Chapter 5: (Development and validation ofH'PLC methodfor Setainefrom Jlchyranthes aspera Linn.

DEVELOPMENT AND VALIDATION OF HPLC

METHOD FOR BETAINE FROM
ACHYRANTHES ASPERA LINN.

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 Chapters-. (Development and validation qfWPLC method for betainefrom Achyranthes aspera Linn.

5.1 INTRODUCTION
5.1.1 Plant profile: Achyranthes aspera Linn, is a variable, erect, annual to perennial
herb which can grow up to 30-120 cm tall. It is with angular stem, ribbed, stiff, with
thickened nodes, variably pubescent and reddish brown. The genus name is derived
from Greek word achyr means chaff or bran and the species name refers to the rough
texture of spikes.30"33
5.1.2 Achyranthes aspera Linn.
(a) Classification34:
Kingdom: Plantae
Division: Magnoliophyta - Flowering plants
Class: Magnoliopsida - Dicotyledons
Order: Caryophyllales
Family: Amaranthaceae
Genus: Achyranthes
Species: aspera L.
(b) Vernacular names35'36:
Sanskrit: Apamarga, Mayura
Hindi: Apang, Latjira
Marathi: Aghada, Pandhara-aghada
Kannada: Uttrani
Malayalam: Katalati
Tamil: Nayuruvi
Telugu: Uttarenu
Gujarati: Aghedo
English: Prickly-Chaff Flower
(c) Parts used: Whole plant, leaves, seeds, roots, flowers and fruits.35
(d) Geographical distribution: It is distributed throughout the Old World tropics,
including Malaysia and Australia and introduced into tropical America. In India it is
distributed throughout along roadsides and waste places.37
(e) Botanical description: Erect annual herb, up to 1.5 m high. Leaves opposite, with
short petiolate, lanceolate, silky beneath when young, 1-5 cm long, 0.4-2 cm wide.
Inflorescence long terminal and short axillary spikes, up to 30 cm long, 0.6-1.0 cm
diameter. Flowers bisexual, the lower ones retroflexed. The bracts are glossy which

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 Chapter 5: (Development and validation offflPLC method for betainefrom Achyranthes aspera Linn.

enable the fruit to stick on clothes and woolly skin of animals; small spiny tips,
whitish or purplish. Pollen grains apolar, spherical - polyhydral with obtuse angles.
Diameter 13-16 -urn. Pollen grains polypantoporate (32 pores), each of about 2 urn
diameter and round. Exine 0.7-1.0 urn thick. Sexine as thick as nexine, minutely
granulate Tectum thin, supported by densely spaced bacula. Trichomes unirow,
multiarticulated cells with smooth surface.38
(f) Traditional uses: In the Vedas, Apamarga has been described as a divine
medicine. All ancient ayurvedic texts have attributed a wide range of actions of
apamarga on the human body. It is famous as a herbal lithotriptic agent (that breaks
the urinary stones) and as diuretic. It is also carminative, digestive, expectorant, anti-
inflammatory and a killer of intestinal worms. Having blood-purifying and anti-
endotoxin properties, it is also a bitter tonic. The ground root is given with water for
snake bites, a fresh root piece is used as tooth brush, crushed leaves rubbed on aching
back to cure strained back, juice of fresh leaves is given in diarrohoea. Apamarga is
the chief ingredient of the famous Apamarga Kshara Tailam which is a classic
medicine for the treatment of chronic ear diseases. The juice of fresh powder of
apamarga is also used to treat chronic febrile conditions, bleeding piles, intestinal
worms and post-delivery uterine problems. The average daily dose of its kshara and
juice is up to 2 g and 20 mg daily respectively.39_41
(g) Pharmacology:
Antifertility activity: Gupta et al reported that alkaloidal fraction of alcoholic extract
of the root bark inhibited the response of oxytocin in rat uterus. 42 A 58-kDa protein
(Ap) was isolated from the 50 % ethanolic extract of the roots of Achyranthes aspera
and was evaluated for spermatotoxicity. It was found that the spermatotoxic effects
were comparable to gossypol.43 Study was conducted to determine the most effective
fraction of the hydroethanolic extract of roots of Achyranthes aspera and it was found
that the hexane fraction of the hydroethanolic extract produced the most effective
spermicidal activity.44
Anticancer activity: The methanol extract, alkaloid, non-alkaloid and saponin
fractions of leaves of Achyranthes aspera exhibited significant inhibitory effects on
the Epstein-Barr virus early antigen activation induced by the tumor promoter, 12-0-
tetradecanoylphorbol-13-acetate in Raji cells. The nonalkaloid fraction containing
mainly non-polar components showed the most significant inhibitory activity.

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Antidiabetic activity: Powdered whole plant, aqueous and methanol extracts of

Achyranthes aspera were evaluated for antidiabetic activity. A significant dose-
related hypoglycaemic effect in normal as well as in diabetic rabbits was seen after
oral administration of A. aspera powder. The aqueous and methanol extracts also
lowered blood glucose levels in normal and alloxan diabetic rabbits. No adverse or
side effects were observed at dosages up to 8 g/kg orally for seven days.46
Antiinflammatory activity: The plant is used traditionally for its anti inflammatory
effects. The ethanol extracts of Achyranthes aspera showed significantly inhibition of
paw edema induced by carrageenan and Freund's complete adjuvant at the doses of
100-200 mg/kg.47
Immunity enhancer: Achyranthes aspera was incorporated in artificial fish diet, and
fed to catla Catla catla. After 4 weeks, fish were immunized with bovine serum
albumin (BSA) spleen and blood were sampled on weekly intervals for four times
after immunization. ELISA was used to determine antigen-specific antibody level in
serum. Antigen clearance was determined in spleen by immuno-electron microscopy.
There was significant increase in the BSA specific antibody titers than the untreated
control group throughout the study period. The efficiency of antigen clearance was
also enhanced in C. catla treated with Achyranthes aspera.
(h) Toxicity: Seven days acute toxicity study of whole plant powder did not reveal
any adverse or side effect upto a dosage of 8 g/kg, orally.46The alkaloid isolated from
the plant was tested for its acute, subacute and chronic toxicity in rats. During acute
toxicity test, there was a slight increase in sedation and slight loss in righting region at
6 mg/kg dose level, which became prominent at 7 mg/kg dose level. At higher doses,
significant depletion in righting region, depression in respiration, remarkable increase
in sedation and diarrhea was observed. Subacute toxicity test revealed (5 and 6
mg/kg) a significant increase in sedation and hypnosis, depletion in respiration and
loss of righting reflexes. At 6 mg/kg dose, it also caused remarkable increase in
salivation and diarrhoea. Chronic toxicity showed (3 mg/kg) an increase in sedation,
hypnosis, salivation and diarrhoea. There was a significant depression of respiration
and loss of body weight.
(i) Phytochemistry: The plant contains betaine, achyranthine, hentriacontane,
ecdysterone and two glycosides of oleanolic acid and amino acids. Achyranthine and
betaine are principal alkaloid this plant.3I

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 Chapter 5: (Development and validation ofJfPLC methodforbetainefrom Achyranthes aspera Linn.

