WHO guidelines for the standardization of crude drugs and extracts

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WHO guidelines

Content

WHO guidelines for the standardization of crude drugs and extracts with
special emphasis on pharmacological and toxicological evaluation

» Haemolytic
activity

» Swelling
index

» Foaming index

» Aflatoxins

» Arsenic

» Detection
of Micro-organisms

Determination
of pesticide residues

Ø Chlorides

Ø Phosphates

Objectives

• At the end of this lecture, student
will be able to

Discuss the principle and
procedure involved in the determination of

» Haemolytic
activity

» Swelling
index

» Foaming index

» Aflatoxins

» Arsenic

» Discuss the principle and procedure
involved in the determination of
pesticide residues like

» Chlorides

» Phosphates

» Discuss the principle and procedure
involved in the detection of microorganisms in crude drugs and extracts

Determination of Haemolytic Activity

v Caryophyllaceae,
Araliaceae, Sapindaceae, Primulaceae, and Dioscoreaceae – contain Saponins

v Saponins
– ability to cause haemolysis

v Comparison
with that of a reference material, saponin R, which has a haemolytic activity
of 1000 units per g

v Suspension
of erythrocytes + equal volumes of a serial dilution of the herbal material
extract

v Lowest
concentration to effect complete haemolysis is determined

v Similar
test is carried out simultaneously with Saponin R

Procedure

Erythrocyte suspension

v Fill
a glass-stoppered flask to one tenth of its volume with sodium citrate (36.5
g/l) TS, swirling to ensure sufficient volume of blood freshly from a healthy
ox, shake

v Citrated
blood- can be stored for about 8 days at 2−4 °C

v 1
ml of citrated blood in a 50-ml volumetric flask with phosphate buffer pH 7.4
TS – diluted blood suspension (2% solution)

Reference solution

v 10
mg of Saponin R add phosphate buffer pH 7.4 TS to make 100 ml

Preliminary test

v Serial
dilution – herbal material extract with phosphate buffer pH 7.4 TS and blood
suspension (2%) using four test-tubes

ü Gently
invert – mix

ü Shake
again after a 30-minute interval

ü Allow
to stand for six hours at room temperature

v Examine
the tubes

v Record
the dilution at which total haemolysis has occurred

Ø Total
heamolysis – clear, red solution without any deposit of erythrocytes

If total haemolysis

Ø only
in tube 4: original herbal material extract directly for the main
test

Ø In
tubes 3 and 4: two-fold dilution of the original herbal material
extract

Ø In
tubes 2, 3 and 4: five-fold dilution of the original herbal material
extract

Ø In
all four tubes: ten-fold dilution and carry out the preliminary
test again

Ø Not
observed in any of the tubes: Repeat the preliminary test using a more
concentrated herbal material extract

Main test

v Prepare
a serial dilution – undiluted or diluted herbal extracts

v Blood
suspension (2%) using 13 test-tubes

v Observe
the results after 24 hours

Calculate

ü Amount
of herbal material in g or ml that produces total haemolysis

ü Quantity
of saponin R in g that produces total haemolysis

Calculation of haemolytic activity of the herbal material

1000=a/b

1000 = the defined haemolytic activity of saponin R in
relation to ox blood

a = quantity of saponin R that produces total haemolysis (g)

b = quantity of herbal material that produces total
haemolysis (g)

Determination of Swelling Index

ü Swelling
properties

Especially
gums, appreciable amount of mucilage, pectin or hemicellulose

ü Swelling
index

Volume
in ml taken up by the swelling of 1 g of herbal material under specified
conditions

Procedure

ü Atleast
three simultaneously determinations

Accurately weighed
sample

25-ml glass-stoppered
measuring cylinder (Internal diameter around 16 mm, length of graduated portion
about 125 mm marked in 0.2- ml divisions from 0 to 25 ml)

Add 25 ml of water,
Shake thoroughly every 10 minutes for 1 hour

Allow to stand for 3
hours at room temperature

Measure the volume in
ml occupied by the material, including any sticky mucilage

Calculate the mean
value

Determination of Foaming Index

Ø Saponins
– persistent foam – shaken

Ø Foaming
ability – measured in terms of Foaming
Index

Procedure

1 g of coarsely
powdered herbal material

Transfer to a 500-ml
conical flask

100 ml of boiling
water

Moderate boiling for
30 minutes

Cool and filter into
a 100-ml volumetric flask

Add sufficient water
through the filter to dilute to volume

Pour the decoction
into 10 stoppered test-tubes (height 16 cm, diameter 16 mm) (successive
portions of 1 ml, 2 ml, 3 ml up to 10 ml) adjust the volume with water to 10 ml

