Heavy Metals and Microbial Contamination in Medicines. Learn more at https://www.alfachemic.com/testinglab/applicationnote/Heavy-Metals-and-Microbial-Contamination.html

1. Heavy Metals and Microbial
Contamination in Medicines
With the rapid population ageing of the world, the medicine and health problems of urban
elder in many countries such as Japan and China are standing out. For the human
society, drug is a special commodity, which is characterized by complexity of types,
medical specificity, and strict quality standards. It is not like other goods with quality
grade to divide such as excellent, first class or second class. There is the only difference
between compliance and non-compliance in medicines. Generally, all medicines, whether
they are synthetic or of plant origin, should meet the basic requirements of safety and
effectiveness. However, the most serious drug pollution is heavy metal or microbial
pollution in the production and storage of medicines. Therefore, heavy metals and
microbial contamination become the key factors for their quality assessment and quality
control to ensure the drug safety, purity, potency and efficacy.
Heavy Metals Analysis
Usually heavy metals refer to a general term of all metals that can be reacted with
thioacetamide, hydrogen sulfide or sodium sulfide to produce a color change in the pH
3.5 of acetate buffer solution, These metals include silver, lead, mercury, copper,
cadmium, bismuth, antimony, tin, arsenic, nickel, cobalt, zinc, and so on. The
contamination of these heavy metals to the drugs exists in raw materials, excipients and
preparations and so on. For example, some mineral drugs themselves contain heavy
metals, such as cinnabar and realgar, and some other herbal medicines are accumulated
heavy metals during planting due to the pollution from the water or soil, while sometimes
we need to use some heavy metals to act as carrier or catalyst in the production of
excipients and preparations, which may directly lead to high levels of heavy metals in the
drugs. No matter how the contamination by toxic metals either be accidental or
intentional, these toxic metals can pose clinically relevant dangers for the health of the
user and should therefore be limited. For example, when the mercury content is up to
200 ug/L in the blood, it can lead to the renal dysfunction, tremor and even paralysis; Tin

2. and its organic compounds are deadly poisonous, of which more than 250 mg / kg
content can lead to severe brain edema; Cadmium is a commonly known poison
substance that can accumulate in the body for a long time, which has been identified as
IA carcinogen by International agency for research on cancer (IARC). All in all, the
contamination of drugs by toxic metals is a threat to human body and it is necessary to
be qualitative and quantitative analysis for our health life. Generally, the main methods
commonly used for detection heavy metals are atomic absorption spectrophotometry
(AAS), inductively coupled plasma (ICP) and neutron activation analysis (NAA).
Atomic Absorption Spectrometry (AAS):
Atomic Absorption Spectrometry (AAS)
Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the
quantitative determination of chemical elements using the absorption of optical radiation
(light) by free atoms in the gaseous state. There are four kinds of atomizer: flame
atomizer, graphite furnace atomizer, hydride generation atomizer and cold vapor
atomizer.
Flame atomic absorption spectrometry (FAAS) have many merits, such as low cost, easy
operation, fast analysis speed, good selectivity, and the signal is stable in the
measurement of high concentration heavy metal elements. It is the most widely adopted
method to detect the heavy metal in recent years.
Graphite furnace atomic absorption spectrometry (GFAAS) has the advantages of high
sensitivity and low detection limit. The relative standard deviation (RSD) of the measured
values can usually be controlled within 5%. At present, it is one of the most important

3. methods for the determination of trace heavy metal elements. When GFAAS is used for
the determination of trace heavy metal elements, it often needs to add appropriate matrix
modifier to eliminate the interference of the matrix, and make the analyte to be fully
released to reduce the ash loss.
Hydride generation-atomic absorption spectrometry (HG-AAS) is an efficient separation,
enrichment and sampling technique, which measures elements by reacting with reducing
agents in an acidic environment at room temperature to form gaseous hydride or atomic
vapor, and then separate from the substrate. This method can greatly improve the
sensitivity and injection efficiency, and significantly reduce the matrix interference, which
can determine the elements such as arsenic, germanium, tin, selenium, bismuth,
antimony, mercury, lead and tellurium.
Cold vapor-atomic absorption spectrometry (CV-AAS) is a specific method to determine
the micro-content element of mercury. This method for measuring and tracing mercury
has high sensitivity and accuracy features. Dai yihua used CV-AAS for detecting mercury
in medicinal materials, of which the detection limit is 0.334 ug/g, the RSD is less than 5%,
and the recovery is between 98% and 104%.
Inductively Coupled Plasma Methods (ICP-AES, ICP-MS):
Inductively coupled plasma methods include inductively coupled plasma atomic emission
spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS).
Inductively coupled plasma (ICP) as a kind of the atom spectrum analysis technique
began in the 1960s, and by the 1980s, theory and application entered its mature stage. It
is an analytical technique mainly used for the detection of trace metals.
ICP-AES ICP-MS

