Journal of Chromatography B, 981–982 (2015) 40–47
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of tapentadol and tapentadol-O-glucuronide in human serum samples by UPLC–MS/MS Vera Hillewaert a,∗ , Klaus Pusecker b , Luc Sips a , Tom Verhaeghe a , Ronald de Vries a , Manfred Langhans b , Rolf Terlinden b , Philip Timmerman a a b
Department of Bioanalysis, Janssen Research and Development, Turnhoutseweg 30, B-2340 Beerse, Belgium Grünenthal GmbH, Zieglerstraße 6, 52078 Aachen, Germany
a r t i c l e
i n f o
Article history: Received 2 June 2014 Accepted 14 December 2014 Available online 8 January 2015 Keywords: Tapentadol Serum Mass spectrometry LC–MS/MS UPLC Assay validation
a b s t r a c t Tapentadol is a novel, centrally acting analgesic with 2 mechanisms of action, MOR agonism and noradrenaline (NA) reuptake inhibition in a single molecule. It is the first member of a new therapeutic class, MOR-NRI. A high throughput liquid chromatography-tandem mass spectrometric (LC–MS/MS) assay was developed and validated for the quantitative analysis of tapentadol and its O-glucuronide metabolite in human serum. Simultaneous quantification was deemed to be challenging because of the large difference in concentrations between tapentadol and its O-glucuronide metabolite in clinical samples. Therefore, a method was established using a common processed sample, but with different injection volumes and chromatographic conditions for each analyte. Tapentadol and tapentadol-O-glucuronide were determined by protein precipitation of 0.100 ml of the samples with acetonitrile. The internal standards used are D6 -tapentadol and D6 -tapentadol-O-glucuronide. The validated concentration range was 0.200–200 ng/ml (tapentadol) and 10.0–10,000 ng/ml (tapentadol-O-glucuronide). Chromatographic separation was achieved by gradient elution on a Waters Acquity UPLC BEH C18 (1.7 m, 2.1 × 50 mm) column, with mobile phase consisting of 0.01 M ammonium formate (adjusted to pH 4 using formic acid) (A) and methanol (B). A separate injection was done for measurement of each analyte, with a different gradient and run time. The analytes were detected by using an electrospray ion source on a triple quadrupole mass spectrometer operating in positive ionization mode. The run time was 1.6 min for tapentadol and 1.5 min for tapentadol-O-glucuronide. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of serum samples in clinical trials. The validated method was used for analysis of tapentadol in over 17,000 samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tapentadol is a novel, centrally acting opioid analgesic for the treatment of moderate to severe acute or chronic pain, such as pain following injury or surgery, painful diabetic peripheral neuropathy and chronic low back pain. It has been approved as immediaterelease and as extended-release tablets by the United States Food and Drug administration, by Health Canada and a European decentralized registration procedure was successfully completed for the European Union countries. Tapentadol differs from classic opioids such as morphine or oxycodone in a number of important ways. It combines 2 mechanisms of action, opioid receptor (MOR) agonism and noradrenaline
∗ Corresponding author. Tel.: +32 14605820. E-mail address: [email protected] (V. Hillewaert). http://dx.doi.org/10.1016/j.jchromb.2014.12.013 1570-0232/© 2015 Elsevier B.V. All rights reserved.
