Perspective
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Stabilization of clinical samples collected for quantitative bioanalysis – a reflection from the European Bioanalysis Forum
In bioanalysis of small molecules, the analyte concentration in the measured samples should reflect the concentration during sample collection. Precautions may be needed to prevent over- or under-estimation of the obtained result. This might require the addition of stabilizers to prevent degradation or nonspecific binding. For unstable drugs, it is essential to know how analytes can be stabilized before the start of the clinical study. Although the stabilization methods are well documented, the impact of the stabilization on the clinical workflow is not properly addressed. Already during method development, the clinical implications in terms of personnel safety, ease of use, training possibilities and staff capacity should be taken into account, and validation of the bioanalytical method should reflect collection procedures.
During PK studies, drug and metabolite levels are commonly monitored in biological matrices. As analysis is usually not performed immediately after collection of the samples, it is a requirement to ensure that the concentration results obtained after storage and processing reflect the concentrations of the samples directly after they were collected. Several issues may affect the analyte concentration during sampling and storage, such as analyte instability, adsorption to containers or collection materials and nonhomogeneous distribution within the sample. Even when the analyte itself is stable, metabolites may not be and they may undergo back conversion (reversion) to the analyte resulting in an overestimation of the measured concentration in addition, sometimes, endogenous compounds have been shown to degrade to products that may interfere with the method [1] . To prevent over- or under-estimation of the concentrations, these effects need to be understood and controlled. Unfortunately, the stability of analytes or metabolites cannot always be guaranteed by applying appropriate storage and processing conditions, but may require the addition of stabilizers. As instability is often non reversible, the need for stabilizers
10.4155/BIO.14.290 © 2015 Future Science Ltd
should be investigated before the start of the clinical sample collection. Several papers have been published describing the reasons for compound instability, in several matrices, such as thermal instability, (auto) oxidation, pH instability, and so on [2–7] (see Table 1 for a comprehensive overview of known bioanalytical instability issues with certain chemical classes). Also, several papers, including original journal publications [8–10] , regulatory guidelines [11–15] and white papers [16–19] have been published over the past decade that provide guidance to the bioanalytical scientist on how to approach stability assessment in the validation of bioanalytical methods. Conducting incurred sample stability at an early stage of method development has been recognized as a powerful tool to investigate sample integrity [20] . Recently, the Global Bioanalysis Consortium (GBC) published a review article with recommendations on the best practice for the assessment of analyte stability [8] . Despite the attention of the bioanalytical community, stabilization of potentially unstable drugs or metabolites in relation to the consequences for sample collection at the clinical site to ensure representative samples are taken is not well described and papers
Bioanalysis (2015) 7(3), 333–343
Martijn Hilhorst*,1, Peter van Amsterdam2, Katja Heinig3, Elke Zwanziger3 & Richard Abbott4 Bioanalytical Laboratory, PRA Health Sciences, Westerbrink 3, 9405 BJ Assen, The Netherlands 2 Abbott Healthcare Products, Weesp, The Netherlands 3 F Hoffmann-La Roche Ltd, Basel, Switzerland 4 Bayer Healthcare, Wuppertal, Germany *Author for correspondence: Tel.: +31 592 303 441 Fax: +31 592 303 223 [email protected] 1
part of
ISSN 1757-6180
333
Perspective Hilhorst, van Amsterdam, Heinig, Zwanziger & Abbott
Table 1. Overview of most commonly occurring stability issues. Compound
Stability issue(s)
Acylglucuronide metabolites
Hydrolysis to parent drug, acylmigration
Lactones (e.g., statin drugs)
Reversible hydrolysis to hydroxy acid
Prodrugs (alkyl esters)
Conversion into active drug by cleavage of ester bonds by esterases
Amides
Hydrolysis to acid
Amines
Reversible N-oxidation
Oxidizable compounds (folates, catecholamines)
Oxidative degradation
Peptides/small proteins (neuropeptides, hormones, GLP1 analogues)
Potential dipeptidyl peptidase IV-catalyzed N-terminal degradation
Cytostatic nucleosides (cytarabine, gemcitabine)
Enzymatic deamination
Thiols (captopril)
Dimerization
Enantiomers
Racemization
Cis/trans isomers
Interconversion
Alkylating agents (anticancer drugs)
Unexpected metabolic transformations
documenting proper strategies are limited. Stabilization of clinical samples can be straightforward from a laboratory point of view, but can result in difficulties at the clinical site as it may require the use of toxic material, pipetting expertise, or intensive resources. As stabilization can often be done in various ways (such as flash freezing instead of addition of an antioxidant), frequent communication between the laboratory and the clinical staff is essential to provide a strategy that does not only guarantee reliable bioanalysis results, but is also practical in the clinical setting. It must be ensured that the conditions and materials used in the clinic are identical to those used during method validation in the laboratory. It is the aim of the current paper to increase awareness and provide reflections on the scientific appropriateness and the required practicalities that are potentially needed. The paper focuses on the analysis of small molecules although the proposed strategies may also be applicable for large molecules. An overview is given of the most common reasons for sample instability and the implications for sample collection strategies. It is not the scope of this article to discuss sample integrity Key terms Adsorption: Process by which analytes binds to a surface. Back conversion: Process during which a degradation product reverts to its original structure. Stabilizers: Additives preventing degradation or nonspecific binding. Antioxidant: Chemical that prevents oxidation. Enzyme inhibitors: Compounds reacting with enzymes to reduce their activity.
