A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of γ-hydroxybutyrate and its precursors in forensic whole blood

Forensic Science International 222 (2012) 352–359

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A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of g-hydroxybutyrate and its precursors in forensic whole blood Lambert K. Sørensen *, Jørgen B. Hasselstrøm Section for Toxicology and Drug Analysis, Department of Forensic Medicine, Aarhus University, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 April 2012 Received in revised form 17 July 2012 Accepted 28 July 2012 Available online 20 August 2012

A liquid-chromatography–tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC–ESI-MS/MS) was developed for the simultaneous determination of g-hydroxybutyric acid (GHB), g-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in human ante-mortem and post-mortem whole blood. The blood proteins were precipitated using a mixture of methanol and acetonitrile, and the extract was cleaned-up by passage through a polymeric strong cation exchange sorbent. Separation of the analytes and their structural isomers was obtained using a column with a zwitterionic stationary phase. Matrix-matched calibrants, combined with isotope dilution, were used for quantitative analysis. GHB was determined in both positive and negative ion modes. The relative intralaboratory reproducibility standard deviations were better than 10% and 6% for blood samples at concentrations of 2 mg/L and 20–150 mg/L, respectively. The mean true extraction recoveries were 80% for GHB and greater than 90% for GBL and 1,4-BD at concentration levels of 20–50 mg/L. The limits of detection were approximately 0.5 mg/L for GHB and GBL, and 0.02 mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1 mg/L for GHB and GBL, and less than 0.1 mg/L for 1,4-BD. GBL was unstable in whole blood freshly preserved with a sodium fluoride oxalate mixture, but the stability could be improved significantly by preservation with a sodium fluoride citrate EDTA mixture. ß 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Gamma-hydroxybutyrate (GHB) Gamma-butyrolactone (GBL) 1,4-Butanediol Whole blood LC–MS/MS

1. Introduction The drug g-hydroxybutyric acid (GHB) is a short-chain hydroxylated fatty acid that is endogenously present in various mammalian tissues, blood and urine. It is a powerful central nervous system depressant and was used in legal medicine as an anaesthetic agent prior to being phased out because of its very steep dose-response curve and various side effects. Today, GHB is mainly used for the treatment of narcolepsy and less frequently during the treatment of alcohol dependence [1]. However, GHB is also abused for recreational purposes and is available as both a colourless, odourless liquid and as a solid material on the illicit market. GHB related fatalities have been reported [2,3] and the drug has been found in both the body fluids and hair from suspected victims of drug-facilitated sexual assaults [4,5]. Although GHB is a controlled drug, the pharmacologic effects of GHB may be obtained indirectly from the intake of the commercially available and legal chemicals g-butyrolactone

* Corresponding author. Tel.: +45 87167500; fax: +45 86125995. E-mail address: [email protected] (L.K. Sørensen). 0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2012.07.017

(GBL) and 1,4-butanediol (1,4-BD). Both are used as industrial solvents and they may be constituents in various cleaners or used during the synthesis of other chemicals (e.g. 1,4-BD is used during the production of tetrahydrofuran). Following the direct intake of GBL, the substance is rapidly converted to GHB by the lactonase enzymes present in the blood of mammals. However, GBL can also be easily converted to GHB by treatment with lye in a simple nonindustrial (kitchen) process. In humans, 1,4-BD is converted to GHB through the combined actions of alcohol and aldehyde dehydrogenases. GHB is rapidly and extensively metabolised in the body. Approximately 1% is excreted unchanged in urine [6]. In forensic toxicology, body fluids are monitored for substances that may have been abused, for instance, by vehicle drivers. However, testing for GHB in biological matrices is challenging because of its polar properties, its equilibrium state with GBL favouring GBL at low pH, and the complete conversion of GLB to GHB at high pH values. Determination of GHB in biological fluids is typically performed by gas chromatography mass spectrometry (GC–MS) after derivatisation of the substance, such as silylation. The use of LC–MS/MS allows for omission of the derivatisation step, but poor retention on analytical columns is often observed. Methods based on LC–MS/MS of the native

