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A novel approach to signal normalisation in atmospheric pressure ionisation mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 399–401
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A novel approach to signal normalisation in atmospheric pressure ionisation mass spectrometry Michael Vogeser a,∗ , Fabian Kirchhoff a , Roland Geyer b a b
Institute of Clinical Chemistry, University of Munich, Munich, Germany Tecan Trading AG, Männedorf, Switzerland
a r t i c l e
i n f o
Article history: Received 27 January 2012 Received in revised form 28 March 2012 Accepted 30 March 2012 Available online 5 April 2012 Keywords: Internal standardisation Post column infusion Ion suppression, atmospheric pressure ionisation (API) Liquid chromatography–tandem mass spectrometry (LC–MS/MS)
a b s t r a c t The aim of our study was to test an alternative principle of signal normalisation in LC–MS/MS. During analyses, post column infusion of the target analyte is done via a T-piece, generating an “area under the analyte peak” (AUP). The ratio of peak area to AUP is assessed as assay response. Acceptable analytical performance of this principle was found for an exemplary analyte. Post-column infusion may allow normalisation of ion suppression not requiring any additional standard compound. This approach can be useful in situations where no appropriate compound is available for classical internal standardisation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Liquid chromatography tandem–mass spectrometry has found an important role in clinical chemistry. In contrast to ligand-binding based assays, method development is typically straightforward with LC–MS/MS. The principle of internal standardisation–ideally involving stable isotope-labelled compounds – enables highest analytical reliability. By this approach, matrix effects which may be relevant in the entire process of extraction, chromatography and mass spectrometric detection can be compensated. For many of the clinically relevant endogenously occurring small molecule analytes (such as steroid hormones), stable isotope labelled compounds are available. This is in contrast to most analytes in therapeutic drug monitoring (TDM) where only few stable isotope labelled compounds are available. Consequently, in most TDM methods “homologues” are used as IS, representing compounds of similar molecular structure with respect to the target analyte. However, already minor differences (as functional groups) in the molecular structure between target analyte and IS can result in substantial differences in a compound’s ionisation and fragmentation behaviour. Whenever the ionisation
∗ Corresponding author at: Institute of Clinical Chemistry, University of Munich, Marchioninistr. 15, 81375 Munich, München, Germany. Tel.: +49 89 7095 3221; fax: +49 89 7095 8888. E-mail address: [email protected] (M. Vogeser). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.03.055
efficacy of a target analyte and such a related internal standard compound, respectively, is modulated in an non-equal manner by matrix constituents, inaccuracy arises in LC–MS/MS methods [1]. Thus, the reliability of LC–MS/MS applications, in particular in the field of TDM, is critically determined by the appropriateness of available candidate IS substances. Therefore, the search for suitable compounds is typically a major challenge in the development of LC–MS/MS methods in TDM today. Unsuited IS compounds (differing “too much” from the target analyte), typically lead to non-linearity of response and/or unacceptable reproducibility. We here describe a novel principle for monitoring and normalisation of signal generation in LC–MS/MS, not requiring any compound in addition to the target analyte. 2. Materials and methods 2.1. Instrumental set-up The analytical principle described herein is based on the continuous post-column infusion of a pure solution of the target analyte throughout an analytical series. This instrument configuration generates a background signal in the signal-trace of the target analyte; the actual peak of the chromatographic separation is superimposed to this background-signal [2]. Despite introducing the reference solution of the target analyte at a continuous rate, the intensity of the background signal is highly variable in LC–MS/MS, displaying the actual modulation of target analyte ionisation by
400
M. Vogeser et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 399–401
Fig. 1. Principle of internal standardisation by continuous post-column infusion of the target analyte and representative chromatogram of HMR 1726 quantification with LC–MS/MS; (A) peak area; (B) area-under-the-peak (AUP).
