REMOVEMARK GEL METHOD OF ANALYSIS - Allium

cepae extract

Allium cepae extract In-House

DESCRIPTION
Allium cepae extract is a reddish brown liquid and has a characteristic odor.
IDENTIFICATION
[Criteria]Identification by TLC
[Method]
1) Amino Acid in Allium cepae extract (1.8 in 1) : use Allium cepae extract as test solution.
Separately, use Allium cepae extract standard (1.8 in 1) as standard solution.
Spot these solutions on a plate of silica gel with a fluorescent indicator for thin layer
chromatography. Develop the plate with a mixture of ethanol 25% ammonia Water (70 :
10 : 20) to a distance of about 10 cm and air-dry the plate. Weigh 0.3 g of ninhydrin, add 90
mL of butanol and 10 mL of glacial acetic acid, spry this solution, and heat at 105C for 10
minutes: the principal spot obtained from the test solution and the spot obtained
from the standard solution have the same Rf value.
2) Flavonoid in Allium cepae extract (1.8 in 1) : use Allium cepae extract as test solution.
Separately, dissolve 10.0mg of Hyperoside and 10.0mg of Rutoside in methanol to
make 25 mL and use this solution as standard solution. Spot these solutions on a plate
of silica gel with a fluorescent indicator for thinlayer chromatography. Develop the
plate with a mixture of ethyl acetate formic acid water (80 : 10 : 10) to a distance
of about 10 cm and air-dry the plate. Spray evenly 1.1% diphenylboryloxyethylamine
TS and then, a 2.5% solution of polyethylene glycol 400 in methanol. Examine
under ultraviolet light (main wavelength: 365 nm): the principal spot obtained from
the test solution and the spot obtained from the standard solution have the same Rf
value.
3) The retention time of main peak of the test solution and the standard solution for
Assay is the same.
pH

[Criteria] 5.2 7.2 (20C)

SPECIFIC GRAVITY 1.000 1.100 (20C)
RESIDUE ON EVAPORATION - 7.0-21.0% (105C, 4 hours)
ASH Not more than 2.0% (2.0g)
CONTENT OF ETHANOL

[Criteria] 13.0 17.0% (v/v).

Gas chromatography
This is a method to determine the Alcohol Number by determining ethanol (C2H5OH) content
(vol %)from a sample measured at 15 C by the following procedures.
(1) Reagent Dehydrated ethanol for Alcohol Number: Dehydrated ethanol with determined
ethanol(C2H5OH) content. The relation between specific gravity,d 1'15 of dehydrated
ethanol
,

and ethanol (C2H5OH)content is 0.797: 99.46 vol %, 0.796: 99.66 vol %, and0.795 : 99.86
vol %.
(2) Preparation of test solution and standard solution: Test solution -Measure accurately a
volume of sample at 15 2 C equivalent to about 5mL of ethanol(C2H5OH), and
add water to make exactly 50mL.Measure accurately 25mL of this solution, add
exactlyl0mL of the internal standard solution, and add water to make 100mL.
Standard solution-Measure accurately 5mL of dehydrated ethanol for Alcohol Number at the
.

same temperatureas the sample, and add water to make exactly 50mL. Measure accurately 25mL of
this solution, addexactly 10mL of the internal standard solution, and addwater to make 100mL.
(3) Procedure: Place 25mL each of the test solutionand the standard solution in a 100-mL,
Narrowmouthed, cylindrical glass bottle sealed tightlywith a rubber closure and aluminum band,
immerse thebottle up to the neck in water, allowed to stand at roomtemperature for more than I
hour in a room with littlechange in temperature, shake gently so as not to splash the solution on the
closure, and allow to stand for 30minutes. Perform the test with ImL each of the gas inthe bottle
with a syringe according to the Gas Chromatographyunder the following conditions, and
calculatethe ratios, QT and QS , of the peak height of ethanol tothat of the internal standard.
Alcohol Number:
OT X 5 ml
Xethanol content of dehydrated ethanol for Alcohol number
Q, X a volume (ml) of a sample 9.406
Internal standard solution -A solution of acetonitrile (3 in 50).
Operating conditions
Detector: A hydrogen flame-ionization detector.
Column: A glass tube, about 3 mm in inside diameterand about 1.5 m in length, packed with 150
pmto180 !Am porous ethylvinyl benzene divinylbenzene copolymer for gas chromatography.
Column temperature: A constant temperature between l 05 C and 115 C.
Carrier gas: Nitrogen.
Flow rate: Adjust the flow rate so that the retentiontime of ethanol is 5 to 10 minutes. Selection of
column: Proceed with lmL of the gasobtained from the standard solution in the bottle underthe
above operating conditions, and calculate the resolution.
Use a column giving elution of ethanol and theinternal standard in this order with a resolution
betweentheir peaks being not less than 2.0.
HEAVY METALS