L.K. Sørensen, J.B. Hasselstrøm / Forensic Science International 222 (2012) 352–359

molecule have been published for the determination of GHB in plasma/serum, [7,8] and for the simultaneous determination of GHB, GBL and 1,4-BD in whole blood and urine [9,10]. These methods use reverse phase chromatography for the separation of the components and acidified mobile phases to obtain some retention of GHB, which restricts the electrospray ionisation (ESI) to be performed in the positive ion mode where only a single abundant product ion is produced. To improve the chromatographic and retention properties, derivatisation has also been applied in the determination of GHB in urine and serum using LC–MS [11]. However, the chromatographic separation of small polar molecules can often be directly improved through the use of hydrophilic interaction liquid chromatography (HILIC) instead of classical reverse phase chromatography. The HILIC conditions allow the chromatography to be performed under neutral conditions. The present LC–MS/MS method is based on the HILIC analysis of extracts that have been cleaned-up using cation-exchange solid phase extraction (SPE) for adsorption of interfering substances. The method was validated as a robust technique, suitable for the determination of GHB, GBL and 1,4-BD in both ante-mortem and post-mortem whole blood samples. Several abundant transition products of GHB were obtained for the proper identification of the substance. 2. Materials and methods 2.1. Chemicals

a-Hydroxybutyric acid (AHB) sodium salt, b-hydroxybutyric acid (BHB) sodium salt, GHB, GBL, 1,4-BD and 2,3-butanediol (2,3-BD) were obtained from Sigma– Aldrich (Schnelldorf, Germany). The isotope analogues, GHB-D6 and GBL-D6, were obtained from Cerilliant (Round Rock, Texas) and 1,4-BD-D4 was obtained from Cambridge Isotope Laboratories (Andover, MA). Whole blood samples, used for calibration, were obtained from the Blood Bank, Aarhus University Hospital (Skejby, Denmark). Formic acid, ammonium acetate and sodium dihydrogen phosphate were purchased from Merck (Darmstadt, Germany). Acetonitrile (MeCN) and methanol (MeOH) of LC–MS grade were purchased from Sigma–Aldrich. Water was purified using a Direct-Q 3 apparatus (Millipore, Bedford, MA). 2.2. Samples Samples of ante-mortem and post-mortem whole blood, used for validation of the method, were obtained from the Department of Forensic Medicine, Aarhus University. Ante-mortem blood samples that were used in the validation study were preserved in Venosafe VF-053SFC32 tubes that contained 6.8 mg of sodium fluoride (NaF) and 15.7 mg of citrate-EDTA buffer ingredients (FC mixture) for a 3 mL draw volume of blood (Terumo Europe, Leuven, Belgium). Post-mortem blood samples were preserved with 200 mg of NaF per 30 mL of blood. Venosafe VF109SFX07 tubes (Terumo Europe) that contained 100 mg of NaF and 22.5 mg of potassium oxalate (FO mixture) for a 9-mL draw volume of blood and Venosafe VF052SDK tubes (Terumo Europe) that contained 3.9 mg K2EDTA for a 2-mL draw volume of blood were included in a stability study on GHB and its precursors in ante-mortem whole blood.

353

were used for the extractions. Autosampler vials made of glass (Mikrolab, Aarhus, Denmark) were used for storage of the final extracts. Other equipment used included pipettes (Biohit, Helsinki, Finland) and a Heraeus Biofuge Pico (Thermo Scientific, Langenselbold, Germany). 2.5. Extraction and clean-up A 200-mL volume of sample was transferred to a disposable 2 mL centrifuge tube. Then, 100 mL of MeOH and 100 mL the IS were added, and the tube contents were mixed gently. Shortly thereafter, a 600-mL volume of MeCN was added and the tube was immediately closed and vigorously vortex-mixed for a few seconds. After a standing time of 5–10 min, the mixture was centrifuged at 10,000  g for 5 min. A volume of 600 mL of the supernatant was mixed with 250 mL of water and was then passed through a CX SPE cartridge with a flow rate of max 0.5 mL/min. The cartridge was previously conditioned sequentially with 1 mL of MeOH, 1 mL of water, 1 mL of a 1 M sodium dihydrogen phosphate solution and 1 mL of water and then dried under full vacuum suction for few seconds. The eluate was collected, and 200 mL of the eluate was mixed with 600 mL of MeCN in an autosampler vial. The total sample dilution factor was 28. 2.6. Calibration The calibrants for both the ante-mortem and post-mortem samples were based on blank donor blood from single persons preserved with FC mixture. The samples were treated using the same procedure with the exception that the 100 mL of MeOH was replaced by 100 mL of the mixed standards of the drug substances. Sample concentrations were obtained at 5, 50, 100, 150 and 200 mg/L of GHB, and 2.5, 25, 50, 75 and 100 mg/L of GBL and 1,4-BD in the original blood sample. In addition, a blank sample (a processed matrix sample without analyte and without IS) and a zero sample (a processed matrix sample with IS) were included to verify the absence of detectable concentrations of the analytes. The calibration curves were created from weighted (1/x) linear regression analysis of the IS-normalised peak areas (analyte area/IS area) and were forced through the origin. 2.7. LC–MS/MS analysis The sample extracts were maintained at 7  2 8C prior to analysis. A 10-mL volume was injected onto a SeQuant ZIC HILIC column running 5% mobile phase A (1 mM ammonium acetate) and 95% mobile phase B (MeCN). The mobile phase was changed through a linear gradient to 50% A and 50% B over 4 min. Then, the gradient was changed to 95% A over 0.2 min. Five minutes after injection, the gradient was returned to 5% A over 0.5 min, and the column was equilibrated for 4.5 min before the next injection resulting in a total runtime of 10 min. The column flow rate was 200 mL/min and the column temperature was maintained at 30  2 8C. During the interval of 0.5–2.5 min, a reagent containing 0.1% formic acid was infused post column with a flow rate of 100 mL/min. The eluent was diverted to waste during the time interval of 4–9 min after injection by using a post-column switch. The source and desolvation temperatures were set at 150 8C and 600 8C, respectively, and the cone and desolvation nitrogen gas flows were set at 50 L/h and 800 L/h, respectively. The mass spectrometer was operated in positive ion mode with a probe voltage of 3 kV and in negative ion mode with a probe voltage of 2.5 kV. The dwell time was 50–64 ms depending on the number of ion transitions processed during the same time period. At least 12 data points were obtained across the peaks. Selected reaction monitoring (SRM) was applied using the conditions shown in Table 1. Argon was used for collision-induced dissociation (CID). The data acquisition and processing were performed using MassLynx 4.1 (Waters). 2.8. Limits of detection and quantification

