Journal of Analytical Toxicology 2014;38:177 –183 doi:10.1093/jat/bku010 Advance Access publication February 11, 2014
Article
Determination of Therapeutic g-Aminobutyric Acid Analogs in Forensic Whole Blood by Hydrophilic Interaction Liquid Chromatography – Electrospray Tandem Mass Spectrometry Lambert K. Sørensen and Jørgen B. Hasselstrøm Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark *Author to whom correspondence should be addressed. Email: [email protected]
Vigabatrin, pregabalin, gabapentin and baclofen are g-aminobutyric acid analogs that are used in the treatment of epileptic seizures (vigabatrin, pregabalin and gabapentin) and spasticity (baclofen). The intake of these drugs may induce adverse reactions and impair the ability of an individual to drive a vehicle. There have also been reports of cases of intoxication and fatalities from overdoses. For rapid and accurate quantification of these drugs in forensic cases, an ultraperformance liquid chromatography tandem mass spectrometry method using pneumatically assisted electrospray ionization has been developed. The technique has been validated on both ante- and postmortem human whole blood. The protein in the blood samples was removed by the addition of a mixture of methanol and acetonitrile, and the extract was ultrafiltered and diluted with acetonitrile. The separation was performed by hydrophilic interaction liquid chromatography. Calibration of the system was achieved through use of matrix-matched calibrants combined with isotope dilution. The lower limits of quantification were 0.02– 0.04 mg/L, and the relative intralaboratory reproducibility standard deviations were 89%. The trueness expressed as the relative bias of the test results was within + 7% at concentrations of 1– 40 mg/L for vigabatrin, pregabalin and gabapentin and of 0.1 –4 mg/L for baclofen.
Introduction Vigabatrin, gabapentin, pregabalin and baclofen are structural analogs of g-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system of mammals. Vigabatrin irreversibly inhibits the transaminase responsible for the catabolism of GABA and is used in the treatment of epileptic seizures. Gabapentin and pregabalin are used in the treatment of partial seizures and neuropathic pain, whereas baclofen is used in the treatment of spasticity. These drugs are small molecules that contain both a primary amino group and a free carboxylic acid group, making them highly polar molecules. Their therapeutic blood concentration ranges are relatively high: 1 – 25 mg/L for vigabatrin, 2 –20 mg/L for gabapentin, 1 –8 mg/L for pregabalin and 0.08 –0.6 mg/L for baclofen (1, 2). Several adverse reactions are associated with the use of vigabatrin, gabapentin, pregabalin and baclofen, including somnolence, dizziness, fatigue and visual disorder (2), which may affect the ability of an individual to drive a vehicle. Cases have been reported where gabapentin was the only detected drug and has apparently caused impaired driving at blood concentrations within the therapeutic range (3). Use of vigabatrin may cause depression (4), and use of gabapentin may be associated with an increased risk of suicidal acts or violent deaths (5). Acute intoxication from an accidental overdose, abuse and attempted suicide
has been reported for gabapentin (6, 7), pregabalin (8, 9) and baclofen (10 –14). Fatal cases due to single or combined drug intake have been reported for vigabatrin (15), gabapentin (16), pregabalin (17) and baclofen (18, 19). Thus, the quantitative determination of the GABA analogs in blood specimens collected from cases of impaired driving, intoxication and suspicious death is highly pertinent for forensic toxicology. Quantification of the GABA analogs, especially of vigabatrin, in blood is more challenging than the quantification of many other therapeutic drugs because of their high polarities. Several methods based on high-performance liquid chromatography combined with fluorescence or UV detection of derivatized analytes have been published for the determination of vigabatrin and other GABA analogs in serum and plasma (20 –29). Recently, direct determination of antiepileptic drugs by ultra-performance liquid chromatography tandem mass spectrometry (UPLC – MS/ MS) using traditional reverse-phase (RP) technology has also been described (30, 31). By using such methods, the retention time (Rt) of vigabatrin is typically in the range of 0.5 – 0.7 min when flow rates of 0.4 mL/min are used in combination with standard UPLC columns. Thus, the elution volume is close to the column void volume, which leaves few possibilities for optimizing selectivity and reducing matrix interferences from complex matrices, such as postmortem blood. However, the retention of highly polar molecules can often be directly improved through the use of hydrophilic interaction liquid chromatography (HILIC) instead of classical RP chromatography. This has also been demonstrated for the determination of some anticonvulsants in serum (32). The present UPLC –MS/MS method, which is based on HILIC, was developed and validated to obtain a simple, sensitive and robust technique that is suitable for the simultaneous quantification of vigabatrin, gabapentin, pregabalin and baclofen in both ante- and postmortem whole blood samples. Experimental Standards and reagents Vigabatrin was purchased from Cerilliant (Round Rock, TX, USA). Pregabalin and gabapentin were obtained from Pfizer (Ann Arbor, MI, USA). Baclofen was purchased from SigmaAldrich (Schnelldorf, Germany). Vigabatrin-13CD2, baclofen-D4, pregabalin-13C3 and gabapentin-D4 were purchased from Cerilliant. Formic acid was purchased from Merck (Darmstadt, Germany). Methanol (MeOH) and acetonitrile (MeCN) were purchased from Sigma-Aldrich. Water was purified using a Direct-Q 3 apparatus (Millipore, Bedford, MA, USA). Separate stock solutions (1 mg/mL) of pregabalin and gabapentin were prepared in MeOH, while a stock solution of
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baclofen (1 mg/mL) was prepared in water. Stock solutions (1 mg/mL) of all other analytes were provided in MeOH. Combined standard solutions to spike the samples and prepare the calibrants were prepared by diluting the stock solutions with 50% MeOH. An internal standard solution of stable isotopelabelled substances (SIL-IS) containing 5 mg/L each of vigabatrin- 13CD2, pregabalin-13C3 and gabapentin-D4, and 0.5 mg/L of baclofen-D4 was also prepared in 50% MeOH. The mobile phases A and B consist 1 mM ammonium acetate and MeCN, respectively. Sample specimen Blank whole blood samples for calibration were obtained from the blood bank at Aarhus University Hospital (Skejby, Denmark). The blank samples of ante- and postmortem whole blood that were used for the method validation were obtained from the Department of Forensic Medicine, University of Aarhus. Antemortem blood was collected and preserved in Venosafe VF-109SFX07 tubes that contained 100 mg of sodium fluoride (NaF) and 22.5 mg of potassium oxalate (FO mixture) for a 9-mL draw volume of blood (Terumo Europe, Leuven, Belgium). Venosafe VF-053SFC32 tubes (Terumo Europe) that contained 6.8 mg of NaF and 15.7 mg of citrate-EDTA buffer ingredients (FC mixture) for a 3-mL draw volume of blood were used in a stability study on the GABA derivatives in whole blood. Postmortem blood samples were preserved with 200 mg of NaF per 30 mL of blood. 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 set at 7 + 28C and a column oven set at 30 + 28C (Waters, Milford, MA, USA). The mass spectrometer was a Waters Xevo TQMS triple – quadrupole instrument with an electrospray ionization (ESI) source. The separation was performed using an Acquity UPLC BEH Amide (1.7 mm, 2.1 mm ID 100 mm) column (Waters). Disposable 2-mL polypropylene Safe-Lock tubes (Eppendorf, Hamburg, Germany) were used for the extractions. Amicon Ultra filter units with a 0.5-mL reservoir and a 30-kDa membrane of regenerated cellulose (Millipore) were used for the ultrafiltration (UF) of the sample extracts. Autosampler vials made of glass (Mikrolab Aarhus, Aarhus, Denmark) were used for the storage of the final extracts. Other equipment that was used included pipettes (Biohit, Helsinki, Finland) and a Heraeus Biofuge Pico (Thermo Scientific, Langenselbold, Germany).
