Complementary approach for accurate determination of carbon isotopic compositions in γ-hydroxybutyric acid using gas chromatography/combustion-isotope ratio mass spectrometry

Article abstract of DOI:10.1002/rcm.8481

Rationale: γ-Hydroxybutyric acid (GHB) is a naturally endogenous neurotransmitter that is popular as a recreational drug due to its sedative, hypnotic, and euphoric effects. GHB derived from endogenous production or exogenous ingestion has been effectively discriminated by carbon isotopic compositions (δ¹³C values) through gas chromatography/combustion-isotope ratio mass spectrometry (GC/C-IRMS). However, an unintended uncertainty of isotopic signatures caused by a wide range of GHB quantities remains unsolved when using only single-isotope corrections of the di-TMS derivative. Methods: The δ¹³C values of the original GHB standard were first determined by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). The δ¹³C values of silylated GHB in concentrations from 10 to 500 ppm were determined by GC/C-IRMS. With respect to the silylated reaction products, the correction of δ¹³C values for the introduced carbons was calculated from a stoichiometric mass balance equation. Results: The results showed a significant quantity-dependent trend in δ¹³C values of introduced carbon (δ¹³C_di-TMS values) with increased GHB standard concentrations (r² = 0.70, p <0.05). We applied a logarithmic equation to determine isotopic data in low-GHB urine specimens from five healthy female volunteers. The δ¹³C_GHB values in urine samples corrected with quantity-dependent δ¹³C_di-TMS values were different by an average of 2.7‰ from those corrected with single δ¹³C_di-TMS values (p <0.05). Conclusions: Our results suggest that the overall residual amount-dependent isotope fractionation should be mathematically corrected by the logarithmic function and this may improve the reliability of isotopic analysis to evaluate the origin of GHB before applying the approach to routine toxicological and forensic studies.

Full text of DOI:10.1002/rcm.8481

Shin Kyung-Hoon (Orcid ID: 0000-0002-3169-4274)
Complementary approach for accurate determination of carbon isotopic compositions
in γ-hydroxybutyric acid using gas chromatography-combustion-isotope ratio mass
spectrometry
Dong-Hun Lee1†, Hyojeong Kim2†, Hee Young Yun1, Meejung Park3, Sanggil Choe3,
Sanghwan In3, Eunmi Kim3, Eunyoung Han2,*, Kyung-Hoon Shin1,* *
1Hanyang University, 55 Hanyangdaehak-ro, Sangrok-gu, Ansan 15588, Republic of Korea
2College of Pharmacy, Duksung Women’s University, 144 Samyang-ro, Dobong-gu, Seoul
01369, Republic of Korea
3National Forensic Service, Republic of Korea
†These authors contributed equally to this study as first authors.
*Corresponding author:
Tel.: +82 02 901 8386
homepage2600@duksung.ac.kr
**Corresponding author:
Tel.: +82 31 400 5536
shinkh@hanyang.ac.kr
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/rcm.8481
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ABSTRACT
RATIONALE: γ-Hydroxybutyric acid (GHB) is a naturally endogenous neurotransmitter but
it is popular as a recreational drug due to its sedative, hypnotic, and euphoric effects. GHB
derived from endogenous production or exogenous ingestion has been effectively
discriminated by carbon isotopic compositions (δ13C values) through gas chromatography-
combustion-isotope ratio mass spectrometry (GC/C-RIMS). However, an unintended
uncertainty of isotopic signatures caused by a wide range of GHB quantities remains unsolved
using single-isotope corrections of di-TMS.
METHODS: The δ13C values of the original GHB standard were first determined by elemental
analyzer-isotope ratio mass spectrometry (EA/IRMS). The δ13C values of silylated GHB in
concentrations from 10 to 500 ppm were determined by GC/C-IRMS. With respect to the
silylated reaction products, the correction of δ13C values for the introduced carbons was
calculated from a stoichiometric mass balance.
RESULTS: The results showed a significant quantity-dependent trend in δ13C values of
introduced carbon (δ13Cdi-TMS values) with increased GHB standard concentrations (R2 = 0. 70,
p < 0.05). We applied the logarithmic equation to determine isotopic data in low-GHB urine
specimens from five healthy female volunteers. The δ13CGHB values in urine samples corrected
with quantity-dependent δ13Cdi-TMS values were different by an average of 2.7‰ from those
corrected with single δ13Cdi-TMS values (p < 0.05).
CONCLUSIONS: Our results suggest that the overall residual amount-dependent isotope
fractionation should be mathematically corrected by the logarithmic function and this may
improve the reliability of isotopic analysis to evaluate the origin of GHB before applying the
approach to routine toxicological and forensic studies.
