Applications of Mass Spectrometry to the

Analysis of Chemical Components Found

in Botanical Dietary Supplements

Alexander J. Krynitsky, Lukas Vaclavik, Rahul Pawar,

Hemlata Tamta, Erich Grundel, Jon Wong and Jun Ma

Office of Regulatory Science, Center for Food Safety and Applied Nutrition,

Food and Drug Administration, College Park MD, USA

Current Studies on Botanical Dietary Supplements

o Characterization of authenticated raw botanical

materials (Example Below).

o Acacia rigidula

o Leaves used in Weight loss products

o Phenethylamine, tyramine and tryptamine derivatives

o Non natural compound found in 9 of 21 dietary supplements

o β-methylphenethylamine

o Can be misidentified as amphetamine using LC-MS

o Independent GC-MS analysis able to confirm presence of β-

methylphenethylamine and absence of amphetamine

Source: R.S. Pawar et. al J. Pharm. Biomed. Anal. 88 (2014) 457-466

Current Studies on Botanical Dietary Supplements (Cont.)

o Toxicity (hepatotoxicity) of specific components.

o Example: pyrrolizidine alkaloids (from comfrey root).

o Adulteration and contamination of herbal dietary

supplements (Discussion to follow)

o Screening for potential active pharmaceutical

ingredients (API) adulterants and analogues

(Discussion to follow)

Herbal dietary supplements

o Sales of herbal dietary supplements

have increased steadily over the last

decade

o Dietary supplements represent

valuable commodities with relatively

high retail price

o Consumers may assume that use of

dietary supplements is a safer and

more natural alternative to traditional

medical treatments

Year

Source: New Hope Natural Media (http://newhope360.com)

Risks

o Adulteration of dietary supplements with active pharmaceutical

ingredients (API’s)

o Potential interactions between natural constituents and

prescription drugs

o Presence of chemical contaminants in dietary supplements

• Plant toxins, mycotoxins

• Pesticides

• Heavy metals, etc.

Source: M.J. Bogusz et al. J. Pharm. Biomed. Anal. 41 (2006) 554.

o The aim is typically to intensify claimed biological effect • High concentration close or above the therapeutic doses

• Addition of multiple API’s

• Withdrawn drugs, designer drugs analogues

Adulteration with API’s

WEIGHT LOSS SUPPLEMENTS (major)

SPORTS SUPPLEMENTS (major)

MALE ENHANCEMENT SUPPLEMENTS (major)

BLOOD SUGAR SUPPORT SUPPLEMENTS (growing)

MOOD/WELL BEING SUPPLEMENTS (re-emerging)

SLEEP AID SUPPLEMENTS (re-emerging)

CARDIOVASCULAR AID SUPPLEMENTS

ANTI-INFLAMMATION SUPPLEMENTS

Courtesy of Dr. Lukas Vaclavik

Adulteration with API’s - Example

Source: M.L. de Calvano et al. Forensic Sci. Int. 204 (2011) 6.

o Adulterants reported in weight loss supplements

ANOREXICS ANXYOLYTICS ANTIDEPRESSANTS DIURETICS LAXATIVES

Amfepramone

Femproporex

Sibutramine

Mazindol

Fenfluramine

Diazepam

Flurazepam

Clonazepam

Alprazolam

Medazepam

Midazolam

Oxazepam

Oxazolam

Fluoxetine

Paroxetine

Sertraline

Bupropion

Furosemide

Spironolactone

Chlorothiazide

Phenolphthalein

Adulteration with API’s - Example

Source: M.L. de Calvano et al. Forensic Sci. Int. 204 (2011) 6.

