Commentary
For reprint orders, please contact [email protected]
Manual chromatographic baseline integration: is it needed, if so when should it be used? “
…when is it appropriate to reject the ‘integrator’ results? If reintegration is necessary, how do you set objective and consistent procedures to minimize bias at the best and fraud at the worst?
”
Keywords: acceptance criteria • consistency • objectivity • regulatory issues • reintegration
Howard M Hill*,1, David Bakes2 & Iain Love2
Scope & overview The objective of this article is to discuss the prevailing opinions on whether chromatographic baselines should be determined by setting the “integrator” (integration algorithm) parameters and accepting this data only or when there are situations where the integrator does not get it right and further evaluation is necessary. The issues arise largely in the second case. When is the data not acceptable, that is, when is it appropriate to reject the ‘integrator’ results? If reintegration is necessary, how do you set objective and consistent procedures to minimize bias at the best and fraud at the worst? While this scenario is driven largely by the measurement of drugs(usually small molecules) in biological fluids for the purposes of defining the pharmacokinetics, there are a growing number of scenarios discussed here, where this is not always the case.
Independent Pharma Consultant, Harrogate, North Yorkshire, UK 2 Huntingdon Life Sciences, Woolley Road, Alconbury, Cambridgeshire, PE28 4HS, UK *Author for correspondence: [email protected]
Regulatory situation The two key publications relevant to bioanalysis are the US FDA [1] and EMA [2] guidance documents on bioanalytical method validation. While the FDA [3] have recently produced an updated draft guidance on this topic, it is not intended to refer to this document in this article. In the coming months it is likely it will be subject to much discussion and change before a definitive version is produced. Subjective interpretation of the current guidance documents is required, as neither offers prescriptive instruction on the subject of peak integration. The FDA Guidance [1] states on page 15 that, “Sample data reintegration: An SOP or guideline for sam-
10.4155/BIO.14.68 © 2014 Future Science Ltd
ple data reintegration should be established. This SOP or guideline should explain the reasons for reintegration and how the reintegration is to be performed. The rationale for reintegration should be clearly described and documented. Original and Reintegration Data should be reported.” “Documentation for reintegrated data: Documentation should include the initial and repeat results, the method used for reintegration, the reported result, assay run identification, the reason for the reintegration, the requestor of the reintegration and the manager authorizing reintegration. Reintegration of a clinical or preclinical sample should be performed only under a predefined SOP.” The EMA document [2] (page 13) is little more succinct or open to interpretation, depending upon your view point, “Chromatogram integration and reintegration should be described in an SOP. Any deviation from this SOP should be discussed in the analytical report. Chromatogram integration parameters and in the case of reintegration, initial and final integration data should be documented at the laboratory and should be available on request.” For further guidance reference is made to the “Reflection paper for Laboratories That Perform The Analysis or Evaluation of Clinical Trial Samples” (EMA/INS/GCP/532137/2010). This document discusses data recording issues and procedures not immediately related to baseline integration. It is evident that both authorities endorse the need for reintegration and both emphasize the need for clear criteria to be applied and a transparent audit trail of who authorized what to be provided in the study
Bioanalysis (2014) 6(9), 1171–1174
1
part of
ISSN 1757-6180
1171
Commentary Hill, Bakes & Love documentation. While not legally binding to bioanalysts, the pharmacopeias, particularly the US Pharmacopiea (USP) [4] provide guidance on what criteria should be assessed in defining good chromatography. However, they do not provide a great deal of guidance on integration issues nor does the FDA related document from 1994 [5] which has been cited many times where chromatographic methods are used. The current situation The decision as to where to draw a baseline on a chromatographic peak, and thereby determine its peak height or area used to construct a calibration curve and related data, has a pivotal impact on an analytical method. Approximately 20 years ago the publications of Dyson [6] and Meyer [7] discussed in some detail the impact on the accuracy of peak ratio measurements caused by ‘inconsistent’ peak shapes. These documents are as relevant today as they were when first published. In 2002, Chromatographia published two papers on this topic, one paper discussed the issues associated with ‘drawing a line’ [8] while another discussed the impact of 21CFR11 [9] on the recording of chromatographic raw data. More recently the Global Bioanalysis Consortium [10] provided some thoughts on ‘Chromatographic Run Quality Assessment’. This was presented by McDougall at the European Bioanalytical Forum(EBF) Consortium meeting in June 2012 (the consortium had over 20 authors representing the European pharmaceutical industry). The presentation also provided a more recent review/collation of relevant publications since circa 2002. Pragmatically the presentation presents the points to consider leaving it to individual laboratories to justify their approach. Nevertheless the issue remains! ‘To reintegrate or not’, and if so what is bad chromatography and what criteria are to be used for reintegration?