[Criteria] Test solution is less darker than comparative solution. (Not more than 300ppm)
[Method]
Wet combustion method: Place 2-5 g of the sampling material into a beaker and add 10-30 ml of
nitric acid. Cover it with watch glass and let it stand overnight. Place it on a heat plate and break it
down gradually by increasing the heat until it stops generating brown smoke. If it does not break
down, add 10 ml of nitric acid. Add 5-10 ml of hydrogen peroxide to dissolve it completely until it
turns light yellow or yellow. Repeat this process with 10-30 ml of nitric acid to prepare standard

test solution.
Measurement: It can
be made using test solution, standard solution and standard test solution by following the Atomic
Absorption Spectrometric method. Using the Atomic Absorption Spectrometric, dilute 1000 mg/L
of standard solutions for each heavy metal with 0.5 mol/L nitric acid to produce a calibration
curve. Correct it with standard test solution and measure optical density (or intensity). Inductively
coupled plasma spectrometer or Inductively coupled plasma-mass spectrometer can be used
instead of Atomic Absorption Spectrometry for measurement.
PESTICIDE RESIDUES
Equipment: Gas Chromatography system
Reagents and test solutions:
1.
2.
3.
4.
5.

Solvents: solvents for testing agrochemical residues or equivalent.

Water: distilled water or equivalent.
Florisil: A Cartridge of Florisil
Filter agents:
Undiliuted standard solutions: dissolve standard and specimen of each agrochemical into
100 ppm acetone.
6. Standard Solutions: Mix and dilute each undiluted standard solution with acetone to
appropriate level.
7. Other reagents: reagents for testing agrochemical residues or special grade reagents.
Preparation of test solution
1. Extraction: Reduce 500-600 g of specimen to fine powder. Mix 5 g of powder with water and
let stand for 4 hours (the amount can be adjusted if necessary). Add 90 ml of acetone to the
mixture; blend it for 5 minutes using a homogenizer; and then filter it under vacuum using a
vacuum pump, a conical flask and a Buchner funnel.
Transfer this solution into a 500 ml separating funnel and add 50 ml of a saturated solution of
sodium chloride and 100 ml of distilled water. Add 70 ml of dichloromethane to the mixture and
shake it vigorously; then allow for separation. Collect bottom layer in another separating funnel.
After adding 70 ml of dichloromethane to the aqueous layer bypassing anhydrous sodium sulphate
under vacuum and then dissolve it in 4 ml of hexane.
2.Purification: Pour 6 ml of hexane into a Florisil-charged cartridge and elute it after 2 minutes.
Pour 6 ml of hexane-acetone mixture into the cartridge and re-elute. Then pour the extracted
solution into the top end of the cartridge and slowly receive the elute after 2 minutes.
While the cartridge is still wet with the solvent, pour 5 ml of hexane-dichloromethaneacetone
mixture (50:48.5:1.5) for prochloraz and thifluzamid, hexane-acetone mixture (7:3) and collect the
elute. Concentrate the elute under vacuum in a water-bath at below 400 C and make a test solution
by dissolving it in 2 ml of acetone, hexane (1 0).
Conducting Test:
Measurement conditions for gas chromatography:
Electron capture detector (GC-ECD)
Column: A silica glass capillary column (inner diameter 0.25 mm and length 30 cm) coated with
5% methyl silicone (in use for gas chromatography) in 0.25 gm thickness and a silica glass