Extraction A 100-mL volume of sample was transferred to a disposable 2 mL centrifuge tube. Then, 100 mL of water, 100 mL of SIL-IS solution and 100 mL of 50% MeOH were added, and the tube contents were gently mixed. Shortly thereafter, a 600-mL volume of MeCN was added, and the tube was immediately closed and vigorously vortexed for a few seconds. After a standing time of 5 –10 min, the mixture was centrifuged at 10,000 g for 5 min. A 300-mL volume of clear supernatant was ultrafiltered at 10,000 g for 5 min. A 100-mL volume of the filtrate was mixed with 900 mL of MeCN in an autosampler vial. Calibration Calibrants based on blank donor blood were used for the construction of seven-point calibration curves. The calibrants were treated according to the above procedure, except that 100 mL of 50% MeOH was replaced by 100 mL of the mixed standards containing the drugs. Samples with concentrations of 1, 5, 10, 20, 30, 40 and 50 mg/L of vigabatrin, pregabalin and gabapentin in the original blood sample were made for calibration. The concentrations of baclofen in these calibrants were 10 times lower than the concentrations of the other three drugs. The calibration curves were created by weighted (1/x) linear regression analysis of the SIL-IS normalized peak areas (analyte area/IS area) and were forced through the origin. LC– MS/MS conditions All sample extracts were kept at 7 + 28C until analysis. A 10-mL volume was injected onto an Acquity UPLC BEH Amide column running with 15% mobile phase A and 85% mobile phase B. The mobile phase was changed by a linear gradient to 30% A over 3 min. Then, the mobile phase was changed to 70% A over 0.1 min. Five minutes after injection, the gradient was returned to 15% A over 0.1 min, and the column was equilibrated for 2.9 min before the next injection, resulting in a total runtime of 8 min. The eluent was diverted to the waste during the intervals of 0 –1.4 and 3.3– 8 min after the injection, using a postcolumn switch. The column flow rate was 400 mL/min, and the column temperature was kept at 30 + 18C. The source and desolvation temperatures were set at 150 and 6008C, respectively, and the cone and desolvation gas flows were set at 50 and 800 L/h, respectively. The mass spectrometer was operated in positive ion mode with a capillary voltage of 3 kV and an extractor potential of 3 V. The dwell time was 20 ms for all ion transitions. Selected reaction monitoring was applied under the conditions shown in Table I. Argon was used for the
Table I Mass Spectrometry Conditions and Retention Times (Rt) Drug
Vigabatrin Pregabalin Gabapentin Baclofen
Transition Q1 (m/z)
Q3 (m/z)
130 160 172 214
71/113/67 97/55/142 137/154/55 116/151/179
Cone voltage (V)
Collision energy (eV)
Relative abundance
Rt of drug (min)
Rt of SIL-IS (min)
12 18 18 16
14/9/18 15/20/11 15/12/22 30/17/14
95/100/15 36/48/100 50/100/26 50/100/37
2.83 1.77 1.82 1.84
2.83 1.77 1.83 1.84
The bold and underlined ions were used for quantification, and the underlined ions were used as the primary qualifiers.
178 Sørensen and Hasselstrøm
collision-induced dissociation. Data acquisition and processing were performed using MassLynx 4.1 (Waters).
matrix effects, the final extracts were spiked with SIL-IS; true extraction recovery ¼ ðAsample spiked before extraction =Amatching SILIS Þ 100=ðAsample spiked after SPE =Amatching SILIS Þ .
Method validation Results and discussion Precision and trueness 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 in a short interval of time) and the intralaboratory 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 ante- and postmortem whole blood samples spiked with vigabatrin, pregabalin and gabapentin at concentrations of 0.02 – 0.04, 1, 10 and 40 mg/L and spiked with baclofen at concentrations of 0.02, 0.1, 1 and 4 mg/L. Duplicate analyses were performed on eight different days. The repeatability and intra-laboratory reproducibility parameters were calculated in accordance with ISO standard 5725-2 (33). The method trueness (i.e., the closeness of agreement between the average value obtained from a large series of test results and the accepted reference value) was determined from the results obtained on the spiked samples in the precision study. The trueness was expressed as the relative bias; relative bias ¼ (mean test result of spiked sample 2 spiked concentration) 100/spiked concentration. Limits of detection and quantification The limit of detection (LOD) was determined using a random selection of 10 different blank samples of antemortem whole blood and 20 different blank samples of postmortem whole blood. The samples were spiked with the individual drugs prior to the extraction to obtain concentrations that were three to six times the signal-to-noise ratio. The LOD was calculated as 2 t0.95 SDB (t0.95 ¼ 1.645), where SDB is the standard deviation of the results obtained from the spiked samples. The lower limit of quantification (LLOQ) was determined from the precision studies at concentrations of 10 SDB. The acceptance criterion was an RSDR,intra-lab of a maximum of 20% and a bias within +20% of the spiked concentration. Matrix effects and true extraction recovery The matrix effects (including ion-suppression and ion-enhancement effects) were investigated on 10 different blank samples of antemortem whole blood and 20 blank samples of postmortem whole blood that were spiked after UF at a level that was equivalent to 10 mg/L of vigabatrin, pregabalin and gabapentin and 1 mg/L of baclofen in the original samples. They were analyzed in attenuating order along with blank samples and the pure standards at the same concentration. The matrix effect from each sample was calculated from the peak areas (A) without IS correction using the closest standards in the series; matrix effect ¼ ðApure standard Aspiked sample Þ 100=Apure standard . The true extraction recoveries were determined from the same blood samples that were spiked to 10 mg/L of vigabatrin, pregabalin and gabapentin and 1 mg/L of baclofen. The standards that were used for the determination of the true recoveries were the same blood samples that were spiked after UF. To compensate for changes in
Extraction and chromatography The blood samples were treated with a mixture of MeOH and MeCN. The MeCN was used as the primary protein denaturing solvent, while MeOH was added to obtain a disperse precipitate. The denatured proteins were precipitated by centrifugation, and particles and dissolved high-molecular-weight substances were removed by filtration through a 30-kDa membrane. The filtrate was diluted with MeCN to avoid solvent effects that would have distorted the chromatography. All substances were ionized by electrospray in the positive mode (ESI(þ)). The dominant precursor (Q1) ions obtained were the protonated molecular ions ([M þ H]þ) and several product (Q3) ions of significant abundance were obtained for all substances. The quantifier and qualifier ions were selected from their abundances and their chromatographic separation from interferences (Table I). Due to the high polarity of vigabatrin, poor retention is obtained on traditional RP column packing. The Rt is typically 0.6 min for 100 mm UPLC columns at a flow rate of 0.4 mL/ min. Using the HILIC separation technique, the elution order is reversed compared with RP chromatography (i.e., the Rt increases with increasing polarity of the analyte), because the more polar compounds will have a stronger interaction with the aqueous layer that is established within the hydrophilic stationary phase than the less polar compounds. An Rt of several minutes is easily obtained for vigabatrin by this technique. Although the other GABA derivatives are less polar, sufficient retention can still be obtained for these substances by HILIC (Figure 1). Pregabalin, gabapentin and baclofen were not baseline separated by the short run time. However, the m/z ratios of the precursor ions were different, and no interferences from insource fragmentation were observed. The gradient was changed to 70% A after elution of the analytes to remove substances more polar than vigabatrin and maintain the selectivity constant from injection to injection.
