PROJECT REPORT

ON
INDUSTRIAL TRAINING
DR. ASHAVIN’S LABS SERVICES
PLOT NO 120 (10 MARLA, INDUSTRIAL AREA) PHASE 2 CHANDIGARH)

GOSWAMI GANESH DUTTA SANTAN DHARMA COLLEGE

SEC 32 CHANDIGARH-160032

Submitted in Partial fulfillment of the requirement for the award of the

Degree
of
M.Sc. APPLIED CHEMISTRTY(PHARMACEUTICAL)
Under the supervision of

Submitted By: Submitted to:

Priyanka Rani Dr. Jasamrit Nayyar
Roll no. 2245858 Head Of The Department
M.Sc. Applied Chemistry Department Of Chemistry
(Pharmaceutical) GGDSD College sector 32-c
Chandigarh
Supervised By:
Dr. Mamta Sharma , Dr. Neha Dhiman & Dr. Geetika Sharma
Depatment Of Chemmistry
G.G.D.S.D College

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 CERTIFICATION

This is to certify that Miss Priyanka, a student of dept. of chemistry, G.G.D.S.D. College,
Chandigarh has completed the compulsory Industrial Training six weeks in Dr. Ashavin’s lab
services, Chandigarh after completion of 1st year in M.sc Applied chemistry

PLACE: CHANDIGARH Dr. Jasamrit Nayyar

DATE:15/04/2023 (Head Of The Department)

Department of Chemistry

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 ACKNOWLEDMENT

Before submitting the training report, it is pleasure to express deep regards to all those who
have being associated with me in the project work.

Words seems to be insufficient in conveying out thanks to those who helped me in this
journey. It indeed gives me immense pleasure to express my gratitude to Dr. Mamta Sharma
(Assistant professor) Dr. Neha Dhiman (Assistant Professor) Dr. Geetika Sharma (Assistant
professor) for their special attention and to all my teachers and guides for helping me and
guiding me in completing the project. I am also thankful to Mr. Ved Prakash Singh and Dr.
Ashvin Aggarwal (dr. Ashavin’s lab) who guided me during the whole journey of my training
period.

I want to take this opportunity to express my sincere thanks and deepest gratitude to my
parents and fellow mates for their support

I want to pay warmer regards to other staff members of Dr. Ashavin’s lab for their cooperative
attitude.

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 ABSTRACT
Impurities in pharmaceutical are the unwanted chemicals that remain with the active
pharmaceutical ingredient (API) or can develop during formulation or upon ageing of both API
and formulation. The presence of unwanted chemicals even in small amount may influence
the efficacy and safety of the pharmaceutical products.

The different pharmacopeia’s such as the British Pharmacopeia (BP), Indian Pharmacopeia
(IP), and the United State Pharmacopeia (USP) have incorporated limits to allowable levels of
impurities present in the API or formulation.

This report gives an overview on quality control process and its use in calculating the impurity
limits in various pharmaceutical companies

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Contents

• Introduction
• Company profile
• Products
• Departments of company
• Impurity introduction
• Structure
• Mechanism of action
• Quality control analysis of nitrosoguanidine
• Ph determination
• HPLC
• Results
• Conclusion
• References

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Introduction
Company profile

Dr. Ashavin’s lab services are technology based, research driven enterprise aim to setup the
world’s largest inventory of drug product and impurity. They aim to enable the research
community in accelerated discovery via synthesis of complex and difficult to make
compounds. they specialize in innovative organic chemistry and synthesis of multiple complex
molecules, metabolites, impurities, compounds. they also undertake synthesis of complex
molecules or difficult to synthesize compounds/intermediates. They have a strong
commitment towards improving our quality systems, their members are remarkably exercised
scientists with impeccable knowledge and comprehensive exposure. Our services are not
limited to the pharmaceuticals, nutraceuticals, agrochemical industries.