Stopper, shake in
lengthwise for 15 seconds, two shakes per second

Allow to stand for 15
minutes

Measure the foam
height

Evaluation of result

If the height of the foam in every tube is less than 1 cm Foaming
index is less than 100

If a height of foam of 1 cm is measured in any
tube, the volume of the herbal material decoction in this tube (a) is used
to determine the index If this tube is the first or second tube in a series, an
intermediate dilution is prepared in a similar manner to obtain a more precise
result

If the height of the foam is more than 1 cm in
every tube Foaming index is over 1000 Repeat the determination using
a new series of dilutions of the decoction

Calculate the foaming index

1000/a

a = the volume in ml of the decoction used for preparing the
dilution in the tube where foaming to a height of 1 cm is observed

Determination of Aflatoxins

ü Only
products that have a history of aflatoxin contamination need to be tested

Tests for aflatoxins

ü Detect
the presence of aflatoxins B1, B2, G1 and G2, which are highly toxic
contaminants in any material of plant origin

Procedure

ü Does
not require the use of toxic solvents
like

Chloroform

Dichloromethane
etc

ü Multifunctional
column

Ø Lipophilic
and charged active sites

Ø High-performance
liquid chromatography (HPLC)

Ø Fluorescence
detection to determine aflatoxins B1, B2, G1 and G2

Advantages of multifunctional column

ü High
total recoveries of aflatoxins B1, B2, G1 and G2 (>85%)

ü Column
can be kept at room temperature for long time prior to use

Standard solutions of aflatoxin B1, B2, G1 and G2 (2.5
ng/ml)

Standard stock solution

Weigh exactly 1.0 mg
each of aflatoxins B1, B2, G1 and G2

Dissolve in 50 ml of toluene-acetonitrile
(9:1) (20 μg/ml)

Keep in a tightly
sealed container and store in refrigerator at 4^OC in dark

Working standard solution

0.5 ml
of standard stock solution added to toluene acetonitrile (9:1) to 200 ml (50
ng/ml)

Standard solution

1.0 ml
of working standard solution, add to toluene acetonitrile (9:1) solution to 20
ml

(Final
standard solution) (2.5 ng/ml)

Standard solution for liquid chromatography analysis

Transfer 0.25 ml of
the final standard solution to a glass centrifuge tube

Evaporate to dryness
at 40^OC or by using a nitrogen air stream

To derivatize aflatoxins B1 and G1 (precolumn
derivatization)

Add 0.1 ml of trifluoroacetic
acid to the residue in the tube

Tightly seal and
shake vigorously

Allow to stand at
room temperature for 15 min in dark

Add 0.4 ml of acetonitrile:
water (1:9)

20-μl portion of the
solution – subjected to liquid chromatography

Preparation of sample

Grind the herbal
material

50-g test sample with
400 ml of acetonitrile-water (9:1)

Extract by shaking
for 30 minutes or by mechanical blender for 5 minutes

Filter or centrifuge

Transfer 5-ml portion
or the top clean layer, to a multifunctional column (MultiSep #228 cartridge
column or Autoprep MF-A)

Flow rate of 1
ml/minute

Aflatoxins present
pass through the column as the first eluate

First 1-ml of the
eluate collected as test solution

Evaporate 0.5 ml of
test solution to dryness at 40^OC or by using a nitrogen air

To derivatize
aflatoxins B1 and G1 (precolumn derivatization)

Add 0.1 ml of
trifluoroacetic acid to the residue in the tube

Tightly seal and
shake vigorously

Allow to stand at
room temperature for 15 min in dark

Add 0.4 ml of
acetonitrile: water (1:9) solution

20-μl portion of the
sample solution – subjected to liquid chromatography

Method

Liquid chromatography conditions

ü Mobile
phase – acetonitrile-methanol-water (1:3:6)

ü De-gas
the mobile phase by sonication

ü Octadecyl-silica
gel (ODS) column ( Inertsil ODS-3 (4.6
mm ID × 250 mm, 3 μm)

