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Writing Guidelines: ANALYTICAL METHODS AND QUANTITATIVE STANDARDS
Below is the outline format for the Analytical section of the monograph. There are examples for Spectrophotometric Assay HPLC, most monographs do not include Spectrophotometric Assasys and these tend not to be favored here in the US, though they are heavily used by PharmEuropa. There is an outline example of a Spectrophotometric Assay form from the Bilberry monograph, and an example of an HPLC method from the valerian monograph. Please adhere to abbreviation formation as well (eg. mL rather than ml).
Give a brief introduction of the methods included.
This section states where the methods come from, what
pharmacopoeias, what journal references, MVP-INA, etc.,
as well as any other general information that may be
applicable. If there are methods from both the European
Pharmacopoeia and the USP and one was chosen over the
other, a statement is included explaining why the method was chosen. If changes were made to any of the
methods during the substantiation process, these changes
are also explained.
Further introduction if necessary
Reagents
Sample Preparation
Standard Preparation
Linearity Range (Not all methods provide this, though they usually state how to calculate it. It is best if we can provide values for this range.)
Chromatographic Conditions
Column: Brand, phase, particle size, dimensions
Column Temperature: If not given in the source method,
assume room temperature
Mobile Phase:
Flow Rate:
Detection:
Injection Volume:
Run Time: May include elution order, retention times.
Peak Identification
System Suitbility
Quality Assurance
Calculation
Discussion of the Chromatogram(s)
Spectrophotometric Assay
Sample Preparation
Absorption
Calculation
Sample Preparation
Procedure
Calculation
Following is a draft of the Introduction and Spectrophotometric
Assay, in the Bilberry Analytical Section. It is followed
by the HPLC method from the Valerian monograph which
has a beefier introduction which might be helpful for
you. The primary sources for the Bilberry methods are
included so that you can see how we pull the information
from the sources and put it into our format.
A thin layer chromatography (TLC/HPTLC) method developed
by CAMAG, Switzerland is presented as a fingerprint
for the qualitative determination of bilberry raw material
and powdered extract. A spectrophotometric assay for
quantifying total anthocyanins in bilberry raw material
was adopted from the European Pharmacopoeia (PharmEuropa
1998) and substantiated by two American Herbal Pharmacopoeia™ collaborating
laboratories. This assay calculates total anthocyanin
content expressed as cyanidin-3-glucoside. Because
this method cannot detect adulteration, it should only
be employed after appropriate methods of plant identification
have ensured authenticity and purity. For quantifying
total anthocyanins in bilberry extract, the pH-differential
spectrophotometric assay published by the Institute
for Nutraceutical Advancement Methods Validation Program
(INA-MVP) (Giusti and Wrolstad 2001) is recommended.
This method is able to detect adulteration by added
colorants such as FD & C Red No. 40, FD & C
Red No. 3, cochineal, and beet powder.
Accurately weigh 1.00 g of the powdered drug and add
95 mL of methanol. Sonicate for 30 minutes and filter
into a 100 mL volumetric flask. Rinse the filter and
adjust to volume with methanol. Prepare a 20-fold dilution
of this solution in a 0.1% V/V solution of HCl in methanol.
Measure the absorbance of the solution at 528 nm using
a 0.1% V/V solution of HCl in methanol as the reagent
blank.
Calculate the content of anthocyanins, expressed as
cyanidin-3-glucoside, from the expression:
A x 2000
772 x m
A = the absorbance of the solution at 528 nm.
m = the mass of the sample in grams
The specific absorbance of cyanidin-3-glucoside
at 528 nm is 772.
The specific absorbance of 3-glucoside-cyanidol is
772.
An AHP-Verified™ reference standard for valerenic
acid is readily available. Standards for acetoxyvalerenic
acid and hydroxyvalerenic acid are available but relatively
expensive. Standards for valerenal are generally not
available. According to the original paper of Hänsel
and Schulz, valerenal was assumed to possess the same
molecular extinction coefficient as valerenic acid.
This was not confirmed.
For quantitative analysis of valerenic acid, a modification
of the method of Hänsel and Schulz was adopted
(Hänsel and Schulz 1982). This same method formed
the basis for the method proposed by the Pharmacopeial
Convention (USP) for inclusion in the National Formulary
(Pharmacopeial Forum 1998). It provides good separation
of valerenic acid, acetoxyvalerenic acid, and hydroxyvalerenic
acid, and the aldehyde valerenal. In Europe, and in
some analytical laboratories in the United States,
total valerenic acid content is calculated as the sum
of these compounds. In the USP proposal, only valerenic
acid content is determined. These differences in calculating
valerenic acid content cause confusion and incongruities
in the marketplace. Calculation of total valerenic
acid values (the sum of valerenic acid, acetoxyvalerenic
acid, hydroxyvalerenic acid, and valerenal) is more
representative of effective valerian products than
determination of valerenic acid alone. Additionally,
some analytical laboratories calculate total valerenic
acids using each reference standard while others calculate
total valerenic acids based on the assumption that
the extinction coefficients for each compound are the
same. The extinction coefficients of each compound
are not the same. However, calculating total valerenic
acids in this manner provides a more accurate determination
of total valerenic acid values than the calculation
of valerenic acid alone. For a more accurate determination
of total valerenic acids, laboratories are encouraged
to determine the extinction coefficients of the three
primary compounds. Valerenic acid is available and
is relatively inexpensive. Standards for acetoxyvalerenic
acid and hydroxy valerenic acid are available and are
relatively expensive. Standards for valerenal are not
available but it is considered to have the same extinction
coefficient as valerenic acid.