They believe in the power of research and the need to stay competent and dynamic. In this
challenging and competitive environment, our business driven by a strong commitment and
operates with the highest standards of ethics and integrity THE CATAYST IN MAKING YOUR
REASEARCH WORK. Their corporate strategy aims to achieve SUPERIOR CUSTOMER DELIGHT
with extraordinary standards in every aspect of the business process and rapid
internationalization of company’s business. This involves all aspects like the process, people,
sales, business level standards, research and development activities etc.

Services
Pharmaceutical impurity standards
• Supplier and manufacturer of pharmacopeial and non-pharmacopeial impurity
standards
• Supplier and manufacturer of drug metabolites/ in house impurity standards

Technology development services

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 • Development and optimization of synthetic routes
• Support in all aspects of process development

CRAMS

• Capabilities to provide a wide range of CRAMS starting from route selection.

• Synthesis of a wide variety of organic compounds on the milligram to kilogram

Intellectual property rights (IPR)

• Patent landscaping and mapping/ freedom to operate/non infringing analysis.

• Invalidity study/patentability study.

All impurity standards are supplied with certificate of analysis and characterized by:

• Chromatographic purity (HPLC or GC)

• Nuclear magnetic resonance
• Mass spectrum

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 Various departments

Department

Quality Quality
Research and
Assurance Packaging
Control Development

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Quality control department
QC is the part of GMP concerned with sampling, specification and testing. It ensures that the
product is pure and safe. Before consuming any type of medicine, they need to be tested and
approved for consumption in prepared pharmaceutical laboratories, so that they can be sold
and consumed by the population. In this article, we will see in some topics how these
processes work and their main function.

The main objective of quality control in the Pharmaceutical Industry is to test the drugs in
their various stages of production, verifying that they are able to proceed to the next stage
and release the manufacturing process in accordance with the regulations and specifications
required for consumption.

Company’s Analytical Expertise

• HPLC

Company’s capacities of stability studies

• Control on storage and management

• Development and validation of stability indicating methods

• Stability chamber with backup reach in chamber

Instruments used in qc lab

• Sonicator

• Balance meter

• Weighing balance

• HPLC

• PH meter

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 A

Analytical research department Dr. Ashavin’s lab

HPLC (High Performance Liquid Chromatography)

HPLC is an abbreviation for High Performance Liquid Chromatography. "Chromatography" is
a technique for separation, "chromatogram" is the result of chromatography, and
"chromatograph" is the instrument used to conduct chromatography.

Among the various technologies developed for chromatography, devices dedicated for
molecular separation called columns and high-performance pumps for delivering solvent at a
stable flow rate are some of the key components of chromatographs. As related technologies
became more sophisticated, the system commonly referred to as High Performance Liquid
Chromatography, simply became referred to as "LC". Nowadays, Ultra High-
Performance Liquid Chromatography (UHPLC), capable of high-speed analysis, has also
become more wide-spread.

Only compounds dissolved in solvents can be analysed with HPLC. HPLC separates compounds
dissolved in a liquid sample and allows qualitative and quantitative analysis of what
components and how much of each component are contained in the sample.

Fig.1 shows a basic overview of the HPLC process. The solvent used to separate components
in a liquid sample for HPLC analysis is called the mobile phase. The mobile phase is delivered
to a separation column, otherwise known as the stationary phase, and then to the detector

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at a stable flow rate controlled by the solvent delivery pump. A certain amount of sample is
injected into the column and the compounds contained in the sample are separated. The
compounds separated in the column are detected by a detector downstream of the column
and each compound is identified and quantified.

Fig.1 Overview of HPLC

2 The Apparatus of HPLC

The “Basic Overview of the HPLC process"(As shown in Fig.1) and its mechanisms have now
been covered. Going into more detail, HPLC consists of a variety of components, including a
solvent delivery pump, a degassing unit, a sample injector, a column oven, a detector, and a
data processor. Fig.2 shows the HPLC flow diagram and the role of each component.

