Ion-pair Reversed-phase HPLC Research Articles

Reversed-phase HPLC/EC determination of folate in citrus juice by direct injection with column switching

The method utilized a two-position, 10-port valve that enabled direct injection of filtered citrus juice into an HPLC system. separation was by reversed-phase ion-pair HPLC with selective amperometric detection. Treatment with a folate polyglutamate hydrolase enzyme provides evidence for the existence of 5-methyl-THFpolyglutamates in Citrus juices.

Control de pigmentos clorofílicos y carotenoides por HPLC en el aceite de oliva virgen

The use of solid phase extraction columns of octadecyl (C 18 ) to get a fat free pigment extract in combination with reversed phase ion pair HPLC permits the separation of chlorophylls and its derivates, carotenoids, xantophylis and metalochlorophyllic complexes in a maximum period of one hour.

Indirect Determination of Phosphate, Silicate, and Arsenate by HPLC-AES

The indirect determination of phosphate, silicate, and arsenate is performed by separation of their heteropoly acids by using ion-pair reverse-phase HPLC (IP-RPHPLC) and monitoring the molybdenum emission from a DCP with an echelle spectrometer and a charge-injection device (CID) detector. Limits of detection found for phosphate, silicate, and arsenate are 26, 31, and 52 ng/mL, respectively. The detection limit for arsenate is slightly degraded due to its proximity to the excess molybdate peak. These results represent an improvement in the determination for these nonmetals over results for direct aspiration into an emission source and show excellent linearity over three orders of magnitude.

Quantitative Analysis of β-Blockers in Human Plasma by Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Using a Microbore Column

Abstract HPLC assays for four β-blockers (namely atenolol, labetalol, metoprolol and propranolol) in human plasma have been developed using a reversed-phase microbore column. Sodium dodecyl sulphate (SDS) was chosen as the pairing ion. SDS concentration was varied to provide acceptable retention for the drugs of interest and good resolution from plasma interfering peaks. Based on these results, a general reversed-phase ion-pair HPLC elution scheme using microbore column (ODS-Hypersil) with fluorescence or UV detection was developed. the detection limits were 10, 5, 5 and 1 ng/ml for atenolol, labetalol, metoprolol and propranolol, respectively. the coefficients of variation for the drugs studied were less than 10%. These methods have been shown to be suitable for routine analysis in bioavailability studies and for studying the placental transfer of β-blockers in both human placental perfusion and the animal models.

High Performance Liquid Chromatographic Method for the Determination of Diphenhydramine in Liquid and Solid Drug Dosage Forms and Its Application to Stability Testing

A simple, precise, rugged, stability-indicating method for diphenhydramine in liquid and solid drug preparations based on reversed phase ion-pair HPLC is described. The eluent used with the C8-bonded phase column, was acetonitrile/0.005 M aq. hexanesulfonic acid/acetic acid (70/30/1,v/v). The liquid product was simply dissolved in acetonitrile/water/acetic acid (70/30/1,v/v) and filtered; the solid product was briefly ground, dissolved in the same solvent, and filtered. The precision (rsd) of the method for diphenhydramine in the liquid sample is 0.52%, and 0.50% for the solid sample. The method is linear up to 200 μg/mL. Based on spiking studies, the recoveries are 100.1% for the liquid sample, and 99.2% for the solid sample. The method was applied to the study of the thermal stability of the drug by following the degradation of diphenhydramine in the two products at temperatures in the 42°C to 62°C range for up to 16 weeks. Shelf lives of the products were determined assuming zero and first order kinetics of decomposition, and are at least 163 days for the liquid product, and at least 255 days for the solid product.

Characterization of some natural and semi-synthetic betaxanthins

Betalamic acid was conjugated with a variety of amino acids, both protein and non-protein, to yield a series of semi-synthetic betaxanthin standards. Retention characteristics for reverse phase-ion pair HPLC chromatography and optical properties are described for these standards.

The bioavailability and nonlinear clearance of (-)-carbovir in the rat.

