Natural products and quality control (208)

(Applications in QC of Herbal Drugs)

By Dr. Rana Mohamed
Email: rana.mohamed@pharma.cu.edu.eg
Office hrs: Thursday 10-12
 Content:
A. Chemoprofiling using several chromatographic techniques
- Chemoprofiling of simple herbal products.
- Quantification of chemical markers in natural products.
- Chemoprofiling of polyherbal formulations.
B. Chemical contaminants in herbal drugs
- Microbial & mycotoxins detection
- Pesticide residues analysis
C. Adulteration detection in herbal drugs
- Synthetic substances & other herbal adulteration
D. Advanced approaches in quality control of herbal drugs
 Chemoprofiling and Marker Analysis for Quality Evaluation of Herbal Drugs

Remember……………
 Standardization is a system to ensure that every packet of medicine that is being sold has
the correct amount and will induce its therapeutic effect.
 Chemical profiling establishes a characteristic chemical pattern for a plant material, its
fractions or extracts using suitable chromatographic techniques.
 This process leading to a chromatographic fingerprinting of the plant in which marker
compounds or (“fingerprints”) are used to evaluate authenticity or quality and to ensure
the efficacy and safety of the natural health products.
 Marker compound is a chemical constituent of a botanical raw material, drug substance, or
drug product that is used for identification and/or quality control purposes e.g., silymarin
in milk thistle and ginsenosides from ginseng.
 Chemoprofiling of different ginger products by TLC using three gingerol homologs

 Quantitative estimation of gingerols in methanolic extract of different ginger marketed

samples using TLC fingerprinting.
 The results showed interesting differences in the quantities of all the three major
gingerols.

Chemical structures of gingerol homologs.

 Chemoprofiling of different ginger products by TLC using three gingerol homologs
std std std std
6-gingerol
Ginger samples 8-gingerol
visualized at 254 nm 10-gingerol
Some samples were substituted
(no gingerols)

Ginger samples
Pure samples with the three
visualized at 366 nm
gingerols are present

Some samples were exhausted or

Ginger samples inferior quality not all gingerols are
visualized at 450 nm detected

TLC of Standard tracks: 3, 6, 9, 12

 HPTLC Fingerprint Profile of different Curcuma batches

 Three Curcuma markers: curcumin, demethoxycurcumin and bisdemethoxycurcumin were

determined using HPTLC.
 This method could be used for identification and authentication purposes in order to prevent
adulteration by other species which doesn’t contain curcminoids.
exhausted
std curcuma or other
extract √ species with less
curcuminoids

exhausted
curcuma or
adulterant

1 2 3 4 5
Determination of hypericin and hyperforin content in different Hypericum species

• Three Hypericum species were extracted with methanol and analyzed by HPLC/PDA, and
compared to the official species H. perforatum.
• Hypericin and hyperforin were used as external reference standards.

 The investigated species can be easily distinguished from H.

perforatum using this chromatographic technique Explain??

STD hypericum sp-1

hypericum sp-2

H. perforatum
hypericum sp-3
 Observations

 Wide-ranging differences were observed in hypericin and hyperforin content among

the species.
 H. perforatum (the official plant showed nearly equal concentrations of both
constituents.
 While, sp1 and sp2 showed wide difference in their concentrations and hypericin was
absent in the extract of sp 3.
 Fingerprinting of Sennosides in C. senna leaves and pods using HPTLC.

 According to European Pharmacopoeia (Ph. Eur.),

the official parts used of two Cassia senna are the
leaves and pods.
 The main active constituents are the dianthrone
glucosides (mainly sennoside A and B) and they are
higher in pods than in leaves.
 Therefore, for quality control purposes HPTLC method
using sennosides as markers was developed.
 Standard senna leaves and pods dry extracts were
used as test controls.
  The chromatograms of C. senna
Chromatograms of different batches of C. senna leaves and pods
using HPTLC. leaves and pods are very similar in
C. senna leaves C. senna pods their profile.
Std extracts  Exceptions are the blue fluorescent
zones [c, d] and red fluorescent in
between and above them which
STD differ distinctly.
 The references sennosides A and B
showed yellowish or yellowish
brown fluorescent bands (f, g, h
zones).
Sennoside A and B
 The main difference also, the higher
concentrations of sennosides in the
pods than in the leaves.
 Identification of Echinacea Species by HPTLC

 TLC method is presented for identification of Echinacea species

(root or herb) and their common adulterants using markers from
β-sitosterol
different chemical classes.

