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Response to Comment on “Direct monitoring of exogenous GHB in body fluids by NMR spectroscopy”: several issues to consider when quantifying GHB in biological matrices Martina Palomino-Schätzlein, Alan David Brailsford, Teodor Parella, and Míriam Pérez-Trujillo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04473 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 1, 2017
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Response to
Comment on “Direct monitoring of exogenous GHB in body fluids by NMR spectroscopy”: several issues to consider when quantifying GHB in biological matrices, by Simona Pichini, Francesco Paolo Busardò. Martina Palomino-Schätzlein,† Alan D. Brailsford⊥, Teodor Parella§, Míriam Pérez-Trujillo§*
†NMR Facility, Centro de Investigación Príncipe Felipe (CIPF), C. Eduardo Primo Yúfera 3, 46012 Valencia, Spain ⊥Department of Forensic Sciences and Drug Monitoring, Drug Control Centre, King’s College London, SE1 9NH London, United Kingdom §Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Barcelona, Spain
In response to the letter from Pichini and Busardò, we would like to stress that we do not suggest NMR as an alternative technique to GC-MS or LC-MS, but complementary to them (which could provide additional valuable information), as was stated in the last sentence of the conclusions of the published article.
· In the introduction we quoted the cut-off discriminant limit of 10 mg/L, since this is the one that we mentioned later in the Results and Discussion section Quantifying Exogenous GHB in Urine by 1H qNMR, without specifying that this is for urine. We see that it can be confusing without specifying that this corresponds to urine solely (though in the reference given, reference 18, Castro et al. 2014, this is contemplated). It would be more accurate and avoid confusion if the bold text was included: “(10 mg/L in urine and 5 mg/L in blood)...” in the Introduction section and “discriminant limit of 10 mg/L (ca. 0.10 mM) in urine has been defined...” in the section Quantifying Exogenous GHB in Urine by 1H qNMR. · Regarding the section Quantifying Exogenous GHB in Urine by 1H qNMR, showing the results of experiments of quantification in urine, we agree with Dr. Pichini and Dr. Busardò that the
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section deserves a clarification. The value given in the manuscript (“With standard equipment for metabolomics studies (500-600 MHz magnets with cryo or conventional probes) concentrations up to 0.1 mM could be detected and quantified.48”) does not correspond to the detection limit of the method. We wanted to state that metabolite concentration values of the order of 0.1 mM (cut-off discriminant limit for urine) have been previously quantified by 1D 1
H qNMR. However, lower metabolite concentrations have been detected and quantified by 1H
qNMR as shown in recent literature (1,2). Metabolite concentration values in the low µM range (e.g. 1 and 10 µM in urine and serum respectively) have been detected and quantified using similar NMR equipment (1,2). It was not the intention of this first work to make a complete/broad study about quantification of GHB in biofluids by NMR, but to show that NMR yielded interesting results in the urine example shown, in accordance with those yielded previously by another technique (GC-MS). Concerning the availability of NMR spectrometers, it is true that currently they are not generally present in forensic laboratories, but this situation could change in the next years. For instance, the development of increasingly sophisticated bench-top NMR spectrometers could make available NMR spectroscopy even in forensic toxicology laboratories. Precisely for this reason, studies as ours, presenting forensic analysis based on NMR spectroscopy highlighting the valuable complementary information that this technique can provide, are essential. On the other hand, NMR spectrometers are largely present in universities, research institutions and companies, which can proportionate a quick sample analysis service. In our opinion, the lack of equipment (when this can be available in other laboratories) should never be a reason to prevent advancing or doing complementary analyses if they could help to solve the problem.
· Lyophilization is a technique widely applied for the conservation of biological samples well known to preserve the chemical and biological characteristics of the sample (3), especially if the sample is reconstituted in the initial solvent (water in our case). Problems may arise with compounds that are volatile or quite insoluble, which is not the case for GHB. Also, the freezing process that is carried out as part of the lyophilization will not have a significant impact on GHB concentration, as previous studies about the stability of freeze-thaw cycles with GHB have shown (4,5). Furthermore, GHB is even commercialized in lyophilized form (Bühlman Laboratoires AG). Though in principle, nothing indicates that GHB would be affected by lyophilization, we have performed an experiment comparing the 1H NMR spectra of a same sample of urine containing GHB, prepared and analyzed following the procedure described in the manuscript, being the difference between them that in case (a) it was not ACS Paragon Plus Environment
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lyophilized and in case (b) it was. As shown, the intensity of H3 GHB signal was not affected (Figure 1).
