Liquid chromatographic determination of methylxanthines and catechins in herbal preparations containing guaraná

Herbal preparations derived from the dried seeds of guaraná (Paullinia cupana) have become a popular nutritional supplement used for stimulatory purposes. Once considered a drug substance in the United States, guaraná currently is classified as a food additive and dietary supplement. The pharmacological activity of guaraná-containing products is primarily due to methylxanthine alkaloids. For guaraná preparations, methylxanthine levels and, more significantly, the presence of several polyphenol compounds (i.e., catechins) provide phytochemical markers of authenticity. Methylxanthines and polyphenols are extracted from sample matrix with a heated phosphate buffer-methanol solution, the cooled extract is filtered, and the extract is injected into the liquid chromatographic (LC) system. A Nova-Pak C18 column eluted with phosphate buffer-methanol mobile phase (pH = 3.50) and monitored at 272 nm gave satisfactory resolution for the methylxanthines theobromine, theophylline, caffeine and the polyphenols (+)-catechin and (-)-epicatechin. Twenty-four products including dried seeds, dried paste, seed powders, tablets, and capsule formulations were assayed and conclusions were drawn about their authenticity. The LC system responded linearly to methylxanthines over the 100-fold range in concentration from 0.043 to 4.30 micrograms/mL for theobromine and caffeine and from 0.041 to 4.10 micrograms/mL for theophylline. Precision data for the 3 methylxanthines obtained from 10 different products (n = 5) gave relative standard deviation (RSD) values of 1.18-15.52% within a concentration range of 0.01-52.28 mg/g. Recoveries of methylxanthines from fortified products varied from 87.5 to 120.0%. The response for catechins was linear over a 200-fold range in concentration of 0.05-10.0 micrograms/mL. Precision data from 5 products (n = 5) gave RSD values of 1.08-5.54% within a concentration range of 0.34-32.65 mg/g. Recoveries from these products ranged from 87.7 to 109.7%. Results and chromatographic profiles for 14 commercial products in solid dosage form indicate that a number of these products may not contain authentic guaraná as an active ingredient or contain less than the declared quantity of guaraná. The proposed procedure also was applied to 2 carbonated soft drinks and a sample of maté.     

Liquid chromatographic determination of ivermectin in feed

An analytical method for determining ivermectin in feed at 0.50-3 ppm is presented. The method is based on liquid chromatographic measurement after sample preparation by adsorption chromatography on alumina and solid-phase extraction. Two complete, final, finished medicated feeds and the corresponding control feeds used in their preparation were analyzed. Recoveries from feeds fortified at 50-150% of the 2 ppm ivermectin use concentration also were determined. Mean recoveries from replicate analyses ranged from 90 to 100%, and coefficients of variation (CVs) were less than 4.5%. No significant interferences were found in control feeds. The pooled distribution of individual analytical results (n = 100) gave a mean recovery of 100%, a recovery range of 90-111%, and an overall CV of 5.5%. Resolution of the total variance into its 2 components gave a within-laboratory CV of 4.1% and a between-laboratory CV of 3.4%. There was no significant difference in recoveries among laboratories, days, concentrations, and feed base or between fortified and medicated feeds (P > 0.2).     

High-pressure liquid chromatographic determination of thiothixene in pharmaceutical formulations

A method was developed for the quantitative determination of thiothixene in pharmaceutical formulations by high-pressure liquid chromatography after dissolution of the sample in methanol.     

Spectrophotometric determination of diphenhydramine. HC1 in pharmaceutical preparations

A new spectrophotometric method has been developed for determining diphenhydramine. HC1, based on solvent extraction into chloroform of the complex formed with bromocresol green. The complex solution in chloroform showed maximum absorption at 415 nm and obeyed Beer's law over the concentration range of 3.0-12.0 microgram/ml. The molar absorptivity of the complex was 2.02 x 10(4). Complex formation and extraction was complete and quantitative over the pH range from 2 to 5. The ratio of diphenhydramine to bromocresol green was 1:1. Excipients, coloring matter, flavoring agents, and other substances likely to be present in diphenhydramine preparations do not interfere in the determination. Direct determinations in tablet, capsule, sirup, and lotion preparations were carried out satisfactorily, and the average recovery was 100 +/- 1.0%.     