A. Alkylamides and sterols

 Alkylamides such as dodeca-2,4,8,10-tetraenoic acid isobutylamide

and β-sitosterol were used as markers.
 Detection is performed after derivatization with p-anisaldehyde
reagent, in white light for different Echinacea species (root or herb)
and also for common adulterants.

Dodeca-2,4,8,10-tetraenoic acid
isobutylamide
 HPTLC Fingerprinting using Alkylamides and sterols

 A zone of dodeca 2,4,8,10-tetraenoic acid roots

herbs
isobutylamide is obtained from the roots of
all Echinacea species except no. 4. !!
 For the herbs this zone is either missing STD
or very weak. STD

 species no. 4 also showed olive zone in

the upper third of the chromatogram and
a dark zone in the lower third.
E. purpurea E. purpurea
 A zone at the position of β-sitosterol is
dodeca 2,4,8,10-tetraenoic acid
obtained for all samples. isobutylamide β-sitosterol
 Adulteration detection using Alkylamides and sterols
 Typical adulterants can clearly be distinguished from Echinacea by their
chromatographic fingerprint after derivatization with anisaldehyde reagent.

Echinacea species common adulterants

STD STD
STD STD

β-sitosterol
dodeca-2,4,8,10-tetraenoic acid
isobutylamide
 Herbs
B. Phenylpropanoids & Phenolic acids Roots

E. purpurea herb
STD STD
 Echinacoside, chlorogenic acid, caftaric acid,

E. purpurea root
cynarin, chicoric acid, and caffeic acid are
suitable marker compounds.
 Visualization is achieved in UV light after
derivatization with natural products reagent.
 Distinguishable profiles are obtained from the
roots of different species.
 A red zone of chlorophyll at the solvent front
was obtained for the samples of the Herba
caftaric, echinacoside,
drugs only. cynarin, chlorogenic,
caffeic acid
chicoric acid
HPTLC Fingerprinting using phenylpropanoids
 Adulteration detection using phenylpropanoids

 All investigated adulterants of Echinacea give a significantly different fingerprint

chromatogram and can thus be identified.
common adulterants
Echinacea species STD STD STD
STD

caftaric, cynarin, echinacoside, chlorogenic,

chicoric acid caffeic acid
 C. Fructofuranosides

 Fructofuranosides, such as inulin Inulin Saccharose

polysaccharides and saccharose, can be used to

evaluate the quality of Echinaceae.
 Visualization is possible by use of aniline–
diphenylamine reagent.
 Similar profiles were obtained for all the samples
investigated, although concentrations were
different.
 Improper storage, microbial and enzymatic
HPTLC Fingerprinting using Fructofuranosides
degradation, and addition of carbohydrate-
based fillers will alter the profiles significantly.
 Quantitative Estimation of Piperine marker in Pipers Using HPLC

 Piperine is the main therapeutically active constituent of Black pepper and Long pepper
fruits.
 A HPLC method was used for the analysis of piperine in both species.
● Since piperine shows UV absorption 343 nm so plate scanned at 343nm.
● The % w/w yield of piperine was 8.76 for Black pepper and for Long pepper 4.96.

Piperine
Black pepper Long pepper
Quantitative Estimation of Piperine marker in Pipers Using HPLC

Calibration curve of piperine

Piperine

Black pepper
Long pepper
Abs. 1.54
Abs. 0.899
 Standardization of Biomarkers in Polyherbal Formulation Entoban Capsules by HPTLC

 Entoban capsules have been used to eradicate worms from the gastrointestinal tract.
 It is a polyherbal formulation.
 Gallic acid is a common phytoconstituent present in Entoban capsules with antimicrobial activity.
While, Berberine is a specific marker from B. aristata with antibacterial and anti-inflammatory
activities.
 Therefore, both can be helpful in the routine quality control of the capsule.
 HPTLC was used for the quantitative estimation of biomarkers in Entoban capsules
Entoban capsules gallic acid Entoban capsules
14.5 min 16.5 min berberine
14.5 min
16.5 min
 Contaminants in Herbal drugs