Keywords: γ-hydroxybutyric acid (GHB), gas chromatography-combustion-isotope ratio mass
spectrometry (GC/C-IRMS), carbon isotopic compositions (δ13C values), silylation, isotopic
fractionation
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Introduction
γ-Hydroxybutyric acid (GHB) is an endogenous organic acid that is a primary inhibitory
neurotransmitter in the central nervous system 1,2. Since its first synthesis in 1964, GHB has
been prescribed for treatment of narcolepsy and alcohol dependence in some countries (e.g.,
USA, Italy, and Austria) 3,4. However, GHB often has been illegally abused or misused due to
its strong anesthetic effect in drug-facilitated crimes such as robberies, sexual assaults, and
fraudulent gambling 5–7. Thus, together with GHB presenting a serious social problem,
identification of endogenous and exogenous GHB is a critical issue with regard to supporting
toxicological and forensic investigations.
Several approaches have been attempted to distinguish between exogenous and
endogenous GHB. For instance, quantitative approaches to confirm the origin of GHB have
been conducted by establishing a reference range of naturally occurring GHB concentrations
in tissues (such as human blood, urine, and saliva 8–10) and testing the cut-off against excessive
quantities by exogenous ingestion 8,10. The quantitative approach may be problematic due to
rapid metabolization of GHB to γ-aminobutyric acid (GABA) and/or succinic semialdehyde
2,11 and a possible time gap of several hours between drug ingestion and sample collection. On
the other hand, the carbon isotopic compositions (δ13C values) of GHB have been employed to
separate blood samples derived from endogenous GHB production and those from exogenous
abuse, showing a significant isotopic difference (17.1 ± 1.7‰) 12,13, because the isotope
approach can be independent of sample quantity. The isotopic approach is known to effectively
distinguish various carbon sources, which reflect dietary or non-dietary origin, as well as GHB
synthesis routes via naturally occurring chemical precursors such as γ-butyrolactone and 1,4-
butanediol 12–15. Accordingly, the use of GHB δ13C values can be an alternative way to
overcome the limitations of a quantitative approach 12,13,15
.
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With the isotopic approach, using gas chromatography-combustion-isotope ratio mass
spectrometry (GC/C-IRMS), methodological developments have improved the accuracy of
isotopic measurements 16–18. For instance, multiple factors involved with sample derivatization,
injection, chromatography, combustion, and ionization procedures can decrease the accuracy
of isotopic data 16,19. Furthermore, sample quantities can modify the δ13C values of C-
containing compounds such as amino acids 20 and phenols 16. Such issues have been evaluated
by (1) measuring samples and standard compounds of interest under identical set-up conditions,
(2) using quantities of standards similar to those of samples, and (3) calibrating samples relative
to standards 18,21. However, studies that involve the matching of standard quantities to sample
16
quantities are tedious and costly . It was recently recommended a best fit function of the
dependence of isotopic change on standard quantity should be determined 16,20, reflecting an
exact range of sample concentrations. Nevertheless, a quantity-dependent strategy has not yet
been explored with regard to GHB source evaluation, since traditional approaches assume no
effect of analyte quantities on isotopic signals. When considering an extremely variable
spectrum of GHB abundance in biological samples 22–24, a quantity-dependent correction could
be a better option for tracing GHB origins through δ13C values.
In this study, our objectives were to reduce the analytical uncertainty regarding sample
quantity-dependent isotopic signals and improve the potential for GHB source identification in
GC/C-IRMS systems. For this, we assessed the variation of isotopic signals over a variable
GHB standard range of 10 to 500 ppm and found the best-fit functions that could correct
isotopic changes that occurred during the analytical process. We than evaluated a valid method
for determining accurate δ13CGHB values of GHB quantities using a quantity-dependent
equation. Our approach will help determine the origin of GHB for various biological samples
using GC/C-IRMS.
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Experimental
Reagents and chemicals
The GHB standard was purchased from the Ministry of Food and Drug Safety (MFDS),
Chungcheongbuk-do, South Korea. All chemical reagents for purification and analysis were of
HPLC grade. The 99% BSTFA+1% TMCS used as a derivatization regent was purchased from
Sigma-Aldrich (St. Louis, MO, USA). A certified n-alkane mixture standard (A6 mixture) was
supplied by Indiana University (Bloomington, IN, USA).
