Determination of the Composition of Natural Products by HPLC with Charged Aerosol Detection Ian Acworth, Bruce Bailey, Paul Gamache, and John Waraska Thermo Fisher Scientific, Chelmsford, MA, USA Introduction Natural products contain a great diversity of compounds that can show extreme variation in their physicochemical properties. Analysis of active components can be challenging, as not all contain a chromophore or can be ionized, thereby limiting the use of UV absorbance and mass spectrometry, respectively. Charged aerosol detection is a sensitive, universal (nonselective) approach that can measure any nonvolatile and many semivolatile compounds. A number of isocratic and gradient HPLC-Thermo Scientific Dionex Corona CAD Charged Aerosol Detector methods have been developed and evaluated for the measurement of analytes from a variety of natural products, including black cohosh, ginkgo biloba, ginseng, soy, and stevia. Analytes showed consistent response independent of chemical structure (typically < 1% variability between compounds). All methods had a wide dynamic range (four orders of magnitude), good sensitivity (typically low ng levels of detection), and excellent reproducibility (RSDs typically < 2%) even at low detection levels. FIGURE 1. Charged aerosol detection principle. 1 2 1 Black Cohosh Mobile Phase A:.1% Formic acid Mobile Phase B: 1% Acetonitrile Gradient: Time (min) 6 6 66 8 % A 8 8 8 % B 2 1 1 2 2 Flow Rate: 1. ml/min.6 2 mm, µm in 1 ml methanol for min. The mixture was then passed through a Centrex.2 µm nylon filter FIGURE 2. Separation of metabolites in black cohosh. 8 6 9.2 7 1. Liquid eluent enters from 7. Charged gas collides with HPLC system particles and charge is 2. Pneumatic nebulization occurs transfered. Small droplets enter drying tube 8. High mobility species are. Large droplets exit to drain removed by an ion trap. Dried particles enter mixing 9. Remaining charged particles chamber pass to a collector where the 1 2 6 6. A secondary gas stream charge is measured by a very passes over corona needle sensitive electrometer 1. Signal transferred to 2772 chromatographic software 2682-1
Ginseng Mobile Phase A: Water Mobile Phase B: Acetonitrile Gradient: Time (min)..1. % A 8 8 8 % B 2 6 2 2 Flow Rate:.8 ml/min.6 2 mm, µm in 1 ml methanol for min. The mixture was then passed through a Centrex.2 µm nylon filter FIGURE. Reproducibility of ginseng analysis (five replicates). Ginkgo Biloba Mobile Phase A: % Acetonitrile in.1% trifluoroacetic acid Mobile Phase B: 7% Acetonitrile in.1% trifluoroacetic acid Gradient: Time (min) % A 1 2 2 1 % B 7 7 Flow Rate: 1. ml/min.6 2 mm, µm in 1 ml methanol for min. The mixture was then passed through a Centrex.2 µm nylon filter FIGURE. Reproducibility of ginko biloba analysis (five replicates)...18 8 1 12 1 16 18 2 22 2 26 28 2772 8 12 16 2 2 28 2 6 2772 2 Determination of the Composition of Natural Products by HPLC with Charged Aerosol Detection
Soy Saponins Mobile Phase A:.1% Trifluoroacetic acid Mobile Phase B: 1 % Acetonitrile Gradient: Time (min) 6 6 % A 9 1 9 % B 1 9 1 Flow Rate: 1. ml/min.6 2 mm, µm The sample ( mg powder) was dissolved in 2 ml water in a ml volumetric flask. The mixture was sonicated for 1 1 min. Ethanol (2 ml) was then added and the now warm solution was cooled. The solution was brought to ml by the addition of ethanol. The solution was then passed through a Centrex.2 µm nylon filter FIGURE. Separation and detection of soy saponins. Phytoesterogens Isoflavones Mobile Phase: 1% (v/v) Acetonitrile in.1% (v/v) acetic acid Flow Rate: 1. ml/min.6 mm, µm Standards (1 mg/ml) were prepared in DMSO. Dilutions were made in mobile phase FIGURE 6. Phytoestrogen standards (2 ng each on column, five replicate injections). Inset: 1 ng injections of each standard. 6 Daidzin Glycitin 2 21 Daidzin Glycitin Genistin. 1. 2....26 Genistin 2 1 2 27727 1 2 6 27726
