Determination of Ephedrine Alkaloids in Botanicals and Dietary Supplements by HPLC-UV: Collaborative Study

Abstract An international collaborative study was conducted of a high-performance liquid chromatography (HPLC)-UV method for the determination of the major (ephedrine [EP] and pseudoephedrine [PS]) and minor (norephedrine [NE], norpseudoephedrine [NP], methylephedrine [ME], and methylpseudoephedrine [MP]) alkaloids in selected dietary supplements representative of the commercially available products. Ten collaborating laboratories determined the ephedrine-type alkaloid content in 8 blind replicate samples. Five products contained ephedra ground herb or ephedra extract. These 5 products included ground botanical raw material of Ephedra sinica, a common powdered extract of Ephedra sinica, a finished product containing only Ephedra sinica ground botanical raw material, a complex multicomponent dietary supplement containing Ma Huang, and a high-protein chocolate flavored drink mix containing Ma Huang extract. In addition, collaborating laboratories received a negative control and negative control spiked with ephedrine alkaloids at high and low levels for recovery studies. Test extracts were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. Repeatability relative standard deviations (RSD r) ranged from 0.64–3.0% for EP and 2.0–6.6% for PS, excluding the high protein drink mix. Reproducibility relative standard deviations (RSD R) ranged from 2.1–6.6% for EP and 9.0–11.4% for PS, excluding the high protein drink mix. Recoveries ranged from 84.7–87.2% for EP and 84.6–98.2% for PS. The data developed for the minor alkaloids are more variable with generally unsatisfactory HORRATS (i.e., >2). However, since these alkaloids generally add little to the total alkaloid content of the products, the method gives satisfactory results in measuring total alkaloid content (RSD r 0.85–3.13%; RSDR 2.03–10.97%, HORRAT 0.69–3.23, exclusive of the results from the high protein drink). On the basis of these results, the method is recommended for Official First Action for determination of EP and PS in dietary supplements exclusive of the high protein drinks.

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Method for the Determination of β-Carotene in Supplements and Raw Materials by Reversed-Phase Liquid Chromatography: Single Laboratory Validation

Journal of AOAC International ◽

10.1093/jaoac/87.5.1070 ◽

2004 ◽

Vol 87 (5) ◽

pp. 1070-1082 ◽

Cited By ~ 25

Author(s):

Joseph Schierle ◽

Bernd Pietsch ◽

Alan Ceresa ◽

Christian Fizet ◽

Edward H Waysek

Keyword(s):

Liquid Chromatography ◽

Dietary Supplements ◽

Raw Materials ◽

Collaborative Study ◽

Reversed Phase ◽

Raw Material ◽

Relative Standard ◽

Single Laboratory ◽

Β Carotene

Abstract A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-β-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of β-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane–ethanol. Oily suspensions were directly dissolved in dichloromethane–ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of α-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1–50 μg β-carotene/mL. The main geometrical isomers of β-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as α-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total β-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2–4.4%. Recoveries determined for supplements and beadlet raw material spiked with β-carotene levels of 10 μg to 100 mg/test portion and 0.2–40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-β-carotene in dietary supplements.

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Determination of Lycopene in Dietary Supplements and Raw Materials by High-Performance Liquid Chromatography: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/93.2.499 ◽

2010 ◽

Vol 93 (2) ◽

pp. 499-509 ◽

Cited By ~ 1

Author(s):

Edward H Waysek ◽

Joseph Schierle ◽

Andre Duesterloh ◽

Jayant Deshpande ◽

John Austad ◽

...

Keyword(s):

