Improved Universal Approach to Measure Natural …...Improved Universal Approach to Measure Natural Products in a Variety of Botanical and Supplements Marc Plante, Ian N. Acworth,

Improved Universal Approach to Measure Natural Products in a Variety of Botanical and SupplementsMarc Plante, Ian N. Acworth, Bruce A. Bailey, Qi Zhang, and David Thomas Thermo Fisher Scientific, Chelmsford, MA, USA

2 Improved Universal Approach to Measure Natural Products in a Variety of Botanical and Supplements

I d U i l A h t M N t l P d t i V i t f B tImproved Universal Approach to Measure Natural Products in a Variety of BotImproved Universal Approach to Measure Natural Products in a Variety of BotMarc Plante Ian N Acworth Bruce A Bailey Qi Zhang and David ThomasMarc Plante, Ian N. Acworth, Bruce A. Bailey, Qi Zhang, and David Thomas Thermo Fisher Scientific Chelmsford MA USAThermo Fisher Scientific, Chelmsford, MA, USA

Overview Methods HoodiaC l A C8Overview

Purpose: To evaluate the application of HPLC with a charged aerosol detector forMethodsLi id Ch h

Column: Accucore C8Column Temp : 40 oCPurpose: To evaluate the application of HPLC with a charged aerosol detector for

the measurement of natural products and botanicals.Liquid Chromatography HPLC System: Thermo Scientific™ Dionex™ UltiMate™ 3000 RS system with:

Column Temp.: 40 CFlow Rate: 2.0 mL/mint e easu e e t o atu a p oducts a d bota ca s

Methods: HPLC methods were developed for the analysis of black cohosh carallumaHPLC System: Thermo Scientific Dionex UltiMate 3000 RS system with:

- Diode Array Detector DAD-3000RS and a Mobile Phase A: Deionized waM bil Ph B A t it ilMethods: HPLC methods were developed for the analysis of black cohosh, caralluma,

bacopa, milk thistle, ashwagandha, and hoodia. - Thermo Scientific™ Dionex™ Corona™ Veo™ Charged Aerosol Detector:

Mobile Phase B: Acetonitrile,Gradient: 40%B to 47%

Results: HPLC with charged aerosol detection is sensitive (low ng levels on-column),Detector:

Evaporation Temperature: 35 or 50 °CGradient: 40%B to 47%Injection Vol.: 10 µLg ( g ),

has a wide dynamic range and minimal inter-analyte response variability. It is ideal for i l t th t l k h h

p pPower function: 1.00Data collection rate: 2 Hz

Sample Prep.: Place 50 mg2 L t if t b dd 1 Lmeasuring analytes that lack a chromophore. Data collection rate: 2 Hz

Th S i tifi ™ Di ™ Ch l ™ Ch t h D t S t S ft 7 22 mL centrifuge tube, add 1 mL m5 mL volumetric flask Repeat two

IntroductionThermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System Software, 7.2 5 mL volumetric flask. Repeat two

to volume with methanol. CentrifuIntroduction Black CohoshColumn: Thermo Scientific™ Accucore™ C18 column

a glass HPLC autosampler vial.Botanicals contain a great diversity of compounds that exhibit wide variation in their ph sicochemical properties Altho gh no single anal tical method is a ailable to Results and Discu

Column: Thermo Scientific™ Accucore™ C18 column, 2.1 × 150 mm, 2.6 µm physicochemical properties. Although no single analytical method is available to

measure all potentially active components, HPLC with charged aerosol detection is aResults and DiscuExtracts of black cohosh have been

, µColumn Temp: 40 oCmeasure all potentially active components, HPLC with charged aerosol detection is a

nearly universal approach that nonselectively measures any nonvolatile and many Extracts of black cohosh have beenmenopause. The active ingredients

Flow Rate: 0.5 mL/minMobile Phase A: Deionized watersemivolatile compounds; that is, CAD does not require that analytes be ionizable (as

required for mass spectrometry) or contain a chromophore (as required for UV

menopause. The active ingredients27-deoxyactein, actein, cimiracemo

Mobile Phase A: Deionized water Mobile Phase B: Acetonitrile, Fisher Scientific™ Optima LCMS required for mass spectrometry) or contain a chromophore (as required for UV

spectrophotometry).do not possess chromophores abovlow abundant analytes in a black co

, pGradient: 30% B to 40% B in 12 min; 40% to 60% B from 12–30 min; 60% -

95%B f 30 t 40 ispectrophotometry).

