ALN-01

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AOAC Stakeholder Panel on Dietary Supplements Expert Review Panel AOAC Candidate Method #ALN-01 Determination of Aloin A and Aloin B in Aloe vera Raw Materials and Finished Products by High-Performance Liquid Chromatography: Single-Laboratory Validation • Author(s): Paula N. Brown, Ronan Yu, Chiow Hui Kuan, Jamie Finley, Elizabeth M. Mudge, Seven Dentali • Submitted by: Elizabeth Mudge, BCIT • Enclosures: 1 • Submitter notes: None

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

1324 B rown et al . : J ournal of AOAC I nternational V ol . 97, N o . 5, 2014

( d ) Water (H 2

A validated method can also aid in future assessments regarding safety, use, and application of A. vera products.

O).—FW: 18.01 g/mol; CAS No. 7732-18-5,

HPLC grade (Fisher Scientific) or equivalent.

Solutions

Experimental

( a ) Reference standard diluent .—100% methanol. ( b ) Extraction solvent for high concentration test materials .—0.1% (v/v) acetic acid in methanol. ( c ) Extraction solvent for low concentration test materials .—0.1% (v/v) acetic acid in water. ( d ) Mobile phase A .—0.1% (v/v) acetic acid in water (filtered through a 0.2 µm nylon filter). ( e ) Mobile phase B .—0.1% (v/v) acetic acid in acetonitrile. Separate stock solutions of 100 µg/mL aloin A and aloin B are prepared by dissolving 2.83 mg aloin A in 25.0 mL reference standard diluent and 2.74 mg aloin B in 25.0 mL reference standard diluent. The stock solution of aloin A is then diluted to appropriate concentrations for external calibration. The aloin B stock solution is used as a QC sample. ( a ) Aloin A (C 21 H 22 O 9 ).—FW: 418.39 g/mol; CAS No. 8015- 61-0, purity 88.4%, acquired from Chromadex (Irvine, CA), Cat. No. 1625. ( b ) Aloin B (C 21 H 22 O 9 ).—FW: 418.39 g/mol; CAS No. 8015- 61-0, purity 91.3%, acquired from Chromadex, Cat. No. 1626. Reference standards are stored at 4°C until use. ( a ) Analytical balance .—Capable of reading ±0.01 mg (Mettler Toledo, Vancouver, BC, Canada) or equivalent. ( b ) HPLC vials .—Clear, volume 300 µL (Phenomenex, Torrance, CA). ( c ) Centrifuge .—Eppendorf 5810 table top centrifuge (VWR International) or equivalent. ( d ) Centrifuge tubes .—15 mL polypropylene (VWR International). ( e ) Micropipets .—Eppendorf Reference series 100, 200, and 1000 µL (Eppendorf, Mississauga, ON, Canada) or equivalent. ( f ) Solvent bottles .—100, 500, and 1000 mL. ( g ) Syringe filters .—Luer-lok ® type, 0.2 µm PTFE (VWR International). ( h ) Disposable syringes .—3 mL screw-type (VWR International). ( i ) Sonicating water bath .—Bransonic 3510 (VWR International) or equivalent. ( j ) Vortex mixer .—Thermo Scientific Maxi Mix1 (VWR International) or equivalent. Reference Standards Apparatus