“The decision as to where to draw a baseline … has a pivotal impact on an analytical method.” The establishment of methods with “clean” symmetrical peaks has been assisted by the development of LC–MS – including better chromatographic separations and more selectivity with respect to detector technology. In such a case the use of automated systems would seem to be clear. However, with the need to develop methods capable of measuring metabolites/ degradants in the same chromatograms, and the need to seek more and more sensitivity, where the peak shape may not be symmetrical then the situation may not be so clear cut. Increasingly, the need to measure macromolecules in biological fluids by chromatographic techniques can complicate or compromise the ability
1172
Bioanalysis (2014) 6(9)
of automated integration algorithms to produce consistent integration of the non-Gaussian peak shapes often encountered in this field. Moreover, recent techniques developed to characterize and quantify proteins by measuring “multiple peptide” peaks, post hydrolysis, highlight that imposition of a one size fits all scenario is not necessarily realistic. Baseline assignment Data sampling and integration criteria have been discussed in much detail by Dyson [6] and Myers [7] and have been reiterated in the article by Hill et al. [8] and in the GBC presentation by McDougall [10] . McDougall presents several succinct quotes by Dyson, which are relevant to this ongoing debate, “It should be clear now that integrators are like any other tool – an excellent thing in the right hands. What they do best is measure peaks which are suitable for measuring rapidly and without tedium. If all these measurements are worth making then all subsequent calculations are worth noting and perhaps acting upon. As long as integrators use perpendiculars and tangents and draw straight lines, they are of use only in controlled circumstances when chromatography is good. Even then the use of integrators requires vigilance from the operator and skill in assessing and assigning parameters. Integrators cannot improve bad chromatography only analysts can do that and at the end of the day that’s what they are paid for.” The conclusion would appear to be that if you can set the right integration parameters correctly then reintegration should not be necessary. There are however two caveats. Firstly the parameters should be set and validated with as much consideration as is given to developing the chromatographic conditions. Setting default conditions is inappropriate,-to cite McDougall’s presentation, “it is scientifically invalid to assume chromatographic integration parameters must be fixed.” Secondly, chromatography may not be perfect but it may be acceptable, indeed it may be the best that can be achieved, bearing in mind the nature of the matrix, the chemistry of the molecule and complexity of the chromatogram. It is therefore essential that typical examples of the ‘accepted’ chromatography be included as part of the analytical methods documentation to illustrate this. Data sampling This is a widely discussed issue, in publications at least, but is not often addressed in labs where default positions are routinely used. It can be, and in many cases is, a major contributor to data variability [6] and the key parameters should be considered with equal weight as developing the chromatographic conditions. These are sampling frequency, smoothing and filtering. Filtering
future science group
Manual chromatographic baseline integration: is it needed, if so when should it be used?