 Medicinal plants are obtained from natural sources,

which may be wild or cultivated.
 Thereby, they are exposed to environmental
contaminants, such as microbial species, pesticides,
fertilizers, and heavy metals.
 As a result, these contaminants accumulate in the plant
tissues.
 As it is not possible to make herbal materials
completely free from all these contaminants,
specifications have been set up to limit them.
The most common contaminants of medicinal herbs

Chemical Contaminants

Microbial Contaminants

Adulteration & Undeclared

chemical substances
 Microbial contamination

 Sometimes, aerobic bacteria and fungi may be present in plant material due to faulty growing,
harvesting, and storage or processing.
 Microbial contamination in the herbal product can not only deteriorate the active constituents,
but there have been clinical case studies exhibiting serious infections due to consumption of
contaminated herbs.
 Also, chemical contamination by their toxic metabolites, known as endotoxins (Bacteria),
Aflatoxins, and Ochratoxins (Fungi) may take place.
 Microbial contamination Cont.

 Example (1): Assessing the microbiological quality of fennel, chamomile, peppermint teas
(commercially available in tea bags).
 Some tea bags were brewed at 90 °C for 5 minutes according to the manufacturer’s
recommendations, and others were soaked in water taken from a hot-water outlet of an automatic
coffee machine (temperature 67 °C).
 All the infusions were contaminated with non-fermentative Gram-negative bacterial
species, while spore-forming bacteria were most prominent in chamomile and peppermint
teas.
 High temperature (90 °C) decreased the number of moulds in teas.
 However, in the second group of teas, 67 °C was not enough to kill the bacteria and the
total microbial count of aerobic microbes was exceeded the pharmacopoeial limits.
 Microbial contamination Cont.

Example (2): A study of medicinal herbs collected from a Brazilian market showed that
more than 50 % of samples exceeded the microbial count limits set by the US
Pharmacopoeia.
 The highest mould burden was observed in leaves, rhizomes and seeds.
 Dominant moulds were from the Aspergillus genus, followed by Penicillium genus.
 After the testing for mycotoxin-producing abilities under in vitro conditions.
 22 % of the isolates were found to produce mycotoxins, of which 43 % aflatoxin, 23 %
ochratoxin, and 35 % citrinin.
 Chemical Contaminants (Mycotoxins)
Example (1): Quantitation of aflatoxins in pistachios and groundnuts using HPLC-FLD method

 The presence of aflatoxins (AFs) in pistachios and groundnuts was confirmed by HPLC-FLD.
 AFs were present in 14.6% of pistachios and 19.2% of groundnuts.

AFs standard solution

AFs standard solution

AFB1 AFB1
contaminated pistachio
contaminated groundnut
AFB2
Chemical Contaminants (mycotoxins) standard solution

Example (2): Simultaneous multi-

mycotoxin determination in nutmeg

 High performance liquid nutmeg samples

chromatography coupled with

fluorescence detection (HPLC-FLD)
was used for simultaneous
determination of aflatoxins (AFB1, nutmeg samples

AFB2, AFG1, AFG2) and ochratoxin A

(OTA) in edible and medicinal nutmeg
samples.
Example (3):

 It seems that liquorice roots favour contamination with y=920808x

ochratoxin A and the results were confirmed using HPLC-
FLD.
 1 gm of each product was investigated, high concentration of
ochratoxin A was found in one sample (Does it exceed the
limits???)
 Ph. Eur. Limits maximum 20 μg per kilogram of herbal drug.
 (Conc x)=10220/920808=0.011μg/g = 11 μg/kg so it doesn’t
exceed the pharmacopoeial limits and will be accepted

liquorice sample Auc 10220

 Chemical contaminants (Pesticides)
 Medicinal plants are liable to contain pesticide
residues, which accumulate from agricultural
practices, such as spraying, treatment of soils
during cultivation, and administration of pesticides
during storage.
 British, American and European
Pharmacopoeias have included methods for
the analysis of pesticides in medicinal products
of plant origin.
 Limit tests for acceptable levels of all these
pesticides and fumigants are of the utmost
necessity to control the quality of plant materials.
 Chemical contaminants (Pesticides)
Sample 1
Example (1): Detection of multiresidue of
pesticides in medicinal plants
• Two commercial samples of Passiflora leaves
11

were analyzed using gas chromatography with Standard

electron-capture and flame photometric
detection (GC–ECD).
• Twenty-three percent of the samples showed the
Sample 2
presence of the organochlorine or
organophosphorus pesticide residue.

