“ROLE OF INSTRUMENTATION IN QUALITY CONTROL IN PHARMACEUTICAL

INDUSTRIES”

A dissertation submitted to

DR.REDDYS LABORATORIES LTD. BANASTHALI VIDHYAPITHFTO UNIT –VI, Baddi BANASTHALI, RAJASTHAN

IN THE PARTIAL FULFILLMENT FOR THE AWARD OF

MASTERS OF SCIENCE IN

PHARMACEUTICAL CHEMISTRY2008-2009

BYSONALI SHARMA

Guide: Mr. Rajesh M Sharma Dr.. Sarvesh PaliwalH.O.D., Associate Professor, Quality control, Deptt. Of Chemistry Dr.Reddy’s lab. Ltd. Pharmaceutical chemistry,FTO Unit-6, Baddi, Banasthali vidhyapith,

TABLE OF CONTENTS

• CERTIFICATE

• ACKNOWLEDGEMENT

• NTRODUCTION Company profile

• QUALITY CONTROL Instrumentation

Karl fischer moisture content detector

Polarimeter

Ultra-violet and visible spectrophotometry

Gas chromatography

High performance liquid chromatography

• QUALITY ASSURANCE

CERTIFICATE

This is to certify that Miss Sonali Sharma, final year student of M. Sc. Pharmaceutical Chemistry, Banasthali Vidyapith, Banasthali, Rajasthan, did her project work on “Role of instrumentation in quality control in pharmaceutical industries” for the partial fulfillment of the award of Master’s Degree. She worked from 12th January to 21st April, 2007 under my supervision in the Dr.Reddy’s Laboratories Ltd. FTO Unit-6, Khol, Baddi - Himachal Pradesh.

The data contained in her report has resulted from the work that she carried out during the tenure of her training program. This work was originally carried out in my laboratory by various workers.

I wish her all the success in her life.

Mr. Rajesh M SharmaHeadQuality Control Dr. Reddy’s Laboratories Ltd.FTO Unit – VI, Khol village, Baddi (HP)

DECLARATION

I hereby declare that the dissertation entitled “Role of instrumentation in quality control in pharmaceutical industries” submitted to Banasthali Vidyapith, Rajasthan and Dr.Reddy’s laboratories ltd. khol baddi, in partial fulfillment for the award of M.Sc. Pharmaceutical Chemistry is a record of research work done by me under supervision and able guidance of Mr. Rajesh sharma, H.O.D quality control.

I also declare that nothing in part or full has been submitted for the award for any other degree, diploma, or fellowship to any other university or institute.

Sonali Sharma

ACKNOWLEDGEMENT

A journey is easier when you travel together. This project report is the

result of five months of work whereby, I accompanied and supported by

many people and its easant time to express my gratitude for all of them.

Fore most, I would like to thank God for His grace and blessings.

The first person I express to my heartiest thanks to Mr. Rajesh M

Sharma, Head, quality control department , for providing me excellent

laboratory facilities, valuable guidance, untiring help during my project

work and constant encouragement during the whole period of my project

work

I wish to express to my sincere gratitude to Mr. Surinder Pal Singh, HR,

Dr.Reddy’s lab, FTO Unit-6, baddi for giving me an opportunity to carry

out my project work in such a prestigious institute.

I am grateful To Mr. Vikrant Singh Parmar, Mr. Ashwin Deep Mishra,

Mr. Ashwani Mahajan, Mr. Bipan Singh Katoch who have provided

support at each and every step of my work.

This acknowledgement seems to be incomplete without the name of Miss.

Jyotsna chauhan, Miss. Priyanka mishra, Miss. Babita. Whose

consistent help has only leaded me to the completion of this project work.

I am also thankful to Mr. Dharmender verma for enriching me during

course of investigations.

I owe my gratitude to Prof. D. Kishore, H.O.D., Deptt. Of Chemistry and

Pharmaceutical Chemistry, Banasthali Vidyapith, Rajasthan and Mr.

Sarvesh K. Paliwal for their kind support throughout the course.

The chain of gratitude would be incomplete, if I forget the name of my

friend Miss. Vaishali jamwal with her I enjoyed a lot at Dr.Reddy’s.

Finally, I express my gratitude to my family members for their blessings

and constant support. Words with me are insufficient to express the

feelings of my heart to acknowledge them for educating me and keeping

me in all comfort without which this work would not have seen the light

of the day at all.

Sonali sharma

COMPANY’S PROFILE

DR.REDDY’S LABORATORIES PROFILE

Dr. Reddy’s aim is to help people lead healthier lives through two parallel objectives:Making medicine affordable and accessible in all parts of the world so that many people as possible benefit from them.

And discovering and developing and commercializing innovative treatment options that satisfy unmet medical needs.With headquartered in India, it is a global pharmaceutical company with a presence in more than 100 countries. Dr. Reddy’s is the first pharmaceutical company in Asia outside of Japan to be listed on the NYSE.

Its strong portfolio of businesses, geographies and products gives an edge in an increasingly competitive global market and allows it to provide affordable medication to the people across the world, regardless of geographic and socio-economic barriers. Dr. Reddy’s is a global, vertically integrated pharmaceutical company with a presence across the value chain, producing and delivering safe, innovative and high quality finished dosage forms, active pharmaceutical ingredients and biological products.

It conducts NCE drug discovery research in the areas of metabolic disorders and cardiovascular indications as research facilities in Atlanta (USA) and Hyderabad (India). Through its custom pharmaceutical services business unit, it provides drug substance and drug product development and manufacturing services on a proprietary basis.

PHARMACEUTICAL CHEMCALS

Active pharmaceutical ingredient: its capabilities span 24 major chemistries including stereo selective synthesis, cryogenics, hydrogenations and cyanations. Its strong IP and analytical skills are evident in 84 US DMFs it has filed, the highest in India and the second highest in the world. Its manufacturing facilities are capable of supporting the product development effort through concurrent scale-up and piloting of feasible route as they are developed by the R&D teams. State-of-the-art equipment and instruments give it’s the edge to compete globally. Its operations are fully integrated through supply chain and ERP systems (SAP R/3), which enable seamless response to customers, all the while keeping the environment around plants clean, green and safe.

Custom pharmaceutical services: In an industry cluttered with manufacturers, CPS stands out because of its understanding of pharmaceutical business and the associated expertise needed. Rather than just being a chemical provider, CPS offers a service mix covering the entire pharmaceutical value chain. It execute cost-effective and time-bound projects for its customers, and provide them with cGMP-complaint products manufactured in FDA-inspected, ISO-certified facilities. A team of experienced project managers ensures smooth progress if projects from initiation to closure in order to avoid any cost and time overruns.

GENERICS

GENERIC FORMULATIONS: Geographic diversifications, cost containment, strengthening its product portfolio and building scale – Dr.Reddy’s is strong in all these aspects in the generic space. It is now the fourth largest in Germany after the acquisition of betapharm, and is constantly looking for opportunities to maximize the potential of its current and future portfolio in different territories across the US and EU. It has the necessary expertise of customer-specific packaging, compliance packaging and anti-counterfeit packaging. In fact Dr.Reddy,s has won several awards globally fir its packaging efforts, including the Asia star, Ameristar and World star awards. Branded formulations: Dr.Reddy’s brand are todays recognized and trusted across several continents. Brands like Omez (omperazole), Nise (nimesulide), Stamlo (Amlodipine), Ciprolet (ciprofloxacin), Enam (Enalapril) and Ketorol (Ketorolac) are leaders in their category in several

countries, with many of them being used by more patients than use the innovators products. Over 1.5 million patients across the world take ‘omez’ for their acid peptic disorders every single day! Entrepreneurship, coupled with the will to make a difference drives our 2,000 strong field to force to reach out to over 210,000 doctors and 115,000 pharmacies in more than 40 countries across the world.