CH3
+ 2 1
H3C N:—CH2—COO"
I
CH 3

Figure 5.1: Structure of betaine Figure 5.2: Structure of achyranthine

5.1.3 Literature review-Isolation of achyranthine

Two methods have been reported claiming isolation of achyranthine from
Achyranthes aspera.
Basu et al isolated water soluble base and chloroform soluble base from Achyranthes
aspera by extracting the plant material with 95 % ethanol. The alcohol extract was
treated with 2 % sulphuric acid. The acid layer was then made alkaline to precipitate
base. The base was extracted with chloroform. The remaining aqueous layer was
treated with sulphuric acid and Dragendorr's reagent to precipitate another base. The
water soluble base was further treated with alcohol to produce colourless crystals of
achyranthine.50
V. K. Kapoor et al used same procedure as Basu et al for isolation of water soluble
base and they designated this base as betaine.51
5.1.4 Literature review- HPLC analysis of achyranthine
No reports are available for HPLC analysis of achyranthine.
5.1.5 Literature review-Isolation of betaine
A number of methods have been reported for isolation of betaine alone or in
combination with other constituents from various medicinal plants and biological
fluids such as urine.
Betaine was isolated as hydrochloride salt from Guayule shrub by Murray and
Walter.52
Betaine was isolated from epigeal part of Chenopodium botrys L. (Jerusalem oak)
using extraction with 10 % NH4OH and methanol followed by column

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 Chapter 5: (Development and validation of!H<PLC method for betainefrom Achyranthes aspera Linn.

chromatography using alumina as stationary phase and methanol as eluent. Isolated

betaine was recrystallized using n-butanol.53
Betaine was also isolated along with choline in anthers and wheat germ.54

Elyakov et al isolated betaine from brown algae using extraction using ethanol.
Ethanol extract thus obtained was treated with water and the aqueous portion was
chromatographed on silica column using ethanol as eluent. Fractions containing
alkaloid was rechromatographed on silica using 50 % alcohol as eluent to get
betaine.55

Betaine was also isolated from aerial parts of Lycium barbarum Linn. The aerial parts
were extracted using methanol. The methanol extract was partitioned between water
and ethyl acetate. The aqueous layer was passed through cation exchanger. The
column was then eluted with 2 N NH4OH and eluent was evaporated, taken in
methanol and filtered. Residue was further purified using preparative TLC using
methanol as mobile phase. The colorless crystalline material thus obtained was
converted to hydrochloride and recrystallized using methanol.56

Glycine betaine was isolated from human urine by precipitation using ammonium
reineckate at acidic pH followed by gel filtration and high-pressure liquid
chromatography.57

5.1.6 Literature review: HPLC analysis of betaine

A number of HPLC methods have been reported for quantification of betaine alone or
in combination with other constituents from various medicinal plants and biological
samples.

Betaine was estimated in sugar and wine using amino bonded silica gel column (150
mm X 4.7 mm, 10 um) as stationary phase and acetonitrile: water (75:25, v/v) as
mobile phase. Flow rate of mobile phase was 1.0 ml/min.

J. Gorham separated betaines and their sulphur analogues by HPLC using strong
cation exchange column (Partisil 10-SCX, 250 mm X 5 mm) as stationary phase and
50 raM of KH2PO4 buffer containing 5 % methanol (pH 4.6) as mobile phase pumped
at 1.5 ml/min. Detection was carried out at 190-200 nm. Betaine eluted at 5.47 min
with total run time of 10 min.59

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 Chapter5: (Development and validation offflPLC methodforbetainefrom JZchyranthes aspera Linn.

J. Wurtman developed normal phase HPLC method for separation of choline

metabolites using silica column, Rainin Microsorb (250 mm X 4.6 mm, 5 um) and
gradient elution with buffer. Flow rate of mobile phase and temperature were kept at
2.7 ml/min and 45 °C, respectively. Retention time of betaine was 10.1 min with total
run time of 60 min.60
HPLC-radioenzymatic assay was used to determine betaine aldehyde and betaine in
rat liver mitochondria using silica column. Chromatographic elution was carried out
using buffer containing 800 ml acetonitrile, 68 ml ethanol, 5 ml of 1.0 M ammonium
acetate: glacial acetic acid buffer (3:2, v/v), 127 ml water and 10 ml of 0.1 M
KH2PO4. Flow rate of mobile phase was kept at 1.5 ml/min.61

Betaine content was estimated in oriental medicine, Jikoppi which is powdered root-
skin ofLycium chinense using Kaseisorb LC NH2 super column (250 mm X 4.6 mm,
5 urn) and mobile phase containing acetonitrile: water (75: 25, v/v) containing 0.1 %
of Brij 35. The flow rate of mobile phase and detection wavelength were kept at 1.0
ft")

ml/min and 210 nm, respectively.

Betaine was determined in biological samples by derivatization using 4'-bromo-

phenacyl triflate. Quantitation was carried out by UV.63

Betaine and N,N-dimethylglycine in human urine were determined by HPLC using

Supelcosil LC-SCX (250 mm X 4.6 mm, 5 um) as stationary phase and KH2PO4 (1
mM/1) and HC1 (0.12 mM/1) as mobile phase. Flow rate was 1.0 ml/min and detection
was carried out at 195 nm. Limit of detection (LOD) was found to be 0.9 ug/ml. %
Recovery of the betaine was greater than 85 %.64

Betaine and N,N-dimethylglycine were analysed in blood and urine by precolumn

derivatization using p-bromophenacyl bromide and crown ether as catalyst. Column
used was Supelcosil LC-SCX (250 mm X 4.6 mm, 5 um) and mobile phase used was
acetonitrile: water (9:1, v/v) containing 22 mM/1 choline. Flow rate was kept at 1.5
ml/min and the detection was carried out at 254 nm. Retention times of the phenacyl
bromide esters of dimethylglycine and betaine were 12.7 and 14.8 min, respectively.
The LOD was found was to be 5 mM /l.65

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 Chapter 5: (Development and validation ofWPLC method for Setainefromjlchyranthes aspera Linn.

Bakalli et al determined betaine content in several feed ingredients like alfalfa, wheat,
wheat middlings, poultry meal by ion chromatography using cation exchange column
(Partisil SCX-10) and buffer containing 50 mM KH2PO4. Detected was carried out at
200 nm with flow rate of mobile phase at 1.5 ml/ min. The method was found to be
linear in the range of 15 to 650 ug/ml. The LOD was found to be 500 ug/ g.66

Aracyl derivatives of betaine were separated using different strong cation exchange
columns, normal phase columns and reversed phase columns. Detection was carried
out at 249 nm and flow rate of mobile phase was maintained at 1.0 ml/min. Column
oven temperature was maintained at 40 °C.67

Betaine was estimated in whole plant of Achyranthes aspera Linn, by reversed phase
HPLC using Zorax column (250 mm X 4.6 mm, 5 um) and mobile phase was
consisting of methanol: 50 mM KH 2 P0 4 buffer pH 4.6 (50:50, v/v) at flow rate of 0.8
ml/min. Detection was carried out at 200 nm. Betaine eluted at 3.492 min. The
method was linear in the range of 1-40 ug/ml. LOD and LOQ were found to be 0.29
and 0.88 ug/ml, respectively.68

Betaine was determined in 70 % methanol extract of Fructus lycii by hydrophilic

interaction liquid chromatography with evaporative light scattering detection using
Kinetex HILIC column (100 mm X 2.1 mm, 2.6 um) and mobile phase consisting of
acetonitrile and 10 mM ammonium formate (pH 3.0): acetonitrile (90:10, v/v) in
gradient mode. The flow rate of mobile phase was maintained at 0.7 ml/min and
column was equilibrated at 27.5 °C. Linearity was observed in the range of 10-250
ug/ml. LOD and LOQ were found to be 3 ug/ml and 10 ug/ml, respectively. Betaine
eluted at retention time of 15 min.

