2018

NPC Natural Product Communications Vol. 13

No. 12
Recent Development of Quality Control Methods for Herbal Derived 1599 - 1606
Drug Preparations
Gunawan Indrayanto

Plant Biotechnology Research Group, Faculty of Pharmacy, Airlangga University, Surabaya 60286, Indonesia

gunawanindrayanto@yahoo.com

Received: July 6th, 2018; Accepted: November 14th, 2018

Pharmaceutical industries should apply rigorous QC (quality control) to ensure the consistency, safety, and efficacy of their herbal derived drug-preparations.
QC must be performed at every stage of the production line i.e. incoming raw materials, extractions, in-process control, finished products and keeping samples.
Due to the complex nature of the chemical content of herbal drugs, two approaches to QC should be taken, that is quantitative determination of the selected
marker(s) compound(s), and metabolite profiling. Contamination of herbal medicines by heavy metals, pesticides, toxic metabolites, microbial toxins,
pathogenic microorganisms and other foreign matter should also be evaluated. A combination of chemical profiling and multivariate analysis (MVA) is
recommended as the QC tool for the botanical identification method (BIM) of herbs, extracts, herb materials, and herbal drug preparations. Microscopic
methods, DNA profiling or chemical marker(s) are not recommended for use as the sole BIM due to the lack of specificity. Only markers that meet certain
criteria i.e. quality active (QA) markers can be utilized as a QC tool. The limit specification range of markers used as QC tools should be described in the
analytical target profile (ATP). To gain reliable results of any analysis that has been performed at any QC laboratory, the analysis method must be validated
according to the newest guidance. Sample detection limit of any toxic compound(s) should be lower than its cut-off value and MPL. The reliability of any
results of analysis of a QC laboratory must be evaluated by using QC-samples for each series of measurements.

Keywords: Chemical-profiling, DNA-profiling, Herbal drugs, QA-marker, Metabolite profiling, Multivariate analysis, Sample detection limit, Validation.