capillary column (inner diameter 0.25 mm and length 30 m) coated with 50% Phenyl and 50%
methyl silicone (in use for gas chromatography) in 0.25 m thickness, or equivalent.
Carrier gases and their quality: Nitrogen 1.0 ml/min
Column Temperature:
Inject the specimen into the column at 80 C. After 2 minutes, increase temperature by 10 c every
minute until the temperature reaches 280 C, and let it stand for 10 minutes.
Injector Temperature: 260 C, Split mode (10:1)
Detector Temperature: 280 C.
Nitrogen Phosphorous detector (GC-NPD)
Column: A silica glass capillary column (inner diameter 0.25 mm and length 30 cm) coated with
5% methyl silicone (in use for gas chromatography) in 0.25 p.m thickness and a silica glass
capillary column (inner diameter 0.25 mm and length 30 m) coated with 50% Phenyl and 50%
methyl silicone (in use for gas chromatography) in 0.25 m thickness, or equivalent.
Carrier gases and their quality: Nitrogen 1.0 ml/min
Column Temperature:
Inject the specimen into the column at 80 C. After 2 minutes, increase temperature by 10 c every
minute until the temperature reaches 280 C, and let it stand for 10 minutes.
Injector Temperature: 260 C, Split mode (10:1) Detector Temperature: 280 C.
Mass selective detector (GC-MSD)
Column: A silica glass capillary column (inner diameter 0.25 mm and length 30 cm) coated with
5% methyl silicone (in use for gas chromatography) in 0.25 um thickness or equivalent.
Carrier gases and their quality: Helium0.9 ml/min
Column Temperature:
Inject the specimen into the column at 100 0 C. After 2 minutes, increase temperature by 10 c
every minute until the temperature reaches 280 C, and let it stand for 15 minutes.
Injector Temperature: 260 C, Split mode (10:1) Detector Temperature: 280 C.
Mobile Phase flow rate: 1.0 ml/min.
Qualitative Test:
1. The retention time for each peak in the chromatogram of the chemicals under
study must correspond to the peak obtained in a standard solution, regardless of
the measurement conditions.
2. Using a GC-MSD detector, components of each agrochemical can be identified by
retention time and mass spectrum.
3. Quantitative Test: Evaluations are based on peak heights and peak areas, obtained
under the same conditions as in the qualitative test.
Equipment: HPLC (UV- Detector)
Reagents and Test Solutions:
1. Solvents: Solvents for testing material
2. Water: distilled water or equivalent
3. Undiluted Standard solutions: Dissolve standard thiram in methanol. Completely dissolve

standard metiram and standard propineb in specimen extract solvent to make 100 mg/L for
immediate use.
4. Standard Solutions: Dilute the undiluted standard solution with extracted solvent adjusted
to the pH 7.0 Make sure that 1 ml of the siluted solution undergo through processes of
preparation of test solution, Extraction and Derivatization before diluting it to an
appropriate concentration for use.
5.
Other reagents: Methyl iodide, tetrabutylammonium
hydrogen
sulfate,
ethylenediaminetetraacetica acid, disodium salt, L-cysteine Hydrochloride Hydrate, or special
grade test solution.
Preparation of test solution
1. Extraction: Reduce 500-600 g of the specimen to fine powder and place 20 g into a
conical flask. Add 0.5 g of L-Cysteine Hydrochloride Hydrate to 80 ml of 0.45 ml/L
Sodium hydroxide solution (the pH: 9.5-9.6), Containing 0.25 mol/L
ethylenediaminetetraacetica acid. Immediately cover it with a stopper and shake it for 10
minutes. Eilter this solution using a glass filter. Rinse the conical flask with 10 ml of the
extracted solvent a few times, and then add this to the filtered solution.
After adding 5 ml of tetrabutylammonium hydrogen sulfate aqueous solution and 10 g of
sodium chloride, shake it. Quickly adjust the pH to 7.0 using 2 mlo/L hydrochloric acid,
before transferring to a 300 ml separating funnel.
2. Derivatization: After putting 40 ml of dichloromethane-hexane mixture (1:1) containing
0.05 mol/L methyl iodide into the above separating funnel, shake it vigorously for 5
minutes and leave it to stand. After transferring the organic solvent layer to a 50 ml of
centrifuge bottle and separating it for 5 minutes at 800 rpm, mix 20 ml with 5 ml of
dichloromethane solution containing 1,2-propanediol and under vacuum extract solvents
from which 1,2- propanediol has been removed under nitrogen flow. Immediately dissolve
this in methanol to make a set amount of test solution.
Conducting Test:
Measurement conditions for HPLC:
Column: A stainless steel column coated with octadecyl silica gel in 51.1.m thickness. Detectors:
UV-Detector (at 272 nm)
Mobile phase: Water-acetonitrile-methanol mixture (65:22:13)
Flow rate: 1.0 ml/min.
Qualitative Test:
The retention time of each peak in the chromatogram of the extract correspond to ther peak
obtained in a standard solution, regardless of the conditions of measurement.
Using a LC/MS/MS components of each agrochemical can be identified by retention time and
mass spectrum.
Quantitative Test:
Evaluations are made using the peak heights and peak areas, obtained under the same conditions as
in the qualitative test. Detections are made in the order of thiram, metiram and propineb.
MICROBIAL LIMIT
Apparatus:
Conical flask 250 ml
Graduated cylinder
100 ml
Petriplates
100 mm diameter
Graduated pipettes
2 ml