Dietary Supplements ◽

High Performance ◽

Raw Materials ◽

Collaborative Study ◽

Negative Control ◽

Study Results ◽

Relative Standard ◽

Lycopene Content ◽

Standard Deviations

Abstract A collaborative study was conducted to evaluate the interlaboratory performance of an LC method for lycopene in dietary supplements and the raw materials commonly used in their manufacture. Twelve laboratories from six countries agreed to participate in the study. Results from 10 laboratories were received and are reported. Five dietary supplements, including both tablets and a softgel capsule with a lycopene content ranging from 25 g to 25 mg per unit, and three raw materials, including gelatin-based beadlets, vegetarian beadlets, and a suspension in oil ranging from 5 to 20 lycopene, were analyzed as blind duplicates. In addition to the commercial products, two positive controls and a negative control were included in the study. For the raw materials studied, the repeatability relative standard deviations (RSDr) ranged from 1.49 to 5.13 for total lycopene, and the reproducibility relative standard deviations (RSDR) ranged from 3.84 to 9.21 with HorRat values from 1.23 to 3.24. For finished products, the RSDr ranged from 1.31 to 4.62, RSDR from 4.28 to 10.5, and HorRat values from 0.79 to 2.07. Corresponding values for all-trans-lycopene were significantly higher. It is recommended that the method be considered for Official First Action for all-trans- and total lycopene in finished products and raw materials.

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Determination of Coenzyme Q10 Content in Raw Materials and Dietary Supplements by High-Performance Liquid Chromatography-UV: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/91.4.702 ◽

2008 ◽

Vol 91 (4) ◽

pp. 702-708 ◽

Cited By ~ 14

Author(s):

Steven Lunetta ◽

Mark Roman ◽

A Chandrah ◽

T Edamura ◽

T Honda ◽

...

Keyword(s):

Dietary Supplements ◽

Coenzyme Q10 ◽

High Performance ◽

Raw Materials ◽

Collaborative Study ◽

Negative Control ◽

Test Material ◽

Relative Standard ◽

Standard Deviations

Abstract An international collaborative study was conducted of a high-performance liquid chromatographic (HPLC)-UV method for the determination of coenzyme Q10 (CoQ10, ubidecarenone) in raw materials and dietary supplements. Ten collaborating laboratories determined the total CoQ10 content in 8 blind duplicate samples. Sample materials included CoQ10 raw material and 4 finished product dietary supplements representing softgels, hardshell gelatin capsules, and chewable wafers. In addition, collaborating laboratories received a negative control and negative control spiked with CoQ10 at low and high levels to determine recovery. Materials were extracted with an acetonitriletetrahydrofuranwater mixture. Ferric chloride was added to the test solutions to ensure all CoQ10 was in the oxidized form. The HPLC analyses were performed on a C18 column using UV detection at 275 nm. Repeatability relative standard deviations (RSDr) ranged from 0.94 to 5.05. Reproducibility relative standard deviations (RSDR) ranged from 3.08 to 17.1, with HorRat values ranging from 1.26 to 5.17. Recoveries ranged from 74.0 to 115. Based on these results, the method is recommended for Official First Action for determination of CoQ10 in raw materials and dietary supplement finished products containing CoQ10 at a concentration of >100 mg CoQ10/g test material.

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Determination of Ephedrine Alkaloids in Dietary Supplements and Botanicals by Liquid Chromatography/Tandem Mass Spectrometry: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/86.4.657 ◽

2003 ◽

Vol 86 (4) ◽

pp. 657-668 ◽

Cited By ~ 8

Author(s):

William A Trujillo ◽

Wendy R Sorenson ◽

J Laurensen ◽

G Luo ◽

R McClanahan ◽

...

Keyword(s):

Liquid Chromatography ◽

Dietary Supplements ◽

Collaborative Study ◽

Internal Standard ◽

Negative Control ◽

Interlaboratory Study ◽

Tandem Mass ◽

Accuracy And Precision ◽

Ephedra Nevadensis

Abstract An interlaboratory study was conducted to evaluate the accuracy and precision of a method for ephedrine-type alkaloids [i.e., norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE), methylephedrine (ME), and methylpseudoephedrine (MPE)] in dietary supplements and botanicals. The amount of ephedrine-type alkaloids present was determined using liquid chromatography with tandem mass selective detection. The samples were diluted to reflect a concentration of 0.0200 to 1.00 μg/mL for each alkaloid. An internal standard was added and the alkaloids were separated using a 5 μm phenyl LC column with an ammonium acetate, glacial acetic acid, acetonitrile, and water mobile phase. Eight blind duplicates of dietary supplements or botanicals were analyzed by 10 collaborators. Included was a negative control, ephedra nevadensis, and negative controls fortified at 2 different levels with each of the 6 ephedrine-type alkaloids. The spike levels were approximately 100 and 1000 μg/g for NE, 100 and 600 μg/g for NPE, 6500 and 65 000 μg/g for E, 1000 and 10 000 μg/g for PE, 300 and 3000 μg/g for ME, and 100 and 1000 μg/g for MPE. On the basis of the accuracy and precision results for this interlaboratory study, it is recommended that this method be adopted Official First Action for the determination of 6 different individual ephedrine-type alkaloids in dietary supplements and botanicals.