2.3. Standards Separate stock solutions containing 1 mg/mL of the active substances were prepared in MeOH and stored at 20  28C. The combined standard solutions for the fortification of the samples and preparation of the calibrants were prepared by diluting the stock solutions with MeOH. An internal standard solution (IS) containing 0.1 mg/ mL of the deuterated analogues of GHB, GBL and 1,4-BD was prepared in MeOH. 2.4. Equipment The liquid chromatography system was a Waters Acquity UPLC system that consisted of a binary pump, an autosampler with a 10- mL sample loop thermostated at 7  2 8C and a column oven thermostated at 30  2 8C (Waters, Milford, MA). The mass spectrometer was a Waters Xevo TQMS triple-quadrupole instrument with an ESI ion source and a programmable infusion pump. The separation was performed using a SeQuant ZIC HILIC (5 mm, 200 A˚, 2.1 mm I.D.  100 mm) column (Merck SeQuant, Umea˚, Sweden). Solid phase extraction (SPE) was performed on a 3-mL Strata-X-C cartridge containing 60 mg of a polymeric strong cation exchange (SCX) sorbent (Phenomenex, Torrance, CA). A VacMaster-20 vacuum manifold (Biotage, Uppsala, Sweden) was used during the SPE procedure. Disposable 2-mL polypropylene Safe-Lock tubes (Eppendorf, Hamburg, Germany)

The limits of detection (LODs) were determined using a random selection of 20 different samples of ante-mortem whole blood and 20 different samples of postmortem blood. The samples were spiked prior to extraction with the individual substances in order to obtain concentrations that were approximately three to five times the signal/noise ratio. The LODs were calculated as 2  t0.95  SDB (t0.95 = 1.645), where SDB is the standard deviation of the results obtained from the spiked samples. The lower limits of quantification (LLOQs) were calculated as 10 times the SDB of the quantifier ions. 2.9. Precision, trueness and recovery The repeatability standard deviation (SDr) (i.e., the variability of independent analytical results obtained by the same operator using the same apparatus under the same conditions on the same test sample and in a short interval of time) and the intra-laboratory reproducibility standard deviation (SDR,intra-lab) (i.e., the variability of independent analytical results obtained on the same test sample in the same laboratory by different operators on different days) were determined on blank control samples of ante-mortem whole blood spiked to levels of 2, 20 and 75 mg/L of GBL and 1,4-BD, and 2, 20 and 150 mg/L of GHB and from post-mortem samples with an endogenous content of GHB. Duplicate analyses were performed on eight

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different days. The repeatability and intra-laboratory reproducibility parameters were calculated in accordance with ISO standard 5725–2 [12]. The matrix effects (including ion-suppression and ion-enhancement effects) were investigated on 20 blank samples of both ante-mortem and post-mortem blood that were spiked after SPE to a level that was equivalent to 20 mg/L in the original samples. They were analysed in attenuating order along with the pure standards at the same concentration level; the matrix effect from each sample was calculated from the peak areas (As) without IS correction using the closest standards in the series; matrix effect ¼ ðA¯ pure standard  Afortified sample Þ  100 = A¯ pure standard . The true extraction recoveries were determined from 20 samples of each type that were spiked to a level of 20 mg/L. The standards used for the determination of the true recoveries were the same blank samples that were spiked after SPE. Finally, the trueness of the method, which is the closeness in agreement between the average value obtained from a large series of test results and the accepted reference value, was determined from 20 different blank samples of both ante-mortem and post-mortem blood that had each been spiked with GBL, 1,4-BD and GHB to a level of 20 mg/L with the exception of GHB in post-mortem blood that was spiked with 50 mg/L. These samples were different from the samples used in the investigation of ion-suppression effects.

