Concentrations of ephedra alkaloids and caffeine in commercial dietary supplements

Dietary supplements that contain Ma Huang (ephedra alkaloids) and guarana (caffeine) are widely marketed and used in the U.S. for weight loss and athletic performance enhancement, despite a lack of adequate research on the pharmacology of these botanical stimulants. We developed and applied a novel liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to quantitate the various ephedra alkaloids found in dietary supplements that contain Ephedra species. The quantities of ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedine, methylpseudoephedrine, and caffeine were determined for 35 commercial dietary supplements and compared with the amounts listed on the product labels. The total ephedra alkaloid content ranged from 5.97 mg to 29.3 mg per serving. Two supplement brands did not list the quantity of ephedra alkaloids on the label, and four did not list the amount of caffeine per serving. Of the products tested, 31% contained > 110% of the total ephedra alkaloids listed on the label, and 6% of the supplements contained < 90% of the listed amount. For caffeine, 86% of the product lots that listed the caffeine amount contained less than 90% of the labeled quantity. No products contained > 110% of the declared caffeine content. The total ephedra alkaloid content varied significantly from lot to lot in 5 of 9 products. Three product brands contained proportions of alkaloids that exceeded amounts reported for E. sinica, including one that was 98% ephedrine, one that had 10% norpseudoephedrine, and one that contained an average of 13% methylephedrine. We conclude that product inconsistency is common among some commercially available dietary supplements that contain ephedra alkaloids and caffeine.     

Determination of methazolamide concentrations in human biological fluids using high performance liquid chromatography

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma. In vivo, methazolamide readily distributes into red blood cells. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid–liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 μm reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 μg ml⁻¹, concentrations reflective of those expected in vivo. Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1–7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.     

The use of polymerase chain reaction (PCR) for the identification of ephedra DNA in dietary supplements

As part of a continuing research effort to develop chemical and genetic authentication profiles of botanicals, an investigation was performed with the goal to detect, identify and verify Ephedra sinica Stapf DNA in dietary supplements such as plant mixtures and tablets/capsules. We amplified and sequenced the chloroplast psbA-trnH spacer from 21 Ephedra spp. and from two of their closest relatives, Gnetum gnemon L. and Welwitschia mirabilis Hook. Based on sequence comparisons, we identified regions unique to all of the Ephedra spp. samples analyzed. We concluded that the psbA-trnH spacer sequence could be used as a molecular marker. Based on this spacer sequence, we designed Ephedra spp.-specific primers that can help to identify Ephedra spp. DNA in plant mixtures containing as little as 1/1,000 part of Ephedra spp. tissue. We used a DNA extraction method that allows for quick DNA isolation from plant mixtures for PCR analysis.     

Determination of monoureides in biological fluids by high-pressure-liquid-chromatography

A sensitive and specific method for the simultaneous determination of bromisoval, carbromal, and methaqualone is described. The drugs are adsorbed from serum onto charcoal at pH 11 and eluted from it with organic solvent. The eluate is separated by high-pressure liquid-chromatography on reverse phase (RP 18) column using acetonitrile: water (26 74 by volume) as mobile phase. The eluted drugs are detected by uv-absorption at 210 nm. The method is sensitive, specific, precise, and accurate.     

Determination of Arduan and its desacetyl metabolites in biological fluids

The authors elaborated a method for the determination of Arduan and its desacetyl metabolites in biological fluids. The method is based on the use of labelled Arduan and on the determination of radioactivity of the parent drug and metabolites separated by ion-pair TLC in the development system of chloroform-dichloromethane-methanol 6 : 2 : 2 (v/v) 3% NaI (w/v).     

Determination of non-steroidal anti-inflammatory drugs in biological fluids.

The present paper summarizes the methods of determination of non-steroidal anti-inflammatory drugs in biological fluids. CZE, HPLC, HPTLC, GC-MS are analytical techniques, which are capable of a separation of arylpropionic acids.     

Determination of guanfacine in biological fluids by electron-capture GLC.

A quantitative electron-capture GLC method with good sensitivity is described for the determination of guanfacine in human plasma and urine. By condensing its amidino group with hexafluoroacetylacetone, guanfacine is converted to a pyrimidino derivative with better GLC properties than the parent drug. This method allows the determination of guanfacine in plasma and urine at concentrations as low as 0.5 ng/ml and was applied successfully to measurement of plasma levels in humans after therapeutic dosing.     

Determination of procainamide and N-acetylprocainamide in biological fluids by high-pressure liquid chromatography

A modification of a high-pressure liquid chromatographic method for the simultaneous determination of procainamide and N-acetylprocainamide in plasma is described. The deficieicies in the specificity of the existing method were overcome by replacing the cation-exchange column and the mobile phase. The recovery and reproducibility of both procainamide and N-acetylprocainamide from human, dog, and rat plasma and urine spiked with either compound were excellent in the concentration range of 0.05--10 microgram/ml for plasma and 0.5--20 microgram/ml for urine. The comparison of this method with a specific extraction method for sets of plasma samples from human subjects and rats receiving N-acetylprocainamide and procainamide, respectively, showed no statistically significant difference.