Principle This method is used to detect and quantify aloin A and aloin B in A. vera leaf juice, dietary supplements, and finished products by HPLC with a diode array detector (DAD). This method has been summarized in the American Herbal Pharmacopeia (AHP) monograph for A. vera leaf, leaf juice, and inner leaf juice (17). The focus of the validation study is the diastereomers aloin A (barbaloin) and aloin B (isobarbaloin), although other anthraquinone-type compounds are known to exist in A. vera containing materials. These diastereomers are present in the latex of the plant, which is removed during processing of raw materials into juices and other finished products since recent studies have shown adverse effects when they are consumed in sufficient quantities. The validation consisted of nine test materials. ( a ) A. vera inner leaf dry juice powder A .—Obtained from Tampa Bay Analytical Research (Tampa Bay, FL). ( b ) A. vera inner leaf dry juice powder B .—Obtained from Tampa Bay Analytical Research. ( c ) A. vera nondecolorized leaf dry juice powder .—Obtained fromAloecorp (Ontario, Canada). ( d ) A. vera decolorized inner leaf dry juice powder .— Obtained fromAloecorp. ( e ) A. vera leaf juice powder capsule .—Obtained fromTampa Bay Analytical Research. ( f ) A. vera nondecolorized leaf juice .—Obtained from Aloecorp. ( g ) A. vera decolorized leaf juice .—Obtained fromAloecorp. ( h ) A. vera nondecolorized inner leaf juice .—Obtained from Aloecorp. ( i ) A. vera nondecolorized inner leaf juice concentrate .— Obtained fromAloecorp. All solid test samples were stored in a desiccator at room temperature, and liquid test samples were stored at 4°C. For capsules, to minimize variability due to nonuniformity between dosage units, contents of 20 capsules were combined and homogenized prior to analysis. ).—Formula weight (FW): 60.05 g/mol; CAS No. 64.19-7, ACS reagent grade (Fisher Scientific, Ottawa, ON, Canada) or equivalent. ( b ) Methanol (CH 4 O).—FW: 32.04 g/mol; CAS No. 67-65-1, HPLC grade (VWR International, Mississauga, ON, Canada) or equivalent. ( c ) Acetonitrile (C 2 H 3 N).—FW: 60.10 g/mol, CAS No. 75- 05-08, HPLC grade (VWR International) or equivalent. Test Samples Reagents and Supplies ( a ) Acetic acid, glacial (C 2 H 4 O 2

( k ) Volumetric flasks .—Class A; 2, 5, and 10 mL. ( l ) Volumetric pipets .—Class A; 1, 2, 4, and 8 mL.

HPLC Conditions

( a ) Instrument .—The HPLC system must be equipped with a DAD or UV detector, sample injection system, pump capable of delivering constant flow up to 400 bar, temperature-controlled column compartment, and computing data processor. ( b ) Autosampler temperature .—Room temperature.

B rown et al .: J ournal of AOAC I nternational V ol . 97, N o . 5, 2014 1325

( c ) Analytical column .—Phenomenex (Torrance, CA) Kinetex 2.6 µm C 18 , 100 × 4.60 mm id, 2.6 µm particle size. ( d ) Column temperature .—30°C. ( e ) Detector conditions .—Monitor at 357 nm (4 nm bandwidth, no reference). ( f ) Flow rate .—1.850 mL/min. ( g ) Injection volume .—15 µL with methanol wash. ( h ) Run time .—18 min. ( i ) Isocratic elution conditions .— See Table 1. The calculation used to determine aloin concentration is as follows: Concentration, mg/mL: C = (A – B)/D where A = peak area (mAU × seconds), B = intercept of the calibration curve, and D = slope of the calibration curve. To quantify the individual aloins on a % (w/w) basis, the following calculation is used: Aloin, % (w/w) = [(C)(FV)(D)(100%)]/W where C = concentration (µg/mL) from linear regression analysis, FV = the final volume (mL) of the sample preparation, D = the dilution factor of the sample preparation, and W = the sample weight (mg). For the validation study, the following equations were used for evaluating precision: × 100 where SD(r) = population SD (σ/ n , where σ = sum of squares and n = number of replicates). PRSD r (RSD r calculated, %): PRSD r = 2C –0.15 where C = the concentration of the analyte expressed as a mass fraction. HorRat value: HorRat = RSD r PRSD r Within-day precision average and SDs are of four data points within a day, whereas within-laboratory precision average and SDs are 12 data points over 3 days (separate batches on 3 days). Calculations RSD r (found, %): RSD r = SD(r) mean ( a ) Liquid and juice samples .—( 1 ) Highly concentrated (>50 µg/mL) or viscous samples–mix thoroughly. Dilute 1:1 (v/v) with 0.1% acetic acid in methanol. Centrifuge at 5000 rpm for 3 min. ( 2 ) Filter supernatants and low concentrated juices through 0.2 µm PTFE filters into HPLC vials for analysis. ( b ) Extraction of powders and capsules .—( 1 ) The total capsule weight was determined by weighing 20 capsules. Combine and homogenize the contents of 20 capsules into a conical tube. ( 2 ) Weigh 100.0 ± 1.0 mg A. vera powder into a 15 mL conical centrifuge tube. Add 1.0 mL of 0.1% acetic acid in water and sonicate for 5 min three times, vortexing for 30 s between intervals. Preparation of Test Samples

