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Appendices
Appendix I Sampling
Appendix I: Sampling
The sampling here refers to the method used for selecting CMM sample for examination. As the
representativeness of sampling directly affects the conclusions of the examination, recommended sampling
procedures are detailed in the following paragraphs for reference.
(1) Before sampling, it is necessary to inspect each container or package to see whether the name, source,
specification and packaging of CMM are correct. Examine the intactness, cleanliness and any water
trace of the package. Check any contamination by moulds or of foreign matter, and make detailed notes.
Any abnormal package should be sampled separately.
(2) The following are the general requirements for random CMM sampling in a consignment –
(a) For packages less than five : sample every package
(b) For packages of 5–100 : sample five packages
(c) For packages of 100–1000 : sample 5%
(d) For packages over 1000 : sample 50 packages plus 1% of those in excess of 1000
(e) For precious CMM : sample every package regardless of the number
(3) Take CMM by portions from different parts of the selected packages and mix them into one pooled
sample. For small amount of material, the quantity of the sample taken should not be less than three
times of the amount required for tests; for large amount of material, the quantity of the sample taken is
recommended as follows –
(a) Common CMM : 100–500 g
(b) Powdered CMM : 25 g
(c) Precious CMM : 5–10 g
(4) One third of the samples are used for analysis; one third for verification and the remaining for retention
for at least a year.
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Appendix II General Quality Control Method
Appendix II: General Quality Control Method
General quality control method includes "Description", "Identification", "Tests", "Extractives" and "Assay".
A scheme for the examination of CMM is outlined in the paragraphs below.
(1) Follow the method for sampling of CMM as set out in Appendix I for CMM examinations.
(2) Whenever necessary, use a reference herb that complies with the requirements listed in the Appendix of
the current edition of Pharmacopoeia of the Peoples’ Republic of China to verify the results of the
examinations.
(3) "Description" refers to the macroscopic and organoleptic characteristics including form, size, colour,
texture, fracture, gross internal structures, odour/smell, taste, and other relevant information of CMM
samples. Safety precautions should be taken in handling the samples.
(a) "Form" refers to the shape of dried CMM samples. In general, it is observed without preliminary
treatment while wrinkled herbs, leaves or flowers can be moistened or softened and spread for the
examination. For some fruits and seeds, the pericarp or seed coat can be softened and removed, if
necessary before the examination of the inner characteristics.
(b) "Size" indicates the length, diameter and thickness of CMM samples, measured by a millimeter
ruler. A few variations from the defined values are acceptable. For fine seeds, arrange 10 seeds
closely in a row on a piece of paper with a millimeter scale, measure and calculate the average
value.
(c) The "colour" of CMM samples is observed in daylight. For a description of a combination of two
colours, the latter is the main colour. For example, in ‘yellowish-brown’, the main colour is brown.
(d) The "surface characters", "texture" and "gross internal structure" (including fracture
characteristics) of CMM samples are observed without preliminary treatment. If the striations of
the fracture surface are difficult to observe, it should be re-examined with a smooth cut surface.
(e) The "odour/smell" of CMM samples can be examined by smelling directly, or after fracturing
and rubbing. When necessary, the examination can be carried out after the samples are moistened
with hot water.
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(f) The "flavour" of CMM samples can be examined by tasting a small amount of sample directly or
by tasting its water extract.
(4) "Identification" refers to the verification of CMM samples by means of microscopic examination of
cross sections and powders, physical and chemical tests and chromatographic analysis.
(a) "Microscopic identification" refers to the observation of the characteristics of structural features,
cells and ergastic substances in section, powder, disintegrated tissue or surface slides of
CMM samples. It is usually carried out by making slides in an appropriate way as detailed in
Appendix III.
(b) "Physicochemical identification" refers to the testing of the representative constituents in the
samples by physical or chemical methods.
(c) "Chromatographic identification" refers to the identification of samples by means of TLC,
HPLC or GC.
(5) "Tests" refers to the qualitative and quantitative detection of heavy metals, pesticide residues, mycotoxins
(aflatoxins), foreign matter, ash, water content and other chemical components in the CMM which should
be monitored.
(6) "Extractives" refers to the soluble contents of a CMM as extracted by water, ethanol or other appropriate
solvents.
(7) "Assay" refers to the quantitative determination of the active ingredients or markers of CMM samples.
(8) Chemical reference substances of high purity should be used. For Assay, the purity should not be less
than 97%.
Appendix II General Quality Control Method
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Appendix III: Microscopic Identification
Microscopic identification is a method using a microscope to identify the characters of structural features,
cells and ergastic substances in section, powder, disintegrated tissue or surface slides of CMM samples. Select
a representative CMM sample and conduct the examination in accordance with the methods described below.
(1) Preparation of cross and/or longitudinal sections slides –Select a representative sample for examination.
After softening, cut the material with a razor blade or a sliding microtome to a thickness of 10–20 µm.
Examine the sample under a microscope after treated with glycerol-acetic acid TS, chloral hydrate TS or
other suitable TS. Embed the material in hard paraffin for cutting, when necessary.
(2) Preparation of powder slides – Spread a small quantity of the powder on a slide, treated with glycerol-
acetic acid TS, chloral hydrate TS, or other suitable TS and conduct the examination.
(3) Preparation of surface slides – After moistening and softening, cut the sample apart or tear out the
epidermis, add suitable TS and conduct the examination.
(4) Maceration and preparation of slides – Cut or slice the sample into small pieces of about 2 mm in
thickness for maceration. Depending on the nature of the material, one of the following three methods is
used –
(a) Potassium hydroxide method – Place the sample in a test tube, and add an adequate quantity of
aqueous potassium hydroxide solution (5%, v/v), then heat until the residue can be easily separated
when pressed with a glass rod. Decant the alkaline solution and wash the residue with water,
transfer a small amount of macerated material onto a slide and tease it out with a needle. Mount in
dilute glycerine and examine under a microscope.
(b) Chromic-nitric acid method – Place the sample in a test tube and add an adequate quantity of
chromic-nitric acid TS, then let stand until the material can be easily separated when pressing with
a glass rod. Decant the acidic solution, wash the residue with water, and prepare the slide as
directed above in (4)(a).
(c) Potassium chlorate method – Place the sample in a test tube, add dilute nitric acid (50%, v/v)
and a small quantity of potassium chlorate; warm gently until the effervescence subsides, then add
small amount of potassium chlorate to maintain a slight effervescence. When the tissue shows a
tendency to disintegrate, break the material with a glass rod. Decant the acidic solution, wash the
macerated material with water and prepare the slide as directed above in (4)(a).
Appendix III Microscopic Identification
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For CMM samples with only a few or scattered woody tissues or with parenchyma tissues, use potassium
hydroxide method. Whereas, for hard material mainly with woody tissues or woody tissues grouped into
bundles, use chromic-nitric acid or potassium chlorate method.
(5) Measurements of sizes of cell and its contents – Under a microscope, use an ocular micrometer to
measure the sizes of cell and cell contents of specimens. Place the ocular micrometer scale in the eyepiece
of the microscope, then calibrate with a stage micrometer. For the calibration, turn the eyepiece and
move the stage micrometer to make the divisions on the two scales parallel and their left "O" lines
coincide, then look for another line which coincides to the right.
The value (in micrometer) of one ocular micrometer division can be calculated on the basis of divisions
of the two micrometer scales between the coinciding lines.
To measure the object, multiply the number of object-measuring divisions of ocular micrometer by the
value (in micrometer) of each division. In general, it is carried out under a high power objective, but a
low power objective would be more convenient to measure the lengths of longer fibres and non-glandular
hairs, etc.
Record the maximal and minimal values (in micrometer), values slightly higher or lower than those
specified in the individual monograph are acceptable.
(6) Histochemical detection of cell walls –
(a) Lignified cell wall – Add 1–2 drops of phloroglucinol TS to the specimen on the slide, allow to
stand for a moment, then add 1 drop of hydrochloric acid. Lignified cell walls are stained red or
purplish-red according to the extent of lignification.
(b) Suberized or cuticular cell wall – Add 1–2 drops of Sudan III TS to the specimen on the slide,
allow it to stand for a few minutes or warm gently. Suberized or cuticular cell walls are stained
orange-red or red.
(c) Cellulose cell wall – Add 1–2 drops of zinc chloride-iodine TS and allow to stand for few minutes;
alternatively, add 1–2 drops of iodine TS, allow to stand for a while, and then add dilute sulphuric
acid (66%, v/v). Cellulose cell walls are stained blue or purple.
(d) Siliceous cell wall – Add 1–2 drops of sulphuric acid, no change is observed.
Appendix III Microscopic Identification
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(7) Histochemical detection of cell contents –
(a) Starch – Add iodine TS, a blue or purple colour is observed.
(b) Aleurone – (i) Add iodine TS, a brown or yellowish-brown colour is observed; (ii) Add mercuric
nitrate TS, a brick red colour is observed. If the material is oily, render it fat-free by immersing in
and washing with ether or petroleum ether before carrying out the test.
(c) Fatty oil, volatile oil or resin – (i) Add Sudan III TS, an orange-red, red or purplish-red colour is
observed; and (ii) Irrigate the material with ethanol (90%), volatile oils are dissolved in the solvent,
while fatty oils are insoluble (except castor oil and croton oil).
(d) Inulin – Add α-naphthol in ethanol (10%, w/v) and then add sulphuric acid, the crystals of inulin
turn purplish-red and dissolve rapidly.
(e) Mucilage – Add ruthenium red TS, a red colour is observed.
(f) Calcium oxalate crystals – (i) Insoluble in dilute acetic acid (6%, v/v), soluble in dilute
hydrochloric acid (9.5–10.5%, v/v) without effervescence; (ii) Dissolve gradually in dilute sulphuric
acid (50%, v/v), needle crystals of calcium sulphate appear after stand for a moment.
(g) Calcium carbonate (stalactile) – Soluble in dilute hydrochloric acid (9.5–10.5%, v/v) with
effervescence.