Herbal derived drug preparations or herbal medicines have been
used worldwide for centuries. Herbal medicines can contain single
or multiple herbs in a single drug preparation. That is why herbal
medicines could have a complex mixture of many herbs that contain
many chemical compounds [1, 2]. According to the WHO
guidelines [3] herbal medicines are herbs, herbal materials, herbal
preparations and finished herbal products. Herbs include crude
materials that can derive from lichen, fungi, algae and parts of
higher plant materials, which may be entire, fragmented or
powdered, whilst herbal materials include fresh juices, gums, fixed
oils, essential oils, resins and dry powders of herbs. These materials
may be processed by steaming, roasting, backing with honey or
alcoholic beverages. Herbal preparations may include comminuted
or cut herbal materials, or extracts, tinctures and fatty oils of herbal
materials. Finished herbal products and mixtures of herbal products
may contain excipients in addition to the active ingredients i.e.
herbal preparations made from one or more herbs or their extracts.
If more than one herb is used, the term “mixture herbal product”
was used. The term "Botanical" of FDA [4] means products that
include plant materials, algae, macroscopic fungi, and combinations
thereof; it does not include products that contain animals or animal
parts, GMO plants, products produced by fermentation of yeast,
bacteria, plant cells, or other microscopic organisms, and highly
purified compounds either derived from nature or chemically
modified. Products that contain chemically defined isolated
constituents, or a mixture thereof, are not herbal medicine products
[5]. Botanicals can be available as two different products i.e. herbal
medicinal products or as plant food supplements (PFS) [6].
In Indonesia, herbal medicines were generally called Obat
tradisional, and comprised: (1) non standardized traditional drugs
(Jamu), (2) standardized herbal drugs (Obat Herbal Terstandard
or OHT). This meant herbal drugs that had their safety and
efficacy already proved by pre-clinical studies, and (3)
Phytopharmaceuticals (Fitofarmasi), that could be defined as (2)
with additional clinical studies. Nowadays, herbal medicines in
Indonesia are produced by many pharmaceutical companies, and
according to the BPOM (Indonesian National Agency of Drug and
Food Control), 872 companies were registered in 2005; the growth
rate of these products in Indonesia was estimated at 20-30%
annually [7, 8].
Due to the increasing application of herbal medicines or herbal
drugs globally, also in Indonesia, in the last decade, QC of the drug
preparations derived from herbs is very important. This QC can be
used to monitor their efficacy and toxicity [1, 9, 10]. QC should be
performed at all stages of the production line of the herbal derived
products i.e. incoming raw materials (herbs, herbal materials,
extracts, excipients), in-process control (IPC), finished products
(FP) and keeping samples (KS) of FP (Figure 1).General guidelines
for evaluating the quality of herbs and herbal derived drugs have
been issued by many countries and organizations e.g. EMA [5, 11],
FDA [4], AOAC [12], WHO [13], USP Herbal Medicine
Compendium [14], Hong Kong Chinese Materia Medical Standard
[15], Farmakope Herbal Indonesia (Indonesian Herbal
Pharmacopoeia) [16], American Herbal Products Association [17],
and British Pharmacopoeia (BP) 2018 [18]. Unfortunately the
described official methods are very general, and only have specific
application for certain herbs or herb materials. It seems the
described methods could not be applied directly for analyzing
mixtures of herbals in the product preparations. A specific method
of analysis, as the QC tool, should be self-developed and validated
by the pharmaceutical companies for each of their herbal product
preparations.
Some interesting review articles on QC methods for herbal drugs
have been published. Jiang et al. in 2010 [19] described some
analytical approaches for the quality control (QC) of traditional
Chinese medicines. A strategy for QC for Chinese medicine was
proposed by Li et al. in 2011. [20]. Pan et al. (2013) [21] proposed
1600 Natural Product Communications Vol. 13 (12) 2018
an integration process analytical technology of herbal drugs from
raw materials to finished extracts or active ingredients using a
combination of spectroscopy methods and multivariate analysis
(MVA). General analytical validation methods for the analysis of
botanical drugs were reviewed and discussed by Brown and Lister
[22], whilst validation chromatographic methods of analyses for
specific applications for herbal medicines were reviewed recently
by the author [23], Chen et al. (2017) proposed a holistic strategy
for quality and safety control for traditional Chinese medicines [24].
Abdel-Tawab described very general method assessments for
botanicals as PFS products [6].
In the last three years some important new developments in QC
methods for herbal medicines have been published: (1) new
descriptions of markers as QC tools, and the methods of
determination; (2) application of chemical profiles for the
determination of certain herbs in herbal medicines, qualitatively and
quantitatively; (3) determination of sample detection limit and its
verification; and (4) newer statistical techniques of validation
methods. Unfortunately, those new aspects were not discussed in
detail in the previously described reviews [6, 19-24]. This present
article will discuss these new aspects and other recent developments
that could be applied, and be useful as QC tools for the
pharmaceutical industry.
Sampling methods in manufacturing processes of herbal drugs:
Sampling is the most essential part of QC in the pharmaceutical
industry; if the samples are not representative, the whole QC
processes is useless. USP <561> [25] has been described as the