3. Results and discussion 3.1. Precursor ions and transition products Electrospray ionisation was applied in positive ion mode (ESI(+)) for GBL and 1,4-BD, and the dominant Q1 ions were the protonated molecular ions ([M + H]+). Significant abundances of two product ions were obtained for both substances (Table 1). Electrospray ionisation of GHB was possible in both positive and negative ion mode. Three abundant transition products were obtained in ESI() (Table 1). However, only one product ion of significant abundance was obtained in ESI(+). Effective ionisation of GBL required acidic conditions, whereas the ionisation of 1,4BD was only slightly improved in the presence of acids. The optimal acidic conditions for GBL were obtained by post column infusion of 0.1% formic acid. Without this infusion, the abundance of the GBL product ions was reduced by approximately 30 times. However, the acidic conditions were not optimal for the ionisation of GHB in ESI() as the responses were reduced almost 8 times. Therefore the infusion was stopped before the GHB eluted from the column (more details on the chromatography are given in Section 3.3). In ESI(+), the ionisation of GHB was not significantly influenced by the presence of acids. Because of the lack of more than one abundant product ion in ESI(+), an in source conversion of GHB to GBL has been used for the qualification of the presence of GHB in ESI(+) mode [9]. This in source conversion of GHB to GBL was also observed in the present study but it was not considered suitable for qualification because of the relative low conversion factor (6%). The relative abundances of the transition products were constant in the calibrated range, i.e. the average RSDs of the ion ratios across the calibration points in the precision study were less than 3% for all analytes. The RSDs of the ion ratios obtained from ante-mortem and post-mortem samples (20 of each) were 8% for GBL, 3% for 1,4-DB and 2% for GHB at a concentration of 20 mg/L.

3.2. Extraction and clean-up The blood samples were extracted using a mixture of MeOH and MeCN. The methanol was added to obtain a disperse precipitate of the protein-containing blood components. Because of the hydrophilic properties of the analytes and the lack of ionic functional groups, except in the case of GHB, it was not possible to perform a selective clean-up using SPE for all of the analytes. However, it was possible to remove a significant portion of the dissolved matrix components by eluting the extract through a polymeric SCX sorbent. Sodium was used as counter-ion to avoid conversion of GHB to GBL. If this clean-up was not applied, skewed peaks for GHB and a noisier baseline were in some cases observed. The stability of the blood-based calibrants at 7  2 8C and 20  2 8C was tested daily over a period of five days. The calibrants were extracted according to the procedure with the exception that the internal standard was not added to the blood sample at the beginning but instead was added to subsamples of the extracts on the day of the LC–MS/MS analysis. The slopes of the calibration curves for GLB and 1,4-BD did not change significantly during the five days of storage at 7 8C. However, GHB was not stable in solution. The slope of the calibration curve was reduced by approximately 50% after storing for five days at 7 8C. No significant changes in the slopes were observed during this storage period at 20 8C. GHB-D6 showed similar instability as GHB at 7 8C, and when the IS was added prior to extraction, there were no significant changes in the slopes of the calibration curves observed during five days of storage. 3.3. Chromatography Using reverse-phase LC, the typical elution order is GHB, 1,4-BD followed by GBL. As the substances are very hydrophilic, only limited possibilities are available to control the retention times (RT), especially for GHB. By using the HILIC technique, the elution order is reversed, which makes it easier to optimise the RT of GHB by adjusting the composition of the mobile phase. It was possible to obtain almost baseline separation between GBL, 2,3-BD, 1,4-BD, AHB, BHB and GHB within a 4 min elution period (Fig. 1). In ESI() mode, AHB, but not BHB, produced a significant response at the same transitions as GHB. Conversely, in ESI(+) mode, BHB, but not AHB, produced a significant response at the same transitions as GHB. The HILIC technique required neutral elution conditions for optimal separation of the substances. Neutral elution conditions are also optimal in preventing interconversion of GBL and GHB, but are not optimal for the ionisation of GBL. Therefore, 0.1% formic acid was infused in the fluid stream between the column and the ESI probe. No detectable carry-over was observed when the samples that had been spiked with 500 mg/L of the individual substances and blank control samples were analysed in attenuated order. A typical chromatogram of the blank blood samples is shown in Fig. 2.