INNOVATION

DISCOVERY REASEARCH: It has put in place a state-of-the-art, fully integrated discovery infrastructure to strengthen its effort to discover and develop therapeutically useful new chemical entities (NCEs) and market them globally. Its two discovery research centers one in Atlanta. USA and other in Hyderabad, India have more than 300 hundred scientists actively involved in a number of drug discovery and clinical developments programs.

Specialty pharmaceuticals: Its specialty pharmaceuticals business deals with assets like acquired proprietary technologies, internally developed proprietary drug- delivery platforms, and current internal compounds under pre-clinical and clinical development. Its initial global therapeutic area focus is on dermatology and oncology, two therapeutic areas that best leverage its internal assets. A key component of the strategy in this area is strong, targeted business development effort to accelerate market entry.

Biopharmaceuticals: Its biologics development center span an area of 36,000 sq. ft., with development and manufacturing suites foe both E.coli and mammalian cell culture. It caters to the highest development standards of cGMP, GLP and applicable levels of bio-safety. Grafeel (Filgrastim), its first biologics product to enter the market, enjoys a market share of almost 50% in India and has able to reach many more patients than the innovators product due to its affordability. Its second product Reditux (Rituximab) is the first biosimilar monoclonal antibody to be developed and launched anywhere in the world.

PHARMA SERVICES AND ACTIVE INGREDIENTS

The global API business of Dr. Reddy’s serves generics and innovator companies through its API (Active pharmaceutical ingredients, also

called ‘bulk actives’) and CPS (Custom pharmaceutical services) businesses respectively.

Dr. Reddy’s began APIs operations in 1984 and started with a single drug in a 60-tonn facility near Hyderabad, in India. In 1986, the first consignment of that drug, methyldopa, was shipped to West Germany. Its emphasis on high quality and R&D led to USFDA inspection of our manufacturing facilities before long. Since then, Dr. Reddy’s has come a long way and today has a wide portfolio of APIs. Its core strength in APIs has continued to increase significantly over the years. It has the largest number of US DMF submissions from India and are among the top five API players globally. Its global API business offers over 100 molecules to customers across the world.

Its CPS business, formed in 2001, serves more than five big pharmaceutical companies and over 25 emerging pharma companies today. The CPS business got a boost in 2005 with the acquisition of Roche’s API manufacturing unit in Mexico. This facility added a niche capability of steroid API manufacture to DRL’s capability portfolio. Today, Dr. Reddy’s is the largest CPS player from India. Its CPS business provide a unique opportunity for innovator companies worldwide to make use of its technical expertise, world-class infrastructure, and flexibility to bring their medicines to the market quickly and economically. Dr. Reddy’s has emerged as a trusted supplier of value added advanced intermediates and APIs to generic as well as innovator companies.

The discovery and development of new active ingredients for a medicine is a long and expensive process which is why a patent protects a new active ingredients for a certain a time. When the patent expires, other pharmaceuticals companies market medicine with the same active ingredient

KARL FISCHER MOISTURE CONTENT DETECTOR

POLARIMETER

ULTRA-VIOLET AND VISIBLE SPECTROPHOTOMETRY

GAS CHROMATOGRAPHY

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

QUALITY CONTROL

Quality is important in every product or service but it is

vital in medicine as it involves life. Unlike ordinary consumer

goods there can be and there is no 'Second' quality in drugs.

Quality control is a concept, which strives to produce a perfect

product by series of measures designed to prevent and estimate

errors of different stages of production. As a mater of fact it is

built in from the time of inception of the thought to make a

product, to the time, it is finally made and sent out with an ok

quality report. In popular practices the quality of medicines or

pharmaceutical product is assured through quality control. It is,

therefore, essential that quality assurance department must adopt

"Good Laboratory Practice" to ensure reliability and accuracy of

results given out by them.

The assurance of the quality and the reliability of

pharmaceuticals, together with their careful control are out

moral obligations arising from the humanism towards the sick

human beings. Consequently, the manufacture and the control of

the drugs are very responsible tasks and they need substantial

knowledge of the science.

The decisions to release or reject a product are bared upon one or two types of control actions or combination of both. Providing sample analytical procedures for complex formulations is a matter of foremost importance.

QUALITY CONTROL:-

QC department is just like the backbone of the company because

the quality and the feasibility to stand and the market are

checked here. Products manufactured are checked for various

parameters required to be fulfilled before they are dispatched to

the market.

Quality control department consists of the analysis results from

three different labs.

Instrumentation Lab :- It is also called as the quality insurance

lab and it mainly deals with the chromatographic techniques

lime High Performance Liquid Chromatography (HPLC) and

Gas Chromatography to mark the potency and the efficiency of

the drug, This lab deals with the following experiments:-

Assays: - This is mainly done to check for the potency of

the drug produced.

Residual Substrate Estimation: - This test is performed to

check for the residual substances present in the resultant

drug.

Reaction Monitoring :- This test is of utmost importance as

through this test we continuously keep a check on the

reaction carried out in the reactor by analyzing the

intermediated formed at different step

QUALITY CONTROL AND ITS RESPONSIBILITIES

Quality control is the part of good manufacturing practices

concerned with sampling specifications & testing & with the

organization, documentation and release procedures which

ensure that the necessary and relevant tests are actually carried

out and that materials are not released for user nor products for

sale or supply until their quality has been judged to be

satisfactory. Quality control is not confined to laboratory

operations but must be involved to laboratory concerning the

quality of the product.

Each holder of the manufacturing authorization should

have a quality control department. The independence of quality

control from production is considered fundamental. The quality

control department should be independent than the other.

The basic requirements for quality control are as follows:

1. Adequate facilities, trained personnel and approved

procedure must be available for sampling inspecting and

testing starting materials, packing materials and

intermediate, bulk and finished products and where

appropriate for monitoring environmental conditions for

GMP purpose.

2. Sample of starting materials, packing materials,

intermediate products bulk products and finished

products must be taken by methods and personal approved

of b the quality control department.

3. Test methods must be validated.

4. Record must be made demonstrating that all the required

sampling, inspecting and testing procedures have actually

been carried out and that any deviations have been fully

recorded and investigated.

The quality control department as a whole will also have

other duties, such as to establish validate and implement are

quality control procedures, to evaluate, maintain and store the

reference standards for substances to ensures the correct labeling

of containers of materials & products to ensure that the stability

of the active pharmaceutical ingredients and products is

monitored, to participate in the investigation product and to

participate in the investigation products and to participate in

environmental monitoring all there operations should be carried

out in accordance with written procedures and where necessary,

recorded.

QUALITY SYSTEM

The basic elements of quality and systemic action. The

quality system involves all phase from initial identification to

final satisfaction of requirements and customers expect action.

The first thing that a manufacturer will like to know is what product should be manufactured. The next steps follow as under:

Procurement of raw materials

↓

Process planning

↓

Production

↓

Inspection & test

↓

Packing

↓

Storage

↓

Sales & Distribution

KARL FISCHER MOISTURE CONTENT DETECTOR

For the determination of small amounts of water, "Karl

Fischer" in (1935) proposed a reagent, is known of Karl Fischer

reagent.

Principle In this method, the action of sulphur dioxide upon a

solution of Iodine in a mixture of anhydrous pyridine and

anhydrous methanol. Water reacts with this reagent in a two

stage process in which one molecule of iodine disappears for

each molecule of water.

CH3OH + SO2 + RN → RNH – SO3R

R5HNSO3R + 2RN + I2 + H2O → (RNH) SO4R + 2 (RNH) I

The end point of the reaction is conveniently determined

electrometrically I2 generate electrometrically from I-.

When iodine comes in contact with water the reaction as:

I2 + SO2 + H2O → HI + H2SO4-

The amount of water in the sample is calculated by measuring

the current needed for electrochemical generation of iodine from

I-.

