Single-laboratory validated method for determination of nordihydroguaiaretic acid in chaparral-containing dietary supplements.

Nordihydroguaiaretic acid (NDGA) occurs naturally in chaparral (Larrea tridentate Coville), a plant which commonly grows in the Southwest United States and has been used for medicinal purposes by Native Americans indigenous to that region. In addition to its traditional use as a tea, manufacturers of dietary supplements have marketed chaparral-containing products in a variety of formulations. Because of the hepatotoxicity of NDGA, and its occurrence in regulated products, we have developed a method for the determination of NDGA in dietary supplements and have tested this method in several dietary supplement formulations. Products were extracted with 80% methanol, filtered, and analyzed by high-performance liquid chromatography. NDGA was detected and determined with both a diode array detector and negative-ion electrospray. Fragmentation in the triple-quadrupole mass spectrometer was obtained by collisional activation of the [M-H](-) ion. Collisional activation produced sufficient fragmentation to provide unambiguous identification. Lack of a stable isotope labeled internal standard has led us to compare quantitations based on UV detection with quantitations based on tandem mass spectrometry (MS/MS). Presence of NDGA was confirmed in several dietary supplement products. Quantitative results from the 2 detection methods were comparable for most products. The limit of quantitation using MS/MS was lower and fewer interferences were observed, although UV detection provided better linearity.

An isotopically labeled internal standard liquid chromatography-tandem mass spectrometry method for determination of ephedrine alkaloids and synephrine in dietary supplements.

We report a simplified analytical procedure for determination of ephedrine alkaloids and synephrine in dietary supplements. Cleanup by simple filtration, when combined with tandem mass spectrometry (MS/MS) detection, provided results comparable to our published method with solid phase extraction (SPE) cleanup and single-stage MS detection with in-source fragmentation. We also compared three mass spectrometric experimental configurations: electrospray (ESI) and atmospheric pressure chemical ionization (APCI) with MS/MS and APCI-MS with fragmentation provided by increasing cone voltage. Because these methods used one isotopically labeled internal standard to determine several different analytes, quantitation errors may arise from susceptibility to ionization suppression caused by the matrix. We therefore compared the results obtained by ESI and APCI ionization.

Preparation and characterization of a suite of ephedra-containing standard reference materials.

The National Institute of Standards and Technology, the U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Food Safety and Applied Nutrition, and the National Institutes of Health, Office of Dietary Supplements, are collaborating to produce a series of Standard Reference Materials (SRMs) for dietary supplements. A suite of ephedra materials is the first in the series, and this paper describes the acquisition, preparation, and value assignment of these materials: SRMs 3240 Ephedra sinica Stapf Aerial Parts, 3241 E. sinica Stapf Native Extract, 3242 E. sinica Stapf Commercial Extract, 3243 Ephedra-Containing Solid Oral Dosage Form, and 3244 Ephedra-Containing Protein Powder. Values are assigned for ephedrine alkaloids and toxic elements in all 5 materials. Values are assigned for other analytes (e.g., caffeine, nutrient elements, proximates, etc.) in some of the materials, as appropriate. Materials in this suite of SRMs are intended for use as primary control materials when values are assigned to in-house (secondary) control materials and for validation of analytical methods for the measurement of alkaloids, toxic elements, and, in the case of SRM 3244, nutrients in similar materials.

Determination of ephedrine alkaloids in dietary supplement standard reference materials.

A suite of five ephedra-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for ephedrine alkaloids, synephrine, caffeine, and selected toxic trace elements. The materials represent a variety of natural, extracted, and processed sample matrixes that provide different analytical challenges. The constituents have been determined by multiple independent methods with measurements performed by NIST and by three collaborating laboratories. The methods utilized different sample extraction and cleanup steps in addition to different instrumental analytical techniques and approaches to quantification. In addition, food-matrix proximates were determined by National Food Processor Association laboratories for one of the ephedra-containing SRMs. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials.

Analysis of coffee for the presence of acrylamide by LC-MS/MS.

A variety of popular instant, ground, and brewed coffees were analyzed using a modified liquid chromatography-tandem mass spectrometry (LC-MS/MS) method specifically developed for the determination of acrylamide in foods. Coffee test portions were spiked with 13C3-labeled acrylamide as an internal standard prior to their extraction and cleanup. Ground coffees (1 g) and instant coffees (0.5 g) were extracted by shaking with 9 mL of water for 20 min. Brewed coffee test portions (9 mL) were taken through the cleanup procedure without further dilution with extraction solvent. Coffee test portions were cleaned up by passing 1.5 mL first through an Oasis HLB (hydrophilic/lipophilic copolymer sorbent) solid phase extraction (SPE) cartridge and then a Bond Elut-Accucat (cation and anion exchange sorbent) SPE cartridge. The cleaned up extracts were analyzed by positive ion electrospray LC-MS/MS. The MS/MS data was used to detect, confirm, and quantitate acrylamide. The limit of quantitation of the method was 10 ng/g for ground and instant coffees and 1.0 ng/mL for brewed coffee. The levels of acrylamide ranged from 45 to 374 ng/g in unbrewed coffee grounds, from 172 to 539 ng/g in instant coffee crystals, and from 6 to 16 ng/mL in brewed coffee.

Rugged LC-MS/MS survey analysis for acrylamide in foods.

The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.

Determination of ephedrine alkaloids and synephrine in dietary supplements by column-switching cation exchange high-performance liquid chromatography with scanning-wavelength ultraviolet and fluorescence detection.

An HPLC method with on-line cleanup coupled to the separation column is described for determination of (-)-norephedrine, (+)-norpseudoephedrine, (-)-ephedrine, (+)-pseudoephedrine, (-)-N-methylephedrine, (+)-N-methylpseudoephedrine, and (+/-)-synephrine in finished dietary supplement products. Test portions were extracted in acidified aqueous acetone. A filtered aliquot was cleaned up on a strong cation exchange (SCX) precolumn that later was automatically coupled to the SCX analytical column. Measurement was by full-scan UV spectra for confirmation of identity by spectral matching and real-time integration of three wavelength signals for multiple quantitation. (+/-)-Synephrine was also quantitated by native fluorescence. Recovery averaged 95-100%. Determination of the major ingredients (-)-ephedrine, (+)-pseudoephedrine, and (+/-)-synephrine compared favorably to findings by an independent LC-MS analysis for a set of 25 samples. The results of a survey were reported for total ephedrine alkaloid and synephrine content and were compared to content declaration, for approximately 48 finished products.