(h) Silica – Insoluble in sulphuric acid.
(8) Preparation of test solutions (TS) for microscopic analysis –
(a) Chloral hydrate TS – Dissolve 50.0 g chloral hydrate in a mixture of 15 mL of water and 10 mL
of glycerine.
(b) Cuoxam TS – Add a sufficient amount of water to 0.5 g of copper carbonate and grind in a mortar,
then add 10 mL of strong ammonia solution to dissolve.
(c) Ferric chloride TS – Dissolve 1.0 g of ferric chloride in water and make up to 100 mL.
Appendix III Microscopic Identification
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(d) Fuchsin glycerine gelatin – Dissolve 1.0 g of animal gelatin in 6 mL of water, then add 7 mL of
glycerine and heat with gentle stirring until well mixed; after filter through a piece of gauze into a
Petri dish, add sufficient amount of basic fuchsin solution (dissolve 0.1 g of basic fuchsin in
600 mL of absolute ethanol and 80 mL of camphor oil), then mix well and allow to solidify.
(e) Glycerol-acetic acid TS – Mix well equal volumes of glycerine, glacial acetic acid and water.
(f) Iodine TS – Use 0.1 M iodine solution directly.
(g) Mercuric nitrate TS – Add 3 mL of fuming nitric acid to 4.5 g of mercury, when the reaction is
completed, dilute with an equal volume of water. Preserved in an amber-coloured glass bottle
with a glass stopper and protected from light.
(h) α-Naphthol TS – To 10.5 mL of a solution of α-naphthol in ethanol (15%, w/v), gently add
6.5 mL of sulphuric acid and mix well, then add 40.5 mL of ethanol and 4 mL of water, mix well.
(i) Phloroglucinol TS – Dissolve 1.0 g of phloroglucinol in 100 mL of ethanol (90%) and then filter.
Preserved in an amber-coloured glass bottle and protected from light.
(j) Ruthenium red TS – Add a sufficient quantity of ruthenium red to 1–2 mL of aqueous sodium
acetate solution (10%, w/v) to make a wine red colour. Freshly prepare the solution.
(k) Sudan III TS – Dissolve 0.01 g of Sudan III in 5 mL of ethanol (90%), then add 5 mL of glycerine
and mix well. Preserved in an amber-coloured glass bottle, use within 2 months.
(l) Tissue-disintegrating solution (chromic-nitric acid TS) – (i) Add 10 mL of nitric acid to
100 mL of water and mix well; and (ii) Dissolve 10 g of chromic trioxide in 100 mL of water. Mix
equal volumes of the above solutions (i) and (ii) prior to use.
(m) Zinc chloride-iodine TS – Dissolve 8.0 g of potassium iodide in 8.5 mL of water and add 2.5 g of
anhydrous zinc chloride, the mixture is then saturated with sufficient quantity of iodine. Preserved
in an amber-coloured glass bottle with a glass stopper.
Appendix III Microscopic Identification
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Appendix IV(A) Chromatography – Thin-Layer Chromatography (TLC)
Appendix IV(A): Chromatography - Thin-Layer Chromatography (TLC)
TLC is a separation technique in which a stationary phase consisting of an appropriate material is spread in a
uniform thin layer on a support (plate) of glass, plastic or aluminum film. Standard and test solutions are
applied separately on the plate and the components are separated by the developing solvent systems. For the
identification of CMM, separated spots obtained from the test solution are compared with the corresponding
spots obtained from the chemical reference substance(s) in the chromatogram.
(1) Apparatus and materials –
(a) TLC plates – The most commonly used coated plates are silica gel G, silica gel F254
, HPTLC
silica gel F254
, silica gel H and silica gel HF254
. Diatomaceous earth, diatomaceous earth G, aluminum
oxide, aluminum oxide G, microcrystalline cellulose and microcrystalline cellulose F254
etc., can
be used as well. Coated plates with the size of 10�5 cm; 10�10 cm; 10�15 cm; 20�10 cm
or 20� 20 cm are commonly used.
(b) Application devices – Micropipettes, micro-syringes, calibrated capillaries or other suitable
application devices can be used for the proper application of solutions to the plates.
(c) Developing chamber – A tank of size suitable for the plates, with a tightly fitting lid and with a
flat bottom or twin trough is usually used.
(d) Spray reagents – Spray reagent for the detection of spots is specified in the individual monograph.
(e) Ultraviolet (UV) light source – An emitting light source in the UV range is used for the examination
of spots in the chromatogram.
(2) Procedure –
(a) Saturation of the developing chamber – Unless otherwise specified, carry out the chromatography
in a saturated chamber. To achieve saturation, pour sufficient amount of the developing solvents
into the developing chamber, replace the lid and allow it to stand for 15-30 min at room temperature.
If necessary, line the inner walls of the developing chamber with filter paper strips, the lower
edges of the filter papers should be immersed in the developing solvents.
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(b) Applying the standard and test solutions – Apply separately the prescribed volumes of the
standard and test solutions in small portions to obtain spots (less than 3 mm in diameter) or bands
on a line parallel to, and 15 mm from, the lower edge of the plate. Pay attention not to apply spots
or bands less than 15 mm from the sides of the plate and no disturbance of each other should
occur.
(c) Developing a chromatogram – Place the plate in the chromatographic tank after the solvent has
evaporated from the applied solutions, ensuring that the sample line are 5 mm above the surface of
the developing solvents. Then cover the chamber tightly with a lid. Remove the plate from the
chamber when the developing solvents have moved over the distance as prescribed in the individual
monograph. Dry the plate and visualize the chromatogram under visible light and/or UV light as
specified in the individual monograph.
(d) Interpretation of the chromatogram – Compare the principal spots or bands observed from the
test solution with the corresponding spots or bands observed from the standard solutions. For
positive identification, the sample must give spots or bands with chromatographic characteristics,
including the colour and the Rf value, similar to those of the chemical reference substances when
observed under visible light and/or UV light as specified in the individual monograph.
The Rf value is defined as the ratio of the distance from the point of application to the centre of the
spot or band to the distance travelled by the solvent front from the point of application:
Rf
=Distance from the point of application to the centre of the spot or band
Distance travelled by the solvent front from the point of application
Appendix IV(A) Chromatography – Thin-Layer Chromatography (TLC)
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Appendix IV(B) Chromatography –High-Performance Liquid Chromatography (HPLC)
Appendix IV (B): Chromatography – High-Performance Liquid Chromatography (HPLC)
HPLC is a separation technique consisting of a solid stationary phase and a liquid mobile phase. The sample
is injected through an injector and carried into the column by the mobile phase, the components are separated
on the stationary phase and pass through the detector in succession, a chromatogram is recorded.
(1) Preparation of test sample – Powder the CMM sample and pass through a No.2 sieve before analysis.
The quantity of the sample to be powdered should be of at least five times as much as those needed for
the analysis.
(2) General requirements for the apparatus – Set up the stationary and mobile phases of the HPLC as
specified in the individual monograph. One of the most commonly used packing material is ODS
chemically bonded to silica. Ion exchange resins are used for ion exchange chromatography and porous
silica or polymers are used for size exclusion chromatography. The column is usually maintained at
room temperature and an UV photometer is used as a detector.
The types of stationary phase, mobile phase and detector as specified in the individual monograph should
not be varied. Other parameters may be varied to fit for the performance of the system suitability test
when necessary.
(3) System suitability test – This is to test the suitability of the instruments according to the requirements
prescribed in the individual monograph. By using specified chemical reference substances, adjust the
following parameters to comply with the requirements specified in the individual monographs, i.e. to
match the n value, the repeatability, the R value and the T value of the column.
(a) Number of theoretical plates of the column (n) – The n value is a measure of the column efficiency.
It should not be less than the value specified in the individual monograph. The n value is calculated
by using the following equation –
Where tR
= the retention time of the marker peak in the standard solution or analyte
peak in the test solution,
Wh / 2
= the peak width at half-height of the marker peak in the standard solution
or analyte peak in the test solution.
n = 5.54t
R
W h / 2
( ) 2
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(b) Repeatability – The repeatability is expressed as an estimated RSD of at least five replicate
injections of the standard solution. The RSD of the peak area and the retention time should comply
with the requirements specified in the individual monograph.
(c) Resolution factor (R) – To ensure the accuracy of quantitative analysis, the R value (Fig. 1) of the
analyte peak with the adjacent peak must be larger than 1.5, unless otherwise specified. The R
value is calculated by using the following equation –
R =2( t
R2 - t
R1 )
W1 + W
2
Where tR1
and tR2
= the retention times of two adjacent peaks 1 and 2, respectively,
W1 and W
2= the widths of two adjacent peaks 1 and 2, respectively.
(d) Tailing factor (T) – It is necessary to inspect the T value (Fig. 2) of the peak, especially when
using the peak height method. It should comply with the requirement specified in the individual
monograph. The T value is calculated by using the following equation –
T =W
0.05h
2d1
Where W0.05h
= the peak width at 0.05 of the peak height,
d1
= the distance between the perpendicular line passing through the peak
maximum and the leading edge of the peak at 0.05 of the peak height.
(4) Quantitative procedure – Set up the HPLC system according to the procedures described in the
manufacturer’s manuals. Under the recommended HPLC conditions, establish the calibration curves by
injecting an appropriate amount of standard solutions of a series of concentrations into the HPLC system
for analysis. Identify the analyte peaks in the chromatogram of the test solution by comparing their
retention times with those of the peaks of the chemical reference substances in the chromatogram of the
standard solution obtained under the same HPLC conditions as specified in the procedure. Alternatively,
spike an appropriate amount of chemical reference substance in one of the analyzing samples to verify
the identified peak.
