Analysis of minocycline by high-performance liquid chromatography in tissue and serum

A sensitive and rapid reversed-phase high-performance liquid chromatography assay can be used to accurately determine serum and tissue minocycline concentrations. Minocycline is a broad spectrum tetracycline derivative with many applications. Tissue and serum samples were obtained from guinea pigs that had received either topical or intravenous minocycline. Samples were extracted using a Sep-Pak C₁₈ cartridge and were injected into a μBondapak C₁₈ column with an isocratic methanol mobile phase. Samples were analyzed using UV detection and produced sharp peaks with a retention time of 2.5 min. The lower limit of detection was 100 ng and drug recovery was 61%. This method greatly facilitated the analysis of minocycline while allowing for sensitivity.     

Estimation of 26-hydroxycholesterol in serum by high-performance liquid chromatography and its measurement in patients with atherosclerosis

A method for analysing 26-hydroxycholesterol (26OHC) in serum and tissue samples using solid phase extraction and high-performance liquid chromatography is reported. This procedure was used to measure the levels of 26OHC in the sera of apparently healthy subjects and of 18 patients with angiographically proven atherosclerosis. Sixteen of the patients had levels within or below the range detected in the apparently healthy subjects (125-294 ng/ml), indicating that high 26OHC levels cannot be a major factor in the development of atherosclerosis. However, when the patients and the normal subjects were combined in a group, there was a significant positive correlation (r = 0.54, P less than 0.01) between serum cholesterol and serum 26OHC, and that correlation approached significance for each of the individual groups (P = 0.06 for each group). These results suggest that there is an association between cholesterol and 26OHC levels in human serum.     

Micro-determination of tobramycin in serum by high-performance liquid chromatography with ultraviolet detection

A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-μl samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5–16 mg/l. The absolute recovery of tobramycin is 41%.The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm × 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)—acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Rapid analysis and quantitation of PCR products by high-performance liquid chromatography

HPLC utilizing a high efficiency anion-exchange column provides a rapid and easily automated technique for the qualitative and quantitative analysis of subnanogram to microgram amounts of DNA fragments generated by the PCR. The accuracy, precision and linearity of this method exceed those attainable with electrophoretic techniques. In addition, because of the nondestructive nature of HPLC, the desired PCR amplification product can be purified for subsequent utilization. Thus, liquid chromatography extends the utility of the PCR technique to those applications requiring precise quantitation.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 × 4.0 mm I.D., 5 μm particle size) protected by a guard column (Perisorb RP-18, 30 × 4.0 mm I.D., 30–40 μm particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5–100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) − 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0–97.8%; method comparison c(bioassay) = 1.092c(HPLC) − 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

Quantitation of harringtonine and homoharringtonine in serum by high-performance liquid chromatography

Harringtonine and homoharringtonine are naturally occurring alkaloids with demonstrated antineoplastic activity against certain types of leukemias in cell cultures, experimental animals, and initial clinical trials. Sample preparation consists of addition of the internal standard (one compound used as the internal standard for the other), solvent extraction with methylene chloride, washing with ammonium formate, and evaporation to dryness. The residue is dissolved in the mobile phase (40% methanol—60% 0.1M ammonium formate) and an aliquot is chromatographed on μC₁₈ reversed-phase column (flow-rate 1.5 ml/min). Peaks are detected with a spectrophotofluorimeter by monitoring the emission at 320 nm with excitation wavelength of 280 nm. Limit of detection is 10 ng/ml (20 nM) for both compounds; reproducible quantitation can be made to 30 ng/ml (60 nM).     

Clinicopathologic features in colorectal cancer patients with microsatellite instability

The microsatellite instability (MSI) mutational pathway is critical to carcinogenesis in a small but significant proportion of colorectal cancers. While MSI is identified in most cancers in individuals with hereditary non-polyposis colorectal cancer, the majority of MSI tumors are found in individuals with sporadic disease. Colorectal cancers arising as a result of MSI have distinct clinicopathologic features distinguishing them from those with microsatellite stability. MSI colorectal cancers affect a larger percentage of women, are usually localized proximal to the splenic flexure, and have a higher incidence of synchronous and metachronous tumors. They are associated with a mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like inflammatory response, and a higher grade but lower stage. Overall survival is better in individuals with MSI. The benefit of chemotherapy in MSI colorectal cancers, with and without lymph node metastases, remains unclear.     

Determination of pyridinium crosslinks in plasma and serum by high-performance liquid chromatography

A chromatographic method for the determination of pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in serum and plasma is described. The analytical procedure involved plasma or serum purification by ultrafiltration (20 000 relative molecular mass cut-off) under centrifugation at 2500 g for 4 h, as an innovative step. Analysis was done by isocratic high-performance liquid chromatography with fluorescence detection. The linearity of the method was tested from 0.6 to 15 pmol/ml and 0.12 to 3 pmol/ml for Pyr and Dpyr, respectively. The detection limit was 60 fmol/ml for both crosslinks. Except for Dpyr in plasma (coefficient of variation 19.9%), intra-assay variation was always below 10% in serum and plasma. The method has been applied to the quantification of crosslinks in serum and plasma of healthy volunteers and also in mouse and rat plasma. Serum proved to be the most suitable biological fluid for the systemic measurement of these compounds in humans and under the experimental conditions used, contained an average of 3.62 ± 0.65 and 0.7 ± 0.18 pmol/ml Pyr and Dpyr, respectively.     

