Sensitive High Pressure Liquid Chromatography Research Articles

The bioavailability of ofloxacin from several formulations

The bioavailability of ofloxacin after a single dose of one of two tablet formulations (200 or 400 mg) or a liquid formulation (1.67 mg/ml) was investigated in 24 healthy male volunteers in an open randomized, crossover design study with a 5-day wash-out period between doses. Plasma concentrations of ofloxacin were determined at various times after administration by a sensitive and specific High Pressure Liquid Chromatography (HPLC) method. Ofloxacin was well absorbed after each administration, although somewhat more slowly after the tablet formulations than the solution. Mean AUC values of 31.8, 31.3, and 31.3 micrograms.hr/ml were calculated after administration of the solution, 2 x 200 mg tablets and a 400 mg tablet, respectively. Thus, the bioavailability of the tablets was in excess of 98% that of the liquid reference. Mean Cmax values of 4.4, 3.7 and 3.7 micrograms/ml were observed at Tmax values of 0.8, 1.6 and 1.8 hours after administration of the solution, 2 x 200 mg tablets and a 400 mg tablet, respectively. The drug was well tolerated and no adverse effects necessitating subject withdrawal were noted during the study.

Stability of Ketoconazole in Ethanolic Solutions

AbstractThe stability of the antifungal drug Ketoconazole (Kz) in an ethanolic solution was examined. The ethanolic solutions of Kz were placed in either clear or amber glass bottles and stored either at room temperature or at 8 C for 29 days. Kz was assayed using, a sensitive simple high pressure liquid chromatography procedure. Results demonstrated no statistically significant difference between or among the test solutions over time, regardless of the storage conditions or the concentrations (P>0.05).

Effects of smoking and oral contraception on plasma β-carotene levels in healthy women

Oral contraceptive use and smoking have been known to affect plasma vitamin levels. Total carotenoids have been studied with spectrophotometry, a relatively insensitive technique. In this study plasma concentrations of β-carotene and retinol were measured in coded samples by sensitive high-pressure liquid chromatography in a cross-sectional study of 149 normal healthy women attending a family planning clinic. At the time of recruitment in the morning, a general health questionnaire was administered for patient age, methods of contraception, smoking habits, and food intake at breakfast. Of the 149 enrolled volunteers, 88 were oral contraceptive users and 61 were not users. Among users, 21 smoked cigarettes, and there were 18 smokers among nonusers. Oral contraceptive users had significantly lower plasma concentrations of β-carotene (p < 0.001) and higher retinol levels (p < 0.0001). Plasma β-carotene or retinol levels did not differ among users of intrauterine contraceptive devices or barrier methods of contraception. No association was noted between the plasma levels of these two micronutrients and age > or <30 years. Cigarette smoking alone was associated with significantly reduced plasma β-carotene levels in nonusers (p < 0.001). Combined cigarette smoking and oral contraceptive usage were associated with low plasma β-carotene levels; the results appear to be additive. These findings suggest a possible synergistic effect on plasma β-carotene levels from the use of both cigarette smoking and oral contraception.

Evidence for the involvement of prostaglandins in modulation of acetylcholine release from canine bronchial tissue

A sensitive high pressure liquid chromatography (HPLC) method was used to measure acetylcholine release from canine bronchial tissue in response to electrical stimulation. Indomethacin enhanced the efflux of acetylcholine, an effect that was reversed by the addition of prostaglandin E 2 (PGE 2). Prostaglandin D 2 (PGD 2) had no effect on acetylcholine output. These data suggest that prostaglandins, possibly PGE 2, are involved in a negative feedback control mechanism for acetylcholine release in canine bronchi.