Prepare a 5-point calibration curve by plotting the peak areas of the chemical reference substance against
the corresponding concentrations (in milligram per litre) of the standard solutions. Obtain the slope, y-
intercept, the regression equation and the r2 value from the calibration curve. With the calibration curve
Appendix IV(B) Chromatography –High-Performance Liquid Chromatography (HPLC)
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of the corresponding chemical reference substance, calculate the concentration (in milligram per litre) of
the analyte in the test solution by using the following equation –
Concentration of the analyte =A - I
m
Where A = the peak area of the analyte in the test solution,
I = the y-intercept of the 5-point calibration curve,
m = the slope of the 5-point calibration curve.
Calculate the percentage content of the analyte in the sample by using the following equation –
Content (%) of the analyte =C � V � D
10000 W
Where C = the concentration, in mg/L, of the analyte in the test solution,
D = dilution factor, if any,
V = the final make-up volume, in mL, of the test solution,
W = the weight, in g, of the sample used for the preparation of the test solution.
Appendix IV(B) Chromatography –High-Performance Liquid Chromatography (HPLC)
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Figure 2 Parameters for calculation of tailing factor (T)
Appendix IV(B) Chromatography –High-Performance Liquid Chromatography (HPLC)
Figure 1 Parameters for calculation of resolution factor (R)
tR1 t
R2
W1 W
2
Wh / 2
W0.05h
d1
0.05
h
h
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Appendix IV (C): Chromatography – Gas Chromatography (GC)
GC is a separation technique consisting of gaseous mobile phase and a solid or immobilized liquid stationary
phase. The sample is introduced through the sample injection port, heated and vaporized, and carried into the
column by a carrier gas. Components of the test sample are separated in the column and pass through the
detector in succession, a chromatogram is thus recorded.
(1) General requirements of the apparatus – Unless otherwise specified, nitrogen is employed as a carrier
gas. A packed column or a capillary column may be employed for the test. A packed column is made of
stainless steel or glass. The stationary phase of the column consists of active adsorbent, porous polymer
beads or inert solid supports impregnated with liquid phase. A capillary column is made of glass or
quartz with internal diameter of 0.2 or 0.32 mm. The stationary phase may be coated or chemically
bonded to the inner surface of a column or supporting materials. The temperature of the sample injection
port is usually set at 30–50°C higher than that of the column itself. The volume of solution injected is
usually no more than several micro-litres, the smaller the diameter of the column, the smaller the volume
of solution is injected. Flame-ionization detector, electron-capture detector and mass spectrometric detector
can be used to detect the separated components. The temperature of the detector is higher than that of the
column itself, usually set at about 250–350°C, but never below 100°C. This will avoid condensation of
the moisture.
Parameters including the type of the detector, stationary phase and the supporting material of the column
as specified in the individual monograph should not be varied. Other parameters may be varied to fit for
the performance of the system suitability test. These include the internal diameter and the length of the
column; the commercial brand and size of the supporting materials; the concentration of the liquid
stationary phase; the flow rate of the carrier gas; the temperature of the column; the quantity of the
injecting and the sensitivity of the detector, etc.
(2) System suitability test – The criteria for assessing the suitability of the system are the same as those set
out in Appendix IV(B).
(3) Procedure – The procedure is the same as those set out in Appendix IV(B). Pay special attention to the
effect of the change in room temperature and the injection time.
Appendix IV(C) Chromatography – Gas Chromatography (GC)
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Appendix V Determination of Heavy Metals
Appendix V: Determination of Heavy Metals
Heavy metals refer to the heavy metals and their respective compounds arising from external contamination,
and are absorbed and accumulated in CMM. Arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) are
those heavy metals with relatively high toxicity to human beings.
Method –
(1) Analysis of heavy metals – The analytical procedures must be validated and satisfy with all of the
following criteria –
(a) the selected method is suitable for the analysis of the targeted heavy metals;
(b) the limits of detection and quantification are determined for each targeted heavy metal;
(c) the limit of quantification for each targeted heavy metal is set at 0.05 mg/kg;
(d) the recovery for each targeted heavy metal is between 75 and 125%;
(e) the repeatability of the method is less than 15% RSD; and
(f) a linear response is obtained from the analytical detector within the calibration range.
(2) Reagents – All reagents used should be of analytical grade or equivalent and free from any contaminant
which may interfere with the analysis.
(3) Apparatus – Before using the laboratory wares which have been in contact with the samples, the standard
and test solutions, clean them with dilute acids and then rinse them with distilled and de-ionized water.
(4) Preparation of test sample – Take a representative CMM sample and cut it into pieces, if necessary,
before grinding. Powder the sample before the analysis. Whenever possible, the quantity of sample to be
powdered should be of at least five times as much as those needed for the analysis.
(5) Procedure – The following procedures are applicable for the quantitative detection of As, Cd, Pb and Hg
contents in CMM samples. It may have to modify the procedures for the analysis of some samples.
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(a) Microwave assisted acid digestion – Weigh accurately 0.5 g of the sample in a PTFE microwave
digestion vessel, add 7.5 mL of nitric acid. Allow the vessel to stand a while until the reaction
ceases, then seal all vessels properly and place the completed vessel modules in the turntable of
the microwave unit. Start the digestion programme, the selection of low-pressure or high-pressure
microwave assisted acid digestion depends on the type of microwave digestion vessel available in
the individual laboratory. Upon the completion of the programme, cool the mixture and vent the
vessel manually. Transfer the digested sample solution to a 50-mL volumetric flask and make up
to the mark with water, then transfer the solution to a centrifuge tube and centrifuge for 5 min.
Pipette 10 mL of this solution into another 50-mL volumetric flask and make up to the mark with
water. This is the test solution for subsequent instrumental analysis.
(b) Quantitative analysis – Quantify the heavy metals by using ICP-MS with indium (In) as an
internal standard. Internal standards other than In can also be used provided that the method is
properly validated.
Use an ICP-MS system that satisfies with all of the following criteria –
• a resolution better than or equal to 0.7 amu at 10% peak height;
• a mass range from at least 6 to 240 amu and a mass accuracy of ±0.05 amu; and
• a data system that allows correction for isobaric interferences and with an application of internal
standard technique.
Prepare at least four standard solutions in dilute nitric acid (3%, v/v) containing all the targeted
heavy metals at concentrations suitable for plotting calibration curves.
Note: The concentration of the internal standard in the test solution should be same as those in
the standard solutions.
The suggested operation parameters are as follows –
Nebulizer Gas Flow : ~ 0.9 L/min
Auxiliary Gas Flow : ~ 1.2 L/min
Plasma Gas Flow : ~ 15 L/min
Integration Time : 1000 ms
ICP RF Power : 1200 W
Detector : Dual mode
Scan Mode : Peak hopping
Appendix V Determination of Heavy Metals
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Set up and optimize the ICP-MS according to the manufacturer’s recommended procedures.
Calibrate the instrument with the mixed standard solution. Prior to data collection, flush the system
with rinse blank until the signal level returns to the calibration blank level. Where appropriate,
several isotopes of an element may be monitored with appropriate signal correction to counter-
check the presence of spectral interferences. The isotopes recommended for monitoring the heavy
metals are listed in Table 1. The calculation for As, Cd and Hg contents should be based on the
signals of isotopes of 75 m/z, 114 m/z and 202 m/z, respectively. For Pb, the calculation should be
based on the summation of the signal of the isotopes 206 m/z, 207 m/z, and 208 m/z.
Table 1 Recommended isotopes for monitoring the heavy metals
Heavy Metal Isotope for Monitoring (m/z)
Arsenic 75
Cadmium 111, 114
Lead 206, 207, 208
Mercury 200, 202
Limits – The amount of heavy metals in CMM samples should comply with the limits listed in Table 2 below,
unless otherwise specified.
Table 2 The recommended limits of heavy metals in CMM samples
Heavy Metal Limit (Not more than)
Arsenic 2.0 mg/kg
Cadmium 0.3 mg/kg
Lead 5.0 mg/kg
Mercury 0.2 mg/kg
Appendix V Determination of Heavy Metals
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Appendix VI Determination of Pesticide Residues
Appendix VI: Determination of Pesticide Residues
Pesticide is a synthetic chemical, a natural or biological substance, or a mixture thereof, used for prevention,
termination and/or control of diseases, pests, grass or other living things which are hazardous to agriculture
and forestry; or for regulation of the growth of plants and pests in an intended way.
The targeted pesticides for the analysis of pesticide residues in CMM are listed as follows –
(a) Aldrin and Dieldrin (sum of)
(b) Chlordane (sum of cis-, trans- and oxychlordane)
(c) Dichlorodiphenyltrichloroethane (DDT) [sum of p,p’-DDT, o,p’-DDT,
p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE) and
p,p’-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p’-TDE)]
(d) Endrin
(e) Heptachlor (sum of heptachlor and heptachlor epoxide)
(f) Hexachlorobenzene
(g) Hexachlorocyclohexane isomers (α-, β- and δ-hexachlorocyclohexane)
(h) Lindane (γ-hexachlorocyclohexane)
(i) Quintozene (sum of quintozene, pentachloroaniline and methyl pentachlorophenyl sulphide)
Method –
(1) Analysis of pesticide residues – The analytical procedures must be validated and satisfy with all of the
following criteria –
(a) the selected method is suitable for the analysis of the targeted pesticides;
(b) the limits of detection and quantification are determined for each targeted pesticide;
(c) the limit of quantification for each targeted pesticide is 0.02 mg/kg. Except for cis-chlordane,
trans-chlordane and oxychlordane, each of which is set at 0.01 mg/kg;
(d) the recovery for each targeted pesticide is between 70 and 120%;
(e) the repeatability of the method is less than 15% RSD; and
(f) a linear response is obtained from the analytical detector within the calibration range.
A - 18
(2) Reagents – All reagents used should be of analytical grade or equivalent and free from any contaminant
which may interfere with the analysis. Suitable blank tests should be conducted to demonstrate no
occurrence of contamination of the pesticide residues.