Betaine was analysed in Chinese Lycium by HPLC coupled with evaporative light
scattering detector. Column used was HILIC (250 mm x 4.6 mm, 5 um) and mobile
phase used was methanol: 10 mM ammonium acetate buffer pH 6.0 (75:25, v/v).
Flow rate of mobile phase was kept at 1.0 ml/min.70

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 Chapter 5: (Development and validation ofJfPLC method for betainefrom Achyranthes aspera Linn.

5.1.7 Selection of betaine as marker compound

Whole plant of Achyranthes aspera contains two major alkaloids i,e betaine and
achyranthine. There are number of methods reported for isolation of betaine from
medicinal plants like Chenopodium botrys and Lycium barbarum and biological fluid
such as urine. These methods use either tedious repeated column chromatography or
cation exchange chromatography. Also few HPLC methods are reported for
determination of betaine but these methods use either ion-exchange chromatography,
hydrophilic interaction liquid chromatography, buffers in mobile phase or tedious
precolumn derivatization techniques. Hence, the present work discusses a simple
method for isolation of betaine from seeds of Achyranthes aspera and development
and validation of a new, rapid and simple reversed phase HPLC method for
quantitative determination of betaine in purified extract of seeds of Achyranthes
aspera.

5.2 ISOLATION OF ACHYRANTHINE/BETAINE FROM DRIED SEEDS OF

ACHYRANTHES ASPERA
5.2.1 Procurement of seeds of Achyranthes aspera
Dried seeds of Achyranthes aspera were procured from three different geographical
sources.
Sample 1: Seeds of Achyranthes aspera were collected from Valsad district of Gujarat
in November 2009.
Sample 2: Seeds of Achyranthes aspera were collected from Virudunagar district of
Tamilnadu in November 2009.
Sample 3: Seeds of Achyranthes aspera were purchased from local market of
Mumbai, Maharashtra in November 2009.
5.2.2 Authentication of dried seeds of Achyranthes aspera
Different herbaria of sample 1, sample 2 and sample 3 were deposited at Botanical
Survey of India (BSI), Pune under names AAGSOD4, AATSOD5 and AAMSOD6,
respectively and authenticated.
5.2.3 Extraction of alkaloids
The plant of Achyranthes aspera contains two principal alkaloids, achyranthine and
betaine. Achyranthine, chemically l-methylpyrrolidine-3-carboxylic acid, is bioactive
marker having anti-inflammatory and anti-arthritic activities. No reports are available
for isolation of achyranthine in its stable form. Hence, initially isolation of
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 Chapter 5: (Development and validation of'MPLCmethodforbetainefrom ^.chyranthes aspera Linn.

achyranthine was under taken. Extraction of alkaloid-achyranthine was carried by

solvent extraction technique. Extraction of alkaloid was carried out by two methods.
Method 1: 20 g of powdered seeds were defatted with 200 ml of petroleum ether
(60°-80 °C). Defatted seeds were moistened with ammonia (25 %) for an hour and
then extracted with 200 ml chloroform. Chloroform layer was evaporated to obtain a
sticky extract which was designated as CM1. This sticky extract, CM1 was further
extracted with methanol. The methanol extract thus obtained was designated as
MM1.
Method 2: 20 g of powdered seeds were defatted with 200 ml of petroleum ether
(60°-80 °C). Defatted seeds were extracted with 200 ml of distilled water. Water
extract was designated as WM2. Approximately 1-2 ml of water extract (WM2) was
kept aside and remaining water extract was evaporated to l/3 rd volume. To this
concentrated water extract, ammonia solution (25 %) was added in 1: 1 proportion.
Above mixture was extracted with chloroform. Chloroform layer and ammoniacal
layer were separated. Chloroform layer was designated as CM2 and water soluble
ammoniacal layer was designated as AM2.
Preliminary phytochemical screening of all five fractions (MM1, CM1, WM2, AM2
and CM2) was carried out. All these fractions showed the presence of alkaloids,
carbohydrates and glycosides.
5.2.4 HPLC studies of fractions
HPLC studies of all five fractions were carried out to know the number of
components present in each fraction. All fractions were evaporated to dryness and
dissolved in HPLC grade methanol, sonicated for 10 min, filtered and subjected to
HPLC analysis.
Various combinations of methanol- water, acetonitrile-water and acetonitrile-
trifluoroacetic acid were tried as mobile phase in isocratic mode and gradient mode.
Mobile phase composed of acetonitrile along with trifluoroacetic acid (TFA) in
gradient mode gave best resolution with maximum number of peaks. TFA is the most
commonly used as ion pairing agent at low concentrations in reversed phase HPLC
because it sharpens the peaks and improves resolution. It has low absorption at
detection wavelengths.
The following chromatographic conditions were optimized and used:
Column: Unisphere CI8 column

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 ChapterS: (DeveCopment and vaCidation ofMPLC methodfor hetainefrom JZchyranthes aspera Linn.

Column Dimensions: 250 mm X 4.6 mm, 5pm

Mobile Phase: Solvent A: 0.1% TFA, Solvent B: Acetonitrile
Gradient (Time/%A):- 0/90, 20/10, 22/90, 30/90
Flow Rate: l.Oml/min
Temperature: 40 °C
Detection Wavelength: 254 nm
Concentration of sample: lO.Omg/ml
Injection volume: 20 ul

Figures 5.3, 5.4, 5.5, 5.6 & 5.7 show LC chromatograms of MM1, CM1, WM2, AM2
and CM2 respectively.

Figure 5.3: HPLC chromatogram of fraction MM1

HPLC analysis of fraction MM1 revealed four major peaks with retention time of
3.014 min, 5.379 min, 9.389 min and 9.889 min (Figure 5.3).

D A D 1 C. S>g=254.4 RefcoH (ANTVIRAL 07061002 D)

mAU : s %
80- <>
<>
70-

60-
722

eg
50-
8 ~
o * <r T>

40- c

_ CM
30-

20-
A,
,0
~:
J^ii LJ WV H
~^-\- — - ~
0-
^—
i r i
j 5 10 15 20 25 rrvr

Figure 5.4: HPLC chromatogram of fraction CM1

(DeveCopment and VaCidation ofWPLC Methodsfor Marker compounds and (Eioactive as per ICK
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 Chapter 5: (Development and validation ofWPLC methodfor Setaine from JlcHyrantfies aspera Linn.

HPLC analysis of fraction CM1 revealed five major peaks with retention time of
2.982 min, 5.379 min, 9.391 min, 9.899 min and 11.722 min (Figure 5.4).

Figure 5.5: HPLC chromatogram of fraction WM2

HPLC analysis of fraction WM2 revealed seven major peaks with retention time of
2.982 min, 5.390 min, 5.790 min, 9.404 min, 9.903 min and 11.258 min and 11.735
min (Figure 5.5).

D A D l C. Sig=254.4 R e b o f f (ANTVIRAL 0 4 0 6 1 0 1 6 D)
mAUj £
1

200- sn
m
175-
3.031

150-
904

01
125-
_ is
100 J tri Hs~
07

75-
9
Si |
2 304

5 0 -,
in

25
-. oj
l^jj^ si
0- ,!vJ v — J
5 10 15 20 25 mir

Figure 5.6: HPLC chromatogram of fraction AM2

HPLC analysis of fraction AM2 revealed five major peaks with retention time of
3.031 min, 5.394 min, 5.791 min, 9.405 min and 9.904 min (Figure 5.6).

