ALN-01

B rown et al .: J ournal of AOAC I nternational V ol . 97, N o . 5, 2014  1323

DIETARY SUPPLEMENTS

Determination of Aloin A and Aloin B in Aloe vera Raw Materials and Finished Products by High-Performance Liquid Chromatography: Single-Laboratory Validation P aula N. B rown , R onan Y u , C hiow H ui K uan , J amie F inley , and E lizabeth M. M udge BC Institute of Technology, Centre for Applied Research and Innovation, Department of Natural Health and Food Products Research, 3700 Willingdon Ave, Burnaby, BC, V5G 3H2 S teven D entali Herbalife International of America, Inc., 990 W. 190th St, Suite 650, Torrance, CA 90502

of burns, dermatitis, and fungal infections (2). Furthermore, they have been used as an ingredient in cosmetics, and the latex of this and other species is generally known for its laxative properties (3). The hydroxyanthrone derivates or anthraquinones are active ingredients known to be responsible for this cathartic effect (4). The anthraquinones, mainly aloins (15–40%), are a mixture of aloin A (barbaloin) and aloin B (isobarbaloin), which are mainly present in the latex of A. vera  (5). When ingested, aloins are hydrolyzed and reduced to the active metabolite (aloe-emodin-9-anthrone), which acts as an irritant in the gastrointestinal tract, producing the purging and cleansing reaction (6). Pharmacological studies have shown that the consumption of aloin-containing products causes acute and chronic adverse reactions such as abdominal pain, electrolyte disturbances, and even hepatitis (7–9). In 2002, the U.S. Food and Drug Administration recommended that aloe laxatives no longer be considered generally recognized as safe over-the-counter drugs and further pharmacological and toxicological investigations are necessary. In early 2011, the National Toxicological Program concluded a 2-year animal study on the toxic and carcinogenic effects of nondecolorized whole leaf A. vera juice in drinking water and found clear evidence of carcinogenic activity in male and female rodents (10). In North America, the International Science Aloe Council has set an industry guideline of less than or equal to 10 ppm total aloins at single-strength concentrations to be considered safe for ingredients in products intended for oral consumption (11). In order to meet this standard, commercial producers and manufacturers follow manufacturing procedures that process A. vera leaf to remove the latex either through physical leaf rind removal and/or decolorization. In this way, finished products can be certified to contain minute to nondetectable levels of aloins. To support a program of this nature, a validated analytical method that is reliable and suitable for its intended purpose of determining and quantifying aloin A and aloin B in A. vera juice and finished products is essential. Although there are several analytical methods available (12–16) that can detect and quantify aloins, none of these methods have addressed performance characteristics such as accuracy, sensitivity, specificity, reproducibility, LOD, LOQ, linearity, and range of the test method using AOAC INTERNATIONAL guidelines. By having a validated method, A. vera ingredients and products can be examined with a degree of confidence that they contain no more than the permissible aloin level set as an industry standard.

Received January 27, 2013. Accepted by APApril 10, 2014. Corresponding author’s e-mail: paula_brown@bcit.ca DOI: 10.5740/jaoacint.13-028 Mill.), more commonly known as A. vera. The plant is a native of southern and eastern Africa and was subsequently introduced to the northern part of Africa, the Arabian Peninsula, China, Mediterranean countries, and the West Indies (1). Historically, A. vera plants were cultivated for the treatment A single-laboratory validation (SLV) was conducted on an HPLC method for the detection and quantification of aloin A and aloin B in Aloe vera raw materials and finished products. An extraction procedure using sonication with an acidified solvent was used for solid test materials while liquid test materials only required dilution, if necessary, prior to filtration and analysis. Separation was achieved using a fused core C 18 column in 18 min under isocratic elution conditions allowing for a single analyte (aloin A) calibration curve to quantify both aloins. Adequate chromatographic resolution (Rs >1) was achieved for aloin A and aloin B. The calibration curves for aloin A exhibited coefficients of determination (r 2 ) of >99.9% over the linear range of 0.3–50 µg/mL. The LOD values were 0.092 and 0.087 µg/mL, and LOQ 0.23 and 0.21 µg/mL for aloin A and aloin B, respectively. Repeatability studies were performed on nine test materials on each of 3 separate days, with five of the test materials determined to be above the LOQ having repeatability RSD (RSD r ) values ranging from 0.61 to 6.30%. Method accuracy was determined through a spike recovery study on both liquid and solid matrixes at three different levels: low, medium, and high. For both aloins, the recovery in the liquid matrix ranged from 92.7 to 106.3% with an RSD r of 0.15 to 4.30%, while for the solid matrix, the recovery ranged from 84.4 to 108.9% with an RSD r of 0.23 to 3.84%. Based on the results of the SLV study, it is recommended that this method be evaluated for reproducibility through a collaborative study. T he genus Aloe comprises more than 100 species of semitropical perennial flowering plants. The most common of these is A. vera (L.) Burm. F. ( A. barbadensis