(3) Apparatus – All apparatus to be used should be thoroughly cleaned to ensure that they are free from any
pesticides. Soak the apparatus in a solution of phosphate-free detergent for at least 16 h, then rinse them
with a large quantity of distilled water and wash them with acetone.
(4) Preparation of test sample – Take a representative CMM sample and cut it into pieces, if necessary,
before grinding. Powder the sample before the analysis. Whenever possible, the quantity of the sample to
be powdered should be of at least five times as much as those needed for the analysis.
(5) Procedure – The following procedures are applicable for the quantitative detection of pesticide residues
in CMM samples. It may have to modify the procedures for the analysis of some samples. Wherever
possible, it is necessary to use a second capillary column with different polarities and/or MS to confirm
the analytical results.
(a) Extraction – Weigh accurately 10.0 g of the blended sample powder, add about 4.0 g of anhydrous
sodium sulphate and about 100 mL of ethyl acetate. Sonicate in pulse mode by using an ultrasonic
processor for 3 min. Allow the solids to settle and then filter the supernatant solution and collect
the filtrate. Repeat the extraction twice each with 50 mL of ethyl acetate. Combine the filtrates
and the washings and then evaporate to near dryness in a rotary evaporator at about 35°C. Dissolve
the residue in 10 mL of a mixture of dichloromethane and cyclohexane (1:1, v/v) (Solution A).
(b) Clean-up –
(i) Gel permeation chromatography – The chromatographic procedure may be carried out by
using –
• a Bio-beads S-X3 glass column, 60 g in weight and 43 cm in length, or equivalent; and
• a mixture of dichloromethane and cyclohexane (1:1, v/v) as the mobile phase.
Performance of the column – Inject a solution containing corn oil (about 25 mg/mL), bis(2-
ethylhexyl)phthalate (about 1 mg/mL), methoxychlor (about 0.2 mg/mL) and perylene (about
0.02 mg/mL) and proceed with the chromatography. The column is not suitable unless the
resolution of any adjacent peaks is ≥ 0.85. If necessary, calibrate the column using a solution
containing the pesticides [at a suitable concentration and in a mixture of dichloromethane
and cyclohexane (1:1, v/v)] with the lowest molecular weight (for example pentachloroaniline)
Appendix VI Determination of Pesticide Residues
A - 19
and that with the highest molecular weight (for example oxychlordane). Determine which
fractions of the eluate contain the target pesticides.
Purification of the test solution – To 10 mL of solution A, add about 1.0 g of anhydrous
sodium sulphate, centrifuge the mixture and get the supernatant layer. Inject an appropriate
volume of the extract and proceed with the chromatography. Collect the fraction as determined
above. Concentrate the solution in a rotary evaporator on a water bath at a temperature below
35°C until the solvent has almost completely evaporated. Then dissolve the residue in 1 mL
of hexane (Solution B).
(ii) Solid phase extraction – The chromatographic procedure may be carried out by using –
• a florisil solid phase extraction column, 75–150 µm in diameter and 1000 mg in weight,
or equivalent; and
• a solution of diethyl ether in hexane (15%, v/v) as the eluting solvent.
If necessary, calibrate the column by using a solution in hexane containing suitable
concentrations of the targeted pesticides. Determine the fractions of the targeted pesticides
from the eluate.
Pack about 10 mm of anhydrous sodium sulphate on the top of the florisil column. Condition
the column with about 5 mL of hexane. Transfer quantitatively solution B onto the florisil
column and proceed with the chromatography. Collect the eluate (Solution C).
(c) Quantitative and qualitative analysis – Examined by GC using 2,4,5,6-tetrachloro-m-xylene as
an internal standard. Another internal standard may be needed if interferences occur.
Use the gas chromatograph that satisfies with all of the following criteria –
• the R value of any analyte peak with the adjacent peak: > 1.5;
• the n value: ≥ 100000 for the peak of α-hexachlorocyclohexane; and
• the RSD of the peak area: ≤ 5%.
Solution (1): Prepare at least five standard solutions in isooctane containing 2,4,5,6-
tetrachloro-m-xylene and all the targeted pesticides at concentrations suitable for plotting
calibration curves.
Appendix VI Determination of Pesticide Residues
A - 20
Solution (2): Concentrate solution C in a stream of nitrogen to almost dryness and dilute to
1 mL with isooctane containing 2,4,5,6-tetrachloro-m-xylene as an internal standard [Notes
1 and 2].
Note 1: The concentration of the internal standard in the test solution should be same as
those in the standard solutions.
Note 2: The sulphuric acid treatment in combination with copper powder treatment may
prove useful to remove certain matrix interference arisen from the sample matrix. However,
this treatment will destroy or remove certain targeted pesticides such as aldrin, dieldrin,
endrin, heptachlor epoxide, methyl pentachlorophenyl sulphide and pentachloroaniline.
The chromatographic procedure may be carried out by using –
• a capillary column (0.25 mm � 30 m) of which the internal wall is covered with (14%-
cyanopropylphenyl)-methylpolysiloxan in a layer about 0.25 µm thick;
• a second capillary column of different polarities (0.25 mm � 30 m) of which the internal
wall is covered with (5%-phenyl)-methylpolysiloxane in a layer about 0.25 µm thick;
• nitrogen as the carrier gas;
• an electron-capture detector; and
• a device allowing split/splitless injection. After maintaining the temperature of the column
at 100ºC for 2 min, raise it to 165ºC at a rate of 10ºC/min and maintain at this temperature
for 10 min. Raise the temperature to 230ºC at a rate of 3ºC/min and afterward to 280ºC at
a rate of 15ºC/min, then maintain at this temperature for 10 min. Maintain the temperature
of the injector port at 210ºC and the temperature of the detector at 300ºC.
In the prescribed conditions, inject 1 µL or other appropriate volume of each solution and
record the chromatograms. The reference RRTs of the targeted pesticides obtained are listed
in Table 1. Calculate the content of each targeted pesticide from its peak area and concentration.
Appendix VI Determination of Pesticide Residues
A - 21
The results obtained can be confirmed by GC-MS.
The chromatographic procedure may be carried out by using –
• a capillary column (0.25 mm � 30 m) of which the internal wall is covered with (35%-
phenyl)-methylpolysiloxane in a layer about 0.25 µm thick;
• helium as the carrier gas;
• a mass selective detector capable of operating in a scan mode or selective ion mode (m/z
of the monitoring ions for the targeted pesticides are listed in Table 2 for reference); and
• a device allowing split/splitless injection. Maintain the temperature of the column at
100ºC for 2 min, raise to 160ºC at a rate of 15ºC/min and afterward to 270ºC at a rate of
5ºC/min, then maintain at this temperature for 10 min. Maintain the temperature of the
injector port at 250ºC and the temperature of the ion source at 230ºC.
In the prescribed conditions, inject 1 µL or other appropriate volume of each solution and
record the chromatograms. The reference RRTs of the targeted pesticides obtained are listed
in Table 2.
Appendix VI Determination of Pesticide Residues
A - 22
Table 1 The reference RRTs of the targeted pesticides obtained by GC
RRT
Pesticide [column used: 0.25 mm � 30 m,
(14%-cyanopropylphenyl)-methylpolysiloxane of 0.25-µm thick]
Hexachlorobenzene 1.24
α-Hexachlorocyclohexane 1.55
Quintozene 1.64
Lindane 1.83
Heptachlor 1.94
Pentachloroaniline 2.01
Aldrin 2.09
Methyl pentachlorophenyl sulphide 2.10
β-Hexachlorocyclohexane 2.32
Oxychlordane 2.41
δ-Hexachlorocyclohexane 2.43
Heptachlor epoxide 2.50
trans-Chlordane 2.67
cis-Chlordane 2.71
p,p’-DDE 2.76
Dieldrin 2.82
Endrin 2.92
o,p’-DDT 2.98
p,p’-TDE 3.15
p,p’-DDT 3.21
Appendix VI Determination of Pesticide Residues
A - 23
Table 2 The reference RRTs and the monitoring ions of the targeted pesticides obtained by GC-MS
Pesticide RRTPrimary Ion, Secondary Ion,
m/z m/z
Hexachlorobenzene 1.18 284 286, 282
α-Hexachlorocyclohexane 1.22 181 183, 217
Quintozene 1.32 237 249, 214
Lindane 1.35 183 217, 221
β-Hexachlorocyclohexane 1.45 181 183, 217
Heptachlor 1.48 272 274, 270
Pentachloroaniline 1.49 265 267, 263
δ-Hexachlorocyclohexane 1.55 181 183, 217
Aldrin 1.58 263 265, 261
Methyl pentachlorophenyl sulphide 1.63 296 246, 263
Oxychlordane 1.74 185 387, 237
Heptachlor epoxide 1.79 353 355, 351
trans-Chlordane 1.87 373 375, 377, 371
cis-Chlordane 1.91 373 375, 377, 371
p,p’-DDE 2.00 246 316, 248
Dieldrin 2.03 263 261, 265
Endrin 2.14 263 265, 281
o,p’-DDT 2.17 235 237, 165
p,p’-TDE 2.20 235 237, 165
p,p’-DDT 2.30 235 237, 165
Appendix VI Determination of Pesticide Residues
A - 24
Limits – The amount of pesticide residues in CMM samples should comply with the limits listed in Table 3
below.
Table 3 The recommended limits of pesticide residues in CMM samples
Pesticide Limit (Not more than)
Aldrin and Dieldrin (sum of) 0.05 mg/kg
Chlordane (sum of cis-, trans- and oxychlordane) 0.05 mg/kg
DDT (sum of p,p’-DDT, o,p’-DDT, p,p’-DDE and p,p’-TDE) 1.0 mg/kg
Endrin 0.05 mg/kg
Heptachlor (sum of heptachlor and heptachlor epoxide) 0.05 mg/kg
Hexachlorobenzene 0.1 mg/kg
Hexachlorocyclohexane isomers (α-, β- and δ- hexachlorocyclohexane) 0.3 mg/kg
Lindane (γ-hexachlorocyclohexane) 0.6 mg/kg
Quintozene (sum of quintozene, pentachloroaniline and methyl1.0 mg/kg
pentachlorophenyl sulphide)
Appendix VI Determination of Pesticide Residues
A - 25
Appendix VII Determination of Mycotoxins (Aflatoxins)
Appendix VII: Determination of Mycotoxins (Aflatoxins)
Mycotoxins, including aflatoxins, refer to the toxic metabolites generated by molds and/or fungi. Since there
is concern over the contamination of aflatoxins in CMM, a harmonized method is developed to study the
contents of aflatoxins B1, B
2, G
1 and G
2 in CMM.