Method performance parameters The mean matrix effects were ,15% for all of the substances (Table II). The mean true recoveries were .89%, and no significant differences between ante- and postmortem blood were observed (Table II). The LODs were 10 mg/L for the two most sensitive transition products of all the substances in both the ante- and the postmortem blood (Table III). The RSDR,intra-lab was ,8% at a concentration of 1 mg/L and ,4% at concentrations of 10 mg/L or higher (Table IV). The RSDR,intra-lab was also ,8% for baclofen at a concentration of 0.1 mg/L. According to the general rule of Horwitz (34), the obtained precision figures are considered acceptable. The Horwitz equation states that the RSDR between laboratories should be ,16 and 11% at concentrations of 1 and 10 mg/L, respectively. For intralaboratory data, the imprecision would normally be 25 – 50% lower. The relative bias determined at low, medium and high Therapeutic GABA Analogs in Forensic Whole Blood 179
Figure 1. Chromatograms of the ESI(þ) product ions in the extract of an antemortem whole blood sample spiked with 1 mg/L of vigabatrin, pregabalin and gabapentin and 0.1 mg/ mL of baclofen.
concentrations was within 210 –5% of the spiked concentration (Table IV). The LLOQ criteria were fulfilled at concentrations of 0.02 mg/L of gabapentin and baclofen, 0.03 mg/L of pregabalin and 0.04 mg/L of vigabatrin (Table IV). In comparison, LLOQ 180 Sørensen and Hasselstrøm
values of 0.02, 0.01, 0.02 and 0.02 mg/L were obtained for gabapentin, baclofen, pregabalin and vigabatrin when the LLOQ values were calculated as 10 SDB using the data obtained in the LOD study. The upper limits of quantification are 50 mg/L
for vigabatrin, pregabalin and gabapentin and 5 mg/L for baclofen, which correspond to the concentrations of the highest calibrant. The selectivity of the method against endogenous interference was demonstrated by the analysis of 10 different blank samples of antemortem blood and 10 different blank samples of postmortem blood that were independent from the samples used for the LOD determination. No interferences were observed that would impact the trueness of the method at the LLOQ level. Whole blood samples were also spiked with 67 frequently detected
Table II Matrix Effects and True Extraction Recoveries Obtained from the Single Analyses of the Spiked Antemortem Whole Blood (AMB) and Postmortem Whole Blood (PMB) Samples (n ¼ 10 for AMB, n ¼ 20 for PMB) Drug
Vigabatrin Pregabalin Gabapentin Baclofen
Spiked conc. (mg/L)
10 10 10 1
Matrix effect, mean (+SD) (%)
True extraction recovery, mean (+SD) (%)
AMB
PMB
AMB
PMB
215 (+7) 27 (+9) 29 (+10) 25 (+9)
24 (+6) 2 (+16) 22 (+18) 6 (+11)
92 95 96 89
91 (+7) 97 (+6) 96 (+5) 93 (+10)
(+5) (+3) (+1) (+4)
Matrix-matched standards used in the calculation of the recoveries were the samples spiked after the extraction and clean-up.
Table III LODs Determined in Antemortem Whole Blood (AMB) and Postmortem Whole Blood (PMB) Samples (n ¼ 10 for AMB, n ¼ 20 for PMB) Drug
Vigabatrin
Pregabalin
Gabapentin
Baclofen
Transition Q1/Q3 (m/z)
130/67 130/71 130/113 160/55 160/97 160/142 172/55 172/137 172/154 214/116 214/151 214/179
Spiked conc. (mg/L)
50 10 10 10 10 10 10 10 10 5 5 5
Result mean (+SD) (mg/L)
LOD (mg/L)
AMB
PMB
AMB
PMB
44 12 11 9 11 10 11 10 10 4 4 4
50 12 12 11 9 10 12 11 11 6 6 6
30 10 10 7 7 7 7 7 10 7 7 7
26 10 10 10 10 10 10 7 7 7 7 7
(+9) (+3) (+3) (+2) (+2) (+2) (+2) (+2) (+3) (+2) (+2) (+2)
(+8) (+3) (+3) (+3) (+3) (+3) (+3) (+2) (+2) (+2) (+2) (+2)
licit and illicit drugs and metabolites (Table V). The substances were tested at the high end of the concentration range that is normally found in blood. Finally, the selectivity was tested against less frequently or rarely detected drugs, which are characterized by having molecular weights (MW) that are close to the analytes: metformin (MW 129.2), tranexaminic acid (MW 157.2), buformin (MW 157.2), hydralazine (MW 160.2), chloroxazone (MW 169.6), levetiracetam (MW 170.2), metronidazole (MW 171.2), zonisamide (MW 212.2) and butobarbital (MW 212.3). Buformin and hydralazine were tested at a concentration of 1 mg/L in the blood, whereas the other drugs were tested at a concentration of 50 mg/L. No interferences from all of these drugs were observed on the chromatograms. The calibration curves were created using weighted linear regression analysis to improve the variance homogeneity over the calibrated concentration range. From the residual plots, the weighting factor 1/x appeared suitable. Because no significant interferences from endogenous substances were present on the chromatograms, the calibration curve was forced through the origin to improve reproducibility at low concentrations. The difference in the slopes of the calibration curves prepared from pure solvent calibrants and matrix-matched calibrants (eight independent pairs from the precision study) was ,3% for vigabatrin and ,2% for the other substances. This difference was not statistically significant at a 95% confidence level. The ruggedness of the calibration would most likely not be affected by using pure calibrants because the differences between the Rt of the analytes and the Rt of the corresponding SIL-IS were very small compared with the peak band widths (Table I). However, because of the simple sample pretreatment procedure, matrix-matched calibrants were used. The R 2 values of the eight calibration curves obtained in the precision study were 0.998 + 0.002 (mean + SD) for baclofen and 0.999 + 0.002 for the other substances. Stability of analytes in blood and extracts The stability of the substances in whole blood freshly preserved with FO and FC mixtures (n ¼ 2 of each) was tested at storage temperatures of 20 + 28C, 5 + 28C and 220 + 28C for 8 days. The samples included in the stability test were spiked with the drugs at concentrations of 1 mg/L of baclofen and 10 mg/L of the other analytes. The samples were analyzed when they were