( 3 ) Centrifuge at 5000 rpm for 3 min. ( 4 ) The supernatant is then filtered through 0.2 µm PTFE filter to an HPLC vial. ( 5 ) Highly concentrated (>50 µg/mL) or viscous samples.— Extract 100.0 mg Aloe powder with 1.0 mL 0.1% acetic acid in methanol first, gently vortex, and then add 1.0 mL 0.1% acetic acid in water. Sonicate for 5 min three times; vortex for 30 s between intervals. Centrifuge at 5000 rpm (4500 × g ) for 3 min prior to filtering with 0.2 µm PTFE filter into an HPLC vial. If samples are not analyzed immediately after preparation, they are refrigerated at 4°C prior to analysis to prevent degradation of aloins for 72 h. ( a ) Aloin A calibration curve .—Because the HPLC method uses an isocratic elution, only aloinAis required as the calibration standard for both aloins. A seven point calibration curve is generated from the 100 µg/mL stock solution as described below. ( 1 ) Linearity 1 (50 µg/mL) .—500 µL aloin A stock solution was pipetted into a 2.0 mL HPLC vial and diluted with 500 µL methanol and mixed well. ( 2 ) Linearity 2 (25 µg/mL).— 250 µL aloin A stock solution was diluted with 750 µL methanol and mixed well. ( 3 ) Linearity 3 (10 µg/mL).— 100 µL aloin A stock solution was diluted with 900 µL methanol and mixed well. ( 4 ) Linearity 4 (5 µg/mL) .—100 µL Linearity 1 was diluted with 900 µL methanol and mixed well. ( 5 ) Linearity 5 (1 µg/mL) .—100 µL Linearity 3 was diluted with 900 µL methanol and mixed well. ( 6 ) Linearity 6 (0.5 µg/mL) .—100 µL Linearity 4 was diluted with 900 µL methanol and mixed well. ( 7 ) Linearity 7 (0.3 µg/mL).— 300 µL Linearity 5 was diluted with 700 µL methanol and mixed well. ( b ) QC sample .—Amixed standard of 25 µg/mL aloin A and B is used for QC. Prepare the QC standard by combining 1.0 mL 100 µg/mL aloin A stock solution and 1 mL 100 µg/mL aloin B stock solution. Dilute the aloin A and B mixture with 2.0 mL of 0.1% acetic acid in water. Table 1. The isocratic elution conditions for the separation of aloin A and aloin B with column cleanup and post time re-equilibration Time, min Mobile Phase B, % 0.0 17 8.0 17 12.0 100 13.0 100 14.0 17 18.0 17 Preparation of Calibration and Quality Control Solutions

Single-Laboratory Validation (SLV) Parameters

This method was validated according to the guidelines of AOAC INTERNATIONAL SLV criteria (18). ( a ) Selectivity .—The selectivity of themethodwas established by injecting each of the aloins to show chromatographic resolution between analytes.

1326 B rown et al . : J ournal of AOAC I nternational V ol . 97, N o . 5, 2014 ( b ) Linearity .—Linearity was evaluated using a seven-point standard calibration curve of aloin A. Calibration curves were plotted and linear regression was used to determine the slope and y-intercept. Linearity was also confirmed visually by generating trendlines using a Microsoft Excel program (Microsoft, Mississauga, ON, Canada). 25 30 35