(Development and Validation offfPLC Methodsfor Marker compounds and'(Bioactive as per IQK
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 Chapter 5: (Development and validation ofJCPLC method for betaine from JAchyranthes aspera Linn.

D A D 1 E. Sig=310 4 Ref=off ( A N T V I R A L . 0 4 0 6 1 0 1 4 D i

BO

a
40

30

2C

10
A* JU * ^ / U ' V A M A M ^ _ ^ J ^
c -HV-
-10

Figure 5.7: HPLC chromatogram of fraction CM2

HPLC analysis of fraction CM2 revealed three major peaks with retention time of
9.403 min, 10.313 min and 10.743 min (Figure 5.7).
5.2.5 LC-MS studies of fractions
All fractions were subjected to LC-MS to find presence of achyranthine (M+= 129,
Molecular weight is 129 g/mol).
Following experimental conditions were used for LC-MS:
A 10 ul aliquot of the sample was introduced into a LC-MS instrument with a split
ratio 4:1 to a photo diode array detector and 250 ul/min to MS. The flow rate was 1.0
ml/min. The dry gas temperature was 200 C. The mass spectrometer was run in
positive electron spray ionization (ESI) mode with mass/charge (m/z) ratio in the
range of 0-2000 m/z.
For LC-MS studies, LC profiles of each extracts and mass spectra of major peaks in
each extract are given in figures 5.8-5.40.

01<MM-1>LCMS*_61_01_62548 d: UV Chromatogram. 254 nm

400 125
11 12.1- 207
200
21.1
. *^AJM •
0

Figure 5.8: LC profile of LC-MS analysis of fraction MM1

(DeveCopment and VaCidation offPPLC Methodsfor Marker compounds and (Bioactive as per ICK
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 Chapter 5: (Development and validation ofJfPLC method for 6etainefrom Jlchyranthes aspera Linn.

Intens •MS 27min#121

x105
6

118

2-

75 3
191 273 f 494 600
n. • ••' ''

Figure 5.9: Mass spectrum of the peak eluting at 2.7 min of fraction MM1

0* •MS. 12.0min#544

4
579
3

1- 7:J7 107 0 1133

433 8 821 QQR
7
.5 137 280 ? 3 9
1 487 545 .fl 689 871 1346
, i 1 . i . . 11 . ^ HI, I I
n
200 400 600 800 1000 1200 m/z
Figure 5.10: Mass spectrum of the peak eluting at 12.0 min of fraction MM1

Intens •MS. 125min#564

x105'

2.0

579
15-

1.0-

648
0.5
75 433 921 1165
209 330 I I j 7
96751799 , 9960 1267
1 0 3 1 1092
1364
^ • - i ^ l " I - -1

Figure 5.11: Mass spectrum of the peak eluting at 12.5 min of fraction MM1

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 Chapter 5: (Development and validation ofWPLC method for betainefrom Jichyranthes aspera Linn.

ttP- •MS. 15.8min#716

3-

724

1-
433
579
75 5
\T\. 235 329 387 ]° LL. 658 790 850 1007 1093 1209
0 • < • ) * • — - ~ 4 * —J— ^— r-
200 400 600 800 1000 1200 nvi
Figure 5.12: Mass spectrum of the peak eluting at 15.8 min of fraction MM1

Intens. +MS. 20.7min#941

xlO5

10

329
0.8- 439
639
0.6-

0.4
75
149
1252
0 2i 249 385 7
I 495 5f6 ^ . ft 841 899
EiiUMJL.il. 1181

Figure 5.13: Mass spectrum of the peak eluting at 20.7 min of fraction MM1

Yfi •MS 21 1 min #961

2.0

1 5
439

10-
329

6C19
05-
7 r 121 205 249 . 383 1 543583 700 753 810 868 963 1068
no .li V Lihllf. J »JLi.[.. L • • '

200 400 600 800 1000 1200 rrvz

Figure 5.14: Mass spectrum of the peak eluting at 21.1 min of fraction MM1

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 Chapter 5: (Development and validation ofJdPLC method for Setaine from JZchyranthes aspera Linn.

02|CM-1}LCMS* 62 0162549d: UV Chromatogram. 254 nm

12.1- "12.5 _U9
60-
15.8
28 23.3
40

20-

L \Jt*uJ^ V\
Figure 5.15: LC profile of LC-MS analysis of fraction CM1

tens •MS Z8min#123

x106
15

118
1.0

05-

. 75 273 329
fin-

Figure 5.16: Mass spectrum of the peak eluting at 2.8 min of fraction CM1

M •MS, 121min#540

57S
390

•HI-

700 1072

1165
1331
I . ...I II . i . , ,1 I
200 400 600 800 1000 1200 m.z

Figure 5.17: Mass spectrum of the peak eluting at 12.1 min of fraction CM1

(Development and'Validation ofHTLCMethods for9Aarker compounds and(Rioactive asperlCH

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 Chapter 5: (Development and validation ofJt&LC method for betainefrom JZchyranthes aspera Linn.

Inters •MS. 12.5min#560

xlO5
1.5

10
579

648 798 1333

0.5
825 1084
75 610 1254
1023
329 433 111160
e
97 1 « , 246 I. 383 I L
ill T I i *" T ,
Figure 5.18: Mass spectrum of the peak eluting at 12.5 min of fraction CM1

+MS. 14.9min#672

786

329
75
2

cm 1012
6 7
149 2)9 279 ^427 579 | 724 , 1058
1320

200 400 600 800 1000 1200 nVz

Figure 5.19: Mass spectrum of the peak eluting at 14.9 min of fraction CM1

it ens •MS. 15.8min#709

x105

08
724

06-

0.4-

329
02-. 857 1059
75 433
579
i 649
1217 1322
_ | W.-k.luil -• . U J L UII. LIIIi
II, • III j j j ^ L'-hUlJ... ' • ' • - • • • ' •

Figure 5.20: Mass spectrum of the peak eluting at 15.8 min of fraction CM1

(Development and Validation offPPLC Methodsfor Marker compounds and(Bioactive as per ICK
guidelines 46
 Chapter 5: (DeveCopment and vaddation ofl{(PLC methodfor 6etainefrom Jichyranthes aspera Linn.

250 500 750 1000 1250 1500 1750 2000 m/Z

Figure 5.21: Mass spectrum of the peak eluting at 23.3 min of fraction CM1

05(WM-2)LCMS*_65_01_62552.d: UV Chromatogram. 254 nm

12.0
200 125 152

23.2

too HO 157
28

JLJJL—• i i i T 1 T J - T I I ^f^^™ ^ ^ - ^ * ^ ^ ^ T I I ' ' I

5 10 15 20 25 30 Time [min
Figure 5.22: LC profile of LC-MS analysis of fraction WM2

Mere.. +MS 28min#121

xlO6'
1.25- li M
100:

0.75:

050

025:
34184566 _
nmi
Figure 5.23: Mass spectrum of the peak eluting at 2.8 min of fraction WM2

(DeveCopment and'VaCidation ofJftPLC'Methodsfor'Markercompounds and ftioactive asperICK

guideCines 47
 Chapter 5: <Deve[opment and variation ofJd'PLC methodfor Setaine from Jlchyranthes aspera Linn.