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 Fig.2 HPLC Flow Diagram

As for HPLC, the pump delivers the mobile phase at a controlled flow rate(a). Air can easily
dissolve in the mobile phase under the standard atmospheric pressure in which we live in. If
the mobile phase contains air bubbles and enters the delivery pump, troubles such as flow
rate fluctuations and baseline noise/drift may occur. The degassing unit helps prevent this
issue by removing air bubbles in the mobile phase(b). After the dissolved air has been
removed, the mobile phase is delivered to the column. The sample injector then introduces a
standard solution or sample solution into the mobile phase (c). Temperature fluctuations can
affect the separation of compounds in the column. The column is placed in a column oven to
keep the temperature constant(d). Compounds eluted from the column are detected by a
detector which is placed downstream of the column(e). A workstation processes the signal
from the detector to obtain a chromatogram to identify and quantify the compounds

3 HPLC Separation

HPLC can separate and detect each compound by the difference of each compound's speed
through the column. Fig.3 shows an example of HPLC separation.

There are two phases for HPLC: the mobile phase and the stationary phase. The mobile phase
is the liquid that dissolves the target compound. The stationary phase is the part of a column
that interacts with the target compound.

In the column, the stronger the affinity (e.g.; van der waals force) between the component
and the mobile phase, the faster the component moves through the column along with the

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mobile phase. On the other hand, the stronger the affinity with the stationary phase, the
slower it moves through the column. Fig. 3 shows an example in which the yellow component
has a strong affinity with the mobile phase and moves quickly through the column, while the
pink component has a strong affinity with the stationary phase and moves through slowly.
The elution speed in the column depends on the affinity between the compound and the
stationary phase.

Fig.3 An Example of HPLC Separation

Chromatogram

The word "chromatogram" means a plot obtained via chromatography. Fig.4 shows an
example of a chromatogram. The chromatogram is a two-dimensional plot with the vertical
axis showing concentration in terms of the detector signal intensity and the horizontal axis
representing the analysis time. When no compounds are eluted from the column, a line
parallel to the horizontal axis is plotted. This is called the baseline. The detector responds
based on the concentration of the target compound in the elution band. The obtained plot is
more like the shape of a bell rather than a triangle. This shape is called a “peak”.

Retention time (tR) is the time interval between sample injection point and the apex of the
peak. The required time for non-retained compounds (compounds with no interaction for the
stationary phase) to go from the injector to the detector is called the dead time (t0).

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The peak height (h) is the vertical distance between a peak's apex and the baseline, and the
peak area (A) coloured in light blue is the area enclosed by the peak and baseline. These
results will be used for the qualitative and quantitative analysis of a sample's components.

Fig.4 Chromatogram and Related Terms

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Quality Assurance

Quality assurance (QA) is any systematic process of determining whether a product or service
meets specified requirements.

QA establishes and maintains set requirements for developing or manufacturing reliable

products. A quality assurance system is meant to increase customer confidence and a
company's credibility, while also improving work processes and efficiency, and it enables a
company to better compete with others.

The ISO (International Organization for Standardization) is a driving force behind QA practices
and mapping the processes used to implement QA. QA is often paired with the ISO
9000 international standard. Many companies use ISO 9000 to ensure that their quality
assurance system is in place and effective.

The concept of QA as a formalized practice started in the manufacturing industry, and it has
since spread to most industries, including software development.

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Quality Control Analysis of Nitrosoguanidine
INTRODUCTION (Nitrosoguanidine)
Methylnitronitrosoguanidine (MNNG] or MNG, NTG when referred to colloquially as
nitrosoguanidine) is a biochemical tool used experimentally as
[1] 6 4
a carcinogen and mutagen. It acts by adding alkyl groups to the O of guanine and O of
thymine, which can lead to transition mutations between GC and AT. These changes do not
cause a heavy distortion in the double helix of DNA and thus are hard to detect by the DNA
mismatch repair system.
One of the earliest uses of methylnitronitrosoguanidine was in 1985. A group of scientists
tested whether or not the chemical composition of methylnitronitrosoguanidine would
directly affect the growth of tumors and cancer cells in rats.
In the experiment, the cancer cells from a Japanese cancer patient were injected into 8 rats.
The biochemical tool and showed a decline of cancer cells in a few of the rats' bodies.
In organic chemistry, MNNG is used as a source of diazomethane when reacted with
aqueous potassium hydroxide.
MNNG is a probable human carcinogen listed as an IARC Group 2A carcinogen.