Table 1 Mass spectrometric conditions and relative retention times (RRTs). The bold ions were used for quantification. The underlined ions were used as qualifiers. In matrix extracts, the response of the ESI() transition m/z 103/85 was approximately half of the response of the ESI(+) transition m/z 105/87. Substance

GBL 1,4-BD GHB GHB GBL-D6 1,4-BD-D4 GHB-D6 GHB-D6

ESI mode

+ +  + + +  +

Transition Q1 (m/z)

Q3 (m/z)

87 91 103 105 93 95 109 111

45/43 73/55 85/57/101 87/69 49 77 90 93

Cone voltage (V)

Collision energy (eV)

Relative abundance

RRT

20 22 20 12 20 20 20 20

10/8 6/10 8/12/12 7/8 10 6 8 7

100/65 100/47 100/93/32 100/2

0.427 0.568 0.998 0.998 0.428 0.570 1.000 1.000

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The gradient was changed to 95% A after elution of the analytes in order to clean the column and to maintain the selectivity constant from injection to injection. According to the column manufacturer, it should be possible to improve the selectivity of the column by flushing with an aqueous salt solution between injections. We verified the manufacturer’s recommendation by flushing the column with a 100 mM NH4Ac solution. In general, however, it was not necessary to do this when the described gradient was used.

2.05

100 %

2,3-BD

0

3.4. Matrix effects and quantification The matrix effects of the blood samples on the ionisation of GLB and 1,4-BD were close to zero (Table 2). For the more polar GHB, an ion suppression of approximately 30% was observed in ESI(+). The same level of ion suppression was reported by Johansen and Windberg [9]. However, the ion suppression was much higher in ESI(). This was investigated in more detail through the continuous infusion of GHB at a concentration of 6 mg/L using a

m/z 91 > 73 ESI+

1,4-BD

1.86

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

2.05

100

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

1.53

100

3.00

3.20

3.40

3.60

In source produced GBL

GBL

3.56

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

%

3.56

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

% %

3.60

3.80

Time

3.55

m/z 105 > 87 1.23 ESI+ 1.35 1.20

1.40

1.62

1.60

BHB

1.85

1.80

2.21 2.33 2.41

2.00

2.20

2.40

GHB

3.38

2.60

2.80

3.00

3.20

3.40

3.60

3.80

3.55

100

0

3.80

m/z 87 > 43 ESE+

In source produced GBL

GBL

100

0

3.60

1.54

100

0

3.80

m/z 87 > 45 ESE+

% 0

3.80

m/z 91 > 55 ESI+

1,4-BD

2,3-BD

% 0

355

m/z 103 > 85 ESI1.20

1.40

3.13

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

GHB

AHB

3.20

3.40

3.60

3.80

%

100

0

m/z 103 > 57 ESI1.20

1.40

AHB 1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

%

GHB

3.60

3.80

3.39

100

0

3.40

3.55

m/z 103 > 59 ESI1.20

1.40

BHB 1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

3.80

Time

Fig. 1. Chromatograms of the quantifier and qualifier product ions in the extract of ante-mortem whole blood that was spiked with 50 mg/L of GBL, 1,4-BD, 2,3-BD, AHB, BHB and GHB. A chromatogram of the transition m/z 103/59 for BHB is included to show the retention time of this substance.

L.K. Sørensen, J.B. Hasselstrøm / Forensic Science International 222 (2012) 352–359

