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Talanta

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Determination of bioactive nonvolatile ginger constituents in dietarysupplements by a rapid and economic HPLC method: Analytical methoddevelopment and single-laboratory validationHong Youa,⁎, Bailey Irelanda, Michael Moeszingera, Haoshu Zhanga, Laura Snowb, Scott Krepichb,Vivian Takagawac

a Eurofins Scientific, Inc., 1365 Redwood Way, Petaluma, CA 94954, United Statesb Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501, United Statesc ChromaDex, Inc., 10005 Muirlands Blvd Suite #G, Irvine, CA 92618, United States

A R T I C L E I N F O

Keywords:Nonvolatile ginger constituentsAOAC validationGingerolsShogaols6-paradolZingerone

A B S T R A C T

Most of the validated methods for ginger-containing dietary supplements have long run time and low sensitivityand only analyze gingerols and shogaols. 6-Paradol and zingerone become popular in modern dietary supple-ment industry as bioactive ginger constituents. Therefore, we developed an efficient HPLC-UV/Vis method toanalyze all above major constituents. Compared to 282/280 nm used by the current compendial United StatesPharmacopeia (USP) monograph method and International Organization for Standardization (ISO) 13685-1997method, detection wavelength was optimized to 230 nm which showed a higher sensitivity (signal-to-noise ratio)and better peak resolution. For measuring the ginger constituents in AOAC required matrices, the method wasdemonstrated to be selective, linear (R2 > 0.999), specific, accurate (91.1–103.2% spike recovery rate) andprecise (RSDr < 5%, RSDR < 8%). Among 10 commercial ginger-containing samples that we screened using thismethod, the results were 80–123% of the products’ labeling value. The HPLC running time was successfullyshortened from 29 min (USP method) and 40 min (ISO method) to 12 min without the need of using an expensiveMass Spectrometer for analyte separation. The method is the first method that meets all AOAC SMPR 2017.12requirements and therefore has the potential to be adopted as a consensus industrial reference method formeeting FDA's cGMP Compliance for the manufacture and quality control of dietary supplements and in-gredients.

1. Introduction

Ginger (Zingiber officinale) rhizome is used in several systems ofmedicine, including traditional Chinese medicine, Ayurveda andWestern herbal medicine. It has been used to treat a great variety ofdisorders including colds and flu, dyspepsia, flatulence and colic, mi-graine, nausea, rheumatic disorders, and vomiting [1]. Gingerols arethe major bioactive nonvolatile pungent constituents of fresh gingerrhizome [2]. Gingerols are mainly converted to zingerone and shogaolsduring drying, prolonged storage, processing, and cooking [3]. Theshogaols can then be partially transformed to paradols upon cooking ormetabolized to paradols in the animals’ body after being consumed andabsorbed by digestive system [3]. Major ingredients of ginger-con-taining dietary supplements such as ginger rhizome, powder extract,

and oleoresin contain the non-volatile components including gingerols,paradols, shogaols and zingerone. Previous methods and publicationsfocused on analytical methods that only quantify gingerols and sho-gaols [4–7]. However, compared to other ginger non-volatile con-stituents, 6-paradol has shown better anti-hyperglycemic assay activityin stimulating glucose utilization of 3T3-L1 adipocytes and C2C12myotubes [3]. In a separate study, 6-paradol was suggested as a ther-apeutic agent to effectively protect the brain after cerebral ischemia,likely by attenuating neuroinflammation in microglia [8]. As an alka-loid with strong antioxidantive activity, zingerone has been proven tohave an ONOO(-) peroxynitrite scavenging ability, which indicates itscellular defensive potential against related-symptoms in several humandiseases such as stroke, Alzheimer's disease, and atherosclerosis [9]. Byincreasing systemic superoxide dismutase activity, zingerone was also

https://doi.org/10.1016/j.talanta.2018.10.075Received 25 September 2018; Received in revised form 22 October 2018; Accepted 22 October 2018

⁎ Correspondence to: Eurofins Supplement Analysis Center, Eurofins Scientific, Inc., 1365 Redwood Way, Petaluma, CA, 94954, United States.E-mail addresses: hongyou@eurofinsus.com (H. You), baileyireland@eurofinsus.com (B. Ireland), michaelmoeszinger@eurofinsus.com (M. Moeszinger),

haoshuzhang@eurofinsus.com (H. Zhang), LauraSn@phenomenex.com (L. Snow), ScottK@phenomenex.com (S. Krepich), viviant@chromadex.com (V. Takagawa).