Induction of closely linked multiple mutation by nitrosoguadine

N- methyl -N-nitro -N-nitrosoguanidine (nitrosoguanidine) is a powerful and widely used

mutagen. Using synchronized populations of Escherichia coli, we have shown that large
numbers of mutations occur at specific loci when these loci are replicated. This specificity
makes it possible to use nitrosoguanidine to direct mutagenesis, to study the mode of
replication of the chromosome and to map the chromosome and to map the the chromosome.
The addition of 100ug nitrosoguanidine per ml. of growing culture causes a complete and
abrupt cessation of DNA synthesis. Movement of the application region is therefore halted for
the entire treatment period. It is treated cell in buffer as a further precaution to stop replication
to minimize lethality .

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Selective mutagenesis at the replication region implies that if multiple mutations are induced
by nitrosoguanidine, they will be closely linked. examination revealed that azide resistance
clones induced by nitrosoguanidine for the presence of other mutations and find a large excess
of these at genes closely linked to azide.

Explosives components (nitro organic compounds) continue to be produced in large quantities

and therefore are subject to regulations by environmental agencies. These compounds are, for
the most part, non-volatile and sparingly soluble in water. The main concern, from an
environmental standpoint, thus becomes the contamination of aquifers, both surface water
and groundwater. The objective of this work was to develop analytical methods for the analysis
of low concentrations of nitroguanidine in surface and groundwaters. Due to the high polarity
of nitroguanidine, the collection of this explosive on solid sorbents failed. However, it was
possible to concentrate nitroguanidine in aqueous samples by rotary evaporation at 50°C. The
nitroguanidine was analysed by high performance liquid chromatography (HPLC) with
electrochemical detection at a hanging mercury drop electrode (HMDE) positioned at -1.2 volts
vs an Ag/AgCl reference electrode. Provided the samples are not too complex, voltammetry,
particularly differential pulse voltammetry, offers a rapid, direct and sensitive method for the
analysis of nitroguanidine. From voltammetry, it was established that nitroguanidine is reduced
via an irreversible diffusion controlled 4 e- process. Nitrated organic compounds are the most
widely used explosives components. These compounds have been and continue to be
produced in large quantities, and are therefore, subject to regulation by environmental
agencies. For the most part, these compounds are non-volatile but sparingly soluble in water.
The main concern, from an environmental standpoint, thus becomes contamination of aquifers,
both surface water and groundwater, in and near facilities producing, handling, and storing
explosives. While various toxicology studies have been carried out on these compounds (1-5) s
there exists no definitive toxicology data base for the establishment of acceptable levels of
aquifer contamination. Furthermore, reliable analytical methodology for the determination of
these components in aqueous samples is lacking. We have previously reported on the
application of solid sorbent collection techniques to the analysis of several explosives in water
by high performance liquid chromatography (HPLC) with reductive electrochemical detection
(6). This report summarizes our results on the determination of nitroguanidine in aqueous
samples by HPLC/electrochemical detection (EC) and voltammetry. Nitroguanidine can be
produced from guanidine, which is the strongest organic base known (7). Nitroguanidine is
used as a component of some munitions. It is about as powerful as 2,4,6-trinitrotoluene (TNT)
and explodes without producing a flash (7). While elaborate procedures have been developed
for the analysis of process streams for the production of this substance (8), there appears to be
a limited number of analytical. procedures for the measurement of low concentrations of
nitroguanidine in surface and groundwaters. In addition, the relatively high solubility of
nitroguanidine in water indicates high probability of contamination of the aqueous
environment.