356

1.84

%

100

0

1.20

100

1.60

1.20

2.02 2.13

1.80

2.00

1.80 1.85 1.87 1.90

2.33

2.20

2.42 2.47

2.40

1.40

1.60

1.23 1.29

1.53 1.59

2.65

2.60

2.80

3.00

3.20

3.40

3.60

2.04

m/z 91 > 55 ESI+

2.44

2.19 2.25

2.54

1.80 1.79 1.86

0

2.00

1.94

2.20 2.17 2.24

2.40 2.35

2.60

2.80

3.00

3.20

3.40

3.60

1.24

1.31

1.60

1.44

1.54

2.66

1.80

1.59

1.85

2.00

2.20

1.99

2.19

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

0

%

100

0

%

100

0

RT of GBL 1.20

1.40

1.60

1.80

2.40

2.60

1.40

3.00

% %

100

0

3.20

3.40

3.60

3.31

2.77

1.60

1.80

2.00

2.20

2.40

2.60

2.93

2.80

3.02

3.00

3.49

3.15 3.19

3.20

3.40

Time

3.80

RT of GHB

3.37 3.42

m/z 105 > 87 ESI+

1.20

2.80

3.70 3.75 3.92 3.97 3.62

3.60

3.80

RT of GHB

3.13

m/z 103 > 101 ESI1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

3.14

100

0

3.80

m/z 87 > 43 ESE+

2.37 2.44 2.54

%

100

1.40

3.80

m/z 87 > 45 ESE+

2.54

RT of GBL 1.20

3.80

RT of 1,4-BD

1.57 1.64

1.33 1.42

m/z 91 > 73 ESI+

%

100

1.40

1.24

%

1.18

0

1.46 1.57 1.62

1.20 1.30

RT of 1,4-BD

1.87 1.91

m/z 103 > 85 ESI1.20

1.40

RT of GHB

2.93 2.95 3.29 3.39

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.58

3.60

1.40

3.71

3.92

3.80

RT of GHB

m/z 103 > 57 ESI1.20

3.80

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

3.80

Time

Fig. 2. Chromatograms of the quantifier and qualifier product ions in the extract of a typical blank ante-mortem whole blood sample. The expected retention times of GBL, 1,4BD and GHB are marked with arrows.

flow rate of 50 mL/min after injecting an extract of a blank blood sample (Fig. 3). Basically, the ESI() transition m/z 103/85 was more sensitive than the ESI(+) transition m/z 105/87, but at the RT of GHB, significant matrix effects caused a reduction of the signal in ESI(). The gradient itself also caused a reduction in the signal, but the effect of the matrix induced an almost 80% reduction. In spite of this reduction, ESI() tended to result in better LODs and precision figures than ESI(+). The insignificant effects of the ion suppression on method accuracy may arise from a combination of the

following: (a) the RT of GHB was in the relatively flat region of the suppression curve and the suppression was mostly constant from sample to sample and (b) an efficient correction could be obtained by using GHB-D6 as an internal standard because the RTs of GHB and GHB-D6 were very close, only differing by 0.01 min. The matrix effect on GHB could be reduced to approximately 40% in ESI() by an additional clean-up in which GHB was adsorbed to a silica-based strong anion exchange (SAX) sorbent (data not shown). However, the matrix effect was unchanged in ESI(+).

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Table 2 Matrix effects, true recoveries and method trueness obtained from single determinations of spiked ante-mortem blood (AMB) and post-mortem blood (PMB) samples (n = 20 for each sample type). Matrix-matched standards used in the calculation of the recovery were the same samples spiked after SPE. Substance

Spiked concentrationa mg/L

GBL 1,4-BD GHB ESI() GHB ESI(+)

20/20 20/20 20/50 20/50

a

Matrix effect mean (SD), %

True recovery mean (SD), %

Trueness mean (SD), %

AMB

AMB

PMB

AMB

PMB

102 96 84 80

102 92 76 80

98 103 99 103

98 100 101 106

4 4 79 35

PMB

(4) (5) (1) (4)

5 2 79 24

(7) (6) (1) (7)

(8) (7) (6) (4)

(9) (7) (5) (6)

(3) (3) (4) (3)

(7) (5) (7) (7)

Ante-mortem/post-mortem.

3.5. Method performance parameters The mean true recoveries of GLB and 1,4-BD were greater than 90% for both ante-mortem and post-mortem blood (Table 2). The mean true recovery of GHB was approximately 80%. The trueness of the method, as determined from 20 additional samples each of ante-mortem and post-mortem blood, was close to 100% (Table 2). The LODs of the quantifier and qualifier ions were approximately 0.5 mg/L for GHB and GBL, and 0.02 mg/L for 1,4-BD in antemortem blood (Table 3). The corresponding LLOQs were less than 1 mg/mL for GHB and GBL, and less than 0.1 mg/L for 1,4-BD. Different cut-off levels have been proposed by different investigators due to an endogenous content of GHB in blood. In practice, cut-off levels in the range of 10–20 mg/L are often used, although a cut-off level of 4 mg/L has recently been proposed for ante-mortem blood [13]. In post-mortem blood, the LODs were estimated to be less than 2 mg/L for GBL and less than 0.1 mg/L for 1,4-BD (Table 3). The LOD for GHB could not be precisely determined due to the high endogenous content of this substance. The mean endogenous level of the tested post-mortem samples was 17 mg/L (range 1–34 mg/ L), which is consistent with the results reported by Elliott [14] and within the major distribution reported by Kintz et al. [15]. The RSDr and RSDR,intra-lab values obtained in the precision study were below 10% at a concentration of 2 mg/L and below 6% at a concentration level of 20–150 mg/L for ante-mortem samples (Table 4). The RSDR,intra-lab for GHB determined from post-mortem samples containing 14–20 mg/L endogenous GHB was less than 9% in