2I- → I2 + 2e-

Apparatus A titration vessel of about 60ml capacity is fitted with two

platinum electrodes about 0.05 sq. cm in area and about 2.5cm

apart, a nitrogen inlet tube, a stopper which accommodates the

burette tip and vent tube protected by a suitable desiccant such

as phosphorous pent oxide or silica gel. The substance being

examined is introduced through an Intel which can be closed by

a ground stopper. Stripping is done by magnetically. The air in

the entire system should be kept dry during the titration.

The voltage across the electrodes may be obtained from a

1.5volt dry cell and a variable resistance of about 200ohms. The

resistance is adjusted so that an initial current passes through the

electrodes connected in series to an ammeter. On adding the

reagent the needle of the ammeter shows a deflection but return

immediately to its starting position. At the end point of the

titration a slight excess of the reagent produces a deflection

which persists for not less than half a minute.

Procedure The determination of small amount of moisture content involves

three steps.

1. Neutralization of methanol

a) Fill the titration of vessel with methanol in order to

dip the Pt. electrode in it.

b) Start the titration in order to neutralize the methanol.

2. K.F. Factor

a) Add about 150 to 200ml of purify water in titration.

b) Start the titration and not the volume of K.F. reagent

used.

K.F.Factor = Wt. of purified water

Volume of K.F.Reagent used

Limit = 5-7mg/ml.

Units = mg/ml

3. Moisture Content

a) Wt. amount of sample according to STP add it in

titration vessel.

b) Stir it for one minute

c) Titrate it with K.F. reagent, note the volume of K.F.

Reagent.

V × F × 100

% of moisture content = ----------------W × 1000

V = Vol. of K.F.R. used with sample.

F = water equivalence factor

W = weight of sample.

CalibrationStandardization of KF Reagent

1) Remove all the liquid from KF apparatus vessel, rinse it

with methanol twice and transfer about 30 ml methanol

into vessel.

2) Neutralization to end point with KF reagent.

3) Weight accurately about 150mg of disodium tartrate

transfer it into KF vessel, wait for about 10-15 seconds.

Start addition of KF reagent record the volume of KF

reagent consumed.

Calculate the KF factor by the formula W × 1000 × 15.66

F is mg/ml = --------------------------V × 100

W = weight of disodium tartrate in g

V = Volume of KF reagent consumed in ml.

Note:

Disodium tartrate can be replaced with water for standardization with the change in step no. 3 ice weight accurately about 30-6 mg of water, transfer it into KF vessel,

start addition of KF reagent record the vol. of KF reagent consumed and calculate the factor by formula.

W × 1000F mg/ml = --------------

V

W = weight of water in g

V = vol. of KF reagent in ml.

POLARIMETER

Many pharmaceutical substance are optically active i.e.,

they rotate an incident plane of polarized light so that the

transmitted light emerges at a measurable angle to the plane of

the incident light. This property is characteristic of some crystals

and of many pharmaceutical liquids or solutions of solids. The

property is the result of presence of one or more asymmetric

centers usually a carbon atom with four different asymmetric

centers. The only convenient means for distinguishing optically

active isomers from each other is polarimeter.

Substances that show optical rotation are optically active.

Those that rotate light in a clockwise direction as viewed

towards the light source are dextrorotatory. Those that rotate

light in the opposite direction are called laevorotatory.

The general equation used in polarimeter is:

[D]λ = 100 α

I. C

[D] = specific rotation at wavelength λ

t = temperature

a = observed rotation in degrees

I = path length in decimeter

C = concentration of the substance

Polarimeter was performed using an instrument where the

extent of the optical rotation is estimated by visual matching of

the intensity of split fields. For this reason the D-line of the

sodium lamp at the visible wavelength of 589nm was most often

employed.

Instrument:

Optical System Provide basic optical rotation information. It contains a

polarizer section, sample chamber and analyzer section.

Power Module It transforms the 110 or 220v a/c line power into the AC

and DC voltage for the system.

Procedure:

Take a polarimeter tube that has been selected, cleaned

and tested.

Fill the tube with the solvent used to dissolve the optically

active sample and place in the sample chamber.

Press zero reset button.

Remove the solvent from the tube and rinse at least three

times with sample solution.

Fill with sample solution and place in sample chamber.

After achieving polarimetric balance, record the display

reading.

Applications The physicochemical properties of non-super

imposable chiral substances rotating plane polarized light in

opposite directions to the same extent, enantiomers are identical

except for this property and in their reactions with other chiral

substances. Enantiomers often exhibit different pharmacological

and toxicological properties, owing to the fact that biological

receptors and enzymes themselves are chiral. Many article from

nature source, such as amino acids, proteins, alkaloids,

antibiotics, glycosides, and sugars, exist as chiral compounds.

Synthesis of such compounds from non-chiral materials results

in equal no. of enantiomers, recreates. Recemates have net null

optical rotation and their physical properties may differ from

those of the componentanatomies.

ULTRA VOILET AND VISIBLE

SPECTROPHOTOMETRY

When radiation is passed through a layer of a solution

containing an absorbing substance, part of the radiation is

absorbed; the intensity of the radiation emerging from the

solution is less than the intensity of radiation entering it. The

magnitude of the absorption is expressed in terms of the

absorbance, A, defined by the expression.

A = log10 (I10/I)

Where I0 is the intensity of the radiation passing into the

absorbing layer and I is the intensity of the radiation passing out

of it. The absorbance depends on the concentration of the

absorbing substance in the solution and the thickness of the

absorbing layer taken for measurement. For convenience of

reference and for ease in calculations, the absorbance of a 1-cm

layer of a 1% w/v solution is adopted for several substances and

evaluated by the expression.

A (1%, 1-cm) = A/C l

Where c is the concentration of the absorbing substance

expressed as percentage w/v and I is the thickness of the

absorbing layer in cm. The value of A (1%, 1-cm) at a particular

wavelength in a given solvent is a property of the absorbing

substance.

Apparatus A ultra-violet and visible spectrophotomer, suitable for

measuring in the ultra-violet and visible range of the spectrum

consist of an optical system capable of producing

monochromatic light in the range 200 to 800mm and a device

suitable for measuring the absorbance.

Two empty cells used for the solution being examined and the reference liquid must have the same spectral characteristic. Where double beam recording instruments are used, the solvent cell is placed in the reference beam.

Control of Wavelength

Verify the wavelength scale using the absorption maxima

of holmium per chlorate solution, the line of hydrogen or

deuterium discharge lamp or the lines of a mercury vapour are

shown below:

The permitted tolerance is ± 1 nm for the range 200 to

400nm and ± 3 nm for the range 400 to 600nm.

241.15nm (Ho) 404.66 nm (Hg) 253.70nm (Hg) 535.83 nm (Hg) 287.15 nm (Ho) 486.00 nm (Dβ ) 302.25nm (Hg) 486.10 nm (Hβ ) 313.16 nm (Hg) 536.30 nm (Ho) 334.15 nm (Hg) 546.07 nm (Hg) 361.50 nm (Hg) 576.96 nm (Hg) 365.48 nm (Hg) 579.07 nm (Hg)

Control of Absorbances

Check the absorbance using potassium dichromate solution UV at the wavelength indicated in Table1, which gives for each wavelengths the exact value of a (1%, 1cm) and the permitted limits.

Table – 1

Wavelength (nm) A (1%, 1cm) Maximum

tolerance 235 124.5 122.9 to 126.2257 144.0 142.4 to 145.7313 48.6 47.0 to 50.3350 106.6 104.9 to 108.2

Limit of stray light

Stray light may be detected at a given wavelength with

suitable filters or solutions; for example, the absorbance of a

1.2% w/v solution of potassium chloride at a path length of 1cm

should be greater then 2.0 at about 200nm when compared with

water as reference liquid.

