High-pressure Liquid Chromatographic Assay Research Articles

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.

Smoking-induced changes in nicotine disposition: application of a new HPLC assay for nicotine and its metabolites.

A sensitive, rapid high-pressure liquid chromatographic assay was developed to compare the disposition of an intravenous dose of 14C-nicotine in normal, carefully matched smokers and nonsmokers. The elimination half-lifes of nicotine and cotinine were shorter in smokers than in nonsmokers. Also consistent with an inductive effect of smoking was the increased nicotine elimination rate constant in smokers, but smoking induced more complex kinetic changes: nicotine volume of distribution was diminished in smokers, whereas nicotine clearance and area under the concentration-time curve were unchanged. The presence of nicotine and its principal metabolites in a morning specimen of urine obtained from nonsmokers before 14C-nicotine administration suggests ubiquitous, passive exposure to and absorption of chemicals present in cigarette smoke.

Biosynthesis of the Indole Alkaloids. Cell‐free Systems from Catharanthus roseus Plants

AbstractCell‐free systems from Catharanthus roseus plants are utilized for various studies relating to the biosynthesis of indole alkaloids. Tryptamine (5) and secologanin (6), two fundamental building units, are shown to be incorporated into the alkaloid vindoline (7). In another study, catharanthine (18) and vindoline (7) are utilized by this enzyme system and coupled to the important bisindole biointermediate 3′,4′‐anhydrovinblastineThe previously [20] used name for 17, 3′, 4′‐dehydrovinblastine, is incorrect. (17). The latter substance is, in turn, incorporated and converted to the natural alkaloids leurosine (8), Catharine (9) and vinblastine (10), thereby providing information about the biosynthesis of these complex molecules. High pressure liquid chromatography assay of the enzymic mixture sheds light on the enzymes involved in the coupling of 18 and 7.

Maternal and fetal pharmacokinetics of moxalactam given intrapartum

Two grams of moxalactam was given intravenously to 28 women at high risk for infection following cesarean delivery. After a mean time of 48 minutes from infusion, maternal sera, cord sera, and uterine tissue obtained at delivery had concentrations of moxalactam of 62 μg/ml, 22.2 μg/ml, and 9.6 μg/gm, respectively. The maternal serum half-time was calculated to be 2.1 hours. R and S epimeric distribution was determined in these sera and tissues, and the mean R/S ratios were 0.95, 0.93, and 1.22 for the three groups, respectively. The significance of these observations is discussed. A new method in which a high-pressure liquid chromatographic assay is used is described, and results are compared to those obtained with the microbiological assay. The high-pressure liquid chromatographic method was found to be quick, accurate, and reproducible.

Stability of dobutamine hydrochloride in selected large-volume parenterals

Stability of dobutamine hydrochloride when mixed with large-volume parenteral solutions was assessed. Dobutamine hydrochloride was added to large-volume solutions of 5% dextrose injection, 0.9% sodium chloride injection, lactated Ringer's injection, and 5% dextrose and 0.45% sodium chloride injection, in both glass and polyvinyl chloride containers; the initial concentration was 1 mg/ml. After 0.25, 1, 3, 8, 24, and 48 hours, the concentration of dobutamine hydrochloride was determined by high-pressure liquid chromatography assay, and each solution was visually examined for evidence of haze, precipitation, color change, or evolution of gas. Concentration of dobutamine hydrochloride in the samples did not exhibit any appreciable change over the 48-hour period, and no HPLC peaks indicating degradation products were noted. Color changes were observed in some of the solutions, but no other visual changes occurred. There were no apparent differences in stability between the admixtures packaged in glass and those in polyvinyl chloride bags. At the concentration studied, dobutamine hydrochloride is stable in the admixtures tested for a minimum of 48 hours.

A sensitive high-pressure liquid chromatographic method for the determination of propranolol in microliter serum samples

A high-pressure liquid chromatographic assay capable of quantitating subnanogram per milliliter concentrations of propranolol in microliter volume of serum was developed. Propranolol was extracted from serum with hexane at pH 9.7. Further sample clean up was achieved by back extraction. Separation of propranolol from its metabolites and normal serum constituents was performed on 10 μm C18 reversed-phase column packing. High sensitivity was attained with fluorescence detection using a low UV excitation wavelength. This assay is suitable for use in pharmacokinetic studies involving small laboratory animals.