Methods –
(1) Analysis of aflatoxins – The analytical procedures must be validated and satisfy with all of the following
criteria –
(a) the selected method is suitable for the analysis of the targeted aflatoxins and is not susceptible to
the interference from co-extractives;
(b) the limits of detection and quantification are determined for each aflatoxin;
(c) the limit of quantification for each targeted aflatoxin is set at 0.3 µg/kg;
(d) the recovery for each targeted aflatoxin is between 50 and 120%;
(e) the repeatability of the method is less than 15% RSD; and
(f) a linear response is obtained from the analytical detector within the calibration range.
(2) Reagents – All reagents used should be of analytical grade or equivalent. Methanol and acetonitrile used
should be at least of HPLC grade.
(3) Apparatus – All apparatus to be used should be thoroughly cleaned to ensure that they are free from any
aflatoxins. Soak the laboratory wares in a solution of household bleach (10%, v/v) for at least 12 h and
then wash them with distilled water.
(4) Preparation of test sample – Take a representative CMM sample and cut it into pieces, if necessary,
before grinding. Powder the sample before the analysis. Whenever possible, the quantity of the sample to
be powdered should be of at least five times as much as those needed for the analysis.
(5) Procedure – The analysis of aflatoxins is based on the detection of the characteristic fluorescence emitted
by aflatoxins B1, B
2, G
1, and G
2 after iodine derivatization and UV excitation. It may have to modify the
procedures for the analysis of some samples.
A - 26
(a) Extraction – Weigh accurately 15.0 g of the blended sample, add 3 g of sodium chloride and
75 mL of a mixture of methanol and water (7:3, v/v). Homogenize the mixture for about 2 min and
centrifuge for about 10 min. Check the pH of the extract. Transfer accurately 15 mL of the
supernatant solution of the centrifuged mixture to an amber bottle and reduce to about 5 mL with
a gentle stream of nitrogen at about 60˚C on a water bath [Note 1]. Make up the resultant solution
to 50 mL with a solvent that is compatible with the performance of the immunoaffinity column as
suggested by the manufacturer [Note 2]. Centrifuge the solution for about 10 min and filter through
a glass-fibre filter paper by suction. Collect the filtrate as the test solution.
Note 1: If the sample absorbs solvent significantly (i.e. the volume of the resultant supernatant is
less than 40 mL), repeat the extraction as directed above. However, use 5.0 g of sodium chloride
and 125 mL of a mixture of methanol and water (7:3, v/v) instead, and transfer 25 mL of the
supernatant solution.
Note 2: When handling samples that produce extracts which might affect the normal functioning
of the immunoaffinity column, seek manufacturer’s advice to ensure the column performance. Pay
particular attention to the suggested pH working range of the column and the solvent used for
diluting the sample extract prior to column clean-up.
(b) Clean-up by immunoaffinity column chromatography –
Chromatographic procedure – It may be carried out by using –
• an immunoaffinity column containing antibodies specific for aflatoxins B1, B
2, G
1 and G
2; and
• methanol as the eluting solution.
Performance test of the column – Pass an appropriate volume of the mixed standard solution
containing aflatoxins B1, B
2, G
1 and G
2 through the column, then follow the procedure as described
below ‘Clean-up of the test solution’, and the recovery of the aflatoxins B1, B
2, G
1 and G
2 should
be at least 90%, 80%, 90% and 60% respectively.
Clean-up of the test solution [Note 3] – Condition the column according to the manufacturer’s
instruction, then pass a selected volume of the test solution through the column at a flow rate of
about 3 mL/min. Wash the column with 10 mL of eluent, as recommended by the manufacturer, at
a flow rate of about 3 mL/min, then wash dry the column by passing 10 mL of air through the
column. Elute the column with 1.5 mL of methanol followed by 10 mL of air. Collect all eluates in
a 2-mL volumetric flask and make up to the mark with water.
Appendix VII Determination of Mycotoxins (Aflatoxins)
A - 27
Note 3: It may have to modify the clean-up procedures for different brands of immunoaffinity
column. Please refer to manufacturer’s instruction.
(c) Quantitative analysis – Use a HPLC system that satisfies with all of the following criteria –
• the R value of any analyte peak with the adjacent peak: > 1.5;
• the n value of any analyte peak: ≥ 7000; and
• the RSD of peak area: ≤ 5%.
Individual aflatoxin standard stock solutions – Determine the concentration of individual
aflatoxin stock standard solution (about 10 mg/L) in a mixture of benzene and acetonitrile (98:2,
v/v) by UV spectroscopy according to the following equation –
Concentration of aflatoxin (mg/L) =A
350 � M
w � 1000
ε
Where A350
= the absorbance of the aflatoxin at a wavelength of maximum absorption
close to 350 nm,
Mw
= the molecular weight of the aflatoxin (Table 1),
ε = the molar absorptivity of the aflatoxin in benzene-acetonitrile solution
(Table 1).
Table 1 The molecular weights (Mw) and molar absorptivities (ε) of the aflatoxins
Aflatoxin Molecular Weight ( Mw) Molar Absorptivity ( ε)
B1
312 19800
B2
314 20900
G1
328 17100
G2
330 18200
Mixed aflatoxins standard solutions – Prepare at least five standard solutions in a mixture of
methanol and water (7:3, v/v) containing all the targeted aflatoxins at concentrations suitable for
plotting calibration curves.
Chromatographic procedure – It may be carried out by using –
• a LC column (4.6 � 250 mm) packed with particles of octadecylsilyl groups modified silica
(5 µm in diameter);
Appendix VII Determination of Mycotoxins (Aflatoxins)
A - 28
• a post-column reactor system with the reaction temperature set at 70˚C and 0.5 mM iodine
solution as post-column derivatization reagent;
• a mixture of distilled water, acetonitrile and methanol (3:1:1, v/v) as the mobile phase; and
• a fluorescence detector: λext
= 360 nm and λem
= 450 nm.
Set the flow rate of the mobile phase (LC column) as 1.0 mL/min and the flow rate of the post-
column derivatization reagent as 0.3 mL/min. Under such conditions, aflatoxins are eluted in the
order of G2, G
1, B
2, and B
1 with retention times of about 13, 16, 18, and 23 min, respectively. If
necessary, adjust the retention times by changing the solvent composition. Calculate the content
of each aflatoxin from its peak area and concentration.
Note: Soak all used laboratory wares in a 10% solution of household bleach overnight before
reuse or disposal.
Limits – The amount of aflatoxin B1 and the total amount of aflatoxins (B
1, B
2, G
1 and G
2) in CMM samples
should comply with the limits listed in Table 2 below.
Table 2 The recommended limits of aflatoxins in CMM samples
Aflatoxin Limit (Not more than)
Aflatoxin B1
5 µg/kg
Aflatoxins (sum of B1, B
2, G
1 and G
2 ) 10 µg/kg
Appendix VII Determination of Mycotoxins (Aflatoxins)
A - 29
Appendix VIII Determination of Foreign Matter
Appendix VIII: Determination of Foreign Matter
Foreign matter is material consisting of any of the following –
(1) The biological origin of which is the same as that specified in the monograph concerned but the appearance
or botanical part is different.
(2) The biological origin of which differs from that specified in the monograph concerned.
(3) Foreign mineral matters such as stones, sand, lumps of soil.
Method and Procedure –
(1) Weigh 100–500 g of CMM sample and spread in a thin layer. Sort the foreign matter into groups either
by visual inspection, using a magnifying lens (5–10�), or with the help of a suitable sieve.
(2) Weigh each group of foreign matter separately, and calculate the percentage of foreign matter in the
weight of CMM sample.
Note 1: In case of close resemblance between the foreign matters and the bulk sample in appearance, use
microscopic, physical or chemical methods to identify the foreign matter.
Note 2: For large-sized sample, cut it off when necessary, so as to examine any signs of spoilt or contamination
by insects and moulds.
Limits – The amount of foreign matter in CMM samples should not be more than the percentage specified in
the individual monograph.
A - 30
Appendix IX Determination of Ash
Appendix IX: Determination of Ash
Method and Procedure –
(1) Total ash –
(a) Pulverize CMM sample, pass through a No.2 sieve and mix well. Accurately weigh 2–3 g (3–5 g
for the determination of acid-insoluble ash) of the powdered sample in a tared crucible (to the
nearest 0.01 g). Ignite the sample gently until completely carbonized, keep it from burning, then
gradually increase the temperature to 500–600°C. Continue the ignition until a constant weight of
carbon-free ash is obtained. Calculate the percentage of total ash in the weight of CMM sample.
(b) If a carbon-free ash cannot be obtained in this way, cool the crucible, and moisten the residue with
hot water or 2 mL of aqueous ammonium nitrate solution (10%, v/v), then dry the residue on a
water bath. Ignite the residue again as directed above until a carbon-free ash is obtained.
(2) Acid-insoluble ash –
To a crucible containing the total ash, add 10 mL of dilute hydrochloric acid (10%, v/v), cover with a
watch glass and gently heat for 10 min on a water bath. Rinse the watch glass with 5 mL of hot water and
add the rinsing to the crucible. Transfer the insoluble matter and rinse the remaining residues from the
crucible onto an ashless filter paper, wash with hot water until the filtrate is free of chlorides. Transfer the
ashless filter paper containing the insoluble matter to the original crucible, dry and ignite to constant
weight. Calculate the percentage of acid-insoluble ash in the weight of CMM sample.
