API, Impurities and Regulatory aspects

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The impurities in pharmaceuticals are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs) or develop during formulation or upon aging of both API and formulation. The presence of these unwanted chemicals even in trace amount may influence the efficacy and safety of pharmaceutical product
Impurities is defined as an entity of drug substances or drug product that is not chemical entity defined as drug substances an excipients or other additives to drugproduct.

The control of pharmaceutical impurities is currently a critical issue to the pharmaceutical industry. Structure elucidation of pharmaceutical impurities is an important part of the drug product development process. Impurities can have unwanted pharmacological or toxicological effects that seriously impact product quality and patient safety. Potential sources and mechanisms of impurity formation are discussed for both drugs. The International Conference on Harmonization (ICH) has formulated a workable guideline regarding the control of impurities. In this review, a description of different types and origins of impurities in relation to ICH guidelines and, degradation routes, including specific examples, are presented. The article further discusses measures regarding the control of impurities in pharmaceuticals substance and drug product applications.

Impurities in pharmaceuticals are the unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during formulation, or upon aging of both API and formulated APIs to medicines. The presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products.

According to ICH, an impurity in a drug substance is defined as-“any component of the new drug substance that is not the chemical entity defined as the new drug substance”. There is an ever increasing interest in impurities present in APIs recently, not only purity profile but also impurity profile has become essential as per various regulatory requirements. The presence of the unwanted chemicals, even in small amount, may influence the efficacy and safety of the pharmaceutical products.

“In the pharmaceutical world, an impurity is considered as any other organic material, besides the drug substance, or ingredients, arise out of synthesis or unwanted chemicals that remains with API’s”

The control of pharmaceutical impurities is currently a critical issue to the pharmaceutical industry. The International Conference on Harmonization (ICH) has formulated a workable guideline regarding the control of impurities.

Impurities have been named differently or classified as per the ICH guidelines as follows:

A] Common names
1. By-products
2. Degradation products
3. Interaction products
4. Intermediates
5. Penultimate intermediates
6. Related products
7. Transformation products

B] United State Pharmacopeia
The United States Pharmacopoeia (USP) classifies impurities in various sections:
1. Impurities in Official Articles
2. Ordinary Impurities
3. Organic Volatile Impurities

C] ICH Terminology
According to ICH guidelines, impurities in the drug substance produced by chemical synthesis can broadly be classified into following three categories –
1. Organic Impurities (Process and Drug related)
2. Inorganic Impurities
3. Residual Solvents

Organic impurities may arise during the manufacturing process and or storage of the drug substance may be identified or unidentified, volatile or non-volatile, and may include
1. Starting materials or intermediates
2. By-products
3. Degradation products

Impurities are found in API’s unless, a proper care is taken in every step involved throughout the multi-step synthesis for example; in paracetamol bulk, there is a limit test for p-aminophenol, which could be a starting material for one manufacturer or be an intermediate for the others. Impurities can also be formed by degradation of the end product during manufacturing of the bulk drugs.

The degradation of penicillin and cephalosporin are well-known examples of degradation products. The presence of a β-lactam ring as well as that of an a-amino in the C6 or C7 side chain plays a critical role in their degradation.

The primary objectives of process chemical research are the development of efficient, scalable, and safe reproducible synthetic routes to drug candidates within the developmental space and acting as a framework for commercial production in order to meet the requirement of various regulatory agencies. Therefore, assessment and control of the impurities in a drug substance and drug product are important aspects of drug development for the development team to obtain various marketing approvals. It is extremely challenging for an organic chemist to identify the impurities which are formed in very small quantities in a drug substance and wearisome if the product is nonpharmacopeial. A study describes the formation, identification, synthesis, and characterization of impurities found in the preparation of API. A study will help a synthetic organic chemist to understand the potential impurities in API synthesis and thereby obtain the pure compound.
Care to taken ensure that desired drug metabolism, safety and clinical studies are not jeopardized by inconsistent purity or impurities having potential harmful toxicological properties,
As regulatory guidelines promulgated by the International Conference on Harmonization (ICH)(1) dictate rigorous identification of impurities at levels of 0.1%,
It is important to develop commercially viable processes for drug substance manufacture to allow greater and more affordable access in the health care sector. In regard to the process development of drug substances, it is essential to know the origin and method of control of any unwanted substances present in it. The limit should be controlled under the threshold of toxicological concern (TTC) for the purpose of ensuring safety and efficacy of the drug and to meet the requirements of various drug regulatory agencies.(2,3)
The impurities in drug substances mostly come from starting substrates, reagents, solvents, and side reactions of the synthetic route employed. Therefore, assessment and control of the undesired substances is an essential aspect of the drug development journey, with special consideration of patient health risk.(4,5)
The isolation/synthesis and characterization of process-related critical impurities (more difficult to control under the desired regulatory limits) of any drug substance in order to evaluate their origin/fate and thereafter their control strategies in the developed process as per International Council for Harmonisation (ICH) guidelines.(4)
The goal of pharmaceutical development is to develop process understanding and control which will yield procedures that consistently deliver products possessing the desired key quality attributes. To achieve this, the quality by design (QbD) paradigm has been employed in combination with process-risk assessment strategies to systematically gather knowledge through the application of sound scientific approaches.(6)
Ganzer et al. recently published an article about critical process parameters and API synthesis.(7) The article presented an in-depth discussion of a stepwise, process risk assessment approach to facilitate the identification and understanding of critical quality attributes, process parameters, and in-process controls. The primary benefit of working within the QbD conceptual framework and employing process risk assessment strategies is the reproducible delivery of high-quality active pharmaceutical ingredient (API). However, a secondary benefit is the ability to obtain regulatory flexibility with respect to filing requirements.(8)
The control of impurities observed in an API is critical in delivering an API of high quality. Identification and understanding of the mechanism of formation of process-related impurities are critical pieces of information required for the development of control strategies. In addition, to ensure a continuing supply of API for drug product clinical manufacture, timely identification of key impurities is essential. These synthesis-related impurities and their precursors are considered as critical impurities because they directly affect the quality and impurity profile of the API. It is our practice that critical impurities be identified if practicable. Therefore, the timely identification of critical impurities becomes an integral part of process development.
There are different approaches to the identification of impurities. Described, herein, a general strategy that we have used in our laboratory, which leads to the rapid identification of impurities. To identify the structure of a low-level unknown impurity, we usually use liquid chromatography/mass spectrometry (LC/MS)/high-resolution MS (HRMS) and tandem MS (MS/MS) for molecular weight (MW) determination, elemental composition, and fragmentation patterns. On the basis of the mass spectrometric data and knowledge of the process chemistry, one or more possible structure(s) may be assigned for the impurity, with definitive structure information obtained by inspection of the HPLC retention time, UV spectrum, and MS profile of an authentic compound.
If an authentic sample is not available, the isolation of a pure sample of the impurity is undertaken for structure elucidation using NMR spectroscopy. The isolation of low-level impurities is usually conducted using preparative HPLC chromatography
 1 ICH Q3A Impurities in New Drug Substances, R2International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)Geneva, Switzerland, October 2006http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3A_R2/Step4/Q3A_R2__Guideline.pdf.
  • 2. Patil, G. D.; Kshirsagar, S. W.Shinde, S. B.Patil, P. S.Deshpande, M. S.Chaudhari, A. T.Sonawane, S. P.Maikap, G. C.Gurjar, M. K.Identification, Synthesis, and Strategy For Minimization of Potential Impurities Observed In Raltegravir Potassium Drug SubstanceOrg. Process Res. Dev. 2012161422– 1429DOI: 10.1021/op300077m
  • 3. Huang, Y.; Ye, Q.Guo, Z.Palaniswamy, V. A.Grosso, J. A. Identification of Critical Process Impurities and Their Impact on Process Research and DevelopmentOrg. Process Res. Dev.200812632– 636DOI: 10.1021/op800067v

4. ICH Harmonised Tripartite Guideline Q3A(R): Impurities in New Drug SubstancesInternational Conference on HarmonizationGeneva2002.

5. Mishra, B.Thakur, A.Mahata, P. P. Pharmaceutical Impurities: A ReviewInt. J. Pharm. Chem.20155 (7), 232– 239

6 International Conference on Harmonisation (ICH) Guidelines; Q8, Pharmaceutical Development, 2005; Q9, Quality Risk Management, 2006.

GanzerW. R.MaternaJ. A.MitchellM. B.WallL. K. Pharm. Technol. 2005July 21–12.

NasrM. Drug Information Association Annual Meeting, Philadelphia, PA, June 19, 2006; Pharmaceutical Quality Assessment System (PQAS) in the 21st Century, 2006.


Genotoxic impurities: the new ICH M7 addendum to calculation of compound-specific acceptable intakes

Genotoxic impurities: the new ICH M7 addendum to calculation of compound-specific acceptable intakes

The draft for a guideline ICH M7(R1) published recently supplements the ICH-M7 guideline published last year. Read more about the calculation of compound-specific acceptable intakes of genotoxic impurities.

The final document of the ICH-Guideline M7 was published in June 2014. It describes the procedure for evaluating the genotoxic potential of impurities in medicinal products (see also our news Final ICH M7 Guideline on Genotoxic Impurities published dated 23 July 2014).

An important approach to the risk characterisation of impurities is the TTC concept (TTC = threshold of toxicological concern). According to this approach the exposure to a mutagenic impurity having the concentration of 1.5 µg per adult person per day is considered to be associated with a negligible risk. It can be used as default evaluation approach to most pharmaceuticals for long-term treatment (> 10 years) and where no carcinogenicity data are available (classes 2 and 3). According to ICH M7 the TTC concept should not be used where sufficient carcinogenicity data exist. Instead the data should be used to calculate or derive compound-specific acceptable intakes.

Now the ICH published an addendum to Guideline M7 with the title “Application of the principles of the ICH M7 Guideline to calculation of compound-specific acceptable intakes” (“Addendum to ICH M7”; short name “M7(R1)”).

This addendum describes the basis for calculating the acceptable intakes for 15 substances in total that are common and widespread in pharmaceutical manufacturing. These substances are known to have mutagenic/carcinogenic potential (ICH M7(R1) contains comprehensive references on the toxicology of these substances). The calculations of the AI (acceptable intake) or PDE (permitted daily exposure) values are partly based on a linear extrapolation from the TD50 values as well as on toxicological data on the non-linear dose-response curve of the relevant substances.

ICH M7(R1) has the status of a draft consensus guideline (step 2 document). The draft guideline was published on the EMA “Scientific Guidelines” site as step 2b document on 4 August 2015 for consultation (deadline for comments: 3 February 2016)


Controlling Residual Arylboronic Acids as Potential Genotoxic Impurities in APIs

Arylboronic acids, but not the corresponding deboronated arenes, recently have been found to be weakly mutagenic in microbial assays [1].  Hence arylboronic acids may be considered potentially genotoxic impurities, and controlling the levels of residual arylboronic acids in APIs could become a regulatory requirement.  The issues should be decided by toxicology studies for the specific arylboronic acids in question.