Talanta 194 (2019) 795–802

Available online 25 October 20180039-9140/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

T

Table1

Test

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IDcode

Matrices

Sampleform

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purity)

251

Rhiz

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pow

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mat

eria

lGinger

USP

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0%G

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vola

tile

ging

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sa

099

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act

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nstit

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prod

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(665

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tabl

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%gi

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0.03

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580

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101

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0.58

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986

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100%

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H. You et al. Talanta 194 (2019) 795–802

796

suggested to have possible value in the treatment of Parkinson's disease[10]. The dietary supplement industry has recognized the market of 6-paradol and zingerone and has made them the major bioactive com-ponents in commercially available dietary supplements (Table 1;Sample 101 and 648). The AOAC SMPRs (Standard Method Perfor-mance Requirement) are developed by working groups that are com-posed of stakeholders from academia, industry, and non-profit organi-zations and designed to serve as guidance for the development of aconsensus industrial reference method for meeting FDA's cGMP com-pliances of analyzing dietary supplements and ingredients. Targeting tomeet the AOAC SMPR 2017.12 (Quantitation of Select NonvolatileGinger Constituents) [11] requirements for ginger-containing dietaryingredient and supplements, the method in development is designed toidentify and quantitate 6-paradol and zingerone, in addition to thetraditional target nonvolatile ginger pungent constituents including 6-,8- and 10-gingerols and 6-, 8- and 10-shogaols. The details of SMPR2017.12 are discussed later.

2. Material and methods

2.1. Reagents and materials

Standard 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaoland 10-shogaol were provided by ChromaDex (Irvine, CA, USA).Zingerone, cellulase (cellulase from Aspergillus sp.; 1200 IU/mL;Enzyme Commission (EC) Number 3.2.1.4; Sigma Product # C2605)and citric acid (reagent grade) were obtained from Sigma-Aldrich (St.Louis, MO, USA). 6-Paradol was donated by Dalton Research (Toronto,Canada). HPLC grade methanol and acetonitrile were purchased fromFisher Scientific (Pittsburgh, PA, USA).

Ten samples (Table 1.) in the forms of both dietary supplements(tablet, capsule, softgel, etc.) and dietary ingredients (rhizome powder,dry extract, and oleoresin) were assigned by random ID codes and usedfor validation studies. Sample 251, 099, 986, 740, and 692 were kinddonations from their manufactures. Other samples were purchased fromwww.iHerb.com. A ginger rhizome powder sample (Botanical Re-ference Materials 30290-5) was obtained from ChromaDex (Irvine, CA,USA). Samples were stored in room temperature prior to testing.

2.2. Preparation of standard solution

Standard stock solution was prepared by accurately weighing andtransferring 2.5 ± 0.3 mg of 6-gingerol, 0.75 ± 0.08 mg of 8-gingerol,1.0 ± 0.1 mg of 10-gingerol, 1.0 ± 0.1 mg of 6-shogaol,0.75 ± 0.08 mg of 8-shogaol, 1.0 ± 0.1 mg of 10-shogaol,0.5 ± 0.1 mg of 6-paradol and 1.0 ± 0.1 mg of zingerone to a 20-mLamber VOA vial and then diluting with 20.0 mL of methanol. Stocksolution was stored in freezer until use. This solution may be stored in -20 ± 2 °C freezer for up to 50 days (stability data not shown). 6-pointcalibration curve was prepared by diluting the standard stock solution.

2.3. Optimization of extraction

To optimize the extraction conditions, the ginger rhizome powdersample was used as study material. Extraction solvents (methanol;methanol:water (80%:20% v/v); methanol:water (50%:50% v/v);ethanol, ethyl acetate; dichloromethane-methanol (50%:50% v/v))were tested for the extraction efficiency optimization using 1 h (soni-cate 30 min, vortex 5 s, sonicate another 30 min) sonication.

After determining the extraction solvent, acidic environment andenzyme application in extraction solvent were also studied using thesame ginger rhizome powder sample. The treatments included:

• Control: 80%methanol/20%water was used as extraction solvent.• Acidic extraction: Adjusted water pH to 5 using citric acid solution

before mixing with methanol for making the 80%methanol/20%

water diluent. The rest of the sample preparation was the same asControl.

• Published enzymatic method [12]: Used 0.5% v/w cellulase solutionfor each gram of ginger powder and the reaction occurs in pH = 5water. Incubated sample for 1 h in 50 °C waterbath before addingmethanol for further extraction. Final methanol/water ratio was80%/20%. The rest of the sample preparation was the same ascontrol.

• Optimal enzymatic method: Reaction conditions were designedbased on the enzyme manufacturer's recommendations (emailcommunication with Sigma-Aldridge). 0.5% v/w cellulase solutionwas used for each gram of ginger powder and the reaction occurs inpH = 8 water. Incubated sample for 1 h in 60 °C waterbath beforeadding methanol for further extraction. Final methanol/water ratiowas 80%/20%. Rest of sample preparations was the same as control.