matrix compounds eluting from the chromatographic system. This post-column infusion set-up is widely used to investigate the ion-suppression characteristics of LC–MS/MS methods. Upon injection of a biological, matrix-based sample, ionisation yield typically decreases drastically for a time period of seconds to minutes due to matrix constituents. During method development, it is tried to minimise the extent of this ion-suppression range (in particular by efficient sample preparation) and to obtain elution of the target analyte outside this range by appropriate chromatographic retention of the analyte. Notably, the extent of the ion-suppression range can be variable from sample to sample. The key feature of the analytical principle described here is that a peak integration line is drawn and that in – a second step – the area of the quadrangle below this integration line (characterizing the background signal) is recorded. A representative chromatogram applying post-column infusion is shown in Fig. 1. The “area above the peak integration line” is denoted with A, the “area under the peak” (AUP) with B. (A time interval of ion suppression resulting from residual sample matrix components and preceding the analyte peak can be observed in a typical pattern.) The ratio of the analyte peak area to the AUP can be calculated as the response of the analysis (A/B; Fig. 1). In case of ion-suppression occurring during the target analyte peak elution, the area of both, the peak above the integration line and the AUP will be diminished alike, resulting in an unaffected ratio of the two areas (i.e. response). Thus, any sample-individual ion suppression (or enhancement) effect should be compensated by this instrumental configuration and mode of data analysis. 2.2. Evaluation In order to test this latter principle of quantification involving continuous post-column infusion without use of a separate IS substance, we performed experiments on an exemplary analyte, the drug metabolite HMR1726 (also referred to as A//1726 or teriflunomide). This compound is the active metabolite of leflunomide, an immunosuppressant drug used for the treatment of autoimmune disorders [3,4]. The detailed mass spectrometric and chromatographic conditions are given as Supplemental file. We performed a validation protocol including four independent series in order to investigate the performance of this quantification method. Calibrator samples and quality control samples were prepared by spiking drug free serum. Furthermore, to study the
precision of the method, aliquots of pooled leftover serum from patients treated with leflunomide were analysed in fourfold determination in each series. 3. Results and discussion Applying normalisation by post-column infusion, linear calibration functions were observed in all series (r > 0.99). Accuracy and imprecision was below 15% for the QC samples and the serum pool (Table 1). In most cases, LC–MS/MS methods applying classical internal standardisation realise a clearly higher degree of accuracy and precision, however, the validation results were in accordance with the FDA guidelines for bioanalytical method validation [5]. Indeed, for a LC–MS/MS method employing isotope dilution internal standardisation for the quantification of HMR 1726 CVs ≤ 7.1% have been observed [4]. In five analyses among the total of 64 performed during our method validation, atypical ion suppression patterns – displayed by a low AUP – were observed in a sporadic manner. In these cases, both, the peak area and the AUP of the affected QC samples were substantially smaller compared to all other analyses of the same sample. This may be explained by concomitant elution of substances interfering with the processes of ionisation which have cumulated within the chromatographic system. Using the quantitative post-column infusion principle of normalisation we obtained results in accordance with the expected concentrations in all these five cases. While post-column infusion of a solution of a target analyte is widely used in the context of the validation of LC–MS/MS methods with respect to ionisation modulation characteristics, the approach to use this set-up for normalisation of API signal generation within Table 1 Results of the method validation study for the quantification of HMR 1726 in serum by LC–MS/MS applying the principle of continuous post-column infusion for normalisation of the efficacy of atmospheric pressure ionisation (fourfold determination in four series).