Several approaches have been successful in removing boronic acids.  Diethanolaminomethyl polystyrene (DEAM-PS) [2],[3] and immobilized catechol [4] have been used to scavenge boronic acids.  Complex formation with diethanolamine may solubilize residual boronic acids in mother liquors.  Since arylboronic acids ionize similarly to phenols, basic washes of an API solution may remove arylboronic acids.  A selective crystallization can purge an arylboronic acid from the API.

The best means to control residual aryl boronic acids in APIs at the ppm level may be to decompose them through deboronation.  Sterically hindered, electron-rich aryl boronates and heteroaromatic boronates are especially prone to deboronation [5],[6],[7].  Deboronation of a hindered difluorobenzeneboronic acid was accelerated in the presence of Pd, and increased in the presence of H2O [8].  Kuivila and co-workers found that protodeboronation of 2,6-dimethoxybenzeneboronic acid was slowest about pH 5, and rapid under more acidic or basic conditions [9].  Butters and co-workers found that protodeboronation occurred for an arylboronate anion, but not for the corresponding arylboronic acid [10].  Amgen researchers found that deboronation of an ortho-substituted arylboronic acid was fastest in the presence of K2CO3 [11].  The Snieckus group found that deboronation occurred readily when pinacol was added to 4-pyridylboronic acid [12].  Percec and co-workers found that deboronation of neopentylglycol boronates, especially an ortho-substituted arylboronic acid ester, was catalyzed by nickel species[13].  Kuivila and co-workers found that CuCl2 catalyzed the deboronation of 2,6-dimethoxybenzeneboronic acid and other arylboronic acids, with formation of the corresponding aryl chlorides [14].  Unfortunately, adding reagents to a reaction mixture increases the burdens of analysis and impurity removal, but additives such as these may accelerate deboronation in difficult cases.  Simply extending the reaction conditions, which are generally basic for efficient Suzuki coupling, or heating with some amount of aqueous hydroxide are probably the preferred treatments to decompose an arylboronic acid.  By knowing the kinetics of the decomposition of the arylboronic acid it may be possible to show by QbD that analyses for the residual arylboronic acid in an API are not necessary.

[1] O’Donovan, M. R.; Mee, C. D.; Fenner, S.; Teasdale, A.; Phillips, D. H. Mutat. Res.: Genet. Toxicol. Environ. Mutagen. 2011, 724(1-2), 1.

[2] Antonow, D.; Cooper, N.; Howard, P. W.; Thurston, D. E. J. Comb. Chem. 2007, 9, 437.

[3] Hall, D. G.; Tailor, J.; Gravel, M. Angew. Chem. Int. Ed. 1999, 38, 3064.

[4] Yang, W.; Gao, X.; Springsteen, G.; Wang, B. Tetrahedron Lett. 2002, 43, 6339.

[5] Goodson, F. E.; Wallow, T. I.; Novak, B. M. Organic Syntheses; Vol. 74; Smith, A. B., III, Ed.; Organic Syntheses, Inc.; 1997; p. 64.

[6] Baudoin, O.; Cesario, M.; Guénard, D.; Guéritte, F. J. Org. Chem. 2002, 67, 1199.

[7] Dai, Q.; Xu, D.; Lim, K.; Harvey, R. G. J. Org. Chem. 2007, 72, 4856.

[8] Cammidge, A. N.; Crépy, K. V. L. J. Org. Chem. 2003, 68, 6832.

[9] Kuivila, H. G.; Reuwer, J. F., Jr.; Mangranite, J. A. Can. J. Chem. 1963, 41, 3081.

[10] Butters, M.; Harvey, J. N.; Jover, J.; Lennox, A. J. J.; Lloyd-Jones, G. C.; Murray, P. M.Angew. Chem. Int. Ed. 2010, 49, 5156.

[11] Achmatowicz, M.; Thiel, O. R.; Wheeler, P.; Bernard, C.; Huang, J.; Larsen, R. D.; Faul, M. M.J. Org. Chem. 2009, 74, 795.

[12] Alessi, M.; Larkin, A. L.; Ogilvie, K. A.; Green, L. A.; Lai, S.; Lopez, S.; Snieckus, V. J. Org. Chem. 2007, 72, 1588.

[13] Moldoveanu, C.; Wilson, D. A.; Wilson, C. J.; Leowanawat, P.; Resmerita, A.-M.; Liu, C.; Rosen, B. M.; Percec, J. Org. Chem. 2010, 75, 5438.

[14] Kuivila, H. G.; Reuwer, J. F., Jr.; Mangravite, J. A. J. Am. Chem. Soc. 1964, 86, 2666.


The usefulness of boronic acids and their esters is well documented.

Over the last decade much interest has been shown in the field of boron chemistry particularly in the inorganic aspect. Boronic acids, RB(OH)2, have been one such compound that has attracted a widespread interest over the last decade. They have been involved in various fields, such as sugar chemistry, thermoplastics, enzymes and various other areas in chemistry and biology.

Until now, the synthesis of these compounds has usually involved the reaction of Grignards or organolithium derivatives with trialkyl borates. Although widely applicable, this method normally works well only at low temperatures and is limited to substrates lacking functional groups which could react with the organometallic intermediates.(synthesis will be discussed later)

Figure 1-1: Some common boronic acids.

Boronic esters, RB(OR¢ )2, have also made great strides in research over the last decade. They are being used in macrocyclic chemistry, carbohydrate chemistry, and as molecular recognition compounds.

Figure 1-2: Some common boronic esters.






Before I begin to talk about boronic acids I feel that it is fair to discuss the properties of these compounds first. By doing so, it will be easier to understand why these compounds react in the manner they do.


  • Acid Strength


Phenylboronic acid is about three times as strong as boric acid which is a weak acid. This is in agreement with the substitution effect in acetic acid. Since any effect that causes a reduction in electron density on the proton of the oxygen strengthens the acid, then an effect that increases the electron density on the proton weakens the acid. When a substitution of an ortho hydrogen by an alkyl or phenyl occurs a decrease in the strength of the phenylboronic acid occurs. A meta or para susbstitution on the other hand results in only a slight decrease in strength. When electronegative substituents replace a proton of the oxygen, the strength of the acid increases considerably. This increase occurs in the following manner:

NO2 > F > Cl > Br > COOH


  • Substitution in the Aromatic Nucleus


When treated with a concentrated solution of NaOH, n-Butylboronic acid gives a hydrated sodium salt and a calcium salt. This substitution in the aromatic nucleus forms the structure of the barium salt of p-carboxyphenylboronic acid.

Figure 1-2: Structure of the barium salt of p-carboxyphenylboronic acid


  • Anhydride formation


Another property of boronic acids is that they form anyhydrides easily. Formation of the anhydride is very important in determining the melting-point of the particular boronic acid. This is main reason why the melting point of boronic acids are not frequently misquoted. Phenylboronic acid has a melting point of ~218oC, and was first prepared in 1882. Boronic anhydrides are very easily hydolysed, and readily form esters of the same acid. This is done by heating with an alcohol or phenol.



Figure 1-3: Formation of esters via boronic acids

History of Boronic Acids:

The chemistry of boronic acids is a relatively new topic with respect to chemistry. Early accounts of boronic acid synthesis involved the production of diboronic acids. This synthesis was accomplished by using tetrahydrofuran as the solvent to attain a higher temperature for the completion of metallation, and a Grignard reagent was formed in the process.

Figure 1-4: Formation of diboronic acid.

The Importance of the Grignard reagent:

Formation of the Grignard reagent in the above step is essential to the formation of the diboronic acid. By insertion of the Grignard a polarization occurs causing there to be a slight negative charge on the carbon of the phenyl group and a slight positive charge on the Mg. This causes the phenyl group to act as a nucleophile easily react with (MeO)3B.

Another technique that was used in the early years of boronic acid synthesis also involved the formation of the Grignard reagent. A general procedure was to attach an alkyl group or aryl group to boron by use of a boron trihalide, and a Grignard reagent to supply a hydorcarbon group. Another method that was used in the early years was Khotinsky’s method. This involved the formation of a carbon-boron bond by interaction with an alkyl borate at 0oC.


B(OR)3(in ether) added to ArMgBr ® ArB(OR)2 ® ArB(OH)2

The use of boron triflouride and borontrichloride have also been used to form boronic acids when reacted with Grignard reagents.

BF3(Cl,Br) + RMgBr ® RBF2(Cl,Br) ® RB(OH)2+ 2HF(Cl,Br)

Modern day Synthesis and important reactions of Boronic Acids:


Today there are numerous ways to synthesize boronic acids, and modifications continue to appear. One such technique involves the preparation of aryl boronic acid by heating a mixture of trialkyl borate with magnesium and the aryl bromide. The reaction requires heat which starts the reaction at ~150oC and by ~230oC the magnesium disappears after heating it for 2-3 hours under reflux. After the usual procedure of hydolysis the phenyl boronic acid was produced and isolated. The yield attained was between 33-44 %. The advantage of using bromobenzene over chlorobenzene is that you don’t have to heat the mixture as long. Otherwise, the same results are obtained.

Figure 1-5: The Cowie reaction used to synthesize aryl boronic acids.

Tri-n-butyl borate even reacts with dibromobenzene to produce a diboronic acid. However, this method is not preferred because the yield is substantially lower at 16%. On the otherhand, p-Dichlorobenzene responded poorly to the same reaction.

Figure 1-6: Synthesis of diboronic acid via dibromobenzene.


As was mentioned earlier when attempting to synthesize boronic acids, anhydrides can be easily formed. One such example occurs when attempting to prepare o-phenyldiboronic acid via the above method. A phenylboronic anhydride is produced with a mixture of olefins. This result is unexpected since hydrolysis of the later formed trimer will produce a phenylboronic acid. The exact mechanism of this reaction is unknown, but the most probable one is given below.





Figure 1-7: Probable mechanism of phenylboronic anhydride synthesis.


There are numerous other ways to synthesize boronic acids. As a result there are numerous groups of boronic acids. The first group which will discussed are the alpha-Aminoboronic acids. Although there are several examples of aminomethylboronate syntheses, the most important is probably the boronate amino acid isotere. This complex is a useful inhibitor of the serine chymotrypsin . Thus, it has triggered a great interest in both synthetic and medicinal use.




Figure 1-8: Formation of alpha-Aminoboronic acid

Another group of boronic acids that have vastly been studied are the alpha-haloboronic acids. These are produced by hydolysis of halomethyl boranes.