To further evaluate the impacts of changing 7 factors in the analy-tical procedures, a Youden Ruggedness Trial [13] was conducted.Challenging factors were listed in Table 1S (Supplementary data), and 8aliquots of the ginger rhizome powder sample were extracted and testedusing different factor combinations as shown in Table 2S(Supplementary data).

2.4. Sample preparation

In dietary supplements and ingredients sample, estimated levels oftotal nonvolatile ginger constituents may vary from 0.03% w/w to 50%w/w. They were determined from the dietary supplement formulationor dietary ingredient supplier's certificate of analysis. Because of thelarge range and importance of sample/diluent ratio (shown later in the3.2.3 Optimization of extraction studies), the amount to weigh for eachsample (Table 2) was adjusted based on the expected level and the mid-level of the calibration curve. To assure peak area lied within the curve,the sample's weight was calculated to target levels 2–4 of the Calibra-tion Solution's concentration. The maximum sample weight was1000 mg for all the matrices to avoid the oversaturation of diluent.

(a) Rhizome powder and rhizome dry extract – e.g. for ginger rhizomepowder that has an expected level of 1% total nonvolatile gingerconstituents, 60 ± 6 mg (Table 2) sample was accurately weighedand transferred into a 20-mL amber VOA vial. Before weighing,mixed the sample thoroughly until homogenized. Samples weregrinded if necessary.

(b) Tablet and capsule – composited 10 tablets or capsules (removeshells), grinded the samples until homogenized. E.g. for a tablet thathas 3% ginger powder and an expected level of 0.03% total non-volatile ginger constituents, 1000 ± 100 mg (Table 2) wasweighed into a 20-mL amber VOA vial.

(c) Liquid capsule, softgel capsule, and oleoresin– a Pasteur pipette,positive displacement pipette, or other appropriate tool was used totransfer sample (for softgel liquid capsule, mix and composite the

Table 2Sample preparation instructions for samples with different matrices and ex-pected levels.

Exampleestimated level,% w/w

Rhizomepowder,rhizome dryextract

Tablet, capsule Liquid capsule,softgel capsule, andoleoresin

Example sample's weight, mg

0.03 N/A 1000 ± 100 1000 ± 250.3 N/A 1000 ± 100 250 ± 251 60 ± 6 300 ± 30 75 ± 85 12 ± 3 60 ± 6 15 ± 250 1.2 ± 0.3 6 ± 0.6 1.5 ± 0.2

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content of 5 capsules before sample transfer). E.g. for gingeroleoresin that has an expected level as 50% total nonvolatile gingerconstituents, 1.5 ± 0.2 mg (Table 2) was weighed into a 20-mLamber VOA vial using a microbalance.

After weighing, each sample was diluted with 10.0 mL of 80% me-thanol: 20% water (the water was acidified with citric acid, pH = 5),vortexed for 1 ± 0.1 min, sonicated for 30 ± 3 min in cold water (ifthe sonicator has no temperature control function, use icebags to keepthe temperature low, ≤ 30 °C), vortexed for 5 s, sonicated for another30 ± 3 min in cold water and filtered through a 0.45-µm PTFE filterinto an amber autosampler vial for future analysis.

2.5. HPLC analysis

HPLC (Agilent; Santa Clara, CA, USA) equipped with a binary pumpand a diode-array detector (190–400 nm) was employed for the ana-lyses. A Kinetex C18 column (5 µm, 150 ×3 mm) with a SecurityGuardUltra Cartridge (Phenomenex, Torrance, CA, USA) was used for thechromatographic separation. The optimal instrument conditions are:injection volume of 5.0 μL, column temperature at 30 °C, flow rate at1.1 mL/minute and detection wavelength at 230 nm. The gradientprogram with water as mobile phase A and acetonitrile as mobile phaseB is listed below: 0–1.5 min, hold 35% B; 1.5–1.8 min, from 35% to 60%B; 1.8–5 min, hold 60% B; 5–6.5 min, from 60% to 100% B; 6.5–9 min,hold 100% B; 9–9.1 min, 100–35% B; 9.1–12 min, hold 35% B. The totalrun time was 12.0 min.

2.6. Single-laboratory validation parameters

2.6.1. SelectivityFor major target matrices (ginger rhizome dietary ingredient, and

tablet and capsule dietary supplements), method selectivity was de-monstrated by running “Placebo” samples expected to be free of non-volatile ginger constituents. Galangal (Kaempferia galangal, AromaticGinger) root was used as a placebo of ginger rhizome matrix because oftheir morphological similarities. Galangal root is also considered as anadulterant of ginger rhizome [14]. An excipient blend placebo wasformulated (50% maltodextrin, 42% hydroxypropyl methyl cellulose,5% stearic acid, 2% magnesium stearate, and 1% silicon dioxide) andtested to evaluate potential chromatographic interferences from thefiller ingredients used in common tablet and capsule dietary supple-ments.

