Determination of serum retinol by reversed-phase high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic method was developed for the determination of serum levels of retinol in humans. A direct serum injection technique after deproteinisation was used to avoid lengthy pretreatment steps which can result in degradation of retinol during analysis. The column used was CLC-ODS, the mobile phase was acetonitrile-water and detection wavelength was 328 nm. Deterioration in column performance was not observed even after injection of 300 samples. The lower detection limit was 10 μg/l. On analyzing a serum pool six times, a C.V of 0.7% was obtained. The method is quantitative, reproducible, rapid and highly accurate for routine analysis.     

Selective analysis for adenosine using reversed-phase high-pressure liquid chromatography

A high-pressure liquid chromatographic procedure for the selective determination of adenosine in the presence of other nucleic acid components is reported. Reversed-phase microparticle columns and an isocratic elution mode of dilute potassium dihydrophosphate and anhydrous methanol were used. The analysis is specific for adenosine and is achieved in less than 10 min. An example of the use of this analysis in a biomedical study is reported.     

Some applications of reversed-phase high-performance liquid chromatography to oligosaccharide separations

Oligosaccharide separation on reversed-phase high-performance liquid chormatographic columns have been examined using a range of aqueous solvents. Addition of anionic, cationic and non-ionic surfactants, tetramethyl urea and organic solvents to the mobile phase cause faster elution of oligosaccharides, and allow the separation of the larger oligomers in an acceptable time. Addition of neutral, inorganic salts increase the retention factors considerably, and allows good resolution of some compounds poorly resolved in water alone.The mechanism operating in the separations approximates to that invoked in the solvophobic theory of reversed-phase chromatography. There is some evidence also of hydrogen bond effects. The improvements described should prove useful in the isolation and analysis of neutral oligosaccharides in general, and in structural analyses of polysaccharides in paritcular.     

Determination of phenylalanine in serum using reversed-phase liquid chromatography and fluorescence detection

A liquid chromatography procedure is reported for determining phenylalanine in small volumes of serum. A 10-μl volume of serum was deproteinized with ethanol and an aliquot was derivatized with dansyl chloride reagent. The dansylated phenylalanine and the norleucine internal standard were separated using reversed-phase chromatography and measured with a fluorescence detector. Linearity was excellent over the range 50–800 mg/l. Within-run precision was better than 4%. Total analysis time including chromatography was approximately 40 min. As little as 300 pg of dansylated phenylalanine was detected.     

An ultraperformance liquid chromatography method for the normal-phase separation of lipids

An ultraperformance liquid chromatography method using normal-phase solvents, a silica column, and evaporative light-scattering detection is presented. The method is based on a quaternary gradient profile and is capable of resolving the major neutral and polar lipids present in plasma and animal tissue in under 5 min, with a total cycle time of 11 min. Limits of quantitation for 7 different lipid classes were on the order of 200 ng of material on column which enables an accurate analysis from as little as 20 μL of plasma or 50 mg of tissue for typical samples. Intraday and interday precision for the determination of the major lipid classes in human plasma ranged from 3.6 to 10.5% CV with a variability in retention time of less than 6%. The utility of the method is demonstrated through the separation and quantitation of lipids in mouse plasma, liver, and heart tissue.     

Assay for taurine conjugates of bile acids in serum by reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a new high-performance liquid chromatographic (HPLC) procedure for quantifying taurine conjugates of bile acids in serum. The technique involved three basic steps. The first removed free amino acids via solid-phase extraction of the serum. The second step involved the reaction of the extracted serum with the enzyme choloylycine hydrolase, which liberated the taurine from the conjugated bile acids. The third step was the reversed-phase HPLC separation of o-phthalicdicarboxaldehhyde derivatives of taurine. The assay provides a simple technique for determination of the total amount of taurine-conjugated bile acids in serum.     

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm.Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.     

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 μg/ml. Recoveries varied from 91 to 10% with coefficients of variation ranging from 0.387 to 7.95%.     

Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C(18) reversed-phase column (250 mm x 4.6 mm I.D., particle size 5 microm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6-99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2-5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.     