• l U a ^ A d l
•MS 110min #490

;
4::

176.7
1045.5
6705
32? 9 854.3
13523

LMmm 250 500 750 1000

i l)i | . > , , ,11,11

1250 1500
16027

1750 2000 m/2

Figure 5.24: M a s s spectrum of the peak eluting at 11.0 min of fraction W M 2

Figure 5.25: Mass spectrum of the peak eluting at 12.0 min of fraction W M 2

x10^ •MS.IZ5min#562

125: 4449

100: 1169.7
0.75
6302
0.50
3027
0.25 795 2 907.6
1689.3 2016.4 -
000 i—;—i—i—i—"i—i—i—i—i—i—r-i—i—:—i—r
250 500 750 1000
1000 1250 1500 1750 2000 mi
Figure 5.26: Mass spectrum of the peak eluting at 12.5 min of fraction WM2

(Dex>elopment and'Vafidation ofJfVLC Methodsfor Marker compounds and<Bioactive as per ICK

CjuideCines 48
 Chapter 5: (Development and validation ofJtfPLC method for betainefrom Jichyranthes aspera Linn.

Intens., +MS. 15.2min#682

xlO 5
724.5
61

2-
292.8 1046.1
146.9 ,| 432.6 628.7 1213.7
.*_L it^.
Figure 5.27: Mass spectrum of the peak eluting at 15.2 min of fraction WM2

^^^^|A^^M^tf^^^^MAriB^MdhrfM|^Mjft|^^te
x1# +MS.15.8min#709

1.25
724.5
1.00

0.75}

0.50
578.6
0.25} 176.8 308.9 432.7
308.9 ***•' I
^L,.,9?',4 , 12592 1455.9
250 500 750 1000 1250 1500 1750 2000 ml
Figure 5.28: Mass spectrum of the peak eluting at 15.8 min of fraction WM2

Intens. • M S 23 2 m i n * 1 0 4 9
x10 5

6390

L
^ - 4 ^ ! •••_.'

250
)• !•" M i'
500
^1 • l | •' *
750
l< ii
1065 2
1250 1750 2000

Figure 5.29: Mass spectrum of the peak eluting at 23.2 min of fraction WM2

(Development and Validation ofWPLC Methodsfor Marker compounds andftioactive as per 1CK
guidelines 49
 Chapter 5: (DeveCopment and validation ofJ{<PLC method for 6etainefromJlchyranthes aspera Linn.

u
04AM-2,iLCMS+_64_0!_6255l d: UV Chromatogram, 254 nm
|mAU)

300

28 12.0
200 125 197

100

A hL^»j>
00 5 10 15 20
20 25 30 Time (min
Figure 5.30: LC profile of LC-MS analysis of fraction AM2

Mens.. +MS.28min#121
xlO 6
1.25:
118
1.00-

0.75:

0.50-

0.25-
273 543
000- m—n—i—i—1.» I I

Figure 5.31: Mass spectrum of the peak eluting at 2.8 min of fraction AM2

m +MS 12. Imin #543

2.0 579

1.5

1.0-

0.5- 1093
351 433 ^ 964
75 205 1322 1S&S
00 U jlkmtiT, ili,i ii U , - J' i i, • , I , •
25C 500 750 1000 1250 1500 1750 2000 mfe
Figure 5.32: Mass spectrum of the peak eluting at 12.1 min of fraction AM2

(DeveCopment and Validation ofHVLCMethods forMarker compounds and(Bioactive asperICK

guidelines 50
 Chapter 5: (Development and validation oftCPLC method for Setainefromjlchyranthes aspera Linn.

Irttens. +MS.12.5min#562
x105

25' 579

20

15

10

433 672 792 Q4P

0.54 11Qfl
75,49
nn
ill
Figure 5.33: Mass spectrum of the peak eluting at 12.5 min of fraction AM2

+MS 19.7min#892
54
329
4
639
:-

2 75
439
149 1226
H 203 522 764839 1420
o mm !

250
y,
500
^ I M I , in ,•,,„„),,
750 1000
1055

1250
i,
1500
1561
1750 2000 m/2

Figure 5.34: Mass spectrum of the peak eluting at 19.7 min of fraction AM2

03(CM-2)LCMS+_63_01_62550 d: UV Chromatogram 254 nm

125 23.3
12.1
3D 158
-2.8
22

10

0 J- WuJuW wu^/kv.
r
•10 T T " T- T T T * * ~~I f'"' ' T' T' T ' T I t ! p — r — T ™ T'" T T' T * I » p.».T- T> >, „r, , j .

0 5 10 15 20 25 30 Time [min]
Figure 5.35: LC profile of LC-MS analysis fraction CM2

(Development and Validation ofJf<PLC Methodsfor Marker compounds and<3ioactive asperICK

51
guidelines 7 0 9 ^
 Chapters: (Development and'validation ofJtfPLC method for Setainefrom JAchyranthes aspera Linn.

Intens., +MS.28min#121
X105-

18
3-

:-

1-
329 ACI
73 191 1 4
J 7 570 803 —
•;v

Figure 5.36: Mass spectrum of the peak eluting at 2.8 min of fraction CM2

lift •MS. 121 min #535

5
-??

579
1355
1010.
1093

1264
1481 1592
u.M»..ii< ,i, ; r , '—l 1 1 1 1 1 r 1
—f
250 500 750 1000 1250 1500 1750 2000 mlz
Figure 5.37: Mass spectrum of the peak eluting at 12.1 min of fraction CM2

Figure 5.38: Mass spectrum of the peak eluting at 12.5 min of fraction CM2

(Development and'Validation ofJftPLC'Methods forMarker compounds and(Bioactive as perICK

guidelines 52
 Chapter 5: (Development and validation of'K'PLC method for betainefrom Jlchyranthes aspera Linn.

xlO* +MS.15.8min#704

3
1084

2-

628 802 1182

75 179 329

952 1319
1669
7—1 1 r^ 1 r-
250 500 750 1000 1250 1500
1500 1750
1750 2000 miz

Figure 5.39: Mass spectrum of the peak eluting at 15.8 min of fraction CM2

Figure 5.40: Mass spectrum of the peak eluting at 23.2 min of fraction CM2

In all mass spectra, there was no peak having m/z value 129 (M+) or 130 (M++H)
corresponding to molecular weight of achyranthine. All fractions revealed the
presence of a compound eluting at mean retention time of 2.80 min having m/z 118
which is corresponding to betaine (molecular weight 117 g/mol). Betaine was present
in all the fractions, but achyranthine was found to be absent. To evaluate reason
behind this, a synthetic form of achyranthine (1-methyl pyrrolidine-3-carboxylic acid)
was purchased from Alinda Chemical Ltd., Russia. Synthetic achyranthine was
semisolid yellowish compound which was highly hygroscopic. Attempts were made
to develop HPLC method using synthetic achyranthine but the compound was not
sufficiently stable to develop the method. Synthetic achyranthine was found to be
soluble in water and methanol. Various combinations water with methanol or
acetonitrile were tried as mobile phase but none of these was optimum to carry out the
analysis. HPLC chromatograms showed the presence of many peaks when solution of

(Development and Validation ofJfPLC Methodsfor Marker compounds and<Bioactive as per ICH
guidelines S3
 Chapter 5: (Development and validation of!K<PLC method for betainefrom jlchyranthes aspera Linn.