The pharmacokinetics and bioavailability of (+/-)-carbovir, a carbocyclic nucleoside active against human immunodeficiency virus, have been described previously. To determine the bioavailability of (-)-carbovir, the biologically active enantiomer, four male Sprague-Dawley rats received 18 mg/kg of (-)-carbovir through the jugular vein and 54 mg/kg orally. Following the pilot studies, five rats were randomly assigned to receive (-)-carbovir in a three-way crossover design as either a single 18-mg/kg iv bolus, a single 54-mg/kg oral dose, or a single iv infusion of 18 mg/kg to achieve a target steady-state concentration (Css) of 1 micrograms/ml, the peak concentration after an oral dose. Blood and urine samples were analyzed by an improved ion-paired reversed-phase HPLC method with fluorescence detection. Blood concentrations of (-)-carbovir declined in a biphasic manner after the iv bolus dose. The terminal half-life was 116 and 106 min after the iv bolus and oral dose, respectively. The blood/plasma distribution ratio was approximately 1.0 in the range of 1 to 10 micrograms/ml of (-)-carbovir in blood. The free fraction in serum was concentration dependent. Significant differences in the renal, nonrenal, and total-body clearances after the iv bolus and iv infusion suggested nonlinear elimination of (-)-carbovir. The oral bioavailabilities derived from blood data were significantly different when the iv bolus was used as a reference rather than the iv infusion. However, the bioavailabilities were not significantly different when the total urinary excretion of unchanged (-)-carbovir after iv bolus or infusion was used as a reference.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Alveolar and Peritoneal Macrophages Mediate Fungistasis Independently of L-Arginine Oxidation to Nitrite or Nitrate

Human alveolar macrophages (HAM) from 28 normal volunteers were found to inhibit replication of Cryptococcus neoformans. Conditions under which fungistasis occurred were different than those required for mouse peritoneal macrophage-mediated fungistasis. Inhibition of fungal replication by mouse peritoneal macrophages (MPM) requires that the macrophages are activated and that the cocultures of C. neoformans and macrophages be done in the presence of serum, L-arginine, and endotoxin. During MPM-mediated fungistasis and tumor cell killing, L-arginine is oxidized to NO2-, NO3-, and L-citrulline. In addition, MPM have arginase activity that converts L-arginine to L-ornithine and urea. HAM-mediated fungistasis was similar to that mediated by MPM in terms of the serum requirement, but HAM did not require L-arginine or endotoxin. HAM did not produce NO2- or NO3- detectable by colorimetric and bioassay, nor did HAM produce L-citrulline or L-ornithine from 14C-radiolabeled L-arginine as detectable by reverse-phase ion-pairing HPLC of macrophage-C. neoformans coculture supernatants. HAM had no detectable arginase activity, hence there was no evidence for L-arginine nitrogen metabolism in HAM. HAM-mediated fungistasis was not enhanced by endotoxin or by recombinant human interferon-gamma (rHIFN-gamma). The combination of endotoxin and rHIFN-gamma inhibited the fungistatic effect of HAM. Human peritoneal macrophages (HPM) from women undergoing laparoscopy were tested for fungistasis and L-arginine nitrogen oxidation. Partial inhibition of cryptococcal replication occurred; however, there was no evidence of L-arginine metabolism to NO2- or NO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation Kinetics of an Antiasthmatic, 2-[(4-Hydroxyphenyl)amino]-5-methoxybenzenemethanol, and Stabilization with Ascorbic Acid

A novel antiasthmatic, 2-[(4-hydroxyphenyl)amino]-5-methoxybenzenemethanol (1), oxidizes to the corresponding iminoquinone in aqueous solutions. The reaction was monitored by a paired-ion reversed-phase HPLC method. The oxidation rate was highly dependent on the solution pH, with a large rate increase occurring above pH 6.1. In nonaqueous solution, the oxidation reaction was significantly slower. Ascorbic acid protects 1 from oxidation. The aqueous solution of the decomposed product is reduced to 1 in the presence of ascorbic acid.