sampling method for samples of botanical origin or herbs. All
containers must be sampledif the number of containers (n) = 1-10;
11 containers if n = 11-19; if n containers> 19, the number of
containers to be sampled is 10 + n/10. Samples must be taken from
upper, middle and lower sections of the container. If the plant
materials consist of parts < 1 cm or less or powdered materials, a
sampling device can be used; if samples have parts > 1 cm, samples
can be withdrawn by hand. For containers with < 1 kg materials,
after mixing, sufficient quantity of samples can taken out; for
containers 1-5 kg, a sample must be withdrawn from upper, middle
and lower parts; if container is > 5 kg, 250 g samples must be
withdrawn from upper, middle and lower parts, and after mixing, a
portion taken for testing. WHO [13] recommended a procedure for
sampling of herbs as follows: for containers or packaging n= 5, all
must be sampled; n = 6-50, a sample should be taken from each of
five containers; n >50, take 10% for sample, rounding up the
number of units to the nearest multiple of 10, e.g. if n = 52 this
would be sampled as n = 60. General sampling methods that have
been explained in detail in USP 41 <1010> [26] can be applied for
samples at each stage of the production line in the manufacturing
processes of herbal derived drugs in the pharmaceutical industry; in
this case, a random sampling method is usually recommended.
Chemical profiling method as QC tool for herbal medicines:
Confirming the identity of the incoming herbs or extracts is a
crucial step in producing good quality products for herbal medicines
(Figure 1). General methods for identification of plant materials or
herbs by using macroscopic-, microscopic-, and chromatographic-
methods are described officially e.g. by WHO [13], USP Herbal
Medicines Compendium [14], Indonesian Herbal Pharmacopeia
[16], American Herbal Product Association [17], BP 2018 [18] and
USP 41 <563> [27]. Our experiences showed that the application of
only macroscopic- and microscopic-methods for identification of
commercial herbs or herbal materials could not be utilized, due to
the lack of specificity, and so additional chemical- and/or DNA-
profiling methods of identification needed to be performed. These
Indrayanto
macroscopic- and microscopic-methods are characteristic, but not
specific. TLC methods for identification of articles of botanical
origin are explained by USP 41 <203>[28], and <1064> [29].
Evaluation of the HPTLC methods were performed by comparing
visually the chromatograms of samples and authentic botanical
reference material (BRM); the sequences of zones (spots) that have
specific positions (Rf), colors and intensity (TLC profiles or finger
printings) are evaluated and compared. Evaluation of TLC/HPLC
chromatograms by visual means only (i.e. comparing Rf, Rt, peak
intensity and color), as described in some guidance as shown above,
could be very subjective and difficult. Identical Rf/Rt, colors and
intensities of two spots/peaks does not mean identical compounds.
That is why it is recommended to evaluate the chromatograms
(HPLC/TLC) by using a DAD detector or a TLC-scanner
(densitometry). Using these, the identity and purity of the target
peak(s) could be evaluated by comparison with an authentic
standard. Evaluation of TLC-spots can also be performed by using a
video-densitometric method, but unfortunately, in this method, the
identity and purity of the peaks cannot be evaluated. Recently
Frommenwiller et al. [30] applied peak profiles (PPI) directly from
the finger printing images of the TLC spots for evaluating samples
of Angelica gigas root. For confirming the chemical structure of the
target spot/peak of the TLC [31] or HPLC unambiguously, the
chromatographic system must be coupled with a MS detector.
.
The main disadvantage of applying chromatographic methods is
that they are time consuming; this is due to various reasons i.e.
samples must be extracted with a selected solvent, the stability of
the sample in this selected solvent must be evaluated, and all target
peaks must be evaluated regarding their identity and purity [23]. To
solve this problem, the application of direct methods (MS, FT-IR,
NIR, and Raman) for identification of herbal materials and extracts
in drug preparations could be preferred and very interesting. Some
recent publications reported the application of direct MS combined
with multivariate analysis (MVA) for identifying herbal powdered
materials, their adulterants and related herbs [32-34]. It seemed that
direct MS identification of plants or herbs was more specific
compared with DNA profiling, especially for closely related herbal
species [35, 36]. The drawback of the application of LC-MS/MS- or
direct MS-profiling is the relatively high price of the equipment and
its operation cost; not many pharmaceutical companies have utilized
this equipment. The application of ATR-FT-IR methods were
preferred for direct identification and quantitative estimation of
herbs that have been widely used in China; this is due to their
relatively low operation cost, and so it is recommended to apply
ATR-FT-IR methods as QC tools for pharmaceutical companies,
especially for developing countries. Implementation of IR methods
for QC of traditional Chinese medicines has been described and
reviewed by Sun et al. [37]. QC methods for herbal drugs have been
published and reviewed using FT-IR and Raman spectroscopy [38,
39]. Different grades of Panax notoginseng powders can be directly
differentiated by using FT-IR and 2D FT-IR [40]. A combination of
ATR-FTIR micro-spectroscopy imaging can be applied directly for
identifying complex mixtures of powdered herbal drugs [41].
Chemical constituents of herbs and herb materials are very complex
and that is why the evaluation of the chemical profiles
(chromatography and spectroscopy) should be performed in
combination with MVA i.e. PCA, HCA, PLS-DA, SIMCA etc. It