reuptake inhibition (NRI) in a single molecule. It shows only moderate affinity for MOR and offers a second, non-opioid mechanism of action (NRI) that contributes to the analgesic effect [1–3]. Due to the lower MOR affinity opioid-typical side effects are reduced (literature). This results in much lower discontinuation rates. Tapentadol is well tolerated. Excretion is mainly via urine, for a large part as conjugated metabolites, for a lesser part as other metabolites, and almost no unchanged tapentadol is excreted. Hence it is eliminated by metabolism, mainly via direct glucuronidation (phase II metabolism) to tapentadol-O-glucuronide [4,5]. Many classic opioids form active metabolites that contribute to analgesic and/or side effects. Tapentadol does not have active metabolites, so there is no resulting variability in analgesic effect. In the literature, recent analytical methods were summarized [6], listing only few publications in which an analytical method for tapentadol determination is described. An LC–MS/MS method
V. Hillewaert et al. / J. Chromatogr. B 981–982 (2015) 40–47
[7] using solid phase extraction was developed for oral fluid and urine with a limit of quantification of 10 and 50 ng/ml, an HPLC/FL method [8] using liquid/liquid extraction was developed for canine plasma with a limit of quantification of 1 ng/ml, and a UPLC–MS/MS method [9] using hydrolysis was also described for urine samples with a limit of quantification of 50 ng/ml. For the bioanalytical support of clinical studies, in 2000 separate methods were developed for the measurement of tapentadol and its O-glucuronide metabolite in human serum at Grünenthal (GRT), Aachen, Germany. In 2005, a combined method for the quantification of tapentadol and tapentadol-O-glucuronide in serum was developed and validated at the bioanalytical group of Janssen Research & Development in Beerse, Belgium. The large difference in the calibration range for tapentadol (low concentrations) and tapentadol-O-glucuronide (high concentrations) required detuning of the tapentadol-O-glucuronide to decrease its sensitivity to keep the response in the linear range of the detector. There was also fluctuation in the internal standard areas for tapentadol-Oglucuronide. For this reason, and because the clinical program did not require analysis of the glucuronide in each study, the combined method was adapted. The throughput was increased by using UPLC instead of HPLC, resulting in a reduced run time from 5.5 to 1.6 min, and two separate injections are done out of the same processed sample, one for tapentadol measurement and one for tapentadol-Oglucuronide measurement. The validated range remained the same. All methods were based on protein precipitation; they differed in chromatographic conditions and detection. In this publication, the high throughput UPLC–MS/MS method with one sample preparation and two separate injections is described. The method was optimized to obtain a good chromatographic separation between tapentadol and two phase 2 metabolites, tapentadol-sulfate and tapentadol-O-glucuronide in, order to prevent impact on the tapentadol quantification due to in-source fragmentation of the phase 2 metabolites. Also, investigations were conducted to ensure that the phase 2 metabolites would not degrade to tapentadol during sample handling, storage and extraction. The assay was validated in two laboratories according to current Guidelines on bioanalytical Method Validation. The assay been used for the support of a number of clinical studies, in which more than 17,000 samples have been analyzed. 2. Experimental 2.1. Chemicals and reagents Reference standards for tapentadol, tapentadol-O-glucuronide and tapentadol-sulfate and internal standards D6 -tapentadol and D6 -tapentadol-O-glucuronide were supplied by Grünenthal (Aachen, Germany). Tapentadol has two chiral centers and is manufactured as the RR stereoisomer. Methanol and acetonitrile were of spectrophotometric grade (Merck, Darmstadt, Germany). Deionized water was prepared using a Milli-Q water purification system (Millipore, Billerica, MA, USA). Ammonium formate (Acros, Geel, Belgium) and formic acid (Merck, Darmstadt, Germany) in analytical grade were used. Blank human serum was obtained from Bioreclamation (Hicksville, NY, USA). Chemical structures of the analytes and internal standards are shown in Fig. 1. 2.2. Standard solutions, calibration curve and QC samples Stock solutions of tapentadol, tapentadol-O-glucuronide, D6 tapentadol and D6 -tapentadol-O-glucuronide (100 g/ml) were prepared in methanol. The stock solutions of tapentadol and
41
Fig. 1. Structures of tapentadol and internal standard (D6 -tapentadol), tapentadol O-glucuronide and internal standard (D6 -tapentadol-O-glucuronide) and tapentadol sulfate.
