Subscriber access provided by READING UNIV
Comment
Comment on “Direct monitoring of exogenous GHB in body fluids by NMR spectroscopy”: several issue to consider when quantifying GHB in biological matrices. Simona Pichini, and Francesco Paolo Busardò Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03450 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 1, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Comment on “Direct monitoring of exogenous GHB in body fluids by NMR spectroscopy”: several issue to consider when quantifying GHB in biological matrices. Simona Pichini1, Francesco Paolo Busardò2 1 2
National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy Unit of Forensic Toxicology (UoFT), Department of Anatomical, Histological, Forensic and
Orthopedic Sciences, Sapienza University, Rome, Italy
Corresponding Author: Simona Pichini, PhD Head – Analytical Pharmacotoxicology Unit National Centre on Addiction and Doping, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161, Rome, Italy Tel +390649906545 Fax +390649902016 Email:
[email protected] We have read with great interest the article of Palomino-Schätzlein and colleagues [1] entitled “Direct monitoring of exogenous GHB in body fluids by NMR spectroscopy”, where the authors investigated the feasibility and suitability of NMR spectroscopy for the identification and quantification of exogenous GHB in urine and serum. In addition, the potential of NMR-based metabolomics for the identification of biomarkers indicative of GHB consumption was also evaluated. In our opinion, the study of Palomino-Schätzlein et al. gives rise to some points worthy of attention, discussion and clarification: -
One of the major challenges of GHB (γ-Hydroxybutyric acid) in forensic toxicology is represented by the fact that this compound is a naturally occurring in biological matrices. Therefore, when GHB is administered, special caution has to be paid in discriminating between endogenous and exogenous values. To overcome this issue, set-up GHB cut-offs play a crucial role. Palomino-Schätzlein et al. state that “Many studies establish a cut-off discriminant limit (10 mg/L, 0.1 mM) to distinguish external exposure from endogenous values”. However, this is only partially true because the reported cut-off is the one for urine, ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
both in ante- and post-mortem samples [2]. For blood samples, coming from living subjects, the cut-off presently used is 5 mg/L. This latter cut-off can be considered “precautionary”, because in reality endogenous GHB is usually detected in blood in the range of 0.5 – 1 mg/L, even if concentrations can increase depending on different storage conditions [3]. -
In the method proposed by Palomino-Schätzlein and colleagues “concentrations up to the 0.1 mM (10 mg/L) could be detected and quantified” which is significantly higher of that provided by GC-MS/MS or LC-MS/MS methods, which can quantify even less than 0.5 mg/L. Therefore, the application of this method does not allow to detect positive serum GHB cases with values below 10 mg/L [4,5].
-
In forensic practice, when cases of GHB-facilitated sexual assault occur, the victim goes to the emergency department usually some hours (or even some days) after the assault, when GHB for its rapid metabolism cannot be detected anymore in blood and urine or can be detected at very low concentrations. Therefore, it is crucial to have methods sensitive enough to distinguish between endogenous values and exogenous administration, both in conventional and non-conventional matrices, such as hair [2,3]. In this concern, we wish to underline that NMR technology is not generally available in laboratories that specialize in forensic toxicology and accordingly receive forensic samples for GHB analysis, such as in date-rape, DUID cases and sudden death investigations.
-
The storage condition applied by Palomino-Schätzlein et al. includes the lyophilization of serum and urine samples and the reconstitution of specimens in D2O prior to the analysis. However, it is advisable to perform experiments assessing if this process could interfere with GHB stability, which is known to be affected by different storage conditions [6]. Furthermore, always concerning GHB stability, in the description of the experimental design, it is apparent that NMR analysis was done on urine and serum samples collected some time before 2012 and used for previous GC-MS study [7]. It would have been interesting to compare the results obtain in the present NMR study with those from the original GC-MS method. In this way, the NMR method could have been cross-validated and verified for its reliability.