ü Column
temperature: 40⁰C

ü Flow
rate – 1 ml/minute

ü Aflatoxin
and its derivatives detected at the excitation and emission wavelengths of 365
nm and 450 nm, respectively

ü Injection
volume is 20 μl

If impurity peak overlaps the peaks corresponding to
aflatoxins – alternative liquid chromatography conditions

Alternative liquid chromatography conditions

ü Mobile
phase – methanol-water (3:7)

ü De-gas
mobile phase by sonication

ü Fluorocarbonated
column (Wako-pack Fluofix 120E)

ü Column
temperature 40⁰C

ü Flow
rate – 1 ml/minute

ü Aflatoxin
and its derivatives are detected at the excitation and emission wavelengths of
365 nm and 450 nm, respectively

ü Injection
volume is 20 μl

Interpretation of the results

ü Compare
the retention time of peak area or peak heights standard and sample

ü If
sample bigger than standard – positive

Determination of Arsenic

Limit test for arsenic

ü Abundant
in nature

ü Test
method uses colorimetry, NOT toxic mercuric bromide paper

ü Method
uses N-N-diethylmethyldithiocarbamate in pyridine and it reacts
with hydrogen arsenide to afford a red–purple complex

ü Limit
expressed in terms of arsenic (III) trioxide (As₂O₃)

Preparation of test solution

Sample
in crucible of platinum, quartz or porcelain

Add
10 ml of magnesium nitrate hexahydrate in ethanol (95) (1 in 10)

Burn
ethanol, heat gradually, ignite to incinerate

If
carbonized material still remains

Moisten
with a small quantity of nitric acid and ignite again

After
cooling, add 3 ml of hydrochloric acid

Heat
in a water bath to dissolve the residue

(Test
solution)

Standard solutions

Absorbing solution for hydrogen arsenide

Dissolve 0.50 g of silver N,N-diethyldithiocarbamate
in pyridine to make 100 ml (protected from light, in a cold place)

Standard arsenic stock solution

Weigh accurately
0.100 g of finely powdered arsenic (III) trioxide

Add 5 ml of NaOH
solution

Add dilute H₂SO₄
to neutralize

Add a further 10 ml
of dilute H₂SO₄

Add freshly boiled
and cooled water- make exactly 1000 ml

Standard arsenic solution

Pipette 10 ml of
standard arsenic stock solution

Add 10 ml of dilute H₂SO₄

Add freshly boiled
and cooled water – make exactly 1000 ml

(Each ml of the
solution contains 1 μg of arsenic (III) trioxide (A_s2O₃))

Procedure

Unless otherwise specified, use the mentioned apparatus

Test solution in the
generator bottle A

Add 1 drop of methyl
orange

Neutralize with
ammonia, ammonia solution or dilute HCl

Add 5 ml of dilute
hydrochloric acid (1 in 2)

Add 5 ml potassium
iodide

Allow to stand for
2–3 minutes

Add 5 ml of acidic
tin (II) chloride

Allow to stand for 10
minutes

Add water to make 40
ml

Add 2 g of zinc for
arsenic analysis and immediately connect the rubber stopper H fitted with B and
C with the generator bottle A

Transfer 5 ml of
absorbing solution for hydrogen arsenide to the absorber tube D

Insert the tip of C
to the bottom of the absorber tube D

Immerse the generator
bottle A up to the shoulder in water maintained at 25 °C

Allow to stand for 1
hour

Disconnect the
absorber tube

Add pyridine to make
5 ml, if necessary

Observe the colour of
the absorbing solution

Colour produced is
not more intense than the standard colour

Determination of pesticide residues

Methods for the determination of pesticide residues

ü Chromatography
(mostly column and gas), Coupled with MS

ü Samples
are extracted by a standard procedure

ü These
techniques are not universally applicable

ü Some
pesticides –

Ø Satisfactorily
carried through the extraction and clean-up procedures

Ø Recovered
with a poor yield & some are lost entirely

Ø After
chromatography, the separations may not always be complete

Ø Pesticides
may decompose or metabolize

Methods for the determination of pesticide residues

ü Spectrum
of pesticides to be tested –

Ø Dependent
on the specific pesticides used on the herbal material

Ø History
of use of persistent pesticides in the region

Ø If
the pesticide is known or can be identified- established method for particular
pesticide residue should be employed