An Improved High-Pressure Liquid Chromatographic Assay for Secobarbital in Serum

A high-pressure liquid chromatographic method for the analysis of secobarbital in serum was developed. Secobarbital was extracted from buffered serum (pH 5.5) with a solvent mix of hexane-ether-n-propanol. 5-(4-Methylphenyl)-5-phenylhydantoin was added as an internal standard. Separation of secobarbital and internal standard from serum constituents and other drugs was achieved on a 5-μm C-18 reversed-phase column using an acetonitrile-phosphate buffer (pH 4.4) mobile phase. The eluent was monitored at 195nm. The sensitivity limit of the assay was ~0.02 μg/ml with 0.5ml of serum sample. The application of this method to pharmacokinetic studies in pediatric patients was demonstrated.

Stability of stored methacholine chloride solutions: clinically useful information.

Methacholine inhalation challenge (MIC) has been shown to be an extremely useful diagnostic test. Because a decrease in the time and expense involved in the preparation of methacholine chloride solutions might encourage more laboratories to perform MIC, we assessed the stability of several different concentrations of methacholine chloride in solution over a period of 4 months. We used and compared 2 different assay techniques: a high pressure liquid chromatography assay and a colorimetric assay. Comparable results were obtained by both assays and demonstrated that methacholine solutions stored either at room temperature or at 4 degrees C showed no significant decomposition over a period of 4 months. From our results, we conclude that: (1) methacholine chloride solutions are much more stable than stated in the Merck Manual, (2) the original data of MacDonald and coworkers on the stability of methacholine chloride solution are accurate, (3) our high pressure liquid chromatography method is an accurate and highly specific technique for measuring methacholine chloride solutions. The major clinical implication of our results is that the time and cost necessary to prepare methacholine chloride solutions is much less than previously thought. This should encourage a more widespread use of this important diagnostic technique for the demonstration of bronchial hyperreactivity.

High-Pressure Liquid Chromatographic Assays for Ticarcillin in Serum and Urine

Rapid and sensitive high-pressure liquid chromatographic (HPLC) assays for ticarcillin in serum and urine have been developed. Sample pretreatment and optimized chromatographic conditions are presented for a C-18-bonded reversed-phase column used in an internal standard assay method. Ticarcillin has a retention time of ~5.3min at a flow rate of 1.5ml/min for a mobile phase of acetonitrile-aqueous 0.06 M sodium biphosphate, pH 2.05, (50.5:100). In a two-step extraction procedure, the ticarcillin extraction efficiencies from serum and urine were 76.1 ± 4.7 and 80.9 ± 3.2%, respectively. The assay sensitivity limit for ticarcillin in these fluids is ~1.0 μg/ml. A comparison is made of the HPLC and microbiological assay results for ticarcillin in 20 different but equally divided serum samples obtained from two volunteers.

Improved High-Pressure Liquid Chromatographic Method for the Analysis of Erythromycin in Solid Dosage Forms

A stability-indicating high-pressure liquid chromatographic (HPLC) method for the assay of erythromycin in enteric film-coated tablets was developed. The method used a reversed-phase column at 70° with a mobile phase of acetonitrile-methanol-0.2 M ammonium acetate-water (45:10:10:35) at pH 7.0. The column effluent was monitored at 215nm. Several reversed-phase columns were evaluated for the analysis of erythromycin. The HPLC method was also applicable for the analysis of salts and esters of erythromycin. The linearity and precision of the HPLC assay method for erythromycin in the solid dosage form were examined by spiking erythromycin into a tablet placebo at 60-120% of the label. The recovery of erythromycin was 99.9% with a relative standard deviation of <1%'. The correction factors to express the results of HPLC in terms of antimicrobial bioequivalency against Staphylococcus aureus ATCC 6538P for erythromycins A, B, and C were determined to be 1.0,0.92, and 0.48, respectively. Eight lots of tablets were assayed by the HPLC method, and the results, expressed in terms of erythromycin bioequivalency, showed no statistically significant difference from those of the microbiological assay method.