334
Bioanalysis (2015) 7(3)
during the sample processing and analysis in the laboratory. Also, the paper does not give a comprehensive overview of all stability problems that can be encountered as the strategies in the clinic to control sample integrity are often similar. A clear distinction is made between plasma and urine stabilization as the approach is different. Furthermore, the article describes the regulatory consequences of the suggested stabilization strategy and provides solutions to avoid pitfalls during method validation. Regulatory requirements In order to obtain drug approval, for pivotal studies, bioanalytical methods should meet the criteria as outlined in the relevant bioanalytical method validation guidelines [11–15] . Other clinical studies require bioanalytical methods (e.g., urine analyses) to be at least scientifically validated to demonstrate the method is fit for purpose [21,22] . It is essential that whatever measures taken to stabilize clinical samples during (scientific) method validation are closely mimicked during clinical sample collection to ensure the validity of the results. This means that conditions in the clinic such as storage temperature, light exposure, collection tubes used, concentration of stabilizer and anticoagulant need to be the same as used during method validation in the laboratory. This might be obvious, but commercially available tubes used in the clinic may contain different concentrations or amounts of stabilizer than the commercially obtained blank (often collected in bags) plasma in the laboratory. Furthermore, during sample collection, insufficient whole blood might be collected resulting in a stabilizer concentration higher than used during the validation. Light exposure is
future science group
Stabilization of clinical samples collected for quantitative bioanalysis
also a parameter that is easily overlooked. Seasonal or latitude differences, differences in freezers (walk-in or closed) can lead to biased results when light sensitive analytes are to be measured and samples are stored in transparent boxes. The materials and start and end times of storage and processing should be well documented to demonstrate that the correct measures were taken. of In case exact volumes of stabilizer need to be added to an exact volume of matrix in a clinical setting, it is necessary to ensure that the pipetting is done correctly. This can be done by documented training of clinical personnel. As the addition of stabilizer will influence the total volume of the sample, it is important to ensure that the volume increase is accounted for in the validation study. In general, communication between the laboratory and the clinic and the review of sample collection manuals is of critical importance. This is easily overlooked or sacrificed when method development is not conducted well before the sample collection. Practical considerations for the clinic As instability often is a nonreversible process, it is important that the stability is investigated before the start of the clinical study as stabilization may require the use of special conditions or addition of reagents during sample collection in the clinic. It is not always realized by the laboratory that the addition of stabilizer to a collected sample can have a large implication on the workflow in the clinic. In general, the manual addition of stabilizers to collection tubes should be avoided if possible. As a general principle, defining sampling conditions are part of the method validation and a first strategy usually is to freeze the samples as soon as possible and to analyze them within the validated stability period upon arrival at the bioanalytical laboratory. In Phase I clinical trials, the samples are often measured within weeks of the end of the study or after each shipment that arrives at the laboratory. Adding stabilizers to avoid a long-term stability effect may not be needed when stored at a sufficient low temperature. If the analytes require stabilization, it is important to consider that in general, clinical personnel are not trained in pipetting accurate volumes. This can be solved by hands on training by laboratory personnel. The addition of stabilizers, is also time consuming. For example, if 10 μl of stabilizer needs to be added to 1 ml of plasma, this requires two pipetting steps. In the first step, 10 μl of stabilizer is added to an empty tube and in the second step, 1 ml of plasma is pipetted into the tube. As an error made during pipetting is relatively high at low volumes, it is recommended to avoid the pipetting of small volumes and prepare the plasma collection tube in the laboratory that will be
future science group
Perspective
involved in the analysis of the collected samples. If stabilizers need to be added to the whole blood collection tube, it is important not to break the vacuum. Small amounts of stabilizer can be added using an injection needle penetrating the septum of the tube while maintaining the vacuum. Note that blood cannot be acidified to pH
For reprint orders, please contact [email protected]
Stabilization of clinical samples collected for quantitative bioanalysis – a reflection from the European Bioanalysis Forum
In bioanalysis of small molecules, the analyte concentration in the measured samples should reflect the concentration during sample collection. Precautions may be needed to prevent over- or under-estimation of the obtained result. This might require the addition of stabilizers to prevent degradation or nonspecific binding. For unstable drugs, it is essential to know how analytes can be stabilized before the start of the clinical study. Although the stabilization methods are well documented, the impact of the stabilization on the clinical workflow is not properly addressed. Already during method development, the clinical implications in terms of personnel safety, ease of use, training possibilities and staff capacity should be taken into account, and validation of the bioanalytical method should reflect collection procedures.
