Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Enzymic inactivation of substance P in the central nervous system.

A membrane-bound 'substance P degrading enzyme' (EC 3.4.24.-) from human brain has been purified to apparent homogeneity. The enzyme was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the rate of disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme is a thermolabile, neutral metallo-endopeptidase with a relative molecular mass of about 50000. It cleaves substance P between Gln6-Phe7, Phe7-Phe8 and Phe8-Gly9, with a ratio of 0.7:1:1. The breakdown products have been identified by a combination of reverse-phase high-performance liquid chromatography and amino acid analysis. A similar cleavage pattern of substance P has also been demonstrated in a synaptic membrane fraction prepared from rat brain, indicating that a 'substance P-degrading enzyme' is the major peptidase responsible for inactivating the peptide in rat brain membranes. The properties of this enzyme distinguished it from previously described peptidases for which substance P is a substrate. Its high selectivity and its affinity for substance P, among many other neuropeptides, suggest that it may be involved in the physiological inactivation of the peptide by neural tissues.

Reversed-Phase High-Performance Liquid Chromatography Analysis of Apigenin and its Glucosides in Flowers of Matricaria chamomilla and Chamomile Extracts

Quantitative estimation of the flavones apigenin, apigenin-7-glucoside and apigenin-7-acetylglucoside in ligulate florets of Matricaria chamomilla was performed by HPLC using a reverse-phase column and eluting with acetonitrile/water, acetic acid system. These flavonoids were detected at 335 nm. Apigenin and its glucosides were not found in the tubular florets. The method was also applied to the estimation of these flavonoids in chamomile extracts.

Reversed-Phase High-Performance Liquid Chromatographic Analysis of Liothyronine Sodium and Levothyroxine Sodium in Tablet Formulations: Preliminary Studies on Dissolution and Content Uniformity

Levothyroxine sodium was estimated from tablet formulations of levothyroxine sodium and liotrix (liothyronine sodium-levothyroxine sodium combination tablet). The procedure consisted of the addition of 3,3′,5′-triiodothyronine as the internal standard to the pulverized sample, followed by an acidic butanol extraction, evaporation, and injection onto a μBondapak reversed-phase high-performance liquid chromatographic column. The eluent was methanol-water-phosphoric acid (50:50:0.1), and the effluent was monitored by UV detection at 254 nm. A standard linear calibration curve was obtained for direct standard solutions equivalent to 18-225 μg of levothyroxine sodium/tablet. The procedure is sensitive enough for single-tablet analysis. Using this procedure, content uniformity studies were performed on liothyronine sodium tablets, levothyroxine sodium tablets, and liotrix tablets. The procedure also was adapted for conducting dissolution studies on levothyroxine sodium tablets in deionized water using the rotating-paddle method.

Paired-ion reversed-phase high-performance liquid chromatography of human and rat calcitonin

Improved methods for isolation, characterization, and quantitation of immunochemically heterogeneous forms of calcitonin (CT) in tissue and plasma must be developed before the biological origins and clinical importance of CT moieties can be elucidated. We are now proposing reversed-phase high-performance liquid chromatography (RP-HPLC) as one possible means of achieving high recovery, high resolution of CT moieties. In this paper we report RP-HPLC analyses of trace amounts of radiolabeled and unlabeled synthetic human and rat CT. We have systematically evaluated our application of RP-HPLC by employing several elution modes, including isocratic and gradient elution, as well as several elution reagents. We determined that high recovery and high resolution were best achieved with alkyl ion-pairing reagents, such as tetrabutylammonium phosphate, pH 7.5, or sodium sulfonyl hexane, pH 3.5. The most sensitive UV detection of trace amounts of CT was achieved with tetrabutylammonium phosphate buffer (TBAP). We recommend for RP-HPLC of CT a C 18-bonded silica column and elution with a 20-min linear gradient of methanol-water (20:80 to 80:20, v/v) containing 0.005 M TBAP. Combined with appropriate extraction procedures, such as silica adsorption or immuno-adsorbant chromatography, this paired-ion RP-HPLC method can be an important aid in achieving more accurate and extensive information about CT moieties in biological samples. This method will also allow the rapid, optical detection and quantitation of CT moieties recovered from tissues, and perhaps from plasmas.