Resolution power

When prescribed in a monograph, record the spectrum of

a 0.02% v/v solution of toluene in hexane UV. The ratio of the

absorbance at the maximum at about 269nm to then at the

minimum at about 266nm is not less than 1.5 unless otherwise

specified in the monograph.

Spectral slit width

When measuring when measuring the absorbance at

absorption maximum the spectral slit width must be small

compared with the half-width of the absorption band otherwise

erroneously low absorbance will be measured. Particular care is

needed for certain substances and the instrumental slit width

used should always be such that further reduction does not result

in an increased absorbance reading.

Cells

The absorbance of the cells intended to contain the

solution to be examined and the reference liquid, when filled

with the same solvent, should be identical. If this is not the case,

an appropriate correction must be applied. The tolerance on the

path length of the cells used is ± 0.005cm. Cells should be

cleaned and handled with great care.

Solvents

In measuring the absorbance of a solution at a given

wavelength, the absorbance of the reference cell and its contents

should not exceed 0.4 and should preferably be less than 0.2

when measured with reference to air at the same wavelength.

The solvent in the reference cell should be of the same lot as

that used to prepare the solution and must be free from fluoresce

at the wavelength of measurement. Ethanol (95%), ethanol,

methanol and cyclohexane, used as solvents, should have an

absorbance, measured in a 1-cm cell at about 240nm with

reference to water, not exceeding 0.10.

Determination of absorbance

Unless otherwise directed measure the absorbance at the

prescribed wavelength using a path length of 1cm at 240 to 260.

If necessary, this path length may be varied provided that

compliance with Beer's law has been shown over the range in

question. Unless otherwise prescribed, carry out the

measurements with reference to the solvent used to prepare the

solution being examined. In certain cases measurement are

carried out with reference to a mixture of reagents, detail of

which are given in the individual monograph.

A statement in an assay or test of the wavelength at which

maximum absorption occurs implies that the maximum occurs

either precisely at or within ± 2nm of the given wavelength.

Likewise a statement in a test of the absorbance, A at a given

wavelength or at the maximum at about a specified wavelength

implies that the measured absorbance is within ±3% of the

stated value.

When an assay or test prescribes the use of a reference

substance, make the spectrophotometer measurements with the

solution prepared from the reference substance by the official

directions and then with the corresponding solution prepared

from the substance being examined. Carry out the second

measurement as quickly as possible after the first, using the

same experimental conditions.

Unless otherwise specified, the requirements in the

monographs for light absorption in the tests and assays apply to

the dried or anhydrous material, where a standard is given for

loss on drying or content of water, respectively. Similar

considerations apply where standards are given for solvent

content. In calculating the result, the loss on drying or contents

of water or solvent, determined by the methods specified in the

monograph, is taken into account.

LOD (Loss on Drying

It is the loss in % w/w resulting from water and volatile

matter of any kind that can be driven off under

specified conditions. The test is carried out on

a well mixed sample of the substance. If the

substance is the form of large crystals, reduce

the size by rapid crusting to a powder.

Method

Weigh a glass stoppered, shallow weighing bottle

that has been dried under the same conditions to be

employed in the determination. Transfer to the bottle the

quantity of the sample specified in the individual

monograph, cover it and accurately weigh the bottle and

contents. Distribute the sample as evenly as practicable

by gentle sidewise shaking to a depth not exceeding

10mm. Place the loaded bottle in the drying chamber

(over or desiccator) as directed in the monograph, remove

the stopper and leave it also in the chamber. Dry the

sample to constant weight or for the specified time and at

the temperature indicated in the monograph. After drying

is completed, open the drying chamber, close the bottle

promptly and allow it to cool to R.T. (where applicable) in

a desiccators. Weigh the bottle and contents.

Calculation

LOD (Loss on Drying)

Wt of empty bottle = x g

Wt of bottle + sample = y g

Therefore, Wt of sample = y – x g = zg

After Drying,

Wt. of bottle + sample = a g

Wt of sample = a – x

= w g

W × 100% of LOD = ------------

z

= % w/w

Determination of Sulphated Ash

Heat a silica or platinum crucible to redness for 10

min, allow to cool in a desiccator and weigh, transfer to

the crucible 1g of the substance being examined and

weigh the crucible and the contents accurately. Ignite,

gently at first, until the substance is thoroughly charred

cool, moisten the residue with the 1 ml of H2SO4, heat

gently until the white fumes are no longer evolved and

ignite at 8000C±250C until all black particles have

disappeared. Conduct the ignition in a place protected

from air currents. Allow the crucible to cool, add a few

drops of H2SO4 and heat. Ignite as before, allow cooling

and weighing. Repeat the operation until 2 successive

weighing do not differ by more than 0.5mg.

Calculation

Sulphated Ash

Wt. of Empty Crucible = x g

Wt. of empty crucible + sample = y g

Wt. of sample taken = y – x = z g

Wt. of Crucible after ignition = w g

Sulphated Ash = w – x = A g

A × 100% of Sulphated ash = ------------

Z

= B % w/w

Water AllianceModel No. Detector 2487Separation Module 2695

GAS CHROMATOGRAPHY

GAS CHROMATOGRAPHY

In 1952 year James and martin invented gas liquid

chromatography and 1949-50 cremeratal fabricated the first

complete gas chromatography in their laboratory at the

University of Innsbruck Austria.

Chromatography technique is simply a method of

separation of volatile substance which is present in very minute

amount and the separation is done by adsorption and partition

process on the different adsorption column.

Gas chromatography refers to a physical process by which

a mixture is separated into its constituents by moving gas phase

over stationery adsorbent.

The gas chromatography separation occurs by partitioning

a sample between a mobile gas phase and this layer of non

volatile. Liquid coated on an inert support.

PRINCIPLE

When a gas vapors comes in contact with adsorbent a

certain amount of it get adsorbed on the solid surface according

to freundlich law

X/m = kc1/n

x = mass of gas

m = mass of adsorbent

k = constant

c = concentration

Instrument

Carrier Gas

Helium, nitrogen, hydrogen and argon are generally used

as carrier gas. The choice of carrier gas depends upon

availability; purity and inertness towards sample and stationary

phase carrier gas flow rate generally range between 20-100

ml/min. The gas flow control is achieved by use of pressure

regulator and flow controller.

To avoid contamination carrier gas should be pure ad gas

purity can be improved by filtering it. For nitrogen as carrier gas

filtering process.

This sequence prevents any hydrocarbons present in the

gas stream from reaching the oxygen filter.

Sample Injection System

To introduce a liquid sample a syringe is used. The liquid

sample is injected through a self sealing septum into head of

Carrier gas

Hydro carbon filter

Moisture filter

Oxygen filter

Head space

column and solid sample in a suitable solvent according to STP

and inject through head space.

Head Space

A head space sample is normally prepared in a vial

containing the sample, solvent, a matrix modifier and the head

space. Volatile components from complex sample mixture can

be extracted from non volatile sample components and isolated

in the head space or gas portion of a sample vial. A sample of

the gas in the head space is injected into a G-C system for

separation of all of the volatile components.

Phases of headspace vial

The gas phase is commonly referred to as the headspace

and lies above the condensed sample phase.

The sample phase contains the compound of interest. It is

usually in the form of a liquid or solid in combination with a

dilution solvent and matrix modifier.

Once the sample phase is introduced into the vial and the

vial is sealed, volatile component diffuses into the gas phase

until the head space has reached a state of equilibrium. The

sample is than taken from the headspace has reached a state of

equilibrium. The sample is then taken from the headspace.