Aloin B

Aloin A

( c ) LODand LOQ .—The LODand LOQ for the quantification of aloin A and aloin B were determined using the International Union of Pure and Applied Chemistry (IUPAC) method. This method, recommended byAOAC INTERNATIONAL, uses data sets from at least seven replicate injections of the blank matrix. The LOD is defined as the mean response plus 3x the SD, and the LOQ is the mean plus 10x the SD. ( d ) Precision (repeatability; precision study) .—Precision was evaluated by analyzing multiple replicates of each test sample. Four replicate preparations of each test material were prepared and analyzed on 3 separate days, thereby having 12 replicates of each test material prepared. The within-day, between-day, and total SDs for aloin A and aloin B for each of the nine test materials were calculated and consequently used to generate HorRat values by using the Horwitz formula for each of the test material to summarize the precision of the validation study (19). Acceptable HorRat values are 0.1 to 2.0. ( e ) Accuracy .—Accuracy of the method was assessed using both solid and liquid aloe test samples that were determined not to contain any detectable aloins. Stock solutions of known concentrations were spiked in both liquid and solid materials in triplicate at three levels that correspond to 10% (low), 100% (medium), and 200% (high) of aloin A and aloin B expected in typical aloin raw materials and finished products. For the solid matrix recovery study, a commercial spray-dried A. vera powder was spiked to expected concentrations of 4, 40, and 80 µg/mL. An A. vera inner fillet juice was spiked to expected concentrations of 1, 10, and 20 µg/mL for the liquid matrix. The samples were processed as per the Preparation of Test Samples section above, taking into account the volume of spike solution added. ( f ) Stability .—The stability of the aloin A and aloin B was determined by comparing the concentration of the reference standards either acidified with 0.1% acetic acid or nonacidified at room temperature over time. Each aloin was evaluated by storing the solutions at room temperature (18–22°C). Identical reference standards were aliquotted in two sets having four replicates each: one set was acidified and the other set nonacidified and analyzed at a given time (t) point (t = 0, 4, 8, 24, 48, and 240 h). Stability was determined by comparing the peak areas to time = 0 where the assumption of no degradation was made. Optimization Studies Prior to the method validation, the extraction and chromatography parameters were investigated through optimization studies. For the extraction optimization, parameters such as extraction solvent, the addition of acid, sonication time, and the effect of heat were explored. For the chromatography method, parameters such as analytical column length and type, mobile phase composition, gradient versus isocratic elution, peak resolution, and run time were factors that were Results and Discussion

Absorbance (mAU)

Time (min)

Figure 1. Figure 1. Chromatogram of an aloin A and aloin B reference standard.

Aloin B

Aloin A

Absorbance (mAU)

Time (min) Figure 2. Chromatogram of an untreated A. vera juice product.

Figure 2.

examined to provide the best resolution and shortest run time for the separation of aloin A and aloin B. The optimization of the method was carried out using both aloin reference standards and A. vera test materials.

Method Validation Results–Performance Characteristics

Identification of aloin A and aloin B in the test materials was possible by comparing the retention times of the aloins from the reference standards. An isocratic elution was used for the analysis of aloin A and aloin B, where the retention order is aloin B followed by aloin A. Representative chromatograms of the reference standards and test material are displayed in Figures 1 and 2. Quantification of the analytes was carried out by a simple linear regression analysis using quadruplicate samples prepared on three separate days at seven concentration levels. The analytical range used for the following aloins are listed above in the section on Preparation of Calibration and QC Solutions . Table 2. The LOD and LOQ by the IUPAC method Analyte LOD, µg/mL LOQ, µg/mL Aloin A 0.09 0.23 Aloin B 0.09 0.21

B rown et al .: J ournal of AOAC I nternational V ol . 97, N o . 5, 2014 1327

Table 3. Precision results summary of the test materials

Analyte (Aloin)

RSDr, %

HorRat (0.3–2.0) a

Mean b

Matrix

TM1 – Inner leaf juice dry powder A

A B

TM2 – Inner leaf dry juice powder B

1.5

6.62

6.33

2.0

6.62

8.64

TM3 – Nondecolorized leaf dry juice powder

1.4

26.20

4.89

1.8

24.80

6.30

TM4 – Decolorized inner leaf dry juice powder

TM5 – Powder capsule

1.0

4.73

4.44

0.6

4.73

2.74

TM6 – Nondecolorized leaf juice

0.3

23335

1.21

0.3

23265

1.09

TM7 – Decolorized leaf juice

TM8 – Nondecolorized inner leaf juice

1.3

13889

4.92

1.2

14000

4.51

TM9 – Decolorized inner leaf juice concentrate

a HorRat values between 0.3 and 2.0 are considered acceptable according to AOAC guidelines for SLVs. b µg/g For solid samples and µg/mL for liquid samples.

materials. It should be noted that quantification of the treated test materials (i.e., low level aloin content) was done using a four- point calibration curve (i.e., 0.3, 0.5, 1, and 5 µg/mL) since linear regression does not give equal weight to the calibration points especially at low levels when a seven-point calibration curve is used. The r 2 for the four-point calibration curve was ≥99.9%. Alternatively, a weighted least square calibration curve could have been used using all seven points, which would produce results comparable to the four-point calibration curve.