Figure 1-9: Formation of alpha-haloboronic acids

The aryl boronic acids undergo protodeboronation on treatment with carboxylic acids, such as HCO2H. The reaction proceeds via the formation of a six-membered cyclic transition state.



Figure 1-10: The protodebromonation of aryl boronic acids.


Synthetic routes to boronic acid synthesis:


There are six general synthetic routes to boronic acid synthesis. The first used, and in many ways the least likely, was the slow partial oxidation of the spontaneously inflammable trialkylboranes followed by the hydrolysis of the ester.


Et3B + O2 ® EtB(OEt)2 ® EtB(OH)2


Ph3B + O2 ® PhB(OPh)2 ® PhB(OH)2

The ready availability of many trialkylboranes makes this an attractive route but it is still relatively used very little. Dialkyl borinic acids can also be oxidized to produce boronic acids.


R2B(OH) + 1/2O2 ® RB(OH)2 + R(OH)

A second route to produce boronic acids involves the acid hydolysis of trialkyl- or triaryl-boranes. This method involves the change of these substituted boranes to borinic, boronic, and boric acids respectively.



R3B ® R2B(OH) ® RB(OH)2® B(OH)3

The third synthetic route is via halogenation and subsequent hydrolysis. While the appropriate alkyldihalogenborane intermediates can be prepared via the fourth route; the reaction of organometallics on boron trihalides.

Br2 H2O

Bu3B ® BuBBr2 + Bu2BBr ® BuB(OH)2 + Bu2B(OH)

I2 H2O air

R3B ® R2BI ® R2B(OH) ® RB(OH)2


BCl3 + Ar2Hg ® ArHgCl + ArBCl2 ® ArB(OH)2


R3B + BX3 ® RBX2 + R2BX® RB(OH)2+ R2B(OH)

By far the most widely used method for preparing boronic acids is the reaction of aryl or alkyl Grignard reagents on organo-orthoborates or boron halides. The preferred starting materials include B(OMe)3, B(OBu)3 and BF3.

500C H3O+

B(OR)3 + ArMgX ® [ArB(OR)3MgX] ® ArB(OH)2

Organolithium compounds have also been used to synthesize boronic acids.


B(OR)3 + R’Li ® [R’B(OR)3]Li ® R’B(OH)2

The final route to synthesize boronic acids involves the aromatic substitution of an existing boronic acids by conventional organic techniques; this is particularly important since the Grignard or organolithium syntheses just mentioned cannot be carried out in the presence of other reactive functional groups such as nitro, amino, carboxy, sulphono, etc. A typical sequence of reactions is as follows:


Figure 1-11: Formation of boronic acids via aromatic substitution.




Handling, Toxicity, and Analysis of Boronic Acids:

Since there are numerous boronic acids I will analyse each of the above headings in terms of just one boronic acid, namely benzene boronic acid. Benzene boronic acid like most boronic acids are sensitive to oxygen and are susceptible to hydolysis. When handling benzene boronic acid one must adapt to the chemical and physical characteristics of that particular compound.

The toxicity of benzene boronic acid is fairly high. It has an lethal dosage number of 50.(LD 50) That is, 50% of the rats that took a dosage of 740 mg/kg died. The dosage of 740 mg/kg is the approximate amount of benzene boronic acid that causes intake of it to be lethal.

The analysis of volatile boronic acids can be achieved by vapor phase chromatography provided that inert liquid and stationary phases are used, protic sites are converted to trimethylsilyl groups and injection port temperatures are regulated such that thermal degradation of the molecules does not occur. As a general rule, the retention time of boronic acids on a nonpolar column is similar to that of the corresponding hydrocarbon. Analytical determinations of boronic acids depend on complete cleavage of the boron-carbon bond.(cleavage of both if diboronic acid) Cleavage can occur via oxidation, or use of a variety of reagents. These reagents include NaOH, alkaline hydrogen peroxide, sulphuric acid, etc. Also, it appears that some boronic acids can be titrated by NaOH in the presence of mannitol using phenolphthalein as an indicator.


Applications of Boronic Acids and recent research:

Boronic acids have been used for various reasons. Alkyl boronic acids have been used for research and developnment purposes. These include the nonyl and dodecyl boronic acids. These boronic acids are termed as ‘bacteriastatic’, ‘fungistatic’and ‘surface active’. They are used as gasoline additives, polymer components and stabilizers for aromatic amines. These properties are of importance to slime organisms in the manufacturing of paper, and to minimize bacterial damage in paint, leathers, and polymers. They are also used in the dehydrated form, (RBO)3, as potential oil additives for stabilization against oxidation and polymerization. Alkyl boronic acids are also used in motor fuels, particularly in race cars.

Recently, boronic acids have been used as reagents in Suzuki coupling reactions to produce aryl substituted pyrrolo benzodiazepines(PBDs). These compounds have been used as gene targeting vectors and as antitumour and diagnostic agents.


Figure 1-12: Use of boronic acids to produce PBD via Suzuki Coupling.

Tandem Suzuki coupling-cyclisation with 2-Formylthiophene-3-boronic acid, was used to synthesise the thieno [2,3-c]-1,7-naphthyridine ring system.

Palladium catalysed Suzuki-type cross coupling of an iodocyclopropane with Thiophene-3-boronic acid, promoted by cesium fluoride.

Asymmetric boronic acid Mannich reaction of Furan-2-boronic acid, 15297, with an aldehyde in the presence of a chiral amine template.

Figure 1-13: New Boronic acids reactions.




There are numerous ways to synthesize boronic esters. Among these involve b -eliminations. b -eliminations are oxidative processes in which the boron becomes bonded to oxygen or another electronegative element, carbon loses an electronegative ligand at the b -position, and a carbon-carbon p -bond is formed. It is not remarkable that b -eliminations of boron and an electronegative atom occur, but boron is inusual for a relatively metallic element in that b -eliminations tend to be very slow. There are various types of b -eliminations; these includeb -elimination of halides, vinylic chlorides, alkoxides, oxide anions and azides.

Boronic esters can be easily made by the interaction of boronic acids with a hydoxy-compound. In this reaction water is removed as an azeotrope.

RB(OH)2 + 2R’OH ® RB(OR’)2 + 2H2O

Esters of alkyl boronic acids are in most cases more slowly hydrolysed than esters of arylboronic acid, or of boric acid, and indeed di-n-butylboronate. As a consequence they can be isolated from the Grignard system because of this relative stability. As a rule of thumb, boronic esters are far from being hydrolytically stable, although they have good thermal stability.

When aryl and alkyl groups are of ordinary reactivity then both esterification and hydolysis involves B-O fission, and not C-O fission. The is because(+)-2-methylheptanol is isolated unchanged in rotatory power after being converted into and obatined from the ester of phenylboronic acid. This optical result is confirmed when an (+)-alcohol is reacted with and phenylboron dichloride.

Figure 1-12: Ester preparation via a (+)-alcohol and a phenylboron dichloride.

Cyclic esters are an important part of boronic ester chemistry. One such method to prepare these involves the mixing of dihydric alcohols and phenols. Halogeno-ester preparation involves mutual replacement reactions. These produce both alkyl-and arylboronic halogeno-esters, and the reaction occurs quickly.

n-BuB(OBun)2 + BCl3 ® n-BuBCl(OBun) + BunOBCl2


Figure 1-13: Synthesis of halogeno-esters.


a -amino boronic esters:

Secondary amines react readily with iodomethylboronic esters to produce stable (dialkylamino)methyl boronic esters. These reactions are illustrated by the conversion of pinacol iodomethylboronate to piperidine and triethylamine deritvatives.


Figure 1-14: Synthesis of piperidine and triethyl amine derivatives


Air Oxidation:

Cyclic boronic acids are generally stable in air, and can be stored for a number of days or weeks without protection from atmospheric oxygen. But, this is misguiding because autooxidations can consume the entire sample within a relatively short time. Many accounts have been recorded where people used boronic esters for a number of weeks, but after a few months of storage the entire compound decomposed due to exposure to air by a hole in the bottle. Thus, a rubber septum cap is not sufficient for long-tem storage. Analytical samples of boronic esters should be stored in a screwcap vials. But, should be replaced every year if one intends to store these esters for a long time.

Reactions of boronic esters with lithium compounds:

The reaction of esters with lithium compounds is a complex process which involves several factors to consider. Among these are the choice of chiral diol, protection of the a-halo boronic ester, catalysis of the reaction, etc. When making the choice of the chiral diol, depending on the chiral diol used, there may be two different stereochemical outcomes that may result. One such case is when one uses two different borate complexes. That is, the borate complex derived from pinanediol alkylboronate rearranges stereoselectivity, but the borate complex from the pinanediol(dichloromethyl)borate yields a gross mixture of diastereomeric a-chloro boronic esters.


Figure 1-15: Reactions of esters with Lithium compounds.

Figure 1-16: New Boronic ester reactions.





  • Fieser & Fieser. Reagents for organic synthesis. Wiley-Interscience. New York, 1969.
  • Gerrard,W. The Organic Chemistry of Boron. Academic Press. New


York, 1969.

3. Herbert & Brown. Hydroboration. W.A. Benjamin, INC. New York, 1962.

4. Muetterties, E. The Chemistry of Boron and Its Compounds. John Wiley & Sons, 1967.

  1. Proctor, G. Asymmetric Synthesis. Oxford Science Publications. New York, 1996.



  • Abel et al. Comprehensive organometallic Chemistry II-A review of the literature 1982-1994. Volume 6,7, and 12. Pergamon Press. United Kingdom, 1995.
  • Greenwood N.N. and Earnshaw. Chemistry of the Elements. Pergamon Press. New York, 1984.
  • Katritzky et al. Comprhensive Organic Functional Group Transformations. Volumes 1,2,4. Pergamon Press. Glasgow, 1995.
  • Wilkenson et al. Comprehensive Organometallic Chemistry. Volumes 1,4. Pergamon Press. New York, 1982.


Web Sites:

  1. http://siri.org/msds/tox/f/q20/q129.html
  2. http://www.yahoo/chemistry/boronic
  3. www.lancastersynthesis.com/html/body_het._boronics.html
  4. www.netscape/chemistry/boronic




  • Howard et al. Synthesis of Novel C7-Aryl Substitued Pyrrolo(2,1-C)(1,4)Benodiazepines(PBDs) via Pro-N-10-Troc Protection and Suzuki Coupling. Bioorganic & Medicinal Chemistry Letters 1998, Vol , Iss 2, pp. 3017-3018.
  • Metteson, D. Functional compatibilities in boronic ester chemistry. Journal of Oganometallic Chemistry. Washington, 1999. (web of science)
  • Nishimura et al. Preparation and Thermal Properties of Thermoplastic Poly(vinyl alcohol) Complexes with Boronic Acids. Kyoto, Japan, 1998.



