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.     

Determination of DNA base composition by reversed-phase high-performance liquid chromatography

Abstract DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 μg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.     

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), in human plasma. Didanosine (2′,3′-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a μBondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)—methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.     

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.     

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas–liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with β-glucuronidase. Separation is achieved using a C₈ reversed-phase column with a methanol—water—phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 μg/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.     

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.     

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.     

Determination of quinolone antibiotics in growth media by reversed-phase high-performance liquid chromatography

A simple, accurate, precise, and versatile high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of three quinolone antibiotics in Mueller–Hinton broth. The fluoroquinolone agents studied were ciprofloxacin, ofloxacin, and sparfloxacin; other quinolone agents have been identified using this method but not validated in this matrix (levofloxacin, clinafloxacin, temafloxacin, and trovafloxacin). In addition, several other biological growth mediums have been investigated (human serum, human urine, Todd–Hewitt growth media, Ensure enteral feeding solution, and Haemophilus growth media). This method uses UV detection (280 nm), a simple, one-step protein precipitation extraction, and separation using a C₁₈ column with an isocratic, ion-pairing mobile phase. An appropriate internal standard was obtained by using another quinolone antibiotic of differing retention time. The calibration curves were linear (r²≥0.999) over a concentration range of 0.0625–20.0 μg/ml with a lower limit of quantification of 0.1 μg/ml. The intra-day and inter-day coefficients of variation were less than 15%.     

Analysis of histidine and urocanic acid isomers by reversed-phase high-performance liquid chromatography

The qualitative separation performance of a C₁₈, C₈ and C₄ reversed-phase column was investigated for the separation of histidine and its metabolites histamine, 1-methyihistamine and trans- and cis-urocanic acid. Trans- and cis-urocanic acid were baseline separated from their precursor histidine on all three columns using isocratic elution with a mobile phase composed of 0.01 M aqueous TEAP pH 3.0 and acetonitrile at a ratio of 98:2 (v/v). However, histidine was not separated from histamine and 1-methyihistamine. Selecting the C₈ column and introducing 0.005 M of the ion pairing reagent 1-octanesulfonic acid sodium salt into the aqueous solution and acetonitrile at a ratio of 90:10 (v/v), significantly improved the separation. The separation was also followed by a change in the retention times and the order of elution. The sequence of elution was histidine, cis-urocanic acid, trans-urocanic acid, histamine and 1-methylhistamine with retention times of 5.58±0.07, 7.03±0.15, 7.92±0.18, 18.77±0.24 and 20.79±0.21 min (mean±SD; n=5). The separation on the C₈ column in the presence of the ion-pairing reagent was further improved with gradient elution that resulted in a reduction in the retention times and elution volumes of histamine and 1-methylhistamine. The detection limits of histidine and trans-urocanic acid at a wavelength of 210 nm and an injection volume of 0.05 ml were 5×10⁻⁸ mol l⁻¹ (n=3). The kinetic of the in-vitro conversion of trans- into the cis-isomer after UV irradiation was depending on the time of exposure and the energy of the light source. UVB light induced a significantly faster conversion than UVA light. TUCA and cUCA samples kept at −25°C were stable for up to 50 weeks. Samples, eluted from human skin showed various concentrations of histidine and trans- and cis-urocanic acid with an average of 1.69±0.33×10⁻⁵ mol l⁻¹, 1.17±0.43×10⁻⁵ mol l⁻¹ and 1.67±0.33×10⁻⁵ mol l⁻¹, respectively (n=8).     

Determination of trovafloxacin,a new quinolone antibiotic,in biological samples by reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C₁₈ column with a mobile phase consisting of 0.04 M H₃PO₄-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 μg/ml and the calibration curves were linear over a concentration range of 0.1 to 20..0 μg/ml (r² = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 μg/ml. Human serum samples could be stored for up to 12 months at −20°C and urine samples could be stored up to 18 months at −80°C.     

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.     

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 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.     

Determination of growth hormone-releasing hexapeptide by reversed-phase high-performance liquid chromatography with electrochemical detection

A novel HPLC method with electrochemical detection is described for the determination of a growth-hormone-releasing hexapeptide (GHRP-6). HPLC conditions, such as the column, mobile phase, and oxidation potential, were optimized for sensitivity and selectivity of analysis. GHRP-6 was separated on a reversed-phase CN column with 37% acetonitrile in 100 mM phosphate buffer (pH 7.0) as the mobile phase. The optimum electrochemical oxidation signal was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to two electroactive tryptophans and a histidine residue. Solid-phase extraction using octadecyl cartridges was optimized for sample cleanup of GHRP-6 from serum samples and the method was successfully applied over the concentration range of 5 to 100 ng/ml of analyte.     

Determination of transdermal sildenafil in nude mouse skin by reversed-phase high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5–500 ng/ml. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.