Buffered Sodium Chloride Peptone Solution pH7.0

Disodiumhydrogen phosphate7.23 g
Potassiumdihydrogen
3.56 g
phosphate
Sodiumchloride
4.3 g
Peptone
:1g
Make the volume to 1000ml distilled water.
All glass ware & media must be sterilized at121Cat15 lbsteampressurefor20 minutes.
A. Total Viable Aerobi cCount/TotalAerobic Microbial Count: NMT 100 cfu / gm
PROCEDURE:
About 10gm of the sample is removed aseptically in a presteri I is eddriedandv, eighed250m1
conical flask.
The flask is again weighed.
The difference between two weights will be the weight of the sample.
1:10dilutionis achieved by adding aseptically 9mlvolumes of sterile warm buffered sodium
chloride peptone solution pH7.Ocontaining 0.5%ofsoya lecithinand4 %ofpolysorbate20 with the
help of measuring cylinder.
Flasks are shaken thoroughly. Aseptically transfer1.0m1ofthe
Solution to 2sets of two petridishes,15m1of sterile Soya bean case in Digest agar maintained at 45
c is poured in one set of petri plates.
Allow the agar to solidify.Incubate the plates in inverted position at 30-35C for 48hrs for bacteria.
To other set of petri plates add 15 ml of sterile liquefied Sabourauds chloramphenicolagar.
Allow to solidify and incubate the plates in inverted position at 20-25 C for 72hrs for Fungal
count. After the incubation period count the number of colonies per plate.
Take
the average number of colonies.Calculate the number of organism per gram or ml of the
Sample by multiplying the dilution factor.
B. Total Fungal Count: NMT 10 cfu / gm
Sample preparation: Use a same sample preparation given in Total bacterial count. Procedure:
For pour plate technique
Add 1 ml of sample prepared as described in the sample preparation in two sterile petri plates and
add about 15 to 20 ml of liquefied sterile Sabouraud Cloramphenicol Agar cooled to 45C. Allow
to solidify the plates. Along with sample run a negative control as a blank for test.
Incubate the plates at 20C to 25C for 5 days.
Interpretation of results: After incubation count number of colonies on the plates. Take the
arithmetic average of the counts and calculate the number of colony forming units per gm or ml
considering the dilution factor.
Testforpathogens
i. TestforEschericida coli: Absent/g Sample preparation and pre-incubation
Using Soya bean case indigest broth as a diluent make lin lO dilution i.e.add 10gm/10m1 sample to
90m1 Soya bean case in digest broth and use 10 ml quantity corresponding to
lgm/lml of product to inoculate 100ml soya bean case in digest broth Incubate at 30-35 c for 18 to
24 hours.
Selection and Subculture
Shake the above enriched broth and transfer 1.0 ml to 100 ml of MacConkeys broth. Incubate at 42
to 44 C for 24-48 hours. Subculture on a plate of MacConkey agar and incubate at 30350c for 18-

