WHO Draft on Analytical Method Validation

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The World Health Organization (WHO) recently published a draft document on analytical method Validation for comment. Read more about the draft “Guidelines on Validation – Appendix 4 Analytical Method Validation“.,15438,Z-PDM_n.html

In June 2016 the World Health Organization (WHO) published a draft document “Guidelines on Validation – Appendix 4 Analytical Method Validation”. Comments on the text should be sent to WHO until July 30, 2016.

The appendix 4 of the published Supplementary guidelines on good manufacturing practices: validation (WHO Technical Report Series, No. 937, 2006, Annex 4) has been revised in view of current trends in validation. The appendix presents some information on the characteristics that should be considered during validation of analytical methods. Approaches other than those specified in the Appendix may be followed and may be acceptable.

The new Appendix 4 is structured as follows (New and revised):

1. Principle (revised):

  • 1.5 The recommendations as provided for in good laboratory practices and guidelines for transfer of technology (WHO Technical Report Series, No. 961, 2011, Annex 7) should be considered, where applicable, when analytical method validation is organized and planned.

2. General (revised):

  • 2.6 The procedure should become part of a continuous verification procedure to demonstrate that it meets the predefined criteria over the life of the procedure.
  • 2.7 Trend analysis and risk assessments should be considered at intervals to ensure that the method is appropriate for its intended application.
  • 2.8 Changes to methods should be managed in accordance with the authorized change control procedure.
  • 2.9 The scope of verification or degree of revalidation depend on the nature of the change(s) and the outcome of risk assessment.
  • 2.11 The data obtained during method validation and verification should be considered covered by good anything practices (GxP) requirements and are expected to follow the principles of good data and record management practices. Their associated metadata are also expected to be retained and subjected to good data and record management practices (WHO Technical Report Series, No. 996, 2016, Annex 5).
  • 2.12 When computerized systems are used to obtain and process data relating to method validation and verification, they should comply to the principles enunciated in Appendix 5 – Validation of computerized systems.
  • 2.13 Adequate attention should be paid to the method of sample preparation.
  • (…)

3. Pharmacopoeial methods
4. Non-pharmacopoeial methods
5. Method validation
6. Method verification (New):

  • 6.1 Method verification should be performed for already validated analytical methods, for example, when it is used on a product for the first time (e.g. in case of a change in API supplier, change in method of synthesis or after reformulation of a drug product).
  • 6.2 Method verification may include only the validation characteristics of relevance to the particular change.
  • (…)

7. Method revalidation (New):

  • 7.1 Methods should be maintained in a validated state over the life of the method. Revalidation (see also ICH Q2) should be considered whenever there are changes made to the analytical method (e.g. changes to mobile phase, column, column temperature, detector).
  • 7.2 In case of repeated SST failures or when obtaining of doubtful results. In such cases an investigation of the root cause should be performed, the appropriate changes made and the method revalidated.
  • 7.3 Periodic revalidation of analytical methods should be considered according to a period that is scientifically justifiable.
  • (…)

8. Method transfer  (New)

  • 8.1 During method transfer, documented evidence should be established to prove that a method has equivalent performance when used in a laboratory different from that where it has been originally validated.
  • (…)
  • 8.3 The two sets of results should be statistically compared and the differences between the two sets of test results should be within an acceptable range.
  • 8.4 Method transfer should be performed before testing of samples for obtaining critical data for a dossier, such as process validation or stability studies or applied for routine use.
  • (…)

9. Characteristics of analytical procedures (revised), 9.3 System suitability testing:

  • 9.3.1 The suitability of the entire system should be confirmed prior to and during method validation tests as well as during the test of samples.
  • 9.3.2 System suitability runs should include only established standards or reference materials of known concentration to provide an appropriate comparator for the potential variability of the instrument.
  • 9.3.3 Where a sample is used for system suitability or a trial run, written procedures should be established and followed and the results of all such trial runs be included in the results and data review process. A sample can be used only if it is a well characterized material. Characterization in such a case should be performed prior to the use of this sample as part of system suitability testing. The sample material or product under test should not be used for trial run purposes or to evaluate suitability of the system (see WHO guidelines on good data and record management practices).

The revised version of appendix 4 parallels certain considerations of the current USP lifecycle approach for analytical method validation. However, QbD concepts and the Analytical Target Profile (ATP) – which is equivalent to the Quality Target Product Profile (QTPP) – have not yet been introduced in the WHO draft.

According to WHO the draft of Appendix 4 will also be placed on the WHO Medicines website under “Current projects“.