Table IV Method Precision and Trueness Estimated for Different Drug Concentrations in Antemortem Whole Blood (AMB) and Postmortem Whole Blood (PMB) Samples Drug
Spiked conc. (mg/L)
Mean result AMB/PMB (mg/L)
RSDr AMB/PMB (%)
RSDR,intra-lab AMB/PMB (%)
Relative bias AMB/PMB (%)
Vigabatrin
0.04 1 10 40 0.03 1 10 40 0.02 1 10 40 0.02 0.1 1 4
0.036/0.039 0.99/0.97 9.98/10.0 39.8/39.7 0.031/0.030 0.96/0.93 9.76/9.84 39.8/39.6 0.021/0.021 0.96/0.95 9.91/10.0 39.6/39.9 0.021/0.021 0.094/0.10 0.98/1.01 3.95/3.99
15/14 2.8/5.9 2.1/1.6 2.5/2.4 16/15 2.7/3.3 1.4/1.7 1.6/1.3 7.3/13 2.4/3.0 1.6/2.1 1.3/1.4 20/14 7.5/6.5 3.3/2.7 2.8/2.2
15/15 4.2/7.7 3.3/3.0 2.8/3.4 16/16 3.3/4.5 3.7/2.3 2.3/1.6 15/16 3.0/4.9 2.4/2.9 1.4/2.1 20/14 7.5/6.5 5.1/2.7 4.0/2.2
210/23 21/23 0/0 21/21 3/0 24/27 22/22 21/21 5/5 24/25 21/0 21/0 5/5 26/0 22/1 21/0
Pregabalin
Gabapentin
Baclofen
Therapeutic GABA Analogs in Forensic Whole Blood 181
Table V Drugs and Metabolites Frequently Detected in Whole Blood Alprazolam 7-Aminoclonazepam 7-Aminoflunitrazepam Amphetamine Barbitale Benzoylecgonine Bromazepam Buprenorphinea Caffeinee Carbamazepinee Chlordiazepoxidec Chlorprothixene Citalopramc Clonazepam Cocaine Codeine Cyclobarbitale Demoxepam Desmethylmirtazapine O-Desmethyltramadolc O-Desmethylvenlafaxine Diazepam Diclofenacd
Etodolace Fentanyla Flunitrazepam Hydromorphon 10-Hydroxycarbazepinee Ibuprofene Ketamine Lamotriginee Lidocainec Lorazepam MDA MDMA Metamphetamine Methadonec Methylphenidate Mirtazapine 6-MAMa Morphine Morphine-3-b-D-glucuronide Morphine-6-b-D-glucuronide Naproxene Nitrazepam
Norbuprenorphinea Nordazepam Noscapinea Olanzapine Oxazepamc Oxycodone Papaverine Paracetamolf Pentobarbitale Phenobarbitale Phenytoine Quetiapine Salicylic acidf Sertraline THCa THC –COOHb THC –OHa Tramadolc Venlafaxine Zolpidem Zopiclone Zuclopenthixol
The interference from these drugs were investigated at concentrations of 0.032 mg/L (a), 0.08 mg/ L (b), 1.6 mg/L (c), 4 mg/L (d), 40 mg/L (e), 80 mg/L ( f ) and 0.8 mg/L (all other substances).
freshly prepared and after 1, 2, 4, 7 and 8 days of storage. No significant decline in concentrations was observed during the storage at ambient temperature. The results (mean + SD) that were obtained for freshly prepared samples preserved with the FO mixture were 9.81 + 0.22 mg/L (vigabatrin), 9.80 + 0.23 mg/L ( pregabalin), 9.72 + 0.08 mg/L (gabapentin) and 1.04 + 0.04 mg/L (baclofen). The corresponding mean results that were obtained after 7 and 8 days of storage at ambient temperature were 10.05 + 0.30, 9.78 + 0.13, 10.00 + 0.26 and 1.06 + 0.06 mg/L. The results obtained for freshly prepared samples that were preserved with the FC mixture were 9.98 + 0.05 mg/L (vigabatrin), 10.07 + 0.07 mg/L ( pregabalin), 9.90 + 0.36 mg/L (gabapentin) and 1.06 + 0.02 mg/L (baclofen). The corresponding mean results that were obtained after 7 and 8 days of storage at ambient temperature were 9.97 + 0.37, 9.83 + 0.12, 9.83 + 0.08 and 1.05 + 0.06 mg/L. For the other storage temperatures, the results obtained after 7 and 8 days of storage were also within +3% of the results obtained from the freshly prepared samples that were preserved with the FO and FC mixtures. The stability of the blood-based calibrants stored at 5 + 28C and 220 + 28C was tested over a period of 8 days. The calibrants were prepared according to the previously described procedure, except the SIL-IS was not added to the blood sample at the beginning but was instead added to the extracts on the day of the UPLC – MS/MS analysis. No significant changes in the slopes of the calibration curves were observed during the 8 days of storage at 5 and 2208C, respectively. The slopes of freshly prepared calibrants (in units of L/mg) were 0.34 (vigabatrin), 0.16 ( pregabalin), 0.35 (gabapentin) and 7.2 (baclofen). The slopes of the calibrants stored at 5 + 28C and 220 + 28C were 0.33 and 0.36 (vigabatrin), 0.16 and 0.17 ( pregabalin), 0.34 and 0.36 (gabapentin) and 7.3 and 7.3 (baclofen). Application of the method The developed method has been applied to 17 clinical and autopsy cases where the presence of the drugs has previously been 182 Sørensen and Hasselstrøm
detected by a screening method based on time-of-flight mass spectrometry or was likely from information about the prescribed medicines. The determined concentrations of gabapentin and baclofen ranged from 0.2 to 19 mg/L (n ¼ 8) and from 0.16 to 0.35 mg/L (n ¼ 2), respectively, which are within the therapeutic ranges. For pregabalin, most samples (n ¼ 6) were in the concentration range of 0.3 –5 mg/L. However, in one autopsy case, the blood concentration of pregabalin was 30 mg/L, which is greatly above the normal therapeutic range. The concerned person, a 49-year-old male, who was found dead in his bed, also had a high blood concentration of venlafaxine (3.6 mg/L) and a mirtazapine concentration (0.27 mg/L) at the high end of the therapeutic range.
Conclusion A sensitive and rapid UPLC –MS/MS method was developed and validated for the determination of vigabatrin, gabapentin, pregabalin and baclofen in ante- and postmortem whole blood. The extraction recoveries of 89 – 97% were obtained by a simple deproteinization procedure using a mixture of MeOH and MeCN. Adequate chromatographic retention of the polar drugs was obtained by HILIC. The matrix effects were relative low (,15%). Isotope dilution of each substance was used to eliminate the influence of differences in the extraction recoveries and ion-suppression effects. The method bias was less than +6% and the imprecision (RSDR,intra-lab) was 8% for concentrations within the therapeutic ranges. The LLOQ values were ,0.1 mg/L. This method is suitable for quantitative investigation in clinical and autopsy cases where the GABA analogs may have caused or contributed to impaired driving, intoxication, attempted suicide or death.