72 hrs.
Interpretation
Growth of pink, non mucoid colonies indicates the possible presence of E. coli. This should be
confirmed by identification test.
Identification test:
Add loopful of colonies from MacConkey agar to5m1 of peptone water Incubate at 42to 44 C for
24 hours&add 0.5m1 of kovac's reagent shake well,&allow to stand
for l minute,
Ifared colour produced in there agent layer,test indicates the presence of E.coli Also streak a
loopful of organism on Levine-eosin-methylene blue agar.Upon examination if the colonies exhibit
metallic sheen on EMB agar confirms the presence ot E.coli.
The product complies with
the
test if there is no growth of such type of colonies or
if the
Identification tests are negative.
Positive Control
Carry out a control test by adding a volume of broth containing not more than100cfuof Escherichia
coliATCC8739organisms,prepared from a 24hourculture in Soya bean case in digest broth to
100m1ofMacconkeybroth. The test is invalid if there sults do not indicate that the control contains
Escherichia coli.
ii. Test for Salmonella:Absent/g
Sample preparation
and
pre-incubation using
Soya bean case indigest broth as a diluent make I in 10 dilution i.e.add 10gm/10m1 sample to
90m1
Soya bean case indigest broth anduse 10 ml quantity corresponding to
lgm/lml of product to inoculate of 100mI soya bean case indigest broth.Incubateat30-35 C forl8
to 24 hours.
Selection and subculture
Transfer0. I ml of the
above enriched broth to 10ml of Rappaport Vassiliadis Salmonella
Enrichment broth and incubate at30-35Cfor18to 48hours.
Subculture on plates of Wilson and Blair's BBS agar and Xylose- lysine deoxycholate agar
Incubate at 30-35C for l8 to 48 hours.
Interpretation
The possible presence of Salmonella on Wilson and Blair's BBS agar is indicated by green colonies
withblackcentreandin48hrscolonies become uniformly black and get surrounded by dark zone and
metallic sheen.
The possible presence of Salmonella on Xylose-lysine deoxycholate agar is indicated by well
developed red colonies with or without black centre. This should be confirmed by identification
test
Identificationtest
Subculture colonies showing
the characteristics given above on triple sugarir on agar and
Inoculate a tube of urea broth and incubate at36Cto38Cfor18 to 24 hours. The formation of
acid& gas in the stab culture with or without concomitant blackening & the absence of acidity
from the growth TSl Agar together with the absence of red colour in the urea broth, indicate the
presence of Salmonella.
The product complies with the test if there is no growth of such type of colonies or if the
Identification tests are negative.
Positive Control
Carry out a control test by adding a volume of broth containing not more than100 cfu of

SalmonellaabonyNCTC6017 organisms, prepared froma24 hour culture in soya bean case in digest
broth.The test is invalid if the results donot indicate that the control contains salmonella.
iii. Test forPseudomonas Aeruginosa:Absent/g
Sample preparation and pre-incubation
Using Soya bean case indigest broth as a diluent makelin lO dilution i.e. add lOgm/10m1 sample to
90m1 Soya bean case in digest broth and use 10 ml quantity
corresponding to
lgm/lml of product to inoculate of l00mI soya bean case indigest broth.Incubateat30-35C