synthetic achyranthine was injected. Thus we could conclude that the molecule was
highly unstable to isolate and to develop HPLC method. Hence, another alkaloid
present in Achyranthes aspera that is betaine was isolated from dried seeds.
5.2.6 Extraction of alkaloids-Betaine
General method for extraction of alkaloids was carried out.71
20 g of powdered seeds were defatted with 200 ml of pet ether (60°-80°C) and then
extracted with 200 ml of acidified ethanol (pH 4 was made by HC1) by heating at 70
°C for 5 h. Acidified solution was filtered and the marc was discarded. To this
alcoholic solution ammonia was added (25 % Exrapure, specific gravity 0.91) till pH
10 to get the precipitate of alkaloids. The precipitates of the crude alkaloids were
filtered and dried (0.0276 g).
5.2.7 Isolation of betaine from Achyranthes aspera
The precipitates of crude alkaloids thus obtained were sonicated with 5 X 50 ml of
distilled water for 20 min. Betaine being water soluble gets extracted in water.
Aqueous layer showed some coloured impurities which were removed by passing the
solution through charcoal column. The charcoal column was washed with 5 X 25 ml
of distilled water. The clear water extracts were pooled and evaporated to dryness to
yield semipuified alkaloid (0.0197 g). Further purification was carried out by
preparative TLC followed by repeated recrystallization using methanol.
5.2.8 Purification of betaine: Semipurified betaine was further purified by
preparative TLC and recrystallization. Following chromatographic conditions and
procedure were used for preparative TLC:
Stationary phase: Silica gel GF254
Mobile phase: Methanol: chloroform (7:3, v/v)
Chamber Saturation Time: 20 min
Development Technique: Ascending development
Sample: Semipurified betaine in methanol
Detection Wavelength: White light
Procedure: Semipurified betaine was dissolved in minimum amount of methanol and
spotted in the form of band on silica plate. The plate was developed using mobile
phase, methanol: chloroform (7:3, v/v). After development the plate was dried, band
at Rf 0.82 corresponding to betaine was scrapped, dissolved in methanol and filtered
through Wattmann filter paper. The procedure was repeated using several silica plates

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 Chapter5: (Development and validation ofWPLC methodfor Betainefrom Jichyranthes aspera Linn.

and the resulting methanol solution was evaporated to dryness (0.0146 g). The
resulted betaine residue was further purified by recrystallization with methanol. For
recrysatllization, various solvents were tried such as water, methanol and absolute
alcohol. The crystals obtained during recrystallization were checked for the yield,
size, shape and melting point. Finally methanol was selected as a solvent for
recrystallization. The yield of purified betaine was 0.0110 g (0.055 % w/w).
5.2.9 TLC studies of isolated betaine
For TLC studies of isolated betaine, several mobile phases were tried and following
mobile phase was selected:
Stationary Phase: Precoated plates of Silica gelGF254 (E. Merck)
Mobile phase: Methanol: Chloroform (7:3, v/v)
Chamber Saturation Time: 15 min
Development Technique: Ascending development
Preparation of sample: Betaine dissolved in methanol
Procedure: Sample was spotted on silica plate with the help of capillary to get narrow
spot. The chamber was saturated with the mobile phase for 15 min and the plate was
developed. After development, the plate was observed under short (254 nm) and long
(366 nm) wavelength. Then the plate was sprayed with Dragendorff s reagent.
Dragendorff s reagent is specific for the detection of alkaloids. Presence of alkalois
was indicated by orange coloured band.
TLC studies of isolated compound showed presence of a well isolated orange band at
Rf of 0.82 indicating the presence of alkaloid (Figure 5. 41).

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 Chapter5: (Development andvalidation ofHPLC methodfor betainefrom Jlchyranthes aspera Linn.

"*" Orange coloured band

Figure 5.41: TLC profile of isolated betaine

5.2.10 Spectral studies of isolated betaine

Before carrying out spectral studies, purity of isolated betaine was checked by HPLC.
The purity of isolated betaine was found to be 97 %. Characterization of isolated
betaine was carried out by spectral studies such as IR, NMR, mass spectroscopy and
spectra were obtained (Figures 5.42, 5.43 and 5.44, respectively).

(Development and Vatidation of'JfPLC Methodsfor Marker compounds and (Bioactive asperICK
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 Chapter5: (Development andvalidation offflPLC method for 6etainefrom JLchyranthes aspera Linn.

l*»<5. &

Figure 5.42: IR spectrum of isolated betaine

Table 5.1: Interpretation of IR spectrum of isolated betaine

Peak No. Wave number (cm"1) Inference

4,5 2980.29, 2947.5 C-H Stretching

7 1624.21 Carboxylate anion Stretching

13 1238.41 C-N Vibration

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 Chapter 5: (Development andvalidation of9f<PLC methodfor Betainefrom Achyranthes aspera Linn.

9 * * ^ y •» - ,
V -• 2 2 T T —

1 \W

i
1 L

Figure 5.43: NMR spectrum of isolated betaine

Table 5.2: Interpretation of NMR spectrum of isolated betaine

Chemical shift
Proton assignment Inference
(5 Value) Reported Value72

3a,3b,3c 3.27 (s, 9 H) Nine methyl protons 3.26 (9 H)

2 3.80 (s, 2H) Methylene protons 3.90 (2H)

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 Chapter 5: (Development and validation ofjf'PLC method for betainefrom fichyranthes aspera Linn.

•MS02-03mh»ilO-i$l

t8

235
»/
140
374
74 96
U—r-L, , .
'if
'i • •' • • ' i • ' r
50 100 150 200 290 303 360 400 nv:

Figure 5.44: Mass spectrum of isolated betaine

Table 5.3: Interpretation of mass spectrum of isolated betaine

Sr. No. m/z values Inference

1. 140 [M+Na]+ peak

2. 118 [M+H]+peak

3. 74 [M+H-COO]+peak

In IR spectroscopy, peaks at wave numbers of 1624.21 cm"1 and 1238.41 cm"1

confirmed the presence of COO" group and C-N stretching, respectively. Proton NMR
confirmed presence of nine equivalent methyl protons at 8 value of 3.27 and two
methylene protons at 5 value of 3.80. Mass spectrum confirmed the presence of M+H
peak at m/z value of 118 confirming the molecular weight (molecular weight of
betaine : 117 gm/mole) of betaine. Based on spectral studies, it was confirmed that the
isolated compound was betaine. Thus an alkaloid, betaine was successfully isolated
from dried seeds of Achyranthes aspera.

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guidelines 59
 Chapter5: (Development and validation qffflPLC methodfor 6etainefrom Jichyranthes aspera Linn.

5.3 DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR

BETAINE
5.3.1 HPLC method development for betaine
Betaine was found to be soluble in water, methanol and combination of water and
acetonitrile hence reversed phase HPLC was the method of choice. Detection
wavelength was selected based on Xmax of compound. To determine X^ax of betaine,
UV spectrum of 10 ug/ml of standard betaine was obtained. It showed absorbance
maximum at 205 nm (figure 5.45). Therefore, all chromatograms were recorded at
205 nm. Column selected was C18 from Waters (300 mm X 3.9 mm, 5 urn). Analysis
was performed at ambient temperature and injection volume was 20 ul. Solvents such
as methanol or acetonitrile were tried in various combinations with water as mobile
phase and effects of these combinations on retention time, the number of theoretical
plates, resolution and peak symmetry were observed. When methanol was used in
combination with water, change in mobile phase combination did not significantly
affect retention time of betaine. At 205 nm, the interference of blank with peak of
interest was observed, therefore it was decided to carry out analysis with acetonitrile
in combination with water. Various combinations of acetonitrile and water were tried
such as acetonitrile: water (75:25, 50: 50, 25:75 and 10:90, v/v). Mobile phase with
higher percentage of acetonitrile produced peak whose symmetry was less than 0.5.
As percentage of acetonitrile decreased the value of peak symmetry increased.
Acetonitrile: water in ratio of 10: 90 gave peak symmetry of 0.6, better peak shape
and optimum retention of betaine than other mobile phase combinations, hence
selected for analysis.