GC–ECD chromatograms of two Passiflora samples, 11 Standard

(1) Hexachlorobenzene, (2) lindane, (3) chlorothalonil, (4)
parathionmethyl, (5) fenitrothion, (6) malathion, (7)
parathion-ethyl, (8) fenthion, (9) a-endosulfan, (10)
methidathion, (11) dieldrin
 Example (2): Determination of multi-pesticide residues in Anise fruits using GC analysis

 Chlorinated hydrocarbons and other

miscellaneous pesticides in the Anise
fruits were analyzed using gas
chromatography coupled with
nitrogen phosphorus detector (NPD).

 one pesticide was above

the limits??
Anise Sample
 ADULTERATIONS OF HERBAL DRUGS

 Adulteration occurs when undesirable materials are

admixed with an herbal medicinal product.
 Example: the addition of starch into ginger, followed by
the addition of a little bit of coloring matter, which
results in a perfect shade of yellow color.
 In some cases, the herbal product is entirely replaced
with a different substance. This is known as
substitution.
 Example: the supply of cheap cottonseed oil in place
of olive oil
 Scientific studies have been able to discover
several adulteration methods.
 Assessing product adulteration of Tongkat Ali using HPLC analysis

 Tongkat Ali is one of the most important plants in Malaysia. The plant extracts (particularly
roots) are widely used as adaptogen besides having antidiabetic and aphrodisiac activities.
 This study assesses the extent of adulteration of Tongkat Ali herbal products using HPLC
analysis for the presence of eurycomanone bioactive marker.
 Unfortunately, Products which were claimed as authentic were found not to contain
eurycomanone and so they are substituted with unkown substances.

Eurycomanone
 std root extract
std eurycomanone

ХХ ХХ

ХХ ХХ
 STD
√
√
Substituted
drugs
√

o Even, the three tested samples which contain eurycomanone, none of them has met the
minimum concentration of eurycomanone as the genuine standard root extract.
o The amount of eurycomanone found in the three samples was 0.01, 0.03 and 0.02%,
respectively, less than 0.2–0.5 asset by pharmacopeia.
 The low concentration of eurycomanone content measured in these products raised a
possibility of another form of adulteration such as addition of undeclared synthetic
phosphodiesterase-5 inhibitors (PDE-5) drugs so as to enhance their efficacy such as
Sildenafil.
 Adulteration of natural product by Addition of Synthetic Principles
A. Adulteration with illegal industrial dyes
 Example: The addition of synthetic colorants such as metanil yellow and sudan red
to traded turmeric.
B. Adulteration by the addition of undeclared Synthetic Drugs
 Example: Cosmetic products have no therapeutic purposes and shall not claim any
therapeutic action.
 However, different topically active drugs e.g. minoxidil, hydrocortisone,
spironolactone, or even oral contraceptives such as progesterone and estrone are
illegally incorporated in anti-alopecia, antihirsutism and anti-acne cosmetics.
 HPLC-UV-DAD and HPLC–ESI-MS assays have been applied to the analysis of some
cosmetic preparations sold on internet web sites and all the examined products
exhibited more than one forbidden compound.
Identification of synthetic dyes as common adulterants in commercial saffron
• Saffron is a highly adulterated spice due to its limited √√
production and high costs.
• Non-saffron plant material is coloured with synthetic
dyes to produce counterfeit saffron.
• Continuous monitoring of the synthetic dyes is
essential because some dyes are not safe for
human consumption.
• 20 commercial saffron samples from several
countries were screened and were found to contain
dyes. Visual appearance of the acetonitrile
• TLC analysis showed adulteration with magenta- and extracts from authentic saffron (AS)
and the market samples of saffron,
pink- coloured dyes. which were suspected to be adulterated
• MS analysis identified the magenta-coloured dye with synthetic dyes (SD1-SD20).
as new fuchsin and the pink-coloured dye as
rhodamine B.
• Both of them are illegal and carcinogenic.
 New fuchsin

A
B Rhodamine B

Preparative TLC analysis of a magenta-coloured and a

pink-coloured saffron sample. The major coloured spots
were extracted with acetonitrile for subsequent HPLC and
MS analyses.
Application of LC–ESI–MS–MS for detection of synthetic adulterants in herbal remedies