is a real-time process where noise is suppressed by electronic means as such the process is irreversible unlike smoothing which takes place post data collection. Sample frequency can be pivotal to the data collection process – rate of collection is governed by baseline noise and peak width. In general a minimum of ten slices are recommended [7] , although taking into account variations in peak shape 20–25 slices per peak has become a generally accepted ‘safer’ option. It is important to note that peak width can be influenced by a number of factors, not least of which is concentration. Setting too low a collection rate can result in loss of peak height while too much data may stress the capacity of the data system. As with all things, getting the balance right is essential. It is important that the interaction of data collection frequency, filtering, smoothing and the setting of integration parameters be evaluated for each method and be continually assessed thoughout the life of the method. Peak height versus peak area Peak area seems to be the common default measurement, indeed the USP [5] in chapter 621 acknowledge that peak areas are the most widely used option. The argument for choosing either peak height or area can vary from the scientific [6] to the pragmatic. Detectors have been divided into mass flow detectors and flow sensitive detectors which justify whether peak area or height should be used, respectively. The latter do not consume the analyte and as such can be used in a hyphenated series. Thus if a UV detector is used, ideally peak height should be chosen to quantify the analyte as it will be little changed by flow rate variability or interferences to the leading and or trailing edges. In general however peak area is used, this is especially so for LC–MS. Nevertheless there may be occasions where problems such as poor peak shape and/or interference and so on may be resolved by using peak height. To reintegrate or not? While it seems that the pragmatic answer is yes Garofolo et al. [11] in a White Paper concluded that, “generally speaking it is accepted by the industry and the regulatory authorities that integration done automatically is preferred but it may sometimes be appropriate to perform manual integration. It was argued that manual integration is generally acceptable as long as it is done before the results are regressed, is justified and documented in a controlled fashion and that good scientific judgment has been used.” This means that criteria for reintegration must be determined and defined a priori. Terminology used when discussing integration varies between workers. The words may be the same but the definitions
future science group
Commentary
may differ. It is essential to develop a consensus with respect to definitions. This is essential in order to drive objective and consistent integration, to satisfy the requirements of good science and moreover, the regulatory authorities. For example, manual integration is often considered to be the subjective practice of a point-and-click action to define the start and end of the “peak”. However, manual integration may also be considered to be the update of an algorithm parameter by the end user to facilitate more consistent integration with respect to analytical background. The former procedure is subjective, may be difficult to reproduce during investigation and require significant justification, whereas the latter is often considered to be more acceptable, although still open to manipulation. This is especially so when multiple attempts are made on the same chromatogram. Equally as important, the term reintegration must be adequately defined. For example, it may be considered that the update of integration parameters is different, depending upon whether regression analysis has been carried out. Thus, ‘integration modification’ may be considered the practice of correcting an inconsistent integration generated by a set of best-fit parameters in the absence of regression data while ‘reintegration’ is the practice of integration of the peak post-regression. In this instance, integration modification may be considered pragmatic and acceptable whereas reintegration may require extra justification and discussion during the reporting phase. If the definition of the terminology used around peak integration is incorporated into SOPs, the discussion between peers, authorizers, auditors and report readers becomes transparent and non-ambiguous. As an additional tool to ensure consistency a compendium of examples of ‘suspect’ chromatography is desirable, be they industry-wide or company-specific, however, consistent application can only be achieved by thorough training and management oversight. Where to next? The primary question to be addressed is whether to modify integration/reintegrate or not. If reintegration is deemed acceptable, how do you decide which samples should be reintegrated and how do you ensure consistency of reintegration? It is essential that the analyst understands the parameters required to assign an appropriate baseline-how do you define what is a good/acceptable baseline? If manual baseline assignment for ‘difficult and distorted’ chromatograms but best peak scenarios is acceptable, it is essential to objectively identify and define these scenarios. Similarly objective and consistent scenarios for carrying out manual baselines should be defined and qualified.
www.future-science.com
1173
Commentary Hill, Bakes & Love Above all it is essential that science reigns over dogma, which may tie the hands of the scientists into developing ‘ingenious’ contortions in order to circumvent the unusual and intractable. Pragmatism does have a place in science but only if objectively and consistently applied. It is a not a substitute for expediency and sloppy science. The current regulatory guidelines provide for objectivity and consistency. However, more and more laboratories are retreating down the road of ‘what the computer says’ is right! It would seem essential therefore for the industry to develop a consistent approach to these problems which reflect a wide variety of scenarios, not just a single symmetrical peak capable of been quantified even at the highest levels of sensitivity. This article attempts to provide an overview of the issues involved in reintegrating chromatographic peaks. The ‘Ask the experts’ article appearing in this issue may References
1174
either emphasize the diversity of opinions or provide an insight into developing an industry consensus towards reintegration that can also be embraced by the regulators. It is hoped that at least it will lead to agreement that reintegration is necessary and that when carried out according to well-developed guidelines, be they industry- or company-specific, it can be regarded as an acceptable procedure. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. 7
Meyer VR. Error sources in the determination of chromatographic methods peak ratios. In: Advances in Chromatography. Brown PR, Grushka E (Eds). Marcel Dekker, New York, NY, USA, 383 (1995).
