Ion-pair High-pressure Liquid Chromatography Research Articles

Ion-pair high-pressure liquid chromatography of cis-trans isomers of retinoic acid in tissues of vitamin A-sufficient rats.

Naturally occurring retinoids were separated by reversed-phase high-pressure liquid chromatography on an octadecylsilane column eluted with acetonitrile-potassium phosphate buffer (pH 7.2) mixtures. The order of elution from a mixture of 500 ng each of the following standards was 4-oxo retinoic acid (RA), retinyl phosphate (RP), 13-cis RA, all-trans RA, retinol, retinal, retinyl acetate, anhydroretinol, and retinyl palmitate. This method was employed to investigate the cis-trans isomerization of RA and its metabolism in vitamin A-sufficient male rats. Rats (200 g) were injected intraperitoneally with 50 muCi of either [10-3H]-all-trans RA (5.4 micrograms) or [11-3H]-13-cis RA (8.8 micrograms) and killed after 0.5 and 3 hr. Blood, liver, kidney, small intestines, and testes were removed and lyophilized. All-trans RA was converted at 0.5 hr after injection to 13-cis RA in all the tissues examined, with the exception of the small intestine; the conversion ranged from 2.4 to 6.9% of the total radioactivity. In addition, all-trans RA was converted to metabolites (17.5--47.7%) of greater polarity than 4-oxo RA. After 3 hr, most of the radioactivity (75--90%) was found in the highly polar metabolites. 13-cis RA was also partially isomerized to the all-trans RA and to the highly polar metabolites by 0.5 and 3 hr after injection. Appreciable radioactivity (10--41%) still resided in the 13-cis RA fraction after 3 hr. These results indicate that 13-cis RA is partially isomerized to all-trans RA and that all-trans RA is rapidly metabolized to highly polar compounds in tissues of vitamin A-sufficient rats.

Determination of Methacholine Chloride by Ion-Pair High-Pressure Liquid Chromatography

Abstract A method of analysis of methacholine chloride is presented, which is adaptable to other choline esters. The method uses ion-pair high-pressure liquid chromatography. Using 1-heptane sulfonic acid optically transparent at low UV wavelengths as the specific ion-pair, it was possible to assay for a specific choline ester using ultraviolet detection at 210 nm without interference by hydrolytic by-products. The method can be used to provide quality control and stability data on methacholine chloride in 0.9% sodium chloride solutions.

H-Pro-[3H]Leu-Gly-NH2: uptake and metabolism in rat brain.

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.

Determination and temperature effects of lidocaine (lignocaine) hydrochloride, epinephrine, methylparaben, 2,6-dimethylaniline, and p-hydroxybenzoic acid in USP lidocaine injection by ion-pair reversed-phase high pressure liquid chromatography.

USP Lidocaine injection was assayed using ion-pair high pressure liquid chromatography with an octylsilane (RP-8) reversed-phase column packing and a mobile phase consisting of D-10-camphorsulfonic acid/methanol/acetic acid/water. The effect of temperature was investigated to determine the optimum temperature for separating the drug components and their degradation products. Lidocaine (lignocaine) hydrochloride, epinephrine, methylparaben, and p-hydroxybenzoic acid were separated at 50 degrees C. 2,6-Dimethylaniline was separated from lidocaine at 15 degrees C. An aliquot of the sample was injected directly into the liquid chromatograph, and after separation the compounds were quantitated by their spectrophotometric response at 254 nm (lidocaine) or 280 nm (lidocaine plus epinephrine).

Determination of Chlorpromazine and Its Sulfoxide and 7-Hydroxy Metabolites by Ion-Pair High Pressure Liquid Chromatography

Abstract An ion-pair HPLC approach with ordinary silica has been applied, with detection by ultraviolet absorption, to the assay of plasma for chlorpromazine and its sulfoxide on the one hand, and for 7-hydroxychlorpromazine (an active metabolite) on the other hand. The respective sample-preparation procedures entail extraction of the plasma with heptane at strongly alkaline pH, or else with diethyl ether at a less alkaline pH and with ensuing back-extraction and re-extraction. For each of the compounds, levels as low as 10 ng.ml−1 are measurable. The conditions adopted are such that specificity and reproducibility are satisfactory although chlorpromazine and its various metabolites, especially 7-hydroxychlorpromazine, are chemically unstable and, moreover, are readily lost onto glass. With the unorthodox separation system adopted, adsorption rather than partition appears to be the dominant mechanism.

