Journal of Chromatography A, 1327 (2014) 160
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Discussion
Microchip capillary electrophoresis–electrospray ionization mass spectrometry analysis of paracetamol metabolites in human urine: An intriguing case Giovanni Lentini a,∗ , Solomon Habtemariam b a b
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via E. Orabona 4, 70126 Bari, Italy Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK
a r t i c l e
i n f o
Article history: Received 25 July 2013 Accepted 20 December 2013 Available online 31 December 2013
a b s t r a c t The strange case of abnormal quantities of unmetabolized paracetamol found in human urine [J. Chromatogr. A 1218 (2011) 739] was examined and an alternative explanation was proposed. © 2013 Elsevier B.V. All rights reserved.
Keywords: Metabolism Capillary electrophoresis Mass spectrometry Glutathione conjugation
We recently read an interesting article appeared in this Journal which presents a relevant example of capillary electrophoresis–electrospray ionization mass spectrometry (CE-ESI/MS) microchip application in metabolism studies [1]. While congratulating the Authors for their novel method development approach, we were intrigued by their data analysis on urine samples collected from two 500 mg paracetamol-treated healthy volunteers. Four peaks were resolved by CE and on the basis of ESI mass spectral analysis, the first one was attributed to the glutathione (GSH) conjugated product while the overlapping second and third peaks were assigned to paracetamol glucuronide and mercapturate. With regard the fourth peak, it was asserted to be paracetamol. Indeed, the Authors correctly stated that ‘the content of unmetabolized paracetamol in urine was seemingly much higher than those of the metabolites’. They also noted that the paracetamol peak was ‘slightly tailing’. As an explanation to these observations, they made two statements: (i) the process of the biotransformation of xenobiotics is complex and ‘can greatly vary between individuals’; (ii) the dose assumed by the two volunteers was ‘relative high’ and, likely, this was the reason for the observed tailing of the paracetamol peak. Starting with this latter statement (ii), it is clearly incorrect: 500 mg of paracetamol is the dose usually adopted for acute administration
of paracetamol and very often even 1000 mg are used. With regard to the first statement (i), we do agree that genetic polymorphism can lead to some outlier results while studying the xenobiotics metabolism, but we find it hard to accept this explanation as just two humans/subjects were enrolled in the study. If we have to come with another hypothesis, why not assume that the fourth peak of their electopherogram, assigned to paracetamol, corresponds to paracetamol sulphate? Being this true, several aspects of the electropherogram would find explanation. First of all, it should be emphasized that paracetamol sulphate is one of the main metabolite of paracetamol [2] and the adopted pre-treatment by solid phase extraction allows the extraction of paracetamol sulphate [3]. Then, considering the low pH of the run buffer, we may accept the higher mobility of GSH conjugate while paracetamol glucuronate and mercapturate would move more slowly towards the cathode. Finally, the sulphate would be completely dissociated, regardless of the pH and, bearing a negative charge, would resist to EOF draining force. This would explain the order of detection while decomposition at the detector might account for both the molecular species revealed (apparently unmetabolized paracetamol) and the peculiar peak tailing observed. What do the Authors think about? References
∗ Corresponding author. Tel.: +39 080 5442744; fax: +39 080 5442050. E-mail addresses: [email protected], [email protected] (G. Lentini). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.12.068
[1] N. Nordman, T. Sikanen, M.-E. Moilanen, S. Aura, T. Kotiaho, S. Franssila, R. Kostiainen, J. Chromatogr. A 1218 (2011) 739. [2] B. Testa, A. Pedretti, G. Vistoli, Drug Discov. Today 17 (2012) 549. [3] I.D. Wilson, J.K. Nicholson, J. Pharmacol. Biomed. Anal. 6 (1988) 151.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Discussion
Microchip capillary electrophoresis–electrospray ionization mass spectrometry analysis of paracetamol metabolites in human urine: An intriguing case Giovanni Lentini a,∗ , Solomon Habtemariam b a b
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via E. Orabona 4, 70126 Bari, Italy Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK
a r t i c l e
i n f o
Article history: Received 25 July 2013 Accepted 20 December 2013 Available online 31 December 2013
a b s t r a c t The strange case of abnormal quantities of unmetabolized paracetamol found in human urine [J. Chromatogr. A 1218 (2011) 739] was examined and an alternative explanation was proposed. © 2013 Elsevier B.V. All rights reserved.
Keywords: Metabolism Capillary electrophoresis Mass spectrometry Glutathione conjugation
We recently read an interesting article appeared in this Journal which presents a relevant example of capillary electrophoresis–electrospray ionization mass spectrometry (CE-ESI/MS) microchip application in metabolism studies [1]. While congratulating the Authors for their novel method development approach, we were intrigued by their data analysis on urine samples collected from two 500 mg paracetamol-treated healthy volunteers. Four peaks were resolved by CE and on the basis of ESI mass spectral analysis, the first one was attributed to the glutathione (GSH) conjugated product while the overlapping second and third peaks were assigned to paracetamol glucuronide and mercapturate. With regard the fourth peak, it was asserted to be paracetamol. Indeed, the Authors correctly stated that ‘the content of unmetabolized paracetamol in urine was seemingly much higher than those of the metabolites’. They also noted that the paracetamol peak was ‘slightly tailing’. As an explanation to these observations, they made two statements: (i) the process of the biotransformation of xenobiotics is complex and ‘can greatly vary between individuals’; (ii) the dose assumed by the two volunteers was ‘relative high’ and, likely, this was the reason for the observed tailing of the paracetamol peak. Starting with this latter statement (ii), it is clearly incorrect: 500 mg of paracetamol is the dose usually adopted for acute administration
of paracetamol and very often even 1000 mg are used. With regard to the first statement (i), we do agree that genetic polymorphism can lead to some outlier results while studying the xenobiotics metabolism, but we find it hard to accept this explanation as just two humans/subjects were enrolled in the study. If we have to come with another hypothesis, why not assume that the fourth peak of their electopherogram, assigned to paracetamol, corresponds to paracetamol sulphate? Being this true, several aspects of the electropherogram would find explanation. First of all, it should be emphasized that paracetamol sulphate is one of the main metabolite of paracetamol [2] and the adopted pre-treatment by solid phase extraction allows the extraction of paracetamol sulphate [3]. Then, considering the low pH of the run buffer, we may accept the higher mobility of GSH conjugate while paracetamol glucuronate and mercapturate would move more slowly towards the cathode. Finally, the sulphate would be completely dissociated, regardless of the pH and, bearing a negative charge, would resist to EOF draining force. This would explain the order of detection while decomposition at the detector might account for both the molecular species revealed (apparently unmetabolized paracetamol) and the peculiar peak tailing observed. What do the Authors think about? References
∗ Corresponding author. Tel.: +39 080 5442744; fax: +39 080 5442050. E-mail addresses: [email protected], [email protected] (G. Lentini). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.12.068
[1] N. Nordman, T. Sikanen, M.-E. Moilanen, S. Aura, T. Kotiaho, S. Franssila, R. Kostiainen, J. Chromatogr. A 1218 (2011) 739. [2] B. Testa, A. Pedretti, G. Vistoli, Drug Discov. Today 17 (2012) 549. [3] I.D. Wilson, J.K. Nicholson, J. Pharmacol. Biomed. Anal. 6 (1988) 151.