Limits – The amount of total ash and acid-insoluble ash in CMM samples should not be more than the
percentages specified in the individual monograph.
A - 31
Appendix X Determination of Water Content
Appendix X: Determination of Water Content
Method – Toluene distillation method is used for the determination of water content in CMM samples.
(1) Preparation of test sample – Prepare a suitable quantity of test sample by cutting (or by using other
appropriate means) CMM sample into pieces of less than 3 mm in length or diameter. Flowers, seeds or
fruits of length or diameter less than 3 mm, can be used directly for the examination.
(2) Reagent – Toluene used in this method should be saturated with water and distilled.
(3) Apparatus – The apparatus (Fig. 1) consists of a 500-mL round-bottomed flask (A); a graduated receiving
tube (B); and a reflux condenser (C) of about 40 cm in length. The apparatus should be cleaned and dried
in an oven before being used.
(4) Procedure – Weigh accurately a quantity of the sample which is expected to give 2–4 mL of water,
transfer to the flask and add about 200 mL of toluene into it. When necessary, add a few pieces of glass
beads. After assembly of the apparatus, fill in the narrow part of the receiving tube with toluene through
the condenser, then heat the flask gently by using an electric heater or other appropriate means. When the
toluene begins to boil, adjust the temperature to allow the distillation proceed at a rate of 2 drops per
second until the water has been completely distilled. Rinse the inside of the condenser with toluene.
Continue the distillation for five more minutes, then remove the apparatus away from the heat and allow
it to cool to room temperature. Disconnect the apparatus and dislodge any droplets of water that adhere
to the wall of the receiving tube.
Allow the receiving tube to stand a while until the water and toluene are completely separated [Note].
Record the volume of water and calculate the percentage of water content in the weight of CMM sample.
Note: A small amount of methylene blue may be added to form a bluish aqueous layer to facilitate
observation.
Limits – The water content in CMM samples should not be more than the percentage specified in the individual
monograph.
A - 32
Appendix X Determination of Water Content
Figure 1 Apparatus for the determination of water content in CMM samples
A. Round-bottomed flask
B. Graduated receiving tube
C. Reflux condenser
C
B
A
A - 33
Appendix XI Determination of Extractives
Appendix XI: Determination of Extractives
Method and Procedure –
(1) Determination of water-soluble extractives – Pulverize CMM sample, pass through a No.2 sieve and
mix well.
(a) Cold extraction method – Place 4.0 g of the powdered sample, accurately weighed, in a
250–300 mL conical flask with a stopper. Accurately add 100 mL of water, insert the stopper and
extract for 24 h. Shake frequently during the first 6 h, then allow to stand for 18 h. Filter rapidly
through a dry filter. Accurately transfer 20 mL of the filtrate to an evaporating dish, previously
dried to constant weight, and evaporate to almost dryness on a water bath, then dry at 105°C for
3 h. Cool in a desiccator for 30 min, and then weigh immediately and accurately. Calculate the
percentage of water-soluble extractives with reference to the dried CMM sample.
(b) Hot extraction method – Place 2.0–4.0 g of the powdered sample, accurately weighed, in a 100–
250 mL conical flask with a stopper. Accurately add 50–100 mL of water, insert the stopper and
weigh. Allow to stand for 1 h. Attach a reflux condenser to the flask and boil gently for 1 h, then
cool to room temperature and weigh, readjust to the original weight with water. Shake and filter
through a dry filter. Accurately transfer 25 mL of the filtrate to an evaporating dish, previously
dried to constant weight, and evaporate to dryness on a water bath, then dry at 105°C for 3 h. Cool
in a desiccator for 30 min, and then weigh immediately and accurately. Calculate the percentage
of water-soluble extractives with reference to the dried CMM sample.
(2) Determination of ethanol-soluble extractives –
(a) Cold extraction method – Proceed as in section 1(a) except by using ethanol (70%) in lieu of
water as solvent.
(b) Hot extraction method – Proceed as in section 1(b) except by using ethanol (70%) in lieu of
water as solvent.
Limits – The amount of water-soluble extractives and ethanol-soluble extractives in CMM samples should
not be less than the percentages specified in the individual monograph.
A - 34
Appendix XII Gas Chromatographic and High-Performance LiquidChromatographic Fingerprinting
Appendix XII: Gas Chromatographic and High-Performance Liquid Chromatographic
Fingerprinting
GC and HPLC fingerprinting refers to the identification of CMM samples by the examination of the GC and
HPLC chromatograms of their solvent extracts.
GC and HPLC fingerprinting have the merits of high selectivity, high sensitivity, high separation rate and
requiring only a short analytical time with a small amount of sample. In general, one or more chemical markers
and characteristic peaks can be identified in the chromatographic fingerprinting.
Method – Establishment of a chromatographic fingerprinting
(1) Appropriate extraction method and chromatographic conditions should be selected in accordance with
the nature of the active ingredients or markers contained in CMM samples. The chromatographic
fingerprinting should contain as many well resolved peaks as possible in order to provide adequate
information for the identification. The recommended run time for every GC and HPLC chromatographic
fingerprinting is not more than 60 min.
(2) In the chromatographic fingerprinting established, a well resolved peak corresponding to an available
chemical reference substance can be used as a marker peak for the calculation of the RRTs of other peaks
in the same chromatogram. When necessary, more than one marker peak may be chosen.
(3) The RRT of a characteristic peak is calculated based on the retention time of a chosen marker peak by
using the following equation –
RRT =Retention time of the characteristic peak
Retention time of the marker peak
(4) The reference fingerprint chromatogram, marker peak, characteristic peaks and acceptable ranges of a
sample is drawn up based on the test results of at least 10 batches of representative samples examined in
duplicate (i.e. a total of 20 sets of data).
(5) A fingerprint chromatogram of a sample is then established by using the above procedure. For positive
identification, the sample must give all the characteristic peaks with the RRTs falling within the acceptable
range of the corresponding peaks in the reference fingerprint chromatogram as specified in the individual
monograph.
A - 35
Appendix XIII Detection of Aristolochic Acid I
Appendix XIII: Detection of Aristolochic Acid I
Aristolochic Acid I (AAI) is a known nephrotoxin and potential carcinogen that is commonly present in herbs
derived from plants belonging to the genera of Aristolochia and Asarum of the family Aristolochiaceae. In
view of this, the Department of Health announced that importation and sale of Chinese herbs containing AAI
were prohibited starting from 1st June, 2004. There are unclear factors in local Chinese herbal market that may
lead to inappropriate or misuse of the herbs containing Aristolochic Acid. It is suggested that the trader may
apply the following method to detect AAI in the suspected Chinese herbs.
Method
Carry out the method as directed in Appendix IV(B).
Standard solution
Aristolochic acid I standard stock solution, Std-Stock (50 mg/L)
Weigh accurately 5.0 mg of aristolochic acid I CRS and dissolve in 100 mL of methanol.
Aristolochic acid I standard solution for detection
Measure accurately the volume of aristolochic acid I Std-Stock, dilute with methanol to produce solutions of
0.05 and 5 mg/L for aristolochic acid I.
Test solution
Weigh accurately 0.5 g of the powdered sample and put into a 50-mL centrifugal tube, and add 8 mL of
methanol. Sonicate (490 W) the mixture for 30 min. Centrifuge at about 1800 � g for 10 min. Transfer the
supernatant to a 25-mL volumetric flask. Repeat twice. Combine the extracts and make up to the mark with
methanol. Mix and filter through a 0.45-µm RC filter.
Chromatographic system
The liquid chromatograph is equipped with a detector (396 nm) and a column (4.6 � 250 mm) packed with
ODS bonded silica gel (5 µm particle size). The flow rate is about 1.0 mL/min. The mobile phase is a mixture
of acetonitrile and 1% acetic acid (52:48, v/v). The elution time is about 25 minutes.
System suitability requirements
Perform at least five replicate injections each with 10 µL of aristolochic acid I (standard solution for detection,
5 mg/L). The requirements of the system suitability parameters are as follows: the RSD of the peak area of
aristolochic acid I should not be more than 3.0%; the RSD of the retention time of aristolochic acid I peak
should not be more than 2.0%; the column efficiency determined from aristolochic acid I peak should not be
less than 10000 theoretical plates.
A - 36
Appendix XIII Detection of Aristolochic Acid I
Procedure
Separately inject aristolochic acid I (standard solution for detection, 0.05 mg/L) and the test solution (10 µL
each) into the HPLC system and record the chromatograms. Identify aristolochic acid I peak in the chromato-
gram of the test solution by comparing its retention time with that in the chromatogram of aristolochic acid I
(standard solution for detection). The retention times of aristolochic acid I peaks from the two chromatograms
should not differ by more than 2.0%.
Limit
Aristolochic acid I (C17
H11
NO7) should not be detected in CMM samples, unless otherwise specified.
A - 37
Appendix XIV Detection of Aconitine, Hypaconitine and Mesaconitine
Appendix XIV: Detection of Aconitine, Hypaconitine and Mesaconitine
The Aconitum diester alkaloids mainly include aconitine, mesaconitine and hypaconitine. It is commonly
present in herbs derived from plants belonging to the genera of Aconitum of the family Ranunculaceae. The
major toxicity expresses in cardiotoxicity and neurotoxicity. To reduce the toxicity of the Chinese Materia
Medica, an appropriate processing method should be adopted in order to reduce the total content of Aconitum
diester alkaloids. In view of this, Chinese herbs containing aconitine, mesaconitine and hypaconitine should
be examined and the limits of the above compounds should be established.
Method
Carry out the method as directed in Appendix IV(B).
Standard solutions
Aconitine standard stock solution, Std-Stock (1000 mg/L)
Weigh accurately 5.0 mg of aconitine CRS and dissolve in 5 mL of 0.01 M hydrochloric acid.