Determination of Methylenediphosphonic Acid by Ion-Pairing High Pressure Liquid Chromatography with Refractive Index Detection

Determination of Methylenediphosphonic Acid by Ion-Pairing High Pressure Liquid Chromatography with Refractive Index Detection

Separation of folate derivatives by in situ paired-ion high-pressure liquid chromatography

Separation of folate derivatives by in situ paired-ion high-pressure liquid chromatography

Determination of isoprenaline by ion-pair high-pressure liquid chromatography

Determination of isoprenaline by ion-pair high-pressure liquid chromatography

Paired-Ion High Pressure Liquid Chromatography of Methotrexate and Metabolites in Biological Fluids

Abstract A paired-ion high pressure liquid chromatography procedure (HPLC) is described for the separation of methotrexate and its major metabolites (7-hydroxymethotrexate, 4-amino-4-deoxy-NlO-methylpteroic acid, methotrexate diglutamate, methotrexate triglutamate), and therapy-related compounds (Diamox and 5-methyltetrahydrofolate). The mobile phase consisted of a 70% solution of 5 mM hexanesulfonic acid, pH 3.75, and 30% methanol eluted on a reverse phase column or columns and monitored at 305 nm or 280 nm UV absorption. The lower limit of sensitivity for methotrexate and 7-hydroxymethotrexate in plasma was 2 ng/ml.

Applications of Paired Ion High-Pressure Liquid Chromatography: Quantitative Determination of Potassium Guaiacolsulfonate and Other Ingredients in Cough Syrups

A method for the quantitative determination of potassium guaiacolsulfonate and phenylephrine hydrochloride in commercial dosage forms was developed. The method is based on paired ion high-pressure liquid chromatography with tetrabutylammonium as the counterion. The method not only separates potassium guaiacolsulfonate from phenylephrine hydrochloride but also from some other ingredients: chlorpheniramine maleate, sodium benzoate, colors, and flavors. Furthermore, two isomers of potassium guaiacolsulfonate, potassium salts of 4- and 5-guaiacolsulfonic acid, also separate from each other. The method was tried on five different commercial dosage forms (all with different colors) with excellent results on three. In the other two samples, which also contained codeine, there may have been a stability problem.

Preparation, isolation, analysis, and characterization of 3-benzo[ a]pyrenyl-β- d-glucopyranosiduronic acid: A metabolite of 3-hydroxybenzo[ a]pyrene with potentially high carcinogenic activity

The aglycone, 3-hydroxybenzo[ a]pyrene, was metabolized to 3-benzo[ a]pyrenyl-β- d-glucopyranosiduronic acid in the presence of uridine 5′-diphosphoglucuronic acid and rabbit liver microsomes. The course of the biosynthetic reaction was followed by fluorimetry and reverse-phase, paired-ion high pressure liquid chromatography (HPLC). Also, the HPLC system was used to analyze for glucuronide and 3-hydroxybenzo[ a]pyrene during the isolation procedure. The existence of a glucuronide of 3-hydroxybenzo[ a]pyrene was determined by radiotracer and enzymic techniques, utilizing the HPLC system. Field desorption and direct inlet mass spectral techniques were used to characterize the 3-hydroxybenzo[ a]pyrene glucuronide.

Applications of Paired Ion High-Pressure Liquid Chromatography to Catecholamines and Phenylephrine

Paired ion high-pressure liquid chromatography was useful for separating intact catecholamines (epinephrine, isoproterenol, levodopa, and methyldopa) and phenylephrine from some of their decomposition products. Furthermore, methyldopa was separated from either hydrochlorothiazide or levodopa. Under identical conditions without I-heptanesulfonie acid (a counterion for paired ion chromatography) in the mobile phase, these separations were not possible. Paired ion chromatography also was tried successfully for the quantitative determinations of isoproterenol, levodopa, methyldopa, and phenylephrine in some dosage forms. The results were compared with the results obtained using some literature methods.

Quantitative Determinations of Two Decongestants and an Antihistamine in Combination Using Paired Ion High-Pressure Liquid Chromatography

A single method for the quantitative determinations of three active ingredients, phenylephrine hydrochloride, phenylpropanolamine hydrochloride, and brompheniramine maleate, and one inactive ingredient (sodium benzoate) in a commercial product for colds is reported. The method is based on paired ion high-pressure liquid chromatography using 1-heptanesulfonic acid as the counterion. It is accurate and precise. The relative standard deviations based on six readings are reported. This method is sensitive; less than 1μg of each ingredient can be assayed. The peak area of each ingredient is related to its concentration.