2.6.2. System suitabilityTo demonstrate the overall chromatographic system suitability, five

replicate injections of the reference material solution were performedfor all 8 nonvolatile ginger constituents. Peak retention time, peak area,and peak shape were analyzed. %RSD (relative standard deviation) ofpeak area, %RSD on retention time, USP tailing factor, and the relativeretention time of each analyte to 6-gingerol were calculated. As thequality control criteria for system suitability, reference material peakarea %RSD must be ≤ 2.5. Reference material retention time %RSDmust be ≤ 2.5. USP tailing factor of the reference material peak must beless than 2.0 for all analyte peaks.

2.6.3. LinearitySix calibration solutions were injected at the beginning of each in-

jection sequence. The calibration curves of the method cover the rangeapproximately 0.5–50 µg/mL for zingerone; 1.2–120 µg/mL for 6-gin-gerol; 0.35–35 µg/mL for 8-gingerol; 0.5–50 µg/mL for 6-shogaol;0.25–25 µg/mL for 6-paradol; 0.5–50 µg/mL for 10-gingerol;0.35–35 µg/mL for 8-shogaol; 0.5–50 µg/mL for 10-shogaol. The cor-relation coefficients, calibration equation slopes, and y-intercepts wereautomatically generated by the HPLC data processing software (e.g.Agilent ChemStation). The blank was not considered as a part of the

calibration curve. Each calibration curve was made up of 6 data points,and the resulting R2 coefficients for all curves must exceed the re-quirement of NLT 0.999.

2.6.4. LOD and LOQThe International Union of Pure and Applied Chemistry (IUPAC)

method (under the recommendation by AOAC INERNATIONSIONAL)was used to determine the LOD (limit of detection) and LOQ (limit ofquantification) of select nonvolatile ginger constituents. The data setfrom seven replications of blank matrix (prepared separately) fromgalangal root was used. LOD is defined as the sum of mean response andthree times the standard deviation. LOQ is defined as the sum of meanresponse and ten times the standard deviation. The instrument's limits(mg/mL) are divided by the sample concentration to convert to a % w/w basis.

2.6.5. AccuracyTo demonstrate method accuracy, galangal root (considered as the

“Placebo” of ginger rhizome) and the excipient blend (considered as the“Placebo” of tablet and capsule dietary supplements) were spiked withnonvolatile ginger constituent reference material solutions at 2 dif-ferent levels. The details of both “Placebo” samples have been describedin Section 2.6.1. The spike recovery experiment was performed byspiking the nonvolatile ginger constituent reference material solution(known concentration) immediately after weighing the samples. Threeseparate sample preparations were conducted (n = 3). The diluent wasadded afterwards to make the total extraction solution volume 10 mL.

2.6.6. PrecisionEight dietary ingredients and supplements (first eight samples in

Table 1) in different matrices were run to demonstrate the repeatabilityand reproducibility of the method. Two NIST SRM samples (sample 740and 692) were tested to further evaluate the accuracy and repeatabilityof the method.

All of ten dietary ingredients and supplements examined for re-peatability were tested in quadruplicate (n = 4) in day 1 by analyst #1on instrument #1. The within-day results, repeatability (RSDr), andHorRatr values for each nonvolatile ginger constituent were then cal-culated for all the materials investigated.

For evaluating method intermediate precision, eight dietary in-gredients (ginger rhizome, dry extract, oleoresin (super critical fluidCO2 soft extract)) and dietary supplements (tablets, capsule, liquidsoftgel capsule) were screened as target matrices. Three runs wereconducted. Two analysts prepared 4 replicates of each sample, on 3separate days, on 3 different instruments (Run 1: Day#1, Analyst#1,Instrument#1; Run 2: Day#2, Analyst#2, Instrument#2; Run 3:Day#3, Analyst#2, Instrument#3). A total of 12 replicate preparations(n = 12) were determined for each type of sample for precision de-termination. The between-day results, reproducibility (RSDR), andHorRatR values were documented.

2.7. Data analysis

Agilent ChemStation was used to automatically determine in-dividual nonvolatile ginger constituents as follows:

% Analyte in sample Area(sample)–Calibration equation interceptCalibration equation slope [Sample, mg/mL]

100%

=×

×

Results were analyzed by using one-way analysis of variance(ANOVA) followed by independent t-tests with Tukey's adjustmentwhen there were significant differences among groups. All data wereanalyzed using Statistix software (version 10.0; Analytical Software,Tallahassee, U.S.).