Simultaneous Measurement of Plasma Catecholamine (Norepinephrine, Epinephrine, and Dopamine) and Free N—Methyl Dopamine (Epinine) Levels, by HPLC with Electrochemical Detection

Abstract Epinine is the active moiety of ibopamine, a cardiovascular prodrug used in congestive heart failure. This catecholic compound shows dopaminergic and adrenergic properties. Moreover the drug seems to affect plasma catecholamine levels in patients with heart failure. Here we present a method developed for the simultaneous determination of epinine and catecholamine plasma levels. Free epinine and catecholamines were extracted from human venous plasma via an alumina adsorption procedure. The extracts underwent an ion-pair reversed-phase HPLC separation with three-electrode coulometry. Quantitation was made by an internal standard method. Coefficients of variation were 0.997). The detection limits were ≤ 5 pg of each catechol after extraction. This method allows about 50 low cost determination to be done in a working day.

A chemical stability study of proguanil hydrochloride

A stability-indicating assay method is described for proguanil based on reversed-phase ion-pairing HPLC. Using this method a study of the stability of proguanil in solution is made. In solution, proguanil decomposes by a first-order reaction. It is shown that the major decomposition product is 4-chloroaniline and that two other minor products can be detected but not identified. First-order rate constants over the temperature range 40–90°C are determined and an activation energy of 90.8 kJ mol −1 derived. The log k-pH profile is determined and indicates maximum stability in the range pH 6–8. From the magnitudes of the rate constants obtained, it is evident that proguanil is a very stable drug in solution. The effect of ultraviolet radiation on proguanil is to produce the same decomposition products. The effects of thermal stress and ultraviolet on a tablet formulation of proguanil (Paludrine ∗) are also studied and these further confirm the stability of this compound to both thermal and photochemical stress.

Ion-Paired Reversed-Phase High-Performance Liquid Chromatography Assay for Determination of Ceftriaxone in Human Plasma and Urine

A rapid, sensitive, and specific ion-paired reversed-phase HPLC assay for ceftriaxone in human plasma and urine is described. Small volumes (50 microL) of sample are deproteinized with acetonitrile and are directly injected on a C18 analytical column. The UV absorbance is monitored at 280 nm. The assay is linear between 1 and 125 micrograms/mL of ceftriaxone, with less than 10% coefficient of both intra- and interday variation. Chromatography was specific for ceftriaxone as endogenous compounds and 30 common drugs did not interfere. The assay was used in open heart surgery patients where potential interference from corticosteroids was overcome.

DNA-synthesis regulation and correlation with inositol trisphosphate levels in cultured bovine retinal capillary pericytes

Inositol phosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in cultured bovine retinal capillary pericytes (BRCP) were quantitated by an ion-pair reverse-phase HPLC. BRCP were grown in media with standard (5 mM) or high (30 mM) glucose, and were either labeled with myo-[2-3H]inositol (20 microCi ml-1) for 60 hr or with dual isotopes (20 microCi ml-1 myo-[2-3H]inositol and 2 microCi ml-1 [14C]glycerol) for 8 hr. In parallel, BRCP in different glucose-media were incubated with 1 microCi ml-1 [3H]thymidine for 4 hr. High glucose significantly suppressed the accumulation of [3H]label in IP, IP2 and IP3, and specifically reduced the incorporation of [14C]glycerol into inositol phospholipids, but not that of neutral lipids and other types of phospholipids. The reduced IP3 level correlated with the decrease in the incorporation of [3H]thymidine into DNA. Both the reduced IP3 formation and DNA synthesis which were induced by high glucose were significantly reversed by adding either myo-inositol or AL1576, an aldose reductase inhibitor (ARI). However, the addition of neither myo-inositol nor ARI stimulated IP3 formation and/or DNA synthesis when BRCP were grown in the standard medium (5 mM glucose). These findings indicate that myo-inositol metabolism and the polyol pathway affect inositol phospholipid-mediated pericyte division in vitro only under the high-glucose condition. These data are compatible with the hypothesis that altered inositol phospholipid metabolism accounts for the loss of pericytes in diabetic retinopathy.

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.