Inappropriate use of metronidazole in gastrointestinal surgery

Pharmacokinetic data suggest that current treatment regimens of metronidazole in abdominal surgery are not always appropriate. We have examined antibiotic concentrations during emergency and elective surgery using a specific and sensitive high pressure liquid chromatography assay. Serum and tissue concentrations were measured after intravenous infusion during intra-abdominal surgery and after suppositories given before appendicectomy. After intravenous dosage, bactericidal concentrations were reached in serum (13.6 +/- 7.8 micrograms/ml), bowel (9.0 +/- 6.6 micrograms/g), tumour (9.9 +/- 7.1 micrograms/g) and subcutaneous fat (4.9 +/- 3.2 micrograms/g). After suppositories the concentrations were: serum 4.6 +/- 2.7 micrograms/ml, appendix 1.1 +/- 0.6 micrograms/g, fat 1.5 +/- 0.9 micrograms/g and peritoneal fluid 4.7 +/- 4.3 micrograms/g. These values were obtained at a mean interval of 86.9 +/- 27.5 min following administration of the drug. Serum concentrations were measured during post-surgical infusion of 500 mg i.v. 8 or 12 hourly. Mean concentrations after 8 hourly doses were 16.3 +/- 4.85 micrograms/ml pre-dose and 28.7 +/- 6.76 micrograms/ml post-dose, with evidence of drug accumulation by detection of metabolites. Twelve hourly infusions gave pre-dose levels of 7.4 +/- 3.86 micrograms/ml and post-dose levels of 17.1 +/- 3.69 micrograms/ml. Metronidazole (500 mg) intravenously at induction of anaesthetic gives effective prophylactic concentrations in all tissues including tumour, but a metronidazole 1 g suppository before appendicectomy does not provide reliable tissue concentrations. Metronidazole (500 mg) i.v. 12 hourly gives effective bactericidal concentrations of the drug and is more economical.

Intracellular trafficking of cell surface sialoglycoconjugates.

Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.

Identification of fluorescent glycopeptide derivatives by two consecutive high pressure liquid chromatographic procedures.

Reversed phase high pressure liquid chromatography (HPLC) was used to separate individual components of the complex glycopeptide antibiotic teicoplanin in microgram quantities with gradient elution. Each of eight different fractions was then subjected to a specific and highly sensitive HPLC method, which has been developed for the determination of teicoplanin concentrations in biological specimens. This analytical procedure includes precolumn derivatization with fluorescamine and isocratic elution. The fluorescent teicoplanin derivatives were identified by comparing their retention times in both HPLC procedures. Derivatization resulted in increased hydrophobicity and improved chromatographic separation, but the order of elution of the different compounds was not changed. The antimicrobial activity of the individual underivatized fractions correlated with their respective contents of total teicoplanin A2, whereas the pseudo-aglycone A3 appeared less active. Similar techniques have the potential to be applied to other complex glycopeptide antibiotics.

Successful treatment with aerosolized pentamidine of Pneumocystis carinii pneumonia in rats.

We examined both the therapeutic efficacy and tissue distribution of aerosolized pentamidine in immunosuppressed rats with Pneumocystis carinii pneumonia. In rats immunosuppressed by 5 weeks of pretreatment with dexamethasone, a 2-week course of 5 mg of aerosolized pentamidine per kg per day, administered free or encapsulated in the drug carrier system (liposomes), eradicated P. carinii pneumonia in 75% of treated animals. At this dose, extrapulmonary drug uptake as measured by a sensitive high-pressure liquid chromatography assay was negligible. No significant differences in tissue distribution were noted between aerosolized free and liposome-encapsulated pentamidine. In rats receiving dexamethasone for 6 weeks prior to treatment with pentamidine, both lung uptake and therapeutic efficacy of aerosolized pentamidine (5 mg/kg per day) were substantially reduced. Aerosolized pentamidine appears to be an effective therapy for P. carinii pneumonia in rats and produces significantly lower extrapulmonary drug deposition than parenteral administration. The severity of P. carinii involvement at the time of treatment influences both the level of drug delivery to the lung and the response to aerosolized pentamidine therapy.

DETERMINATION OF CYTOSINE‐CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 290 nm) and UV-B irradiation (290-320 nm) in the 2'-deoxy-[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol. 52, 519-524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10-20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.

Studies on aminophylline disposition I. A rapid and sensitive HPLC assay for ethylenediamine in plasma and urine.

A simple and rapid assay for ethylenediamine in plasma and urine is described. Ethylenediamine is treated with m-toluoyl chloride, yielding its N,N'-di(m-toluoyl) derivative, which is extracted into dichloromethane and assayed by reversed-phase high pressure liquid chromatography (HPLC) with u.v. detection. Quantitation is achieved with reference to the corresponding derivative of cadaverine as internal standard. The assay is reproducible, and the lower limit of detection is 0.05 microgram ml-1. Calibration curves in plasma and urine are linear over the concentration range 0.05-100 micrograms ml-1. The assay has been applied to the analysis of ethylenediamine in plasma and urine following the administration of aminophylline orally and intravenously to a volunteer.

Specific determination of pentamethylmelamine and its demethylated metabolites in human plasma by high pressure liquid chromatography.