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Therapeutic GABA Analogs in Forensic Whole Blood 183
Article
Determination of Therapeutic g-Aminobutyric Acid Analogs in Forensic Whole Blood by Hydrophilic Interaction Liquid Chromatography – Electrospray Tandem Mass Spectrometry Lambert K. Sørensen and Jørgen B. Hasselstrøm Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark *Author to whom correspondence should be addressed. Email: [email protected]
Vigabatrin, pregabalin, gabapentin and baclofen are g-aminobutyric acid analogs that are used in the treatment of epileptic seizures (vigabatrin, pregabalin and gabapentin) and spasticity (baclofen). The intake of these drugs may induce adverse reactions and impair the ability of an individual to drive a vehicle. There have also been reports of cases of intoxication and fatalities from overdoses. For rapid and accurate quantification of these drugs in forensic cases, an ultraperformance liquid chromatography tandem mass spectrometry method using pneumatically assisted electrospray ionization has been developed. The technique has been validated on both ante- and postmortem human whole blood. The protein in the blood samples was removed by the addition of a mixture of methanol and acetonitrile, and the extract was ultrafiltered and diluted with acetonitrile. The separation was performed by hydrophilic interaction liquid chromatography. Calibration of the system was achieved through use of matrix-matched calibrants combined with isotope dilution. The lower limits of quantification were 0.02– 0.04 mg/L, and the relative intralaboratory reproducibility standard deviations were 89%. The trueness expressed as the relative bias of the test results was within + 7% at concentrations of 1– 40 mg/L for vigabatrin, pregabalin and gabapentin and of 0.1 –4 mg/L for baclofen.
Introduction Vigabatrin, gabapentin, pregabalin and baclofen are structural analogs of g-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system of mammals. Vigabatrin irreversibly inhibits the transaminase responsible for the catabolism of GABA and is used in the treatment of epileptic seizures. Gabapentin and pregabalin are used in the treatment of partial seizures and neuropathic pain, whereas baclofen is used in the treatment of spasticity. These drugs are small molecules that contain both a primary amino group and a free carboxylic acid group, making them highly polar molecules. Their therapeutic blood concentration ranges are relatively high: 1 – 25 mg/L for vigabatrin, 2 –20 mg/L for gabapentin, 1 –8 mg/L for pregabalin and 0.08 –0.6 mg/L for baclofen (1, 2). Several adverse reactions are associated with the use of vigabatrin, gabapentin, pregabalin and baclofen, including somnolence, dizziness, fatigue and visual disorder (2), which may affect the ability of an individual to drive a vehicle. Cases have been reported where gabapentin was the only detected drug and has apparently caused impaired driving at blood concentrations within the therapeutic range (3). Use of vigabatrin may cause depression (4), and use of gabapentin may be associated with an increased risk of suicidal acts or violent deaths (5). Acute intoxication from an accidental overdose, abuse and attempted suicide
has been reported for gabapentin (6, 7), pregabalin (8, 9) and baclofen (10 –14). Fatal cases due to single or combined drug intake have been reported for vigabatrin (15), gabapentin (16), pregabalin (17) and baclofen (18, 19). Thus, the quantitative determination of the GABA analogs in blood specimens collected from cases of impaired driving, intoxication and suspicious death is highly pertinent for forensic toxicology. Quantification of the GABA analogs, especially of vigabatrin, in blood is more challenging than the quantification of many other therapeutic drugs because of their high polarities. Several methods based on high-performance liquid chromatography combined with fluorescence or UV detection of derivatized analytes have been published for the determination of vigabatrin and other GABA analogs in serum and plasma (20 –29). Recently, direct determination of antiepileptic drugs by ultra-performance liquid chromatography tandem mass spectrometry (UPLC – MS/ MS) using traditional reverse-phase (RP) technology has also been described (30, 31). By using such methods, the retention time (Rt) of vigabatrin is typically in the range of 0.5 – 0.7 min when flow rates of 0.4 mL/min are used in combination with standard UPLC columns. Thus, the elution volume is close to the column void volume, which leaves few possibilities for optimizing selectivity and reducing matrix interferences from complex matrices, such as postmortem blood. However, the retention of highly polar molecules can often be directly improved through the use of hydrophilic interaction liquid chromatography (HILIC) instead of classical RP chromatography. This has also been demonstrated for the determination of some anticonvulsants in serum (32). The present UPLC –MS/MS method, which is based on HILIC, was developed and validated to obtain a simple, sensitive and robust technique that is suitable for the simultaneous quantification of vigabatrin, gabapentin, pregabalin and baclofen in both ante- and postmortem whole blood samples. Experimental Standards and reagents Vigabatrin was purchased from Cerilliant (Round Rock, TX, USA). Pregabalin and gabapentin were obtained from Pfizer (Ann Arbor, MI, USA). Baclofen was purchased from SigmaAldrich (Schnelldorf, Germany). Vigabatrin-13CD2, baclofen-D4, pregabalin-13C3 and gabapentin-D4 were purchased from Cerilliant. Formic acid was purchased from Merck (Darmstadt, Germany). Methanol (MeOH) and acetonitrile (MeCN) were purchased from Sigma-Aldrich. Water was purified using a Direct-Q 3 apparatus (Millipore, Bedford, MA, USA). Separate stock solutions (1 mg/mL) of pregabalin and gabapentin were prepared in MeOH, while a stock solution of
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baclofen (1 mg/mL) was prepared in water. Stock solutions (1 mg/mL) of all other analytes were provided in MeOH. Combined standard solutions to spike the samples and prepare the calibrants were prepared by diluting the stock solutions with 50% MeOH. An internal standard solution of stable isotopelabelled substances (SIL-IS) containing 5 mg/L each of vigabatrin- 13CD2, pregabalin-13C3 and gabapentin-D4, and 0.5 mg/L of baclofen-D4 was also prepared in 50% MeOH. The mobile phases A and B consist 1 mM ammonium acetate and MeCN, respectively. Sample specimen Blank whole blood samples for calibration were obtained from the blood bank at Aarhus University Hospital (Skejby, Denmark). The blank samples of ante- and postmortem whole blood that were used for the method validation were obtained from the Department of Forensic Medicine, University of Aarhus. Antemortem blood was collected and preserved in Venosafe VF-109SFX07 tubes that contained 100 mg of sodium fluoride (NaF) and 22.5 mg of potassium oxalate (FO mixture) for a 9-mL draw volume of blood (Terumo Europe, Leuven, Belgium). Venosafe VF-053SFC32 tubes (Terumo Europe) that contained 6.8 mg of NaF and 15.7 mg of citrate-EDTA buffer ingredients (FC mixture) for a 3-mL draw volume of blood were used in a stability study on the GABA derivatives in whole blood. Postmortem blood samples were preserved with 200 mg of NaF per 30 mL of blood. 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 set at 7 + 28C and a column oven set at 30 + 28C (Waters, Milford, MA, USA). The mass spectrometer was a Waters Xevo TQMS triple – quadrupole instrument with an electrospray ionization (ESI) source. The separation was performed using an Acquity UPLC BEH Amide (1.7 mm, 2.1 mm ID 100 mm) column (Waters). Disposable 2-mL polypropylene Safe-Lock tubes (Eppendorf, Hamburg, Germany) were used for the extractions. Amicon Ultra filter units with a 0.5-mL reservoir and a 30-kDa membrane of regenerated cellulose (Millipore) were used for the ultrafiltration (UF) of the sample extracts. Autosampler vials made of glass (Mikrolab Aarhus, Aarhus, Denmark) were used for the storage of the final extracts. Other equipment that was used included pipettes (Biohit, Helsinki, Finland) and a Heraeus Biofuge Pico (Thermo Scientific, Langenselbold, Germany).