4. ICP-AES also referred to as inductively coupled plasma optical emission spectrometry
(ICP-OES). It is a type of emission spectroscopy that uses the inductively coupled
plasma to produce excited atoms and ions that emit electromagnetic radiation at
wavelengths characteristic of a particular element. ICP-AES shortens the analysis time
and improves the analysis efficiency, and can simultaneously determine various metallic
elements, which make up the defect of atomic absorption spectrometry. Therefore, it has
some advantages in the analysis of trace elements due to its high sensitivity, low
detection limit, high precision, wide linear range, multi-element simultaneous analysis
and small matrix effect. It has become one of the most effective methods for the
detection of metallic elements, and is widely used in the detection of heavy metal content
in various medicinal materials. ICP-MS is also an elemental analysis method that uses
the plasma as ion source, and can simultaneously determine multiple elements, which
has high sensitivity and can be combined with other chromatography to analyze element
valence state.
Atomic Fluorescence Spectrometry (AFS):
The principle of AFS is that when the sample is excited by radiation energy, the atomic
vapor of the metal will produce fluorescence effect and can be measured through the
strength of fluorescence. Atomic fluorescence spectrometry has high accuracy without
requiring separation, enrichment and other steps, but only have fluorescence effect on
special metal ions. Therefore it cannot detect all metal ions, which limits the application of
this detection technology.
Microbial Contaminantion Analysis
Medicines may be associated with a broad variety of microbial contaminants,
represented by bacteria, fungi, and viruses. Inevitably, this microbiological exerts an
important impact on the overall quality of medicines. The survival of any microorganisms
in the sterile preparation may contribute to serious or fatal injuries for the drug user, and
the aseptic testing of drug is the highest requirement for the microbiological limit testing
in drug quality management. The pharmacopoeia of many countries has described in

5. detail about the microbiological detection of drugs, and formed consistent detection
standards and operating procedures, which judge the product sterility by visual inspection
the turbidity of culture medium. With the progress of science and technology, many kinds
of new and high techniques have been widely recognized and applied in the field of
microbial identification and classification, such as bioluminescence detection, laser
scattering detection, mass spectrometry, PCR, DNA sequencing, and so on.
Adenosine Triphosphate (ATP) Luminescence Detection
Adenosine triphosphate measurement based on bioluminescence is a rapid method for
detecting contamination in injectable preparations. ATP exists in all organisms, which can
react with luciferase and generate the photons. ATP can be quantitatively analyzed by
the intensity of photons which are detected by a highly sensitive detector. However, this
method still requires a long time for detection, and the reagents are expensive and the
instrument needs high requirements for cleanness. Although the experimental results are
directly related to the luminous intensity of ATP, different growth environments and states
of microorganisms caused the outputs of ATP to be different, which increases the
suspiciousness between the experimental results and microbial quantity.
PCR/ Gen-probe
It mainly includes protein analysis and nucleic acid analysis (plasmid analysis,
hybridization, PCR, gene sequence analysis). Microbial protein analysis is mainly based
on the characteristic electrophoresis figure which is produced by microbial protein
electrophoresis under standard conditions. As the expression of protein is affected by
environmental factors, it is necessary to require the standard growth environment for the
bacterial strain. Nucleic acid analysis is studied on the basis of DNA. It can quickly
determine the classification of a certain microorganism or establish a new classification
for special microorganisms that are difficult to culture.
Flow Cytometry
This detection technology is based on the functional level of single cells or other
biological particles to conduct the quantitative analysis and separation tests. It can

6. analyze thousands of cells in a very fast speed, and can simultaneously get multiple
parameters from a measured cell. It has advantages of high speed, high precision and
good accuracy compared with the traditional spectroscopy and becomes the most
advanced quantitative analysis cell technology in the contemporary.
References
1. Kunle, Oluyemisi F, Egharevba. (2012) ‘Standardization of herbal medicines - A
review’, Int. J. Biodvers. Conserv, 4(3):101-112.
2. A Decool,V Goury, A Tlbl,S Chbaud, F Vmcent, et al. (1991) ‘Detection of bacterial
adenosine triphosphate through bioluminescence, applied to a rapid sterility test of
injectable preparations’, Analytzca Chlmrca Acta, 255: 423-425.
3. Chollet R, Kukuczka M,Halter N,et al. (2008) ‘Rapid detection and enumeration of
contaminants by ATP bioluminescence using the milliflex rapid microbiology detection
and enumeration system’, Journal of Rapid Methods & Automation in Microbiology,16(3):
256-272.
4. Kaleta E J,Clark A E,Cherkaoui A,et al. (2011) ‘Comparative analysis of PCR-
electrospray ionization/mass spectrometry (MS) and MALDI-TOF/MS for the identification
of bacteria and yeast from positive blood culture bottles’, Clinical chemistry, 57(7): 1057-
1067.
5. Gunasekera T S, Attfield P V,Veal D A. (2000) ‘A flow cytometry method for rapid
detection and enumeration of total bacteria in milk’, Applied and environmental
microbiology, 66(3): 1228-1232.
6. J Wang, JN Li, LY Hong, (2015) ‘Recent advances in inspection of heavy metals
present as pollutant in medicines’, PTCA, 51(7): 1043-1047.
Alfa Chemistry Testing Lab is the world’s leading third-party testing company, which
provides one-stop pharmaceutical analysis testing solutions for manufactures, suppliers,
retailers, and consumers.

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