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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• Withdrawn drugs, designer drugs analogues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)
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