for18

to24 hours
Selection and Subculture
Sub culture on a plate of Cetrimide agar and incubateat30-35cforl8to 72 hours.
Interpretation
A greenish colour colony indicates the possibility of presence of Pseudomonasaeruginosa. This
should be confirmed by identification test.
Identification test :Oxidase Test
Place2to3dropsoffreshly prepared1.0%w/v solution of N,N,N, N-tetramethy1-4phenylenediaminedihydrochlorideonapieceofWhatmanNo.1
filterpaperand
smearwith
the
suspected colony.lf a purple colour is produced within five to tenseconds the test is positive. The
test is invalid if the results do not indicate that the control contains Pseudomonasaeruginosa.
The product complies with
the
test if there Is no growth of such type of colonies
or if the Identification tests are negative.
Positive control
Carry out a control test by sub culturing Pseudomonasaeruginosa ATCC9027 prepared from a
24hrs old broth culture on cetrimide agar.The test is invalid if there sults do not indicate
that
the
control contains Pseudomonasaeruginosa.
Carry out a control test by adding a volume of broth containing not more than100 cfu of
SalmonellaabonyNCTC6017 organisms, prepared froma24 hour culture in soya bean casein digest
broth.The test is invalid if the results do not indicate that the control contains salmonella.
iv. Test for Stap hylococcus A ureus:Absent/g
Sample preparation and pre-incubation
Using Soya bean case in digest broth as a diluent make 1 in 1O dilution i.e.add 1 Ogm/10m1
sample to 90m1 Soyabeancase in digest broth and use 10 ml quantity
corresponding to
lgm/Iml of product to inoculate of 100mI soyabeancase in digest broth.Incubateat 30-35 C for
18 to24 hours
Selection and Subculture
Sub cultureon a plate of Mannitol salt agar and incubate at30-35 cfor18 to 72hours.
Interpretation
The possible presence of S.aureus is indicated by the growth of yellow/white
Colonies surrounded by yellow zone.This is confirmed by identification test.
Identification test:Examinethere sulting growth by Gram's stain and apply coagulase test.
Gram+vecocci (in clusters) indicate the presence of Staphylococcusaureus.
CoagulaseTest
Transfer representative suspected colonies from agar surface of the Mannitol salt agar to tubes
containing 0.5 ml often a mamalian preferably rabbit or horse plasma with or without additives.
Incubate at370C and examine the tubes at suitable intervals upto24hours.lfcoagulation in any
degree is observed the test is positive.

Positive control For Coagulase Test

Carry out a control test by inoculating 0.5 ml of mammalian plasma with colonies of a 24hrs
Old culture of StaphylococcusaureusATCC6538.0n incubation at 37 C,coagulation of the plasma
must be observed.
Negative control For Coagulase Test
Incubate a nun inoculated tube of 0.5mI plasma at 37 C.No coagulation must be observed up to 24
hrs.
The product complies with
the
testi f there is no growth of such type of colonies or
if the identification tests are negative
.
Positive Control
Carry out a control test by sub culturing Staphylococcus aureusATCC653 8prepared from 24 hrs
old broth culture on Mannitol Salt agar.The test is invalid if there sults do not indicate that the
control contains Staphylococcusaureus.
Limit:
Total Bacterial count Not more than 100 cfu / gm Total Fungal Count Not more than 10 cfu / gm
Pathogens Should be absent
ASSAY Weigh 100 g of Allium cepae extract, add 200 mL of water and 1 Og of sodium chloride,
and extract four times with each 200 mL of ethyl acetate. Combine the all ethyl acetate layers and
evaporate to dryness. To the residue, add 10 mL of methanol to dissolve, filter, and use this
solution as Test solution. Separately, accurately weigh 2.0 mg of Spiraeoside standard, add
methanol to make 10 mL, and use this solution as Standard
solution. Perform the test with 200 each of the test solution and the standard solution as
directed under Thin-layer Chromatography and Determine the peak areas, Ar and As, of
spiraeoside from the test solution and the standard solution, respectively.
Amount (mg) of spiraeoside(C21 H20012: 464.38) in Allium cepae extract (1.8 in 1) = = Amount
(mg) of Spiraeoside standard x Ar As
Operating conditions
Detector: An ultraviolet absorption photometer (wavelength: 280 rim).
Column: A stainless steel column, about 4 mm in internal diameter and 15 cm in length, packed
with octadecylsilanized silica gel for liquid chromatography (5 1.1m in particle diameter).
Mobile phase : A solution (0.2% formic acid), B (methanol)
Time (minutes)
0- 15
15 17
17 - 19
19 - 21

A solution (%)
65 45
45 10
10
10 - 65

Flow rate : 1.0 mL/min. Limit: NLT 20.0 gg/g

B solution (%)
35 55
55 90
90
90-35