Rapid sample preparation method for the determination of chloramphenicol in swine muscle by high-performance liquid chromatography

A simple and rapid sample preparation method for the determination of chloramphenicol in swine muscle tissue at the 10 micrograms/kg level is described. The method comprises sonication-aided extraction with ethyl acetate, addition of hexane to the extract and cleaning up and concentration of the extract on a small column packed with silica gel. Analysis was performed by high-performance liquid chromatography on a ChromSep column with ChromSpher C8 using acetonitrile-sodium acetate buffer as the mobile phase. Detection was performed at 280 nm. Mean recoveries from spiked muscle samples were 79 +/- 3% (10-50 micrograms/kg). The distribution of chloramphenicol in different muscle and fatty tissues from a pig to which a single dose of chloramphenicol was administered was also investigated.     

Improved method for the determination of serotonin in plasma by high-performance liquid chromatography using on-line sample pre-treatment

An improved method for the determination of serotonin in platelet-rich plasma (PRP) and platelet-poor plasma (PPP), by reversed-phase high-performance liquid chromatography with electrochemical detection and direct plasma injection, is described. The chromatographic system comprises a strong cation-exchange pre-column and a C18 analytical column. The method is selective, rapid, simple and sensitive, and offers good reproducibility and recovery. Reference values for serotonin concentrations in healthy adults (n = 10) are 31 nM for PPP and 6 nmol per 10(9) platelets for PRP. The conditions used for the preparation of PRP and PPP may influence the serotonin concentration in PRP.     

Immunoaffinity chromatography for the sample pretreatment of Taxus plant and cell extracts prior to analysis of taxanes by high-performance liquid chromatography

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.     

Automated determination of polyamines by high-performance liquid chromatography with simple sample preparation

Recently, a new fully endcapped reversed-phase packing material, Inertsil, was introduced, especially suitable for the determination of basic compounds. We used this packing material to separate o-phthaldialdehyde (OPA) derivatives of amino acid derivatives completely from the OPA derivatives of spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine (CAD). The obtained separation made the commonly used off-line extraction procedure redundant and thus an on-line sample clean-up was introduced. This enabled automation of the procedure resulting in a better reproducibility and a more efficient use of equipment. Furthermore, no studies are required to determine the extraction recovery.

The present method has a cycle time of 30 min. A linear response for each polyamine was found up to 250 pmol, with an R² ranging from 0.9981 (SPM) to 0.9998 (CAD). The limit of detection, calculated at a signal-to-noise ratio of 3, was 0.1 pmol, corresponding to a plasma concentration of 0.1 μmol/l. The coefficient of variation (CV) for the peak area was below 3% and for retention times below 0.5% (n=15).

In order to evaluate the applicability of the method, three different types of sample were chromatographed, e.g. urine (obtained from healthy human volunteers), pig plasma and sulfosalicylic acid homogenates of pig intestine biopsies. Tissue homogenates and urine-specimen could easily be quantitated, while plasma concentrations were just above the limit of detection, resulting in a plasma CV ranging from 4.8% (SPM) to 13.6% (SPD) and a tissue CV ranging from 2.1% (SPM) to 8.5% (CAD), The urinary CVs were not determined.

In conclusion, the present method provides an easy way to measure polyamine concentrations for most applications.     

Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions

A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C₁₈ column by gradient elution that used mixtures of trifluoroacetic acid (TFA)–acetonitrile and TFA–water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.     

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.     

Performance of experimental sample injectors for high-performance liquid chromatography microcolumns

An experimental injector for HPLC microcolumns and a 3-nl conductivity detector connected directly to the injector outlet with a 19-nl tube were used to study injector dispersion, guide the design of improved injectors, and suggest appropriate injection techniques. With regard to the small injection volumes required when no on-column concentration technique is used, we show that in some circumstances: (i) there are two volumes to be considered, the sample volume (that which is intended to be injected) and the effective injection volume (that which contains all the sample after it has completely emerged from the injector). Due to dispersion, the latter is often many times the former. An injector performance factor is defined as the ratio of the two volumes. (ii) A smaller sample chamber volume in an injector does not necessarily produce a proportionately smaller effective injection volume, in which case there is a reduction of peak height that degrades sensitivity without a commensurate reduction in peak width that would improve resolution. (iii) Adjusting the geometry of the sample chamber and stator passage can significantly improve injector performance, as illustrated for sample volumes from 2 nl to 1 microl. (iv) In some cases, reducing the diameter of an injector passageway in an attempt to reduce dispersion actually causes performance to worsen.     

Improved high-performance liquid chromatography method for quantitation of proline and hydroxyproline in biological materials

Numerous high-performance liquid chromatography systems have been described for the determination of hydroxyproline (Hyp) and proline (Pro) levels in biological materials. These methods are generally complicated and have shortcomings in applicability due to poor separation, low sensitivity or derivatization-associated problems. The large number of chemical components present in biological samples further complicates the analysis of Hyp which usually occurs in extremely low concentrations. The present investigation describes the development of a simple highly sensitive derivatization method which results in good separation of peaks and which is capable of quantitating less than 10 pmol of Hyp and Pro in complex test systems. The method is based on removal of o-phthalaldehyde (OPA) derivatives of primary amino acids using reversed-phase chromatography, pre-column derivatization with OPA and phenylisothiocyanate, and detection of derivatized Hyp and Pro using a UV detection system. The procedure yields good peaks and a 93% recovery of Hyp and Pro provided that the analysis is initiated within 5 min of completion of OPA derivatization. While a 93% recovery of Pro was obtained up to 100 min post-derivatization with OPA, the recovery of Hyp is decreased to approximately 80% within the same time interval.     