o Adulterants reported in weight loss supplements

ANTIDEPRESSANTS DIURETICS LAXATIVES

Amfepramone

Femproporex

Sibutramine

Mazindol

Fenfluramine

Diazepam

Flurazepam

Clonazepam

Alprazolam

Medazepam

Midazolam

Oxazepam

Oxazolam

Fluoxetine

Paroxetine

Sertraline

Bupropion

Furosemide

Spironolactone

Chlorothiazide

Phenolphthalein

ANOREXICS ANXYOLYTICS ANOREXICS ANXYOLYTICS

Contamination with plant toxins

o Usually a result of accidental (unintentional) substitution of plant

material containing toxins

o May also be performed intentionally, especially in case

authentic plants are either not available or too expensive

ARISTOLOCHIC ACID IN HERBAL

SLIMMING PILLS

ANISATIN IN HERBAL TEA

EPHEDRA ALKALOIDS IN DIET AID

SUPPLEMENTS

Aristolochia fanchi Japanese star anise

Ephedra sinica Courtesy of Lukas Vaclavik

Development of LC–MS methods

o Simple and rapid methods for simultaneous determination of

multiple pharmaceuticals, plant toxins / alkaloids

Anti-hypertension drugs

Amlodipine besylate

Benazepril

Chlorthalidone

Clonidine

Felodipine

Metoprolol tartrate

Minoxidil

Phentolamine mesylate

Prazosin

Valsartan

Anti-diabetic drugs

Acetohexamide

Bumetanide

Chloropropamide

Gliclazide

Glipizide

Gliquidone

Glibenclamide

Metformin

Nateglinide

Phenformin

Pioglitazone

Repaglinide

Rosiglitazone

Tolazamide

Tolbutamide

PDE-5 inhibitors

Icariin

Sildenafil

Tadalafil

Vardenafil

Weight-loss drugs

Bumetanide

Fenfluramine

Orlistat

Phentermine

Rimonabant

Sertraline

Sibutramine

Topiramate

NSAIDs

Indomethacin

Ketoprofen

Mefenamic acid

Naproxen

Phenylbutazone

Aconitine alkaloids

Aconitine

Hypaconitine

Mesaconitine

Tropane alkaloids

Atropine

Homatropine

Scopolamine

Tropine

Coumarins

Bergapten

Psoralene

Scopoletine

Umbelliferone

Other toxins and alkaloids

Aristolochic acid I

Aristolochic acid II

Anisatin

Colchicine

Evodiamine

Lycorine

Picrotin

Picrotoxinin

Ricinine

Strychnine

Yohimbine

Cardiac glycosides

Cymarin

Digitoxigenin

Digitoxin

Digoxin

Ouabain

Other drugs

Diazepam

Fluoxetine

Ibuprofen

Phenytoin

Sertraline

Warfarin

Pyrrolizidine alkaloids

Heliotrine

Lycopsamine

Monocrotaline

Monocrotaline N-oxide

Retrorsine

Retrorsine N-oxide

Senecionine

Senecionine N-oxide

Seneciphylline

Seneciphylline N-oxide

Senkirkine

Psychostimulants

Amphetamine

β-Methylphenethyamine

Cathine

1,3-Dimethylamylamine

Ephedrine

Methamphetamine

Norephedrine

Phentermine

Diuretic drugs

Bendroflumethiazide

Hydrochlorothiazide

Hydroflumethiazide

Indapamide

Methyclothiazide

Metolazone

Sample preparation

o Analytes with different physico-chemical properties

o Expected concentrations ranging from µg/kg to g/kg

o Complex matrices (tablets, sofgels, liquids)

PYROLIZIDINE

ALKALOIDS MULTIPLE DRUGS

EXTRACTION

SPE CLEAN-UP

Conditioning

Sample application

Washing

Elution

FILTRATION

LC-MS

EXTRACTION

FILTRATION

LC-MS

Courtesy of Dr. Lukas Vaclavik

Sample preparation

o QuEChERS procedure

o Acetonitrile-water extraction

followed by partition step

o Polar matrix components remain in

aqueous layer

Acetonitrile extract (analytes)