15.8 g/l (QCL) 88.2 g/l (QCM) 180 g/l (QCH) Pool (mean 33.5 g/l)
Accuracy (%; mean)
Inter-assay imprecision (CV %)
112.7 110.4 102.3 –
14.9% 14.0% 7.2% 10.5%
M. Vogeser et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 399–401
analytical is novel. Two previous articles report methods also aiming to realise real-time signal normalisation in LC–MS/MS: Stahnke et al. [6] have reported a similar instrument configuration; in this method, however, not a solution of the target analyte itself but instead one single solution of an unrelated compound was infused for the generation of a reference signal in the analysis of more than 100 different pesticides. Since the modulation of ion generation by matrix compounds or ion source related factors can be highly analyte-specific, we assume that our approach – based on continuous infusion of a solution of the actual target analyte – might be more reliable. Kaufmann and Butcher [7] describe superimposition of analyte spikes during chromatography which is technically far more demanding compared to the technology described here. In the test reported here we used a syringe pump and a T-piece tube connection set-up in order to obtain a continuous introduction of the analyte to the ion source. This is also applicable in gradient methods; in the case of isocratic chromatographic systems, alternatively, the target analyte can as well simply be added to the mobile phase allowing a very simple configuration. At present, an evident shortcoming of our normalisation approach is the fact that positioning of the integration lines and construction of integration areas has to be performed “manually” in the software system. Inclusion of this post-column-infusion monitoring/normalisation principle into MS/MS-quantification software solutions with calculation of the AUP seems desirable. In order to obtain good reproducibility of the AUP it is essential that the chromatographic performance is stable and in particular that any peak tailing is constant. It must be noted that the principle described in this article cannot equalise between-sample variation of autosampler injection volume or of pre-ionisation analyte extraction procedures like solid phase extraction; only the ionisation process of a LC–MS/MS method can be monitored and normalised by this principle. Thus the availability of a related compound to be used as a “classical” internal standard which undergoes the entire analytical process including chromatographic separation certainly remains desirable for any analyte. However, autosampler precision is very high in many systems, so that the application of our principle also seems potentially applicable for clinical and biomedical research analyses in cases where all available candidate internal standard compounds are actually found to be inappropriate.
401
We have described in this article the proof-of-concept for a novel approach which can be useful for the monitoring and normalisation of between sample variations in the ionisation efficacy of LC-API–MS. Our evaluation results and experiences justify further validation of this approach for different analytes and the development of appropriate software solutions for application of this principle also in a routine setting. Further work will have to address the suitability of the approach described herein also in multi-analyte methods and over extended calibration concentration ranges. Furthermore a potential impact of this technology on the analytical sensitivity in trace concentration ranges has to be assessed. However, the principle may probably help to increase the reliability of LC–MS/MS analyses of “orphan” analytes for which appropriate IS compounds are not available. This may include entirely new compounds in the context of drug development, new illicit drugs in clinical toxicology but also endogenous compounds addressed in the development of innovative “metabolomic” multimethods. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2012.03.055. References [1] M. Vogeser, C. Seger, Pitfalls associated with the use of liquid chromatography– tandem mass spectrometry in the clinical laboratory, Clin. Chem. 56 (2010) 1234–1244. [2] T.M. Annesley, Ion suppression in mass spectrometry, Clin. Chem. 49 (2003) 1041–1044. [3] F. Behrens, M. Koehm, H. Burkhardt, Update 2011: leflunomide in rheumatoid arthritis – strengths and weaknesses, Curr. Opin. Rheumatol. 23 (2011) 282–287. [4] H. Rakhila, T. Rozek, A. Hopkins, S. Proudman, L. Cleland, M. James, M. Wiese, Quantitation of total and free teriflunomide (A77 1726) in human plasma by LC–MS/MS, J. Pharmaceut. Biomed. Anal. 55 (2011) 325–331. [5] US Department of Health and Human Services. Guidance for industry – bioanalytical method validation. Food and Drug Administration, Center for Drug Evaluation and Research, 2001. [6] H. Stahnke, T. Reemtsma, L. Alder, Compensation of matrix effects by postcolumn infusion of a monitor substance in multiresidue analysis with LC–MS/MS, Anal. Chem. 81 (2009) 2185–2192. [7] A. Kaufmann, P. Butcher, Segmented post-column addition; a concept for continuous response control of liquid chromatography/mass spectrometry peaks affected by signal suppression/enhancement, Rapid. Commun. Mass. Spectrom. 19 (2005) 611–617.