Interferences in a high pressure liquid chromatographic assay of theophylline.

Interference by salicylic acid was noted in a high pressure liquid chromatographic assay of theophylline in serum. The acid eluted very close to theophylline in a mobile phase consisting of 0.01 M acetate buffer, pH 4.0, containing 28% methanol on a C-18 reverse-phase column. The two peaks could be resolved by switching to a mobile phase containing 18% methanol, 1.6% acetonitrile, and 1.6% acetic acid in water. However, in this mobile phase traces of the extracting solvent, ethyl acetate, caused a sharpening of the theophylline peak leading to spuriously high results. The problem was overcome by using chloroform to extract the theophylline from serum.

Clinical pharmacokinetics of cefotaxime in patients with normal and reduced renal function.

Two analytic methods for measuring cefotaxime, microbiologic and high-pressure liquid chromatography (HPLC), were compared in normal healthy subjects and in patients with impaired renal function. Results of studies showed that in healthy subjects the levels of desacetylcefotaxime are low relative to those of cefotaxime and that either a bioassay or HPLC is adequately specific for determination of pharmacokinetic parameters. However, in patients with decreased renal function, desacetylcefotaxime reaches much higher levels than in healthy subjects. Thus, the less specific bioassay produces misleading data on serum and urine concentrations in these patients, and the HPLC assay of cefotaxime is recommended for estimating pharmacokinetic parameters in patients with decreased renal function. In healthy subjects and in patients with a creatinine clearance greater than 20 ml/min, neither cefotaxime nor its metabolites accumulates after multiple dosing. If creatinine clearance is less than 20 ml/min, metabolites begin to accumulate. It is recommended that until further data are obtained the dose of cefotaxime given to patients with an estimated creatinine clearance of less than 20 ml/min be half that given to patients with values greater than 20 ml/min.

High-Pressure Liquid Chromatographic Assay of Cloxacillin in Serum and Urine

Two rapid, specific, and sensitive high-pressure liquid chromatographic (HPLC) assays were developed for cloxacillin in serum and urine. A reversed-phase column (RP-8) was selected for use with two different sets of HPLC conditions and sample pretreatment procedures. Cloxacillin extraction efficiencies are reported from serum and urine. Equations are presented for linear relationships between peak height or peak area ratios of cloxacillin to nafcίllin (internal standard) and the cloxacillin concentration over a range of 0–80 μg/ml. The sensitivity limit of these assays was ~0.3 μg/ml of a standard solution for one method and 0.05 μg/ml for the other HPLC assay.

Benzidine-congener-based azo dyes: assays for purity and residues in feces from dosed rats.

Analytical methods were required to determine purities of benzidine-congener-based azo dyes and residues of the intact dyes in feces from rats before valid metabolism studies of such compounds could be conducted. A procedure is described for purity assays based on reduction of the dyes with stannous chloride followed by gas chromatography of the released free amine. Sixteen different samples of commercial dyes based on three benzidine congeners were assayed; purities ranged from 26.4 to 83.4%. Several dyes were also shown to be partially purified by cold water washes. A method to determine two intact dyes in feces from dosed rats, which consisted of extraction with dimethylformamide: water, clean-up by a rapid procedure using an octadecylsilane column, and quantification by ion-pair high-pressure liquid chromatography (HPLC) is reported. minimum detectable levels of both dyes in feces are 0.2 ppm. Excretion profiles based on parallel HPLC and radioassays of feces from rats dosed with 14C-labeled Direct Blue 15 and Direct Red 2 are presented. Based on radioassays, about 74% of each dose was excreted via the feces; however, HPLC assays showed that only about 11% of each dose was present as intact dye in the excrement.

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.

Pharmacokinetics of mitomycin C in rabbit and human.