 Adulteration of natural herbal medicines with undeclared synthetic drugs is a common

and dangerous phenomenon of alternative medicine.
 Most common synthetic adulterants in herbal remedies were detected using high-
pressure liquid chromatography-electrospray tandem mass spectrometry (LC–ESI–
MS–MS).
 Drugs belonging to various pharmacological classes (analgesic, antidiabetic,
aphrodisiacs, corticosteroids and weight reducing compounds) were studied.
 The drugs were isolated from herbal remedies using methanol extraction.
 Drug-free herbal remedy spiked with various pharmaceuticals commonly adulterated herbal
preparations was used for internal standard testing.
 Several undeclared drugs were identified in “herbal” remedies, like e.g. sildenafil,
tadalafil, testosterone, or glibenclamide
Examined herbal remedies from traditional markets Analytical findings

“Herbal” remedy against diabetes, containing glibenclamide

Standard Glibenclamide
Japanese remedy “for female sexuality”, containing testosterone decanoate

Testosterone decanoate standard

The extract from “herbal” capsules “PhytoAndro”, containing sildenafil.

Sildenafil standard
B
 Analysis of the skin irritant p-phenylenediamine (PPD) in henna products using mass
spectrometry

• Henna stains keratin, present in hair, skin and fingernails, a red-orange or rust colour.
• Producers of temporary tattoos mix the aromatic amine compound, para phenylenediamine
(PPD) into natural henna to create ‘black henna’ that rapidly stains the skin black.
• However, PPD may cause severe delayed hypersensitivity reactions following skin contact.
• Eleven of henna samples, originating from various countries, tested positive for PPD when
henna products were screened using LC-MS analysis.

PPD
 Where ‘s PPD??

a. UPLC-MS chromatogram of a henna sample containing PPD. b. The mass spectrum of PPD.
Adulteration of synthetic PDE-5 inhibitors viz., sildenafil and tadalafil in marketed herbal aphrodisiacs

 Herbal aphrodisiacs have a long traditional history in Ayurveda. However, their effectiveness are
milder and a prolonged treatment with these therapies is required to cure the problem.
 With fast onset and quick relief phosphodiesterase-5 (PDE-5) inhibitors were able to overshine
these traditional medicines and generated huge profits for their manufacturers.
 Therefore, in order to increase their sale from herbal aphrodisiacs some of the manufacturers have
resorted to malpractices of adulterating their herbal formulations with phosphodiesterase-5
inhibitors.
 Thus, Fifteen herbal aphrodisiac preparations were analyzed using HPLC. Five of them were
found to be adulterated with sildenafil citrate while tadalafil was detected in 14 preparations.

tadalafil

sildenafil
HPLC graph of sildenafil.

HPLC graph of adulterated formula1

HPLC graph of tadalafil

HPLC graph of adulterated formula 2

 Identification and Determination of Synthetic Pharmaceuticals as Adulterants in Eight Common
Herbal Weight Loss Supplements

 Adulterated herbal weight loss products with undeclared synthetic drugs are
common and responsible for many serious health damages.
 Different synthetic adulterants were detected by LC/MS in eight common herbal
weight loss supplements, which are currently sold in the markets through the
satellite channels and internet, without mentioning on the labels.
 Despite the manufacturer’s claim that their products contained only the extracts
of the plants mentioned on the label, but those are contained other synthetic
substances.
 Sibutramine, phenolphthalein, bumetanide, phenytoin, caffeine,
pseudoephedrine, theobromine and amfepramone were found in eight products.
 Detection and determination of undeclared synthetic caffeine in weight
loss formulations using HPLC-MS
 Caffeine is present in products marketed for weight loss, with the purpose of increasing
thermogenesis and lipid metabolism. The dosage declared by the product manufacturer, or
even its presence, is not always correctly described on the label.
 The undeclared synthetic caffeine in weight loss formulations was confirmed by an
HPLC-MS method by evaluating the ion fragments at 195.1 m/z (molecular ion),138.1 m/z
and 110.1 m/z fragment ions of caffeine.
 From 100 products purchased through internet, 17 contained caffeine.
 The users of these products reported here could have increased sleep latency and a
reduced sleep duration, particularly when consumed close to bedtime.
 Caffeine consumption acutely increases blood pressure, heart rate, causes tremors and
arrhythmias. Moreover, the consumer may use other medicines resulting in deleterious
interactions.
Declared Composition Brand Name

Slendesta

Adventra z

!!!
???