1
US FDA, Center for Drug Evaluation and Veterinary Medicine. Guidance for Industry, Bioanalytical Methods Validation. FDA, Rockville, MD, USA (2001).
8
2
European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Bioanalytical Methods Validation. European Medicines Agency, London, UK (2012).
Hill HM, Smeraglia J, Brodie RR, Smith GT. Where do you draw the line? Points to consider when implementing the FDA method guidance on quantifying chromatographic peaks. Chromatographia Suppl. 55, S79–S81 (2002).
9
3
US Department of Health and Human Services, US FDA. Draft Guidance for Industry, Bioanalytical Methods Validation. FDA, Rockville, MD, USA (2013).
McDowall RD. The impact of 21CFR 11(electronic records and electronic signatures final rule) on bioanalysis. Chromatographia Suppl. 55, S85-S94 (2002).
10
4
United States Pharmacopiea. United States Pharmacopieal Convention, Edition 36 1 May. United States Pharmacopiea, Rockville, MD, USA (2013).
McDougall S. Chromatographic run quality assessment. Presented at: European Bioanalysis Forum 3rd Focus Meeting “Hatching”. Brussels, Belgium, 12–13 June 2012.
11
5
Center for Drug Evaluation and Research. Reviewer Guidance, Validation of Chromatographic Methods. Center for Drug Evaluation and Research, Silver Spring, MD, USA (2014).
Garofolo F, Rocci ML, Dumont I et al. 2011 White Paper, on recent issues in bioanalysis and regulatory findings from audits and inspections. Bioanalysis 3(18), 2081–2096 (2011).
6
Dyson N. Chromatographic Integration Methods. Royal Society of Chemistry, Cambridge, UK (1990).
Bioanalysis (2014) 6(9)
future science group
For reprint orders, please contact [email protected]
Manual chromatographic baseline integration: is it needed, if so when should it be used? “
…when is it appropriate to reject the ‘integrator’ results? If reintegration is necessary, how do you set objective and consistent procedures to minimize bias at the best and fraud at the worst?
”
Keywords: acceptance criteria • consistency • objectivity • regulatory issues • reintegration
Howard M Hill*,1, David Bakes2 & Iain Love2
Scope & overview The objective of this article is to discuss the prevailing opinions on whether chromatographic baselines should be determined by setting the “integrator” (integration algorithm) parameters and accepting this data only or when there are situations where the integrator does not get it right and further evaluation is necessary. The issues arise largely in the second case. When is the data not acceptable, that is, when is it appropriate to reject the ‘integrator’ results? If reintegration is necessary, how do you set objective and consistent procedures to minimize bias at the best and fraud at the worst? While this scenario is driven largely by the measurement of drugs(usually small molecules) in biological fluids for the purposes of defining the pharmacokinetics, there are a growing number of scenarios discussed here, where this is not always the case.