Elemental impurities – A database to facilitate the risk assessment of active ingredients and excipients

One of the main demands of the Guideline ICH Q3D is to carry out risk assessments on metallic impurities. A database with analytical data provides a valuable support. Learn more about the data sharing using the new elemental impurities database.


Released in December 2014, the ICH Q3D Guideline on Elemental Impurities contains extensive specifications for the control of a total of 24 elements (21 metals, 3 metalloids) that can be present as impurities in pharmaceutical products. Main sources can be

  • Active ingredients
  • Excipients (including water)
  • Processing auxiliaries and catalysts
  • Production equipment
  • Container and closure systems

The Guideline ICH Q3D calls for a risk assessment with regard to the presence of metallic impurities in various dosage forms, taking into account the respective limit values. The main factors of influence are to be included (see fishbone diagram on p. 6 of the Guideline). The risks identified in a comprehensive analysis have then to be categorized in a meaningful and justifiable manner.

The data for the content of metallic impurities, e.g. in excipients (for this purpose there is a study conducted by the FDA) or of migratable impurities in container / closure systems (there exists a Literature review in the PDA journal of pharmaceutical science and technology) is rather thin. And the sources of information can only be found through extensive research. The greatest treasure of information is located in the databases of several pharmaceutical and API manufacturers which have carried out analytical studies already.

To merge these data and information and to make them available to all interested companies in the form of a database, representatives of eight major companies have joined forces to an “Elemental Impurities Pharma Consortium”. This group was formed in October 2013, after a Conference on “Elemental Impurities” conducted by the Joint Pharmaceutical Analysis Group (JPAG).

The database that is currently established under the auspices of the EI Pharma Consortium, now comprises analytical data on elemental impurities from over 100 different materials (pharmaceutical excipients, dyes, etc.), which were provided by other companies. These data are anonymized, so that interested users of the database can not recognize the specific origin of the information.

The benefit for the user increases to the same extent as the database grows, which basically means for the companies that have to implement one of the main requirements of the ICH Q3 Guideline – to carry out a risk assessment. The timeframe for this is tight: for medicinal products still to be approved the provisions of ICH Q3D need to be fulfilled by June 2016. Already approved products have to comply from December 2017 (see also our news “Industry Coalition” gives practical advice for the control of elemental impurities in active substances and excipients).

Note : At the Impurities Forum from 16-18 June 2015 in Prague you will receive more information about this topic. Andrew Teasdale, one of the initiators of the Consortium, will report about the database and the possibilities to use it.




read at



methodology provides a risk-based approach to residual solvent
analysis that considers a patient’s exposure to a solvent residue
in the drug product. Solvents have been classified based on their
potential health risks into three main classes:
1. Class 1: Solvents should not be used because of the
unacceptable toxicities or deleterious environmental effects.
2. Class 2: Solvents should be limited because of inherent
3. Class 3: Solvents may be regarded as less toxic and of lower
risk to human health.
Testing is only required for those solvents used in the
manufacturing or purification process of drug substances, excipients
or products. This allows each company to determine which solvents
it uses in production and develop testing procedures that address
their specific needs. It is the responsibility of the drug manufacturer
to qualify the purity of all the components used in the manufacturing
of the drug product. This would pertain to items such as excipients,
of which some contain residual levels of Class 1 solvents by nature
of the manufacturing process and/or nature of the starting materials
(e.g. ethyl cellulose). The new 467 monograph provides an optional
method to determine when residual solvent testing is required for
Class 2 solvents. Each Class 2 solvent is assigned a permitted daily
exposure (PDE) limit, which is the pharmaceutically acceptable
intake level of a residual solvent.
The USP has provided a method for the identification, control,
and quantification of Class 1 and 2 residual solvents. The method
calls for a gas chromatographic (GC) analysis with flame ionization
detection (FID) and a headspace injection from either water or
organic diluent. The monograph has suggested two procedures:
Procedure A G43 (Zebron ZB-624) phase and Procedure B G16
(Zebron ZB-WAXplus) phase. Procedure A should be used first. If
a compound is determined to be above the specified concentration
limit, then Procedure B should be used to confirm its identity.
Since there are known co-elutions on both phases, the orthogonal
selectivity ensures that co-elutions on one phase will be resolved
on the other. Neither procedure is quantitative, so to determine
the concentration the monograph specifies Procedure C, which
utilizes whichever phase will give the fewest co-elutions. Class
3 solvents may be determined by 731-Loss on Drying unless the
level is expected to be >5000 ppm or 50 mg. If the loss on drying
is >0.5 %, then a water deterrmination should be performed using
921-Water Determination.
One of the most important considerations is that, once
implemented, the new method will pertain to all currently marketed
drug products as well as those in development and clinical trials8

United States Pharmacopoeia (USP):
In 1988, the United States Pharmacopoeia (USP) provided
control limits and testing criteria for seven organic volatile impurities
(OVIs) under official monograph 4678
. According to USP, testing
should be conducted only if a manufacturer has indicated the
possible presence of a solvent in a product. Testing may be avoided
when a manufacturer has assurance, based on the knowledge of
the manufacturing process and controlled handling, shipping, and
storage of the product, that no potential exists for specific solvents
to be present and that the product, if tested, will comply with the
accepted limit. Items shipped in airtight containers (such as those
used for food additives) can be considered not to have acquired
any solvents during transportation2
The compounds are chosen based on relative toxicity and only
applied to drug substances and some excipients8
. In addition, a
test for ethylene oxide is conducted if specified in the individual
monograph. Unless otherwise specified in the individual monograph,
the acceptable limit for ethylene oxide is 10 ppm. USP does not
address all other solvents mentioned in the ICH guideline2
In an effort to harmonize with the International Conference
for Harmonization (ICH), the USP has proposed the adoption of
a slightly modified version of ICH (Q3C) methodology, which has
been scheduled for implementation on July 1, 2007. The ICH Q3C

Organic Volatile Impurities
Of the solvents targeted in USP 26 General Chapter 467, only
methylene chloride may appear in bulk pharmaceutical products
manufactured by Pfizer at the Kalamazoo plant. For those products
where OVI testing is required, our material will meet the compendial
limits for methylene chloride and other solvents that may be added
to the target list in the future.
No OVI requirement exists in the USP 26 monograph
for Triamcinolone, but Triamcinolone from Pfizer meets the
requirements of USP 26 General Chapter 467.

Residual solvents in pharmaceuticals, commonly known as
organic volatile impurities (OVIs), are chemicals that are either
used or produced during the manufacture of active pharmaceutical
ingredients (APIs), excipients and drug products1, 2
Organic solvents play an essential role in drug-substance and
excipient manufacture (e.g., reaction, separation and purification)
and in drug-product formulation (e.g., granulation and coating) 3
Some organic solvents are often used during the synthesis of active
pharmaceutical ingredients and excipients or during the preparation
of drug products to enhance the yield, increase solubility or aid
. These process solvents cannot be completely
removed by practical manufacturing practices such as freeze–drying
and drying at high temperature under vacuum. Therefore, some
residual solvents may remain in drug substance material4
. Typically,
the final purification step in many pharmaceutical drug-substance
processes involves a crystallization step, and the crystals thus
formed can entrap a finite amount of solvent from the mother liquor
that may cause degradation of the drug, OVIs may also contaminate
the products during packaging, storage in warehouses and/or during
While solvents play a key role in the production of
pharmaceuticals, there is also a downside, as many of the
solvents used have toxic or environmentally hazardous properties.
Complete removal of residual levels of solvents is impractical from a
manufacturing standpoint, so it is inevitable that traces will remain inthe final product. The presence of these unwanted chemicals even
in small amounts may influence the efficacy, safety and stability of
the pharmaceutical products. Because residual solvents have no
therapeutic benefits but may be hazardous to human health and
the environment, it must be ensured that they are either not present
in products or are only present below recommended acceptable
levels. It is a drug manufacturer’s responsibility to ensure that any
OVIs present in the final product are not harmful to humans and
that medicinal products do not contain levels of residual solvents
higher than recommended safety limits. Solvents known to cause
unacceptable toxicity should be avoided unless their use can be
justified on the basis of a risk-benefit assessment2
. Because of their
proven or potential toxicity, the level of residual solvents is controlled
through national and international guidelines, for example, through
the FDA and International Conference on Harmonization.

“All drug substances, excipients, and products are subject to
relevant control of residual solvents, even when no test is specified
in the individual monograph.”
Regulatory and Compliance Environment
One of the essential aspects of pharmaceutical manufacturing
is regulatory compliance, which typically encompasses two aspects.
The first is compliance with private sets of standards based on
an applicant filing with a regulatory agency, which requires the
applicant to report the determined residual solvent levels in a
number of representative batches of pharmaceutical product to
establish typical levels of solvent contamination that can routinely
be achieved. Based on a statistical evaluation of the reported
data, a specification is agreed for solvents used in the final step of
the process and a decision made on whether testing is required
for solvent used at earlier stages in the process. To arrive at a
specification that is a measure of the routine performance of the
process, regulatory agencies require numerical data rather than
reporting compliance with a limit test.

Internationally, there has been a need to establish regulatory
standard guidelines. In 1997, The International Conference on
Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH), through its Q3C Expert
working group formed by regulators from the three ICH regions,
industry representatives and interested parties/observers, finalized
the Q3C guideline on residual solvents. Essentially, ICH has
consistently proposed that limits on organic solvents be set at levels
that can be justified by existing safety and toxicity data, and also kept
proposed limits within the level achievable by normal manufacturing
processes and within current analytic capabilities.
The second aspect is compliance with public standards set
by Pharmacopoeias from the three ICH regions (United States
Pharmacopoeia (USP), European Pharmacopoeia (Ph. Eur.) and
Japanese Pharmacopoeia (JP)) and also with local pharmacopoeias
from countries outside the ICH regions. In the recent past, guidelines
for organic residual solvents for public standards have generally
been vague and not up-to-date. The pharmacopoeial approach
was typically a limit test for residual solvents, employing standard
. The USP set the official limits in USP 23rd edition in the
general chapter 467, Organic Volatile Impurities5
. Very early on,
the Ph. Eur. employed the ICH Q3C regulatory approach and
updated the acceptance limits but kept the methodology as a limit
test based on standard addition. The general method in Ph. Eur. for
Identification and Control of Residual Solvents in drug substances
defines a general procedure and describes two complementary gas
chromatography (GC) conditions for identifying unknown solvents.
‘‘System A’’ is recommended for general use and is equivalent
to ‘‘Methods IV and V’’ of the USP for analysis of volatile organic
impurities ‘‘System B’’ is used to confirm identification and to solve
co-elutions. Implementation of this general method is a subject of
debate in the pharmaceutical industry due to its limited selectivity
and sensitivity3
. Historically, until its 27th edition, the USP restricted
its listing of residual solvents to those of Class 1 and neglected to