seemed very difficult or impossible to evaluate chromatographic- or
spectroscopic-profiles of samples without any combination with
MVA. Chromatographic and spectroscopy profiling/fingerprinting
that is mostly combined with MVA have been widely used for the
evaluation or QC of herb/plant materials; some good examples have
been described in previous publications [2, 19, 34, 42-44]. Some
Quality control methods for herbal drugs
factors that could affect the chemical profiles are the quality of
samples, method of drying, method of extraction, equipment,
analytical method and its validation. Identical chemical profiles of
samples will be shown by a tight clustering in the score plot of the
PCA. For QC purposes, the identity of the (incoming) herb raw
materials or extract generally can be evaluated by using a pattern
recognition technique i.e. soft independent modeling of class
analogy (SIMCA) [23, 45]; other methods e.g. linear discriminant
analysis (LDA) or support vector machines classification (SVMC)
can be applied [46, 47]. Quantitative determination of certain herbs
in the drug preparations can be performed by using PLS regression;
for example that given by Wang et al. [36]. Quantitative
determination of components of the mixture of herbal medicines
can also be performed by using the ratio of two specific peaks of
FT-IR [37]. The processing of chemical profiles (metabolite
profiling or metabolomics) data (e.g. LC/GC-MS, NMR) can be
completed using a wide range of free on-line software [48]; our
experience showed that sophisticated LC-HR MS/MS equipment
generally has already been completed with MVA software.
Similarity analysis of chromatograms or FT-IR spectra of samples
and BRM can also be performed by calculating their correlation
coefficient (r) and congruence (c) values; values of r and c close to
one means that the chemical profiles are identical; the threshold
value of r should be > 0.990 [49, 50]. Identical chemical profiles of
two samples indicate that the samples have identical qualitative and
quantitative biochemicals, and so the two samples are
phytoequivalent to each other; this means that the two samples will
have equivalent bioactivities or toxicities.
AOAC [12] described the validation of a botanical identification
method (BIM). For validating the BIM for certain applications,
specified inferior test materials (SITM) and specified superior test
materials (SSTM) should be prepared and determined. SITM was a
botanical (herb) mixture of materials that has the maximum
concentration of target material (herb) that is considered
unacceptable (negative result) as specified by its standard method
requirements (SMPR), whilst SSTM was a mixture of herbs which
have minimum concentrations of target herb that is considered
acceptable (positive result). For preparing SITM and SSTM the
author recommends using official BRM (if any), or herb that was
cultivated in a defined location and has been scientifically
identified.According to USP 41 <1039>[51] any chemical method
that is combined with chemometrics analysis should demonstrate
the capability to identify correctly or classify the herb samples, so
receiver operating characteristic (ROC) curves should be
constructed. ROC curves can be constructed by using values of
true-positive rate (TPR) versus false-positive rate over a range
decision threshold; a good identification method should generate an
ROC curve with the area under the curve (AUC) close to one.
Samples with 0% and 100% of SSTM can be used to select the BIM
of certain herbs based on the ROC.The validity of a BIM method
that is determined by a ROC curve should be validated using a
probability of identification (POI) method according to AOAC
Appendix K [12].The ideal goalof a validation method is the
discrimination of SSTM and SITM with a specified degree of
confidence (e.g. p = 0.05); 1-POI at 0% of SSTM was a false-
positive fraction, whilst 1-POI at 100% of SSTM was a false-
negative fraction. Acceptance criteria according to this guidance of
AOAC were: 1-side (95%) confidence interval (CI) at 0% SSTM
should be below 10% and 100% should be above 90%; CI can be
calculated according to the previous publication [52]. Detailed
discussion of this validation method was already described and
discussed by the guideline of the AOAC [12].
Natural Product Communications Vol. 13 (12) 2018 1601
A chemical profiling method (chromatography and spectroscopy)
combined with MVA can also be applied as a QC tool for either
evaluation or standardization of IPC- and FP-samples during
production. For validating the method, laboratory-made
preparations (LM) of IPC and FP should be prepared as training sets
for building the SIMCA models for qualitative analysis.
Quantitative estimation of certain herbs in IPC- and FP-samples can
be performed by using PLS regression. The validity of the models
can be tested using cross-validation, or by other LM preparations or
herb/food materials [53-55]. Evaluations of chemical profile of
samples and LM preparations for each stage of production can also
be performed by using a similarity method (using r or c),as
described above. Due to the relatively fast analysis-time and low-
operation-cost, the application of direct ATR-FT-IR combination
with chemometric evaluation is strongly recommended as the QC
tool for herbs or extracts in herbal drug preparations. A combination
of second derivative and two-dimensional (2D)-FT-IR with MVA
can be applied if 1D-ATR-FT-IR failed to show a good ROC. For
QC purposes using chemical profiling methods, the availability of
BRM, standardized extracts and standardized LM sample of each
stage of production are needed. Standardization of extracts or LM
sample, which will be used for QC, should be performed by using
more sophisticated equipment such as LC/GC- MS (MS).
DNA profiling as identification method for herbs: Identification