For analysis of crude valerian root, weigh 2 g of
finely powdered root material and transfer to a 100
mL volumetric flask. Dilute to volume with methanol:water
(80:20) and sonicate for 30 minutes. Filter a portion
through a 0.45 µm filter into an HPLC vial, or
centrifuge to obtain a clear test solution.
For analysis of powdered extracts, weigh 100 mg of
extract into a 10 mL volumetric flask. Dilute to volume
with methanol:water (80:20) and sonicate for 15 minutes.
Filter a portion through a 0.45 µm filter into
an HPLC vial, or centrifuge to obtain a clear test
solution.
Accurately weigh 5 mg of valerenic acid standard (Indofine
Chemical Company; United States Pharmacopeial Convention
Rockville, MD) into a 100 mL volumetric flask. Dilute
to volume with methanol:water (80:20) and sonicate
for 15 minutes.
The standard and sample are stable when stored in
amber vials and are refrigerated.
Column: C-18, 5 µm, 4.6 x
250 mm (Alltech Hypersil).
Mobile Phase: Methanol:0.5% phosphoric acid (80:20).
Flow Rate: 1.5 mL/minute.
Detection: 225 nm.
Injection Volume: 20 µL.
Run Time: 15 minutes.
Elution Order: Hydroxyvalerenic acid, acetoxyvalerenic
acid, valerenic acid, valerenal.
Calculate the percentage of valerenic acid using the
following formula.
100V (C/W)(ru/rs)
V is the volume in mL of the sample preparation. C
is the concentration in mg per mL of the standard solution.
W is the weight in mg of valerian used to prepare the
sample solution. ru and rs are the peak responses obtained
from the sample solution and the standard solution
respectively.
In some cases, a reference standard may not be commercially
available. In these cases, we can either provide isolation
instructions (if available), utilize an internal standard
(such as capsacin for gingerol) or give relative values
(such as valerenal in relationship to valerenic acid).
There are three primary methods applicable for assaying
volatile oil content of valerian root: AOAC International,
European Pharmacopoeia, and the United States Pharmacopeia.
Each of these is similar; however, the procedures provided
by AOAC have been detailed in such a manner as to minimize
variables that can lead to differences in volatile
oil yields and maximize reproducibility (Ertl 1997).
Because the determination of volatile oil is the primary
qualitative and quantitative marker for effective valerian
products, this method has been adopted (see Ertl Journal
AOAC Int 80(4): 1-6, 1997).
For thin layer chromatography analysis,
the method of the European Pharmacopoeia (1998 supplement)
and the method proposed by the United States Pharmacopeial
Convention (USP) (Pharmacopeial Forum 1998) were compared.
The method of the European Pharmacopoeia method was
preferred for analysis of valerenic acid, a primary
marker compound of V. officinalis. Improvements were
made to the sample preparation.
Shake 0.2 g of freshly powdered valerian
in a test tube with 5 mL dichloromethane for 1 minute.
Allow the mixture to stand for 5 minutes and then filter.
Wash the filter with 2 mL of dichloromethane. Evaporate
the combined filtrate and washing to dryness on a water
bath. Dissolve the residue in 0.2 mL of dichloromethane
and transfer the solution into a small sample vial.
Dissolve 1 mg of valerenic acid (available from Indofine
Chemical Company, Somerville, NJ; United States Pharmacopeial
Convention, Rockville, MD) in 0.5 mL of dichloromethane.
Prepare HCl-acetic acid reagent (1:4) carefully mixing
20 mL of glacial acetic acid with 80 mL of concentrated
hydrochloric acid.
Prepare anisaldehyde-sulfuric acid reagent by slowly
adding 9 mL of 98% H2SO4 to an ice cooled mixture of
85 mL of methanol and 10 mL of glacial acetic acid.
To this solution add 0.5 mL of anisaldehyde and mix
well. The anisaldehyde-sulfuric acid reagent is colorless
and should be stored in a refrigerator. If a color
develops, the reagent must be discarded.
HPTLC
plates 10 x 10 cm silica gel 60 with fluorescence
indicator (EM Science, Whatman, Machery & Nagel,
or equivalent).
Hexane:ethyl acetate:glacial acetic acid (65:35:0.5).
3 µl volumes of both sample
solution and standard are applied each as a 10 mm
band. Space bands 6 mm apart. Application position
should be 8 mm from the lower edge of the plate.
a) UV 254 nm.
b) Spray plate with the HCl-acetic acid reagent, dry
in stream of cold air, heat to 110 ??qC for 5 minutes.
Inspect plate in visible light and under UV 366 nm.
c) Spray the plate with the HCl-acetic acid reagent,
dry in stream of cold air, place on plate heater (or
in oven) at 120 ??qC for 2 minutes (or until color of
standard has developed).
Valerenic acid = 0.48. Following application of the
HCl-acetic acid reagent, this band appears as a very
faint violet color in visible light and as a weak fluorescent
band under UV 366 nm. Following subsequent application
of the anisaldehyde-sulfuric acid reagent, valerenic
acid appears as a strong dark blue band. |