consider the wide range of organic solvents used routinely in the
pharmaceutical industry. Furthermore, the limits stated for Class 1
solvents like benzene, chloroform, 1, 4-dioxane, methylene chloride,
and 1, 1, 1-trichloroethane are in the range 2–600 (ppm) and are
therefore not in concordance with the ICH guideline. Residual
solvent testing using GC has been included in the pharmacopeias
for almost 20 years, while residual solvent-test methods have
been reported in the literature since before that. With USP 28, the
public standard for residual solvents was updated to comply with
the ICH Q3C guideline, but the methodology (the same limit-test
approach as Ph. Eur.) and the targeted monographs were not
considered appropriate by industry and regulators, leading to a
notice postponing implementation in USP 296
ICh Guideline
The objective of this guidance is to recommend acceptable
amounts for residual solvents in pharmaceuticals for the safety of
the patient. The guidance recommends use of less toxic solvents
and describes levels considered to be toxicologically acceptable
for some residual solvents.
Residual solvents in pharmaceuticals are defined here as
‘organic volatile chemicals that are used or produced in the
manufacture of drug substances or excipients, or in the preparation
of drug products’. This guidance does not address solvents
deliberately used as excipients nor does it address solvates.
However, the content of solvents in such products should be
evaluated and justified.
Since there is no therapeutic benefit from residual solvents,
all residual solvents should be removed to the extent possible to
meet product specifications, good manufacturing practices, or other
quality-based requirements. Drug products should contain no higher
levels of residual solvents than can be supported by safety data.
Some solvents that are known to cause unacceptable toxicities
(Class 1) should be avoided in the production of drug substances,
excipients, or drug products unless their use can be strongly justified
in a risk-benefit assessment. Some solvents associated with less
severe toxicity (Class 2) should be limited in order to protect patients
from potential adverse effects. Ideally, less toxic solvents (Class 3)
should be used where practical7

Scope of the Guidance
Residual solvents in drug substances, excipients, and drug
products are within the scope of this guidance. Therefore, testing
should be performed for residual solvents when production or
purification processes are known to result in the presence of such
solvents. It is only necessary to test for solvents that are used or
produced in the manufacture or purification of drug substances,
excipients, or drug products. Although manufacturers may choose
to test the drug product, a cumulative method may be used to
calculate the residual solvent levels in the drug product from the
levels in the ingredients used to produce the drug product. If the
calculation results in a level equal to or below that recommended
in this guidance, no testing of the drug product for residual solvents
need be considered. If, however, the calculated level is above the
recommended level, the drug product should be tested to ascertain
whether the formulation process has reduced the relevant solvent
level to within the acceptable amount. Drug product should also be
tested if a solvent is used during its manufacture.
This guidance does not apply to potential new drug substances,
excipients, or drug products used during the clinical research
stages of development, nor does it apply to existing marketed
drug products. The guidance applies to all dosage forms androutes of administration. Higher levels of residual solvents may be
acceptable in certain cases such as short-term (30 days or less)
or topical application. Justification for these levels should be made
on a case-by-case basis7
Classification of Residual Solvents
OVIs are classified into three classes on the basis of their
toxicity level and the degree to which they can be considered
an environmental hazard. The list provided in the guideline is
not exhaustive, and one should evaluate the synthesis and
manufacturing processes for all possible residual solvents.
The term, tolerable daily intake (TDI), is used by the International
Program on Chemical Safety (IPCS) to describe exposure limits
of toxic chemicals and the term, acceptable daily intake (ADI), is
used by the World Health Organization (WHO) and other national
and international health authorities and institutes. The new term,
permitted daily exposure (PDE), is defined in the present guidance
as a pharmaceutically acceptable intake of residual solvents to avoid
confusion of differing values for ADI’s of the same substance7
Residual solvents are classified on the basis
of risk assessment:
1. Class 1 solvents (Solvents to be avoided): Known human
carcinogens, strongly suspected human carcinogens, and
environmental hazards.
2. Class 2 solvents (Solvents to be limited): Non-genotoxic
animal carcinogens or possible causative agents of other
irreversible toxicity such as neurotoxicity or teratogenicity.3. Class 3 solvents (Solvents with low toxic potential): Solvents
with low toxic potential to man; no health-based exposure limit
is needed. Class 3 solvents have PDE’s of 50 milligrams (mg)
or more per day.
4. Class 4 solvents (Solvents for which no adequate
toxicological data was found): No adequate toxicological
data on which to base a PDE (permitted dose exposure) was
Environmental Regulation of Organic Volatile
Several of the residual solvents frequently used in the
production of pharmaceuticals are listed as toxic chemicals in
Environmental Health Criteria (EHC) monographs and in the
Integrated Risk Information System (IRIS). The objectives of such
groups as the International Programme on Chemical Safety (IPCS),
the U.S. Environmental Protection Agency (EPA), and the U.S.
Food and Drug Administration (FDA) include the determination
of acceptable exposure levels. The goal is protection of human
health and maintenance of environmental integrity against the
possible deleterious effects of chemicals resulting from long-term
environmental exposure. The methods involved in the estimation
of maximum safe exposure limits are usually based on long-term
studies. When long-term study data are unavailable, shorter term
study data can be used with modification of the approach such as
use of larger safety factors. The approach described therein relates
primarily to long-term or lifetime exposure of the general population
in the ambient environment (i.e., ambient air, food, drinking water,
and other media) 7
Limits of Residual Solvents
Solvents to Be Avoided: Solvents in Class 1 (Table 1) should
not be employed in the manufacture of drug substances, excipients,and drug products because of their unacceptable toxicity or their
deleterious environmental effect. However, if their use is unavoidable
in order to produce a drug product with a significant therapeutic
advance, then their levels should be restricted as shown in Table
1, unless otherwise justified. The solvent 1, 1, 1-Trichloroethane
is included in Table 1 because it is an environmental hazard. The
stated limit of 1,500 ppm is based on a review of the safety data

Analysis of Residual Solvent in
The analysis of residual solvents is an essential part in the
quality control of drug substances used in preclinical or clinical
trials as well as for use in commercial drug products. Residual
solvent analysis of bulk drug substance and finished pharmaceutical
products is necessary for a number of reasons such as –
1. High levels of residual organic solvents represent a risk to human
health because of their toxicity.
2. Residual organic solvents also play a role in the physicochemical
properties of the bulk drug substance. Crystalline nature of the
bulk drug substance can be affected. Differences in the crystal
structure of the bulk drug may lead to changes in dissolution
properties and problems with formulation of the finished
3. Finally, residual organic solvents can create odor problems
and color changes in the finished product and, thus, can lead
to consumer complaints.
4. Often, the main purpose for residual solvent testing is in its use
as a monitoring check for further drying of bulk pharmaceuticals
or as a final check of a finished product.

5. Testing for solvent content in intermediates may need to be
performed if a critical amount of residual solvent(s) remaining
in the intermediate can alter the next step of the process.
6. Knowledge of the solvent content in the starting materials may
help to the development chemist to understand the synthetic
routes and predict potential process related impurities.
7. Knowing the solvents used in the process allows the development
chemist to look for possible compound- solvent interactions
which can lead to the formation of impurities5, 16
Residual solvent analysis can be performed with a large array of
analytical techniques17. The most popular, and the most appropriate,
specific solvent analysis is testing by gas chromatography (GC).
Modern capillary-column gas chromatographs can separate a large
number of volatile components, permitting identification through
retention characteristics and detection at ppm levels using a broad
range of detectors5
.Gas chromatographic testing can be categorized
into three main procedures according to the means of introducing
the sample into the instrument. A direct gas chromatographic
procedure is one in which a portion of the actual drug substance
or formulation is injected into a GC system. The drug substance
is usually dissolved in an appropriate solvent and loaded into a
syringe and injected. Headspace analysis, on the other hand, is
an indirect testing procedure. The analysis is conducted when a
volume of gas above the drug substance or formulation is collected
and analyzed by a gas chromatograph. Finally, solid-phase microextraction (SPME) is making much progress in recent years for
residual solvent testing. In SPME, a silica fiber coated with a sorbent
is used to collect and concentrate the volatile solvents. The volatiles
are then thermally desorbed in the inlet of the gas chromatograph
and analyzed18
Many alternatives to gas chromatography have been used to
determine the level of residual solvent in pharmaceutical products.
Many of these procedures are either nonspecific—that is, the
solvents are not identified—or they have high detection limits, so
they are inappropriate for the detailed product characterization
required for a regulatory submission. The oldest and simplest
method for determining the quantity of volatile residue is measuring

the weight loss of a sample during heating. LOD method is widely
used, particularly for Class 3 solvents, due to its simplicity and
ease of introduction into even the most basic analytical laboratory5
Another approach is to use thermogravimetric analysis (TGA),
which is a well-known method for the quantitative analysis of the
loss of volatile components from a sample18. Spectroscopic and
spectrometric methods have generally lacked the low detection
limits needed for toxic residual solvents, although the detection limits
would be applicable for ICH class 2 and 3 solvents. In the case of
Infrared Spectroscopy (IR), a detection limit above 100 ppm and
lack of accuracy at low concentrations of residual solvent has been
reported. For NMR also high detection limit has been reported5
Whenever organic solvents are used in the production of
pharmaceutical products, especially in the last processing steps,
the content of residual solvent in the final product should be
analyzed. The complete removal of residual level of these solvents
is impracticable and traces always remain in the final products.
The presence of these residual solvents even in small amounts
has a negative influence not only on the quality of products but
also on human health. Acceptability of residual solvents seems to
be best judged following the ICH residual solvent guideline which
is adopted by the USP, EP and JP; it classifies the solvent into
four groups. In class 1 are included the most toxic solvents which,
unless strongly justified, should be avoided. For the toxic solvents
of class 2, the limits are expressed as concentrations (ppm) and
additionally in the case of known daily drug intake, by the very
important ‘permitted daily exposure’ (PDE). The class 3 includes
the solvents with low toxic potential for which the general limit is
set at 0.5%. The class 4 includes solvents for which no adequate
toxicological data was found.