Members of the ECA Academy are able to access the new WHO Guidelines on Validation – Appendix 4 Analytical Method Validation in the ECA Members Area.


////////WHO Draft, Analytical Method Validation

USP Chapter Visual Inspection of Injections published

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The long-awaited USP Chapter <1790> regarding the 100% visual control of injections has been issued in the Pharmacopeial Forum 41(1) for commenting. Read on.–1790–Visual-Inspection-of-Injections-published_40007,9087,9200,Z-PEM_n.html

The long-awaited USP Chapter <1790> regarding the 100% visual control of injectables has now been issued as a first draft in the Pharmacopeial Forum 41(1) for commenting.

The new chapter is comprised of the following sub-chapters:
1. Scope
2. Introduction
3. Typical Inspection Process Flow
4. Inspection Life-Cycle
5. Interpretation of Results
6. Inspection Methods and Technologies
7. Qualification and Validation of Inspection Processes
8. Conclusions and Recommendations
9. References

This new informative chapter is applied to the manual, the half-automatic and the fully-automated inspection of parenterals. It mainly aims at controlling particles (>50 µm), but also comprises indications to further defects like cracks in primary containers or poorly fitting stoppers. In Chapter 2 there are also general statements regarding the patient risk due to particulate matter with regards to the size and type of the particulate impurity and the patient’s condition or age. It is interesting that this is expanded in Chapter 4 where possible particle sources (stopper, glass, silicon etc.) are mentioned together with the request to prevent any generation of particles. The Sub-chapter 4.2.1 aims at avoiding of intrinsic particles already in product development – e.g. through the prevention of glass delamination, by choosing appropriate formulations and according stability studies. Supplementary, Chapter 4.3 is dedicated the removal of particles, e.g. by washing primary containers and the associated particle depletion studies.

As already described in the USP Chapter <790> the AQL testing is supposed to be part of the evaluation of a batch. For that purpose samples are drawn from the good proportion of the tested batch according to defined sampling plans. These samples are then tested again to evaluate the quality of the preceeding 100% control. The AQL limits named exemplarily in Chapter <17990> are more strict, though, as those in the ECA Best Practice Paper for the visual control.

Some practical tips are contained in Chapter 5. For instance, it is suggested there to enhance the illumination to 10.000 Lux and to possibly screen the containers from the back when testing brown glass or plastic containers as a visual control for these containers is difficult to conduct.

Chapter 7 (Qualification/Validation of inspection processes) is mainly directed towards the manual visual inspection. It comprises tips for the creation of test sets and the qualification as well as the re-qualification of personnel. The application of Knapp tests for determining the detection rates is also mentioned there. However, there are only very few tips for the fully-automated inspection, and there are no details referring to the qualification or re-qualification of fully-automated inspection processes.

The draft of the new Chapter <1790> is available online on the USP website. You will only need to register, which is free of charge, though. The deadline for comments is the 31 March 2015.

EU Commission issues new EU GMP Guide Chapter 6

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EU Commission issues new EU GMP Guide Chapter 6
In early April 2014, the EU Commission has published a new Chapter 6 Quality Control. The new chapter will become effective on 1 October 2014. Read more in the GMP news to the new EU GMP chapter quality control.



EU Commission issues new EU GMP Guide Chapter 6

In early April 2014, the EU Commission has published a new chapter 6 Quality Control. The new chapter will become effective on 1 October 2014. The main reasons for the changes (as e.g. the insertion of a new chapter on transfer of analytical methods) were already subject of our news Revision of the EU GMP Guide: EU Commission Proposal for Chapter 6 – Quality Control).

The now published document comprises different additions. For instance, the following was added in 6.5. Good Quality Control Laboratory Practices: “laboratory equipment should not be routinely moved between high risk areas to avoid accidental cross-contamination. In particular, the microbiological laboratory should be arranged so as to minimize risk of cross-contamination.”

According to 6.9 now all Out of Trend (OOT) and all Out of Specification (OOS) results have to be considered and need to undergo an investigation. Also, in 6.12. a new requirement asks for a sampling plan based on risk assessment. Further content supplements (in addition to others) deal with the subject of reference standards. The newly inserted text under 6.20 states “… their qualification and certification as such should be clearly stated and documented. Whenever compendial reference standards from an officially recognised source exist, these should preferably be used as primary reference standards unless fully justified (the use of secondary standards is permitted once their traceability to primary standards has been demonstrated and is documented). These compendial materials should be used”.

The regulations with regard to “Technical Transfer of Testing Methods” starting with point 6.37 up to 6.41 are completely new.