Sample matrix

Aqueous layer

Salts

ACETONITRILE-

WATER EXTRACT QuEChERS

EXTRACT

Sample preparation

TABLETS / CAPSULES -Homogenized sample (1g) soaked

with 1% FA in H2O (10mL) for 30 min

-Addition of MeCN (10mL) and extraction by shaking (30 min)

-Addition of salts (4g MgSO4 & NaCl)

-Hand-shaking (1 min)

-Centrifugation (4,500 rpm, 5 min)

-dSPE of MeCN extract (2mL) with C18 sorbent (100mg) and

MgSO4 (300mg)

-Filtration

SALTS

WATER

MATRIX

ACETONITRILE

(analytes)

TABLETS / CAPSULES SOFTGELS -Shaking of homogenized sample (1g)

with n-hexane (4 mL)

-Addition of 1% FA in H2O (10mL)

-Addition of MeCN (10mL) and extraction by shaking (30

min)

-Addition of salts (4g MgSO4 & NaCl)

-Hand-shaking (1 min)

-Centrifugation (4,500 rpm, 5 min)

-dSPE of MeCN extract (2mL) with C18 sorbent (100mg) and

MgSO4 (300mg)

-Filtration

SALTS

WATER

MATRIX

ACETONITRILE

(analytes)

n-HEXANE

SOFTGELS LIQUIDS -Addition of MeCN containing 1% FA

(10mL) to liquid sample (10mL)

-Extraction by shaking (30 min)

-Addition of salts (4g MgSO4 & NaCl)

-Hand-shaking (1 min)

-Centrifugation (4,500 rpm, 5 min)

-dSPE of MeCN extract (2mL) with C18 sorbent (100mg) and

MgSO4 (300mg)

-Filtration

SALTS

SAMPLE

ACETONITRILE

(analytes)

LIQUIDS

Liquid chromatography

o UPHLC separation

• Column: Acquity UPLC HSS T3 reversed phase column

(100 mm x 2.1 mm i.d., 1.7 µm particle size)

• Mobile phase: A – 5 mM ammonium formate + 0.1% formic acid in water

B – 5 mM ammonium formate + 0.1% formic acid in methanol

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0

50

100

0 5 10 15

CO

MP

ON

EN

T B

(%

)

FL

OW

(m

L/m

in)

TIME (min)

GRADIENT ELUTION

• Analytes eluted in 0.7 - 10.5 min

• Typical peak widths: 5 - 12 s

• RT stability: 0.15–3.5% (RSD, n = 50)

Instrumentation - HR MS

Q Exactive hybrid mass spectrometer

Accela UHPLC system

o Hybrid HR mass spectrometer

o Quadrupole-orbital ion trap / ESI

source / UHPLC system

o RP 17,500 – 140,000 FWHM

o Enables to obtain HR product ion

spectra of selected precursor ion

Source: Thermo Scientific Inc.

MS detection - QExactive (1)

o ESI+, full MS – data dependent MS/MS acquisition mode

o Inclusion list (m/z, retention time window, collision energy)

Data dependent-MS/MS

scan triggered from a

m/z inclusion list

Return to full scan until

m/z from the Inclusion list

is triggered

FULL MS

dd-MS/MS

Source: Figure provided by Dr. J. Wong

Identification and confirmation - HRMS

Product ion spectrum: Chloropropamide (NCE = 20%)

Parent ion

0

20

40

60

80

100

Rela

tive a

bundance (

%)

7.37

0

20

40

60

80

100 191.9884

174.9618 60.0809

277.0412

0

20

40

60

80

100 277.0412

SPIKE 500 µg/kg into DS intended for BLOOD PRESSURE MGMT EIC: Chloropropamide (m/z 277.0408)

7.2 7.3 7.4 7.5

Time (min)

0

20

40

60

80

100 7.36

50 100 150 200 250 300 m/z

0

20

40

60

80

100 191.9884

277.1989

174.9618 60.0810

149.0239

137.0601 245.0815 83.0494 201.1644

277.0 277.1 277.2 m/z

0

20

40

60

80

100 277.1989

277.1422

277.0412

277.2535

Parent ion

Interferences

Rela

tive a

bundance (

%)