1. Michulec M., Wardenki, W.; Development of headspace solid-
phase micro-extraction-gas chromatography method for the
determination of solvent residues in edible oils and pharmaceuticals,
J. Chromatogr, 2005; 1071: 119-124.
2. Dwivedi A. M., Residual solvent analysis in pharmaceuticals.
Pharmaceutical Technology 2002; 42-46.
3. Camarasu C., Unknown residual solvents-identification in
drug products by headspace solid phase microextraction gas
chromatography and mass spectroscopy, Chromatographia 2002;
56: S131-S135.
4. Rocheleau M J., Measuring residual solvents in pharmaceutical
samples using fast gas chromatography techniques, J. Chromatogr.
B 2004; 805: 77-86.
5. B’Hymer C., Residual solvent testing: A review of gas chromatographic
and alternative techniques, Pharm. Res. 2003; 20, 337-343.
6. Otero, R., Carrera, G., Static headspace gas chromatographic
method for quantitative determination of residual solvents
in pharmaceutical drug substances according to European
pharmacopoeia requirements, J. Chromatogr. A 2004; 1057: 193-
7. ICH Q3(C), Impurities: residual solvents, 1997.
8. Countrymen, S. Understanding the revision to USP monograph 467;
residual solvents, phenomenex Inc. Torrance, CA, USA, 2007.
9. General chapters 466; «Ordinary impurities» and 1086, «Impurities
in official articles,» in USP 28–NF 23. US Pharmacopoeia. 12601
Twin brook Parkway, Rockville, Maryland 20852, USA, 2004.
10. European pharmacopoeia, Identification and control of residual
solvents (2.4.24), directorate for the quality of medicines of the

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We are providing here details regarding how to write a standard operating procedure SOP for a WHO GMP Pharmaceutical Manufacturing unit. I am giving here a example SOP which will give you a exact idea ,so that you can write SOP of your company your self. I am keeping an example of a microbiology department for this purpose.


SOP No001
Page 2 of 4
Version: 1.4

Date: 02/09/20011

Revise by: 02/09/2014
Written by:M  S DHONI
Revised by : MS DHONI
Authorised by:MS DHONI
This is a guide to the format & writing content for including in a Standard Operating Procedures prepared in Microbiology & Biotechnology Departments.
Use clear, simple, direct wording in short sentences
Write procedures as chronological sequences
Use ‘shall’ or ‘must’ for mandatory actions and ‘should’ for advisory actions
Procedures should reflect current practice
If any section is not applicable to the procedure, include “N/A” under the heading –DO NOT leave blank.The final SOP should consist, as far as possible, of one single electronic master file.
A Standard Operating Procedure begins with an Introduction and does not include a preamble.
1. Introduction: (what the intention of SOP )
This introduction part provides background information on the procedure given in the
SOP. This may include the reason for carrying out a certain task, a brief explanation of the
interactions or theory behind a particular procedure or tast or process, and when it may be appropriate to conduct the procedure described.
Eg: The introduction for a SOP on ‘Protein Electrophoresis’:
This SOP discusses the procedure and safety guidelines for electrophoresis of Proteins.
When an electric charge is applied to an agarose gel, Proteins migrates through the gel matrix at a rate inversely proportional to the log10 of the number of amino acids and (if it is a DNA then it is number Bases).and depending up on the moleculer structure ,Super-helical, nicked circular, and linear DNA migrate at different rates relative to each other, and the relative mobility varies depending on many factors. DNA is visualised by the addition of a dye that intercalates between the stacked base pairs of the DNA molecule. Upon exposure to light of a specific wavelength, DNA-dye complexes emit fluorescent or luminescent light. Traditionally ethidium bromide dye has been used to visualise DNA. However, ethidium bromide is a strong mutagen and is being replaced by new non-mutagenic dyes with similar properties. SYBR Safe, a non-toxic dye, should now be used in place of ethidium bromide in this laboratory.
2. Scope(where this SOP applied)

This section states the circumstances under which the SOP is applied. Mention should be made of the extent or limitations of the SOP. If applicable, refer to associated regulatory or legislative information related to the work area or procedure described in the SOP.
Eg 1: This procedure applies to all staff in the Department of Microbiology .and to any visitors working in the Department.
Eg 2: This SOP sets out procedures for the cleaning, disinfection and sterilization of instruments and equipment, and maintenance of associated environments in a research laboratory. It may be suitable for application to instruments and equipment use in a veterinary practice. It does not apply to items intended by the manufacturer for single use only, nor to items that may be contaminated with unconventional infective agents, eg. Creutzfeldt.
3. Safety
This section must include all warnings of safety risks associated with performing the
procedure. These include but are not restricted to:
· Any general precautions or issues that need to be taken into consideration.
· Chemical Hazards: obtaining and reading of any relevant MSDSs, general or special
storage conditions; licences and permits required.
· Physical Hazards: RSI issues, use of trolleys, carrying of materials/loads, etc.
· Radiation Hazards: monitoring, wearing of badges, details of shielding required.
· Biological hazards: use of Class II cabinets; aerosol production;
requirements as determined by who guidelines and expanded for OGTR certified and QAP accredited laboratories.
· Use of PPE and any extra items that are identified as needed for the procedure.
· Accidents and spills: clearly stated instructions as to the steps that need to be taken,
precautions required and the correct method of “mopping” up and
disposal. First-aid procedure if applicable. Reporting procedure to supervisor and/or
Department Safety Officer, using Incident Reports .
4. Licences and PermitsThis section details all permits and licences that must be obtained before the procedure is
carried out. All permits and licences must be valid. Conditions required to comply with the
permits and licences should be noted on the SOP.
Permits and Licences may include:
Quarantine Approved Premises Certifications,licence for work and certification for facility, Poisons Licence and Poisons Control Plan, Dangerous Goods notification, who guidelines , etc
5. Training and Competency
· List all training needs required for the SOP and pre-requisites (ie. training in other
procedures or items of equipment, knowledge of other SOPs) required for this procedure.
Eg 1: Subculture of Microorganisms (non-pathogenic): The trainee must have already mastered an understanding of and have been given instruction in the use of asceptic technique.
Eg 2: Use of Departmental FACS Machine: Users are required to be registered and must undergo training by the FACS Facility Manager to obtain registration.
· Records must be kept of the personnel trained against this procedure. State where training records are kept.
· The trainee must be assessed to determine if they are competent to perform the
procedure. Competency may be assessed by close observation of the trainee by an
approved trainer along with either a verbal exam or completion of a written assessment
by the trainee. The method by which competency is determined should be stated in this
section, eg. by observation, written quiz, oral examination, continuous record of correct
results, etc.
6. Risk Assessments
Risk assessments must be performed on any procedure that has an element of hazard.
These assessments must be carried out by a staff member that has been trained in Risk Management (courses run by Faculty EHS Officer). Risk assessments should be kept as part of a Work Group Risk Register. Any assessments associated with a SOP should be referenced in the Risk Assessments section of the SOP, and should be attached to a hardcopy of the SOP document. If the SOP is to be stored as an electronic file only, then the risk assessments should be attached as part of the electronic copy of the SOP.
7. Equipment and Maintenance / Handling and Storage / LabellingIdentify any special equipment used, describe location of equipment, location of instruction
manuals and any particular cleaning strategies. Service and maintenance details may also be
included here. Alternatively, use this place to describe the storage and handling of chemicals/ biologicals, etc. State any special handling and transport procedures.
Eg: Transporting a gel around department requires microbiological gel to be placed in a sealed container and one glove removed to avoid contaminating doors and lifts.
Give details of any specific labelling required. A template or picture of the label can be
8. Operating Procedures
The operating instructions or methodology must be written in a recipe-like manner – an
ordered list of clear, concise instructions or action steps.
If applicable include:
· Location where procedure will be done (eg. lab, bench, Biosafety cabinet, fume hood).
· Labelling requirements at the steps where the need arises.
· Troubleshooting information.
· Clear instructions on cleaning and/or decontaminating work area, equipment and other
materials used in the procedure.
· State any routine maintenance requirements.
9. Controls and Calibrations
Any internal or external quality control procedures or issues should be documented here.
Provide any calibrations required for specific items (pH meter, micropipettes). Routine steps
should also be included at the appropriate step in Section 8.
Waste DisposalState specific waste disposal procedures for all items in the SOP. Include type of waste
containers to be used and any special transport or labelling requirements.
11. Relevant Documents / ReferencesRelevant external documents and procedures may apply to the SOP. Examples: WHO Guidelines,website, manufacturer’s operating instructions, scientific literature.
List complete details of any external documents or websites relevant to or required for the
SOP. Risk assessments applicable to the SOP are listed in Section 6.
12. Signage / Summaries / TemplatesAny signs or summaries specifically relating to the SOP should be included here. This
includes simple step-by-step notices, warning signs, and templates for use in conjunction
with the SOP, eg. for recording data or carrying out audits. Include information such as the
number of copies of a sign or notice that needs to be printed, if these need to be laminated,
and where these need to be displayed in appropriate areas or on relevant equipment.
13. Appended MaterialIf the SOP is to be stored as a hardcopy with appended documents (which do not form part of
the electronic SOP file), then a detailed list of all appended material is to be recorded in this
section. This acts as a check in case this material is separated from the SOP, and aids in
assessing the complete SOP when revision is required.The entire SOP, including appended items, should be kept as one complete document.Document control details (ie. SOP number, version, date, etc) should be shown on all the pages of a SOP, including the pages which contain appended material.

Analytical Method Validation


1.0     Purpose     :   To lay down a procedure for Analytical Method Validation.
2.0     Objective   :  To provide documented procedure for Analytical Method Validation.
3.0     Scope        :   To define role/responsibility of various persons responsible for Analytical Method Validation.
4.0     Responsibility    :
·        Primary        :         Officer QA/ QC
·        Secondary    :         Manager QA/ QC
5.0      Procedure   :
·        General Concepts
Ø      Validation is the act of demonstrating and documenting a procedure that operates effectively.
Ø      The discussion of the validation of analytical procedures is directed to the four most common types of analytical procedure:
R           Identification tests
R           Quantitative tests for impurities content
R           Limit tests for the control of impurities
R           Quantitative tests of the active moiety in samples of drug substance or drug product or other selected components in the drug product.
Ø      Typical validation characteristics which should be considered are:
R           Accuracy
R           Precision
R           Specificity
R           Quantitation Limit
R           Linearity and Range
R           Robustness
·        Method Validation Parameter for the assay of —:
Ø      Linearity: —– to be analyzed as per proposed method. The results obtain is used to statistically evaluate for coefficient of determination (r2), standard error of estimate and y intercept.
Ø      Precision: Precision of the chemical method is ascertained by carrying out the analysis as per the procedure and as per normal weight taken for analysis. Repeat the analysis five times. Calculate the % assay, mean assay, % Deviation and % relative standard deviation and %RSD.
Ø      Accuracy: Accuracy of the method is ascertained by standard addition method at 3 levels. Standard quantity equivalent to 80%, 100% and 120% is to be added in sample.
·        Method Validation Parameter for residual solvent by GC for —:
Ø      Specificity: Resolution of the analyte peak from the nearest peak: Solution of each of the analyte was injected separately and their retention time is noted. The standard working solution containing a mixture of the component being analyze is also injected and each of analyte peaks is check for its resolution from the nearest.
Ø      Precision:
R     Repeatability: Six replicate injections of standard solution for system precision should analyze as per the proposed method and from the chromatograms obtained the percentage % RSD is calculated.
R     Intermediate precision: The purpose of this test is to demonstrate the intermediate precision of the method when method is executed by a different analyst and on different day. Results obtained will be compared.
Ø      Linearity and Range: Solution of analyte solvent, having different concentration should make separate from L.O.Q. concentration, which is 50% to 150%. The result obtained is statistically evaluated for coefficient of determination (r2), standard error of estimate and y intercept.
Ø      LOD & LOQ:
R     The limit of Detection (L.O.D.) was calculated as per below equation:
                      LOD          =              3.3     X       SD
R     The limit of Quantification (L.O.Q.) was calculated as per below equation:
                                                LOQ         =              10      X     SD
Ø      Accuracy / % Recovery (By Standard Addition Method): Accuracy of the method was ascertained by standard addition method at 3 levels.
R     Standard solution quantity equivalent to 50%, 100% and 150% are added in sample.
R     The solutions amount is analyzed by the proposed method and chromatogram obtained.
R     The amount recover by the method is compared to the amount added. Percent deviation is calculated at each levels and a grand average across all the levels are also calculated.
Methanol standard concentration ––  3000 ppm
Acetic acid standard concentration –– 5000 ppm
DMF standard concentration ––          880  ppm
Ø      Robustness:
R     The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters.
R     If measurements are susceptible to variation in analytical conditions, the analytical condition should be suitably controlled or a precautionary statement should be included in the procedure.