During PK studies, drug and metabolite levels are commonly monitored in biological matrices. As analysis is usually not performed immediately after collection of the samples, it is a requirement to ensure that the concentration results obtained after storage and processing reflect the concentrations of the samples directly after they were collected. Several issues may affect the analyte concentration during sampling and storage, such as analyte instability, adsorption to containers or collection materials and nonhomogeneous distribution within the sample. Even when the analyte itself is stable, metabolites may not be and they may undergo back conversion (reversion) to the analyte resulting in an overestimation of the measured concentration in addition, sometimes, endogenous compounds have been shown to degrade to products that may interfere with the method [1] . To prevent over- or under-estimation of the concentrations, these effects need to be understood and controlled. Unfortunately, the stability of analytes or metabolites cannot always be guaranteed by applying appropriate storage and processing conditions, but may require the addition of stabilizers. As instability is often non reversible, the need for stabilizers
10.4155/BIO.14.290 © 2015 Future Science Ltd
should be investigated before the start of the clinical sample collection. Several papers have been published describing the reasons for compound instability, in several matrices, such as thermal instability, (auto) oxidation, pH instability, and so on [2–7] (see Table 1 for a comprehensive overview of known bioanalytical instability issues with certain chemical classes). Also, several papers, including original journal publications [8–10] , regulatory guidelines [11–15] and white papers [16–19] have been published over the past decade that provide guidance to the bioanalytical scientist on how to approach stability assessment in the validation of bioanalytical methods. Conducting incurred sample stability at an early stage of method development has been recognized as a powerful tool to investigate sample integrity [20] . Recently, the Global Bioanalysis Consortium (GBC) published a review article with recommendations on the best practice for the assessment of analyte stability [8] . Despite the attention of the bioanalytical community, stabilization of potentially unstable drugs or metabolites in relation to the consequences for sample collection at the clinical site to ensure representative samples are taken is not well described and papers
Bioanalysis (2015) 7(3), 333–343
Martijn Hilhorst*,1, Peter van Amsterdam2, Katja Heinig3, Elke Zwanziger3 & Richard Abbott4 Bioanalytical Laboratory, PRA Health Sciences, Westerbrink 3, 9405 BJ Assen, The Netherlands 2 Abbott Healthcare Products, Weesp, The Netherlands 3 F Hoffmann-La Roche Ltd, Basel, Switzerland 4 Bayer Healthcare, Wuppertal, Germany *Author for correspondence: Tel.: +31 592 303 441 Fax: +31 592 303 223 [email protected] 1
part of
ISSN 1757-6180
333
Perspective Hilhorst, van Amsterdam, Heinig, Zwanziger & Abbott
Table 1. Overview of most commonly occurring stability issues. Compound
Stability issue(s)
Acylglucuronide metabolites
Hydrolysis to parent drug, acylmigration
Lactones (e.g., statin drugs)
Reversible hydrolysis to hydroxy acid
Prodrugs (alkyl esters)
Conversion into active drug by cleavage of ester bonds by esterases
Amides
Hydrolysis to acid
Amines
Reversible N-oxidation
Oxidizable compounds (folates, catecholamines)
Oxidative degradation
Peptides/small proteins (neuropeptides, hormones, GLP1 analogues)
Potential dipeptidyl peptidase IV-catalyzed N-terminal degradation
Cytostatic nucleosides (cytarabine, gemcitabine)
Enzymatic deamination
Thiols (captopril)
Dimerization
Enantiomers
Racemization
Cis/trans isomers
Interconversion
Alkylating agents (anticancer drugs)
Unexpected metabolic transformations
documenting proper strategies are limited. Stabilization of clinical samples can be straightforward from a laboratory point of view, but can result in difficulties at the clinical site as it may require the use of toxic material, pipetting expertise, or intensive resources. As stabilization can often be done in various ways (such as flash freezing instead of addition of an antioxidant), frequent communication between the laboratory and the clinical staff is essential to provide a strategy that does not only guarantee reliable bioanalysis results, but is also practical in the clinical setting. It must be ensured that the conditions and materials used in the clinic are identical to those used during method validation in the laboratory. It is the aim of the current paper to increase awareness and provide reflections on the scientific appropriateness and the required practicalities that are potentially needed. The paper focuses on the analysis of small molecules although the proposed strategies may also be applicable for large molecules. An overview is given of the most common reasons for sample instability and the implications for sample collection strategies. It is not the scope of this article to discuss sample integrity Key terms Adsorption: Process by which analytes binds to a surface. Back conversion: Process during which a degradation product reverts to its original structure. Stabilizers: Additives preventing degradation or nonspecific binding. Antioxidant: Chemical that prevents oxidation. Enzyme inhibitors: Compounds reacting with enzymes to reduce their activity.
