AOACSPDSMethods-2017Awardsv2

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K line et al .: J ournal of AOAC I nternational V ol . 100, N o . 3, 2017  1

DIETARY SUPPLEMENTS

Quantitative Analysis of Aloins and Aloin-Emodin in Aloe Vera Raw Materials and Finished Products Using High- Performance Liquid Chromatography: Single-Laboratory Validation, First Action 2016.09 D avid K line , V icha R itruthai , and S ilva B abajanian Herbalife International of America, 950 West 190th St, Torrance, CA 90502 Q uanyin G ao 1 and P rashant I ngle Herbalife Manufacturing, LLC, Quality Control Department, 20481 Crescent Bay Dr, Lake Forest, CA 92630 P eter C hang and G ary S wanson Herbalife International of America, 990 West 190th St, Torrance, CA 90502

A single-laboratory validation study is described for a method of quantitative analysis of aloins (aloins A and B) and aloe-emodin in aloe vera raw materials and finished products. This method used HPLC coupled with UV detection at 380 nm for the aloins and 430 nm for aloe-emodin. The advantage of this test method is that the target analytes are concentrated from the sample matrix (either liquid or solid form) using stepwise liquid–liquid extraction (water–ethyl acetate–methanol), followed by solvent evaporation and reconstitution. This sample preparation process is suitable for different forms of products. The concentrating step for aloins and aloe-emodin has enhanced the method quantitation level to 20 parts per billion (ppb). Reversed-phase chromatography using a 250 × 4.6 mm column under gradient elution conditions was used. Mobile phase A is 0.1% acetic acid in water and mobile phase B is 0.1% acetic acid in acetonitrile. The HPLC run starts with a 20% mobile phase B that reaches 35% at 13 min. From 13 to 30 min, mobile phase B is increased from 35 to 100%. From 30 to 40 min, mobile phase B is changed from 100% back to the initial condition of 20% for re-equilibration. The flow rate is 1 mL/min, with a 100 µL injection volume. Baseline separation (R s > 2.0) for aloins A and B and aloe-emodin was observed under this chromatographic condition. This test method was validated with raw materials of aloe vera 5× (liquid) and aloe vera 200× (powder) and finished products of aloe concentrate (liquid) and aloe (powder). The linearity of the method was studied from 10 to 500 ppb for aloins A and B and aloe-emodin, with correlation coefficients of 0.999964, 0.999957, and Received November 16, 2016. Accepted by AP January 17, 2017. This method was approved by the AOAC Expert Review Panel for Aloin as First Action. The Expert Review Panel for Aloin Methods invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. 1 Corresponding author’s e-mail: quanying@herbalife.com DOI: 10.5740/jaoacint.16-0387

0.999980, respectively. The test method was proven to be specific, precise, accurate, rugged, and suitable for the intended quantitative analysis of aloins and aloe-emodin in raw materials and finished products. The S/N for aloins A and B and aloe-emodin at 10 ppb level were 12, 10, and 8, respectively, indicating our conservative LOD level at 10 ppb (the typical LOD level S/N is about 3). The S/N for aloins A and B and aloe-emodin at the 20 ppb level were 17, 14, and 16, respectively, indicating our conservative LOQ level at 20 ppb (the typical LOQ level S/N is about 10). The stock standard solution of a mixture of aloins and aloe-emodin and a working standard solution were found to be stable for at least 19 days when stored refrigerated at 2–8°C, with a recovery of 100 ± 5%. A loe vera has long been used in health foods and dietary supplements (1, 2). Aloin A (barbaloin), aloin B (isobarbaloin), and aloe-emodin are natural components in aloe vera and are referred to as anthraquinones (3). These compounds are removed during the raw material manufacturing process because recent literature indicates adverse effects if the compounds are consumed in sufficient quantities (4). In 2011, the International Aloe Science Council provided an industry guideline limiting the amount of aloins present in aloe products for oral consumption to less than 10 parts per million (ppm; Figure 1; 4). In 2015,AOAC StandardMethod Performance Requirements called for “Methods for the Determination of Aloin A and Aloin B in Dietary Supplement Products and Ingredients” (5) with a low parts-per-billion (ppb) level quantitation limit. To support this AOAC initiative and to QC raw materials and finished products of aloe vera, it was essential to develop and validate a method to quantitate aloins Aand B. Several analytical methods are published in the literature for the detection and quantitation of aloins; however, these lack the required performance characteristics of accuracy, precision, specificity, linearity, and LOD and LOQ (6–11). Our test method had conservative LOQ levels at 20 ppb with nearly twice the response (S/N) of what is usually required using a conventional-sized HPLC column (250 × 4.6 mm) and HPLC instrument. In using an ultra- performance LC system and smaller particle-size columns, it is possible that the LOQ level may reach single-digit ppb levels.