SPIKE 500 µg/kg into DS intended for ANTI-INFLAMMATION Product ion spectrum: Chloropropamide (NCE = 20%) EIC: Chloropropamide (m/z 277.0408)

o Interference-free extracted ion chromatograms and product ion spectra typically obtained

o Interfering ions co-isolated by the quadrupole and their fragments were resolved from

diagnostic ions of analytes

Courtesy of Dr. Lukas Vaclavik

Source: Method Validation and Quality Control Procedures for Pesticides Residues Analysis in Food and Feed, Document No. SANCO/12495/2011

IDENTIFICATION/CONFIRMATION REQUIREMENTS - HRMS

• ≥ 2 diagnostic ions (preferably including the

quasi molecular ion). Mass accuracy below

5 ppm, at least one fragment ion

• Requirements can be met for all analytes in

complex matrices

• Mass accuracy in full MS and dd-MS/MS mode

was below 3 and 5 ppm, respectively

10

20

13 11

42

0

5

10

15

20

25

30

35

40

45

1 2 3 4 ≥ 5

Num

ber

of a

naly

tes

Number of fragment ions (RI ≥ 10)

60 80 100 120 140 160 180

m/z

0

20

40

60

80

100

Rela

tive a

bundance (

%)

148.1122

166.1228

Product ion spectrum:

Ephedrine (NCE = 10%)

DS for BLOOD PRESSURE MGMT

SPIKE 100 µg/kg

50 100 150 200 250

m/z

0

20

40

60

80

100

Rela

tive a

bundance

(%

)

116.1072

268.1910 74.0602

98.0966

159.0807

121.0650 191.1069

133.0650

226.1441

197.1328

Product ion spectrum:

Metoprolol tartrate (NCE = 40%)

DS for BLOOD PRESSURE MGMT

SPIKE 100 µg/kg

0

20

40

60

80

100

4 5 6 7 8 9

Rec

over

y (%

)

pH of aqueous extract

1,3-DMAAMonocrotalineMonocrotaline N-oxide

OPTIMIZATION – EXTRACTION / CLEAN-UP

• Extraction at lower pH improves

recoveries of pyrrolizidine alkaloids, their

N-oxides and some other analytes

• Defatting slightly decreases recoveries of

some non-polar analytes

• C18 sorbent does not bind target analytes

2

5

89

0

2

1

93

0

0 20 40 60 80 100

0 - 40

40-70

70-120

>120

Number of analytes

Rec

over

y (%

)

No defattingDefatting

DEFATTING (tested on softgels)

2

0

94

0

7

22

67

0

0 20 40 60 80 100

0 - 40

40-70

70-120

>120

Number of analytes

Ave

rage

rec

over

y -

all m

atric

es

(%)

dSPE PSA/MgSO4

dSPE C18/MgSO4

dSPE (all matrices)

DS intended for BLOOD PRESSURE MGMT (tablet)

NO FA 1% FA 2% FA

Courtesy of Dr. Lukas Vaclavik

• Detectability of analytes is strongly matrix-

dependent

Criteria for estimation of LOQ:

• At least 8 full MS scans at RT of analyte

• Signal-to-noise ratio ≥ 9 (when noise was

present in the extracted ion chromatogram)

• Analyte intensity > threshold for triggering

of product ion scan

• Criteria for analyte identification passed

DETECTABILITY

11

55

16

5

9

0

29

43

7

17

0

22

49

6

18

0 20 40 60

<10

≥10-50

≥50-100

≥100-250

≥250

Number of analytes

LOQ

(µ

g/kg

)

SOFTGELS

TABLETS/CAPSULES

LIQUIDS

Courtesy of Dr. Lukas Vaclavik

2

1

93

0

2

1

93

0

2

5

89

0

0 20 40 60 80 100

0-40

40-70

70-120

>120

Number of analytes

RE

CO

VE

RY

(%

)