ESI(), but somewhat higher in ESI(+). That is considered acceptable when compared to the rule of Horwitz [16]. According to the Horwitz equation, RSDR values of 14% and 8% between laboratories would be acceptable at concentration levels of 2 and 75 mg/L respectively. From the standard analyses of quality control samples over a three-month period, RSDR,intra-lab values of less than 4% were obtained for all substances in ante-mortem blood spiked to a level of 20 mg/L. As shown earlier, this method is selective for the structural isomers 2,3-BD, AHB and BHB that may be present in blood samples. From a list of approximately 1000 substances, covering therapeutic drugs having a licit medical purpose and drugs used illicitly, that are frequently or occasionally analysed by our institution, this method was also evaluated for other potential interfering substances, e.g., substances differing by less than one mass unit from the analytes. There were no substances with potential interference found. The calibration curves were created using weighted regression analysis. The R2 values obtained in the precision study from eight independent test series and analysed within a three week period were generally better than 0.996 (overall mean 0.9990, SD 0.0008) for the transitions used in the quantification. The calibration curves of 1,4-BD, GHB ESI() and GHB ESI(+) could be considered to be linear with slopes of 0.062, 0.042 and 0.052 L/mg respectively (independent variable: blood concentration; dependent variable: peak area ratio of analyte and internal standard). However, for GBL a minor but statistically significant improvement in the standard

100

ESI(-) m/z 103/105

%

Dotted line: Reduction in response caused by the LC gradient in ESI(-)

RT of GHB 3.34

ESI(+) m/z 105/107

0 1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

3.80

Time

Fig. 3. Injection of an extracted blank whole blood sample during post-column infusion (50 mL/min) of a 6 mg/L GHB standard prepared in water.

L.K. Sørensen, J.B. Hasselstrøm / Forensic Science International 222 (2012) 352–359

358

Table 3 Limits of detection determined in ante-mortem blood (AMB) and post-mortem blood (PMB) samples (n = 20 for each sample type). Q3 product ion, m/z

Substance

Spiked concentrationa, mg/L

Measured concentration, mean (SD), mg/L PMB

AMB 45 43 45 43 73 55 73 55 101 85 57 101 85 57 87 87

GBL

1,4-BD

GHB ESI()

GHB ESI(+) a

0.5/1.0 0.5/1.0 0/0 0/0 0.05/0.1 0.05/0.1 0/0 0/0 0.5/– 0.5/– 0.5/– 0/0 0/0 0/0 0.5/– 0/0

0.73 (0.074) 0.76 (0.11) 0.30 (0.067) 0.35 (0.072) 0.045 (0.0055) 0.047 (0.0054) 0.005 (0.0014) 0.005 (0.0023) 0.49 (0.18) 0.40 (0.087) 0.54 (0.14) 0.095 (0.10) 0.080 (0.058) 0.084 (0.14) 0.40 (0.20) 0.10 (0.10)

3.6. Stability of GBL, 1,4-BD and GHB in blood samples The stability of GBL, 1,4-BD and GHB in the blank blood bank samples that were freshly preserved with FO and FC mixtures, which resulted in sample pHs of ca. 7.4 and 5.9, respectively, was tested at storage temperatures of 20  2 8C for 7 days, and 5  2 8C and 20  2 8C for 3 weeks. Samples were individually spiked with a single substance at a concentration of 15 mg/L. Both 1,4-BD and GHB were stable at 20  2 8C for at least 7 days, and at 5  2 8C and 20  2 8C for at least 3 weeks when the samples were preserved with FO or FC mixtures. However, GBL was very unstable in nonpreserved blood and in blood freshly preserved with the FO mixture. In less than 30 min most of the GBL was converted into GHB at ambient temperature. The GBL was more stable in blood that had been preserved with FC mixture, but in the investigated cases, approximately half of the GBL was converted to GBL within 7 days when the samples were stored at ambient temperature (Fig. 4). At

0.3 0.4

1.5 1.8

0.02 0.02

0.04 0.03

0.6 0.3 0.5 16 (7.9) 17 (8.7) 17 (8.5) 0.7 16 (5.0)

GBL

1,4-BD

GHB ESI()

GHB ESI(+)

Matrix

AMB AMB AMB AMB AMB AMB AMB AMB AMB PMB PMB AMB AMB AMB PMB PMB

Spiked concentration, mg/L

RSDra, %

2 20 75 2 20 75 2 20 150 14c 20c 2 20 150 14c 20c

9.2 4.2 3.7 2.7 5.0 4.1 7.1 3.3 4.4 6.3 4.8 5.5 6.1 4.8 6.5 3.4

19 17

RSDR,intra-labb, %

15 9.2 4.2 4.2 4.8 5.0 5.6 8.6 3.7 5.3 8.8 6.8 12 6.1 5.1 14 12

Relative standard deviation of repeatability. Relative standard deviation of intra-laboratory reproducibility. Endogenous content of GHB.