Presented are several HPLC/UHPLC methods with charged aerosol detection thatlow-abundant analytes in a black co95%B from 30 to 40 min.

Injection Vol : 2 µLPresented are several HPLC/UHPLC methods with charged aerosol detection that have been improved to increase speed and sensitivity. The improved methods were

Injection Vol.: 2 µLSample Prep.: Sonicate 300 mg of sample in 40 mL methanol in 50 mL vol. flask FIGURE 2. Comparison of charge

evaluated for the measurement of phytochemicals extracted from a variety of botanical d h b l l t i l di t it l id f bl k h h (Ci i if

p p g pfor 15 min. Cool to room temperature and bring to volume with methanol. Centrifuge at 13 000 f 15 i T f t t t b l HPLC t l i l

determination of triterpene glycoand herbal supplements including: triterpene glycosides from black cohosh (Cimicifugaracemosa) and Bacopa monnieri; oxypregnane glycosides from Caralluma fimbriata;

13,000 rpm for 15 min. Transfer supernatant to an amber glass HPLC autosampler vial. racemosa) and Bacopa monnieri; oxypregnane glycosides from Caralluma fimbriata; flavonolignans from milk thistle (Silybum marianum); steroidal lactones from Caralluma 4.00

Ashwagandha (Withania somniferateroidal and oxypregnane glycosides from Hoodiagordonii

Column: Accucore C8 column, 4.6 × 150 mm; 2.6 µm C l T 45 oC

3.00

A]gordonii.

A l t h d i t t i d d t f h i l t t (t i llColumn Temp.: 45 oCFlow Rate: 2 0 mL/min 2.00en

t [pA

Analytes showed consistent response independent of chemical structure (typically < 10% variability between compounds corrected for gradient elution) All methods had a

Flow Rate: 2.0 mL/minMobile Phase A: Deionized water

1 00Cur

re

10% variability between compounds corrected for gradient elution). All methods had a wide dynamic range (~four orders of magnitude), good sensitivity (typically low ng Mobile Phase B: Acetonitrile, Fisher Scientific™ Optima LCMS

Gradient: 40%B to 47%B in 2 5 min; to 95%B in 12 5 min; hold 10 min1.00C

y g ( g ) g y ( yp y glevels of detection), and excellent reproducibility (RSDs typically < 2%) even at low d t ti l l C ti d t f UV d t ti ill l b di d

Gradient: 40%B to 47%B in 2.5 min; to 95%B in 12.5 min; hold 10 minInjection Vol.: 10 µL 0 50U

]

detection levels. Comparative data from UV detection will also be discussed. Injection Vol.: 10 µLSample Prep.: Sonicate 1 tablet from commercial health supplement in 20 mL

-0.508.00[m

AU

The charged aerosol detector is a sensitive, mass-based detector, especially well-suited for the determination of any nonvolatile analyte independent of chemical

methanol for 15 min. Centrifuge at 13,000 rpm for 10 min. Transfer supernatant to a glass HPLC autosampler vial 6 000

nm

suited for the determination of any nonvolatile analyte independent of chemical characteristics. As shown in Figure 1, the detector uses nebulization to create aerosol

supernatant to a glass HPLC autosampler vial. 6.00

@ 2

2

g ,droplets. The mobile phase evaporates in the drying tube, leaving analyte particles,

hi h b h d i th i i h b Th h i th d bBacopa 4.00

ance

which become charged in the mixing chamber. The charge is then measured by a highly sensitive electrometer providing reproducible nanogram-level sensitivity

Column: Accucore column C18, 2.1 × 150 mm; 2.6 µm Column Temp : 45 oC

2.00

bsor

ba

a highly sensitive electrometer, providing reproducible, nanogram-level sensitivity. This technology has greater sensitivity and precision than ELSD and RI, and it is

Column Temp.: 45 oCFlow Rate: 0.50 mL/min

Ab

gy g y psimpler to operate than a mass spectrometer (MS). Mobile Phase A: Deionized water

M bil Ph B A t it il Fi h S i tifi ™ O ti LCMS0.0 5.0 10.0 15.0 2

-0.50

RMobile Phase B: Acetonitrile, Fisher Scientific™ Optima LCMSGradient: 28%B

R

Th dibl t C ll fi b iFIGURE 1. Schematic showing how charged aerosol detection works.