Extraction A 100-mL volume of sample was transferred to a disposable 2 mL centrifuge tube. Then, 100 mL of water, 100 mL of SIL-IS solution and 100 mL of 50% MeOH were added, and the tube contents were gently mixed. Shortly thereafter, a 600-mL volume of MeCN was added, and the tube was immediately closed and vigorously vortexed for a few seconds. After a standing time of 5 –10 min, the mixture was centrifuged at 10,000 g for 5 min. A 300-mL volume of clear supernatant was ultrafiltered at 10,000 g for 5 min. A 100-mL volume of the filtrate was mixed with 900 mL of MeCN in an autosampler vial. Calibration Calibrants based on blank donor blood were used for the construction of seven-point calibration curves. The calibrants were treated according to the above procedure, except that 100 mL of 50% MeOH was replaced by 100 mL of the mixed standards containing the drugs. Samples with concentrations of 1, 5, 10, 20, 30, 40 and 50 mg/L of vigabatrin, pregabalin and gabapentin in the original blood sample were made for calibration. The concentrations of baclofen in these calibrants were 10 times lower than the concentrations of the other three drugs. The calibration curves were created by weighted (1/x) linear regression analysis of the SIL-IS normalized peak areas (analyte area/IS area) and were forced through the origin. LC– MS/MS conditions All sample extracts were kept at 7 + 28C until analysis. A 10-mL volume was injected onto an Acquity UPLC BEH Amide column running with 15% mobile phase A and 85% mobile phase B. The mobile phase was changed by a linear gradient to 30% A over 3 min. Then, the mobile phase was changed to 70% A over 0.1 min. Five minutes after injection, the gradient was returned to 15% A over 0.1 min, and the column was equilibrated for 2.9 min before the next injection, resulting in a total runtime of 8 min. The eluent was diverted to the waste during the intervals of 0 –1.4 and 3.3– 8 min after the injection, using a postcolumn switch. The column flow rate was 400 mL/min, and the column temperature was kept at 30 + 18C. The source and desolvation temperatures were set at 150 and 6008C, respectively, and the cone and desolvation gas flows were set at 50 and 800 L/h, respectively. The mass spectrometer was operated in positive ion mode with a capillary voltage of 3 kV and an extractor potential of 3 V. The dwell time was 20 ms for all ion transitions. Selected reaction monitoring was applied under the conditions shown in Table I. Argon was used for the
Table I Mass Spectrometry Conditions and Retention Times (Rt) Drug
Vigabatrin Pregabalin Gabapentin Baclofen
Transition Q1 (m/z)
Q3 (m/z)
130 160 172 214
71/113/67 97/55/142 137/154/55 116/151/179
Cone voltage (V)
Collision energy (eV)
Relative abundance
Rt of drug (min)
Rt of SIL-IS (min)
12 18 18 16
14/9/18 15/20/11 15/12/22 30/17/14
95/100/15 36/48/100 50/100/26 50/100/37
2.83 1.77 1.82 1.84
2.83 1.77 1.83 1.84
The bold and underlined ions were used for quantification, and the underlined ions were used as the primary qualifiers.
178 Sørensen and Hasselstrøm
collision-induced dissociation. Data acquisition and processing were performed using MassLynx 4.1 (Waters).
matrix effects, the final extracts were spiked with SIL-IS; true extraction recovery ¼ ðAsample spiked before extraction =Amatching SILIS Þ 100=ðAsample spiked after SPE =Amatching SILIS Þ .
Method validation Results and discussion Precision and trueness 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 in a short interval of time) and the intralaboratory 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 ante- and postmortem whole blood samples spiked with vigabatrin, pregabalin and gabapentin at concentrations of 0.02 – 0.04, 1, 10 and 40 mg/L and spiked with baclofen at concentrations of 0.02, 0.1, 1 and 4 mg/L. Duplicate analyses were performed on eight different days. The repeatability and intra-laboratory reproducibility parameters were calculated in accordance with ISO standard 5725-2 (33). The method trueness (i.e., the closeness of agreement between the average value obtained from a large series of test results and the accepted reference value) was determined from the results obtained on the spiked samples in the precision study. The trueness was expressed as the relative bias; relative bias ¼ (mean test result of spiked sample 2 spiked concentration) 100/spiked concentration. Limits of detection and quantification The limit of detection (LOD) was determined using a random selection of 10 different blank samples of antemortem whole blood and 20 different blank samples of postmortem whole blood. The samples were spiked with the individual drugs prior to the extraction to obtain concentrations that were three to six times the signal-to-noise ratio. The LOD was calculated as 2 t0.95 SDB (t0.95 ¼ 1.645), where SDB is the standard deviation of the results obtained from the spiked samples. The lower limit of quantification (LLOQ) was determined from the precision studies at concentrations of 10 SDB. The acceptance criterion was an RSDR,intra-lab of a maximum of 20% and a bias within +20% of the spiked concentration. Matrix effects and true extraction recovery The matrix effects (including ion-suppression and ion-enhancement effects) were investigated on 10 different blank samples of antemortem whole blood and 20 blank samples of postmortem whole blood that were spiked after UF at a level that was equivalent to 10 mg/L of vigabatrin, pregabalin and gabapentin and 1 mg/L of baclofen in the original samples. They were analyzed in attenuating order along with blank samples and the pure standards at the same concentration. The matrix effect from each sample was calculated from the peak areas (A) without IS correction using the closest standards in the series; matrix effect ¼ ðApure standard Aspiked sample Þ 100=Apure standard . The true extraction recoveries were determined from the same blood samples that were spiked to 10 mg/L of vigabatrin, pregabalin and gabapentin and 1 mg/L of baclofen. The standards that were used for the determination of the true recoveries were the same blood samples that were spiked after UF. To compensate for changes in
Extraction and chromatography The blood samples were treated with a mixture of MeOH and MeCN. The MeCN was used as the primary protein denaturing solvent, while MeOH was added to obtain a disperse precipitate. The denatured proteins were precipitated by centrifugation, and particles and dissolved high-molecular-weight substances were removed by filtration through a 30-kDa membrane. The filtrate was diluted with MeCN to avoid solvent effects that would have distorted the chromatography. All substances were ionized by electrospray in the positive mode (ESI(þ)). The dominant precursor (Q1) ions obtained were the protonated molecular ions ([M þ H]þ) and several product (Q3) ions of significant abundance were obtained for all substances. The quantifier and qualifier ions were selected from their abundances and their chromatographic separation from interferences (Table I). Due to the high polarity of vigabatrin, poor retention is obtained on traditional RP column packing. The Rt is typically 0.6 min for 100 mm UPLC columns at a flow rate of 0.4 mL/ min. Using the HILIC separation technique, the elution order is reversed compared with RP chromatography (i.e., the Rt increases with increasing polarity of the analyte), because the more polar compounds will have a stronger interaction with the aqueous layer that is established within the hydrophilic stationary phase than the less polar compounds. An Rt of several minutes is easily obtained for vigabatrin by this technique. Although the other GABA derivatives are less polar, sufficient retention can still be obtained for these substances by HILIC (Figure 1). Pregabalin, gabapentin and baclofen were not baseline separated by the short run time. However, the m/z ratios of the precursor ions were different, and no interferences from insource fragmentation were observed. The gradient was changed to 70% A after elution of the analytes to remove substances more polar than vigabatrin and maintain the selectivity constant from injection to injection.