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Determination of Campesterol, Stigmasterol, and Beta-Sitosterol in Saw Palmetto Raw Materials and Dietary Supplements by Gas Chromatography: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/90.3.670 ◽

2007 ◽

Vol 90 (3) ◽

pp. 670-678 ◽

Cited By ~ 15

Author(s):

Wendy R Sorenson ◽

Darryl Sullivan ◽

S Baugh ◽

M Collison ◽

R Das ◽

...

Keyword(s):

Gas Chromatography ◽

Dietary Supplements ◽

Raw Materials ◽

Collaborative Study ◽

Unsaponifiable Fraction ◽

Saw Palmetto ◽

Relative Standard ◽

Sample Test ◽

Hydrogen Flame

Abstract An interlaboratory study was conducted to evaluate a method for the determination of campesterol, stigmasterol, and beta-sitosterol in saw palmetto raw materials and dietary supplements at levels >1.00 mg/100 g based on a 23 g sample. Test samples were saponified at high temperature with ethanolic KOH solution. The unsaponifiable fraction containing phytosterols (campesterol, stigmasterol, and beta-sitosterol) was extracted with toluene. Phytosterols were derivatized to trimethylsilyl ethers and then quantified by gas chromatography with hydrogen flame ionization detection. Twelve blind duplicates, one of which was fortified, were successfully analyzed by 10 collaborators. Recoveries were obtained for the sample that was fortified. The results were 99.8, 111, and 111% for campesterol, stigmasterol, and beta-sitosterol, respectively. For repeatability, the relative standard deviation (RSDr) ranged from 3.93 to 17.3% for campesterol, 3.56 to 22.7% for stigmasterol, and 3.70 to 43.9% for beta-sitosterol. For reproducibility, the RSDR ranged from 7.97 to 22.6%, 0 to 26.7%, and 5.27 to 43.9% for campesterol, stigmasterol, and beta-sitosterol, respectively. Overall, the Study Director approved 5 materials with acceptable HorRat values for campesterol, stigmasterol, and beta-sitosterol ranging from 1.02 to 2.16.

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Determination of Aconitum Alkaloids in Dietary Supplements and Raw Botanical Materials by Liquid Chromatography/UV Detection with Confirmation by Liquid Chromatography/Tandem Mass Spectrometry: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/92.1.111 ◽

2009 ◽

Vol 92 (1) ◽

pp. 111-118 ◽

Cited By ~ 6

Author(s):

Siu-Kay Wong ◽

C T Che ◽

H-Z Guo ◽

S Ji ◽

J-H Kim ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Dietary Supplements ◽

Collaborative Study ◽

Uv Detection ◽

Tandem Mass ◽

Aconitum Alkaloids ◽

Relative Standard

Abstract An interlaboratory study was conducted to evaluate a method for the determination of 3 Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in raw botanical material and dietary supplements. The alkaloids were extracted with diethyl ether in the presence of ammonia. After cleanup by solid-phase extraction to remove matrix interferences, the alkaloids were determined by reversed-phase liquid chromatography (LC)/UV detection at 235 nm with confirmation by LC/tandem mass spectrometry (MS/MS). A total of 14 blind duplicates were successfully analyzed by 12 collaborators. For repeatability, the relative standard deviation (RSDr) values ranged from 1.9 to 16.7, and for reproducibility, the RSDR values ranged from 6.5 to 33. The HorRat values were all <2 with only one exception at 2.3. All collaborating laboratories had calibration curves with correlation coefficients of >0.998. In addition, 6 collaborators performed the confirmation and were able to verify the identities of the alkaloids by using LC/MS/MS.