Phytoestrogens Coumestans and Mammalian Lignans Mobile Phase: % (v/v) Acetonitrile in.1% (v/v) acetic acid Flow Rate: 1. ml/min.6 mm, µm Standards (1 mg/ml) were prepared in DMSO. Dilutions were made in 2% acetonitrile. Stevia Mobile Phase A: DI water, acetonitrile, TFA (9::.1) Mobile Phase B: Acetonitrile, DI water (9:) Gradient: Time (min) % A 9 9 1 1 9 9 % B 9 9 Flow Rate: 1. ml/min.6 2 mm, µm UV Detection: 21 nm FIGURE 7. Phytoestrogen standards (2 ng each on column, five replicate injections). Inset: ng injections of each standard. 12 Enterodiol Coumestrol 2 1 2 27728 6 2 Enterolactone Enterodiol 1 2 Coumestrol Enterolactone FIGURE 8. Selected portion of chromatogram of SweetLeaf Stevia Extract at ~86 ng on column run with UV at 21 nm and the Charged Aerosol Detector in series (top). Overlay of response curves for rebaudioside A (Reb A) and stevioside from ~ to 1 ng on column each (bottom). Average of injections, each fit to a linear regression. (peak area) 6 mv -1 mau Reb D Reb A Reb C Reb A Reb C Reb B CAD -26 1 1 1 16 17 18 19 2 21 16 1 12 1 8 6 2 1 2 6 Mass on Column (ng) Rebaudioside: R 2 =.9986 : R 2 =.9978 UV 268 Determination of the Composition of Natural Products by HPLC with Charged Aerosol Detection
Detector Linear Correlation Coefficients Rebaudioside A CAD.9986.9978 UV at 21.9992.999 Detector Limit of Detection, s/n = (mass on column) Rebaudioside A Isosteviol CAD ng ng 6 ng UV at 21 6 ng 6 ng > 9 ng Conclusions The need for universal HPLC detection in analytical laboratories is widespread. While several detection technologies (e.g., low wavelength UV, refractive index, evaporative light scattering, chemiluminescent nitrogen detectors) are currently being used, there is significant room for improvement in performance characteristics (i.e., sensitivity, dynamic range, consistency of response factors, and gradient or solvent compatibility). The Corona CAD detector. was developed to help address the many challenges of universal detection. This novel technology offers many benefits to analytical scientists, including universal detection of nonvolatile analytes with response independent of chemical properties, a wide dynamic response range, high sensitivity, and good precision. These characteristics, along with reliability and simple operation, make this a superior detector for the measurement of numerous natural products. 211 Thermo Fisher Scientific, Inc. SweetLeaf is a registered trademark of United American Industries, Inc. Centrex is a trademark of Whatman International, Ltd. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Dionex Products: 1228 Titan Way, PO Box 6, Sunnyvale, CA 988-6, (8) 77-7 North America: U.S./Canada (87) 29-7 South America: Brazil () 11 71 1 Europe: Austria () 616 1 2, Benelux (1) 2 68 9768 (2) 29 Denmark () 6 6 9 9, France () 1 9 1 1, Germany (9) 6112 991 Ireland () 6 6, Italy (9) 2 1 62 1267, Sweden (6) 8 7 8, Switzerland (1) 62 2 9966, United Kingdom () 1276 691722 Asia Pacific: Australia (61) 2 92 2, China (82) 228 282, India (91) 22 276 27, Japan (81) 688 121, Korea (82) 2 26 28, Singapore (6) 6289 119, Taiwan (886) 2 87 66 www.thermoscientific.com/dionex LPN 29-1 1/11