Column

Two type of columns are used in GC:

1.)Packed column

2.)Capillary column

Packed column are 1.5-10m in lengths and have an internal

diameter of 2-4mm. The tubing is usually made of stainless steel

or glass and contains a packing of finely divided inert, solid

support material that is coated with a liquid or solid stationary

phase. The nature of coating material determines the type of

materials will be mostly adsorbed.

Capillary columns have a very small internal diameter, on the

order of a few tenths of millimeters, and lengths between 25-

60m are common. The inner column walls are coated with the

action material. Most capillary columns are made of fused silica

with a polyimide outer coating. These columns are flexible, so a

very long column can be wound into a small coil.

Detector

Flame ionization detectors are generally used in GC.

Flame ionization detector require a flame for which the common

fuel chosen is hydrogen gas normally carrier gas used is

nitrogen gas. Both these gases are at appropriate flow rates will

enter the detector unit. Air oxygen for combustion is supplied

separately to base flame gases burning in hydrogen flame

contain very small concentration of free ions and e- resulting

from ionization. This gives an electrical conductivity which is

small. But when organic vapors enter the flamer, the

conductivity rises and this can be amplified and recorded. ECD

is also used as detector.

Computing Device:

The output from the detector is usually fed into a

potentiometer recorder or integrator cum printer plotters. These

recording devices produce a straight line when no gas/vapour is

being eluted from the column by the carrier gas

CALIBRATION

Procedure

1) Condition the column at initial oven temp.

2) In case of auto injector inject standard for six times

otherwise inject for once.

Acceptance criteria

Peak response ≥ 10. 00

RSD for area of 6 injections ≤3.0% for auto injector.

For head space sampler calibration

Adjust the chromatographic condition as per standard testing

procedure.

Procedure

1) Prepare six vials as per standard testing procedure and

inject from each 1ml gaseous phase.

2) Determine relative standard deviation of retention time

and area of peaks due to ethanol.

3) Prepare one empty vials and inject 1ml. record the

chromatogram

%age carry over = Area of ethanol in carry over injection

x100 Area of

ethanol in previous injection

Acceptance criteria

1) RSD for area of six preparations not more than 3%.

2) RSD for retention times not more than 1%.

3) % Carry over not more than 1%.

Residual solvent estimation by GC

Residual solvent in pharmaceutical are defined here as organic

volatile chemicals that are used or produced in the drug

substances during the preparation of drug product. The solvents

are not completely removed by practical manufacturing

techniques.

Residual solvents are usually organic volatile impurities that

mainly include solvents as methanol, ethanol, methylene

dichloride, toluene, etc.

Residual solvent estimation

Method of analysis:

1) Instrumentation

a) GC with flame ionization detector.

b) Head space

c) Data handling system

d) Fused silica capillary column

2) GC parameters (SET acc to STP)

a) Oven temp. -1

b) Time -1

c) Rate

d) Injection temp.

e) Oven temp.

f) Time 2

g) FID temp.

h) Carrier gas N2 flow

3) Headspace parameter

a) Oven temp.

b) Needle temp

c) Transfer temp.

d) GC cycle temp.

e) Thermo state time

f) Pressurize time

) Thermo state time

f) Pressurize time

g) Inject time

h) Withdrawal time

i) Head space mode

j) Vial venting

k) Head space carrier pressure

l) Split

Procedure

i) Prepare the standard as mentioned in STP of residual

solvent

ii) Note the area counts of eluting peaks form

chromatographic report.

Calculation

Product (ppm): AT x DS x P x 1000

AS DT

Where,

AT: Average area count of the peak of the reagent used

in sample solution.

AS: Average area count of the peak of the reagent used in

standard solution.

DS: Dilution factor of the standard preparation.

P: Purity in % of reagent standard.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High performance liquid chromatography is a form of column

chromatography used frequently in biochemistry and analytical

chemistry. It is some times referred to as high pressure liquid

chromatography. HPLC is used to separate components of a

mixture by using a verity of chemical interaction between the

substance being analyzed and chromatography column.

Principle

HPLC enables to evaluate components of a mixture by virtue of

their differential distribution between the mobile phase and the

stationary phase, migration of solute component can only occur

when it is only dissolved in the mobile phase. Thus the solute

that have a high distribution into the stationary phase will elute

more slowly than those that distribute more readily into the

mobile phase and the two will therefore undergo

chromatographic separation.

The separation of components by chromatography depends on

the deference in their equilibrium depends on the differences in

their equilibrium distribution co efficient (K) between the

stationery and mobile phases:

K = Cs/ Cm

Where Cs and Cm are the concentration of the solute in the

stationery and mobile phases respectively solute with high K

values. Elute more slowly than those with low K value.

SYSTEM SUITABILITY

Column efficiency is defined in terms of number of theoretical

plates per meter (N) by the expression.

N = 5.54 VR/LWh2

VR = Distance along the base line between the pt. of

injection and

A ⊥ drop from maximum of peak.

L = Length of column in meters.

Wh = Width of the peak at half peak height.

Resolution factor (Russ) between the measured peaks on the

chromatogram should be greater than 1.0 and is given by

1.18 (VRb – VRS)

Rs = ----------------------

Whs + Whb

Where VRb and VRs are the distances along the base line between

the point of injection and ⊥ drawn from the maximum of two

adjacent peaks, Whs and Whb are the peak widths measured at

half peak height.

Tailing factor of peak = Wx/2A

Wx is the width of the peak of one twentieth of the peak height,

a – distance between the ⊥dropped form the peak maximum.

Apparatus

Solvent delivery system (Pump)

The pump is one of the most important component of

HPLC since is performance affect directly effect retention time,

reproducibility and detector sensitivity. The pumps deliver

steady stream of solvent from the reservoir to the detector

through the column. The pump can deliver solvent at a pressure

up to 6000 PSI.

Several types of pumps available for use with HPLC

analysis they are reciprocating piston pumps, syringe type pump

and constant pressure pumps.

The mobile phase acts as a carrier for the sample solution.

A sample solution is injected into the mobile phase of an assay

through injector part. The eluting power of mobile phase is

determined by its overall polarity, the polarity of stationary

phase and nature of the sample component.

Pumps and for quantitative analysis should be constructed

for material inert to corrosive mobile phase components and be

capable of delivering the mobile phase at a const. rate with

minimal fluctuations over extended period of time.

Injector

After dissolution in mobile phase and other suitable

solutions, compounds to be injected either manually by syringe

or loop injectors, or automatically by auto samples. The latter

consist of a carousel or rack to hold sample vials with tops that

have a pierce able Septum or Stopper and an injection device to

transfer sample from the vials to a loop from which is it loaded

into chromatograph.

A syringe can be used for manual injections of samplers

through septum when column head pressure is less than 70

atmospheres. At higher pressure an injection valve is essential.

Stop flow injections and micro volume injection sampling

valves are used.

Detector

Many compendia HPLC methods require the use of

spectrophotometric detectors such as detector consist of a flow

through cell mounted at the end of column. A beam of UV

radiation passes through the flow cell and into detector. As

compound elute from the column, they pass through the cell and

absorb the radiation, resulting in measurable energy level

changes. Fixed, variable and multiwave length detectors are

widely available.

Data collection devices

Modern data station receive and store detector output and

print out chromatographs complete with peak heights, peak

areas, and sample identification and method varieties. They are

also used to program the liquid chromatograph, controlling most

variables and providing for long periods of unattended

operations.

Columns

The columns are made from precision bore polished

stainless steel taking, typical dimensions being 10 to 30cm long

and 4 to 5mm internal diameter. The packing used in modern

HPLC columns consist of small, rigid, particle having a narrow

particle – size distribution.