General aspects of analytical methodology

ü Samples
should be tested as quickly as possible after collection

ü If
stored – should be preferably in airtight containers under refrigeration

ü Water
content – limited to 15% and below

ü Light
cause degradation of many pesticides

ü Type
of container or wrapping material used should not interfere

ü Solvents
and reagents used

Ø Purified
solvents or to be distilled

Ø Blank
determinations – should be carried out

ü Simplest and quickest procedure should be used

ü Process
of concentrating solutions – avoid loss
of pesticide residues

Determination of total chlorine and phosphorus

Most pesticides contain organically bound chlorine or
phosphorus

Procedure

Preparation of samples

Herbal
material to fine powder

Extract
with a mixture of water and acetonitrile

(Most
pesticides soluble in this mixture, while most cellular constituents are
sparingly soluble and hence removed)

Then
transfer pesticide to light petroleum

Pesticides
with chlorine – further purification required
Pesticides with phosphorus – further purification by column chromatography

Eluted
with mixtures of light petroleum and ether

Preparation of the column

ü Use
Florisil R grade 60/100 PR

ü Activated
at 650 °C

ü Prepare
a Florisil column (external diameter 22 mm) with 10 cm of activated Florisil
topped with about 1 cm of anhydrous sodium sulfate

ü Pre-wet
column with 40–50 ml of light petroleum

ü Place
a graduated flask under the column to receive the eluate

Method

Grind
the material

Place
20–50 g of the ground sample into a blender

Add
350 ml of acetonitrile with a water content of 35%

Blend
for 5 minutes at high speed, filter

Transfer
the filtrate to a 250-ml measuring cylinder and record the volume

Transfer
the measured filtrate to separating funnel + 100 ml of light petroleum Shake
vigorously

Add 10 ml of sodium
chloride (40%) and 600 ml of water

Separating funnel in
horizontal position, mix vigorously for 30–45 seconds

Allow to separate,
discard the aqueous layer

Wash the solvent
layer with two 100-ml portions of water

Transfer the solvent
layer to a 100-ml glass-stoppered cylinder

Record the volume

Add about 15 g of
anhydrous sodium sulfate & shake vigorously

Transfer the extract
directly to a Florisil column

Flow rate not more
than 5 ml/minute

Rinse the cylinder
with two 5 ml of light petroleum & transfer to the column

Elute at the same
rate with 200 ml of ether/light petroleum (TS1)

Change the receiver
& elute with 200 ml of ether/light petroleum (TS2)

Again change the
receiver and elute with 200 ml of ether/light petroleum (TS3)

Evaporate each eluate
to a suitable volume for further testing

Method

First elute

Chlorinated pesticides (aldrin, DDE, TDE, HCH, heptachlor,
heptachlor epoxide, lindane, methoxychlor), polychlorinated biphenyls (PCB),
and phosphated pesticides (carbophenothion, ethion and fenchlorphos)

Second elute

Chlorinated pesticides (dieldrin and endrin) and phosphated
pesticides (methyl parathion and parathion)

Third elute

Phosphated pesticide (malathion)

Combustion of the organic matter

Combustion of the
organic matter in oxygen – preparatory step for the determination of
chlorine and phosphorus

Pesticide extracted
from sample and purified

Extract is
concentrated, evaporated to dryness

Transferred to a
sample holder

Burnt in suitable
conical flask flushed with oxygen

Gases produced during
combustion – absorbed in a suitable solution

Absorbed chlorine
– determined as chloride (colorimetry)

Absorbed
phosphorus – orthophosphate (colorimetry)