Gradient: 28%BInjection Vol.: 1 µL

The edible cactus Caralluma fimbriasuppress hunger and enhance end

1 101 10

FIGURE 1. Schematic showing how charged aerosol detection works. j µSample Prep.: Standard mixture dissolved in 25:75 (v/v) water:acetonitrile.

suppress hunger and enhance endoxypregnane glycosides. As with th1

3101

310 yp g g y

oxypregnane glycosides are not weCh d l d t ti d t t33 Milk Thistle

Column: Thermo Scientific™ Acclaim™ 120 C18 columnCharged aerosol detection detects showing less baseline rise due to th

22Column: Thermo Scientific™ Acclaim™ 120 C18 column,

2.1 × 100 mm, 2.2 µmshowing less baseline rise due to th

, µColumn Temp: 40 oCFl R t 0 50 L/ i FIGURE 3 HPLC-Charged aerosoFlow Rate: 0.50 mL/minMobile Phase A: 3 mM ammonium formate 0 3% formic acid in water: MeOH 80:20 v:v

FIGURE 3. HPLC-Charged aerosofimbriata leaf extract.Mobile Phase A: 3 mM ammonium formate, 0.3% formic acid in water: MeOH 80:20 v:v

Mobile Phase B: 3 mM ammonium formate, 0.3% formic acid in water: MeOH 20:80 v:v 10.0pA

88Gradient: 15% B to 45% B in 4.4 minI j ti V l 2 L8

98

9Injection Vol.: 2 µLSample Prep : Sonicate 70 mg of dry milk thistle extract in 100 mL methanol in a

4 64 6Sample Prep.: Sonicate 70 mg of dry milk thistle extract in 100 mL methanol in a

250 mL amber glass bottle for 20 min. Centrifuge an aliquot at 5000 rpm for 10 min. Transfer supernatant to a glass HPLC vial.

55 1 Liquid eluent enters from HPLC systemAshwagandhaC C 5 05 75 7

1. Liquid eluent enters from HPLC system2. Pneumatic nebulization occurs3 Small droplets enter drying tube

Column: Accucore C8 column, 4.6 × 150 mm; 2.6 µm Column Temp : 45 oC

5.0

3. Small droplets enter drying tube4. Large droplets exit to drain


5. Dried particles enter mixing chamber6. Gas stream passes over corona needle Mobile Phase A: Deionized water

M bil Ph B A t it il O ti LCMSp

7. Charged gas collides with particles andcharge is transferred

Mobile Phase B: Acetonitrile, Optima LCMSGradient: 20%B to 50%B in 6 min; to 95%B in 10 min; hold 10 mincharge is transferred

8. High mobility species are removed9 Charge is measured by a highly sensitive

Gradient: 20%B to 50%B in 6 min; to 95%B in 10 min; hold 10 minInjection Vol.: 1 µL9. Charge is measured by a highly sensitive

electrometer

j µSample Prep.: Sonicate 625 mg powder from commercial capsule in 10 mL

th l f 30 i C t if t t t 13 000 f 10 i0.0

10. Signal transferred to chromatographicsoftware

ethanol for 30 min. Centrifuge extract at 13,000 rpm for 10 min and transfer supernatant to a glass HPLC autosampler vial -1.0and transfer supernatant to a glass HPLC autosampler vial. 0.1 5.0 10.

i l d S l tanicals and Supplementsanicals and Supplements

8 l 4 6 150 2 6In Ayervedic medicine Brahmi (Bacopa monnieri) is purported to enhance mind power Withania somnifera is a woody shrub in the nightshade family (Solanacea) native to India.

Known as ashwagandha it is used in Ayurvedic medicine as a strengthening tonic and8 column, 4.6 × 150 mm; 2.6 µm (Medhya effect) and improve all aspects of mental functioning, including comprehension memory and recollection Among the many bioactive ingredients found

Known as ashwagandha, it is used in Ayurvedic medicine as a strengthening tonic and to cool the body, typically by drinking a cup of hot milk containing the powdered root. comprehension, memory and recollection. Among the many bioactive ingredients found

in Bacopa monnieri are the triterpene saponins separated in Figure 4, including y, yp y y g p g p

Purported active compounds include steroidal lactones isolated from the root, some of which are well resolved by HPLC as depicted in Figure 6 The figure shows that chargedater

O ti LCMSBacoside A3, Bacopaside II, Bacopaside X and Bacopasaponin C. Compared to HPLC with low-wavelength UV detection (220 nm) HPLC with charged aerosol detection not

which are well resolved by HPLC, as depicted in Figure 6. The figure shows that charged aerosol detection detects several compounds not readily seen by UV at 230 nm.Optima LCMS