R     One consequence of the robustness should be that a series of system suitability parameters (e.g. resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used.

Microbiology test of water ( IP,BP,USP)

Microbiology test of water ( IP,BP,USP)

Microbiological Test of Water is provided to determine compliance with the requirements given in individual monograph/specifications.
Microbial testing of water includes the estimation of the number of viable aerobic bacteria present in a given quality of water.
Phosphate Buffer pH 7.2
Stock Solution
Dissolve 34 g of monobasic Potassium phosphate in about 500 mL of water contained in a 1 L volumetric flask. Adjust to pH 7.2 ± 0.1 by the addition of 4 % w/v aqueous solution of Sodium hydroxide (about 175 mL), add water to volume, and mix. Dispense, sterilize and store under refrigeration.
For use, dilute the Stock solution with water in the ratio of 1 to 800, and sterilize in an autoclave at 121 OC for about 15 min.
Nutrient Agar Medium
Beef Extract 10.0 g
Peptone 10.0 g
Sodium Chloride 5.0 g
Agar 12.0 g
Water 1000 mL
Dissolve with the aid of heat. Adjust to pH 8.0 to 8.4 with 5 M Sodium Hydroxide and boil for 10 min. Filter , adjust to pH 7.2 to 7.4 and sterilise by maintaining at 115 OC for 30 min.Soyabean Casein Digest agar
Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Agar 12.0 g
Distilled Water 1000 mL
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after Sterilization between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121 OC for about 15min.
MacConkey’s broth
Peptone 20.0 g
sodium Chloride 5.0 g
Sodium Taurocholate 5.0 g
Lactose 10.0 g
Bromocresol purple 10.0 mg
Water 1000 mL
Dissolve the Peptone, the Sodium Chloride taurocholate in the water with the aid of heat. Adjust to pH 8.0 and boil for 20 min. Cool, filter and adjust to pH 7.4. Add the Lactose and the indicator solution, mix and distribute in tubes containing inverted Durham’s tubes. Sterilize by maintaining at 121 OC for 20 min
For double strength medium, use double the quantity of ingredients in the same amount of water.
All the above media should be incubated for 24 h at 37 OC before use. Any contaminated media should be discarded.
Instead of preparing media, use dehydrated media of Hi media can be used. Re hydrate the required quantity as per instructions on the bottle label, dispense in required quantities and sterilize at 15 psi at 121 OC for 20 min.
Perform the Total microbial Count as follows :
Assemble the membrane filtration unit by keeping 0.45 µm filter membrane of the holder of the unit and moisten the membrane with distilled water. Autoclave the entire unit at
121 OC for 15 min. and allow to cool. Transfer 100 mL of water sample to the unit and filter it under aseptic conditions, by applying vacuum.
Transfer the membrane intended for enumeration of total bacterial count onto the surface of nutrient agar OR Soyabean casein digest agar and incubate at 37 OC + 2 °C for 3 days. Repeat the filtration process with 100 mL sample. Observe the plates for development of colonies and report the results as Colony forming units (CFU) per 100 mL sample.
Perform the test for Total microbial count as follows :
1. Transfer aseptically 1 mL of the sample in each of two sterile Petri dishes.
2. Add to each dish approx. 20 mL of sterile nutrient agar / Soyabean casein digest agar previously cooled to about 45OC3. Cover the petridishes and mix the sample with the agar by rotating the dishes 3 times both in clockwise and anti-clockwise directions.
4. Allow the agar to solidify at room temperature.
5. Invert the petridishes and incubate them at 37 OC for 48 h.
6. After incubation, examine the plates for growth and count the number of colony forming units in each plate.
7. The average of both the readings is the total microbial count per mL.
Perform the Total microbial Count as follows .
1. Prepare 1/10 dilution of the water sample by transferring aseptically 10 mL of the sample to 90 mL sterile phosphate buffer pH 7.2
2. Transfer 1 mL from this to two sterile Petri dishes. Add sterile nutrient agar / Soyabean casein digest agar to them in the same manner as above and incubate at 37 OC for 3 days.
3. After incubation, note the number of colony forming units in each plate and calculate the average.
4. Multiply the average count by 10. The result is the Total microbial Count per ml
1. Perform the test for identification of Escherichia coli as per STP No. STP-046-00.
2. Perform the test for most probable number of coliforms as follows. Transfer aseptically 10 mL sample into each of five previously sterilized 10 mL double strength MacConkey’s broth tubes Similarly transfer 1 mL and 0.1 mL sample in 10 mL single strength MacConkey’s broth tube using 5 tubes for each sample quantity. Incubate all tubes at 37 OC for 48 h. and observe for gas production.
3 The number of positive findings of coliform group of organisms should be computed as the combination of positives and recorded in terms of most probable number (MPN) . The MPN for a variety of combinations isgiven in the Table I.
Keep adequate controls of Phosphate buffer pH 7.2, Nutrient agar and MacConkey Broth to confirm the validity of results.