Improved high-performance liquid chromatographic method for analysis of cyclosporin A using an automated sample processor

Transplant patients receiving the immunosuppressive drug cyclosporin A require regular monitoring to maintain levels within a narrow therapeutic range. A stable, accurate and reproducible high-performance liquid chromatographic method for analysis of cyclosporin A in whole blood has been developed using the Varian Advanced Automated Sample Processor. Starting with 200 microliters of blood, absolute recovery of both cyclosporin A and the internal standard was 81% with a detection limit of 12.5 ng/ml. The assay is perfectly linear over the range 0-1000 ng/ml (r2 = 1.0). At a concentration of 250 ng/ml, the coefficient of variation, both between samples and between assays, is 1.87%. Chromatographic cycle time is 10.2 min per sample. Up to eighty samples can be processed by one person in a working day, with final results within 16 h.     

Sample preparation for high-performance liquid chromatography in the clinical laboratory

Techniques for the preparation of clinical specimens for high-performance liquid chromatography are described. Liquid samples containing high enough concentrations of analytes may be injected directly or after centrifugation. Proteins may be removed by precipitation with organic, anionic or cationic precipitants or by ultrafiltration. Compounds having large partition coefficients may be extracted with an organic solvent. Extraction may also be performed following derivatization, ion-suppression, ion-pairing, salt addition or complex formation. Solid phase extraction may be off-line using disposable cartridges or on-line with an Advanced Automated Sample Processor. The advantages and disadvantages of each method and the trends in sample preparation techniques are discussed.     

Electrodialytic sample treatment coupled on-line with high-performance liquid chromatography

Summary An electrodialytic sample treatment method coupled on-line with high-performance liquid chromatography (EDIST-HPLC) is discussed in this paper. The performance of EDIST as a function of the donor-phase (sample solution) flow rate, the voltage applied over the electrodialysis block, and the time of dialysis has been studied using the basic drug ephedrine as a model compound. Enrichment of the analyte by a factor of 10–20 was possible. The determination of human plasma spiked with ephedrine is briefly discussed.     

Microwave-assisted extraction by fast sample preparation for the systematic analysis of additives in polyolefins by high-performance liquid chromatography

This paper presents a new approach for complete and systematic analysis of organic additives in polyolefins. The proposed procedure is a convenient combination of sample preparation, performed by microwave-assisted extraction (MAE), and direct chromatographic evaluation of extract by high-performance liquid chromatography coupled with ultraviolet and evaporative light scattering detection. In particular two microwave-assisted processes are reported and discussed: the one-step MAE, useful for additives with low-medium dipolarity (like stabilizers, flame retardant, antistatics, slip and processing agents), and the two-step MAE, useful for additives with either high dipolarity (like organic salts, antigasfading, antiacid, nucleating agent) or high molecular mass (like polymeric hindered amine light stabilizers). Both the proposed processes have been tested on representative additives in five commercially common polymeric matrices, demonstrating their satisfactory analytical results, in terms of repeatability and percentage recoveries, and their good performances, in terms of safety and time/solvent consumption, in comparison with those of traditional extraction methods.     

Determination of biocatalyst consumption in an aminopeptidase process using automated sample preparation and high-performance liquid chromatography

A rapid and sensitive method has been developed for the determination of the biocatalyst consumption in the chemo-enzymic production of optically pure natural and synthetic alpha-H-amino acids. It is based on automated sample preparation from an enzymic reaction mixture, reversed-phase high-performance liquid chromatographic separation, post-column reaction and fluorimetric detection. The assay procedure has been applied to the enzymic conversion of racemic norvaline amide into L-norvaline, catalysed by an L-specific aminopeptidase from Pseudomonas putida. Both norvaline amide and norvaline can be analysed in a single assay in the low nanogram range. The method yields reproducible results and requires 30 min from the time of sampling the enzymic reaction mixture to quantitation. The reaction mixture is automatically sampled and analysed several times during the course of the reaction. With the results obtained a conversion curve can be constructed from which the exact biocatalyst consumption can be calculated. By adaptation of the mobile phase, the method can also be applied to other amino acid amides used as substrates in the aminopeptidase reaction.     

An improved method for high-performance liquid chromatography of gossypol

Earlier liquid-chromatographic methods for the determination of gossypol, based on highly acidic methanolic solvents, provide broad tailing peaks. The use of acetonitrile with aqueous phosphoric acid/tri-n-butylammonium phosphate at pH 3.5 and a high-resolution radial-compression column gave greatly improved performance and excellent peak shape.     

Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation

A simple assay procedure for tyrosine hydroxylase activity in crude tissue samples was devised that requires minimal sample preparation and use of high-performance liquid chromatography with coulometric electrochemical detection. After incubation of enzyme samples, such as human brain homogenates or rat pheochromocytoma PC12h cells, with L-tyrosine and a tetrahydropterin cofactor, in the presence or absence of p-bromobenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, the reaction was terminated by addition of an equal volume of 0.1 M perchloric acid. For quantitation of L-DOPA produced, the sample was centrifuged, filtered and directly applied to the chromatographic apparatus connected to a coulometric electrochemical detector. This method makes redundant a time-consuming step in the previous methods, purification and concentration of L-DOPA or dopamine using alumina. The reaction conditions for the assay of tyrosine hydroxylase activity in brain homogenates and PC12h cells were re-examined by this method. Both tyrosine hydroxylase samples required a naturally occurring cofactor, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4], catalase and NSD-1055 for the full activity, and tyrosine hydroxylase in human brain homogenates required Fe2+ ions for its full activity. (6R)BH4 proved to be a more effective cofactor than a synthetic cofactor, (6RS)-methyl-5,6,7,8-tetrahydropterin, which is commonly used for this assay.