Adventra z
 pholia magra capsule

caffeine standard

HPLC chromatograms and ESI MS/MS spectrum obtained for the caffeine analysis of (A
and B) pholia magra capsule claimed 100% herbal in comparison to (C and D) caffeine
standard by HPLC-ESI-MS.
 Adulteration of herbal sexual enhancers and slimmers
 Some common synthetic adulterants in some herbal remedies available on the market were
investigated utilizing a reversed-phase HPLC/MS analysis.
 Herbal medicines acting as sexual enhancers and slimming products were tested for the
presence of conceivable adulterants.
 The weight loss products were discovered to be defiled with the effectively withdrawn drug
sibutramine (SIB) and with phenolphthalein (PPH), which has been demonstrated to cause tumors.
 On the other hand, sildenafil (SLD), a medication contraindicated for patients with heart diseases
was found in the herbal product for erectile dysfunction.
 standard SIB standard PPH
standard SLD

Zotreem Plus Slimming Bomb Enjoy

MS spectra of a; standard SIB, b; standard PPH, and c; standard SLD (top), compared with Zotreem Plus,
Slimming Bomb, and Enjoy (bottom), respectively
 Advanced approaches in quality control of herbal drugs
 Chromatographic methods coupled with biochemical detection (in vitro bioassays) have been widely used
for screening active components in herbal medicine.
 Online strategies, which integrated the separation science and bioactivity screening in a single platform,
allowing simultaneous screening and characterization of active compounds.
 TLC and HPLC are the most commonly used techniques.

HPLC separation coupled with on-line bioassay

TLC bioautographic detection of antibacterial drugs

 Investigation of Antibacterial activity of different herbal teas Using TLC-MTT Bioautography
 The separation of the components is performed directly on a TLC plate
 For searching the antibacterial activity, the developed plate is immersed in a
bacterial broth, and bacteria grow directly on its layer during a proper
incubation time.
 Inhibition zones are formed in places where antimicrobial components are
located.
 TLC plates used for chemical derivatization (blank TLC) were visualized using
NP/PEG.
 For bioautography with bacteria, the plates (tested TLC, bioautograms) were
dipped in an aqueous solution of tetrazolium salt (MTT) for 5 s and incubated at
37°C for 2 h.
 The visualization of the inhibition zones of the separated compounds (actives)
was based on dehydrogenase activity of metabolically active bacteria. This enzyme TLC-bioautography testing
converts the yellow color of MTT, into purple formazan. Thus, the inhibition zones antimicrobial substances in
are visible as pale spots against a purple background. plant extracts.
Investigation of Antibacterial activity of different herbal teas Using TLC-MTT Bioautography

apigenin chlorogenic acid

quercetin
kaempferol caffeic acid
luteolin
rosmarinic acid

Detection of flavonoid glycosides in different herbal

teas A) TLC chromatogram, detection with NP/PEG Detection of phenolic acids in different herbal teas A) TLC
reagent B) TLC-MTT bioautogram chromatogram, detection with NP/PEG reagent B) TLC-MTT
bioautogram
Authentication of several Turkish propolis products using HPTLC-DPPH Bioautography

 The TLC plate with samples is developed with the elution solvent and dried.
 It is then sprayed with DPPH solution (pink solution) and the plate is examined in daylight after 30 min.
 Active antioxidant compounds appear as yellow spots against a purple background

HPTLC chromatograms of different

propolis extracts, derivatization: NP/PEG

HPTLC chromatograms of
different propolis extracts,
derivatization: DPPH˙ solution
HPLC with on-line coupled biochemical detection for identification of acetylcholinesterase
inhibitors of anti-Alzheimer’s natural products
 The key point for online biochemical detection based on colorimetric assay is to form
a colored product, which can be detected at visible wavelength.
 Eluting bioactive components react with targeted enzyme in presence of its substrate
and other reagents to give the colored product.

physostigmine

galanthamine
HPLC and Biochemical Detection based on the colorimetric assay for the Authentication
of Three antidiabetic herbal teas through the evaluation of their α-Glucosidase
inhibitory activity

acarbose Std eagle tea

pu-erh tea
spica tea
 Exercise (1): Triacylglycerol (TAG) of Argan oil using HPLC-ESI-MS to detect counterfeit
Argan oil and Argan-oil-based products

 Argan oil is produced in Morocco and it is quite expensive.