Effect of Carrier Dose on the Multiple Tissue Disposition of Doxorubicin Hydrochloride Administered via Magnetic Albumin Microspheres in Rats

The effect of carrier dose on the multiple tissue disposition of doxorubicin hydrochloride has been investigated in rats. The drug was encapsulated in submicron magnetic albumin microspheres using a heat-denaturation technique. The rat tail was used as a target organ. Two groups of animals were administered 2.0 or 0.04 mg/kg of microsphereentrapped drug via the ventral caudal artery, and the predefined tail target site was exposed to a 8000-G magnetic field for 30 min after dosing. In each group, the animals were sacrificed in triplicate over a 48-h period, and their various tissues were analyzed for drug concentration using reversed-phase ion-pair HPLC. The reduction in carrier dose was found to increase drug distribution as well as the targeting efficiency for the target tissue. The drug delivery to heart and liver was reduced. The significance of carrier dose in the targeted delivery of drugs is discussed.

Reversed-phase HPLC separation of plasma norepinephrine, epinephrine, and dopamine, with three-electrode coulometric detection.

We present a method involving minor modifications of previous techniques, in which we use an ion-pair reversed-phase HPLC separation with three-electrode coulometry. Isocratic baseline separations can be obtained in 5 min. The sample preparation procedure, involving extraction with alumina at low temperature, allows good reproducibility (within-run and between-run CVs less than 10%) and improved sensitivity (less than 5 pg of each catecholamine per extract). This method allows approximately 70 low-cost plasma catecholamine analyses to be done in a working day.

High performance liquid chromatographic analysis of time-dependent changes in urinary excretion of indoleamines following tryptophan administration.

Analytical conditions of prepurification extraction and HPLC separation were optimized for determination of urinary serotonin and tryptamine. Under optimal conditions, serotonin, tryptamine and an internal standard were extracted with 15% v/v n-propanol in diethyl ether from urine samples alkalized with a phosphate buffer (0.75 mol/L, pH 10.0), and then they were re-extracted into an HCl solution (0.1 mol/L). Purified indoleamines were simultaneously separated by reversed-phase ion-pair HPLC with native fluorescence detection. Urinary serotonin and tryptamine were selectively determined within about 45 min per sample for the whole procedure. Analytical recovery, reproducibility and detection sensitivity were satisfactory for pursuing time-dependent changes in indoleamine levels. Urinary excretion profiles of serotonin and tryptamine in subjects dosed with L-tryptophan were successfully analyzed by our method.

Ion-pair reversed phase HPLC determination of nucleotides, nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes

Three groups of metabolites were analyzed in extracts of rat hepatocytes by an HPLC method: (i) nucleotides (ATP, ADP, AMP, GTP, GDP, UTP, UDP, IMP, UMP), (ii) nucleosides and nucleobases (adenosine, adenine, guanosine, inosine, xanthine, hypoxanthine, uridine) and (iii) inhibitors of xanthine oxidoreductase (oxypurinol, allopurinol). Perchloric acid extracts were neutralized with K2CO3/triethanolamine and analyzed at 254 nm by reversed-phase ion-pair high-performance liquid chromatography. The nucleotides and their derivatives were separated with a gradient elution using 10 mM NH4H2PO4/2 mM t-butylammonium-phosphate and acetonitrile. Recovery values were estimated for the extraction procedure used. The method was applied to the investigation of nucleotide metabolism of hepatocytes of starved rats at anoxia and reoxygenation.

The separation of [ 32P] inositol phosphates by ion-pair chromatography: Optimization of the method and biological applications

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P 3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [ 3H] inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

HPLC-32P-postlabelling analysis of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine.

A 32P-postlabelling procedure coupled with HPLC has been developed to detect and measure the cyclic nucleic acid adducts 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in DNA. Chloroacetaldehyde-modified DNA containing these adducts was enzymatically digested to 3'-monophosphates and adducts were separated by ion-pair reverse-phase HPLC on PL RP-S prior to 32P-postlabelling with carrier free [gamma-32P]ATP. Following 3'-dephosphorylation with nuclease P1 the resulting [5'-32P]monophosphate adducts were finally resolved by HPLC on PL RP-S and assayed by liquid scintillation counting.