AOACSPDSMethods-2017Awardsv2

line

et al

ournal of

AOAC I

nternational

100, N

3, 2017

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

avid

line

, V

icha

itruthai

and

ilva

abajanian

Herbalife International of America, 950 West 190th St, Torrance, CA 90502

uanyin

and

rashant

ngle

Herbalife Manufacturing, LLC, Quality Control Department, 20481 Crescent Bay Dr, Lake Forest, CA 92630

eter

hang

and

ary

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

> 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

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%.

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.

DIETARY SUPPLEMENTS

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

methodfeedback@aoac.org.

Corresponding author’s e-mail:

quanying@herbalife.com

DOI: 10.5740/jaoacint.16-0387