A sensitive and specific high-pressure liquid chromatographic assay was developed to characterize the plasma elimination and urinary excretion of mitomycin C in humans. Extraction of mitomycin C and an internal standard, porfiromycin, from plasma by chromatography over a non-ionic resin, Porapak Q, yields high recovery of both compounds and facilitates measurement of as little as 5 ng mitomycin C by reversed-phase high-pressure liquid chromatography. The assay was used to characterize the plasma elimination of mitomycin C in rabbits and was shown to be applicable to the characterization of the pharmacokinetics of mitomycin C in humans receiving as little as 8 mg/m2.

Drug-nitrite interactions in human saliva: effects of food constituents on carcinogenic N-nitrosamine formation.

A simple and rapid high-pressure liquid chromatographic assay for monitoring N-nitrosodimethylamine (NDMA) in human saliva was developed. The method was used to study in vitro the effects of common food constituents on NDMA formation in saliva from the interaction of salivary nitrite with aminopyrine and oxytetracycline. Natural phenolic compounds, caffeic acid, and tannic acid, and synthetic additives, erythorbic acid, sorbic acid, propyl gallate, and butylated hydroxytoluene--all inhibited NDMA formation (20-80%). With ascorbic acid, up to 90% inhibition of NDMA synthesis in saliva was observed. In contrast, chlorogenic acid (a phenolic component of coffee) acted as a catalyst (up to 48% increase) of the nitrosamine formation under identical experimental conditions.

Stability kinetics of piperacillin in aqueous solutions

The degradation process of piperacillin in acidic, neutral and alkaline solutions was followed by both high-pressure liquid chromatographic and spectrophotometric assays. Pseudo-first-order rate constants were determined in a variety of buffer solutions. The overall pH-rate profile was determined at 35°C and an ionic strength of 0.5. β-Lactam moiety degradation occurred in acidic media to produce the hydrolysis products. In alkaline solutions, the piperazinyl ring of piperacillin was hydrolyzed about 20 times faster than the β-lactam moiety.

Comparison of the disposition of total and unbound sulfisoxazole after single and multiple dosing

Plasma concentrations of total and unbound sulfisoxazole were followed after single intravenous and oral doses of 1 g sulfisoxazole and during a 500-mg, four-time-a-day dosing regimen in six healthy males, using a specific high pressure liquid chromatographic assay method. Saturable plasma protein binding was observed at total concentrations above 80-100 mg/liter. The clearance of sulfisoxazole was 18.7 +/- 3.9 ml/min for total drug and 232 +/- 64 ml/min for unbound drug. Renal elimination, on the average, accounted for 49% of the clearance of sulfisoxazole. The apparent volume of distribution for total drug was 10.9 +/- 2.0 liters and 136 +/- 36 liters for unbound drug, indicating that sulfisoxazole is primarily distributed extracellularly. Accumulation of N4-acetyl-sulfisoxazole during multiple dosing did not affect the disposition of sulfisoxazole. Adjusting for variable renal clearances between oral and intravenous administration and using the unbound plasma concentrations, the bioavailability for an oral dose of sulfisoxazole was found to be 0.95 +/- 0.04.

Prednisone and prednisolone bioavailability in renal transplant patients

Prednisone and prednisolone are drugs with the potential for therapeutic inequivalence due to bioavailability problems. The objective of our study was to compare the systemic bioavailability of prednisolone from oral prednisone and prednisolone. Nine kidney transplant patients receiving prednisone (12.5 to 22.5 mg per day) were administered, in a randomized fashion, the same dose of oral prednisone (Deltasone), oral prednisolone (Delta-cortef) and intravenous prednisolone (Hydeltrasol). Prednisolone and prednisone levels were measured using a specific high-pressure liquid chromatographic assay. Since prednisolone exhibits dose-dependent pharmacokinetics because of nonlinear plasma protein binding, bioavailability from oral prednisone and oral prednisolone, compared to the intravenous dose, was 84.5 +/- 17.8% and 95.5 +/- 17.6% using unbound drug concentrations. These differences were not statistically significant. Furthermore, no significant differences were observed between the two oral formulations in peak prednisolone levels, time of peak levels or half-life using either total or unbound drug concentrations. The results from our study indicate that both of the oral preparations tested provide similar bioavailability of active prednisolone and the conversion of prednisone to prednisolone occurs rapidly.