WHO guidelines for the standardization of crude drugs and extracts

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 H₂SO₄to 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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WHO guidelines for the standardization of crude drugs and extracts

Prakash surya

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WHO guidelines for the standardization of crude drugs and extracts

Prakash surya

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