Liquid chromatographic determination of safrole in sassafras-derived herbal products

A liquid chromatographic (LC) method was developed for determining safrole in herbal products derived from sassafras (Sassafras albidum), as well as related compounds such as isosafrole and dihydrosafrole. The procedure involves solvent extraction and isolation of analyte by reversed-phase LC with UV detection at 235 nm. Safrole is resolved from related compounds and other sample constituents including thymol, a component of thyme. A linear concentration range of 0.003-0.200 mg/mL was obtained for safrole, isosafrole, and dihydrosafrole. Limits of detection (LOD) and quantitation (LOQ) were e0.0015 and 0.0051 micrograms/mL for safrole, 0.0018 and 0.0061 micrograms/mL for isosafrole, and 0.0038 and 0.0125 micrograms/mL for dihydrosafrole, respectively. Intraday relative standard deviations (RSDs) for safrole (n = 5) from various samples ranged from 1.30 to 5.39% at analyte levels of 0.01-1.5%. Safrole contents of 26 samples including root bark powder, leaves, oils, tea concentrate, herbal extract tinctures, and herbal powder capsules ranged from < LOD for most leaf samples to 92.4% for an oil. Recoveries of safrole from fortified samples ranged from 83.6% for an oil to 117.2% for a tincture preparation. Safrole contents of 0.09-4.66 mg/cup were found for brewed teas prepared from sassafras root bark powders and tinctures.     

Spectrofluorimetric determination of prenalterol hydrochloride in pharmaceutical preparations and biological fluids

A simple and highly sensitive fluorimetric method was developed for the routine determination of prenalterol hydrochloride in bulk, in dosage forms and in biological fluids. The method is based on the fluorescence induced by reaction of the nitroso-derivative of prenalterol hydrochloride with 2-cyanoacetamide in the presence of ammonia. The different experimental parameters were carefully studied and incorporated into the procedure. The fluorescence is measured at 440 nm after excitation at 368 nm. Fluorescence intensity is a linear function of prenalterol hydrochloride concentration over the range of 0.1-2.8 microg x ml(-1) in the solution finally measured. A proposal for the reaction pathway was suggested.     

Gas chromatographic determination of hydrazoic acid

A gas chromatographic procedure was developed for the determination of hydrazoic acid. Two possible columns were evaluated. The limits of detectability were 1.5 ng, or approximately 0.05%, in aqueous solution (w/v) and 2 mg per liter in air.     

Testing of decomposition of pyridinedicarboxylic acid derivatives in selected pharmaceutical preparations

The move to the use of molecular diagnostics in medicine has gathered momentum in the last few years and new methodologies are being sought to reduce the labour component and hence the cost of these molecular diagnostics. A method is described which links PCR to capillary electrophoresis and this allows both a rapid and quantitative diagnosis. In the example provided, the diagnosis of chronic myeloid leukaemia, the method describes improvements in cost per test, greater sensitivity over traditional methods and an estimate of the level of product. The latter points are important in those disorders for which bone marrow transplantation is used as a curative regime in providing earlier detection of relapse.     

High-performance liquid chromatographic determination of bisacodyl in pharmaceutical dosage forms marketed in Australia

HPLC methods have been developed for the assay of bisacodyl in various pharmaceutical forms. The extraction procedures are simple and the HPLC conditions separate bisacodyl from its degradation products. The chromatography was performed using a Merck LiChrospher RP-select B column, a mobile phase of 55% acetonitrile/45% 0.05 M KH2PO4 and detection by UV at 214 nm.     

Liquid chromatographic determination of the antibiotic fumagillin in fish meat samples

A procedure for the determination of fumagillin, an antibiotic of Aspergillus fumigatus in fish meat samples, using reversed-phase high-performance liquid chromatography is described. Liquid chromatography was performed on an octadecylsilane column using acetonitrile-water-phosphoric acid solution as mobile phase, with detection at 350 nm. Two different types of sample preparation were developed, clean-up and enrichment, and the limits of quantification were 100 ng/g and 5 ng/g, respectively, in fish meat. The recovery was 75 +/- 3% in the concentration range 100-500 ng/g. To introduce the methodology and demonstrate its usefulness, a practical experiment was performed. Trouts fed with fumagillin were examined for elimination of fumagillin. After 24 h, the concentration was shown to decrease to below 100 ng/g.     