%B in 2 5 min; to 95%B in 15 min; hold 10 min FIGURE 6 Comparison between charged aerosol detection and UV absorbancewith low wavelength UV detection (220 nm), HPLC with charged aerosol detection not only improves the baseline slope seen with gradient elution, but also offers improved

p y y

%B in 2.5 min; to 95%B in 15 min; hold 10 min. FIGURE 6. Comparison between charged aerosol detection and UV absorbance detection for HPLC separation of Ashwagandha extract.sensitivity.

powder from commercially available capsule into a th l t f 10 i T f t t tethanol, vortex for 10 min. Transfer supernatant to a

o times with fresh methanol Combine extracts and bringFIGURE 4. HPLC-Charged aerosol analysis of Bacopa monnieri extract

o times with fresh methanol. Combine extracts and bring ge at 13,000 rpm for 10 min and transfer supernatant to

6.00A AU

1.20

ussionpA mAU

ussionn used since the 1950s to relieve symptoms of

5.001.00

n used since the 1950s to relieve symptoms of s are believed to include triterpene glycosides including 4.00s are believed to include triterpene glycosides includingoside F, and others. Many of the triterpene glycosides 3.00

0.80

ve 200 nm. Charged aerosol detection easily measures ohosh extract (Figure 2)

3.00

ohosh extract (Figure 2). 2.00 0.60Chargeded aerosol detection to UV detection at 220 nm for 1.00

gAerosolDetectionosides in black cohosh extract.

0 000.40Detection

0.00

-1.00 0.20UV

Hoodigosides are o pregnane steroidal gl cosides ab ndant in Hoodia gordonii and-2.00 Detection

Hoodigosides are oxypregnane steroidal glycosides abundant in Hoodia gordonii andrelated plants native to the deserts of southwestern Africa This plant was used traditionallyCharged 3 00

min0 10

0.00

related plants native to the deserts of southwestern Africa. This plant was used traditionally to ease hunger during long hunting expeditions and enjoys wide use today in dietary

ChargedAerosolD t ti

0.0 5.0 10.0 15.0 20.0 25.0Time [min]

-3.00 -0.10

supplements purported to aid in appetite suppression and weight loss. Figure 7 demonstrates the superior sensitivity of charged aerosol detection for several late eluting

Detection Time [min]

demonstrates the superior sensitivity of charged aerosol detection for several late-elutingcomponents of a commercial Hoodia extract.components of a commercial Hoodia extract.

FIGURE 7 Comparison between charged aerosol detection and UVFIGURE 7. Comparison between charged aerosol detection and UV absorbance detection for HPLC separation of a Hoodia extract.p

UV detectionEvidence suggests that milk thistle extracts may both prevent and repair damage to the liver from toxic chemicals and medications and may be of use in the treatment ofthe liver from toxic chemicals and medications and may be of use in the treatment of mushroom poisoning. The active ingredients are thought to be numerous flavonolignansmushroom poisoning. The active ingredients are thought to be numerous flavonolignans including silybinin, isosilybinin, silychristin and silydianin (Figure 5). Note that the gradient program is extended for several minutes to elute stearic acid and other late eluting formulation components These are poorly detected by UV absorbance but

20.0 25.0 30.0 35.0 40.0 45.0Retention Time [min] eluting formulation components. These are poorly detected by UV absorbance but

should be removed from the column to ensure good long term performance of the HPLC Retention Time [min]

t i d th h t I di b th f d d t should be removed from the column to ensure good long term performance of the HPLC method.ata is used throughout India both as a food and to

urance The active ingredients are believed to includeurance. The active ingredients are believed to includehe triterpenoids in black cohosh, many of the

FIGURE 5 HPLC-Charged aerosol analysis of milk thistle extract

p yell detected by low-wavelength UV absorbance. th l t ith hi h iti it th UV hil l FIGURE 5. HPLC-Charged aerosol analysis of milk thistle extract.the analytes with higher sensitivity than UV while also

he mobile phase gradient (Figure 3)he mobile phase gradient (Figure 3).

ol analysis of oxypregnane glycosides in Caralluma6.00

pAol analysis of oxypregnane glycosides in Caralluma

5.00mAU

10.0

Conclusion4.00

ConclusionCharged aerosol detection offers numerous analytical improvements over low-3 00

UVDetection Charged aerosol detection offers numerous analytical improvements over low

wavelength UV and is of particular use to the analysis of natural products and botanicals 3.00Detection

separated by modern HPLC methods. Charged aerosol detection: Measures any non volatile and many semi volatile species2 00