SOFTGELS

TABLETS/CAPSULES

LIQUIDS

RECOVERY & REPEATABILITY

SPIKING LEVEL: LOQ

• Recoveries for the majority of analytes at

concentrations ≥ LOQ were 70-120%

• Repeatability (RSD, n = 5) was below

25% for all experiments

• Highly polar analytes (metformin, tropine,

ouabain) are not transferred into

acetonitrile layer during partition step

• Recoveries of non-polar analytes are

slightly decreased during defatting step

Courtesy of Dr. Lukas Vaclavik

QUANTIFICATION

Source: Furey, A; Moriarty, M.; Bane, V.; Kinsella, B.; Lehane, M. 2013. Talanta 115: 104 – 122.

• The extent of matrix effects (mainly signal

suppression) differs significantly between

respective herbal dietary supplements

COMPENSATION OF MATRIX EFFECTS

• Matrix-matched external calibration

• Method of standard additions

• Method of internal standardization

MINIMIZATION/REMOVAL OF MATRIX EFFECTS

• Selective extraction

• Effective sample clean-up

• Optimization of chromatographic

separation

• Dilution of sample extract

-100

-80

-60

-40

-20

0

20

0 2 4 6 8 10 12

Mat

rix e

ffect

(%

)

-100

-80

-60

-40

-20

0

20

0 2 4 6 8 10 12

Mat

rix e

ffect

(%

)

-100

-80

-60

-40

-20

0

20

0 2 4 6 8 10 12

Mat

rix e

ffect

(%

)

Retention time (min)

DS intended for MALE ENHANCEMENT (LIQUID)

DS intended for WEIGHT LOSS (SOFTGEL)

DS intended for ANTI-INFLAMMATION (CAPSULE)

QUANTIFICATION – STANDARD ADDITIONS

• Approach provides results and performance

comparable to those obtained by matrix-

matched calibration

• Post-extraction addition at three concentrations

(25, 100, and 500 µg/kg)

• Time-demanding and laborious

3580.9x+3766770 R2=0.9995

0

500000

1000000

1500000

2000000

2500000

-200 -100 0 100 200 300 400 500 600

ADDITION 500 ppb

ADDITION 100 ppb

ADDITION 25 ppb

ANALYTE CONCENTRATION

(105.2 µg/kg)

Concentration (µg/kg)

Pèa

k ar

ea (

coun

ts)

ARISTOLOCHIC ACID I (105 µg/kg):

COMMERCIAL STEPHANIA TETRANDA POWDER EXTRACT

8.2 8.4 8.6 8.8 9.0 9.2 Time (min)

0

20

40

60

80

100

Rela

tive a

bundance (

%)

8.65

9.08

Int.: 2.10E5 AA I

240 260 280 300 320 340 360 m/z

0

20

40

60

80

100

Rela

tive a

bundance (

%)

298.0720

324.0513

342.0620 359.0886

240 260 280 300 320 340 360

0

20

40

60

80

100

Rela

tive a

bundance

(%

)

298.0720

324.0515

256.2281 303.1460 288.1604 342.0618 359.0887

Product ion spectrum:

SAMPLE

Product ion spectrum:

STANDARD

QUANTIFICATION – DILUTION OF SAMPLE EXTRACT

• Successfully applied to quantification of

analytes at higher concentrations

• Dilution followed by quantification using solvent

standards (dilution factors ≥ 25 should be used)

• Straightforward approach

GLIBENCLAMIDE (0.25 mg/g)

DS intended for BLOOD SUGAR MANAGEMENT

-100

-80

-60

-40

-20

0

20

1 10 100 1000 10000

Mat

rix e

ffect

(%

)

Dilution factor (DF)

0

50

100 8.89

0

50

100 8.88

8.6 8.8 9.0 9.2

Time (min)