Preparation of the derivatized GHB standard
GHB purification and silylation were performed according to a previously reported method
12,25. In short, GHB was purified using an SPE-MCX SPE cartridge or an SPE-SAX SPE
cartridge. The eluate was evaporated to dryness under a gentle nitrogen stream at 50 °C. The
residue was reconstituted in water (110 μL) and ultracentrifuged for 15 min at 12,000 rpm in a
microcentrifuge (Smart R17, Hanil Scientific Inc., Gyeonggi-do, South Korea) equipped with
a Microcon filter (cut-off = 30,000 Da). Finally, concentrated GHB was derivatized with 35 L
of 99% BSTFA + 1% TMCS at 70 °C for 25 min (Figure 1). After cooling at room temperature
for 10 min, an aliquot of the silylated fraction was transferred to a GC vial for GC/MS and
GC/C-IRMS analyses.
Preparation of the urine specimens
This study was approved by the Institutional Review Board of Duksung Women’s
University (2017-006-002). Urine samples were collected from five healthy female volunteers
(Asian, aged 27 to 32 years) who provided written informed consent, lifestyle information, and
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dietary patterns (e.g., fish, corn, and beans). Collected urine samples were kept at –50 °C and
were extracted and derivatized as follows using equivalent GHB standard methods 12,13
.
Gas chromatography-mass spectrometry (GC/MS)
GC/MS analysis was performed with a Hewlett-Packard 5890 Series II Plus
chromatograph coupled with a HP 5971 mass-selective detector (MSD) quadrupole mass
spectrometer (both from Agilent Technologies, Santa Clara, CA, USA). GC separation was
achieved on an HP-5MS capillary column (30 m × 0.25 mm i.d. and 0.25 μm film thickness)
from J&W Scientific (Folsom, CA, USA). Helium was used as a carrier gas at a constant flow
rate of 1.0 mL/min (8.8 psi); the injection volume was 1 μL. For the GHB analysis, the oven
temperature was increased from 70 °C (1 min) to 100 °C at 4 °C/min and then to 295 °C (1
min) at 30 °C/min. Electron ionization (EI) mass spectra were recorded with the instrument
autotuned by continuously scanning in the m/z range of 40–450. Data were recorded in full
scan mode (Figure 2), and GHB concentrations were determined from the selected ions (i.e.
m/z 233, 234, and 235). A calibration curve was obtained using seven GHB standard points at
urine concentrations from 1 to 100 μg/mL.
Elemental analyzer-isotope ratio mass spectrometry (EA/IRMS)
To analyze the δ13C values of the original GHB standard, δ13C values were measured of
CO2 generated by an elemental analyzer (vario PYRO cube, Elementar, Hanau, Gemany)
coupled with an isotope ratio mass spectrometer (Isoprime 100, Isoprime/Elementar). Values
were reported per mil (‰) using δ notation relative to the standard Vienna-Pee Dee Belemnite
(VPDB) for carbon. The precision for isotopic determination was better than ±0.1‰.
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Gas chromatography-combustion-isotope ratio mass spectrometry (GC/C-IRMS)
The δ13C values of silylated GHB in the standard and five urine samples were determined
by GC/C-IRMS as described in a previous method 26. An IRMS instrument (visION, Isoprime)
was connected with a Hewlett Packard 7890 N series gas chromatograph (Agilent Technologies)
via a combustion interface (glass tube packed with copper oxide, operated at 850 °C). A fused
silica capillary column (CP-sil 5 CB, 25 m length, 0.32 mm i.d., and 0.12 μm film thickness)
was used with He (at a constant 1.3 mL/min flow rate ) as a carrier gas. Samples were injected
under the same temperature program as for GC/MS analysis. Reference carbon dioxide gas of
known 13C value was pulsed at the beginning and end of each sample run. Standard carbon
isotope ratios were expressed as 13C value per mil relative to Vienna-PeeDee Belemnite
(VPDB) and were calculated according to
(13퐶/ 12퐶)푠푎푚푝푙푒 − (13퐶/ 12퐶)푠푡푎푛푑푎푟푑
δ13C = [
]
(1)
(13퐶/ 12퐶)푠푡푎푛푑푎푟푑
To monitor the accuracy of the measurements, standards with known δ13C values were
analyzed every 5–6 sample runs. Standard deviations of carbon isotope measurements were
generally ±0.4‰ as determined by repeated injections of the standard. Meanwhile, the
correction of δ13C values for silylated carbons was calculated from a stoichiometric mass
balance relationship as follows:
4
6
δ13CGHB di-TMS
=
δ13Coriginal GHB
+
δ13Cdi-TMS
(2)
10
10
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The δ13Coriginal GHB values measured via EA-IRMS were –28.3±0.03‰ throughout the study.