Types of packing are

1. Porous, Polymeric beads

2. Porous – layer beads

3. Total porous

4. Total porous silica particles

There are various columns that are secondary to separating

column or stationary phase

1) Guard column

2) Derivatizing columns

3) Capillary columns

4) Microbore and small bore fast columns

5) Preparatory columns

Solvent

Selection of mobile phase is most important HPLC. In

general, polar materials is separated using partition

chromatography while non polar. Substances are separated using

absorption chromatograph. Better sample separation is achieved

by matching the polarities of the sample and packing and by

using a solvent that has different polarity. Beside polarity,

hydrogen bonding London dispersing forces also affect the

solvent strength.

Note : The presence of air bubbles in the mobile phase which

impair the detector single causing spike on the chromatograph

can be eliminated by degreasing the mobile phase by ultrasonic

vibrations Both single and double detector are commercially

available.

Type of analysis carried by high performance liquid

chromatography:

1.)Chromatographic purity

2.)Related substances(Impurity profile)

3.)Reaction monitoring

4.)Assay

ASSAY :

Assay is done to check the purity level of the product formed

form the substrate or the raw substrate or the raw material used

for its production. Product formed is checked for it spurity level

before it is dispatched. A particular arithmetical value it should

have for the desired parameters as per set by the STP.

Method of analysis:

1. Reagents: Different reagents are depending upon the

solubility of the product being formed.

2. Buffer solution: This solution is prepared to form the

mobile phase for the analysis system.

3. Mobile Phase: Mobile phase according to STP.

4. Chromatography System: Different chromatography

parameters are set for the sample to be analyzed.

* Instrument: HPLC equipped with UP detector.

* Injection volume: A particular amount of the

sample is passed through the column in a particular

time and this parameter is set at very low value

mostly 20µ l.

* Run time: The analysis time is set to get the

required information form the chromatogram

produce through analysis.

REACTION MONITORING:

Production process in the pharma companies has a no. of stages.

After adding the batch to the reactor there are no. of steps it

undergoes to form the required product. Intermediate

compounds formed at each stage. It’s very important to keep a

continuous check on the intermediates formed so that we can

find out whether the reaction is going in right direction or not.

This process of continuous check is known as reaction

monitoring. Definition: Reaction monitoring is done to check

whether the reaction is going in the right direction or not i.e.

required intermediates are formed or not and also to check the

extent up to which the reaction got completed.

HPLC

CALIBRATION OF DETECTOR 2487

CALIBRATION OF 2695 SEPARATION MODULE

Procedure

The following are to be calibrated as per standard operating

procedure.

1) Pump

2) Detector

3) Manual injector

4) Column oven

I) Pump: Pump shall be calibrated for the following

1) Flow rate accuracy

2) F low rate consistency

3) Compositional accuracy

II) Detector: Detector shall be calibrated for the following

1) Wavelength accuracy

2) Detector linearity

3) Detector precision

III) Injector: Injector shall be calibrated for the following

1) Injector accuracy

2) Injector linearity

3) Injector precision and carousel check.

QUALITY ASSURANCE

Quality assurance is a wide ranging concept covering all the matter that individually or collectively influences quality of products with regards to pharmaceuticals, quality assurance may be divided into four major area, quality control, production, distribution and inspection.

The assurance of product quality depends on more than just proper sampling and adequate testing of various components and the finished dosage form. Prime responsibility or maintaining product quality during production rests with the manufacturing department. Removal of responsibility from manufacturing for producing a quality product can result in imperfect composition, such as ingredients missing, sub potent or super potent addition of ingredient, or mix up of ingredients: mistake in packing and fillings, such as product contamination, mislabeling or deficient package; and lack of conformance to product registration. Quality assurance personal must establish control or checkpoints to monitor the quality to the product as it is processed and completion of manufacture. These begin with raw materials and component testing and include in-process, packaging, labeling and finished product testing as well as batch Austin and stability monitoring.

Responsibility of quality assurance department : to formulate system for the implementation of cGMP and, to ensure the preparation, approval and implementation of standard operating procedures, standard cleaning procedures, specifications, standard test procedures, cleaning, validation, protocols, batch manufacturing records, batch packaging records etc. sampling of in process and finished products, reserve samples and stability samples.To review and approve validation protocols, to review changes in product process, equipments, or any other changes as per SOP “change control program”.

To release or reject the batch after reviewing the batch documents to assure that the batch has been manufactured as per the standard batch formula and there are no deviations and deviations, if any, are record and authorized.

To conduct audits of method, results, system and process.

To release the finished products and to maintain the reserve samples of finished product and batch documents.

To investigate market complaints and to maintain market complaints investigation records.

To issue, control, review and retrieval of batch documents.

To investigate and disposition of incidents/OOS and approval of deviations.

To ensure effective storage, retrieval, control and retention of all completed documents.

Parts of quality assurance

1. documentation cell2. In process quality control (IPQC)3. Validation4. Self inspection 5. Training and development

Documentation cell: the documentation cell contains all the master formula, batch production records, and also document of sole of company.

(a) Master formula record: master formula record for each product should be prepared, endorsed and dated by a competent and responsible individual. The master formula shall include the following information’s:

1. The name of the product, a description of the dosage form, and its strength.

2. The complete list of ingredients, designated by whole names and codes sufficiently specific to indicate any special characteristic.

3. The quantity by weight or volume of each ingredient, regardless of whether it appears in the finished product.

4. The standards or specifications of each ingredients used in the product.

5. An appropriate statement concerning any calculated excess of an ingredient.

6. Appropriate statements of theoretic yield at various stages and the termination of processing.

7. Manufacturing and control instructions, specifications, precautions and special notations to be followed.

8. A detailed description of the closures, contrariness, labeling, packaging, and other finishing material.

(b) Batch production record: batch production record should be prepared, maintained and control for each batch of product. The record includes date, specific

code or identification number for each ingredient employee, weights or measures of components and products in the course of processing, result of in-process and control testing, and the endorsement of the individual performing and supervising each step of the operation.

(c) Control of production procedures: to ensure that the product have intended characteristics of identity strength, quality and purity, production and the related in-process quality control procedures should be rigidly followed as required by the master formula record or the batch production record. To a large extent, IPQC is concerned with providing accurate, specific, and definite descriptions of procedures to be employed form the receipt of raw materials to the release of the finished dosage forms.

In process quality control (IPQC)

The important function of the in-process quality assurance program to ensure that the finished dosage form have uniform purity and quality within a batch and between batches.this is accomplished by identifying critical steps in the manufacturing process and controlling them within defined limits.

IPQC: - do the following test to perform quality to product in process

1) weight variation test :2) disintegration test :3) friability test :4) hardness test :5) moisture content test :

QA before start up:-

(1) environmental and microbiological control and sanitation:-

To ensure that finished product meet high standard of quality and purify, and effective sanitation program is required. A successful program must be. Taken within and outside plant to control insects and rodents. Personal cleanliness and proper hair covering are required.

Floor, walls and cooling should be resistant to external forces and should be easily cleaned.

Adequate ventilation, temperature and humidity and water supply are also be checked.

QA should review of monitor the following:Sanitation

Ventilation: - filter condition and changes, pressure gauze, humidity and temperatureWater supply

(2) Manufacturing working formula procedure (MWFP):

A documentation of the component material and processing step to gather with the production operation specifications and equipment to be used, make up the MWFP.

A working formula procedure formula should be prepared for each batch size that is produced.

QA personal must review and check the working formula procedures for each production batch before, during and after production for following detail.

(a) Signature & date if issue given by a responsible or QA employee.

(b) Proper identification by name and dosage form, item no, lot no, effective date of document, reference to the superseded version, amount, lot and code no., of each raw material utilized.

(c) Initializing of each step by two of operator involved.

(d) Starting and finishing time of each operation.

(e) Equipment to be used and etc setup.

(f) Proper labeling of released component and equipment.

RAW MATERIAL: QA should check the origin container of released raw material for cleanliness before takes to production department. They are weighed on environment controlled weighing area and are transferred to secondary container which is properly labeled and are then stored with proper identification.

