Isocratic Reversed-phase High-performance Liquid Research Articles

Measurement of hyperforin a constituent of St. John’s wort in plasma by high-performance liquid chromatography

Hyperforin is a constituent of Hypericum perforatum extracts (St. John’s wort, H. perforatum), which have antidepressant action. Hyperforin was extracted from plasma utilising a solid-phase extraction procedure. Chromatography was performed by isocratic reversed-phase high-performance liquid chromatography with UV end-point detection. The calibration curve was linear over the range 0.15–3 μg per ml of plasma. The sensitivity for hyperforin was 4.5 ng on-column. Mean inter- and intra-assay relative standard deviations over the range of the standard curve were less than 5%. The absolute recovery for hyperforin averaged 97.8%.

Quantitative determination of efavirenz (DMP 266), a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection

Efavirenz is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the quantification of efavirenz in human plasma suitable for therapeutic drug monitoring in plasma is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 246 nm. The method has been validated over the range of 10 to 10 000 ng/ml using a volume of 250 μl of plasma. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.3% for all quality control samples. The lower limit of quantitation is 10 ng/ml and the recovery of efavirenz from human plasma is 106.4% (±1.8%). Frequently co-administered drugs did not interfere with the described methodology. Efavirenz is stable under various relevant storage conditions, for example when stored for 24 h at room temperature. This validated assay is suited for use in pharmacokinetic studies with efavirenz and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz concentrations.

Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C 18 silica column at 55°C using an isocratic mobile phase which contained 27% 1-propanol in a 25 m M potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149→Asp149 or Asn152→Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time.

Improved high-performance liquid chromatographic assay for the determination of “high-energy” phosphates in mammalian skeletal muscle: Application to a single-fibre study in man

A sensitive and reproducible method for the determination of adenine nucleotides (ATP, IMP) and creatine compounds [creatine (Cr), phosphocreatine (PCr)] in freeze–dried single human muscle fibre fragments is presented. The method uses isocratic reversed-phase high-performance liquid chromatography of methanol extracts. Average retention times (min) of ATP, IMP and PCr, Cr from standard solutions were 10.6±0.42, 2.11±0.06 ( n=6) and 10.5±0.31 and 1.19±0.02 ( n=9), respectively. Detection limits in extracts from muscle fibre fragments were 2.0, 1.0, 3.0 and 2.0 mmol/kg dm, respectively. The assay was found successful for analysis of fibre-fragments weighing ≥1 μg.

An Assay for Mandelate Racemase Using High-Performance Liquid Chromatography

Mandelate racemase (EC 5.1.2.2) catalyzes the interconversion of the two stereoisomers of mandelic acid. A fixed-time assay for the quantification of mandelate racemase activity has been developed. The assay involves enzymatic conversion ofR-mandelate toS-mandelate (or the reverse reaction) followed by separation and detection of the substrate and product using isocratic reversed-phase high-performance liquid chromatography on a Sumichiral OA-6100 column and absorbance detection. This method offers an economical and efficient alternative to the existing circular dichroism-based and coupled assays.

Rapid quantification of delavirdine, a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with fluorescence detection

Delavirdine is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of delavirdine in human plasma suitable for drug monitoring in patients is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase, high-performance liquid chromatography coupled with fluorescence detection. The optimal excitation and emission wavelengths are 300 and 425 nm, respectively. The method has been validated over the range of 50–50 000 ng/ml using only 200 μl of plasma samples. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.4% for all quality control samples. The lower limit of quantitation is 50 ng/ml. Recovery of delavirdine from human plasma is 93.8%. Delavirdine is stable under various conditions, for example 1 h at 60°C and one week at 4°C. This validated assay is suited for use in pharmacokinetic studies with delavirdine and can readily be implemented in the setting of a hospital laboratory for the monitoring of delavirdine concentrations.

Determination of hypericin in plasma by high-performance liquid chromatography

Hypericin, a polycyclic dianthroquinone, is one of the characteristic ingredients of Hypericum perforatum extracts (St. John′s wort, HP), which has antidepressant effects. Hypericin and the internal standard (I.S.), dansylamide, were extracted from plasma utilizing solid-phase extraction (SPE). Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with fluorescence end-point detection. The calibration curve was linear over the range 5–100 ng per ml of plasma. The sensitivity for hypericin was 75 pg on column. Mean inter- and intra-assay coefficients of variation (C.V.s) over the range of the standard curve were less than 10%. The absolute recovery for hypericin averaged 72.6%.

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C 4 column maintained at 30°C eluted with 52.5% (w/w) acetonitrile in water, containing 100 m M NaClO 4 and 10 m M HClO 4. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor ( k′) of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1–100 μg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 μg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94±9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.