of botanical samples or herbs using DNA-based profiling has been
described by USP 41 <563>[27]. This method was applied to
distinguish between closely or morphologically similar species;
these included DNA bar coding and DNA (Sanger). DNA bar
coding is a particular DNA sequence-based identification method
that uses a short sequence of specific nuclear plastid DNA loci for
identification of plant species. Botanical identification using DNA
(Sanger) sequencing methods includes marker selection, DNA
extraction, polymerase chain reaction (PCR) primers and
amplification, DNA sequencing and comparison with reference
materials. Detailed discussions that described the applications of
DNA profiling including DNA bar coding, and their limitations for
identification of herbs, were reported inprevious publications [56-
58]. Recently Osathanunkul et al. [59] reported the application of
the DNA bar code method for identifying the herb Tinospora sp in
commercial herbal supplement preparations (tablet and capsule).
Due to many external factors that can affect the (secondary)
metabolites content of a plant both qualitatively and quantitatively,
a QC tool using DNA profiling must be completed using chemical
methods; it is well known that the bioactivity- and toxicity-profiles
of herbal products are dependent on their biochemical content
qualitatively and quantitatively. For this reason, application of DNA
profiling alone as a QC tool is not recommended; plants that have
identical DNA profiles might have different chemical profiles.
Combination of DNA- and metabolite-profiling studies will be very
useful for determining whether the difference in metabolite contents
of herbs or plants was due to the difference in strains of the plant
species or to some external factors.
Marker and its application as the QC tool of herbal medicines:
USP 41 <563> [27] described markers as active, analytical and
negative, whilst EMA [5] described active and analytical markers
only. Markers could also be categorized and summarized as
follows: therapeutic components, toxic components, bioactive
components, main components, synergistic components,
characteristic components and correlative components. However,
due to many external factors that can influence the qualitative and
quantitative chemical profile of plants, the application of simple
marker definition as a QC tool unfortunately cannot be directly
1602 Natural Product Communications Vol. 13 (12) 2018
applied [1, 60]. To overcome this problem Benssousan et al. [1]
proposed a new term i.e. Herbal Chemical Marker Ranking System
(Herb MaRS). In this new system,chemical markers were ranked in
various categories i.e. x, 0, 1, 2, 3, 4 and 5; this ranking was based
on direct observations of therapeutic or toxic effects that are
detectable and measurable for the marker(s) in herb-preparations;
only Herb MaRS that has a ranking of 3-5 can be applied as a QC
tool. Recently, Rivera-Mondragôn et al. [61] reported some
chemical markers that can be applied as QC tools for the genus
Cecropia based on the categories of Herbs MaRS. WHO guidelines
[62] describe a method for selection of the marker(s) as the QC tool;
this guideline describes a marker as a constituent with known
therapeutic and or pharmacological activity, and markers should be
detectable and quantifiable, and so a marker must occur in sufficient
quantity in the herb drugs. If a marker is used for BIM, it must be
specific for one plant or certain plant species and genera. Liu et al.
[63] and Yang et al. [64] proposed the term Q-marker that can be
summarized as follows: (1) a marker is a chemical compound that
exists in herbs in traceable quantity; (2) markers can be qualitatively
and quantitatively determined; (3) markers should be associated
with special bioactivity; (4) the related effect and pharmacokinetics
data of the marker are known; and (5) markers should be related to
traditional use. It can be assumed that WHO’s definition of a
selected marker, and the term Q-marker showed almost the same
descriptions as a Herb MaRS ranked 3-5. For simplification, all
these categories of a marker will be described in this present review
as quality active marker (QA-marker). If for certain herbs the QA-
marker is not yet determined, a combination of chemical profiles,
bioactivity/clinical-profiles and PLS-DA can be performed for
determining the QA-marker. Unfortunately, only few publications
have described the QA-marker for certain herbs that appear
nowadays (2015-2018); and most of these recent publications did
not mention the limit of specification range (λ) of the QA-marker
for specific therapeutic application. For QC purposes, all
commercial drug preparations must have a detailed analytical target
profile (ATP), in which the target concentration τ ± λ of the active
pharmaceutical ingredient (API) must be described. If this limit
specification range is not yet available the pharmaceutical industry
must self-determine the limit specification range for each QA-
marker in all their commercial herbal products.
Constituents of interest or identity markers (for each of the herb’s
monographs) have been described by some guidelines[14-16]. Our
experience showed that it was not easy to select specific chemical
marker(s) for a certain herb or plant; the compound of interest
might not be detected in the described plant, or it could also be
detected in many other plants [1, 60]. Therefore, it is not
recommended to apply this identity marker or compound of interest
as sole BIM for an individual commercial herb(s) or plant
material(s), or in their mixtures. For validation of the BIM by using
a chemical marker, SITM and SSTM must be prepared (section 3),
and this could be very difficult, because the concentrations of
marker(s) in plants can be affected by many external factors [1].
It is well known that chemical constituents of herbal drug