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A novel approach to signal normalisation in atmospheric pressure ionisation mass spectrometry Michael Vogeser a,∗ , Fabian Kirchhoff a , Roland Geyer b a b
Institute of Clinical Chemistry, University of Munich, Munich, Germany Tecan Trading AG, Männedorf, Switzerland
a r t i c l e
i n f o
Article history: Received 27 January 2012 Received in revised form 28 March 2012 Accepted 30 March 2012 Available online 5 April 2012 Keywords: Internal standardisation Post column infusion Ion suppression, atmospheric pressure ionisation (API) Liquid chromatography–tandem mass spectrometry (LC–MS/MS)
a b s t r a c t The aim of our study was to test an alternative principle of signal normalisation in LC–MS/MS. During analyses, post column infusion of the target analyte is done via a T-piece, generating an “area under the analyte peak” (AUP). The ratio of peak area to AUP is assessed as assay response. Acceptable analytical performance of this principle was found for an exemplary analyte. Post-column infusion may allow normalisation of ion suppression not requiring any additional standard compound. This approach can be useful in situations where no appropriate compound is available for classical internal standardisation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Liquid chromatography tandem–mass spectrometry has found an important role in clinical chemistry. In contrast to ligand-binding based assays, method development is typically straightforward with LC–MS/MS. The principle of internal standardisation–ideally involving stable isotope-labelled compounds – enables highest analytical reliability. By this approach, matrix effects which may be relevant in the entire process of extraction, chromatography and mass spectrometric detection can be compensated. For many of the clinically relevant endogenously occurring small molecule analytes (such as steroid hormones), stable isotope labelled compounds are available. This is in contrast to most analytes in therapeutic drug monitoring (TDM) where only few stable isotope labelled compounds are available. Consequently, in most TDM methods “homologues” are used as IS, representing compounds of similar molecular structure with respect to the target analyte. However, already minor differences (as functional groups) in the molecular structure between target analyte and IS can result in substantial differences in a compound’s ionisation and fragmentation behaviour. Whenever the ionisation
∗ Corresponding author at: Institute of Clinical Chemistry, University of Munich, Marchioninistr. 15, 81375 Munich, München, Germany. Tel.: +49 89 7095 3221; fax: +49 89 7095 8888. E-mail address: [email protected] (M. Vogeser). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.03.055
efficacy of a target analyte and such a related internal standard compound, respectively, is modulated in an non-equal manner by matrix constituents, inaccuracy arises in LC–MS/MS methods [1]. Thus, the reliability of LC–MS/MS applications, in particular in the field of TDM, is critically determined by the appropriateness of available candidate IS substances. Therefore, the search for suitable compounds is typically a major challenge in the development of LC–MS/MS methods in TDM today. Unsuited IS compounds (differing “too much” from the target analyte), typically lead to non-linearity of response and/or unacceptable reproducibility. We here describe a novel principle for monitoring and normalisation of signal generation in LC–MS/MS, not requiring any compound in addition to the target analyte. 2. Materials and methods 2.1. Instrumental set-up The analytical principle described herein is based on the continuous post-column infusion of a pure solution of the target analyte throughout an analytical series. This instrument configuration generates a background signal in the signal-trace of the target analyte; the actual peak of the chromatographic separation is superimposed to this background-signal [2]. Despite introducing the reference solution of the target analyte at a continuous rate, the intensity of the background signal is highly variable in LC–MS/MS, displaying the actual modulation of target analyte ionisation by
400
M. Vogeser et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 399–401
Fig. 1. Principle of internal standardisation by continuous post-column infusion of the target analyte and representative chromatogram of HMR 1726 quantification with LC–MS/MS; (A) peak area; (B) area-under-the-peak (AUP).