MANUFACTURING EQUIPMENT: QA personal must ensure that manufacturing equipment is designed and located and maintained so that it facilities through cleaning is suitable for its intended use and minimize contamination. QA personal should as certain that proper equipment and tooling for manufacturing stage is being used. Equipment must be identified by label, lot no., dosage form, item no. etc.

Quality assurance at start- up

(a)Raw material processing: - only released and properly labeled raw material is allowed in the processing area.

(1) QA personnel should be checked and verify the humidity and temperature in area are within limits or not if not then inform it to production department.

(2) QA personnel should verify and document the proper equipmentation, addition of ingredient, mixing, drying and filtering time.

(3) At certain point sample are to be taken to the quality control lab for potency assay.

(b)Compounding:- QA personnel are responsible for as ascertaining that all containers of raw material are properly labeled and stage is the compounding staging area that they are clean and the manufacturing equipment is properly identified as to product, strength stem no.

CGMP requires that IPUA be adequately documented throughout all stages of manufacturing.

© Packaging material control: - QA personnel can check

(1) properties of contains tightness

(2) Moisture and vapour tightness regardless of containers construction.

(3) Physical or chemical changes of contains upon prolonged contract with product.

(4) Toxicity of material needed for the container construction.

(5) Compatibility between contains and product.

(d) Label control:- QA personnel inspects and verifies all packaging component and equipment to be used for

packaging operation to ensure that it has proper identification that the line has been thoroughly cleared and all the material from previous packing operation has been completely removed.

FINISHED PRODUCT CONTROL:-

Specifications: Final testing of finished product is making in the quality control laboratories. These tests are designed to determine compliance with specifications. Thus, the testing of the finished product for compliance with predetermined standards prior to release of the product for packaging, and subsequent distribution is a critical factor for quality assurance. the design of test parameters, procedures, and specification is made during product development.

Quality assurance during packaging operation: - if the quality control dept. confirms that the product complies with the specifications and if QA audit indicates manufacturing operations are satisfactory the product is released to packing department.

(a) QA personnel should periodically inspect the packaging lines and should checked and labeled container for compliance with the written specifications.

(b) QA should perform an independent inspection and select finished preservations samples at random form each lot.

(c) It should be consisting of at beast twice the quantity necessary to perform the entire test required to determine.

(g) Auditing: - GMP requires that the manufacturing process be adequately documented throughout all stages of the operation.

Records keeping area are (a) Individual components, raw material packaging

materials, master formula and procedure. (b) Batch production(c) Packing and labeling operations (d) Lab in process and finished control testing (e) Proper signing and dating by at least two individual

independent for each operation.

Assurance of manufacturing practices(1) Personnel: one criterion from a successful quality

assurance program is the encouragement of quality consciousness in the personnel of the entire company. Proper selection, training, and motivation of production, packaging in the control personnel are vital to produce quality pharmaceutical consistency. The degree to which the desired quality of the product is attainable is proportional to the attitudes or desires of the individual working in production, packaging and control. It is essential that qualified personnel be employed to supervise the formulation, processing, sampling, testing, packaging and labeling of the drug product, and the competent staff be placed in charge of the maintenance of machinery, equipment and sanitations.

(2) Equipments and Buildings: equipments and buildings used in the manufacture, processing, packaging, labeling, storage or control of drug should be suitable design, size, construction and location should be maintained in a clear and orderly manner. The building should provide adequate space for the orderly placement of materials and equipments to minimize any risks of mix-ups or cross contamination.

(3) Control of records: Master formula records Batch production records Control of production procedures

(4) Packing control: at some time before the manufacture of a product is completed, a packaging record bearing an identification number is issued to the packaging section. This record specifies the packaging material to be used, operation to be performed, and the quantity to be packaged. Simultaneously, requisitions and issue for the product to be packaged and or the packaging and printed materials, such as labels, containers, inserts, brochures, cartons and the shipping cases.

VALIDATIONValidation of a process in the demonstration that controlling the critical step of a process results in the products of repeatable attributes (e.g., content uniformity) or causes a reproducible event (e.g., sterilization).

The concept of applying a systems approach to pharmaceutical manufacture and control, requiring validation of the process and qualification of equipment, personnel, and so forth, received considerable impetus when it was recognized.

Types of process validation (1) prospective validation(2) reterospective validation(3) concurrent validation(4) revalidation

Types of equipments qualifications (1) design qualifications (DQ)(2) installation qualification (IQ)

(3) operations qualifications (OQ)(4) performance qualifications

PROCESS VALIDATIONIt would normally be expected that process validation be completed prior to the distribution of a finished product that is intended for sale (prospective validation) where this is not possible, it may be necessary to validate process during routine production (concurrent validation) processes which have been in use for some time without any significant changes may also be validated according to an approved protocol (retrospective validation).

Prospective validation: in prospective validation the validation protocol is executed before the process is put in to commercial use. During the product development phase the production process should be broken down in to individual steps. Each step should be evaluated on the basis or experience or theoretical consideration to determine the critical parameters that may affect the quality of finished product. A series of experiment should be design to determine critically of these factors. Each experiment should be planned and documented fully in an authorized protocol.

All equipments, productions and environment and the analytical testing methods to be used should have been fully validated. Master batch documents can be prepared only after the critical parameters of the process have been identified and machine settings, components specifications and environment conditions have been determined, by using this defined process a series of batches should be produced. In theory, the number of the process runs carried out and observations made should be sufficient to allow the normal extent variations and trends to be establish to provide sufficient data for evaluation. It is generally considered acceptable that three consecutive batches/runs within the final agreed parameters, giving product of the desired quality

would constitute a proper validation of the process. In practice, it may take some considerable time to accumulate these days.Some considerations should be exercised when selecting the process validation strategy. Amongst these should be the use of different lots of active raw materials and major excipients, batches produced on different shifts, the use of different equipments and facilities dedicated of commercial production, operating range of critical process, and a through analysis of the process data in case of prequalification and revalidation.During the processing of the validation batches, extensive sampling and testing should be performed on the product at various stages, and should be documented comprehensively. Detail testing should also be done on the final product in its package.Upon completion of the review, recommendation should be made on the extent of monitoring and the in-process control necessary for routine production. These should be incorporated into the manufacturing record and packaging record or appropriate standard operating procedures. Limits, frequencies and action to be taken in the even to the limits being exceeded should be specified.

Concurrent validation: in using this approach there is the always the risk of having to modify process parameters or specifications over a period of time. This situation often leads to question regarding disposition of the batches that had already been released for the sale subsequently known to have undesired quality characteristics.Concurrent validation may be the practical approach some circumstance. Example:

- When a previously validated process is being transferred to a third party contract manufacturer or to another manufacturing units.

- Where the product is different strength of a previously validated product with the same ratio of active/inactive ingredients.

- When the number of lots evaluated under the retrospective validation were not sufficient to obtain a high degree assurance demonstrating that the process is fully under control.

- When the number batches produced are limited.

Retrospective validation:

In many establishments, processes that are stable and in routine use have not under gone a formally documented validation process. Historical data may be utilized to provide necessary documentary evidence that the processes are validated.

The steps involved in this type of validation still require the preparation of protocol, the reporting of the results of the data review, leading to a conclusion and recommendations.

Retrospective validation is only acceptable for well established detailed process and will be inappropriate where there have been recent changes in the formation of the product, operating procedures, equipments and facility.

The source of data for retrospective validation should includes amongst others, batch documents, process control charts, maintenance log book, process capability studies, finished product results, including trend analysis and stability results.

For the purpose of retrospective validation studies, it is considered acceptable that data for a minimum ten consecutive batches produced to be utilized. When the less than ten batches

are available, it is considered that the data are not sufficient to demonstrate retrospective that the process is fully in control. In such cases the study should be supplemented with concurrent or prospective validation.