preparations are very complex, so the method of choice for the QC
tool must be a chromatographic one (HP/TLC, UPLC, HPLC, GC);
all methods should be validated first, before being applied for
routine application. The most important validation parameter is the
selectivity or specificity of the method; identity and purity of all
target peaks in samples must be evaluated and verified using either
a DAD or MS detector; the Rs-value of the target peak to the
nearest peak should be >1.75. If multiple reactions monitoring
(MRM) by LC-MS/MS can be performed, there is no need to check
the Rs-value of the target peak, if the chemical structures of the
Indrayanto
target marker(s) are known. Important recent developments in
chromatographic validation methods for the compound/marker
approach have been reviewed and discussed recently [23]. A brief
summary of the important developments is presented in Table 1.
Table 1: Summary of the recent developments in validation method for marker approach.
 Correlation coefficient (r) cannot be applied as a sole parameter for expressing the
linearity (even r close to 1)
 Calculated test value Xa/Xpa must be < the lowest concentration level of the
calibration standard
 Accuracy/trueness and precision should be evaluated simultaneously with a single
acceptance criterion
 It is not recommended to evaluate accuracy/trueness and precision independently
 Ra of QA-marker(s) in commercial herbal drugs must be included in their limit
specification range
 Sample's DLa must be determined and verified for each matrix
 Internal quality control must be performed for each series of measurement at QC
laboratory
a
See text for explanation
AOAC published Standard Method Performance Requirements
(SMPRs) for analyzing certain compounds (markers) in certain
preparations. This SMPR described method performance
requirements for certain compounds (recovery, repeatability,
reproducibility, detection limit), but unfortunately a detailed
analytical method was not described. For example, AOAC SMPR
2017.003 [65] described SMPR of proanthocyanidin in cranberry
fruit, juice, beverage, dried cranberry, cranberry sauce, ingredients
and dietary supplement formulations. Monographs of the USP
herbal medicines compendium [14] and other guidelines [15-18]
generally describe the method of analysis and the minimum
concentration criteria of a certain or group of marker(s) in the
specific herbal preparations. The described method of analysis
could be applied for the specific described raw materials or
preparations, but unfortunately cannot be used for analyzing the
marker(s) in other herb preparations or mixed herbal drug
preparations. A valid method of analysis of marker(s) must be
developed for each of the specific herbal drug preparations.
Recently Langer et al. [66] stated that the criteria of minimum
concentration of certain marker(s), or a group of
constituents/markers in certain herbs, described by some guidelines,
cannot be correlated directly with its quality or efficacy, but
sometimes these criteria can be correlated only with the quality of
certain herbs. For correlating the concentrations of certain marker(s)
in herb preparations to its efficacy and safety, quantification of QA-
marker(s) should be carried out to determine whether its
concentration in herb preparations is included in its limit
specification range.
Due to the limited amount and relatively high price of many
authentic standard markers, quantitative determination of marker(s)
in herbal drugs as a QC tool can sometimes be very difficult. Ning
et al.[67], Xu et al. [68] and Zhu et al. [69] proposed quantitative
analysis of multi-components using a single marker only (QAMS),
or single reference standard for determination of multi-components
(SSDMC). This method does not need to have all authentic
standards for the quantification of many markers in herbal drugs.
Those authors proposed a method that used a single marker only as
standard; concentration calculation for other marker(s) can be
performed using a correction or conversion factor (F).
/
Fx = i = 1, 2.........n (1)
/
Σ
Fx = .............................. (2)
𝒏
Cx = Ax. Cs. ( ).................... (3)
where s = standard marker, x = marker x, A = respond detector or peak area; C =
concentration, i = concentration levels, n = number of concentration levels.
Quality control methods for herbal drugs
According to EMA [70], for performing a single point calibration
(equation 3), the concentrations of Cx must be within ± 30% of Cs.
A validation method for QAMS or SSDMC can be performed by
using a standard addition method, as described in our previous
review [23], or by comparing the results of analysis using a
QAMS/SSDMC method to results analysis that used specific
standards for each marker; detailed validation methods for
comparing two procedures have already been described in theUSP
41 <1010> [26].
The results (R) of analysis of any QA-marker(s) in herbal drug
preparations, or the results of recovery evaluations should be
included in the pre-determined specification range (label
claim/target concentration ± λ) that was described in the ATP; R
should be calculated by equation 4 instead of equation 5.
R = Mean ± CI/PI/TI ……….(4)
R = Mean ± SD (RSD)……. .(5)
CI is confidence interval, PI is prediction interval and TI is tolerance interval; detailed
equations are described in the USP 41 <1010> and <1210 [26, 71]. For QC purposes, it
is recommended to apply TI instead of CI or PI [23].
Recently Zhao et al. [9] proposed certain polysaccharides as QA-
markers for a QC tool for a Chinese traditional medicine (TCM),
due to a long history of the application of a water-decoction for
TCM. Shi et al. [66] proposed effective combinatorial markers
(ECM), whilst Guo et al. [10] proposed an effective compounds
combination (EEC) instead of certain markers as a holistic QC tool
for herbal medicine. EMC or EEC was determined using an