Reversed-phase high-performance liquid chromatographic analysis of thiamine phosphate esters at subpicomole levels

Separation and determination of thiamine phosphate esters were achieved by reversed-phase high-performance liquid chromatography (hplc) after conversion to corresponding thiochrome esters. The elution order was thiochrome triphosphate, thiochrome pyrophosphate, and thiochrome monophosphate by a system composed of 25 m m potassium phosphate buffer (pH 8.4) and 2.5% N,N-dimethylformamide. The minimum amount reproducibly detected was 0.05 pmol for each thiochrome phosphate. Thiamine phosphate esters in rat tissues were successfully determined by the reversed-phase hplc after alkaline oxidation of the tissue extract, which resulted in a good agreement in their contents to those obtained by the straight-phase hplc previously reported.

Reversed-phase high-performance liquid chromatographic analysis of methotrexate and 7-hydroxymethotrexate in serum

Reversed-phase high-performance liquid chromatographic analysis of methotrexate and 7-hydroxymethotrexate in serum

Reversed-phase high-performance liquid chromatographic analysis of cinchona alkaloids in pharmaceuticals

An high-performance liquid chromatographic assay for quinidine and dihydroquinidine sulphates in pharmaceutical dosage forms has been developed. The method makes use of a reversed-phase system with a C 18 bonded column and theophylline as an internal standard. Recovery of both compounds is quantitative and the method compares favourably with that of the United States Pharmacopoeia with regard to speed and precision. The method should also be suitable for the control of quinine salts in pharmaceuticals.

High-performance Liquid Chromatographic Determination of Plasma Propylthiouracil

A reversed-phase high-performance liquid chromatographic analysis was developed for propylthiouracil in plasma (1 ml). After protein precipitation with acetonitrile, the solution was diluted with water and injected into a liquid chromatograph equipped with C-18 and C-8 columns in series. The peak area was linear over the 0.25-10-mg/liter range, and the recovery was 101 ± 4.5%. This assay has the advantages of specificity, simplicity, and speed over previously published methods and requires smaller sample volumes. None of 19 drugs tested interfered with the assay.

Use of reversed-phase high-performance liquid chromatographic analysis for the determination of provitamin A carotenes in tomatoes

The usual methods of provitamin A evaluation of foods convert the total pigment amout, determined spectrophotometrically, into vitamin A units. Since the totally inactive lycopene is the major carotenoid in the tomato, such readings result in erroneously high provitamin A values. In view of the recent development of chemically bonded, reversed-phase, microparticulate packings and their use in high-performance liquid chromatography which combines highly accurate and reproducible resolution with the speed and ease of operation, a new method using such a system was developed to isolate carotenoid pigments from tomato samples. A 15-min column separation was thus achieved, dramatically decreasing the analysis time of the classical open column chromatographic procedures, which often result in unresolved and altered fractions due to long-term exposure to oxygen, ligh, solvents and sometimes adsorbent. β-Carotene and lycopene were determined and quantitated in six tomato samples. β-carotene, 100% vitamin A-active, was expressed in International Units of vitamin A. The newly developed method gives a more reliable evaluation of the fruit potency in vitamin A than the methods of the Association of Official Analytical Chemists currently used for food composition tables.

Rapid reversed-phase high-performance liquid chromatographic analysis of steroid products

The optimisation of conditions for the application of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of steroid products is discussed. The influence of temperature and flow-rate on column efficiency are described. At 60 degrees, the reduced velocity required to give maximum efficiency, is approximately twice that found at room temperature, allowing faster through-put. (hv) curves are reported for 5-micron Spherisorb-ODS and Hypersil-ODS material for corticosteroids at 60 degrees. On-column injection techniques are discussed and data presented, showing that for corticosteroids on-column loop injection can be efficient as septum injection using the techniques described.