matrix compounds eluting from the chromatographic system. This post-column infusion set-up is widely used to investigate the ion-suppression characteristics of LC–MS/MS methods. Upon injection of a biological, matrix-based sample, ionisation yield typically decreases drastically for a time period of seconds to minutes due to matrix constituents. During method development, it is tried to minimise the extent of this ion-suppression range (in particular by efficient sample preparation) and to obtain elution of the target analyte outside this range by appropriate chromatographic retention of the analyte. Notably, the extent of the ion-suppression range can be variable from sample to sample. The key feature of the analytical principle described here is that a peak integration line is drawn and that in – a second step – the area of the quadrangle below this integration line (characterizing the background signal) is recorded. A representative chromatogram applying post-column infusion is shown in Fig. 1. The “area above the peak integration line” is denoted with A, the “area under the peak” (AUP) with B. (A time interval of ion suppression resulting from residual sample matrix components and preceding the analyte peak can be observed in a typical pattern.) The ratio of the analyte peak area to the AUP can be calculated as the response of the analysis (A/B; Fig. 1). In case of ion-suppression occurring during the target analyte peak elution, the area of both, the peak above the integration line and the AUP will be diminished alike, resulting in an unaffected ratio of the two areas (i.e. response). Thus, any sample-individual ion suppression (or enhancement) effect should be compensated by this instrumental configuration and mode of data analysis. 2.2. Evaluation In order to test this latter principle of quantification involving continuous post-column infusion without use of a separate IS substance, we performed experiments on an exemplary analyte, the drug metabolite HMR1726 (also referred to as A//1726 or teriflunomide). This compound is the active metabolite of leflunomide, an immunosuppressant drug used for the treatment of autoimmune disorders [3,4]. The detailed mass spectrometric and chromatographic conditions are given as Supplemental file. We performed a validation protocol including four independent series in order to investigate the performance of this quantification method. Calibrator samples and quality control samples were prepared by spiking drug free serum. Furthermore, to study the
precision of the method, aliquots of pooled leftover serum from patients treated with leflunomide were analysed in fourfold determination in each series. 3. Results and discussion Applying normalisation by post-column infusion, linear calibration functions were observed in all series (r > 0.99). Accuracy and imprecision was below 15% for the QC samples and the serum pool (Table 1). In most cases, LC–MS/MS methods applying classical internal standardisation realise a clearly higher degree of accuracy and precision, however, the validation results were in accordance with the FDA guidelines for bioanalytical method validation [5]. Indeed, for a LC–MS/MS method employing isotope dilution internal standardisation for the quantification of HMR 1726 CVs ≤ 7.1% have been observed [4]. In five analyses among the total of 64 performed during our method validation, atypical ion suppression patterns – displayed by a low AUP – were observed in a sporadic manner. In these cases, both, the peak area and the AUP of the affected QC samples were substantially smaller compared to all other analyses of the same sample. This may be explained by concomitant elution of substances interfering with the processes of ionisation which have cumulated within the chromatographic system. Using the quantitative post-column infusion principle of normalisation we obtained results in accordance with the expected concentrations in all these five cases. While post-column infusion of a solution of a target analyte is widely used in the context of the validation of LC–MS/MS methods with respect to ionisation modulation characteristics, the approach to use this set-up for normalisation of API signal generation within Table 1 Results of the method validation study for the quantification of HMR 1726 in serum by LC–MS/MS applying the principle of continuous post-column infusion for normalisation of the efficacy of atmospheric pressure ionisation (fourfold determination in four series).