tapentadol-O-glucuronide were combined and thereafter serially diluted in methanol resulting in spike solutions. The spike solutions were spiked to blank human serum (spike volume
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of tapentadol and tapentadol-O-glucuronide in human serum samples by UPLC–MS/MS Vera Hillewaert a,∗ , Klaus Pusecker b , Luc Sips a , Tom Verhaeghe a , Ronald de Vries a , Manfred Langhans b , Rolf Terlinden b , Philip Timmerman a a b
Department of Bioanalysis, Janssen Research and Development, Turnhoutseweg 30, B-2340 Beerse, Belgium Grünenthal GmbH, Zieglerstraße 6, 52078 Aachen, Germany
a r t i c l e
i n f o
Article history: Received 2 June 2014 Accepted 14 December 2014 Available online 8 January 2015 Keywords: Tapentadol Serum Mass spectrometry LC–MS/MS UPLC Assay validation
a b s t r a c t Tapentadol is a novel, centrally acting analgesic with 2 mechanisms of action, MOR agonism and noradrenaline (NA) reuptake inhibition in a single molecule. It is the first member of a new therapeutic class, MOR-NRI. A high throughput liquid chromatography-tandem mass spectrometric (LC–MS/MS) assay was developed and validated for the quantitative analysis of tapentadol and its O-glucuronide metabolite in human serum. Simultaneous quantification was deemed to be challenging because of the large difference in concentrations between tapentadol and its O-glucuronide metabolite in clinical samples. Therefore, a method was established using a common processed sample, but with different injection volumes and chromatographic conditions for each analyte. Tapentadol and tapentadol-O-glucuronide were determined by protein precipitation of 0.100 ml of the samples with acetonitrile. The internal standards used are D6 -tapentadol and D6 -tapentadol-O-glucuronide. The validated concentration range was 0.200–200 ng/ml (tapentadol) and 10.0–10,000 ng/ml (tapentadol-O-glucuronide). Chromatographic separation was achieved by gradient elution on a Waters Acquity UPLC BEH C18 (1.7 m, 2.1 × 50 mm) column, with mobile phase consisting of 0.01 M ammonium formate (adjusted to pH 4 using formic acid) (A) and methanol (B). A separate injection was done for measurement of each analyte, with a different gradient and run time. The analytes were detected by using an electrospray ion source on a triple quadrupole mass spectrometer operating in positive ionization mode. The run time was 1.6 min for tapentadol and 1.5 min for tapentadol-O-glucuronide. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of serum samples in clinical trials. The validated method was used for analysis of tapentadol in over 17,000 samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tapentadol is a novel, centrally acting opioid analgesic for the treatment of moderate to severe acute or chronic pain, such as pain following injury or surgery, painful diabetic peripheral neuropathy and chronic low back pain. It has been approved as immediaterelease and as extended-release tablets by the United States Food and Drug administration, by Health Canada and a European decentralized registration procedure was successfully completed for the European Union countries. Tapentadol differs from classic opioids such as morphine or oxycodone in a number of important ways. It combines 2 mechanisms of action, opioid receptor (MOR) agonism and noradrenaline
∗ Corresponding author. Tel.: +32 14605820. E-mail address: [email protected] (V. Hillewaert). http://dx.doi.org/10.1016/j.jchromb.2014.12.013 1570-0232/© 2015 Elsevier B.V. All rights reserved.