(animal) bioactivities model. Lee et al. [73] determined 14 markers
simultaneously (Herb MaRS ranked >3) from Chinese medicine
formulations containing a seven herbs mixture; the same concept
using many markers as a QC tool was developed by Yang et al.
(74); these markers were named bioactive chemical markers
(BMC). According to the description of EMC, EEC and BMC,
these categories of marker could also be defined as QA-marker(s).
Analyses of some specific QA- marker(s) from herbal drug
preparations might be used to replace conventional chemical
profiling, but further work is needed to confirm this. In conclusion,
quantification of certain marker(s) as a QC tool for herbal drug
preparations could be well applied and correlated to its efficacy if
the marker(s) is a QA-marker. As a QC tool, the specification limit
of each QA-marker (label claim or target concentration ± λ) should
be determined for every marketed herbal medicine for specific
therapeutic application.
Analysis of chemicals and toxic-compounds in herbal drugs: In
order to ensure the safety of herbal drug preparations,
concentrations of endogenous toxic metabolites, heavy metals,
residual pesticides, mycotoxin, harmful organic additives,
pollutants, chemical drugs and antibiotics in herbal drugs or extracts
should be below the maximum permitted level (MPL) or maximum
residual limits (MRL); this means that the method used must have a
sample detection limit (DL) below its MPL. The amount of
endogenous toxic compounds should be not more than 1.5 µg/day
for carcinogenic and genotoxic compounds [1].
According to guideline VICH GL 49 [75] a sample’s detection limit
must be determined in two steps i.e. first by determination of the
instrumental DL, and then a sample's DL, as described by equations
6 and 7. It is recommended to determine a sample’s DL for each
different matrix. Instrumental QL (quantification limit) was
estimated by 3 Dl. An amount of standard toxic compound at a
concentration of instrumental QL was spiked to the matrix, then the
Natural Product Communications Vol. 13 (12) 2018 1603
sample was analyzed using the proposed method, and the SD was
calculated (n= 7 replications).
Sample DL = t. s........... (6)
Sample QL = 3. DL....... (7)
where t is one-tailed (p = 99%, n-1), and s is the SD of the results of analysis of the
spiked sample,
It is recommended to apply the method of the USP 41 <1210> or
VICH GL49 [71, 75] for determining the instrumental DL. For this,
a linear regression curve that uses relatively low concentrations of
the target toxic compounds in a certain matrix should be
constructed. This method of DL determination was identical to the
that described previously by Funk et al. [76]; in this case,DL = Test
value Xp/Xa (p = 0.05), and QL = 3 DL. Detailed discussion
regarding this Xp/Xa was provided by previous publications [23,
76]. Sample DL that has been calculated by equation 6 must be
verified according to the Eurachem Guide [77]. For this
verification, a cut-off value must be determined with a false-
negative not more than 5%, as a general acceptance criterion.
Values of sample DL must be lower than the cut-off value, and the
cut-off value must be lower than the MPL. Detailed discussion is
presented in a review by the author [23].
General methods for the determination of toxic compounds and
their MPL in herbal drugs, such as heavy metals, pesticides, and
toxins have been described by some guidelines [3, 12-14].Official
methods for the determination of aflatoxins and residual pesticides
have also been described [13, 25],whilst methods for determination
of As, Cd, Pb, Hg total, methyl Hg and other toxic metals are
described by the guidelines of USP <233>[78] and WHO [13].
EMA [64] described in detail a method validation procedure for
pesticide residual analysis. For analysis of chemical drugs in
marketing dietary supplements, official methods from the USP 41
<2251> can be applied [79]; these methods can be used for
detecting chemical drug(s) in herbal medicines. Recently, Thermo
Fischer Scientific described an on-line calculation method i.e. “J
value Calculator” for calculating the MPL of certain metals in the
(herbal) drug preparations [80].
Determination of non-specific parameters and foreign matters:
According to the American Herbal Product Association [81], WHO
[13], and USP <561> [25] herbal products should be free of foreign
matter i.e. parts of herbs other than those named, soil, stones, sand,
dust, glass, plastic, metals and foreign inorganic matter. Herbals
should be examined for the absence of visible signs of
contamination by mold, insects and other animal contamination or
excreta; it should be free of abnormal odor, discoloration, slime or
signs of deterioration. Non-specific parameters that should be
determined are: ash (total ash, acid-insoluble ash and water-soluble
ash), extractable matter (water and ethanol), water and volatile
matter, volatile oils, bitterness value, hemolytic activity, swelling
index, foaming index, tannin total, crude fibers and starch content;
methods of determination have already been described in detail [13,
25]. For botanical extracts, non-specific parameters that should be
determined are residue on evaporation, residual solvents and
alcohol [82]. It seems it is difficult to correlate directly the non-
specific parameters to therapeutic-activity, toxicity and chemical
profiles of the herbal drug preparations. These non-specific
parameters might be applied for thesimple-standardization of the
herbal medicines in which their QA-marker(s) and/or chemical
profiles are not yet determined.
Conclusions and recommendations: For producing good quality
herbal drug preparations, botanicals and phyto-pharmaceuticals, all
incoming raw plant materials, extracts and excipients must be
strictly tested. It means that a completedQC must be performed i.e.
1604 Natural Product Communications Vol. 13 (12) 2018 Indrayanto