15.8 g/l (QCL) 88.2 g/l (QCM) 180 g/l (QCH) Pool (mean 33.5 g/l)
Accuracy (%; mean)
Inter-assay imprecision (CV %)
112.7 110.4 102.3 –
14.9% 14.0% 7.2% 10.5%
M. Vogeser et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 399–401
analytical is novel. Two previous articles report methods also aiming to realise real-time signal normalisation in LC–MS/MS: Stahnke et al. [6] have reported a similar instrument configuration; in this method, however, not a solution of the target analyte itself but instead one single solution of an unrelated compound was infused for the generation of a reference signal in the analysis of more than 100 different pesticides. Since the modulation of ion generation by matrix compounds or ion source related factors can be highly analyte-specific, we assume that our approach – based on continuous infusion of a solution of the actual target analyte – might be more reliable. Kaufmann and Butcher [7] describe superimposition of analyte spikes during chromatography which is technically far more demanding compared to the technology described here. In the test reported here we used a syringe pump and a T-piece tube connection set-up in order to obtain a continuous introduction of the analyte to the ion source. This is also applicable in gradient methods; in the case of isocratic chromatographic systems, alternatively, the target analyte can as well simply be added to the mobile phase allowing a very simple configuration. At present, an evident shortcoming of our normalisation approach is the fact that positioning of the integration lines and construction of integration areas has to be performed “manually” in the software system. Inclusion of this post-column-infusion monitoring/normalisation principle into MS/MS-quantification software solutions with calculation of the AUP seems desirable. In order to obtain good reproducibility of the AUP it is essential that the chromatographic performance is stable and in particular that any peak tailing is constant. It must be noted that the principle described in this article cannot equalise between-sample variation of autosampler injection volume or of pre-ionisation analyte extraction procedures like solid phase extraction; only the ionisation process of a LC–MS/MS method can be monitored and normalised by this principle. Thus the availability of a related compound to be used as a “classical” internal standard which undergoes the entire analytical process including chromatographic separation certainly remains desirable for any analyte. However, autosampler precision is very high in many systems, so that the application of our principle also seems potentially applicable for clinical and biomedical research analyses in cases where all available candidate internal standard compounds are actually found to be inappropriate.
401
We have described in this article the proof-of-concept for a novel approach which can be useful for the monitoring and normalisation of between sample variations in the ionisation efficacy of LC-API–MS. Our evaluation results and experiences justify further validation of this approach for different analytes and the development of appropriate software solutions for application of this principle also in a routine setting. Further work will have to address the suitability of the approach described herein also in multi-analyte methods and over extended calibration concentration ranges. Furthermore a potential impact of this technology on the analytical sensitivity in trace concentration ranges has to be assessed. However, the principle may probably help to increase the reliability of LC–MS/MS analyses of “orphan” analytes for which appropriate IS compounds are not available. This may include entirely new compounds in the context of drug development, new illicit drugs in clinical toxicology but also endogenous compounds addressed in the development of innovative “metabolomic” multimethods. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2012.03.055. References [1] M. Vogeser, C. Seger, Pitfalls associated with the use of liquid chromatography– tandem mass spectrometry in the clinical laboratory, Clin. Chem. 56 (2010) 1234–1244. [2] T.M. Annesley, Ion suppression in mass spectrometry, Clin. Chem. 49 (2003) 1041–1044. [3] F. Behrens, M. Koehm, H. Burkhardt, Update 2011: leflunomide in rheumatoid arthritis – strengths and weaknesses, Curr. Opin. Rheumatol. 23 (2011) 282–287. [4] H. Rakhila, T. Rozek, A. Hopkins, S. Proudman, L. Cleland, M. James, M. Wiese, Quantitation of total and free teriflunomide (A77 1726) in human plasma by LC–MS/MS, J. Pharmaceut. Biomed. Anal. 55 (2011) 325–331. [5] US Department of Health and Human Services. Guidance for industry – bioanalytical method validation. Food and Drug Administration, Center for Drug Evaluation and Research, 2001. [6] H. Stahnke, T. Reemtsma, L. Alder, Compensation of matrix effects by postcolumn infusion of a monitor substance in multiresidue analysis with LC–MS/MS, Anal. Chem. 81 (2009) 2185–2192. [7] A. Kaufmann, P. Butcher, Segmented post-column addition; a concept for continuous response control of liquid chromatography/mass spectrometry peaks affected by signal suppression/enhancement, Rapid. Commun. Mass. Spectrom. 19 (2005) 611–617.