Method performance parameters The mean matrix effects were ,15% for all of the substances (Table II). The mean true recoveries were .89%, and no significant differences between ante- and postmortem blood were observed (Table II). The LODs were 10 mg/L for the two most sensitive transition products of all the substances in both the ante- and the postmortem blood (Table III). The RSDR,intra-lab was ,8% at a concentration of 1 mg/L and ,4% at concentrations of 10 mg/L or higher (Table IV). The RSDR,intra-lab was also ,8% for baclofen at a concentration of 0.1 mg/L. According to the general rule of Horwitz (34), the obtained precision figures are considered acceptable. The Horwitz equation states that the RSDR between laboratories should be ,16 and 11% at concentrations of 1 and 10 mg/L, respectively. For intralaboratory data, the imprecision would normally be 25 – 50% lower. The relative bias determined at low, medium and high Therapeutic GABA Analogs in Forensic Whole Blood 179
Figure 1. Chromatograms of the ESI(þ) product ions in the extract of an antemortem whole blood sample spiked with 1 mg/L of vigabatrin, pregabalin and gabapentin and 0.1 mg/ mL of baclofen.
concentrations was within 210 –5% of the spiked concentration (Table IV). The LLOQ criteria were fulfilled at concentrations of 0.02 mg/L of gabapentin and baclofen, 0.03 mg/L of pregabalin and 0.04 mg/L of vigabatrin (Table IV). In comparison, LLOQ 180 Sørensen and Hasselstrøm
values of 0.02, 0.01, 0.02 and 0.02 mg/L were obtained for gabapentin, baclofen, pregabalin and vigabatrin when the LLOQ values were calculated as 10 SDB using the data obtained in the LOD study. The upper limits of quantification are 50 mg/L
for vigabatrin, pregabalin and gabapentin and 5 mg/L for baclofen, which correspond to the concentrations of the highest calibrant. The selectivity of the method against endogenous interference was demonstrated by the analysis of 10 different blank samples of antemortem blood and 10 different blank samples of postmortem blood that were independent from the samples used for the LOD determination. No interferences were observed that would impact the trueness of the method at the LLOQ level. Whole blood samples were also spiked with 67 frequently detected
Table II Matrix Effects and True Extraction Recoveries Obtained from the Single Analyses of the Spiked Antemortem Whole Blood (AMB) and Postmortem Whole Blood (PMB) Samples (n ¼ 10 for AMB, n ¼ 20 for PMB) Drug
Vigabatrin Pregabalin Gabapentin Baclofen
Spiked conc. (mg/L)
10 10 10 1
Matrix effect, mean (+SD) (%)
True extraction recovery, mean (+SD) (%)
AMB
PMB
AMB
PMB
215 (+7) 27 (+9) 29 (+10) 25 (+9)
24 (+6) 2 (+16) 22 (+18) 6 (+11)
92 95 96 89
91 (+7) 97 (+6) 96 (+5) 93 (+10)
(+5) (+3) (+1) (+4)
Matrix-matched standards used in the calculation of the recoveries were the samples spiked after the extraction and clean-up.
Table III LODs Determined in Antemortem Whole Blood (AMB) and Postmortem Whole Blood (PMB) Samples (n ¼ 10 for AMB, n ¼ 20 for PMB) Drug
Vigabatrin
Pregabalin
Gabapentin
Baclofen
Transition Q1/Q3 (m/z)
130/67 130/71 130/113 160/55 160/97 160/142 172/55 172/137 172/154 214/116 214/151 214/179
Spiked conc. (mg/L)
50 10 10 10 10 10 10 10 10 5 5 5
Result mean (+SD) (mg/L)
LOD (mg/L)
AMB
PMB
AMB
PMB
44 12 11 9 11 10 11 10 10 4 4 4
50 12 12 11 9 10 12 11 11 6 6 6
30 10 10 7 7 7 7 7 10 7 7 7
26 10 10 10 10 10 10 7 7 7 7 7
(+9) (+3) (+3) (+2) (+2) (+2) (+2) (+2) (+3) (+2) (+2) (+2)
(+8) (+3) (+3) (+3) (+3) (+3) (+3) (+2) (+2) (+2) (+2) (+2)
licit and illicit drugs and metabolites (Table V). The substances were tested at the high end of the concentration range that is normally found in blood. Finally, the selectivity was tested against less frequently or rarely detected drugs, which are characterized by having molecular weights (MW) that are close to the analytes: metformin (MW 129.2), tranexaminic acid (MW 157.2), buformin (MW 157.2), hydralazine (MW 160.2), chloroxazone (MW 169.6), levetiracetam (MW 170.2), metronidazole (MW 171.2), zonisamide (MW 212.2) and butobarbital (MW 212.3). Buformin and hydralazine were tested at a concentration of 1 mg/L in the blood, whereas the other drugs were tested at a concentration of 50 mg/L. No interferences from all of these drugs were observed on the chromatograms. The calibration curves were created using weighted linear regression analysis to improve the variance homogeneity over the calibrated concentration range. From the residual plots, the weighting factor 1/x appeared suitable. Because no significant interferences from endogenous substances were present on the chromatograms, the calibration curve was forced through the origin to improve reproducibility at low concentrations. The difference in the slopes of the calibration curves prepared from pure solvent calibrants and matrix-matched calibrants (eight independent pairs from the precision study) was ,3% for vigabatrin and ,2% for the other substances. This difference was not statistically significant at a 95% confidence level. The ruggedness of the calibration would most likely not be affected by using pure calibrants because the differences between the Rt of the analytes and the Rt of the corresponding SIL-IS were very small compared with the peak band widths (Table I). However, because of the simple sample pretreatment procedure, matrix-matched calibrants were used. The R 2 values of the eight calibration curves obtained in the precision study were 0.998 + 0.002 (mean + SD) for baclofen and 0.999 + 0.002 for the other substances. Stability of analytes in blood and extracts The stability of the substances in whole blood freshly preserved with FO and FC mixtures (n ¼ 2 of each) was tested at storage temperatures of 20 + 28C, 5 + 28C and 220 + 28C for 8 days. The samples included in the stability test were spiked with the drugs at concentrations of 1 mg/L of baclofen and 10 mg/L of the other analytes. The samples were analyzed when they were