All attendees of ECA courses and conferences can download a document comparison between the current chapter 6 and the new version in the ECA website members’ area. In this comparison all changes and additions are marked. Otherwise please also see the new Chapter 6 Quality Control for more information.


Risk Assessment of Potentially Genotoxic Impurities within the Framework of Quality by Design

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A strategy for the risk assessment of potentially genotoxic impurities is described that utilizes Quality by Design in an effort to furnish greater process and analytical understanding, ultimately leading to a determination of impurity criticality. By identifying the risks and parameters that most influence those risks, an enhancement of both product and process control is attained that mitigates the potential impact of these impurities. This approach calls for the use of toxicological testing where necessary, chemical fate arguments when possible, multivariate analyses to develop design space, and use of spiking data to support specifications. Strong analytical support, especially with the development of low-level detection methods, is critical. We believe that this strategy not only aids in the development of a robust API process but also delivers on the identification and subsequent mitigation of risks to a class of impurities that are of high interest in the field.

Risk Assessment of Potentially Genotoxic Impurities within the Framework of Quality by Design

Adam R. Looker, Michael P. Ryan, Bobbianna J. Neubert-Langille and Redouan Naji
Org. Process Res. Dev., 2010, 14 (4), pp 1032–1036
pp 1032–1036
Publication Date (Web): April 7, 2010 (Communication)
DOI: 10.1021/op900338g

Risk Assessment of Genotoxic Impurities in Marketed Compounds Administered over a Short-Term Duration: Applications to Oncology Products and Implications for Impurity Control Limits

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Controlling impurities during drug development improves product quality and minimizes safety risks to the patient. Recent regulatory guidance on genotoxic impurities (GTIs) state that identified GTIs are unusually toxic and require lower reporting, identification, and qualification limits than outlined in the International Conference on Harmonization (ICH) guideline “Impurities in New Drug Substances Q3A(R2).” [ ICH Harmonized Tripartite Guideline: Impurities in New Drug Substances (Q3A), (R2); International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), 2006.] Patient safety is always the underlying focus, but the overall impurity control strategy is also driven by appropriate “as low as reasonably practicable” (ALARP)(2)procedures that include assessment of process capability and associated analytical techniques. In combination with ALARP, safe and appropriate GTI levels are currently identified using chronic toxicology-based limits calculated under the standard assumption of 70-years for exposure duration. This paper proposes a risk assessment approach for developing GTI limits based on shorter-term exposure durations by highlighting marketed anticancer compounds with limited dosing schedules (e.g., 2 years). These limits are generally higher than the defaulted threshold of toxicological concern (TTC of 1.5 μg/day) and can result in more easily developed and less complex analytical methods. The described approach does not compromise safety and can potentially speed life-saving medicines to patients.

Org. Process Res. Dev., 2010, 14 (4), pp 986–992
Publication Date (Web): June 7, 2010 (Concept Article)
DOI: 10.1021/op1000226


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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.

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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.
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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
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solvents (2.4.24), directorate for the quality of medicines of the

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A down-scaled fluorimetric determination of the solubility properties of drugs to minimize waste generation

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A miniaturized fluorescence assay on multi-well plates has been developed to study the solubility enhancement effect of (2-hydroxypropyl)-β-cyclodextrin on three anti-tumor alkaloids. The measurement of the fluorescence emission on a multi-well plate format has been proved to be a rapid and efficient technique to evaluate the solubility of pharmaceutical formulations of new drugs that help save time, reagents and wastes in the search for greener analytical strategies.

The proposed methodology was compared with a reference HPLC solubility study and was employed to examine the enhancement of the solubility of camptothecin, luotonin A, and a synthetic derivative of the latter in the presence of (2-hydroxypropyl)-β-cyclodextrin.

Considerable reductions in the time of analysis (almost 50 times faster) and the volume of organic solvents employed (close to 25 times less acetonitrile needed) were achieved. The nature of the inclusion complexes was investigated by analysis of the phase-solubility diagrams obtained by the newly developed method and was complemented with spectrofluorimetry and ESI-MS experiments.

The concentrations of solubilised compounds found by both methodologies were in good agreement (R2 > 0.98). The analytical figures of merit of both methodologies were compared and the adequacy of the proposed method for the development of drug solubilisation studies was discussed.


Green Chem., 2013, 15,2558-2565
DOI: 10.1039/C3GC40974K, Paper
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Corresponding authors
S. D. Química Analítica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal, s/n, 28040-Madrid, Spain
E-mail: ;
Fax: +34 91 394 17 54 ;
Tel: +34 91 394 17 56

A down-scaled high-throughput screening fluorescence assay for solubility studies achieves a 25-times reduction of organic solvent waste.