Observe the sample by physical examination in a 100 ml Nessler’s cylinder. The sample shall be clear.
Check the odour of the sample. Report for any unusual odour.
Principle :
Color is determined by visual comparison of the sample with known concentrations of colored solutions.
Sampling :
Collect representative samples in clean glassware. Make the colour determination within a reasonable period because biological or physical changes occurring in storage may affect color. With naturally colored waters these, changes invariably lead to poor results.
Apparatus : Nessler tubes, matched, 50 mL, of tall form.
Preparation of Standards :
1. Dissolve 1.246 g of Potassium Chloroplatinate [K2PtCl2], [eq. of 500 mg metallic Pt.] and 1.00 g of crystallized cobaltous chloride [CoCl2 6H2O], [eq. to 250 mg of metallic Co] in distilled water with 100 mL conc. HCl and dilute to 1000 ml with distilled water.This stock standard has a colour of 500 units.
2. Prepare standards having colors of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, and 70, by diluting 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 4.5, 5.0, 6.0, and 7.0 mL stock color standard with distilled water to 50 mL in Nessler Tubes.
Protect these colour standards against evaporation and contamination when not in use.
a. Estimation of Intact Sample
Observe sample color by filling a matched nessler’s tube to the 50 mL mark with sample and comparing it with standards. Look vertically downward through tubes toward a white or specular surface placed at such an angle that light is reflected upward through the columns of liquid. If the turbidity is present and has not been removed, report as ‘apparent color’. If the color exceeds 70 units, dilute sample with distilled water to known proportions until the color is within the range of the standards.
b. pH
Measure pH of each sample
Calculation (in case sample has been diluted)
Calculate color units by the following equations :
Color Units = [Estimated Color of diluted sample x 50 ] / mL sample taken for Dilution.
Report color results in whole numbers and record as follows :
Color Units Record to Nearest
0 – 50 1
51 – 100 5
101 – 250 10
251 – 500 20
Report the sample pH.
4. pH
Instrument : The pH meter should be capable of reading with an accuracy of 0.05 pH Units.
Calibration with Standard Buffer solutions : Before sample pH measurement perform calibration of the instrument with pH 4.0 buffer and pH 9.0/9.2 buffer.
Sample measurement : Establish equilibrium between electrodes and sample by stirring sample to ensure homogenity ; Stir gently to minimize carbondioxide entrainment. Measure the sample pH at 25C.
Report pH value to the nearest 0.1 pH unit.
Principle : Ethylenediaminetetracetic acid and its sodium salts [EDTA ] form a chelated soluble complex when added to a solution of certain metal cations. If a small amount of a dye such as Eriochrome BlackT or Calmaginte is added to an aqueous solution containing calcium and magnesium ions at a pH of 10.0 ± 0.1, the solution becomes wine red.
If EDTA is added as a titrant the calcium and magnesium will be complexed, and when all of the magnesium and calcium has been complexed the solution turns from wine red to blue, marking the end point of the titration. Magnesium ion must be present to yield a satisfactory end point.
Titration Precautions
Conduct titration’s at or near normal room temperature. The color change becomes impractically slow as the sample approaches freezing temperature. Indicator decomposition becomes a problem in hot water. Completion to the titration within 5 min. minimizes the tendency for CaCO3 , to precipitate.
Reagents :
Buffer Solution :
1. Dissolve 16.9 g Ammonium Chloride [(NH4Cl)], in 143 mL, conc. ammonium hydroxide [(NH4OH)]. Add 1.25 g magnesium salt of EDTA[commercially available ] and dilute to 250 mL with distilled water.
2. If the magnesium salt of EDTA is unavailable, dissolve 1.179 g disodium salt of ethylenediaminetetraacetic acid dihydrate (analytical reagent grade) and 780 mg magnesium sulfate [(MgSO4.7H2O)] or 644 mg magnesium chloride [(MgCL2.6H2O)] in 50 mL distilled water. Add this solution to 16.9 g NH4Cl and 143 mL of conc. NH4OH with mixing and dilute to 250 mL with distilled water. To attain the highest accuracy, adjust to exact equivalence through appropriate addition of a small amount of EDTA or MgSO4 or MgCl2.
Store Solution 1. and 2. in a plastic or borosilicate glass container for no longer than 1 month. Stopper tightly to prevent loss of ammonia [Nh3 ]
Eriochrome Black T. : Sodium salt of 1-(1-hydroxy-2-naphthylazo)-5-nitro-2-naphthol-4-sulfonic acid; No.203 in the Color Index. Dissolve 0.5 g dye in 100 g 2,2’,2”-nitrilotriethanol (also called triethanolamine) or 2-methoxymethanol (also
called ethylene glycol mono methyl ether). Add 2 drops per 50 mL solution to be titrated. Adjust volume if necessary.
Standard EDTA Titrant, 0.01M : Weigh 3.723 g analytical reagent – grade disodium ethylenediaminetetraacetate dihydrate also called (ethylenedinitrilo) tetraacetic acid disodium salt (EDTA) , dissolve in distilled water, and dilute to 1000 mL , standardize against Standard Calcium Solution as described in method given below.
Standard Calcium Solution : Weigh 1.000 g anhydrous CaCO3 powder [primary standard or special reagent low in heavy metals, alkalis, and magnesium] into a 500 mL erlemeyer flask. Place a funnel in the flask neck and add, a little a time, 1+1 HCl until all CaCO3 has dissoved. Add 200 mL distilled water and boil for few minutes to expel CO2 . Cool, add a few drops of methyl red indicator, and adjust to the intermediate orange color by adding 3N NH4OH or 1+1HCl, as required. Transfer by quantitatively and dilute to 1000 mL with distilled water: 1 mL = 1.00 mg CaCO3.
Titration of Sample
Select a sample volume that requires less than 15 mL EDTA titrant and complete titration within 5 minutes measured from time of buffer addition.
Dilute 25.0 sample to about 50 mL with distilled water in a porcelain casserole or other suitable vessel. Add 1 to 2 mL buffer solution. Usually 1 mL will be sufficient to give a pH of 10.0 to 10.1. The absence of a sharp end-point color change in the titration usually means that an inhibitor must be added at this point or that the indicator has deteriorated.
Add 1 to 2 drops indicator solution or an appropriate amount of dry-powder indicator formulation. Add Standard EDTA titrant slowly, with continuous stirring, until the last reddish tinge disappears. Add the last few drops at 3- to 5-s intervals.
At the end point the solution normally is blue. Daylight or a daylight fluorescent lamp is recommended highly because ordinary incandescent lights tend to product a reddish tinge in the blue at the end point.
Calculation :
Hardness (EDTA) as mg CaCO3 / L =
[mL titration for sample x mg CaCO3 , equivalent to 1.00 m L EDTA titrant]
mL sample.
In a neutral or slightly alkaline solution, potassium chromate can indicate the end point of the silver nitrate titration of chloride. Silver chloride is precipitated quantitatively before red silver chromate is formed.
Apparatus :
– Conical flask, 250 mL
– Burette, 50 mL.
Reagents :
Potassium Chromate Indicator Solution : Dissolve 50 g K2CrO4 in a little distilled water. Add AgNO3 solution until a definite red precipitate is formed. Let stand 12 h, filter, and dilute to 1 L with distilled water.
Standard Silver Nitrate Titrant, 0.0141 M (0.0141N): Dissolve 2.395 g AgNO3 in distilled water and dilute t 1000 mL. Standardize against NaCl by the procedure of “Titration” described below. 1.00 mL = 500 µg of Cl-.
Special Reagents for Removal of Interference :
Aluminium Hydroxide Suspension : Dissolve 125 g aluminium potassium sulfate or aluminum ammonium sulfate, AlK(SO4)2.2H2O or AlNH4(SO4)2.12H2O, in 1 L distilled water. Warm to 60o C and add 55 mL conc. ammonium hydroxide (NH4OH) slowly with stirring. Let stand about 1h, transfer to a large bottle, and wash precipitate by successive additions, with thorough mixing and decanting with distilled water, until free from chloride . When freshly prepared, the suspension occupies a volume of approximately 1L.
Phenolphthalein Indicator Solution
Sodium Hydroxide, NaOH, 1N :
Sulfuric acid, H2SO4, 1N
Hydrogen Peroxide, H2O2, 30% :
Method :
Sample Preparation : Use a 100 ml sample or a suitable portion diluted to 100 mL. If the sample is highly colored, add 3 mL Al(OH)3 suspension, mix, let settle, and filter. If sulfide, sulfite, or thiosulfate is present, add 1 mL H2O2 and stir for 1 min.
Titration :Directly titrate samples in the pH range 7 to 10. Adjust sample pH to 10 with H2SO4 or NaOH if it is not in this range. For adjustment, preferably use a pH meter with a non-chloride-type reference electrode. (If only a clhloride-type electrode is available, determine amount of acid or alkali needed for adjustment discard this sample portion.
Treat a separate portion with required acid or alkali and continue analysis.) Add 1.0 mL indicator solution. Titrate with standard AgNO3 titrant to a pinkish yellow end point. Be consistent in end-point recognition.
Standardize AgNO3 titrant and establish reagent blank value by the titration method outlined above. A blank of 0.2 to 0.3 mL is usual.
Calculation :
mg Cl- / L =
(mL, titration for sample – mL titration for blank) x Normality of AgNO3 x 35.450
mL of sample
Principle :
A well-mixed sample is filtered through a standard glass fiber filter, and the filtrate is evaporated to dryness in a weighed dish and dried to constant weight at 105oC. The increase in dish weight represents the total dissolved solids.
Apparatus :
– Filtration apparatus : One of the following, suitable for the filter
disk selected.
– Membrane filter funnel.
– Gooch crucible, 25 mL to 40 mL capacity, with Gooch crucible
– Filtration apparatus with reservoir and coarse (40- to 60- µm)
– fritted disk as filter support.
– Suction flask, of sufficient capacity for sample size selected.
– Drying oven, for operation at 105 ± 2oC.
Method :
Preparation of glass-fiber filter disk : Insert disk with wrinkled side up into filtration apparatus. Apply vacuum and wash disk with three successive 20 mL
volumes of reagent-grade water. Continue suction to remove all traces of water. Discard washings.
Preparation of Evaporating Dish : If volatile solids are to be measured, ignite cleaned evaporating dish at 550o for 1 h. in a muffle furnace. If only total dissolved solids are to be measured, heat clean dish to 105 ± 2oC for 1 h in an oven . store in desiccator until needed. Weigh immediately before use.
Selection of Filter and Sample Sizes : Choose sample volume to yield between 10 and 200 mg dried residue. If more than 10 minutes are required to complete filtration, increase filter size or decrease sample volume. When very low total suspended solids are encountered (less than 10 mg/L), less dried residue may be collected; compensate by using a high-sensitivity balance (0.002 mg)
Sample Analysis :
Stir sample with a magnetic stirrer and pipette a measured volume onto a glass-fiber filter with applied vacuum. Wash with three successive 1 mL volumes of reagent-grade water, allowing complete drainage between washings, and continue suction for about 3 minutes after filtration is complete. Transfer total filtrate (with washings) to a weighed evaporating dish and evaporate to dryness on a steam bath or in a drying oven.
If necessary, add successive portions to the same dish after evaporation. Dry evaporated sample for at least 1 h. in an oven at 105 ± 2oC, cool in a desiccator to balance temperature, and weigh. Repeat drying cycle of drying, cooling, desiccating, and weighing until a constant weight is obtained or until weight change is less than 4% of previous weight or 0.5 mg, whichever is less. Duplicate determinations should agree within 5% of their average.
Calculation :
mg, total dissolved solids / L (ppm)=
{ [(Weight of dried residue + dish, mg) – Weight of dish, mg] x 1000 }
/sample volume mL
Determine total plate count and absence of pathogens as per the current version


1.0 AIM
To lay down a standard operating procedure for Decontamination of D.M. Water plant.
Maintenance Department
Service floor.
Operator/Maintenance Supervisor concerned.
Maintenance Incharge.
NOTE: Decontamination of D.M. Water plant is done as per recommendation of Q. C. department or once in a month; after regeneration of D.M. Water Plant.
6.1 Decontamination of Mixed Bed Unit
a) Mix 40 ml of formaldehyde solution with 200 ltr of D.M Water.
b) Inject this solution into mixed bed unit (MBU) by dipping tube attached to ejector E-2 into the formaldehyde solution and by opening valve 18, 20 & 22.
c) After completion of formalin injection close the valve 18, 20 & 22.
6.2 Decontamination of anionic exchanger
a) Mix 40 ml of formaldehyde solution with 200 ltr. D. M. Water.
b) Deep the tube attached to ejector into formaldehyde solution.
c) Open valve 4, 5, 6, 7 and adjsut valve 4 to maintain the flow.
d) After completion of injection close the valce 4, 5, 6 & 7
6.3 Decontamination of Cationic exchanger
a) Mix 40 ml of formaldehyde solution with 200 ltr. D.M. Water.
b) Deep the tube attached to ejector into formaldehyde solution.
c) Open valve 1, 2 & 3 and adjust the valve 2 to maintain the flow.
d) After completion of injection close the valce 1, 2, & 3.
6.4 Rinsing of columns
a) Next day start the D.M. Water plant (Ref. SOP-ME- ) and drain the water.
b) Give the sample of D. M. Water to Q.C.
c) After getting OK from Q. C. collect the D. M. Water into D. M. Water collection tank.
After 2 years or when procedure is changed

Genotoxic Impurities In Pharmaceuticals

Decisions to approve, prescribe and consume medicines involve risk/benefit assessments by regulatory agencies, health care professionals and consumers. For serious or life threatening conditions, drugs with higher risks for adverse effects or for serious adverse effects are sometimes acceptable. For example, some life-saving cancer chemotherapies are known human carcinogens. However, if one is suffering from a life threatening tumor, a 5% risk of a secondary, treatment-related tumor is generally considered acceptable. Arguably, the same is not true for impurities found in drug substances and drug products; impurities convey only risk with no associated benefit. Drug impurities might be viewed as “pollutants” in the pharmaceutical world. Much like pollutants in the environment, few people believe that they can be entirely eliminated. The challenge for regulatory agencies is to promulgate standards that assure that unavoidable drug impurities impart no or acceptable levels of risk.

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USP Defers Implementation of Elemental Impurities Provisions

USP Defers Implementation of Elemental Impurities Provisions
USP defers implementation date to work closely with ICH Q3D. USP will also form a new advisory group for implementation of the new general chapters on elemental impurities.
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FDA Issues Draft Guidance on Contract Manufacturing at (www.pharmtech.com)

The draft guidance describes how quality agreements can be used to delineate the responsibilities of contract manufacturers involved in the cGMP manufacture of APIs and finished drug products.

May 28, 2013
By: Patricia Van Arnum
FDA has issued draft guidance, Contract Manufacturing Arrangements for Drugs: Quality Agreements, which describes the agency’s current thinking on defining, establishing, and documenting the responsibilities of each party (or all parties) involved in contract cGMP manufacturing of drugs.