Hypaconitine standard stock solution, Std-Stock (2000 mg/L)
Weigh accurately 10.0 mg of hypaconitine CRS and dissolve in 5 mL of 0.01 M hydrochloric acid.
Mesaconitine standard stock solution, Std-Stock (1000 mg/L)
Weigh accurately 5.0 mg of mesaconitine CRS and dissolve in 5 mL of 0.01 M hydrochloric acid.
Mixed aconitine, hypaconitine and mesaconitine standard solution for detection
Measure accurately the volume of aconitine, hypaconitine and mesaconitine Std-Stock, mix and dilute with
0.01 M hydrochloric acid to produce a series of solutions of 0.5, 1, 2, 3, 4 mg/L for both aconitine and
mesaconitine, and 5, 10, 20, 30, 40 mg/L for hypaconitine.
Test solution
Weigh accurately 0.5 g of the powdered sample and put into a 10-mL centrifugal tube, then add accurately
5 mL of methanol (50%). Sonicate (490 W) the mixture for 60 min. Centrifuge at about 1800 � g for 5 min.
Filter through a 0.45-µm RC filter.
Chromatographic system
The liquid chromatograph is equipped with a detector (240 nm) and a column (4.6 � 250 mm) packed with
ODS bonded silica gel (5 µm particle size, pH: 1-12). The flow rate is about 1.0 mL/min. Programme the
chromatographic system as follows –
Time Ammonium bicarbonate* AcetonitrileElution
(min) solution (%, v/v) (%, v/v)
0 – 60 70 ➔ 45 30 ➔ 55 linear gradient
A - 38
*Ammonium bicarbonate solution
Dissolve 0.2 g of ammonium bicarbonate in 1 L of water and adjust the pH to 10 with 1 mL of ammonia
solution.
System suitability requirements
Perform at least five replicate injections each with 20 µL of hypaconitine (standard solution for detection,
20 mg/L). The requirements of the system suitability parameters are as follows: the RSD of the peak area of
hypaconitine should not be more than 3.0%; the RSD of the retention time of hypaconitine peak should not be
more than 2.0%; the column efficiency determined from hypaconitine peak should not be less than 30000
theoretical plates.
The R value between hypaconitine peak and the closest peak in the chromatogram of the test solution should
not be less than 1.0.
Calibration curve
Inject a series of the mixed aconitine, hypaconitine and mesaconitine (standard solution for detection, 20 µL
each) into the HPLC system and record the chromatograms. Plot the peak areas of aconitine, hypaconitine and
mesaconitine against the corresponding concentrations of the mixed aconitine, hypaconitine and mesaconitine
(standard solution for detection). Obtain the slopes, y-intercepts and the r2 values from the corresponding 5-
point calibration curves.
Procedure
Inject 20 µL of the test solution into the HPLC system and record the chromatogram. Identify aconitine peak,
hypaconitine peak and mesaconitine peak in the chromatogram of the test solution by comparing their reten-
tion times with those in the chromatogram of the mixed aconitine, hypaconitine and mesaconitine (standard
solution for detection). The retention times of aconitine peaks, hypaconitine peaks and mesaconitine peaks
from the two chromatograms should not differ from their counterparts by more than 2.0%. Measure the peak
areas and calculate the concentrations (in milligram per litre) of aconitine, hypaconitine and mesaconitine in
the test solution, and calculate the percentage contents of aconitine, hypaconitine and mesaconitine in the
sample by using the equations indicated in Appendix IV(B). Calculate the sum of the content.
Limit
The total content of aconitine (C34
H47
NO11
), hypaconitine (C33
H45
NO10
) and mesaconitine (C33
H45
NO11
) in
CMM samples should comply with the limit specified in the individual monograph.
Appendix XIV Detection of Aconitine, Hypaconitine and Mesaconitine
A - 39
Appendix XV Determination of Volatile Oil
Appendix XV: Determination of Volatile Oil
Method – Specified apparatus is used to determine the volatile oil in CMM samples.
(1) Preparation of test sample – Pulverize CMM sample, pass through No.2 or No.3 sieves and mix well,
unless otherwise specified.
(2) Apparatus – The apparatus (Fig. 1) consists of a 1000-mL (500-mL or 2000-mL) round-bottomed flask
(A), a volatile oil determination tube (B) and a reflux condenser (C). All parts are connected via ground
glass joints. The measuring tube of B is graduated in 0.1 mL. The apparatus should be cleaned before use
and all parts of apparatus should be tightly connected to avoid the loss of volatile oil.
Note: The volatile oil determination tube should be set vertically. The connecting point between the side
tube and the graduated tube is at a horizontal level.
(3) Procedure
(a) Method A – This method is used to determine the volatile oils with relative density less than 1.0.
Take a quantity of the powdered sample which is expected to give 0.5 – 1.0 mL of volatile oil,
weigh accurately to the nearest 0.01 g, and put into a round-bottomed flask. Add 300 - 500 mL of
water (or appropriate amount) and a few glass beads, shake and mix well. Connect the round-
bottomed flask to a volatile oil determination tube and then connect the volatile oil determination
tube to a reflux condenser. Add water through the top of reflux condenser until the graduated tube
of volatile oil determination tube is filled and overflows to the round-bottomed flask. Heat the
flask gently until boiling by using an electric heating jacket or other appropriate means. Continue
the gentle boiling for about 5 h until the volume of oil does not increase. Stop heating, allow it to
stand for a while. Open the stopcork at the lower part of volatile oil determination tube and run off
the water layer slowly until the oily layer is 5 mm above the zero mark. Allow to stand for at least
1 h, open the stopcock again, run off the remaining water layer carefully until the oily layer is just
on the zero mark. Record the volume of oil in the graduated tube of volatile oil determination tube
and calculate the percentage of volatile oil in CMM sample.
(b) Method B –This method is used to determine the volatile oils with relative density more than 1.0.
Add 300 mL of water and a few pieces of glass beads into a round-bottomed flask. Connect the
round-bottomed flask to volatile oil determination tube. Add water through the top of volatile oil
A - 40
determination tube until the graduated tube is filled and overflows to the round-bottomed flask.
Add 1 mL of xylene by using a pipette and then connect the reflux condenser to volatile oil
determination tube. Heat the flask until boiling, continue the heating to allow the distillation
proceed at a rate that will keep the middle part of the condenser cold. Stop heating after 30 min,
and allow it to stand for at least 15 min. Record the volume of xylene in the graduated tube of
volatile oil determination tube.
Carry out the procedure as described in Method A beginning at the words "Take a quantity of the
powdered sample". Subtract the volume of xylene previously observed from the volume of oily
layer, the difference in volume is taken to be the content of volatile oil, calculate the percentage of
volatile oil in CMM sample.
Limits – The CMM samples contain not less than the percentage of volatile oil specified in the individual
monograph.
Appendix XV Determination of Volatile Oil
A - 41
C
A
1214
11
811
.5 13.5
0.90.8
7
1.4
Unit : cm
Figure 1 Apparatus for the determination of volatile oil in CMM samples
A. Round-bottomed flask
B. Volatile oil determination tube
C. Reflux condenser
B
Appendix XV Determination of Volatile Oil
A - 42
Appendix XVI Storage
Appendix XVI: Storage
Storage refers to the basic conditions required for storing CMM. Unless otherwise specified, CMM should be
stored in dry and clean containers. Some terms for the general storage conditions are specified as follows –
(1) "Protected from light" refers to the storage of CMM in light resistant containers.
(2) "Well closed container" refers to a container which is able to protect CMM from extraneous matters or
from the loss of its contents.
(3) "Tightly closed container" refers to a container which is able to protect CMM from efflorescence,
deliquescence, volatilization or interference of extraneous matters.
(4) "Tightly sealed container" refers to a container which is tightly sealed with suitable material to protect
CMM against contamination and from permeability of air and moisture.
(5) "Cool and dark place" refers to a dark environment, protected from light, where the temperature is 11–
20°C.
(6) "Cold place" refers to an environment where the temperature is 2–10°C.