Table IV Method Precision and Trueness Estimated for Different Drug Concentrations in Antemortem Whole Blood (AMB) and Postmortem Whole Blood (PMB) Samples Drug
Spiked conc. (mg/L)
Mean result AMB/PMB (mg/L)
RSDr AMB/PMB (%)
RSDR,intra-lab AMB/PMB (%)
Relative bias AMB/PMB (%)
Vigabatrin
0.04 1 10 40 0.03 1 10 40 0.02 1 10 40 0.02 0.1 1 4
0.036/0.039 0.99/0.97 9.98/10.0 39.8/39.7 0.031/0.030 0.96/0.93 9.76/9.84 39.8/39.6 0.021/0.021 0.96/0.95 9.91/10.0 39.6/39.9 0.021/0.021 0.094/0.10 0.98/1.01 3.95/3.99
15/14 2.8/5.9 2.1/1.6 2.5/2.4 16/15 2.7/3.3 1.4/1.7 1.6/1.3 7.3/13 2.4/3.0 1.6/2.1 1.3/1.4 20/14 7.5/6.5 3.3/2.7 2.8/2.2
15/15 4.2/7.7 3.3/3.0 2.8/3.4 16/16 3.3/4.5 3.7/2.3 2.3/1.6 15/16 3.0/4.9 2.4/2.9 1.4/2.1 20/14 7.5/6.5 5.1/2.7 4.0/2.2
210/23 21/23 0/0 21/21 3/0 24/27 22/22 21/21 5/5 24/25 21/0 21/0 5/5 26/0 22/1 21/0
Pregabalin
Gabapentin
Baclofen
Therapeutic GABA Analogs in Forensic Whole Blood 181
Table V Drugs and Metabolites Frequently Detected in Whole Blood Alprazolam 7-Aminoclonazepam 7-Aminoflunitrazepam Amphetamine Barbitale Benzoylecgonine Bromazepam Buprenorphinea Caffeinee Carbamazepinee Chlordiazepoxidec Chlorprothixene Citalopramc Clonazepam Cocaine Codeine Cyclobarbitale Demoxepam Desmethylmirtazapine O-Desmethyltramadolc O-Desmethylvenlafaxine Diazepam Diclofenacd
Etodolace Fentanyla Flunitrazepam Hydromorphon 10-Hydroxycarbazepinee Ibuprofene Ketamine Lamotriginee Lidocainec Lorazepam MDA MDMA Metamphetamine Methadonec Methylphenidate Mirtazapine 6-MAMa Morphine Morphine-3-b-D-glucuronide Morphine-6-b-D-glucuronide Naproxene Nitrazepam
Norbuprenorphinea Nordazepam Noscapinea Olanzapine Oxazepamc Oxycodone Papaverine Paracetamolf Pentobarbitale Phenobarbitale Phenytoine Quetiapine Salicylic acidf Sertraline THCa THC –COOHb THC –OHa Tramadolc Venlafaxine Zolpidem Zopiclone Zuclopenthixol
The interference from these drugs were investigated at concentrations of 0.032 mg/L (a), 0.08 mg/ L (b), 1.6 mg/L (c), 4 mg/L (d), 40 mg/L (e), 80 mg/L ( f ) and 0.8 mg/L (all other substances).
freshly prepared and after 1, 2, 4, 7 and 8 days of storage. No significant decline in concentrations was observed during the storage at ambient temperature. The results (mean + SD) that were obtained for freshly prepared samples preserved with the FO mixture were 9.81 + 0.22 mg/L (vigabatrin), 9.80 + 0.23 mg/L ( pregabalin), 9.72 + 0.08 mg/L (gabapentin) and 1.04 + 0.04 mg/L (baclofen). The corresponding mean results that were obtained after 7 and 8 days of storage at ambient temperature were 10.05 + 0.30, 9.78 + 0.13, 10.00 + 0.26 and 1.06 + 0.06 mg/L. The results obtained for freshly prepared samples that were preserved with the FC mixture were 9.98 + 0.05 mg/L (vigabatrin), 10.07 + 0.07 mg/L ( pregabalin), 9.90 + 0.36 mg/L (gabapentin) and 1.06 + 0.02 mg/L (baclofen). The corresponding mean results that were obtained after 7 and 8 days of storage at ambient temperature were 9.97 + 0.37, 9.83 + 0.12, 9.83 + 0.08 and 1.05 + 0.06 mg/L. For the other storage temperatures, the results obtained after 7 and 8 days of storage were also within +3% of the results obtained from the freshly prepared samples that were preserved with the FO and FC mixtures. The stability of the blood-based calibrants stored at 5 + 28C and 220 + 28C was tested over a period of 8 days. The calibrants were prepared according to the previously described procedure, except the SIL-IS was not added to the blood sample at the beginning but was instead added to the extracts on the day of the UPLC – MS/MS analysis. No significant changes in the slopes of the calibration curves were observed during the 8 days of storage at 5 and 2208C, respectively. The slopes of freshly prepared calibrants (in units of L/mg) were 0.34 (vigabatrin), 0.16 ( pregabalin), 0.35 (gabapentin) and 7.2 (baclofen). The slopes of the calibrants stored at 5 + 28C and 220 + 28C were 0.33 and 0.36 (vigabatrin), 0.16 and 0.17 ( pregabalin), 0.34 and 0.36 (gabapentin) and 7.3 and 7.3 (baclofen). Application of the method The developed method has been applied to 17 clinical and autopsy cases where the presence of the drugs has previously been 182 Sørensen and Hasselstrøm
detected by a screening method based on time-of-flight mass spectrometry or was likely from information about the prescribed medicines. The determined concentrations of gabapentin and baclofen ranged from 0.2 to 19 mg/L (n ¼ 8) and from 0.16 to 0.35 mg/L (n ¼ 2), respectively, which are within the therapeutic ranges. For pregabalin, most samples (n ¼ 6) were in the concentration range of 0.3 –5 mg/L. However, in one autopsy case, the blood concentration of pregabalin was 30 mg/L, which is greatly above the normal therapeutic range. The concerned person, a 49-year-old male, who was found dead in his bed, also had a high blood concentration of venlafaxine (3.6 mg/L) and a mirtazapine concentration (0.27 mg/L) at the high end of the therapeutic range.
Conclusion A sensitive and rapid UPLC –MS/MS method was developed and validated for the determination of vigabatrin, gabapentin, pregabalin and baclofen in ante- and postmortem whole blood. The extraction recoveries of 89 – 97% were obtained by a simple deproteinization procedure using a mixture of MeOH and MeCN. Adequate chromatographic retention of the polar drugs was obtained by HILIC. The matrix effects were relative low (,15%). Isotope dilution of each substance was used to eliminate the influence of differences in the extraction recoveries and ion-suppression effects. The method bias was less than +6% and the imprecision (RSDR,intra-lab) was 8% for concentrations within the therapeutic ranges. The LLOQ values were ,0.1 mg/L. This method is suitable for quantitative investigation in clinical and autopsy cases where the GABA analogs may have caused or contributed to impaired driving, intoxication, attempted suicide or death.
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