Simultaneous Analysis of 4- and 5-Series Lipoxygenase and Cytochrome P450 Products from Different Biological Sources by Reversed-Phase High-Performance Liquid Chromatographic Technique

Quantification of lipoxygenase and cytochrome P450 products of both arachidonic acid (AA) and eicosapentaenoic acid (EPA) is of broad interest due to the multiple biological activities of these compounds. Wedeveloped a method combining (i) solid-phase extraction, (ii) isocratic reversed-phase high-performance liquid chromatographic separation, and (iii) online photodiode array detection with spectrum analysis for identification and measurement of all main 4- and 5-series eicosanoids (leukotrienes, hydroxyeicosatetraenoic acids/hydroxyeicosapentaenoic acids, epoxyeicosatrienoic acids) within one run. With these procedures, standard mixtures of AA- and EPA-derived lipid mediators were recovered from different biological liquids, like lung perfusate, human bronchoalveolar lavage fluid, and cell supernatant with linear characteristics for each compound. Recoveries of the different lipid mediators exceeded 80% showing excellent reproducibility. Application of the method to isolated, perfused, and ventilated human lungs challenged with the calcium ionophore A23187 and to human neutrophils stimulated in the presence of arachidonic acid and eicosapentaenoic acid withN-formyl-methionyl-leucyl-phenylalanine demonstrated the generation of a large array of lipoxygenase and cytochrome P450 products. Thus, convenient quantification of 4- and 5-series eicosanoids in fluids of biological interest is achieved by a technique comprising solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, and photodiodearray-based online spectrum analysis of eluting compounds.

Ethyl acetate extraction procedure and isocratic high-performance liquid chromatographic assay for testosterone metabolites in cell microsomes

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for separation of testosterone and its main metabolites over the nominal range 20 to 40 μg/ml and 280 to 4600 ng/ml, respectively. Mobile phase composition (phosphate buffer–methanol–acetonitrile, 50:38.5:11.5) was optimised by studying the influence of numerous chromatographic parameters. The most critical one was the ratio CH 3CN/CH 3OH. Good recoveries (around 90% for all compounds) and an improved specificity were assessed by a double ethyl acetate extraction of biological samples. According to the performance criteria tested, the method could be applied to enzymatic inhibition and induction in vitro studies.

Study of factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)–thiol derivatives by liquid chromatography

A detailed investigation of the factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)–thiol derivatives (i.e. cysteine, homocysteine and cysteinylglycine) is described. Essentially, this assay entails extracting specific thiols by plasma disulphide bond reduction, protein precipitation, sulphydryl compound derivatization with the thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F), and subsequent separation with isocratic reversed-phase high-performance liquid chromatography. By improving the reliability of several analytical parameters (composition of the mobile phase, pretreatment of the sample using different reducing and protein precipitation agents, and optimization of the derivatization of thiols with SBD-F), a number of critical issues can be identified and solved.

Simultaneous measurement of venlafaxine and its major metabolite, oxydesmethylvenlafaxine, in human plasma by high-performance liquid chromatography with coulometric detection and utilisation of solid-phase extraction

Venlafaxine, oxydesmethylvenlafaxine and an internal standard (paroxetine) were extracted from plasma by a solid-phase extraction technique. Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric endpoint detection. The standard curves were linear over the range 0–200 ng/ml for both venlafaxine and oxydesmethylvenlafaxine in plasma. The mean inter- and intra-assay coefficients of variation over the range of the standard curves were less than 10%. The absolute recovery averaged 74% for venlafaxine and 67% for oxydesmethylvenlafaxine. The sensitivity was 0.5 ng for both the analytes. Plasma profiles of the analytes following oral administration of venlafaxine, are presented.

Determination of paclitaxel in human plasma using single solvent extraction prior to isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid–liquid extraction involving a mixture of acetonitrile– n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water–methanol–tetrahydrofuran–ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10–500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6±8.52 and 93.7±5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.

High-Performance Liquid Chromatographic Analysis of Amino Acid- and Peptide-Derived Chloramines

An isocratic reversed-phase high-performance liquid chromatographic method is reported for the analysis of amino acid-and peptide-derived chloramines (these are important intermediates in two very different processes: destruction of microbes by the neutrophil and disinfection of water with chlorine). Specifically, results are reported for the chloramines derived from the following amino acids and dipeptides: taurine, Ala, Gly, Ser, Thr, Phe, Val, AlaGly, GlyGly, PheGly, SerGly, and ValGly. Analyses are performed on a 250 × 4.6-mm C18 column using a buffered water-acetonitrile mixture as the mobile phase. For samples containing hydrophilic chloramines, an ion-pairing agent is added to the mobile phase. Two detection methods are used: direct ultraviolet (UV) detection of the chloramine at 254 nm and indirect UV detection of I3- at 350 nm following postcolumn reaction with iodide. Unexpectedly, the decomposition of amino acid-derived chloramines is found to greatly accelerate during chromatographic elution on a reversed-phase column.