Some of the essential elements for retrospective validation are:

• Batches manufactured for a defined period ( minimum of last ten consecutive batches )

• Number of lots released per year.

• Batch size / strength / manufacture / year / period.

• Master manufacturing/packaging/documents

• Current specification for active materials/finished products.

• List of process deviation, corrective actions and changes to manufacturing documents.

• Data for stability testing for several batches.

• Trend analysis including those for quality related complaints.

Process revalidation

Revalidation provides the evidence that changein a process and/ or the process environments that are introduced do not adversely affect the process characteristics and product quality. Documentation requirement will be the same as for the initial validation of the process.

Revalidation becomes necessary in certain situations. Some of the changes that require revalidation are as follows.

• Changes in raw materials properties such as density, viscosity, particle size, distribution , moisture etc. that may affect the process of product.

• Changes in the source of active raw material manufacture.

• Changes in packing material (primary container and closure system).

• Changes in the process (such as mixing time, drying temperature, and batch size).

• Changes in the equipment (e.g. addition of automatic detection system). Changes of equipment which involves the replacement of equipment on a “like for like” basis would not requires the validation except that this new equipment must be qualified.

• Changes in the plant/facility.

Qualification phases

Qualification of instruments is not a single, continuous process but instead results from many discrete activities. For convenience, these activities have been grouped in to 4 phases of qualification. These phases are described below:

1) design qualification2) installation qualification3) operation qualification 4) performance qualification

DESIGN QUALIFICATION

The design qualification activity is most suitably performed by the instrument developer/manufacturer. Since the instrument design is already in the place for the commercial off the shelf (COTS) systems, the user does not need to repeat all aspects of DQ. However, users should ensure that COTS instruments are suitable for their intended application and that the manufacture has adopted a quality system for developing, manufacturing and testing. Users should also establish that manufacturers and vendors adequately support installation, services and training. Method for ascertaining the manufacturer’s design qualification and as instruments suitability for its intended use depend on the nature of the instrument, the complexity of the proposed application, and the extent of users previous interaction to the manufacturer. Vendor audits or required vendor supplied documentation satisfy the DQ requirement. The required scope and comprehensiveness of the audits and documentation vary with user’s familiarity with the instrument and their previous interactions with the vendor.

INSTALLATION QUALIFICATIONInstallation qualification is a documented collection of activities needed to install an instrument in the users environment. IQ applies to a new, pre-owned and an existing on-site but not previously qualified instrument. The activities and documentation associated with IQ are as follows:-

system description: provide a description for the instrument, including its manufacturer, model, serial number, software version etc. use drawings and flowcharts where appropriate.

Instrument delivery: ensure that the instruments, software, manuals, supplies and any other accessories arrive with the instrument, manuals and documentation should be obtained.

Utilities/facilities/environment: verify that the installation site satisfactorily meets vendor specified environmental requirements. A commonsense judgment for the environment suffices: one need not measure the exact voltage for a standard – voltage instrument or the exact humidity reading for an instrument that will operate at ambient condition.

Network and data storage: some analytical systems users to provide network connections and data storage capabilities at the installation site. If this is the case, connect the instrument to the network and check its functionality.

Assembly and installation: assemble and install the instrument and perform any initial diagnostics and testing. Assembly and installation of a complex instrument are best doneby the vendor of specialized engineer, whereas user can assemble and install simple ones. For example instruments, vendor establish installation tests and guide provide valuable baseline reference for determining instrument acceptance. Any abnormal event observed during assembly and installation merits documenting. If the pre-owned or unqualified existing instruments require assembly and installation, perform the tasks as specified here, and then perform the installation verification procedures described below.

Installation verification: perform the initial diagnostics and testing of the instruments after installation. On obtaining acceptable results, the user (when present) and the installing engineer should confirm that the installation was

successful before proceeding with the next qualification phase.

Operational qualification:

After a successful IQ instrument is ready for OQ testing. The OQ phase may consist of these test parameters:

Fixed parameters: these test measures the instruments nonchanging, fixed parameters such as length, height, weight etc. if the vendor supplied specification for these parameters satisfy the user, he or she may waive the test requirement, however, if the user wants to confirm the parameters, testing can be performed at the users site. Fixed parameters do not change over the life of the instrument and therefore never need redetermining. Note: these tests could also be performed during the IQ phase and, if so, fixed parameters need not tobe redetermined as part of OQ testing.

Secure data storage, backup, and archive: when required, secure data handling, such as storage, back up and archiving should b tested at the user site according to written procedures

Instruments function tests: test important functions to verify that the instruments operates as intended by the manufacturer and required by the user. The user should select important instrument parameters for testing according to the instruments intended use. Vendor supplied information is useful in identifying specifications for these parameters. Test should be designed to evaluate the identified parameters. Users, or their qualified designees, should perform these tests to verify that the instrument meets vendor and users specifications.

PERFORMANCE QUALIFICATIONS

After the IQ AND OQ have been performed, the instruments continued suitability for its intended use is proved through performance qualification. The PQ phase includes these parameters:

Performance checks: set up a test or series of tests to verify an acceptable performance of an instrument for its intended use. PQ test are usually based on the instruments typical on site applications. Some test may resemble those performed during OQ, but the specifications for their results can be set differently if required. PQ test are performed routinely on a working instrument, not just on a new instrument at installation. Therefore, PQ specifications can slightly less rigorous than OQ specifications. Nevertheless, user specification for PQ test should evince trouble free instrument operation vis-à-vis the intended applications. PQ test should be performed independent of the routine analytical testing performed on the instrument. Like OQ testing , the test can be modular or holistic. Since many modules within a system interact, holistic tests generally prove more effective by evaluating the entire system and not just the system individual modules. Testing frequency depends on the ruggedness of the instrument and criticality of the tests performed. Testing may be unscheduled- for example, each time of the instrument is used. Or it may be scheduled to occur at regular intervals; e.g., weekly, monthly and yearly. Experience with the instrument can influence this decision. Generally, the same PQ tests are repeated each time so that a history of the instruments performance can be completed. Some system suitability tests or quality control checks that run concurrently with the test sample also imply that the instrument is performing suitably. However,

through system stability tests can supplement periodic PQ tests, they cannot replace them.

Preventive maintenance and repairs: when PQ test (s) fail to meet specifications, the instrument requires maintenance or repair. For many instruments a periodic preventive maintenance may also be recommended. Revelant PQ test(s) should be repeated after the needed maintenance to repair to ensure that the instruments remains qualified.

Standard operating procedure for operation, calibration and maintenance: establish standard operating procedure to maintain and calibrate the instrument. Use a log book, binder or electronic record to document each maintenance and calibrations activity.

REFERENCES

CONTENTS BOOK NAME PAGE NO. PUBLISHED BYU.V. & Visible

spectroscopy

1) Indian

Pharmacopoeia 1996,

Vol. II,

2) USPNF2006

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Assian edition HPLC 1) Indian

Pharmacopoeia 1996,

Vol. II,

2) USPNF2006

A67 – A68

2644 – 2649

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Assian editionGas Chromatography 1) Indian

Pharmacopoeia 1996,

Vol. II,

2) USPNF2006

A65 – A67

2643 – 2644

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Assian edition Gas Chromatography USPNF 2006 2643 - 2644 Assian editionPolarimeter Indian

Pharmacopoeia 1996,

Vol. II,

554 – 556

A80 – A 82

A93

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1. FDA, “Analytical procedures and methods validation:

chemistry, manufacturing, and controls,” federal Register

(notices) 65 (169), 52, 776-52, 777 (30 August 2000).

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2. Amersham biosciences Inc. text on, “Affinity

chromatography: principles and methods”, 18-1022-29,

1997.

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