Thus the following chromatographic conditions which gave optimum retention time
and peak symmetry were selected.
Column: Ci8 (Waters)
Column Dimensions: 300 mm X 3.9 mm, 5 (im
Mobile Phase: Acetonitrile: Water (10:90, v/v)
Flow rate: 1.0 ml/min
Detection Wavelength: 205 nm
Column Oven Temperature: Room Temperature
Injection Volume: 20 ul

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 Chapter 5: (Development and validation ofjfPLC method for Setaine from Jlchyranthes aspera Linn.

Figure 5.45: UV spectrum of betaine

Figure 5.46 and figure 5.47 represent HPLC chromatograms for standard betaine and
purified extract, respectively. Betaine eluted with mean retention time of 2.793 min.

•o J

T I *

Figure 5.46: HPLC chromatogram of standard betaine

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Quidefines 61
 Chapter 5: (Development and validation ofJ{(PLC method for 6etaine from Achyranthes aspera Linn.

J- s
4"

i-

:-

ftV
i^-.. J
.»-

•»•

i
<> as '» i 2» } n 4 43 -,

Figure 5.47: HPLC chromatogram of isolated betaine from Achyranthes aspera

27,28
5.3.2 Method Validation
The developed method was validated for various parameters such as linearity, limit of
detection (LOD), limit of quantitation (LOQ), accuracy, precision, robustness and
system suitability as per ICH guidelines.
5.3.2.1 Preparation of standard solution
A stock solution of 1000 ug/ml was prepared by dissolving 100 mg of standard
betaine in 100 ml of mobile phase by sonication for 10 min. From this stock solution
working standard of 100 ug/ml was prepared by diluting 10 ml of stock solution upto
100 ml with mobile phase.
5.3.2.2 Preparation of sample solution
Isolated betaine (10 mg) was dissolved in 100 ml of mobile phase by sonication for 10
min to prepare a stock solution of 100 ug/ml. From this, various aliquots were taken
and diluted with appropriate volume of mobile phase to produce different
concentrations which were used to validate the method.
5.3.2.3 Specificity
To assess specificity, two injections of blank (mobile phase), six individual injections
of standard solution (40 ug/ml) and two injections of sample solution (40 ug/ml) were

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 Chapter 5: (Development and validation ofjfPLC method for Betainefrom Achyranthes aspera Linn.

applied before all measurements and any interference from blank or sample was
checked. No interference was observed indicating that the developed method was
specific.
5.3.2.4 System suitability
To assess system suitability, six individual injections of standard solution (40 ug/ml)
and two injections of sample solution (40 ug/ml) were performed before all
measurements and system suitability parameters such as resolution, theoretical plates,
asymmetry and repeatability of the peak area were evaluated. Table 5.4 shows results
of system suitability test of the developed method.

Table 5.4: Results of system suitability parameters for betaine

Peak Number of
Concentration Retention time
Area asymmetry theoretical
(Ug/ml) (Rt) in min
plates (N)
40 2.809 35.5025 0.66 2545
40 2.805 35.7123 0.64 2668
40 2.837 35.2615 0.72 2357
40 2.834 34.9359 0.77 2298
40 2.829 35.8338 0.73 2238
40 2.818 35.4796 0.68 2327
Sample 2.819 35.7981 0.71 2319
Sample 2.820 35.2936 0.74 2350
% RSD 0.9090 %

Number of theoretical plates (N) were more than 2000, resolution (Rs) was more than
2.0, peak asymmetry was less than 2.0 and % relative standard deviation (% RSD)
obtained for six injections of standard solution was less than 1.0 indicating that the
method and instrument were suitable for carrying out analysis.
5.3.2.5 LOD and LOQ: The LOD and LOQ were calculated based on signal to noise
ratio method. Betaine solutions in increasing concentrations were injected until signal
to noise ratio of 3.0 and 10.0 were obtained for determination of LOD and LOQ,
respectively. LOD and LOQ were found to be 5 ug/ml and 15 ug/ml, respectively.

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 Chapter 5: (Development and validation qfJfPLC method for Setaine from Jichyranthes aspera Linn.

5.3.2.6 Linearity and range

To determine the linear relationship, appropriate aliquots of betaine stock solution
were taken in 10 ml volumetric flasks and diluted up to mark with mobile phase to
obtain final concentrations of 15, 20, 30, 40, 50, 60 and 70 ug/ml. Duplicate
injections using 20 ul loop were applied and chromatograms were recorded at 205
nm. Quantitation was carried out by keeping peak area and concentrations of
compound to straight line equation and correlation coefficient (r2) was determined.
The developed method was found to be linear over the range 15-70 ug/ml with
correlation coefficient (r2) of 0.9998 (Table 5.5) (Figure 5.48).

Table 5.5: Results of linearity studies for betaine

Concentration Retention Time Area Average area

(Ug/ml) (min)
15 2.865 12.3740
12.5892
15 2.863 12.8045
20 2.801 17.4799
17.6764
20 2.809 17.8729
30 2.804 26.8622
26.5861
30 2.805 26.3101
40 2.800 35.1101
35.1367
40 2.810 35.1633
50 2.803 44.5941
44.7342
50 2.824 44.8743
60 2.796 53.9291
53.5883
60 2.804 53.2475
70 2.791 61.8752
63.2177
70 2.800 64.5603

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 Chapter 5: (Development and validation of9d<PLC method for Setaine from Achyranthes aspera Linn.

70
y = 0.912x-0.927
60 R2 = 0.999

SO

A
40
r
e 30 • Seriesl
a Linear (Seriesl)
20

10

0
20 40 60 80
Concentration (ug/ml)

Figure 5.48: Graph of linearity studies for betaine

5.3.2.7 Accuracy: The accuracy of the developed method was evaluated through the
analyte recovery test at three concentration levels. Known amount of sample was
spiked with 32, 40 and 48 (ig/ml of the standard solutions of betaine and % recovery
was calculated by following formula:
% Recovery= Measured value /True value X 100.
The mean recoveries were found be 108.35 %, 107.79 % and 107.28 % at 80 %, 100
% and 120 % level, respectively (Table 5.6).

Table 5.6: Results of accuracy studies for betaine

Amount Present3 Amount Found3

Component % Recoverya± SD
(ug/ml) ±SD (ug/ml)± SD
30.8653±0.26 33.4434±0.15 108.3591±1.22%
Betaine 38.6962±0.24 41.7096±0.12 107.7913± 0.97%
46.2987±0.87 49.6580±0.08 107.2807 ± 1.99%
a
n=3, triplicate injections

The recoveries were found to be between 107.28-108.35 % , which is largely within

the 90-110 % range that is considered acceptable. So the developed method was
found to be accurate.
5.3.2.8 Precision: Repeatability (intraday) and intermediate precision (interday) were
determined through analysis (triplicate) of the samples at three levels that is 50 %,

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 Chapter5: (Development andvalidation qfMPLC method for Setainefrom Jichyranthes aspera Linn.