13

Conc., mg/L

Substance

c

PMB

5 8C, approximately 20% and 40% of the GBL was converted after 7 days and 3 weeks, respectively. At 208C, the GBL was stable for at least 3 weeks. Based on observations, the rate of conversion is sample dependent. The conversion is probably due to the activity of lactonase in blood samples, as the conversion was pH dependent and was almost stopped by the addition of 100 mL MeOH to 200 mL blood. Furthermore, observations indicated that the rate of conversion in samples preserved prior to spiking declined by increasing the time lag between preservation and spiking. When blood collection vessels solely containing EDTA were used for sample storage, the conversion of GBL to GHB was also inhibited, but to a lesser extent than with the FC mixture-containing vessels. According to Fishbein and Bessman [17], the lactonase activity is inhibited by EDTA but not efficiently inhibited by NaF. These properties should be considered during the development of sampling strategies when the analysed concentrations of GBL and GHB should resemble the chemical composition of the sample at the sampling time. Because of the higher molecular weight of GHB (MW = 104) compared to GBL (MW = 86), the mass increase in GHB concentration was greater than the mass decrease in GBL concentration. In a study reported by LeBeay et al. [18], elevated GHB concentrations (mean 8.7 mg/L) were observed in blood samples stored for 6–36 months at 20 8C in tubes containing a mixture

Table 4 Average method precision estimated at different drug concentration levels in antemortem blood (AMB) and post-mortem blood (PMB).

b

2.10 (0.46) 2.19 (0.54) 1.34 (0.60) 1.32 (0.60) 0.087 (0.012) 0.074 (0.0082) 0.006 (0.010) 0.007 (0.014)

AMB

Ante-mortem/post-mortem.

error of the residuals was obtained by inclusion of a quadratic term in the calibration model. The coefficients of the linear and the quadratic terms were 0.038 and 0.00002, respectively.

a

LOD, mg/L

11 9 7 5 3 1 -1 0

5

10

15

20

25

Storage time, days Fig. 4. Stability of 15 mg/L GLB in an ante-mortem whole blood sample that was stored at 20 8C (*), 5 8C (~) and 20 8C (&) in Venosafe tubes containing a fluoride-citrate-EDTA additive. The conversion of GLB into GHB at 20 8C (*), 5 8C (~) and 20 8C (&) was monitored by measuring the GHB content.

L.K. Sørensen, J.B. Hasselstrøm / Forensic Science International 222 (2012) 352–359

of trisodium citrate, citric acid and dextrose when compared to tubes containing EDTA. In the present stability study, we used a NaF-citrate-EDTA additive for preservation of the blood samples. No formation of GHB was observed after 3 weeks of storage at 5  2 8C or after 4 months of storage at 20  2 8C. 3.7. Application of the method The developed method has been applied to clinical and autopsy cases with suspected intake of Fantasy. In a clinical case, a blood concentration of GHB of 120 mg/L was found in a person suspected of driving under the influence of drugs. In two recent cases of intoxication that were treated successfully at the local care units, GHB concentrations of 97 and 104 mg/L were detected in venous whole blood. Unfortunately we do not have information on the time lag between intake and sampling. In an autopsy case a 25-year-old male was found unconscious on a lawn, and the person was declared dead after 45 min of unsuccessful resuscitation. According to the investigation the deceased was with three other males and a trivial struggle occurred prior to his death. Both autopsy and toxicological investigation were performed. Apart from minor lesions most likely due to the struggle, the cause of death could not be determined during the autopsy. The toxicological analysis revealed a high GHB concentration of 235 mg/L in the peripheral whole blood, which can result in hypotension, bradycardia, respiratory depression, seizures and lapsing into a coma [19]. A GBL concentration of 6 mg/L was also detected in the blood sample. The effect of GHB may have been increased by the ethanol concentration of 1.75 per mille. The GHB concentration of the stomach content was 250 mg/L. The urine, which was analysed by a separate method [20] contained 2840 mg/L of GHB. A small bottle containing minor residual material of an unknown origin was seized at the scene. The material was dissolved in 2 mL of water and analysed using the current method. The liquid contained 100,000 mg/L of GHB, 680 mg/mL of GBL and 32 mg/L of 1,4-BD. 4. Conclusion A rapid and selective analytical method using a simple extraction and clean-up procedure, which avoids derivatisation and critical steps such as sample treatment under acidic or basic conditions and solvent evaporation, was developed. The chromatography, which is based on the HILIC technique, improved control of the retention of GHB as compared to classical reverse phase chromatography. The ruggedness and reproducibility of the

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