Independent Pharma Consultant, Harrogate, North Yorkshire, UK 2 Huntingdon Life Sciences, Woolley Road, Alconbury, Cambridgeshire, PE28 4HS, UK *Author for correspondence: [email protected]
Regulatory situation The two key publications relevant to bioanalysis are the US FDA [1] and EMA [2] guidance documents on bioanalytical method validation. While the FDA [3] have recently produced an updated draft guidance on this topic, it is not intended to refer to this document in this article. In the coming months it is likely it will be subject to much discussion and change before a definitive version is produced. Subjective interpretation of the current guidance documents is required, as neither offers prescriptive instruction on the subject of peak integration. The FDA Guidance [1] states on page 15 that, “Sample data reintegration: An SOP or guideline for sam-
10.4155/BIO.14.68 © 2014 Future Science Ltd
ple data reintegration should be established. This SOP or guideline should explain the reasons for reintegration and how the reintegration is to be performed. The rationale for reintegration should be clearly described and documented. Original and Reintegration Data should be reported.” “Documentation for reintegrated data: Documentation should include the initial and repeat results, the method used for reintegration, the reported result, assay run identification, the reason for the reintegration, the requestor of the reintegration and the manager authorizing reintegration. Reintegration of a clinical or preclinical sample should be performed only under a predefined SOP.” The EMA document [2] (page 13) is little more succinct or open to interpretation, depending upon your view point, “Chromatogram integration and reintegration should be described in an SOP. Any deviation from this SOP should be discussed in the analytical report. Chromatogram integration parameters and in the case of reintegration, initial and final integration data should be documented at the laboratory and should be available on request.” For further guidance reference is made to the “Reflection paper for Laboratories That Perform The Analysis or Evaluation of Clinical Trial Samples” (EMA/INS/GCP/532137/2010). This document discusses data recording issues and procedures not immediately related to baseline integration. It is evident that both authorities endorse the need for reintegration and both emphasize the need for clear criteria to be applied and a transparent audit trail of who authorized what to be provided in the study
Bioanalysis (2014) 6(9), 1171–1174
1
part of
ISSN 1757-6180
1171
Commentary Hill, Bakes & Love documentation. While not legally binding to bioanalysts, the pharmacopeias, particularly the US Pharmacopiea (USP) [4] provide guidance on what criteria should be assessed in defining good chromatography. However, they do not provide a great deal of guidance on integration issues nor does the FDA related document from 1994 [5] which has been cited many times where chromatographic methods are used. The current situation The decision as to where to draw a baseline on a chromatographic peak, and thereby determine its peak height or area used to construct a calibration curve and related data, has a pivotal impact on an analytical method. Approximately 20 years ago the publications of Dyson [6] and Meyer [7] discussed in some detail the impact on the accuracy of peak ratio measurements caused by ‘inconsistent’ peak shapes. These documents are as relevant today as they were when first published. In 2002, Chromatographia published two papers on this topic, one paper discussed the issues associated with ‘drawing a line’ [8] while another discussed the impact of 21CFR11 [9] on the recording of chromatographic raw data. More recently the Global Bioanalysis Consortium [10] provided some thoughts on ‘Chromatographic Run Quality Assessment’. This was presented by McDougall at the European Bioanalytical Forum(EBF) Consortium meeting in June 2012 (the consortium had over 20 authors representing the European pharmaceutical industry). The presentation also provided a more recent review/collation of relevant publications since circa 2002. Pragmatically the presentation presents the points to consider leaving it to individual laboratories to justify their approach. Nevertheless the issue remains! ‘To reintegrate or not’, and if so what is bad chromatography and what criteria are to be used for reintegration?
“The decision as to where to draw a baseline … has a pivotal impact on an analytical method.” The establishment of methods with “clean” symmetrical peaks has been assisted by the development of LC–MS – including better chromatographic separations and more selectivity with respect to detector technology. In such a case the use of automated systems would seem to be clear. However, with the need to develop methods capable of measuring metabolites/ degradants in the same chromatograms, and the need to seek more and more sensitivity, where the peak shape may not be symmetrical then the situation may not be so clear cut. Increasingly, the need to measure macromolecules in biological fluids by chromatographic techniques can complicate or compromise the ability
1172
Bioanalysis (2014) 6(9)
of automated integration algorithms to produce consistent integration of the non-Gaussian peak shapes often encountered in this field. Moreover, recent techniques developed to characterize and quantify proteins by measuring “multiple peptide” peaks, post hydrolysis, highlight that imposition of a one size fits all scenario is not necessarily realistic. Baseline assignment Data sampling and integration criteria have been discussed in much detail by Dyson [6] and Myers [7] and have been reiterated in the article by Hill et al. [8] and in the GBC presentation by McDougall [10] . McDougall presents several succinct quotes by Dyson, which are relevant to this ongoing debate, “It should be clear now that integrators are like any other tool – an excellent thing in the right hands. What they do best is measure peaks which are suitable for measuring rapidly and without tedium. If all these measurements are worth making then all subsequent calculations are worth noting and perhaps acting upon. As long as integrators use perpendiculars and tangents and draw straight lines, they are of use only in controlled circumstances when chromatography is good. Even then the use of integrators requires vigilance from the operator and skill in assessing and assigning parameters. Integrators cannot improve bad chromatography only analysts can do that and at the end of the day that’s what they are paid for.” The conclusion would appear to be that if you can set the right integration parameters