Combustion Equipment

ü Conical
flask of borosilicate glass

ü Stopper
fused with platinum wire about 1 mm in diameter

ü Free
end of the wire attached to platinum gauze – holding the sample

Sample holder for chlorine-containing residues

ü Halide-free
filter-paper – solid

ü Cone
made from cellulose acetate film – liquid

Sample holder for phosphorus-containing residues

ü Halide-free
filter-paper

Combustion of chlorine-containing residues

Combustion of phosphorus-containing residues

Determination of chloride

Procedure

Equipment

ü Spectrophotometer
at 460 nm

ü Path-lengths
of 2 cm and 10 cm

Method

15 ml of solution
obtained after combustion in a 50-ml conical flask

Add 1 ml of ferric
ammonium sulfate (0.25 mol/l), 3 ml of mercuric thiocyanate

Allow to stand for 10
minutes, transfer to a 2-cm cell

Measure the
absorbance at 460 nm using water as reference cell

Standard

Sodium chloride with
5 μg of chloride per ml

0 ml, 2 ml, 4 ml, 6
ml, 8 ml and 10 ml into a series of 50-ml conical flasks

Dilute to 15 ml with
water

Develop the colour
& measure the absorbance at 460 nm

Plot the absorbances
against the chloride content of the dilutions in μg/ml

Interpolate the
chloride content in the solution of the material under test

Determination of phosphates

Procedure

Phosphomolybdate method

ü Reaction
of phosphate ions with ammonium molybdate to form a molybdophosphate complex

ü Subsequently
reduced to form a strongly blue-coloured molybdenum complex

ü Intensity
of blue colour measured spectrophotometrically

ü Applicable
for the determination of any phosphates

ü Naturally
occurring phosphates – removed during the clean-up procedure

Equipment

ü Spectrophotometer,
absorbance at 820 nm

ü Path-length
– 1 cm

Method

ü Place
7 ml of the solution after combustion in a calibrated 10-ml test-tube

ü Add
2.2 ml of sulfuric acid (300 g/l) & mix

ü Add
0.4 ml of ammonium molybdate (40 g/l) & mix

ü Add
0.4 ml of aminonaphtholsulfonic acid & mix

ü Heat
to 100 °C for 12 minutes

ü Cool
& transfer a portion to a 1-cm cell

ü Measure
the absorbance at 820 nm using water in the reference cell

Standard

Standard
dilutions with a known content of phosphate

Measure
the absorbance at 820 nm

Plot
absorbances against the phosphate content

Interpolate
the phosphate content of the solutions of the material tested

Determination of Microorganisms

ü Total
viable aerobic count determined

Ø Membrane-filtration

Ø Plate
count or serial dilution

ü Aerobic
bacteria and fungi (moulds and yeasts) are determined by the TVC

ü Usually
if TVC exceeds maximum permitted level

Ø Unnecessary
to proceed with determination of specific organisms

Ø Material
should be rejected without further testing

Pretreatment of the test herbal material

ü Depending
on the nature of the crude herbal material

Ø Grind

Ø Dissolve

Ø Dilute

Ø Suspend
or

Ø Emulsify

ü Either
phosphate buffer pH 7.2; buffered sodium chloride-peptone solution, pH 7.0; or
fluid medium, used for the test, is used to suspend or dilute the test specimen
materials with special requirements

Materials containing tannins, antimicrobial substances

ü Test
specimens with antimicrobial activity or contain antimicrobial substances

ü Any
such antimicrobial properties are removed

Water-soluble materials

ü Dissolve
or dilute 10 g or 10 ml in lactose broth or another suitable medium with no
antimicrobial activity

ü Make
the volume to 100 ml with the same medium

ü Adjust
the pH of the suspension to about 7, if required

Non-fatty materials insoluble in water

ü Suspend
10 g or 10 ml of the herbal material in lactose broth or another suitable
medium with no antimicrobial activity

ü Dilute
to 100 ml with the same medium

ü If
required, divide the material, homogenize the suspension mechanically

ü Surfactant
– solution of polysorbate 20 R or 80 R (1mg/ml)

ü Adjust
the pH of the suspension to about 7

Fatty materials

ü Homogenize
10 g or 10 ml of material with 5 g of polysorbate 20 R or 80 R

ü Heat
to a temperature not exceeding 40 °C, if required

ü Mix
carefully while maintaining the temperature in a water-bath or oven

ü Add
85 ml of lactose broth or another suitable medium with no antimicrobial
activity

ü Heat
to 40^OC, Maintain this temperature for the shortest time until an
emulsion is formed

ü Adjust
the pH of the emulsion to about 7

Test procedure: Plate count – For bacteria

ü Petri
dishes 9–10 cm in diameter

ü To
one dish – 1 ml of the pre-treated
herbal material + 15 ml of liquefied casein-soybean digest agar at a
temperature not exceeding 45 °C