5.0 Measures any non-volatile and many semi-volatile species Response is independent of chemical structure

2.00

espo se s depe de t o c e ca st uctu e Allows estimation of analyte amounts even when external standards are not

1 00 available Has excellent sensitivity reproducibility and a dynamic range of over four orders

1.00

Has excellent sensitivity, reproducibility and a dynamic range of over four ordersof magnitude0.00

ChargedA l g

-0.50min0.0

AerosolDetection

© 2013 Thermo Fisher Scientific Inc. All rights reserved. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any

0.0 5.0 10.0 15.0 20.0 25.0Time [min]

DetectionScientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. min

-2.00 15.0 20.0 25.0

Time [min]

3Thermo Scientific Poster Note • PN-70543-AOAC-EN 0914S








































3.00

A]gordonii.


t [pA



1 00Cur

re





]


-0.508.00[m

AU



nm



@ 2

2



ance



2.00

bsor

ba



Ab



-0.50


R




1 101 10



3101

310 yp g g y









98







5.0









electrometer


th l f 30 i C t if t t t 13 000 f 10 i0.0












p y y





6.00A AU

1.20

ussionpA mAU


5.001.00


0.80


3.00



0 000.40Detection

0.00

-1.00 0.20UV



min0 10

0.00



0.0 5.0 10.0 15.0 20.0 25.0Time [min]

-3.00 -0.10















5.00mAU

10.0

Conclusion4.00






2.00



1.00


ChargedA l g

-0.50min0.0

AerosolDetection


0.0 5.0 10.0 15.0 20.0 25.0Time [min]


-2.00 15.0 20.0 25.0

Time [min]









































3.00

A]gordonii.


t [pA



1 00Cur

re





]


-0.508.00[m

AU



nm



@ 2

2



ance



2.00

bsor

ba



Ab



-0.50


R




1 101 10



3101

310 yp g g y









98







5.0









electrometer


th l f 30 i C t if t t t 13 000 f 10 i0.0












p y y





6.00A AU

1.20

ussionpA mAU


5.001.00


0.80


3.00



0 000.40Detection

0.00

-1.00 0.20UV



min0 10

0.00



0.0 5.0 10.0 15.0 20.0 25.0Time [min]

-3.00 -0.10















5.00mAU

10.0

Conclusion4.00






2.00



1.00


ChargedA l g

-0.50min0.0

AerosolDetection


0.0 5.0 10.0 15.0 20.0 25.0Time [min]


-2.00 15.0 20.0 25.0

Time [min]

5Thermo Scientific Poster Note • PN-70543-AOAC-EN 0914S








































3.00

A]gordonii.


t [pA



1 00Cur

re





]


-0.508.00[m

AU



nm



@ 2

2



ance



2.00

bsor

ba



Ab



-0.50


R




1 101 10



3101

310 yp g g y









98







5.0









electrometer


th l f 30 i C t if t t t 13 000 f 10 i0.0












p y y





6.00A AU

1.20

ussionpA mAU


5.001.00


0.80


3.00



0 000.40Detection

0.00

-1.00 0.20UV



min0 10

0.00



0.0 5.0 10.0 15.0 20.0 25.0Time [min]

-3.00 -0.10















5.00mAU

10.0

Conclusion4.00






2.00



1.00


ChargedA l g

-0.50min0.0

AerosolDetection


0.0 5.0 10.0 15.0 20.0 25.0Time [min]


-2.00 15.0 20.0 25.0

Time [min]









































3.00

A]gordonii.


t [pA



1 00Cur

re





]


-0.508.00[m

AU



nm



@ 2

2



ance



2.00

bsor

ba



Ab



-0.50


R




1 101 10



3101

310 yp g g y









98







5.0









electrometer


th l f 30 i C t if t t t 13 000 f 10 i0.0












p y y





6.00A AU

1.20

ussionpA mAU


5.001.00


0.80


3.00



0 000.40Detection

0.00

-1.00 0.20UV



min0 10

0.00



0.0 5.0 10.0 15.0 20.0 25.0Time [min]

-3.00 -0.10















5.00mAU

10.0

Conclusion4.00






2.00



1.00


ChargedA l g

-0.50min0.0

AerosolDetection


0.0 5.0 10.0 15.0 20.0 25.0Time [min]


-2.00 15.0 20.0 25.0

Time [min]

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Documents

Improved Universal Approach to Measure Natural …...Improved Universal Approach to Measure Natural Products in a Variety of Botanical and Supplements Marc Plante, Ian N. Acworth,