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Method for the Determination of Lycopene in Supplements and Raw Material by Reversed-Phase Liquid Chromatography: Single-Laboratory Validation

Journal of AOAC International ◽

10.1093/jaoac/91.6.1284 ◽

2008 ◽

Vol 91 (6) ◽

pp. 1284-1297 ◽

Cited By ~ 6

Author(s):

André Müller ◽

Bernd Pietsch ◽

Nicole Faccin ◽

Joseph Schierle ◽

Edward H Waysek

Keyword(s):

Dietary Supplements ◽

High Performance ◽

Raw Materials ◽

Reversed Phase ◽

Raw Material ◽

Intermediate Precision ◽

Relative Standard ◽

Product Forms ◽

Single Laboratory

Abstract A single-laboratory validation study was conducted for a liquid chromatographic (LC) method for the determination of total and all-trans-lycopene in a variety of dietary supplements and raw materials. Gelatin-based and other water-dispersible beadlets, or tablets, capsules, and softgels containing such product forms, were digested with protease. Alginate formulations and the respective applications were treated with an alkaline sodium EDTA acetate buffer to release lycopene from the matrix. Lycopene and other carotenoids were extracted from the resulting aqueous suspensions with dichloromethane and ethanol. Oily product forms were directly dissolved in dichloromethane and ethanol. The extracts were chromatographed on an isocratic high-performance LC system using a C16 alkylamide modified silica column that provided satisfactory resolution of all-trans-lycopene from its predominant cis-isomers and separated the lycopene isomers from other carotenoids such as - and -carotene, cryptoxanthin, lutein, and zeaxanthin. The within-day precision relative standard deviation (RSD) for the determination of total lycopene ranged from 0.9 to 5.7 over concentration ranges of 50200 g/kg for raw materials and 0.324 g/kg for dietary supplements. The intermediate precision RSD (total RSD) ranged from 0.8 to 8.9. Recoveries obtained for beadlet and tablet material for the different extraction variants ranged from 95.0 to 102.1 at levels of 0.0220 g/kg for tablets and from 95.0 to 101.1 at levels of 1200 g/kg for beadlet material.

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Determination of Hydrastine and Berberine in Goldenseal Raw Materials, Extracts, and Dietary Supplements by High-Performance Liquid Chromatography with UV: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/91.4.694 ◽

2008 ◽

Vol 91 (4) ◽

pp. 694-701 ◽

Cited By ~ 16

Author(s):

Paula N Brown ◽

Mark C Roman ◽

C Chang ◽

C Jin ◽

R Kuriyedath ◽

...

Keyword(s):

Dietary Supplements ◽

High Performance ◽

Raw Materials ◽

Collaborative Study ◽

High Performance Liquid Chromatographic ◽

Hplc Method ◽

Uv Detection ◽

Acetonitrile Solution ◽

Relative Standard

Abstract A multilaboratory collaborative study was conducted on a high-performance liquid chromatographic (HPLC) method utilizing UV detection, previously validated using AOAC single-laboratory validation guidelines for determination of hydrastine and berberine in goldenseal (Hydrastis canadensis L.) raw materials, extracts, and dietary supplements at levels ranging from 0.4 to 6 (w/w). Nine collaborating laboratories determined the hydrastine and berberine content in 8 blind samples. Sample materials included powdered botanical raw materials, whole root material, and 4 finished product dietary supplements containing either goldenseal powdered root material or extract. The materials were extracted with an acidified water and acetonitrile solution. HPLC analyses of the extracts were performed on a C18 column using UV detection at 230 nm. Results for powdered root material and capsule products ranged from about 0.2 (w/w) for each alkaloid to about 4 (w/w) for each alkaloid. Liquid tincture results were approximately 40005000 g/mL for each alkaloid. Reproducibility relative standard deviations (RSDR) for hydrastine ranged from 2.68 to 6.65, with HorRat values ranging from 0.77 to 1.89. RSDR for berberine ranged from 5.66 to 7.68, with HorRat values ranging from 1.32 to 2.12. All finished products containing goldenseal extract yielded HorRat values <2.0. Based on these results, the method is recommended for Official First Action for determination of hydrastine and berberine in goldenseal raw materials and dietary supplement finished products containing powdered goldenseal and goldenseal extract.