Fluorometric determination of octopamine in tissue homogenates by high-performance liquid chromatography

A rapid, sensitive method for quantitative determination of octopamine (a biogenic amine found in both vertebrate and invertebrate nervous tissue) has been developed using reversed-phase high-performance liquid chromatography. The biogenic amine is extracted with perchloric acid from tissue homogenates and derivatized witho-phthalaldehyde (OPT) prior to chromatography. Separation of the fluorescent octopamine-OPT adduct from other biogenic amines was achieved using a Bondapak C₁₈ reversed-phase column and isocratic elution with a methanol-(0.08 mol/liter) acetic acid (5050 by vol, pH 2.9) mobile phase. A variable wavelength fluorometer with an 8-l flow cell was used for detection (excitation 340 nm, 418 nm secondary filter). Linearity ranged from 500 pg to 30 ng injected onto the column. Recovery of internal standard added to tissue homogenates averaged 65.4% with a standard deviation of 3.1%. The method has been used for the determination of octopamine in ganglia ofAplysia californica.This work was supported by the Naval Medical Research and Development Command, National Naval Medical Center, Department of the Navy, Research Task No. ZF51.524.023.1007. The opinions and statements contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large.     

Colorimetric determination of hydroxyurea in human serum using high-performance liquid chromatography

A high-performance liquid chromatography assay for hydroxyurea in human serum was developed based on a commercial colorimetric assay kit for urea (Sigma Diagnostics). Serum (0.5 ml), spiked with methylurea as an internal standard, was treated with 70% perchloric acid. Supernatant (0.2 ml) was combined with 0.7 ml of BUN acid reagent and 0.6 ml of BUN color reagent. The resulting colored reactant (100 μl) was analyzed on a 300×3.9 mm Bondclone 10 C₁₈ column coupled with a UV–Vis detector, at 449 nm. The mobile phase was 13% acetonitrile in water. Retention times of colored derivatives of hydroxyurea and methylurea were 6.5 and 12.2 min, respectively. The log–log calibration curve was linear from 0.0065 to 1.31 mM. Average accuracy was 99.9±4.0% and the intra- and inter-day error of assay did not exceed 11%.     

Analysis of carminomycin in human serum by fluorometric high-performance liquid chromatography

A method is given for the determination of carminomycin (CMM) and a major metabolite carminomycinol (CMMOH) in serum from cancer patients after intravenous administration of carminomycin as the free drug.CMM and CMMOH are extracted from serum with chloroform, the extract evaporated and the residue dissolved in methanol. High-performance liquid chromatography analysis utilized a C₁₈ μBondapak reversed-phase column eluted with 0.1 mol/l acetate buffer (pH 4) — acetonitrile (60:40, v/v) with fluorescence detection. The assay is linear, reproducible, and precise with a limit of detection of 2 ng/ml. Representative serum levels of CMM and CMMOH in a cancer patient are presented.     

Determination of sotalol in human cardiac tissue by high-performance liquid chromatography

A sensitive and quantitative reversed-phase HPLC method for the analysis of -sotalol in human atria, ventricles, blood and plasma was developed. Sotalol was determined in about 100 mg of human right atria, left ventricles, and in 500 μl of blood and plasma samples of patients undergoing coronary bypass surgery or heart transplantation. Patients were taking 80–160 mg of sotalol as an antiarrhythmic agent. Atenolol was used as an internal standard certifying high precision of measurement. Sotalol blood and plasma concentrations correlated linearly to the obtained signals from 26.5 ng/ml to 2.12 μg/ml. Sotalol tissue concentrations showed linearity between 0.27 ng/mg and 10.6 ng/mg wet weight. The limit of quantitation was 0.27 ng/mg at a signal-to-noise ratio of 10. Sotalol was extracted from homogenized tissue with a buffer solution (pH 9) and the remaining pellet was extracted with methanol. The methanol extract was evaporated under nitrogen and reconstituted in buffer (pH 3). The whole extract was cleaned by solid-phase column extraction, eluted with methanol, evaporated again, reconstituted in the mobile phase (acetonitrile-15 mM potassium phosphate buffer pH 3, 17:83, v/v) and injected onto the HPLC column (Spherisorb C₆ column, 5 μm,, 150×4.6 mm I.D). For the detection of sotalol, the UV wavelength was set to 230 nm. Recoveries of sotalol and atenolol in atria and ventricles were 65.6 and 75.0%, respectively. Intra- and inter-assay coefficients of variation for tissue concentrations were 3.38 and 6.14%, respectively. Intra- and inter-assay accuracy for determined tissue sotalol concentrations were 94.9±6.3 and 99.6±4.1%.     

Determination of nucleotide pools in plant tissue by high-performance liquid chromatography

A procedure for the determination of nucleotide pools in plant tissue by HPLC is described. Sample preparation includes the extraction with 0.4 M HClO4, a purification step, which proved to be essential, on a disposable prepacked phenyl-bonded column, neutralization by KOH, and concentration by freeze-drying. The determination of a broad spectrum of ribonucleotides including the ribonucleosides was performed by combining anion-exchange and reversed-phase HPLC. Data are presented for suspension-cultured cells of Nicotiana tabacum and Datura innoxia and for the leaf and root of tobacco.     

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1-20 microg mL(-1) for MTU and 0.1-10 microg mL(-1) for PTU, the detection limits were 0.03 microg mL(-1) for PTU, 0.03 microg mL(-1) for MTU and the quantification limits were 0.1 microg mL(-1) for PTU, 0.1 microg mL(-1) for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.