A - 43
INDEXES
m~ÖÉ=kçK
fK lÑÑáÅá~ä=k~ãÉë fJN
ffK `ÜáåÉëÉ=k~ãÉë fJO
fffK `ÜáåÉëÉ=mÜçåÉíáÅ=k~ãÉë fJP
fsK pÅáÉåíáÑáÅ=k~ãÉë =fJQ
sK `ÜÉãáÅ~ä=oÉÑÉêÉåÅÉ=pìÄëí~åÅÉë fJR
Indexes
Index I Official Names
lÑÑáÅá~ä=k~ãÉë m~ÖÉ=kçK
`~ìäáë=`äÉã~íáÇáë=^êã~åÇáá NN
`çêíÉñ=j~Öåçäá~É=lÑÑáÅáå~äáë ON
cäçë=j~Öåçäá~É PR
eÉêÄ~=aÉëãçÇáá=píóê~ÅáÑçäáá QR
eÉêÄ~=béÜÉÇê~É RT
o~Çáñ=^ÅÜóê~åíÜáë=_áÇÉåí~í~É SV
o~Çáñ=^Åçåáíá=mê~Éé~ê~í~ TV
o~Çáñ=^åÖÉäáÅ~É=mìÄÉëÅÉåíáë UV
o~Çáñ=^ìÅâä~åÇá~É NMN
o~Çáñ=_ìéäÉìêá NNP
o~Çáñ=`çÇçåçéëáë NOR
o~Çáñ=Éí=oÜáòçã~=dÉåíá~å~É NQN
o~Çáñ=Éí=oÜáòçã~=däóÅóêêÜáò~É NRR
o~Çáñ=Éí=oÜáòçã~=oÜÉá NSV
o~Çáñ=m~Éçåá~É=^äÄ~ NUT
o~Çáñ=m~Éçåá~É=oìÄê~ NVT
o~Çáñ=mä~íóÅçÇá ONN
o~Çáñ=mçäóÖçåá=jìäíáÑäçêá OOP
o~Çáñ=p~éçëÜåáâçîá~É OPR
oÜáòçã~=`Üì~åñáçåÖ OQT
oÜáòçã~=`áãáÅáÑìÖ~É ORT
oÜáòçã~=`çéíáÇáë OST
oÜáòçã~=`ìêÅìã~É OUN
oÜáòçã~=Éí=o~Çáñ=kçíçéíÉêóÖáá OVT
I - 1
Index II Chinese Names
`ÜáåÉëÉ=k~ãÉë m~ÖÉ=kçK
�� NSV
�� NN
�� OQT
�� ORT
�� NMN
�� SV
�� NRR
�� NUT
�� OOP
�� NVT
�� PR
�� OPR
�� OVT
�� ON
�� ONN
�� NNP
�� RT
�� OUN
�� OST
�� TV
�� ! QR
�� UV
�� NQN
�� NOR
I - 2
`ÜáåÉëÉ=mÜçåÉíáÅ=k~ãÉë m~ÖÉ=kçK
_~áëÜ~ç NUT
`Ü~áÜì NNP
`ÜáëÜ~ç NVT
`Üì~åãìíçåÖ NN
`Üì~åñáçåÖ OQT
a~Üì~åÖ NSV
a~åÖëÜÉå NOR
aìÜìç UV
bòÜì OUN
c~åÖÑÉåÖ OPR
d~åÅ~ç NRR
dì~åÖàáåèá~åÅ~ç QR
eÉëÜçìïì OOP
eçìéç ON
eì~åÖäá~å OST
gáÉÖÉåÖ ONN
içåÖÇ~å NQN
j~Üì~åÖ RT
jìñá~åÖ NMN
káìñá SV
ná~åÖÜìç OVT
pÜÉåÖã~ ORT
uáåóá PR
wÜáÅÜì~åïì TV
Index III Chinese Phonetic Names
I - 3
pÅáÉåíáÑáÅ=k~ãÉë m~ÖÉ=kçK
^ÅÜóê~åíÜÉë=ÄáÇÉåí~í~=_äK SV
^Åçåáíìã=Å~êãáÅÜ~Éäá=aÉÄñK TV
^åÖÉäáÅ~=éìÄÉëÅÉåë=j~ñáãK=ÑK=ÄáëÉêê~í~=pÜ~å=Éí=vì~å UV
^ìÅâä~åÇá~=ä~éé~=aÉÅåÉK NMN
_ìéäÉìêìã=ÅÜáåÉåëÉ=a`K NNP
`áãáÅáÑìÖ~=ÜÉê~ÅäÉáÑçäá~=hçãK ORT
`äÉã~íáë=~êã~åÇáá=cê~åÅÜK NN
`çÇçåçéëáë=éáäçëìä~=Ecê~åÅÜKF=k~ååÑK NOR
`çÇçåçéëáë=éáäçëìä~=k~ååÑK=î~êK=ãçÇÉëí~=Ek~ååÑKF=iK=qK=pÜÉå NOR
`çÇçåçéëáë=í~åÖëÜÉå=läáîK NOR
`çéíáë=ÅÜáåÉåëáë=cê~åÅÜK OST
`çéíáë=ÇÉäíçáÇÉ~=`KvK`ÜÉåÖ=Éí=eëá~ç OST
`ìêÅìã~=âï~åÖëáÉåëáë=pK=dK=iÉÉ=Éí=`K=cK=iá~åÖ OUN
`ìêÅìã~=éÜ~ÉçÅ~ìäáë=s~äK OUN
`ìêÅìã~=ïÉåóìàáå=vK=eK=`ÜÉå=Éí=`K=iáåÖ OUN
aÉëãçÇáìã=ëíóê~ÅáÑçäáìã=ElëÄKF=jÉêêK QR
béÜÉÇê~=ëáåáÅ~=pí~éÑ RT
dÉåíá~å~=êáÖÉëÅÉåë=cê~åÅÜK NQN
dÉåíá~å~=ëÅ~Äê~=_ÖÉK NQN
däóÅóêêÜáò~=áåÑä~í~=_~íK NRR
däóÅóêêÜáò~=ìê~äÉåëáë=cáëÅÜK NRR
iáÖìëíáÅìã=ÅÜì~åñáçåÖ=eçêíK OQT
j~Öåçäá~=ÄáçåÇáá=m~ãéK PR
j~Öåçäá~=çÑÑáÅáå~äáë=oÉÜÇK=Éí=táäëK ON
j~Öåçäá~=çÑÑáÅáå~äáë=oÉÜÇK=Éí=táäëK=î~êK=ÄáäçÄ~=oÉÜÇK=Éí=táäëK ON
kçíçéíÉêóÖáìã=áåÅáëìã=qáåÖ=Éñ=eK=qK=`Ü~åÖ OVT
m~Éçåá~=ä~ÅíáÑäçê~=m~ääK NUTI=NVT
m~Éçåá~=îÉáíÅÜáá=ióåÅÜ NVT
mä~íóÅçÇçå=Öê~åÇáÑäçêìã=Eg~ÅèKF=^K=a`K ONN
mçäóÖçåìã=ãìäíáÑäçêìã=qÜìåÄK OOP
oÜÉìã=çÑÑáÅáå~äÉ=_~áääK NSV
oÜÉìã=é~äã~íìã=iK NSV
oÜÉìã=í~åÖìíáÅìã=j~ñáãK=Éñ=_~äÑK NSV
p~éçëÜåáâçîá~=Çáî~êáÅ~í~=EqìêÅòKF=pÅÜáëÅÜâK OPR
Index IV Scientific Names
I - 4
`ÜÉãáÅ~ä=oÉÑÉêÉåÅÉ=pìÄëí~åÅÉë m~ÖÉ=kçK
OI=PI=RI=Q’ J=íÉíê~ÜóÇêçñóëíáäÄÉåÉJOJlJβJaJÖäìÅçëáÇÉ OORI=OOUI=OOVI=OPMI=OPOI=OPP
Q’lJβJaJÖäìÅçëóäJRJlJãÉíÜóäîáë~ããáåçä OPTI=OQMJOQR
^ÅçåáíáåÉ ^JPUI=^JPVI=US
^äçÉJÉãçÇáå NTUI=NTVI=NUNI=NUPJNUR
^êáëíçäçÅÜáÅ=~ÅáÇ=f ^JPSI=^JPTI=NV
_ÉåòçóäãÉë~ÅçåáåÉ UNI=UPJUT
_ÉêÄÉêáåÉ=ÅÜäçêáÇÉ OSVI=OTQJOTSI=OTUI=OTV
`ÜêóëçéÜ~åçä NTUI=NTVI=NUNI=NUPJNUR
`çäìãÄá~åÉíáå=~ÅÉí~íÉ VNI=VQI=VS
`çëíìåçäáÇÉ NMPI=NMSJNNN
aÉÜóÇêçÅçëíìë=ä~ÅíçåÉ NMPI=NMSI=NMUJNNN
bÅÇóëíÉêçåÉ TQJTS
bJÑÉêìäáÅ=~ÅáÇ OQVI=OROI=ORQ
bãçÇáå NTUJNUNI=NUPJNURI=ORRI=OOUJOPM
béÜÉÇêáåÉ=ÜóÇêçÅÜäçêáÇÉ SOJSQI=SSI=ST
c~êÖÉëáå PTI=QMI=QO
dÉåíáçéáÅêáå NQUJNRMI=NROJNRQ
dÉêã~ÅêçåÉ OVMJOVOI=OVQJOVS
däóÅóêêÜáòáÅ=~ÅáÇ NRTI=NSOJNSQI=NSSJNSU
eÉëéÉêáÇáå NSJNU
eçåçâáçä OQI=OVI=PNI=PPI=PQ
eóé~ÅçåáíáåÉ ^JPUI=^JPVI=US
fëçÑÉêìäáÅ=~ÅáÇ ORVI=OSOI=OSPI=OSRI=OSS
fëçáãéÉê~íçêáå OVVI=PMOJPMS
fëçîáíÉñáå QTI=ROJRS
iáèìáêáíáå NRTI=NSOJNSQI=NSSJNSU
içÄÉíóçäáå NPQJNPSI=NPUJNQM
j~Öåçäáå PTI=QMJQQ
j~Öåçäçä OQI=OVJPNI=PPI=PQ
jÉë~ÅçåáíáåÉ ^JPUI=^JPVI=US
kçíçéíÉêçä OVVI=PMOI=PMQ
läÉ~åçäáÅ=~ÅáÇ NPI=NSI=TNI=TQI=TTI=TU
lëíÜçäÉ VNI=VQJVSI=VUI=VV
m~ÉçåáÑäçêáå NUVI=NVOJNVSI=NVVI=OMQJOMSI=OMUI=OMV
Index V Chemical Reference Substances
I - 5
Index V Chemical Reference Substances
`ÜÉãáÅ~ä=oÉÑÉêÉåÅÉ=pìÄëí~åÅÉë=EÅçåíáåìÉÇF m~ÖÉ=kçK
m~äã~íáåÉ=ÅÜäçêáÇÉ OSVI=OTQI=OTSI=OTUI=OTV
mÜóëÅáçå NTVI=NUNI=NUPJNURI=OORI=OOUJOPM
mä~íóÅçëáÇÉ=b ONPI=ONSJOON
mêáãJlJÖäìÅçëóäÅáãáÑìÖáå OPTI=OQMI=OQOJOQR
mëÉìÇçÉéÜÉÇêáåÉ=ÜóÇêçÅÜäçêáÇÉ SPI=SQI=SSI=ST
oÜÉáå NTUI=NTVI=NUNI=NUPJNUR
p~áâçë~éçåáå=^ NNRI=NNUI=NOMI=NOOI=NOP
p~áâçë~éçåáå=` NNRI=NNU
p~áâçë~éçåáå=a NNRI=NNUI=NNVI=NOM
wJäáÖìëíáäáÇÉ OQVI=OROJORS
I - 6