Pentamethylmelamine (PMM) and its demethylated metabolites tetra-N2246, tri-N246 and tri-N224 have comparable relative antitumor potencies in animal systems. We have developed a sensitive and specific high pressure liquid chromatography (HPLC) assay for these compounds in the plasma of patients receiving pentamethylmelamine, a cytotoxic S-triazine derivative now undergoing clinical trials. The method involves analysis of an ethyl acetate extract of plasma by a high pressure liquid chromatograph containing a reverse-phase column and ultraviolet detection at 240 nm. The mobile phase is acetonitrile and KH2PO4 buffer (pH 5.5); gradient elution over 25 min yields relative retention times to hexamethylmelamine as internal standard of 0.36, 0.40, 0.54 and 0.70 for tri-N246, tri-N224, tetra-N2246 and PMM. The lower limit of sensitivity for each is 300 ng/ml. Pharmacokinetic studies on four patients receiving a 24-h infusion of PMM (doses 900-3000 mg/m2) showed a mean terminal half-life of PMM of 101 +/- 28 (SD) min, a mean volume of distribution of 2036 +/- 75 ml/kg, and a metabolic clearance rate of 14.2 +/- 3.7 (ml/min)/kg. Mean areas under the plasma-vs-time curves of tri-N246, tri-N224 and tetra-N2246 were 126%, 41% and 56% of PPM, demonstrating extensive formation of these metabolites in man.

Bioavailability and disposition of prednisone and prednisolone from prednisone tablets

AbstractThe relative bioavailability of two 50 mg prednisone tablet formulations was determined in 18 healthy male volunteers who were not pretreated with dexamethasone. Plasma and urine samples were collected over a 24‐h period and their concentrations of prednisone and prednisolone were assayed by a specific and sensitive high pressure liquid chromatography (HPLC) method. Bioavailability was assessed by comparing the areas under the plasma concentration‐time curves and by the relative amounts of prednisone and prednisolone in urine. There were no significant differences between the bioavailability of the film‐coated and standard 50 mg tablets. Eight subjects subsequently received a 40 mg equivalent intravenous dose of prednisolone as prednisolone succinate to provide plasma concentrations of prednisolone similar to concentrations found after oral doses of prednisone. The systemic bioavailability of prednisolone, the active metabolite generated from film‐coated and standard prednisone tablets, was estimated to be 0·77±0·15 and 0·80±0·11, respectively. The nonlinear distribution, the interconversion, and the simultaneous elimination of these drugs, however, complicate any assessment of their relative and absolute bioavailability.

A rapid and sensitive high pressure liquid chromatography assay for deoxyribonucleoside triphosphates in cell extracts

An assay for simultaneous quantitation of deoxyribonucleoside triphosphates (dNTPs) in cell extracts is described. Following destruction of ribonucleotides by treatment with periodate and methylamine, dNTPs are separated by high pressure liquid chromatography and quantitated by their ultraviolet absorbance upon elution. Destruction of ribonucleotides is complete (>99.9%), the resulting mixture is suitable for direct injection without further manipulation, and recovery of dNTPs is reproducible and near quantitative. This technique offers a simple and rapid alternative to either DNA polymerase or thin-layer chromatography assays for dNTPs in cellular extracts.

Rapid and sensitive high-pressure liquid chromatography technique for detecting impurities in benzaldehyde U.S.P.

Rapid and sensitive high-pressure liquid chromatography technique for detecting impurities in benzaldehyde U.S.P.

Use of high-pressure liquid chromatography to determine plasma levels of metronidazole and metabolites after intravenous administration.

A rapid and sensitive high-pressure liquid chromatography assay for metronidazole and its two principle metabolites, 1-(2-hydroxyethyl-2-hydroxymethyl)-5-nitro-imidazole [hydroxy metabolite] and 1-acetic acid-2-methyl-5-metronidazole [acid metabolite], was developed. The retention times observed were 5.7, 3.3, and 4.5 min, respectively. A reverse-phase muC(18) Bondapak column using a solvent system of methanol, acetonitrile, and 0.005 M pH 4 potassium dihydrogen phosphate (4:3:93, vol/vol) was used to achieve separation of the three compounds. Patients receiving metronidazole therapy were given a loading dose of 13.6 mg of drug per kg intravenously over 1 h, followed by a maintenance dose of 1.43 mg/kg per h. The range of metronidazole concentrations observed was 6.8 to 47.5 mug/ml. These levels are well above the minimal inhibitory concentrations of most clinically significant anaerobic bacteria including Bacteroides fragilis. Little of the acid metabolite was observed in the plasma. The concentration of hydroxy metabolite ranged from 1.6 to 16 mug/ml. The latter may represent an additional source of antimicrobial activity since the hydroxy metabolite has approximately 30% the biological activity of metronidazole.