Finally, the δ13Cdi-TMS values were calculated from the δ13CGHB di-TMS values estimated from the
standard GHB measurement by GC/C-IRMS.
Statistical analysis
We used a non-parametric Kruskal-Wallis test to assess statistical significance in urine
GHB among the two δ13Cdi-TMS equations relative to the GHB standard. Significance was
assigned at a p-value < 0.05. Statistical analysis was performed using the SPSS statistical
program (IBM SPSS Statistics 21.0, IBM Corporation, Armonk, NY, USA).
Results and discussion
Isotopic corrections using the best-fit function
To confirm a trend of isotopic fractionation during silylation and isotopic measurements,
we tested variations of δ13C values in the di-TMS derivative-GHB standard (Figure 1), for
abundances ranging from 10 to 500 ppm. Table 1 summarizes the δ13C values of the derivatized
GHB standard at different concentrations. The δ13C values (±SD) of the derivatized GHB
standard ranged from –31.5±0.1‰ to –30.4±0.4‰. Using equation (2), the calculated δ13C
values of the introduced silyl carbon atoms (δ13Cdi-TMS values) ranged from –33.6 to –31.8‰
for the reagent batch. The average δ13Cdi-TMS value was–32.5±0.7‰. Our results indicate that
δ13Cdi-TMS values depend on the range of GHB concentrations. Furthermore, there was a
significant logarithmic regression of the δ13Cdi-TMS values showing positive regression (r2= 0.70,
p <0.05) along with increased concentration (Figure 3). Thus, variations of δ13Cdi-TMS values
were predicted from the following equation:
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δ13Cdi-TMS (‰ VPDB) = 0.42 × In(GHB concentrations) − 34.36
(3)
The precision (SD) of the δ13Cdi-TMS values for our analytical sequence ranged from ±0.2‰ to
±0.7‰ over a broad range of standard quantities, which was better than the precision (±0.7‰)
of the δ13Cdi-TMS values corrected by using a single carbon isotope (Table 1). Furthermore, such
quantity-dependent patterns of isotopic fractionation had previously been reported in TMS-
derivatized organic compounds such as phenols, toluenes, and amino acids by using the GC/C-
IRMS system 16,20. This amount-dependent pattern in δ13Cdi-TMS values may be not fully
explained by the characteristics of the derivatization reaction itself. Because hydroxy groups
are added at the end of an alkylated silyl group during silylation this, theoretically at least, does
not trigger kinetic isotope effects 27,28. In our study, such a phenomenon was observed at certain
GHB standard concentrations (50 ppm to 200 ppm). However, the derivatized GHB standards
at 10 ppm and 500 ppm exhibited the lowest δ13C value (–33.6±0.7‰) and the highest δ13C
value (–31.8±0.3‰), respectively, indicating significant differences from the average (–
32.4±0.1‰) of the other concentrations. This isotopic drift could be due to the kinetic isotope
effect caused by derivatization over a wide range of GHB concentrations (10 ppm and 500
ppm). Another possibility of isotopic drift in our setting could be inferred from the properties
of instrumental performance that are dependent on the quantity of analyte applied 16,18,20. For
instance, after sample injection, several factors (e.g., injector, carrier gas flow rate, and
combustion) could lead to drift of the isotopic signal due to relative variations of the m/z 44 to
45 signal intensities depending on CO2 quantities in the GC/C-IRMS system 16,18,29. In this
study, we confirmed that the δ13C values did not significantly change (less than ±0.2 ‰) under
the adjusted injection amounts of 500 ppm GHB standard. Hence, we may infer that isotopic
changes of GHB standard provide a possibility for kinetic isotope effect under varied
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concentrations in our set-up.
Evaluation of the best-fit function in urine GHB
The δ13CGHB di-TMS values in all detected urine samples ranged from –30.1 to –28.6‰ (SD
= ±0.1 to ±0.3) (Table 1). Based on Equation (3), the corrected δ13Cdi-TMS values ranged from
–34.9 to –34.1‰ in the urine samples (Table 1). Finally, the δ13CGHB values were –24.1 to –
19.5‰ in the urine samples (Figure 4). Until recently, δ13CGHB values were corrected only using
single δ13Cdi-TMS values or known values 12–14 without considering any unintended drift in the
isotopic fractionation during measurement. Previous studies assumed that no isotopic
fractionation occurred during isotopic measurements; thus, the δ13CGHB values were corrected
by a single or known δ13Cdi-TMS value 12. In this study, if a single δ13Cdi-TMS value (–32.5‰ as
an average of all standards) was applied to correct the urine GHB, the δ13CGHB values would
be in the range from –26.1 to –22.2‰ (Figure 4). The δ13CGHB values of the urine samples after
correcting for isotopic fractionations along various standard quantities were significantly
different (by an average of 2.7‰) from those obtained using the single-isotope correction (p <
0.05, Figure 4). This result could be attributed to the isotopic difference (2.2‰) of the δ13Cdi-
TMS values between the two corrections.