specification range for a certain QA-marker is not yet officially

available, the pharmaceutical industry should undertake research to
determine it before commercialization of the product. Criteria of
minimum concentrations of certain markers, as described in some
guidance, unfortunately cannot be applied as a QC tool [66].

FT-IR methods (ATR, 2D, Imaging) combined with MVA are

Figure 1: Complete QC parameters for every stage in the production line of herbal
drugs.
recommended as QC tools for qualitative and/or quantitative
analysis of individual herbs or extracts and in drug preparations.
These methods are relatively cheap, fast and accurate compared
with the chromatographic methods. For application of FT-IR
methods, authentic BRM, standardized QC samples of IP and FP
must be available; SITM and SSTM for every method must be first
determined and validated. The author recommends FT-IR methods
as a QC tool for pharmaceutical companies in developing countries.

Application of a DNA method only as a QC tool is not

evaluating the identity of raw materials and their chemical profiles,
recommended due to the lack of specificity; DNA methods are
quantification of the QA-marker(s) (if any), confirming all toxic
recommended for detection of species substitution or falsification
compounds must be < MPL, and determining other selected non-
[83]. Constituents of interest or identity markers are not
specific parameters; all those measured data of the incoming
recommended to be applied as BIM of any herbs; this is also due to
materials must fulfill the pre-determined specification range or
the lack of specificity.
acceptance criteria. Evaluation of the possible contamination of
toxic compounds for IP samples is not needed. FP and KP samples
To gain reliable results of any analysis performed by a QC
should be evaluated according to the parameters described by their
laboratory, all methods used must be validated according to the
certificates of analysis and/or labels. The QC laboratory of the
latest guidance. In addition, internal quality control (IC) as
pharmaceutical company should be able to assure that the contents
described by Eurachem [77] must be carried out for each series of
of the herbal product are exactly as printed on the label (Figure 1).
measurements by using QC samples. If the results of QC samples
cannot fulfill the acceptance criteria, the results of analysis of the
If a QA-marker in a certain herb is already well specified, there is
whole series of the measurements on that day must be removed or
no need for chemical profiling for this herb. In this case, the result
must be re-analyzed, and a partial or full re-validation of the method
of quantitative analysis of the QA-marker in the herb must be
considered.
included in the pre-determined specification range of the ATP
(target concentration/label claim ± λ). Further work is needed to
Acknowledgments - The author would like to offer thanks for some
determine the best marker categories (Q-marker, Herbs MaRS,
references that have been provided by Ms Febry Ardiana (PT
ECM, EEC, or BMC) that can replace the chemical profiling. If a
Bernofarm, Surabaya, Indonesia).
QA-marker is not yet well specified for a certain herb, chemical
profiling should be performed as the main QC tool.If the limit

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