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 Experimental analysis
All solvents used were "distilled in glass" grade, residue free . Nitroguanidine impurity [H2NC(NH2)-N-
N0,] Fluka AG, Chemische Fabrik CH-9470, Buchs. The stock solution is prepared by dissolving 100 mg
in 100 mL ethanol and is stored at 4°C.

Reagent
0.025 Sodium acetate, pH 6. Sodium acetate (4.1 g) is dissolved in 500 mL distilled water, adjusted to
pH 6 with acetic acid, then diluted to 2 L with distilled water 1-Propanol, distilled in glass

HPLC Mobile Phase

1-Propanol, 0.025 sodium acetate (pH 6) (30/70 v/v). 1-propanol (300 mL) is added to a 1 L volumetric
flask and diluted to the mark with 0.025 5 NaAc solution. This solution is filtered through a 0.45 pm
Nylon-66 filter and added to the 2 L flask for pump A. For 20/80 (v/v), 1-propanol (200 mL) is added to
a 1 L volumetric flask, and diluted to the mark with 0.025 acetate solution. This solution is filtered
through 0.45 pm Nylon-66 filter and added to a 2 L flask for pump B.

Apparatus

Electrochemical detectors used in this work were a Bioanalytical Systems (BAS) Model LC4B
(17-D) dual electrode detector and an EG&G Princeton Applied Research (PAR) Model 310
polarographic detector and (PAR) Model 174A polarographic analyser. The BAS detector was a
BAS TL-6A thin layer cell assembly which consists of a gold-mercury working electrode, stainless
steel counter electrode and a RE-1 Ag/AgCl reference electrode. The reference electrode is
housed downstream in an RC-2A reference electrode compartment. The PAR detector consists
of a hanging mercury drop electrode (medium size). The LC column was a 25 x 0.46 cm C18 (5
pm particle size) Dupont Zorbax . column. The injection valve was a Rheodyne Model 7120
fitted with a 20 pL loop and mounted vertically for sample degassing similar to the method
proposed by Lloyd (9). A Newlett-Packard Model 7045 A X-Y recorder and a Hewlett-Packard
Model 3390 A reporting integrator were used for data collection/readout. A Perkin-Elmer Series
2 liquid chromatograph was fitted with the essential dissolved solvent oxygen removal
apparatus for both pumps (10, ll). A BAS Model MF 4000 flow through pulse damper was
installed between the pump outlet and injection valve. The mobile phase was deoxygenated by
purging with helium at approximately 120 mL/min for 30 minutes followed by continuous
sparging at: 2-4 mL/min.

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Results and discussion

Due to the high polari-ty of nitroguanidine, solid sorbents which were previously used to
collection explosives from water (6) were not applicable to nitroguanidine. These included
Porapak-R, Porapak-S, XAD-4, and Carpark-failed as well. However, it was possible to
concentrate nitroguanidine in water samples by rotary evaporation at 50°C, due to the low
volatility of this compound. A four-day replication study of samples of laboratory water fortified
with nitroguanidine is shown in Table 1. The volume of aqueous sample which was reduced to
a final volume of 10 mL was within the range 100-250 mL depending upon the concentration
of nitroguanidine. In general, reasonable and consistent recoveries were realized over the
concentration range tested. Thus, rotary evaporation appears to be an effective way to
preconcentrate nitroguanidine in very dilute (pg./L) aqueous samples. The chromatographic
separation of nitroguanidine from other polar explosives such as HMX* and RDX"" is shown in
Figure 1. As noted, the explosives are adequately resolved. However, the nitroguanidine is not
well retained by the column and elutes first. For aqueous samples containing other highly polar,
electro reducible substances, interferences (either organic or inorganic) could be a potential
problem. The reduction potential of nitroguanidine is about -1.2 V vs Ag/AgCl reference
electrode. This makes nitroguanidine the most difficult of the explosives to reduce and negates
to some extent the selectivity of the electrochemical detector. Various water samples from an
army ammunition site (Sunflower Army Ammunition Plant) were shipped to ORNL and analysed
for nitroguanidine to further validate the methodology for this compound. The results are
presented in Table 2. Sample 1 was analysed directly. Samples 2-6 were analysed after
concentration. Sample 7 was diluted prior to analysis. While no reference data were available,
the data generated are within historical limits for these particular sampling points at Sunflower
Army Ammunition Plant (SUP). Voltammetry allows a rapid and direct, yet reasonably sensitive
method for analysis of explosives in water. The trinitro aromatic compounds such as tetryl and
TNT are the easiest to reduce. These are followed by the nitrate esters such as nitro-glycerine
and then the nitramines such as HMX, RDX and nitroguanidine. Voltametric analysis is
applicable to mixtures, provided these mixtures are not overly complex. The governing factor
is the relative difference in the reduction potentials of the components. Solvent partition with
methylene chloride

