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.     

Determination of methotrexate in serum by high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method with ultraviolet detection was developed to quantitate methotrexate in serum-based calibrators, controls and patient samples. Sample clean-up was achieved with C₁₈ Sep-Pak Classic cartridges. The chromatographic separation was accomplished on a 5-μm Ultrasphere ODS Beckman column. 8-Chlorotheophylline was used as an internal standard. The method was validated by recovery, linearity, accuracy and precision studies. Two standard curves were constructed to cover the high and the low ends of the calibrator range (0.05–1.0 μmol/l). Response was found linear over the whole range of the calibrator set with a correlation coefficient of 0.999 and 1.00 for the low-level and the high-level curves, respectively. Accuracy varied from 12% at the lowest level to 1.2% at the highest level. The precision study showed a C.V. of 14.4% at the lowest level and 3.3% at the highest level.     

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.     

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 bestatin in serum by high-performance liquid chromatography with fluorescence detection

A simple method for the determination of bestatin and its major metabolite in man, p-hydroxybestatin, in human serum was investigated; the method employs high-performance liquid chromatography with fluorescence detection. Bestatin and p-hydroxybestatin are oxidized to phenylacetaldehyde and p-hydroxyphenylacetaldehyde, respectively, with periodate, which are then converted into fluorescent compounds with 4,5-dimethoxy-1,2-diaminobenzene. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18. The detection limits of bestatin and p-hydroxybestatin are 0.2 and 0.4 μg/ml serum, respectively. This method permits the precise determination of bestatin in serum (20 μl) from patients administered bestatin. p-Hydroxybestatin in serum can not be measured by this method because of its low concentration (less than the detection limit).     

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.     

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.     

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).     

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.     

Determination of monohydroxyethylrutoside in heart tissue by high-performance liquid chromatography with electrochemical detection

7-Monohydroxyethylrutoside (monoHER) is one of the components of the registered drug Venoruton. It showed a good protection against the cardiotoxic effects of doxorubicin. The analysis of monoHER was developed to study the pharmacokinetic profile of the drug in heart tissue. MonoHER was extracted from heart tissue homogenate with methanol. The supernatant was diluted 1:1 (v/v) with 25 mM phosphate buffer and injected onto a reversed-phase ODS column. The mobile phase consisted of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogenphosphate (pH 3.4), 10 mM acetic acid and 36 μM EDTA. The retention time of monoHER was about 5.2 min and no endogenous peaks were interfering. The lower limit of quantification was 0.072 nmol g⁻¹ wet heart tissue. The calibration line was linear up to 24 nmol g⁻¹. The within-day accuracy and precision of the quality controls (0.12, 1.2 and 12.0 nmol g⁻¹) were smaller than 17 and 19%, respectively. The between-day accuracy and precision were better than 6 and 11%, respectively. The recovery of monoHER from heart tissue ranged from 104.1 to 114.3% and was concentration independent. MonoHER was stable in heart tissue when stored at −80°C for 6 months. Repeated injection of monoHER from aliquots of 7.2 nmol g⁻¹ placed on the sample tray at 4°C for 24 h showed a decrease in the concentration of 30.3%. Analyzing sample duplicates in a mirror image sequence could compensate for the influence of this gradual decrease. The small sample volume allowed one to measure monoHER in the hearts of mice.     

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

A procedure for the high-performance liquid chromatographic determination of gentamicin in serum is described using pre-column derivatisation and UV detection. The serum proteins are precipitated with acetonitrile and the gentamicin components in the supernatant are derivatized with 1-fluoro-2,4-dinitrobenzene. The reaction products are chromatographed on a microparticulate C₁₈ reversed-phase column and detected at 365 nm. Sample volumes of 50 μl are sufficient for the determination of gentamicin concentrations in, and well below, the therapeutic range.     

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.     

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.     

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.