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3. Results and discussion

3.1. HPLC detection wavelength selection

UV wavelengths 230 nm, 282 nm, and 200 nm were evaluated in theearly stages of method development. A greater amount of matrix in-terference was found when using 200 nm for screening samples thathave complicate matrices (data not shown). Wavelength 282 nm wasadapted by the USP Ginger Monograph method and wavelength 280 nmwas adapted by the ISO (International Organization forStandardization) 13685:1997 compendial method. Compared to 282/280 nm, 230 nm provided a higher signal-to-noise ratio (Table 3S,Supplementary data) and better peak resolution (Figure. 1S,Supplementary data) for all 8 nonvolatile ginger constituents, especiallyfor 6-, 8-, and 10-shogaols which have 230 nm as their maximum ab-sorbance wavelength (λmax). This feature is of great importance formethod accuracy and precision when analyzing samples that have lowlevel shogaols.

3.2. Experimental design and optimization

3.2.1. Extraction solvent optimizationThe extraction procedure has been thoroughly evaluated for raw

ginger powder and dried aqueous ginger extract by other researchgroups [15]. Compared to methanol: water (50%:50% v/v), water,ethanol, and hexane, methanol was found to be most efficient when 6-,8-, 10-gingerol, and 6-shogaol were considered as analytes of interest. Asingle extraction with a 1 h sonication time (sonicate 30 min, vortex 5 s,sonicate another 30 min) was able to recover as high as ≥98.5% of theanalytes in the raw ginger powder [15]. However, additional solventswere evaluated in our validation study because analytes 8-, 10-shogaol,6-paradol, and zingerone were also screened for this project. Angeliet al. [16] demonstrated that methanol:water (80%:20% v/v) has abetter extraction efficiency than methanol for fresh ginger root. Ethylacetate [4,6] and dichloromethane:methanol [17] were also used forgingerol extraction in some published methods. Ethanol extractionyielded almost 100% higher gingerol-rich oleoresin comparing toacetone extraction [12]. Yudthavorasit et al. [18] confirmed that 1 h(sonicate 30 min, vortex 5 s, sonicate another 30 min) sonication isoptimal for the ginger non-volatile constituents extraction.

The extraction solvent comparison test results were shown in Figure2S (Supplementary data). Methanol: water (80%:20% v/v) has sig-nificantly higher extraction efficiency (p < 0.05) than other extractionsolvent (except pure methanol) when total nonvolatile ginger con-stituent value was evaluated as the outcome of interest (n = 3).

3.2.2. Evaluating acidic environment and enzyme application in extractionsolvent

Sample pretreatment was further tested using the ginger rhizomepowder as study material. Acidic environment (low pH) has been shownto facilitate shogaol production [19]. Therefore, acidic extraction wastested on the basis of using methanol:water (80%:20% v/v) as extrac-tion solvent (pH adjustment was performed on aqueous solution beforemixing with methanol). Moreover, multiple publications showed thebenefits of using enzyme to pretreat ginger rhizome samples for betterextraction efficiency. Amylase, cellulase, pectinase, protease, and vis-cozyme were tested. Cellulase yielded the highest gingerol-rich oleor-esin followed by either alcohol (ethanol) extraction [12] or three phasepartitioning (TPP) separation [20]. Thus, cellulase pretreatment wastested in this study with multiple reaction conditions.

The experiment results did not show significant difference betweeneach treatment groups (Table 4S, Supplementary data). Although thereis no statistical difference between different treatments, acidic condi-tion showed the best extraction efficiency, and therefore was chosen asthe extraction diluent (methanol:water rather than pure methanol wasused because most standard laboratory pH electrodes are not designedto be used in organic solvents) of the final method.

3.2.3. Evaluation of other extraction conditions-Youden Ruggedness TrialThe Youden Ruggedness Trial [13] test results were shown in

Table 5S (Supplementary data) and Fig. 3S (Supplementary data). Totalnonvolatile ginger constituent values were evaluated as the outcomes ofinterest. Among all factors that were being tested, sonication time(Factor A-a) and sample/diluent ratio (Factor G-g) were found to bemost critical. However, the difference between using method factor andchallenging factor were relatively small (0.042% w/w purity differencefor Factor A-a = 3.4% difference from average; 0.052% w/w purity forFactor G-g = 4.3% difference from average). The method was thereforefound to be robust.

3.3. Method validation

A reference material mixture solution (Fig. 1) and eight test samples(Figure 4S, Supplementary data) are shown as representative chroma-tograms. The result chromatograms showed that the method is specificfor the target nonvolatile ginger constituents in the ginger rhizome andtablet and capsule containing ginger, because there is no or negligiblechromatographic interference in the region where nonvolatile gingerconstituents would elute. All required system suitability criteria weremet as shown in Table 6S and 7S (Supplementary data). Calibrationcurves were shown in Fig. 5S (Supplementary data). In the approximate

Fig. 1. An overlaid chromatogram of nonvolatile ginger constituents reference material mixture (red line), galangal root powder sample (green line), and excipientblend placebo (blue line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

H. You et al. Talanta 194 (2019) 795–802

799

range of 0.25–50 μg/mL, the method was found to be linear for differentanalytes (R2 > 0.999).

