High-performance liquid chromatography separation of the (S,S)- and (R,S)-forms of S-adenosyl-l-methionine

S-adenosyl-l-methionine (AdoMet), an important biological cofactor, exists in two chiral forms, (S,S)- and (R,S)-, only the former of which is biologically active. Here, we have developed a chromatographic method to obtain pure (S,S)-AdoMet using a single C18 column.     

Determination of O-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for O⁶-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were constituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O⁶-Benzylguanine peak heights were compared to peak heights of O⁶-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O⁶-benzylguanine.     

Separation by high-performance liquid chromatography of (3R)- and (3S)-beta-leucine as diastereomeric derivatives

For studies on the coenzyme B12-dependent enzyme, leucine-2,3-aminomutase, (3R)- and (3S)-beta-leucines were synthesized. The 10-camphorsulfonamide p-nitrobenzyl esters could be resolved by normal-phase HPLC. A much better separation was obtained by reversed-phase HPLC of the diastereomeric derivatives obtained by treatment of -leucine with Marfey's reagent (N2-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide).     

Analysis of polyamines as their dabsyl derivatives by reversed-phase high-performance liquid chromatography

The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible (436 nm) region. The detection limit for a single dimethylaminoazobenzenesulfonylpolyamine is less than 2 pmol.     

Determination of p-chloronitrobenzene in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of p-chloronitrobenzene (p-CNB) in rat plasma. A plasma sample was deproteinized with methanol containing the internal standard (p-bromonitrobenzene). The resulting methanol eluate obtained after centrifugation was filtered and injected into a high-performance liquid chromatograph (50 μl each). A column packed with 5 μm octadecylsilane (ODS) spherical particles was used with isocratic elution of methanol—water (45:55, v/v) at a flow-rate of 1.0 ml/min. The compounds were detected by ultraviolet absorbance at 280 nm. The retention times of p-CNB and the internal standard were 12.5 and 15.5 min, respectively, at a column oven temperature of 30°C. The results were linear from 0.05 to 100 μg/ml (r = 0.999), and the detection limit was 0.01 μg/ml. The relative error and the coefficient of variation on replicate assays were less than 7 and 10%, respectively, for all concentrations studied. The overall recoveries of p-CNB were between 97 and 105%. Plasma samples could be stored for up to one month at −20°C.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Determination of nadoxolol in human plasma using reversed-phase high-performance liquid chromatography

A rapid, simple and accurate HPLC method is presented for the determination of nadoxolol in human plasma. Nadoxolol from plasma was successfully purified using an Adsorbex column. The samples were chromatographed on a LiChrosorb RP-18 (10 μm) column with methanol—acetonitrile—phosphate buffer (pH 3.3) (70:20:10) as the mobile phase. Detection was carried out at 254 nm. The method was tested for linearity (from 5 to 25 μg/ml), recovery (85%) and precision (C.V. = 4.5%).     

Determination of amidepin in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of amidepin has been developed. The method is based on the extraction of alkaline plasma with diethyl ether—dichloromethane, and the injection into the Supelcosil LC-18 column of the evaporated and reconstituted organic phase. After separation, detection is carried out by a fluorescence detector (excitation at 195 nm with no filter). The limit of detection is 10 ng/ml of plasma. The mean coefficient of variation is 12%. The plasma levels after oral administration and after intravenous administration are shown.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Determination of inulin in plasma and urine by reversed-phase high-performance liquid chromatography

We report a new HPLC procedure for measuring inulin in plasma and urine. Samples after dilution are boiled in mild acidic conditions and then analyzed on a C₁₈ column. Solvent system A is 3.2 mM HCl, pH 2.5, and B is acetonitrile-3.2 mM HCl (60:40, v/v), pH 2.5. The separation is carried out in 8 min with a flow-rate of 1.0 ml/min and the absorbance monitored at 280 nm. The relationship between inulin and the recorded peak area is linear from 0.2 to 3.2 mg/ml with a correlation coefficient of 0.999 for plasma and 0.999 for urine. Within-run precision, measured at three inulin concentrations, ranged from 0.9 to 1.7% in plasma and from 0.8 to 1.2% in urine. Between-run precision varied in plasma from 2.7 to 3.2% and in urine from 3.0 to 3.3%. Analytical recovery ranged from 102 to 107% in plasma and from 101 to 105% in urine, respectively. The method is sensitive, selective and only 30-μl samples are required. Therefore, it could be used to evaluate the glomerular filtration rate even in small babies and to perform studies in animals.     

Simultaneous determination of levomepromazine,midazolam and their major metabolites in human plasma by reversed-phase liquid chromatography

A sensitive and reliable high-performance liquid chromatographic (HPLC) assay is a prerequisite for pharmacokinetic analysis of continuous infusion of levomepromazine adjuvant to midazolam. We developed such a method to determine the levels of levomepromazine, midazolam and their major metabolites (levomepromazinesulfoxide, desmethyl-, didesmethyllevomepromazine, O-desmethyllevomepromazine and alpha-hydroxy-midazolam) simultaneously. Desmethylclomipramine was used as an internal standard (I.S.). The lower limit of quantification of this assay was set for levomepromazine 4.1 microg/l, levomepromazinesulfoxide 4.9 microg/l, O-desmethyllevomepromazine 18.4 microg/l, alpha-hydroxymidazolam 26.6 microg/l, midazolam 23.4 microg/l, didesmethyllevomepromazine 15.8 microg/l, and desmethyllevomepromazine 6.6 microg/l. The between- and within day assay variations were commonly below 5%. The recovery in human plasma for the different analytes varied between 85 and 11%. The accuracy of this assay varied between 95 and 105% for the different concentrations. The linearity of this assay was set between 25 and 800 microg/l (r(2)>0.999 of the regression line). The first results of pharmacokinetic analysis of midazolam indicated that half-life varied between 1.1 and 1.9 h. Pharmacokinetic analysis using a one-compartment model of levomepromazine revealed that the apparent volume of distribution was 4.1+/-2.4 l per kg lean body mass and the metabolic clearance was 309+/-225 l per hour per 70 kg. This assay proved to be robust and reproducible. It can reliably be used for further study of the pharmacokinetics of continuous infusion of levomepromazine.     

Determination of indinavir, an HIV-protease inhibitor, in human plasma by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay has been developed to determine the concentrations of the HIV-protease inhibitor indinavir in human plasma. The sample pretreatment involved a protein precipitation procedure using 100 μl of human plasma and 400 μl of acetonitrile. Chromatography was carried out on an Octadecyl column using a mobile phase of acetonitrile–water (40:60, v/v). The water phase contained 50 mM phosphate buffer pH 6 and 4 g/l tetramethylammoniumchloride. Ultraviolet detection at 210 nm was used. The method has been validated with regard to specificity, detection limit, lower and upper limit of quantitation, recovery, accuracy, and inter- and intra-assay precision. Stability tests under various conditions were performed. The bioanalytical assay is now in use for the determination of indinavir in several clinical pharmacokinetic studies in HIV-infected patients.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C₁₈ column using a mobile phase consisting of water–acetonitrile–1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 μl of plasma, the standard curve was linear from 0.05 to 10.0 μg/ml.