With respect to the complex synthesis routes of GHB (i.e., endogenous vs exogenous),
previous studies reported that GHB concentrations in living people could be influenced by
various factors such as biochemical, drug-ingestion, and dietary effects 12,13,30. In this regard,
source identification of GHB via quantitative methods may face several challenges. For
instance, after ingestion of GHB (25 mg / kg dose), concentrations in urine and blood samples
obtained from some volunteers rapidly decreased to <10 ppm after 4 hrs 23. Actually, the GHB
concentrations preserved in seized urine samples have often yielded low concentrations (less
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15
than 10 ppm) due to unavoidable delays of several hours prior to sample collection . Thus,
the methodology first applied in this study could improve the determination of exogenous GHB
present in varying concentrations (especially low concentrations). Considering increasing
issues related to abused or misuse of GHB, our attempt would enhance the likelihood of
discriminating exogenous GHB from endogenous production by correcting for amount-
dependent isotope fractionation. Thus, we suggest that these approaches could be important for
establishing a valid method to discriminate GHB origins using δ13C values. In addition,
measurement of δ13CGHB values in multiple urine samples (e.g., with a diverse background of
diet sources and/or metabolism by age 12,30) is needed to generalize its use in physiology and
forensic investigation.
Conclusion
In the present study, the δ13CGHB values in urine samples were assessed through the use of
GC/C-IRMS in addition to measuring GHB abundance. With respect to amount-dependent
isotopic fractionation, the δ13Cdi-TMS values exhibited a logarithmic regression (r2= 0.70) with
an increase in GHB standard concentration. The δ13CGHB values in urine samples were
significantly different from those of GHB standards in both correction methods. Moreover, the
urine δ13CGHB values after correcting for the isotopic variations of di-TMS at the GHB
concentrations involved were 2.7‰ higher on average than those in the traditional, simple
correction approach (p < 0.05). Our study supported the possibility that the isotopic approach
could allow source identification for exogenous GHB in biological samples, even at extremely
low GHB quantities. Further studies are required to evaluate the time-series of potential
isotopic fractionations in GHB during various biological processes (e.g., interaction of
endogenous GHB synthesis and metabolism after exogenous ingestion).
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Conflicts of interest
There are no conflicts to declare.
Acknowledgments
This study was supported by research project No. 2017-04 (The development of analysis
method for identification of endogenous and exogenous GHB) of the National Forensic Service
(NFS) and partially by a National Research Foundation of Korea (NRF) grant funded by the
Korean government (MSIP) (No. 2016R1A6A1A03007648 and 2017R1A4A1015393).
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Fig. 1. Chemical structures of GHB and GHB di-TMS.
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Fig. 2. GC/MS chromatogram of GHB obtained from a urine sample
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Fig. 3. The logarithmic relationship (r2 = 0.70) between δ13C value of di-TMS and GHB
concentration. The black dotted line indicates the average δ13C values of introduced di-TMS
measured in the GHB standard.
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Fig. 4. Plot showing the isotopic difference between GHB standards and urine samples. Each
letter indicates a statistically significant group of data (P<0.05) determined using a non-
parametric Kruskal-Wallis test.
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Table 1. Carbon isotope compositions (δ13C values) of γ-hydroxybutyric acid (GHB) + di-TMS
and di-TMS by quantity-dependent and single correction.
di-TMS*
di-TMS
GHB + di-TMS
δ13C (‰
by amount-dependent
correction
by single correction
Concentrations
(ppm)
δ13C (‰
SD (n=3)
VPDB)
δ13C (‰
SD (n=5)
VPDB)
SD (n=3)
VPDB)
10
50
-31.5
-30.7
-30.8
-30.7
-30.4
0.4
0.2
0.1
0.1
0.2
-33.6
-32.4
-32.5
-32.4
-31.8
0.7
0.4
0.2
0.2
0.3
GHB standard
(-28.3±0.03 by
EA-IRMS)
100
200
500
-32.5
0.7
*Calculated from the equation (3) (y = 0.42×In(X1) - 34.36), and then corrected isotopic ratios of GHB using the
mass balance
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