As a part of method specificity evaluation, peak purity was checkedfor each analyte in each sample that was determined in precision andaccuracy tests. A UV–Vis spectrum library (Fig. 6S, Supplementarydata) was established in the HPLC Agilent ChemStation according to theUV–Vis spectra of corresponding bioactive nonvolatile ginger con-stituent reference materials. A peak purity factor (spectrum matchingscore) of 950 was set as the failing threshold in this validation study andany peak that has a peak purity factor < 950 was excluded from theintegration. The LOD and LOQ for the sample with lowest estimatedlevel of nonvolatile ginger constituents are calculated (Table 8S,Supplementary data) as the method limitation. LOQs meet the SMPR2017.12's requirements for LOQ < 0.05% for all analytes (Table 3).

Spike recovery results were summarized in Table 4. The high leveland low level of analytical ranges for ginger containing dietary in-gredient (ginger rhizome) and dietary supplements (tablets and cap-sules) were tested. The reference material spiked levels bracketed theranges as: 0.02–2% w/w for zingerone, 0.06–6% w/w for 6-gingerol,0.02–2% w/w for 8-gingerol, 0.02–2% w/w for 6-shogaol, 0.015–1.5%w/w for 6-paradol, 0.02–2% w/w for 10-gingerol, 0.017–1.7% w/w for8-shogaol, and 0.02–2% w/w for 10-shogaol. The mean recovery value(detected level vs. theoretical level) obtained among different spikedlevels of nonvolatile ginger constituent ranged from 91.1% (zingerone)to 103.2% (8-gingerol) for the galangal root sample, and from 97.2%(zingerone) to 101.5% (8-gingerol) for the excipient blend sample. Allof the results fulfilled the SMPR 2017.12's requirements for recovery(90–107%). The repeatability precision (RSDr) for all analytes amongthe spike recovery test were ≤5%, which is acceptable by the AOACSMPR 2017.12.

Repeatability results (Table 5) showed that all of the total non-volatile ginger constituents, as well as target analytes with levels aboveLOQ, met the AOAC SMPR 2017.12 requirement for repeatability RSDr

(≤5%). Intermediate precision test results (Table 5) showed that all 8samples have their total nonvolatile ginger constituent reproducibilityRSDR acceptable by AOAC SMPR 2017.12 (≤8%). The results ranged

from 0.03% w/w purity for a tablet dietary supplement (Sample 063) to40.2% w/w purity for a super critical fluid CO2 soft extract dietaryingredient (Sample 986). Zingerone's reproducibility RSDR failed AOACSMPR 2017.12 on sample 580 and 986. However, zingerone showedacceptable reproducibility RSDR (3.66%) on Sample 101 that is the onlytarget dietary supplement that has a label claim for zingerone (labelclaim recovery=86%). 6-Paradol's reproducibility RSDR failed AOACSMPR 2017.12 on Sample 251. However, 6-paradol showed acceptablereproducibility RSDR (3.66%) on Sample 648 that is the only targetdietary supplement that has a label claim for 6-paradol (label claimrecovery=100%). Sample 740 and 692 are National Institute of Stan-dards and Technology (NIST) Standard Reference Materials (SRM) 3398(ginger rhizome) and 3399 (ginger dry extract), respectively. Theanalytical results of these 2 samples have been sent to NIST, and theaccuracy of this method was confirmed by NIST through an emailcommunication.

All the required analytes in required matrices have passed the SMPR2017.12 requirements for accuracy and precision (Table 3).

3.4. Application to sample

A variety of dietary ingredients and supplements were tested in therepeatability study using the proposed method. The results were com-parable to their label/manufacture's claims (Table 9S, Supplementarydata). Because the claims may have been developed using differentanalytical methods and terminologies, the recovery results were docu-mented as reference information instead of failing criteria for accuracytest. Among 10 commercial ginger-containing samples that we screenedusing this method, the results were 80–123% of the products’ labelingvalue.

