantoin, creatinine, uric acid, hypoxanthine and xanthine were 1.0, 0.5, 0.5, 0.5 and 0.2 micrograms/ml, respectively, at a signal-to-noise ratio of 3 in a 20-microliters injection volume of tenfold-diluted urine. This sensitivity permits the precise determination of these compounds in ruminants' urine.     

Normal-phase high-performance liquid chromatography of tocopherols and tocotrienols. Comparison of different chromatographic columns

Natural vitamin E is composed of eight different vitamers (alpha-, beta-, gamma- and delta-tocopherols and alpha-, beta-, gamma- and delta-tocotrienols). As these eight vitamers have different antioxidant and biological activities, it is necessary to have quantitative data on each substance separately. The aim of this study was to find universal HPLC columns for the separation of all eight components and to test if a few columns of the same material (different batches) will give reproducible results. Normal-phase HPLC separations of vitamin E compounds in a prepared mixture (containing oat extracts, palm oil and tocopherol standards) were tried on six silica, three amino and one diol columns. As shown by calculations of retention factors (k), separation factors (alpha), numbers of theoretical plates (N) and resolutions (Rs), the best separations were obtained on three silica columns and two amino columns using 4 or 5% dioxane in hexane as the mobile phase as well as on a diol column using 4% tert.-butyl methyl ether in hexane as the mobile phase.     

Adsorbents and columns in analytical high-performance liquid chromatography: a perspective with regard to development and understanding

A brief historical survey is presented on the evaluation of silica adsorbents in analytical HPLC. The theory of analytical HPLC is mostly still being based on the height equivalent to a theoretical plate concept and the van Deemter equation that was derived from gas phase adsorption involving a linear adsorption isotherm and fast mass transfer kinetics. One can obviously wonder whether the use of the van Deemter equation is relevant and valid for the evaluation of the performance of HPLC systems, where most often the liquid solutes involve charged molecules in electrolytes and in very many cases the adsorbates are macromolecules having diffusion coefficients of small magnitude. Instead of the van Deemter equation, a multi-scale modelling approach that involves microscopic and macroscopic dynamic non-linear mass-transfer-rate models should be employed. Furthermore, advanced experimental methods for the characterisation of porous media and the distribution of the density of immobilised active sites (e.g., ligands) on surfaces as well as microscopic pore-network modelling and molecular dynamics modelling and simulation methods could be used for the design of novel adsorbents whose porous structures and immobilised active sites would provide effective mass transport and adsorption rates for realising efficient separations as well as high dynamic capacities when larger throughputs are required.     

The art and science of forming packed analytical high-performance liquid chromatography columns

Columns of packed particles still are the most popular devices for high-performance liquid chromatography (HPLC) separations because of their great utility, excellent performance and wide variety. However, the forming of packed beds for efficient, stable columns traditionally has been an art where the basics of how to form optimum beds generally was not well understood. The recent development of monolith rods was introduced in part to overcome the difficulty of producing stable beds of packing particles. However, these materials are less versatile than packed particle columns. Technology developments in recent years have produced a better understanding among those skilled in the practice of how to form optimized packed beds, and this has led to widely available, high-quality commercial columns. This presentation discusses the developments that led to the present state of column packing technology. Important steps in the packing of efficient, stable beds are described. The key step of selecting the best solvent for the slurry packing method is emphasized. Factors affecting the mechanical stability of packed columns also are discussed. The early art of packing columns now has evolved into a more scientific approach that allows the packing of good columns with a minimum of effort and time.