Figure 1. 1H NMR spectra (NOESY-presat) of a same urine sample containing GHB, which (a) was not and (b) was lyophilized before NMR sample preparation. Spectra acquired at a magnetic field of 600.13 MHz and at 298.0 K of temperature.
As mentioned in the published article, the results concerning the pharmacokinetics of GHB in urine obtained in the present NMR study were compared with those from a previous work conducted by GC-MS. Our samples corresponded to store frozen aliquots of the same sample
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pool (same clinical trial) (“1H qNMR results were in accordance with those reported by
Brailsford et al.,15 in which aliquots of the same sample pool were analyzed... In that study, a Tmax of 1 h, a Cmax of 67.6 mg/L, and similar pharmacokinetic profiles (after 4 h, GHB concentrations were at an endogenous level) were obtained. Our results are also within the same range of other published data.15”).
· Both, approaching the problem using a metabolomics top-down method plus choosing NMR spectroscopy as the analytical platform (with the ability to detect and assign a broad spectrum of very different metabolites in a sample without any previous treatment, no separation or derivatization steps) have greatly contributed to find and identify glycolate as one of the metabolites, the concentration of which significantly increases after GHB intake. We think that it would be highly interesting to confirm this result with other analytical techniques, especially those that are more sensitive and may be able to detect altered glycolate concentrations even at a larger time window. Concerning other GHB metabolites, the interesting work of Petersen et al. 2013 regarding GHB-glucoronide, has been referenced and mentioned in the article (ref. 22, in the introduction and in the section regarding the metabolomic study). Also, we have paid attention to the search of this GHB derivative without observing it (as mentioned in the manuscript). · What we meant with the complete sentence (“NMR spectroscopy allowed the quick monitoring
of exogenous GHB within the almost intact body fluid; simultaneously, it yielded interesting information on the complete matrix, and it would allow one to distinguish between GHB and associated drugs (GBL and BD) by the same analysis”) is that, when these molecules are present in the sample under analysis (i.e. the final collected sample), the technique (NMR) would be able to differentiate them and identify them directly and simultaneously from the same spectrum, coming from an almost intact matrix without prior chromatographic or derivatization steps. We understand that our comment may not be obvious for scientists that are not familiar with NMR. The NMR spectra of real samples of GBL and BD have been previously described (6,7); in the case of GBL even within urine. These molecules, as well as GHB, have been thus characterized by NMR. Both molecules generate NMR signals, under the same experimental conditions, that are different from the signals of GHB (have different chemical shifts), which would allow their differentiation directly by 1D 1H NMR or, when overlapping with other metabolite signals, using deconvolution methods or 2D experiments, which would spread signals in a second dimension and increase spectral dispersion. We agree
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that, though it seems an obvious statement, it is not supported by any experiment (just the data from the references and scientific reasoning) and the conclusions is not the adequate section to write it, but in the discussion.
We thank Dr. Pichini and Dr. Busardò for their constructive comments and questions.
References (1) Bouatra, S.; Aziat, F.; Mandal, R.; Guo, A. C.; Wilson, M. R. et al. PLoS One. 2013, 8 (9), e73076. (2) Psychogios, N.; Hau, D. D.; Peng, J.; Guo, A. C.; Mandal, R.et al. PLoS One. 2011, 6 (2), e16957. (3) Wu, Y.; Wu, M.; Zhang ,Y.; Li, W.; Gao, Y. et al. Amino acids. 2012, 43 (3), 1383-1388. (4) LeBeau, M. A.; Miller, M. L.; Levine, B. Forensic Sci. Int. 2001, 119 (2), 161-167. (5) Chen, M.; Andrenyak, D. M.; Moody, D. E.; Foltz, R. L. J. Anal. Toxicol. 2003, 27 (7), 445-448. (6) Del Signore, A. G.; McGregor, M.; Cho, B. P. J Forensic Sci. 2005, 50 (1), 81-86. (7) Spectral Database for Organic Compounds SDBS (http://sdbs.db.aist.go.jp).
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