4. Conclusion

The validation results showed that the proposed method is selective,linear, accurate, precise, and robust. As shown in Table 3, all SMPR2017.12 requirements were met for required matrices (rhizome

Table 3AOAC SMPR 2017.012 requirements and HPLC method results.a

Parameter Requirement HPLC Method Results

6-Gingerol 8-Gingerol 6-Shogaol 10-Gingerol

Analytical rangeb 0.05–50% w/w 0.02–50% w/wLOQ ≤0.05% w/w 0.0103% w/w 0.0045% w/w 0.0022% w/w 0.0015% w/wRecovery 90–107% 95.9–99.6% 98.3–103.3% 98.7–101.1% 93.7–101.0%RSDr ≤5% ≤2% ≤3% ≤2% ≤2%RSDR ≤8% ≤6% ≤7% ≤4% ≤7%

a Table only summarizes the results of AOAC SMPR 2017.12 required analytes (6-, 8-, 10-gingerols and 6-shoganol) in required matrices (rhizome powder,rhizome dry extract, tablet and capsules).

b Analytical range was calculated from total select nonvolatile ginger constituents contents.

Table 4Spike recovery results for individual nonvolatile ginger constituent in 2 spiking levels.

Matrix Spike level Results Zingerone 6-Gingerol 8-Gingerol 6-Shogaol 6-Paradol 10-Gingerol 8-Shogaol 10-Shogaol Total constitutes

Galangal root Low Mean, % (w/w) 0.022 0.06 0.021 0.022 0.014 0.021 0.016 0.022 0.198RSD, % 1.76% 5.11% 5.48% 4.93% 3.61% 1.70% 4.62% 3.65% 3.36%Mean Recovery 91.11% 95.90% 103.23% 101.03% 95.81% 93.77% 96.15% 99.76% 96.82%

High Mean, % (w/w) 2.33 6.1 1.98 2.18 1.45 2.15 1.68 2.25 20.1RSD, % 2.17% 1.94% 1.83% 2.06% 2.25% 1.85% 2.29% 2.36% 2.04%Mean Recovery 97.99% 98.14% 98.39% 98.70% 99.01% 98.60% 99.26% 101.63% 98.79%

Excipient blend Low Mean, % (w/w) 0.024 0.062 0.021 0.022 0.015 0.022 0.017 0.023 0.206RSD, % 1.19% 0.58% 0.64% 0.47% 1.37% 1.39% 0.59% 0.72% 0.56%Mean Recovery 97.20% 98.70% 101.50% 99.80% 98.70% 100.70% 101.20% 100.30% 99.50%

High Mean, % (w/w) 2.36 6.16 2.01 2.18 1.46 2.2 1.68 2.21 20.3RSD, % 2.22% 2.22% 2.10% 2.36% 2.12% 2.53% 2.23% 2.27% 2.25%Mean Recovery 99.60% 99.60% 100.30% 99.40% 100.60% 101.00% 99.90% 100.20% 99.90%

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powder, dry extract, tablets and capsule dietary supplements). Thismethod avoided extensive sample preparation, only requires onecolumn, had relatively high sensitivity, shortened the run time to12 min, and was able to distinguish eight nonvolatile ginger con-stituents.

Acknowledgement

Authors would like to thank Dr. Michael Chan from the BritishColumbia Institute of Technology for providing valuable assistance indata processing. The funding was provided through an inter-companyResearch & Development Grant.

Appendix A. Supporting information

Supplementary data associated with this article can be found in theonline version at doi:10.1016/j.talanta.2018.10.075.

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Table 5Precision results summary of the test materials.

Sample Analyte Mean,% (w/w)

Repeatability Intermediate Precision

RSDr HorRatr RSDR HorRatR

251a Zingerone < LOQ N/A N/A N/A N/A6-Gingerol 0.554 1.01% 0.231 1.39% 0.3208-Gingerol 0.140 1.23% 0.230 6.80% 1.2686-Shogaol 0.116 1.59% 0.290 2.98% 0.5406-Paradol 0.015 1.51% 0.204 11.11% 1.47610-Gingerol 0.176 1.14% 0.222 3.89% 0.7518-Shogaol 0.025 4.33% 0.631 7.45% 1.07410-Shogaol 0.048 0.96% 0.154 4.03% 0.641Total 1.074 1.15% 0.292 2.52% 0.638

099a Zingerone 0.063 1.64% 0.274 7.14% 1.1826-Gingerol 2.969 0.92% 0.272 5.82% 1.7188-Gingerol 0.523 3.30% 0.752 4.41% 1.0026-Shogaol 0.505 0.82% 0.186 3.98% 0.9006-Paradol 0.075 1.48% 0.254 5.36% 0.91110-Gingerol 0.736 1.22% 0.296 7.37% 1.7648-Shogaol 0.095 0.54% 0.096 8.04% 1.41510-Shogaol 0.210 2.12% 0.422 7.96% 1.579Total 5.177 1.18% 0.381 4.90% 1.571