334
Bioanalysis (2015) 7(3)
during the sample processing and analysis in the laboratory. Also, the paper does not give a comprehensive overview of all stability problems that can be encountered as the strategies in the clinic to control sample integrity are often similar. A clear distinction is made between plasma and urine stabilization as the approach is different. Furthermore, the article describes the regulatory consequences of the suggested stabilization strategy and provides solutions to avoid pitfalls during method validation. Regulatory requirements In order to obtain drug approval, for pivotal studies, bioanalytical methods should meet the criteria as outlined in the relevant bioanalytical method validation guidelines [11–15] . Other clinical studies require bioanalytical methods (e.g., urine analyses) to be at least scientifically validated to demonstrate the method is fit for purpose [21,22] . It is essential that whatever measures taken to stabilize clinical samples during (scientific) method validation are closely mimicked during clinical sample collection to ensure the validity of the results. This means that conditions in the clinic such as storage temperature, light exposure, collection tubes used, concentration of stabilizer and anticoagulant need to be the same as used during method validation in the laboratory. This might be obvious, but commercially available tubes used in the clinic may contain different concentrations or amounts of stabilizer than the commercially obtained blank (often collected in bags) plasma in the laboratory. Furthermore, during sample collection, insufficient whole blood might be collected resulting in a stabilizer concentration higher than used during the validation. Light exposure is
future science group
Stabilization of clinical samples collected for quantitative bioanalysis
also a parameter that is easily overlooked. Seasonal or latitude differences, differences in freezers (walk-in or closed) can lead to biased results when light sensitive analytes are to be measured and samples are stored in transparent boxes. The materials and start and end times of storage and processing should be well documented to demonstrate that the correct measures were taken. of In case exact volumes of stabilizer need to be added to an exact volume of matrix in a clinical setting, it is necessary to ensure that the pipetting is done correctly. This can be done by documented training of clinical personnel. As the addition of stabilizer will influence the total volume of the sample, it is important to ensure that the volume increase is accounted for in the validation study. In general, communication between the laboratory and the clinic and the review of sample collection manuals is of critical importance. This is easily overlooked or sacrificed when method development is not conducted well before the sample collection. Practical considerations for the clinic As instability often is a nonreversible process, it is important that the stability is investigated before the start of the clinical study as stabilization may require the use of special conditions or addition of reagents during sample collection in the clinic. It is not always realized by the laboratory that the addition of stabilizer to a collected sample can have a large implication on the workflow in the clinic. In general, the manual addition of stabilizers to collection tubes should be avoided if possible. As a general principle, defining sampling conditions are part of the method validation and a first strategy usually is to freeze the samples as soon as possible and to analyze them within the validated stability period upon arrival at the bioanalytical laboratory. In Phase I clinical trials, the samples are often measured within weeks of the end of the study or after each shipment that arrives at the laboratory. Adding stabilizers to avoid a long-term stability effect may not be needed when stored at a sufficient low temperature. If the analytes require stabilization, it is important to consider that in general, clinical personnel are not trained in pipetting accurate volumes. This can be solved by hands on training by laboratory personnel. The addition of stabilizers, is also time consuming. For example, if 10 μl of stabilizer needs to be added to 1 ml of plasma, this requires two pipetting steps. In the first step, 10 μl of stabilizer is added to an empty tube and in the second step, 1 ml of plasma is pipetted into the tube. As an error made during pipetting is relatively high at low volumes, it is recommended to avoid the pipetting of small volumes and prepare the plasma collection tube in the laboratory that will be
future science group
Perspective
involved in the analysis of the collected samples. If stabilizers need to be added to the whole blood collection tube, it is important not to break the vacuum. Small amounts of stabilizer can be added using an injection needle penetrating the septum of the tube while maintaining the vacuum. Note that blood cannot be acidified to pH