Simple method for the quantitation of mycophenolic acid in human plasma

A simple and rapid isocratic reversed-phase high-performance liquid chromatographic method using UV detection was developed for the quantitation of mycophenolic acid (MPA) in human plasma. The assay was sufficiently robust to allow the analysis of up to 100 samples in a single analytical run.

Analysis of recombinant human growth hormone directly in osmotic shock fluids

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.

High-performance liquid chromatographic determination of 4-methylpyrazole in plasma and in dialysate

A rapid method for the determination of 4-methylpyrazole (4-MP) levels in plasma and in dialysate by isocratic reversed-phase high-performance liquid chromatography with UV detection is described. The internal standard was the 3-methylpyrazole (3-MP). Plasma sample preparation consisted of a protein precipitation. Dialysate samples were injected without preparation. The method was linear up to 30 mg l −1 in plasma and up to 5 mg l −1 in dialysate. The within-day precisions (C.V.) were less than 4% in plasma and were less than 2% in dialysate. The day-to-day precisions (C.V.) were less than 7% in plasma and were less than 3% in dialysate. This method is easy to perform and has practical interest for clinicians who need to monitor in emergency 4-MP levels in ethylene glycol and methanol poisonings.

GlutathioneS-Transferase-Catalyzed Conjugation of Bioactivated Aflatoxin B1in Rabbit Lung and Liver

Aflatoxin B1(AFB1) requires bioactivation to AFB1-8,9-epoxide for carcinogenicity, and glutathioneS-transferase (GST)-catalyzed conjugation of activated AFB1with glutathione (GSH) is a critical determinant of susceptibility to the mycotoxin. Incubations containing [3H]AFB1, rabbit liver microsomes, an NADPH-generating system, 1 mMGSH, and GST-containing lung or liver cytosol were performed to assess the abilities of lung and liver GSTs to conjugate AFB1-8,9-epoxide. [3H]AFB1–GSH was isolated by isocratic reverse-phase high-performance liquid chromatography (HPLC) and quantitated by liquid scintillation spectroscopy. Maximal [3H]AFB1–GSH formation rates were significantly lower for lung than for liver (0.3 ± 0.1 and 1.7 ± 0.4 nmol/mg/hr, respectively). Immunoprecipitation of rabbit pulmonary cytosolic GSTs with anti-alpha or anti-mu GST antisera decreased [3H]AFB1–GSH production by approximately 45 and 51%, respectively, indicating that alpha-class and mu-class GSTs are of similar importance in catalyzing this reaction in the lung. Because mu-class GSTs comprise only a small proportion of total lung GST content, these enzymes have high specific activity toward AFB1-8,9-epoxide. In contrast, the pi-class GST appeared to play a negligible role. Using a rat liver microsomal system to generate both AFB1exo- andendo-epoxide isomers, and analysis based on chiral HPLC, we found that rabbit liver cytosolic GSTs catalyzed formation of both AFB1exo- andendo-epoxide–GSH conjugates, whereas pulmonary cytosolic GSTs catalyzed formation of only theexostereoisomer at detectable levels. Despite a preference for conjugating the more mutagenic AFB1exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bioactivated AFB1in lung may be an important factor in the susceptibility of the lung to AFB1toxicity.

Separation and identification of astaxanthin esters and chlorophylls in Haematococcus lacustris by HPLC

An isocratic reverse-phase high performance liquid chromatographic method was developed for the separation of carotenoids and chlorophylls from extracts of Haematococcus lacustris. Astaxanthin monoesters are the principal carotenoids in Haematococcus lacustris. Five astaxanthin monoesters, which accounted for 79% of the total carotenoids, were identified and the individual contents were determined by HPLC to be 7.3, 24.9, 6.1, 49.4 and 12.3% of the total astaxanthin monoesters, respectively. The maximum absorption wavelengths of the five astaxanthin monoesters in the mobile phase were all 479 nm, and that of free astaxanthin, the main saponification product of the astaxanthin esters, was 478 nm.

High-performance liquid chromatographic determination of cyclohexylhydantoin and cyclohexylidenehydantoin formed during the synthesis of phenylhydantoin from hydantoin and cyclohexanone

dl-Phenylhydantoin can be converted into d-phenylglycine by an enzymatic process. d-phenylglycine is an important starting material in the production of β-lactams such as semisynthetic penicillins and cephalosporins. In our laboratory, the synthesis of phenylhydantoin was achieved from hydantoin and cyclohexanone in the presence of a base. An efficient and fast isocratic reversed-phase high-performance liquid chromatography method was developed for the determination of phenylhydantoin, cyclohexylhydantoin and cyclohexylidenehydantoin. Quantitative analysis was carried out by an external standard method.