063a Zingerone < LOQ N/A N/A N/A N/A6-Gingerol 0.014 0.86% 0.115 3.16% 0.4198-Gingerol < LOQ N/A N/A N/A N/A6-Shogaol 0.005 0.53% 0.060 3.00% 0.3406-Paradol < LOQ N/A N/A N/A N/A10-Gingerol 0.003 0.65% 0.069 2.68% 0.2858-Shogaol 0.001 2.40% 0.211 5.15% 0.45010-Shogaol 0.002 0.30% 0.029 5.98% 0.580Total 0.026 0.64% 0.092 2.38% 0.345

580a Zingerone 0.011 4.01% 0.517 8.84% 1.1296-Gingerol 1.702 1.25% 0.341 2.48% 0.6728-Gingerol 0.309 1.02% 0.214 6.13% 1.2886-Shogaol 0.338 0.35% 0.075 3.51% 0.7486-Paradol 0.041 1.19% 0.186 5.45% 0.84610-Gingerol 0.456 1.73% 0.386 2.35% 0.5238-Shogaol 0.068 0.48% 0.081 4.16% 0.69610-Shogaol 0.149 0.52% 0.098 5.19% 0.977Total 3.075 0.90% 0.269 1.87% 0.555

101a Zingerone 0.498 0.97% 0.218 3.66% 0.8266-Gingerol < LOQ N/A N/A N/A N/A8-Gingerol < LOQ N/A N/A N/A N/A6-Shogaol < LOQ N/A N/A N/A N/A6-Paradol < LOQ N/A N/A N/A N/A10-Gingerol < LOQ N/A N/A N/A N/A8-Shogaol < LOQ N/A N/A N/A N/A10-Shogaol < LOQ N/A N/A N/A N/ATotal 0.498 0.97% 0.218 3.66% 0.826

986a Zingerone 0.111 2.81% 0.517 12.46% 2.2466-Gingerol 24.419 0.31% 0.124 1.92% 0.7768-Gingerol 4.212 4.11% 1.277 5.18% 1.6106-Shogaol 2.862 0.45% 0.132 3.14% 0.9226-Paradol 0.492 1.03% 0.232 6.59% 1.48510-Gingerol 6.460 0.60% 0.198 3.86% 1.2798-Shogaol 0.538 0.29% 0.068 6.70% 1.53010-Shogaol 1.081 0.56% 0.141 4.74% 1.201Total 40.175 0.12% 0.051 1.19% 0.517

648a Zingerone < LOQ N/A N/A N/A N/A6-Gingerol < LOQ N/A N/A N/A N/A8-Gingerol < LOQ N/A N/A N/A N/A6-Shogaol < LOQ N/A N/A N/A N/A6-Paradol 0.011 1.16% 0.147 3.07% 0.39110-Gingerol < LOQ N/A N/A N/A N/A8-Shogaol < LOQ N/A N/A N/A N/A10-Shogaol < LOQ N/A N/A N/A N/ATotal 0.011 1.16% 0.147 3.07% 0.391

889a Zingerone < LOQ N/A N/A N/A N/A6-Gingerol 2.592 1.54% 0.449 8.18% 2.3648-Gingerol 0.434 0.87% 0.191 7.98% 1.7646-Shogaol 1.064 0.64% 0.161 7.74% 1.9576-Paradol < LOQ N/A N/A N/A N/A10-Gingerol 0.671 0.56% 0.133 6.16% 1.4548-Shogaol 0.274 0.55% 0.113 8.14% 1.68010-Shogaol 0.434 0.42% 0.094 8.49% 1.878Total 5.469 1.05% 0.341 4.14% 1.338

Table 5 (continued)

Sample Analyte Mean,% (w/w)

Repeatability Intermediate Precision

RSDr HorRatr RSDR HorRatR

740b Zingerone < LOQ N/A N/A N/T6-Gingerol 0.212 0.85% 0.1688-Gingerol 0.036 2.52% 0.3836-Shogaol 0.230 0.59% 0.1196-Paradol 0.012 1.46% 0.18710-Gingerol 0.096 1.62% 0.2868-Shogaol 0.064 0.63% 0.10510-Shogaol 0.146 0.38% 0.071Total 0.796 0.48% 0.116

692b Zingerone 0.030 2.87% 0.850 N/T6-Gingerol 1.935 1.75% 0.9718-Gingerol 0.266 1.81% 0.7446-Shogaol 0.497 1.09% 0.4946-Paradol 0.073 3.63% 1.22910-Gingerol 0.543 1.73% 0.7938-Shogaol 0.098 1.18% 0.41910-Shogaol 0.202 1.37% 0.541Total 3.643 1.60% 0.972

N/A = Not applicable; N/T = Not tested.a The mean of each sample (except 740 and 692) was calculated from 12

replicates in the Intermediate Percision test.b the mean values of 740 or 692 were calculated from 4 replicates in the

Repeatability test.

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