-
Taking into consideration the short detection window of exogenous GHB both in blood and in urine, different approaches have been used to detect this complicated compound for a longer period of time. They included continuous efforts to identify new GHB metabolites: GHB-glucuronide, GHB sulphate and β-citryl-glutamic acid [5,8-10]. In this study, for the first time the authors disclosed a significant increase of the concentration of urinary glycolate after GHB ingestion. The same glycolate, previously
ACS Paragon Plus Environment
Page 2 of 4
Page 3 of 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
associated to the metabolism of endogenous GHB, lasted in urine for a longer detection time window than that of exogenous GHB, being an interesting candidate for further studies, proving also its specificity. -
Regarding the conclusions reported by the authors about their method, which “would allow to distinguish between GHB and associated drugs (γ-butyrolactone-GBL, and 1,4butanediol- BD) by the same analysis” it is important to underline that although these analytes have distinct NMR spectra and therefore could be differentiated, the present NMR method focuses only on exogenous GHB following a single dose administration in twelve healthy volunteers, whereas no experiments were performed on GBL and BD. Moreover, differently from other published methods where GHB was detected and quantified with other related compounds such as GBL and GABA (γ-aminobutyric acid) in real biological samples, in this study only GHB, under the form of its sodium salt, was administered and searched. Hence, further studies should be performed including biological samples of subjects who have consumed GBL and/or BD, to assess what is stated by the authors in their conclusions. In routine practice, methods presently used to detect and quantify GHB in different biological matrices by hyphenated chromatographic techniques coupled with mass spectrometry allow a complete separation of GHB from other related analytes and identification by specific ion fragments [5]. In addition, it is important to underline that after ingestion both GBL and BD are very rapidly converted into GHB and only the latter analyte is detected [3].
In summary, we would conclude that, though presenting some limitations, Palomino-Schätzlein et al. study has made a contribution to the direct and indirect detection of exogenous GHB in urine with non- destructive technique such as that of NMR. However, we believe that the points listed in this letter should be considered to improve the proposed technique for a more accurate, reliable detection of exogenous GHB intake, restricted to the biological matrices where analytical limit of quantification allow to precisely discriminate between endogenous and exogenous GHB. In our opinion, once amended the points raised in this comment, the NMR procedure described by Palomino-Schätzlein et al. could complement other analytical approaches currently used for GHB measurement in forensic toxicology, but presently it is not sufficiently settled to replace conventional and more well‐established chromatographic methods in use worldwide. .
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
References: 1. Palomino-Schätzlein, M.; Wang, Y.; Parella, T.; Legido-Quigley, C.; Pérez-Trujillo, M. Anal. Chem. 2017, 89 (16):8343-8350. 2. Busardò, F.P.; Kyriakou, C. Recent Pat. Biotechnol. 2014, 8, (3) 206-214. 3. Busardò, F.P.; Jones, A.W. Curr Neuropharmacol. 2015,13, (1) 7-70. 4. Castro, A.L.; Tarelho, S.; Dias, M.; Reis, F.; Teixeira, H.M.. J. Pharm. Biomed. Anal. 2016,119,139-144 5. Busardò, F.P.; Kyriakou, C.; Marchei, E.; Pacifici, R.; Pedersen, D.S.; Pichini S. J. Pharm. Biomed. Anal. 2017,137, 123-131. 6. Busardò, F.P.; Zaami, S.; Baglio, G.; Indorato, F.; Montana, A.; Giarratana, N.; Kyriakou, C.; Marinelli, E.; Romano G.. Eur. Rev. Med. Pharmacol. Sci. 2015 ,19, (21) 4187-4194. 7. Brailsford, A.D.; Cowan, D.A.; Kicman, A.T.. J Anal Toxicol. 2012, 36, (2) 8-95. 8. Petersen, I,N,; Tortzen, C.; Kristensen, J.L.; Pedersen, D.S.; Breindahl T J. Anal. Toxicol. 2013, 37, (5) 291-297. 9. Hanisch S, Stachel N, Skopp G A potential new metabolite of gamma-hydroxybutyrate: sulfonated gamma-hydroxybutyric acid. Int. J. Legal Med. 2016,130, (2) 411-414. 10. Piper, T.; Mehling, L.M.; Spottke, A.; Heidbreder, A.; Young, P.; Madea, B.;, Hess, C.; Schänzer, W.; Thevis, M. Forensic Sci Int. 2017, 279, 157-164.
ACS Paragon Plus Environment
Page 4 of 4