100 % and 150 % of test concentrations and % relative standard deviation (% RSD)
was determined.
Values of % RSD obtained during precision studies at all level were less than 2.0, it
indicates that the proposed method was precise (Table 5.7).
Table 5.7: Results of intraday and interday precision studies for betaine

Amount level
Component Intraday (% RSD)a Interday (% RSD)a
(ug/ml)
Dayl Dayl Day 2
20 1.5025 0.3534 0.3361
Betaine
40 1.0352 0.7178 1.1830
60 0.4787 0.4327 0.3288
n=3, triplicate injections

5.3.2.9 Robustness: Robustness was evaluated by deliberately changing the method

parameters and their effect on peak area and retention time was observed. Only one
parameter was altered at a time keeping the other parameters constant. The robustness
of the method, related to the variation in retention time, area, and % w/w of betaine in
the sample was evaluated by changing the mobile phase flow rate (0.9, 1.0 and 1.1 ml/
min) and mobile phase composition (acetonitrile: water, 10.5: 89.5, v/v and
acetonitrile: water, 9.5: 90.5, v/v). For each condition, six injections of the standard
solution (40 ug/ml) and two injections of sample (40 ug/ml) were applied.

Table 5.8: Results of robustness study for betaine

% w/w
Mean % RSD
Mean of
Parameters Retention time of
area betaine
(min) area
Mobile phase composition (v/v)
Acetonitrile: Water (10.5:89.5) 2.806 32.9589 1.3635 99.7516
Acetonitrile: Water (10:90) 2.813 34.3811 1.1744 97.9837
Acetonitrile: Water (9.5:90.5) 2.826 33.4416 0.7197 99.1373
Flow rate (ml/min)
0.9 3.127 35.2452 1.0095 98.6233
1.0 2.813 34.3811 1.1744 97.9837
1.1 2.552 27.7383 1.0743 99.3955
a
n=6, six injections

The robustness was estimated using the overall mean, standard deviation and % RSD
for each variable. % RSD was lower than 2.0 for the variables such as mobile phase

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 Chapter5: (Development and validation ofJUPLC method for betainefrom Achyranthes aspera Linn.

composition and flow rate. The present method was found to be robust for the % w/w
of betaine present in the sample (Table 5.8).
5.3.2.10 Stability studies
Stability of the sample solutions was tested after 24, 48 and 72 h after preparation and
storage at 4.0 °C and 25.0 °C separately. Stability was assessed by comparing the
chromatographic parameters of the solutions after storage with the same
characteristics of freshly prepared solutions.
Stability of betaine in the sample solutions was evaluated at 4.0 °C and 25.0 °C for 3
days to verify whether spontaneous degradation occurred. The results were calculated
as the percentage of non-degraded betaine at the specified time intervals. The samples
showed less than 2 % degradation indicating that the samples were stable at 4.0 °C
and 25.0 °C for 3 days (Table 5.9).

Table 5.9: Stability studies of betaine in extract containing Achyranthes aspera

Temperature
Extract 4°C 25 UC
24 h 48 h 72 h 24 h 48 h 72 h
% of betaine in
extract of
99.61 99.43 98.95 99.51 98.70 98.05
Achyranthes
aspera

5.4 QUANTITATIVE ANALYSIS OF EXTRACTS AND FORMULATIONS

CONTAINING ACHYRANTHES ASPERA FOR BETAINE CONTENT
The developed and validated method was used for quantitative determination of
betaine in three extracts and different brands of marketed Ayurvedic tablets
containing Achyranthes aspera.
Different extracts were prepared from seeds of Achyranthes aspera procured from
three different geographical regions of India (Gujarat, Tamilnadu and Maharashtra) as
per section 5.2.6. Marketed tablets containing Achyranthes aspera are polyherbal
formulations mainly used for treatment of kidney stones and other urinary tract
problems.

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 Chapter 5: (Development and validation of^PPLC method for Setainefrom Achyranthes aspera Linn.

To determine the content of betaine present in these tablets, samples were prepared as
given below:
5.4.1 Preparation of sample solutions for analysis of marketed formulations
5.4.1.1 Tablets (Brand I): Twenty tablets were individually weighed; their mean
weight was determined and the tablets were triturated. Accurately weighed 20 g of
tablet triturate was transferred to 250 ml volumetric flask containing 200 ml of
acidified ethanol and extracted. The resulting ethanol extract was treated in similar
manner as discussed in section 5.2.6. The resulting precipitates (1.245 g) were
dissolved in 5 ml of mobile phase, sonicated for 10 min. Accurately measured 0.02 ml
of above solution was further diluted to 10 ml with mobile phase, sonicated, filtered
and injected in HPLC.
5.4.1.2 Tablets (Brand II): Twenty tablets were individually weighed; their mean
weight was determined and the tablets were triturated. Accurately weighed 20 g of
tablet triturate was transferred to 250 ml volumetric flask containing 200 ml of
acidified ethanol and extracted. The resulting ethanol extract was treated in similar
manner as discussed in section 5.2.6. The resulting precipitates (1.156 g) were
dissolved in 5 ml of mobile phase, sonicated for 10 min. Accurately measured 0.02 ml
of above solution was further diluted to 10 ml with mobile phase, sonicated, filtered
and injected in HPLC.
5.4.1.3 Tablets (Brand III): Twenty tablets were individually weighed; their mean
weight was determined and the tablets were triturated. Accurately weighed 20 g of
tablet triturate was transferred to 250 ml volumetric flask containing 200 ml of
acidified ethanol and extracted. The resulting ethanol extract was treated in similar
manner as discussed in section 5.2.6. The resulting precipitates (1.032 g) were
dissolved in 5 ml of mobile phase, sonicated for 10 min. Accurately measured 0.02 ml
of above solution was further diluted to 10 ml with mobile phase, sonicated, filtered
and injected in HPLC.

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 Chapter 5: (Development and validation ofJtfPLC method for betainefrom Achyranthes aspera Linn.

Table 5.10: Percent content of betaine in extracts and formulations containing

Achyranthes aspera

Extracts /Formulations %w/w of betaine8 ±SD %mg of betaine8 ±SD

Extract I (Gujarat) 0.0801±0.01 -
Extract II (Tamilnadu) 0.0688±0.00 -
Extract III (Maharashtra) 0.0566±0.00 -
Tablet (Brand -I) 6.3805±0.82 1.9200±0.24/Tablet
Tablet (Brand-II) 5.8078±0.49 2.1743±0.18/Tablet
Tablet (Brand -III) 18.3382±0.99 5.4627±0.29/Tablet
n=3, triplicate injections

% w/w of betaine in three extracts and three different brands of tablets were
calculated. % w/w of betaine was found to be slightly higher in Extract I (sample
procured from Gujarat). In case of analysis of formulations, brand-Ill of tablets
contained higher amount of betaine (Table 5.10).
A simple, accurate and convenient HPLC method was developed using betaine and
validated as per ICH guidelines. The content of betaine was estimated in three extracts
and three marketed formulations containing Achyranthes aspera. This simple,
accurate and precise validated method can be very well used to determine batch to
batch variations and routine analysis of formulations containing Achyranthes aspera
by herbal manufacturers.

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