correctly then reintegration should not be necessary. There are however two caveats. Firstly the parameters should be set and validated with as much consideration as is given to developing the chromatographic conditions. Setting default conditions is inappropriate,-to cite McDougall’s presentation, “it is scientifically invalid to assume chromatographic integration parameters must be fixed.” Secondly, chromatography may not be perfect but it may be acceptable, indeed it may be the best that can be achieved, bearing in mind the nature of the matrix, the chemistry of the molecule and complexity of the chromatogram. It is therefore essential that typical examples of the ‘accepted’ chromatography be included as part of the analytical methods documentation to illustrate this. Data sampling This is a widely discussed issue, in publications at least, but is not often addressed in labs where default positions are routinely used. It can be, and in many cases is, a major contributor to data variability [6] and the key parameters should be considered with equal weight as developing the chromatographic conditions. These are sampling frequency, smoothing and filtering. Filtering
future science group
Manual chromatographic baseline integration: is it needed, if so when should it be used?
is a real-time process where noise is suppressed by electronic means as such the process is irreversible unlike smoothing which takes place post data collection. Sample frequency can be pivotal to the data collection process – rate of collection is governed by baseline noise and peak width. In general a minimum of ten slices are recommended [7] , although taking into account variations in peak shape 20–25 slices per peak has become a generally accepted ‘safer’ option. It is important to note that peak width can be influenced by a number of factors, not least of which is concentration. Setting too low a collection rate can result in loss of peak height while too much data may stress the capacity of the data system. As with all things, getting the balance right is essential. It is important that the interaction of data collection frequency, filtering, smoothing and the setting of integration parameters be evaluated for each method and be continually assessed thoughout the life of the method. Peak height versus peak area Peak area seems to be the common default measurement, indeed the USP [5] in chapter 621 acknowledge that peak areas are the most widely used option. The argument for choosing either peak height or area can vary from the scientific [6] to the pragmatic. Detectors have been divided into mass flow detectors and flow sensitive detectors which justify whether peak area or height should be used, respectively. The latter do not consume the analyte and as such can be used in a hyphenated series. Thus if a UV detector is used, ideally peak height should be chosen to quantify the analyte as it will be little changed by flow rate variability or interferences to the leading and or trailing edges. In general however peak area is used, this is especially so for LC–MS. Nevertheless there may be occasions where problems such as poor peak shape and/or interference and so on may be resolved by using peak height. To reintegrate or not? While it seems that the pragmatic answer is yes Garofolo et al. [11] in a White Paper concluded that, “generally speaking it is accepted by the industry and the regulatory authorities that integration done automatically is preferred but it may sometimes be appropriate to perform manual integration. It was argued that manual integration is generally acceptable as long as it is done before the results are regressed, is justified and documented in a controlled fashion and that good scientific judgment has been used.” This means that criteria for reintegration must be determined and defined a priori. Terminology used when discussing integration varies between workers. The words may be the same but the definitions
future science group
Commentary
may differ. It is essential to develop a consensus with respect to definitions. This is essential in order to drive objective and consistent integration, to satisfy the requirements of good science and moreover, the regulatory authorities. For example, manual integration is often considered to be the subjective practice of a point-and-click action to define the start and end of the “peak”. However, manual integration may also be considered to be the update of an algorithm parameter by the end user to facilitate more consistent integration with respect to analytical background. The former procedure is subjective, may be difficult to reproduce during investigation and require significant justification, whereas the latter is often considered to be more acceptable, although still open to manipulation. This is especially so when multiple attempts are made on the same chromatogram. Equally as important, the term reintegration must be adequately defined. For example, it may be considered that the update of integration parameters is different, depending upon whether regression analysis has been carried out. Thus, ‘integration modification’ may be considered the practice of correcting an inconsistent integration generated by a set of best-fit parameters in the absence of regression data while ‘reintegration’ is the practice of integration of the peak post-regression. In this instance, integration modification may be considered pragmatic and acceptable whereas reintegration may require extra justification and discussion during the reporting phase. If the definition of the terminology used around peak integration is incorporated into SOPs, the discussion between peers, authorizers, auditors and report readers becomes transparent and non-ambiguous. As an additional tool to ensure consistency a compendium of examples of ‘suspect’ chromatography is desirable, be they industry-wide or company-specific, however, consistent application can only be achieved by thorough training and management oversight. Where to next? The primary question to be addressed is whether to modify integration/reintegrate or not. If reintegration is deemed acceptable, how do you decide which samples should be reintegrated and how do you ensure consistency of reintegration? It is essential that the analyst understands the parameters required to assign an appropriate baseline-how do you define what is a good/acceptable baseline? If manual baseline assignment for ‘difficult and distorted’ chromatograms but best peak scenarios is acceptable, it is essential to objectively identify and define these scenarios. Similarly objective and consistent scenarios for carrying out manual baselines should be defined and qualified.