Quantitative analysis of melphalan and its major hydrolysate in patients and animals by reversed-phase high-performance liquid chromatography

The detection of 4-bis-(2-hydroxyethyl)amino-1-phenylalanine (l-DOH) in blood samples taken from patients after treatment with melphalan [4-bis-(2-chloroethyl)amino-1-phenylalanine, l-PAM] suggests that the quantification of this major hydrolysate of l-PAM can be of considerable importance in l-PAM chemotherapy. A reversed-phase high-performance liquid chromatographic procedure has been developed for the quantitative analysis of both l-PAM and l-DOH in biological samples, with a detection sensitivity of 0.1 ppm. This method provides a distinct separation of l-PAM (retention time 12 min) and l-DOH (retention time 6.5 min), with no interference from the biological background (retention time 1.4–3 min).     

A reversed-phase high-performance liquid chromatography method for analysis of monoclonal antibody-maytansinoid immunoconjugates

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for detection and quantification of free maytansinoid drug in disulfide-linked conjugates between monoclonal antibodies and the maytansinoid drug DM1 (MAb-DM1). Mobile phases and gradient conditions were optimized for separation of several DM1-related free drug species from MAb-DM1 conjugates. The selectivity, linearity, and reproducibility of the method are reported. Reduction of the disulfide-linked DM1 followed by RP-HPLC allowed estimation of purity of MAb-linked DM1 as well as recovery of L-DM1. The method was also used to estimate drug per MAb ratios, which were consistent with those determined by UV spectroscopy.     

Simultaneous determination of azathioprine and 6-mercaptopurine in serum by reversed-phase high-performance liquid chromatography

The simultaneous determination of azathiprine and its metabolite 6-mercaptopurine in serum by reversed-phase high-performance liquid chromatography is described. 6-Mercaptopurine was converted to a derivative, 6-mercaptopurine-N-ethylmaleimide, which is stable against autoxidation, on reaction with N-ethylmaleimide. Since the N-ethylmaleimide derivative was more hydrophobic than the parent compound, it could be extracted into ethyl acetate together with azathioprine and the derivative was retained on the reversed-phase column better than 6-mercaptopurine. In addition, 6-mercaptopurine-N-ethylmaleimide absorbed at the same wavelength (280 nm) as azathioprine. Consequently, this derivatization procedure enabled the simultaneous extraction, separation, and detection of these compounds.     

Determination of cholesteryl 14-methylhexadecanoate in blood serum by reversed-phase high-performance liquid chromatography

A simple reversed-phase HPLC method has been developed for the determination of cholesteryl 14-methylhexadecanoate (CMH) in the blood serum. Lipids are extracted from 0.1 ml of blood serum and after centrifugation, the extract is chromatographed and individual cholesteryl esters, including CMH are separated and eluted with an acetonitrile—2-propanol mixture. The quantification of cholesteryl 14-methylhexadecanoate is precise and highly reproducible and the analysis may be completed within 35 min. The level of CMH in the blood of cancer patients appears to be a useful marker of malignant tumors.     

多维液相色谱及其在生命科学中的应用

介绍了一种适用于复杂体系样品分析的分离技术-多维液相色谱。对该技术的原理、分类、模式的选择做了介绍,并引证了其在蛋白质组研究、药物研究等复杂体系样品分析领域的最新应用及发展。     

Pharmacokinetic studies of amiloride and its analogs using reversed-phase high-performance liquid chromatography

We have studied the pharmacokinetics of amiloride and its analogs. A high-performance liquid chromatographic method has been adapted for the measurement of amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N,N-hexamethylene)amiloride (HMA) in mouse plasma, kidney, liver and tumor tissues. The method uses a C₈ preparative solid-phase column, followed by separation using a reversed-phase C₁₈ column (250×4 mm I.D., 5 μm particle size) with detection by ultraviolet absorption at 365 nm. Reversed-phase separations were performed at ambient temperature using a non-linear gradient method with two different mobile phases: mobile phase A was 100% acetonitrile while mobile phase B was 0.15 M perchloric acid at pH 2.20 (flow-rate was 1.2 ml/min). The retention times for amiloride, benzamil (used as an internal standard), EIPA and HMA are 13.4, 19.5, 21.8 and 23.5 min, respectively. The calibration curves are linear over the range of 0.1–50 μM in plasma and in tissues. The half-lives of amiloride, EIPA and HMA (and their confidence intervals) in plasma after intraperitoneal injection of drugs into mice were 68.8±0.2, 31.2±2.5 and 39.3±7.9 min, respectively. Amiloride was detected as a metabolite of EIPA but not of HMA. When EIPA was injected at a dose of 10 μg/g body weight, it was cleared rapidly from liver, but concentrations > 1 μM were sustained for at least 2 h in murine kidney and in a transplantable tumor.