Materials tested included treated and nontreated powder and liquid/juice.

Selectivity

There was adequate resolution (Rs > 2.0) of the aloins from each other as well as from neighboring eluted compounds.

Linearity

All of the standard curves produced throughout the course of the study appeared linear upon visual inspection. The coefficients of determination (r 2 ) were ≥99.9%, therefore considered linear and acceptable for quantifying the aloins in the different test

LOD and LOQ

By the IUPAC method, the LODs for aloinA and aloin B were

Table 4. Negative control spike recovery results for aloin A and aloin B in a (a) liquid matrix and (b) solid matrix Level 1 Level 2 Level 3 Aloin A Aloin B Aloin A Aloin B Aloin A Aloin B (a) Liquid matrix x Recovery, a % 106.26 95.01 104.74 90.17 105.45 92.70 RSDr, % 4.30 1.83 0.75 0.15 0.84 0.57 (b) Solid matrix x Recovery, % 108.86 91.50 100.20 84.41 105.71 88.06 RSDr, % 0.98 3.84 3.17 0.23 1.95 1.81 a x = Mean value of three replicate samples.

1328 B rown et al . : J ournal of AOAC I nternational V ol . 97, N o . 5, 2014

both 0.09 µg/mL, and the LOQs were 0.23 and 0.21 µg/mL, respectively (Table 2).

References

(1) Haller, J.S. (1990) Bull. N.Y. Acad. Med . 66 , 647–659 (2) Farnsworth, N.R. (1995) NAPRALERT Database , University of Illinois at Chicago, IL, an on-line database available directly through the University of Illinois at Chicago or through the Scientific and Technical Network (STN) of Chemical Abstracts Services (3) Hecht, A. (1981) FDA Consumer Updates (Issue July-Aug) 15 , 26–29 (4) Tyler, V.E., Bradley, L.R., & Robbers, J.E. (1998) in Pharmacognosy , 9th Ed., Lea & Febiger, Philadelphia, PA, pp 62–63 (5) Bradley, P.R. (1992) in British Herbal Compendium, Vol. 1, British Herbal Medicine Association, Bournemouth, UK, pp 199–203 (6) Reynolds, J.E.F. (1993) in Martindale, the Extra Pharmacopoeia , 30th Ed., Pharmaceutical Press, London, UK, p. 903 (7) Blumenthal, M. (1998) in The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines , 12 , 80–81 (8) Beuers, U., Spengler, U., & Pape, G.R. (1991) Lancet , 337–472 (9) Bottenberg, M.M., Wall, G.C., Harvey, R.L., & Habib, S. (2007) Ann. Pharmacother . 41 , 1740–1743. http://dx.doi.org/10.1345/ aph.1K132 (10) NTP Draft Technical Report TR577 (2011) on the Toxicology and Carcinogenesis Studies of a Non-Decolorized Whole leaf extract of Aloe barbadensis Miller ( Aloe vera in F344/N Rats and B6C3F 1 Mice–Drinking Water Study). https://niehs.nih. gov/news/assets/docs_f_o/ntp_draft_technical_report_577_a_ nondecolorized_whole_leaf_extract_of_aloe_barbadensis_ miller_aloe_vera_508.pdf (accessed January 11, 2013) (11) International Aloe Science Council (February 2011) IASC Reference Version 1.0 FAQ. http://www.iasc.org/faq.html (accessed January 11, 2013) (12) Dell’Agli, M., Giavarini, F., Ferraboschi, P., Galli, G., & Bosisio, E. (2007) J. Agric. Food Chem. 55 , 3363–3367. http://dx.doi. org/10.1021/jf070182h (13) El Sohly, M., Gul, W., Avula, B., & Khan, I.A. (2007) J. AOAC Int. 90 , 28–42 (14) Fanali, S., Aturki, Z., D’Orazio, G., Rocco, A., Ferranti, A., Mercolini, L., & Raggi, M.A. (2010) J. Sep. Sci. 33 , 2663–2670. http://dx.doi.org/10.1002/jssc.201000408 (15) Zahn, M., Trinh, T., Jeong, M.L., Wang, D., Abeysinghe, P., Jia, Q., & Ma, W.A (2008) Phytochem. Anal. 19 , 122–126. http:// dx.doi.org/10.1002/pca.1024 (16) Zonta, F., Bogoni, P., Masotti, P., & Micali, G. (1995) J. Chromatogr. A 718 , 99–106. http://dx.doi.org/10.1016/0021- 9673(95)00637-0 (17) Upton, R., Axentiev, P., & Swisher, D. (Eds) (2013) American Herbal Pharmacopoeia and Therapeutic Compendium , AHP, Scott’s Valley, CA, pp 30–33 (18) Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals (2003) AOAC INTERNATIONAL, Gaithersburg MD (19) Wernimont, G.T. (1987) in Use of Statistics to Develop and Evaluate Analytical Methods , W. Spendley (Ed.), AOAC INTERNATIONAL, Gaithersburg, MD