www.future-science.com
1173
Commentary Hill, Bakes & Love Above all it is essential that science reigns over dogma, which may tie the hands of the scientists into developing ‘ingenious’ contortions in order to circumvent the unusual and intractable. Pragmatism does have a place in science but only if objectively and consistently applied. It is a not a substitute for expediency and sloppy science. The current regulatory guidelines provide for objectivity and consistency. However, more and more laboratories are retreating down the road of ‘what the computer says’ is right! It would seem essential therefore for the industry to develop a consistent approach to these problems which reflect a wide variety of scenarios, not just a single symmetrical peak capable of been quantified even at the highest levels of sensitivity. This article attempts to provide an overview of the issues involved in reintegrating chromatographic peaks. The ‘Ask the experts’ article appearing in this issue may References
1174
either emphasize the diversity of opinions or provide an insight into developing an industry consensus towards reintegration that can also be embraced by the regulators. It is hoped that at least it will lead to agreement that reintegration is necessary and that when carried out according to well-developed guidelines, be they industry- or company-specific, it can be regarded as an acceptable procedure. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. 7
Meyer VR. Error sources in the determination of chromatographic methods peak ratios. In: Advances in Chromatography. Brown PR, Grushka E (Eds). Marcel Dekker, New York, NY, USA, 383 (1995).
1
US FDA, Center for Drug Evaluation and Veterinary Medicine. Guidance for Industry, Bioanalytical Methods Validation. FDA, Rockville, MD, USA (2001).
8
2
European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Bioanalytical Methods Validation. European Medicines Agency, London, UK (2012).
Hill HM, Smeraglia J, Brodie RR, Smith GT. Where do you draw the line? Points to consider when implementing the FDA method guidance on quantifying chromatographic peaks. Chromatographia Suppl. 55, S79–S81 (2002).
9
3
US Department of Health and Human Services, US FDA. Draft Guidance for Industry, Bioanalytical Methods Validation. FDA, Rockville, MD, USA (2013).
McDowall RD. The impact of 21CFR 11(electronic records and electronic signatures final rule) on bioanalysis. Chromatographia Suppl. 55, S85-S94 (2002).
10
4
United States Pharmacopiea. United States Pharmacopieal Convention, Edition 36 1 May. United States Pharmacopiea, Rockville, MD, USA (2013).
McDougall S. Chromatographic run quality assessment. Presented at: European Bioanalysis Forum 3rd Focus Meeting “Hatching”. Brussels, Belgium, 12–13 June 2012.
11
5
Center for Drug Evaluation and Research. Reviewer Guidance, Validation of Chromatographic Methods. Center for Drug Evaluation and Research, Silver Spring, MD, USA (2014).
Garofolo F, Rocci ML, Dumont I et al. 2011 White Paper, on recent issues in bioanalysis and regulatory findings from audits and inspections. Bioanalysis 3(18), 2081–2096 (2011).
6
Dyson N. Chromatographic Integration Methods. Royal Society of Chemistry, Cambridge, UK (1990).
Bioanalysis (2014) 6(9)
future science group