ü Alternatively,
spread the material on the surface of the solidified medium

ü Dilute
the material to obtain an colony count of not more than 300, if needed

ü Prepare
at least two dishes using the same dilution

ü Invert
them and incubate them at 30–35 °C for 48–72 hours

ü Count
the number of colonies formed

ü Calculate
the results using the plate with the largest number of colonies, up to a
maximum of 300

Test procedure: Plate count – For fungi

ü Use
Petri dishes 9–10 cm in diameter

ü To
one dish – mixture of 1 ml of the
pretreated material + 15 ml of liquefied Sabouraud glucose agar with
antibiotics or potato dextrose agar with antibiotics, temp NMT 45 °C

ü Alternatively,
spread the pretreated material on the surface of the solidified medium

ü Dilute
the material to obtain an expected colony count of not more than 100

ü Prepare
at least two dishes using the same dilution

ü Incubate
them upright at 20–25 °C for 5 days

ü Count
the number of colonies formed

ü Calculate
the results using the dish with not more than 100 colonies

Membrane filtration

ü Membrane
filters – pore size NMT 0.45 μm

ü Effective
in retaining bacteria

ü Cellulose
nitrate filters – aqueous, oily and weakly alcoholic solutions,

ü Cellulose
acetate filters – strongly alcoholic solutions

Detailed method

ü Filter
10 ml or a solution containing 1 g of the material through two membrane filter
apparatuses

ü If
necessary, dilute the pretreated material to obtain an expected colony count of
10–100

ü Wash
each membrane, with 3 or more successive quantities (100 ml) of a suitable
liquid such as buffered sodium chloride-peptone solution at pH 7.0

ü Fatty
materials – surfactant may be added (Polysorbate 20 R or 80 R)

ü Transfer
one of the membrane filters to the surface of a plate with soybean casein
digest agar (Bacteria)
other to the surface of a plate with Sabouraud glucose agar with
antibiotics (Fungi)

ü Incubate
the plates for 5 days at 30–35 °C (bacteria), 20–25 °C (fungi)

ü Count
the number of colonies formed

ü Calculate
the number of microorganisms per gram or per ml of the material tested

Serial dilution

ü Prepare
a series of 12 tubes each containing 9–10 ml of soybean-casein digest medium

ü To
each of the:

Ø First
group of three tubes: 1 ml of the 1:10 dilution of dissolved,
homogenized material (0.1 g or 0.1 ml of specimen)

Ø Second
group of three tubes: 1 ml of a 1:100 dilution of the
material

Ø Third
group of three tubes: 1 ml of a 1:1000 dilution of the material

Ø Last
three tubes: add 1 ml of the diluent

ü Incubate
at 30–35 °C for at least 5 days

ü No
microbial growth should occur in the last three tubes

ü If
the reading of the results is difficult or uncertain

Ø Subculture
in a liquid or a solid medium

ü Determine
the most probable number of microorganisms per gram or per ml of the material
using table

ü If,
for the first column, the number of tubes showing microbial growth is two or
less, the most probable number of microorganisms per g or per ml is less than
100

Summary

ü Saponins
– ability to cause haemolysis, Lowest concentration to effect complete
haemolysis is determined, Similar test is carried out simultaneously with
Saponin R

ü Swelling
index – Volume in ml taken up by the swelling of 1 g of herbal material
under specified conditions

ü Saponins
– persistent foam – shaken, Foaming ability
– measured in terms of Foaming Index

ü Aflatoxins
– Only products that have a history of aflatoxin contamination need to be
tested

ü Tests
for aflatoxins Detect the presence of aflatoxins B1, B2, G1 and G2, which
are highly toxic contaminants in any material of plant origin

ü Arsenic
– Method uses N-N-diethylmethyldithiocarbamate in pyridine and it reacts with
hydrogen arsenide to afford a red–purple complex

ü Limit
expressed in terms of arsenic (III) trioxide (As₂O₃)

ü Chromatography
(mostly column and gas), Coupled with MS

ü Preparation
of

ü Sample

ü Column

ü Elution

ü Combusted
material

ü Determination
of chlorides (spectrophotometry) and phosphates (phosphomolybdate method)

ü Total
viable aerobic count determined

ü Membrane-filtration

ü Plate
count or serial dilution

ü Pretreatment
of the test herbal material

ü Plate
count for bacteria and fungi

ü Membrane
filtration followed by serial dilution

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