Precision

From the nine test materials used in this study, five test materials (TM2, TM3, TM5, TM6, and TM8) were above the LOQ and had a HorRat range of 0.3 to 2.0 and RSDr values of 0.61–8.64% as summarized in Table 3. Three of the test materials (TM1, TM7, and TM9) were below the LOQ but were detected, and the one test material (TM4) was below the LOD; therefore, no HorRat and RSD r values were reported for these materials.

Accuracy

The average recovery range of aloin A and aloin B in both solid and liquid matrixes was 84.41–108.86% with an RSD r of 0.15–8.64%. Tables 4a and b contain the summarized results for each individual aloin at the three different concentration levels.

Stability of Mixed Standard

The stability of the aloins in reference standards, as well as test material solutions, was found to be higher with the addition of an acid. It was determined during method optimization that 0.1% acetic acid was adequate to provide good peak shape and retain analyte stability over a period of time. Storage of the aloin reference standards with the addition of an acid provided consistent peak area after 48 h when exposed to room temperature conditions with an average difference of 2–3% mAu*s. In the nonacidified solutions, the aloins started to degrade after 8 h at room temperature, and with an average difference of 23–28% mAu*s after 48 h from the initial time point. This method for the determination of aloin A and aloin B in raw materials and select finished products containing A. vera leaf juice was subjected to an SLV study according to AOAC INTERNATIONAL guidelines. All parameters investigated in the validation study were in compliance with the AOAC INTERNATIONAL guidelines. The described method is thus considered to be suitable for its purpose and ready for further validation studies. Conclusions

Acknowledgments

We would like to acknowledge Aloecorp (Ontario, CA), International Aloe Science Council (Silver Spring, MD) and Mark Roman from Tampa BayAnalytical Research (Tampa Bay, FL) for providing the materials for this study. In addition, we would like to acknowledge the ChromaDex for supplying the reference standards used in the validation study.

Aloe SLV Method Supplementary Information Precision: The precision (RSDr) reported in the publication is intermediate precision (different days, calibration curves, solutions, etc); which is more rigorous in comparison to AOACs guidelines for repeatability as specified in the SMPR. Threfore, we have complied the repeatability according to AOAC guidelines below. The repeatability (%RSDr) between replicate samples on the same day for day 1 of the analysis (4 replicate samples) is summarized below for the samples which contained Aloins above the quantitation limit. Repeatability (%RSD) Aloin A Aloin B TM2 3.6 2.8 TM3 0.83 4.3 TM5 1.4 2.1 TM6 0.83 0.74 TM8 0.85 0.51 LOD/LOQ concentration in Samples: The LOD/LOQ reported in the publication is concentration of the calibration solution. The back calculation for liquid and dry matrices are summarized below: Liquid samples (as per manuscript): LOD aloin A &B: 0.09 µ g/mL; LOQ Aloin A: 0.23 µ g/mL, Aloin B: 0.21 µ g/mL. Powders & capsules: LOD Aloin A & B: 0.9 µ g/g; LOQ Aloin A: 2.3 µ g/g, Aloin B: 2.1 µ g/g

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