Flow injection amperometric determination of L-dopa, epinephrine or dopamine in pharmaceutical preparations

OBJECTIVE: The association of systemic lupus erythematosus (SLE) and multiple myeloma (MM) is an uncommon event. We report the relapse of SLE in a patient with a previous history of MM, treated with chemotherapy and, subsequently, with alpha-2b interferon (alpha-2b IFN) as a maintenance therapy. The case is discussed in light of past relevant literature. METHODS: The history and clinical, laboratory and radiographic findings of the patient, as well as the subsequent therapeutic approach are discussed. In our review of the literature, journal articles are identified by Medline search. RESULTS: We describe the case of a woman who developed a multiple myeloma 14 years after a diagnosis of SLE. A careful literature review confirms that the association of these two diseases has been reported only in a few cases. When the plasma cell neoplasia occurred, SLE had been quiescent for several years; the patient was treated with prednisone-melphalan and, subsequently, with alpha-2b IFN as a maintenance therapy. On admission to our department, SLE was in a relapse phase, probably because of IFN treatment. The disease was poorly responsive to steroid therapy and required the use of cytotoxic drugs. CONCLUSIONS: The coexistence of SLE and MM is very rare and the possible pathogenetic mechanisms underlying this association remain unclear. The use of interferon in a patient with an autoimmune disease always invites caution.     

Liquid chromatographic determination of five benzoylurea insecticides in fruit and vegetables

A liquid chromatographic (LC) method was developed to determine 5 benzoylureas--diflubenzuron, hexaflumuron, teflubenzuron, flufenozuron, and lufenuron--in peppers, tomatoes, eggplants, cucumbers, and oranges. Preparation of samples involve extraction with acetone and partitioning into dichloromethane-petroleum ether. A portion of this extract is cleaned up with a solid-phase extraction aminopropyl disposable column. With LC analysis using an RP-8-DB microbore column, acetonitrile-water (70 + 30, v/v) as mobile phase, and photodiode array detection at 254 nm, recovery and repeatability data were collected for the 5 benzoylureas on 4 vegetables and citrus in the range 0.04-2.0 mg/kg. Validated limits of detection and quantitation were 0.01 and 0.04 mg/kg, respectively. The method is reliable for routine analysis of vegetables and fruits.     

Benzoyl peroxide stability in pharmaceutical gel preparations

The storage stability of benzoyl peroxide in the presence of both individual and combined pharmaceutical gel ingredients was investigated. Benzoyl peroxide was quite unstable in the presence of nucleophilic agents and certain acidic substances. At both 30 and 40 degrees storage temperatures, benzoyl peroxide was destroyed rapidly (within 1 month) in the presence of ethanol and acidic chelating agents. The substitution of acetone for ethanol, the elimination of chelating agents, and the addition of sodium hydroxide to gel preparations markedly reduced degradation.     

Liquid chromatographic determination of a non-steroidal oral contraceptive CDRI-85/287 in rat serum

A precise and sensitive high performance liquid chromatographic (HPLC) assay method was developed and validated for the quantitation of 2-[4-(2-piperidinoethoxy) phenyl]-3-phenyl-(2H)-1-benzo(b)pyran (compound CDRI-85/287) in rat serum. This method, applicable to 0.5 ml volumes of serum, was validated according to GLP guidelines. It involved double extraction of serum samples with a mixture of hexane and iso-propanol (98:2 v/v) at alkaline pH and the use of UV detection at 332 nm. Linearity, precision and accuracy were acceptable (5-200 ng ml-1. The absolute recovery was more than 75% and the lower limit of quantitation was 5 ng ml-1. Freeze-thaw stability studies up to four cycles showed no apparent differences in the calculated spiked concentrations. However, in-process stability evaluation showed the stability of the processed samples lasted up to 85 h.