0

50

100 8.87

NO DILUTION

DF 100

DF 10,000

INT: 1.46E9

INT: 2.02E8

INT: 1.76E6

Quantification based on solvent standards

300 350 400 450 500

m/z

0

20

40

60

80

100

Rela

tive a

bundance (

%)

369.0686

494.1534

395.0480

Product ion spectrum:

SAMPLE

LIQUID TABLET SOFTGEL

× × ×

Acquity UPLC system

5500 QTRAP LC-MS system

Instrumentation - LR MS

o Hybrid mass spectrometer

o QqQ-LIT / ESI source / UHPLC system

o Combines capabilities of QqQ and IT

Q0 Q1 Q0 Q1

ION GUIDE

COLLISIONAL FOCUSING

& ION TRAPPING

QUADRUPOLE (Q1)

CURVED

COLL. CELL

QUADRUPOLE (Q3)

Source: AB Sciex

SENSITIVITY IN

PRODUCT ION SCAN

MASS RANGE IN

PRODUCT ION SCAN

MS3 CAPABILITY

NEUTRAL LOSS /

PREC. ION SCAN

ADDITIONAL SCAN

FUNCTIONS

ION TRAP TRIPLE QUAD QTRAP

×

×

×

×

×

×

Performance of the method - DETECTABILITY

7.48 7.52 7.56 7.60 7.64 7.68 7.72 7.76 7.80 7.84 7.88 Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

Inte

nsity,

cps

7.65

7.50 7.55 7.60 7.65 7.70 7.75 7.80

Time (min)

0

50

100

Rela

tive A

bundance

7.66

MS/MS HR MS(/MS)

324.9 > 243.0

324.9 > 91.0 325.1216 ± 2.5 ppm

BLOOD SUGAR MANAGEMENT DS: SPIKE 100 µg/kg

7.92 7.96 8.00 8.04 8.08 8.12 8.16 8.20 8.24 8.28 8.32 Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

Inte

nsity,

cps

8.11

8.0 8.1 8.2 8.3 8.4 Time (min)

0

50

100 R

ela

tive A

bundance

8.16

MS/MS

392.0 > 339.1

392.0 > 91.0

HR MS(/MS)

375.2529 ± 2.5 ppm

ACETOHEXAMIDE

DIGITOXIGENIN

Identification and confirmation - LRMS

2.5 3.0 3.5 4.0 4.5 0.0

2.0e4

4.0e4

6.0e4

Inte

nsi

ty, c

ps

4.20 3.47

4.36

Aristolochic acid II

329.2 > 268.2

329.2 > 163.2

2.5 3.0 3.5 4.0 4.5 Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

Inte

nsi

ty, c

ps

3.48 Aristolochic acid II

329.2 > 268.2 > 238.2

MS2

MS3

Q0 Q1 Q2 Q0 Q1 Q2 Q3/LIT MS2 MS3

Source: Vaclavik et. al Food Additives & Contaminants 31 (2014) 784-791

Post-run screening EXAMPLE: SIBUTRAMINE & ANALOGUES

IN WEIGHT LOSS SUPPLEMENT

Sibutramine

Benzylsibutramine

N-Desmethylsibutramine

N

C H 3

C H 3

C H 3

C H 3

Cl

N C H 3

C H 3

Cl

N H

C H 3

C H 3

C H 3

Cl

Conclusions

o LR and HR MS techniques provide complementary information that

allow identification and confirmation of target analytes

o The developed method allowed for achieving acceptable

performance characteristics in terms of both detectability and

recoveries

o The post-run screening without analytical standards based on

accurate mass measurements can reveal presence of new API’s

and analogues

Acknowledgments

• Lukas Vaclavik, FDA-CFSAN

• Rahul Pawar, FDA-CFSAN

• Hemlata Tamta, FDA-CFSAN

• Erich Grundel, FDA-CFSAN

• Jun Ma, FDA-CFSAN

• Jon W. Wong, FDA-CFSAN

• James B. Wittenberg, FDA-CFSAN

Thank you for your attention!

Courtesy of R. Chris Clark (Coastal Point)