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 Column: Dupont Zorbax ODS (511)

Mobile Phase: 1-propanol: 0.025 M Acetate buffer, PH 6.0, (20:80)

Flow: 0.5 rnL/min

Inj. Vol.: 20 pL

Detection: Au/Hg electrode @ -1.2 V vs Ag/AgCl Peak: 1 Nitroguanidine, 2 mg/L 2 HMX, 2 mg/L
3 RDX, 2 mg/L

Figure 1. HPLC Separation of Nitroguanidine, HMX and RDX with Electrochemical Detection.

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background baselines are also partially compensated for by differential pulse voltammetry. Cyclic
voltammetry, on the other hand, can be carried out faster, typically 100 mv/sec scan rate vs 5 mv/sec
sacn rate for differential pulse. Since most instruments are multipurpose, the preferred technique is
to obtain the cyclic voltammetric information first, then switch to pulse techniques if needed for
increased sensitivity and resolution of voltammetric reduction waves. The samples in Table 2 were
analyzed for nitroguanidine by voltammetry and. the results were in good agreement with the values
obtained by HPLC/EC . Thus, voltammetry offers a rapid and direct way of determining nitroguanidine
in aqueous samples where the concentration may extend into the low milligram per liter range. For an
irreversible single sweep voltammogram, the peak current (i.,) is given by (12) as: i, = 3.01 x
105n(ana)1/2AD1/2Cv1/2 (1) which shows that the peak current is proportional to the parameters
AD1I2Cv1I2 as in the case for a reversible electrode process, and al.so to n(ana)ll2 instead of n312 as

background baselines are also partially compensated for by differential pulse voltammetry. Cyclic
voltammetry, on the other hand, can be carried out faster, typically 100 mv/sec scan rate vs 5 mv/sec
sacn rate for differential pulse. Since most instruments are multipurpose, the preferred technique is
to obtain the cyclic voltammetric information first, then switch to pulse techniques if needed for
increased sensitivity and resolution of voltametric reduction waves. The samples in Table 2 were
analysed for nitroguanidine by voltammetry and. the results were in good agreement with the values

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obtained by HPLC/EC. Thus, voltammetry offers a rapid and direct way of determining nitroguanidine
in aqueous samples where the concentration may extend into the low milligram per litre range. For an
irreversible single sweep voltammogram, the peak current (i.,) is given by (12) as: i, = 3.01 x
105n(ana)1/2AD1/2Cv1/2 (1) which shows that the peak current is proportional to the parameters
AD1I2Cv1I2 as in the case for a reversible electrode process, and al.so (ana)ll2 instead of n312 as for
the reversible case. The term (ana) is related to the peak-half peak potential difference

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24
Conclusion
The quality control testing is assigned to production or quality control depending on the
company basis of large scale and small scale. The quality executives evaluate the quality tests
of the impurities to pass the product into market. These are regularly checked by the RA and
FDA bodies. These tests bring out the errors and misbranded and bad quality products. The
standard of the products are based upon this analysis. It is the duty of the pharmaceutical
companies to manufacture the dosage forms which can sutain rattling and handling the
demands of pharmaceutical companies

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Refrences
• http://www.dr.ashavinslabservices.com
• https://www.shimadzu.com
• http://www.osti.gov.in
• http://pubchem.ncbi.in

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