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Liquid Chromatographic Method for Determination of Diquat and Paraquat Herbicides in Potatoes: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/76.4.881 ◽

1993 ◽

Vol 76 (4) ◽

pp. 881-887 ◽

Cited By ~ 3

Author(s):

Brian L Worobey ◽

◽

F Béraldin ◽

G Bruns ◽

J Embleton ◽

...

Keyword(s):

Ion Pairs ◽

Collaborative Study ◽

Reversed Phase ◽

Negative Control ◽

Silica Cartridge ◽

Liquid Chromatographic Method ◽

Relative Standard ◽

Repeatability And Reproducibility ◽

Liquid Chromatographic

Abstract A liquid chromatographic (LC) method for the determination of diquat and paraquat herbicides/desiccants in potatoes was collaboratively studied in 6 laboratories. Analytes are extracted from 5 g sample with dilute acid by using a microreflux procedure; the hydrolysate is adjusted to pH 9–10 and passed through a disposable silica cartridge for rapid cleanup and preconcentration. Analytes are separated on a reversed-phase LC column and are measured as their heptanesulfonate ion pairs by UV detection. Each collaborator determined diquat and paraquat at 4 levels (0.05,0.1,0.5, and 1.0 ppm) in blind duplicate samples plus 2 blind negative control samples. Potatoes, obtained from each participant’s region, were spiked by the collaborators with unknown aqueous solutions containing no analyte or a mixture of diquat and paraquat standards. Repeatability and reproducibility relative standard deviations (RSDr and RSDR) averaged 17.1 and 29.0%, respectively, for determination of diquat and 10.8 and 29.5%, respectively, for paraquat. For analysis of standard solutions, RSDr and RSDR values were 6.3 and 12.0%, respectively, for diquat and 7.3 and 13.9%, respectively, for paraquat. Accuracy, measured by comparison with true spiking values (absolute recovery) averaged 77.6 and 76.2% for diquat and paraquat, respectively, and ranged from 71.8 to 88.0% for both compounds. The method was adopted first action by AOAC International.

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Indirect and Direct Determination of the Casein Content of Milk by Kjeldahl Nitrogen Analysis: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/81.4.763 ◽

1998 ◽

Vol 81 (4) ◽

pp. 763-774 ◽

Cited By ~ 58

Author(s):

Joanna M Lynch ◽

David M Barbano ◽

J Richard Fleming

Keyword(s):

Standard Deviation ◽

Nitrogen Content ◽

Relative Standard Deviation ◽

Collaborative Study ◽

Direct Determination ◽

Raw Milk ◽

Method Performance ◽

Relative Standard ◽

Milk Casein

Abstract The classic method for determination of milk casein is based on precipitation of casein at pH 4.6. Precipitated milk casein is removed by filtration and the nitrogen content of either the precipitate (direct casein method) or filtrate (noncasein nitrogen; NCN) is determined by Kjeldahl analysis. For the indirect casein method, milk total nitrogen (TN; Method 991.20) is also determined and casein is calculated as TN minus NCN. Ten laboratories tested 9 pairs of blind duplicate raw milk materials with a casein range of 2.42- 3.05℅ by both the direct and indirect casein methods. Statistical performance expressed in protein equivalents (nitrogen ⨯ 6.38) with invalid and outlier data removed was as follows: NCN method (wt%), mean = 0.762, sr = 0.010, SR = 0.016, repeatability relative standard deviation (RSDr) = 1.287℅, reproducibility relative standard deviation (RSDR) = 2.146%; indirect casein method (wt℅), mean = 2.585, repeatability = 0.015, reproducibility = 0.022, RSDr = 0.560℅, RSDR = 0.841; direct casein method (wt℅), mean = 2.575, sr = 0.015, sR = 0.025, RSDr = 0.597℅, RSDR = 0.988℅. Method performance was acceptable and comparable to similar Kjeldahl methods for determining nitrogen content of milk (Methods 991.20, 991.21,991.22, 991.23). The direct casein, indirect casein, and noncasein nitrogen methods have been adopted by AOAC INTERNATIONAL.

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