 In order to assess the TAGs profile of argan oil HPLC coupled with ESI-MS
detector was used.
 Consequently, Five TAGs namely, OOL, LLL, OOS, LLO and OOO were the
main components of argan oil.
 The adulteration of argan oil can be performed by the addition of less
expensive vegetable fats.
 The simultaneous presence of other vegetable fats can be easily ascertained
because they contain triglycerides with molecular mass and RTs different
from that of triglycerides of argan oil.
The characteristic HPLC-ESI/MS
“hand-shape profile” of argan oil
*L: linoleic acid 18:2; oleic acid 18:1;
S: stearic 18:0
 Exercise (1): cont.
 Actually, in cosmetics more than one vegetable fat such as shea butter, coconut, linseed,
sunflower, peanut and almond oils are added to improve the desired formulation properties and
must be declared on the product’s label.
 The profile of certified argan oil was compared to the profile of twenty cosmetics claiming argan
oil as a major or minor ingredient.
 The presence of argan oil was ascertained in most products but in some cases, differences
between the declared and found composition were evidenced !!!

EXPLAIN????
 Exercise (1): cont.
Characteristic peaks of some common used oils identified by LC/MS in analysed fats
Vegetable fat (M+H)+m/z

Argan oil (CAO) 878, 880.7, 882.8, 884.8, 886

Shea butter 886.8, 888.8, 890.8

Coconut oil 605.5, 661.5, 689.6, 717.6

Linseed oil 872.7, 874.7, 876.7

Peanut oil 916.8, 942.8

 Samples 1, 3 and 4 were declared as100% pure argan oils??
 Samples 10 and 11 contained argan oil and shea butter in the ingredient list.
 Sample 13 was assigned with linseed and argan oils.
 Sample 15, listing coconut and argan oils as fatty ingredients.
 Sample 19 declared the presence of both argan oil and peanut oil in the ingredient list.
 Exercise (1): cont.

HPLC-ESI-MS chromatograms of some Argan-oil-based products

m/z= 878, 880.7, 882.8,

884.8, 886, 886.8, m/z= 878, 880.7, 882.8,
888.8, 890.8 884.8, 886, 872.7,
874.7, 876.7
m/z= 878, 880.7,
882.8, 884.8, 886,
605.5, 661.5, 689.6,
717.6

Sample 11
Sample 13 Sample 20
 Exercise (1): cont.
784.3, 812.8, 840.8 , 807.7, 835.8, 863.8, 878, 878, 880.7, 882.8,
605.5, 661.5, 689.6, 880.7, 882.8, 884.8, 886 884.8, 886
717.6, 878, 880.7, 876.7, 874.7
882.8, 884.8, 886

sample 15 sample 19
Sample 3
880.7, 882.8, 884.8, 878, 880.7, 882.8,
886, 901.7, 903.7, 884.8, 886 878, 880.7, 882.8,
905.8, 907.8 884.8, 886, 886.8

sample 4 sample 1 sample 10

Exercise (2): HPLC was used to Standard
analyse three herbal tea samples.

 Fumonisin B1 and B2 are mycotoxins

produced by the fungus Fusarium, which
Sample a
commonly infects the moisture-damaged
herbal products.
 They are detected in two samples only

Which of them?? Sample b

Sample c
 Exercise (3): Quantification of Furosemide by HPLC-DAD as a Co-adulterant in Natural Products

o Two pharmaceutical capsules indicated for treatment of rheumatic and inflammatory

diseases were purchased via the internet.
o Sample A contained in its declared formulation pecan, yellow ipe and sucupira.
o Sample B had in its declared composition Moringa oleifera and Miconia albicans.
o Furosemide standard was analyzed to verify the adulteration.
o The products were tested using HPLC-DAD system.
o The results indicated the presence of the undeclared furosemide compound in the
original formulation of both analyzed samples.
o Sample A, contains ??? mg of furosemide, while sample B contains ??? mg.
Exercise (3): cont.

Abs.=1.1781 Sample A chromatogram

Sample B chromatogram
Abs.=2.3126
mg/ml
 Exercise (4): HPLC Determination of Active Compounds in Fengshiding Capsules

 HPLC method was established for simultaneously determining four bioactive components,
salicin, liquiritin, paeonol, and imperatorin in Fengshiding capsule, a widely used traditional
Chinese medicine for treating rheumatic disease. Reference solution
paeonol
salicin
Find out the biomarkers?
imperatorin
liquiritin

HPLC chromatogram of capsule