reuptake inhibition (NRI) in a single molecule. It shows only moderate affinity for MOR and offers a second, non-opioid mechanism of action (NRI) that contributes to the analgesic effect [1–3]. Due to the lower MOR affinity opioid-typical side effects are reduced (literature). This results in much lower discontinuation rates. Tapentadol is well tolerated. Excretion is mainly via urine, for a large part as conjugated metabolites, for a lesser part as other metabolites, and almost no unchanged tapentadol is excreted. Hence it is eliminated by metabolism, mainly via direct glucuronidation (phase II metabolism) to tapentadol-O-glucuronide [4,5]. Many classic opioids form active metabolites that contribute to analgesic and/or side effects. Tapentadol does not have active metabolites, so there is no resulting variability in analgesic effect. In the literature, recent analytical methods were summarized [6], listing only few publications in which an analytical method for tapentadol determination is described. An LC–MS/MS method
V. Hillewaert et al. / J. Chromatogr. B 981–982 (2015) 40–47
[7] using solid phase extraction was developed for oral fluid and urine with a limit of quantification of 10 and 50 ng/ml, an HPLC/FL method [8] using liquid/liquid extraction was developed for canine plasma with a limit of quantification of 1 ng/ml, and a UPLC–MS/MS method [9] using hydrolysis was also described for urine samples with a limit of quantification of 50 ng/ml. For the bioanalytical support of clinical studies, in 2000 separate methods were developed for the measurement of tapentadol and its O-glucuronide metabolite in human serum at Grünenthal (GRT), Aachen, Germany. In 2005, a combined method for the quantification of tapentadol and tapentadol-O-glucuronide in serum was developed and validated at the bioanalytical group of Janssen Research & Development in Beerse, Belgium. The large difference in the calibration range for tapentadol (low concentrations) and tapentadol-O-glucuronide (high concentrations) required detuning of the tapentadol-O-glucuronide to decrease its sensitivity to keep the response in the linear range of the detector. There was also fluctuation in the internal standard areas for tapentadol-Oglucuronide. For this reason, and because the clinical program did not require analysis of the glucuronide in each study, the combined method was adapted. The throughput was increased by using UPLC instead of HPLC, resulting in a reduced run time from 5.5 to 1.6 min, and two separate injections are done out of the same processed sample, one for tapentadol measurement and one for tapentadol-Oglucuronide measurement. The validated range remained the same. All methods were based on protein precipitation; they differed in chromatographic conditions and detection. In this publication, the high throughput UPLC–MS/MS method with one sample preparation and two separate injections is described. The method was optimized to obtain a good chromatographic separation between tapentadol and two phase 2 metabolites, tapentadol-sulfate and tapentadol-O-glucuronide in, order to prevent impact on the tapentadol quantification due to in-source fragmentation of the phase 2 metabolites. Also, investigations were conducted to ensure that the phase 2 metabolites would not degrade to tapentadol during sample handling, storage and extraction. The assay was validated in two laboratories according to current Guidelines on bioanalytical Method Validation. The assay been used for the support of a number of clinical studies, in which more than 17,000 samples have been analyzed. 2. Experimental 2.1. Chemicals and reagents Reference standards for tapentadol, tapentadol-O-glucuronide and tapentadol-sulfate and internal standards D6 -tapentadol and D6 -tapentadol-O-glucuronide were supplied by Grünenthal (Aachen, Germany). Tapentadol has two chiral centers and is manufactured as the RR stereoisomer. Methanol and acetonitrile were of spectrophotometric grade (Merck, Darmstadt, Germany). Deionized water was prepared using a Milli-Q water purification system (Millipore, Billerica, MA, USA). Ammonium formate (Acros, Geel, Belgium) and formic acid (Merck, Darmstadt, Germany) in analytical grade were used. Blank human serum was obtained from Bioreclamation (Hicksville, NY, USA). Chemical structures of the analytes and internal standards are shown in Fig. 1. 2.2. Standard solutions, calibration curve and QC samples Stock solutions of tapentadol, tapentadol-O-glucuronide, D6 tapentadol and D6 -tapentadol-O-glucuronide (100 g/ml) were prepared in methanol. The stock solutions of tapentadol and
41
Fig. 1. Structures of tapentadol and internal standard (D6 -tapentadol), tapentadol O-glucuronide and internal standard (D6 -tapentadol-O-glucuronide) and tapentadol sulfate.
tapentadol-O-glucuronide were combined and thereafter serially diluted in methanol resulting in spike solutions. The spike solutions were spiked to blank human serum (spike volume
