Isocratic Reversed-phase High-performance Liquid Chromatography Research Articles

COMPARATIVE STUDY OF AMYLOSE AND CELLULOSE DERIVATIZED CHIRAL STATIONARY PHASES IN THE REVERSED-PHASE MODE

A direct, isocratic, and simple reversed-phase HPLC method was described for the separation of enantiomers, the newly synthesized potential drug substance and corresponding optical impurity, employing polysaccharide-based chiral stationary phases (Chiralpak AD-RH and Chiralcel OD-RH). A baseline separation was attained with both columns. These two chiral stationary phases exhibit opposite chiral discrimination patterns concerning the elution order of enantiomers. Chiralpak AD-RH is more suitable for resolving the enantiomers. Low level quantification (0.05%) of the minor enantiomer is achieved. The analytical procedure was successfully applied to the detection of the optical impurity in a potential drug substance. The minor enantiomer was not determined quantitatively because of a trace level of it in the potential drug substances.

Quantitative determination of abacavir (1592U89), a novel nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection

Abacavir is a novel nucleoside reverse transcriptase inhibitor for the treatment of HIV-1 infection. A simple and rapid high-performance liquid chromatographic method for the quantification of abacavir in human plasma suitable for pharmacokinetic research purposes is described. Sample pretreatment consists of protein precipitation with perchloric acid. The supernatant is injected directly into the chromatographic system after centrifugation. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 285 nm. The method has been validated over the range of 20–2000 ng/ml using a volume of 300 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 7.5% for all quality control samples. The lower limit of quantitation is 20 ng/ml and the recovery of abacavir is 88.1% (±1.3%). Frequently coadministered drugs did not interfere with the described methodology. Abacavir is stable in human plasma under various relevant storage conditions, for example when stored for 51 days at −20°C. This validated assay is suited for use in pharmacokinetic studies with abacavir in human plasma and can readily be implemented in the setting of a hospital laboratory for the monitoring of abacavir concentrations.

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%.

High-performance liquid chromatography of methanol released from pectins after its oxidation to formaldehyde and condensation with 2,4-dinitrophenylhydrazine

A procedure was developed to measure the content of methanol in pectins after the base-catalysed hydrolysis of galacturonic acid methyl esters and oxidation of released methanol with potassium permanganate followed by condensation of the resulting formaldehyde (HCHO) with 2,4-dinitrophenylhydrazine (DNPH) dissolved in acetonitrile. The constant yields of resultant formaldehyde 2,4-dinitrophenylhydrazone (HCHO-DNPH derivative) were obtained at molar ratios of DNPH/HCHO higher than 5. The separation of the HCHO-DNPH derivative from DNPH reagent was achieved by isocratic reversed-phase HPLC equipped with the spectrophotometric detector set at a wavelength of 351 nm. The calibration curve was linear in the methanol concentration range between 0.04 and 15 μmol/ml ( R=0.9995). The total recovery from pectin solutions spiked with methanol was equal to 100.6±5.1%.

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.

Simultaneous determination of catecholamines in rat brain tissue by high-performance liquid chromatography

A novel and highly sensitive method has been developed for the determination of catecholamines [noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA)] in brain tissue. The method uses isocratic reversed-phase HPLC with amperometric end-point detection. The calibration curve was linear over the range 10–150 pg on-column. The assay limits of detection for NA, DA, 5-HT, 5-HIAA and HVA were 3.8, 3.8, 6.8, 5 and 7.5 pg on-column, respectively. The mean inter- and intra-assay relative standard deviations (RSDs) over the range of the standard curve were less than 5%. The absolute recoveries averaged 99.1%, 99.5%, 97.7%, 99.5% and 98.8% for NA, DA, 5-HT, 5-HIAA and HVA, respectively.

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%.

SOME ASPECTS OF SIMULTANEOUS DETERMINATION OF PYRIDINOLINES AND DESMOSINES AS COLLAGEN AND ELASTIN RESORPTION MARKERS BY MEANS OF REVERSE PHASE HPLC, UTILIZING FLUORESCENCE AND/OR UV-ABSORPTION MONITORING

Isocratic reverse phase HPLC method was elaborated and optimized for simultaneous determination of pyridinoline (PD), deoxypyridinoline (DPD) and isodesmosine (IDES) in urine and in tissues within approximately 16 minutes. Mobile phase is 0.02 M heptafluorobutyric acid (HFBA) with 0.01 M (NH2)2SO4 and 8 - 12 % acetonitrile (ACN), at pH 1.55, flow rate 0.5 mL/min. and temperature of 40°C. The addition of ammonium sulphate affects positively the quality of chromatographic separation. The glass column 150 × 3 mm is filled with 7 μm spherical silica C18. The concentrations of determined substances are monitored by detector of fluorescence at λEX/λEM = 297/400 nm for both PD and DPD, and at λEX/λEM = 275/320 nm for IDES. Both PD and DPD are measurable with the same sensitivity limit of 200 femtomols; in the case of IDES the determination sensitivity is approximately 5x lower (1 pmol). Since IDES concentration in urine is considerably lower, it means that approximately 25 mL of the sample must be processed and the time of preparatory work is, therefore, considerably increased. In this system, desmosine (DES) is practically not detectable by fluorescence. It was verified by comparison of detection sensitivity in fluorescence and UV-absorption measurements that fluorescence is substantially more sensitive (with the detectors used by us) for the determination of PD, DPD, and IDES. If it is necessary to quantify DES as well, UV-absorption must be measured, and therefore the urine must be concentrated at least 5 times more. HPLC separation itself is very effective, on baseline, and with high number of theoretical plates of the column for all relevant substances. HPLC assays of tissue samples are of higher quality and more transparent than those of urine.

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 m M sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO 4), allows a quantification limit of 2.52 μg ml −1 and a detection limit of 0.83 μg ml −1. Recoveries and absolute recoveries of cefepime from plasma were 96.13–99.44% and 94–102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 μg ml −1 ( n=10).The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.

Detection of clobetasol propionate as an undeclared steroid in zinc pyrithione formulations by high-performance liquid chromatography with rapid-scanning ultraviolet spectroscopy and mass spectrometry

Clobetasol propionate, an anti-inflammatory glucocorticosteroid, was detected in an over-the-counter topical drug product with no indication on the label of this compound as an ingredient. The product was formulated as a topical spray, a cream, or a shampoo and labeled to contain zinc pyrithione as the active ingredient. The finding of clobetasol propionate in the pharmaceutical products was shown by comparison to an authenticated standard of clobetasol propionate by retention time on normal-phase and reversed-phase HPLC, UV spectroscopy, LC–MS and LC–MS–MS. A simple method was developed and validated for the assay of clobetasol propionate by isocratic reversed-phase HPLC. Several lots contained clobetasol propionate at therapeutic levels of 0.02–0.06%. Zinc pyrithione formulations from two other manufacturers were free of clobetasol propionate.

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 m M sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 m M diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di- tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.

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.

Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC–FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2–84.9% at concentrations of 10 μg/l. The R.S.D. values were 1.7–3.9% ( n=8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.

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.