BUPRENORPHINE - Imperial College London



Validated method for the screening and quantification of baclofen, gabapentin and pregabalin in human post-mortem whole blood using protein precipitation and liquid chromatography - tandem mass spectrometry

Abstract

There has been a rapid increase in the number of prescriptions for baclofen (BLF), gabapentin (GBP) and pregabalin (PGL) in the United Kingdom since their introduction to therapy. Recent studies across the European Union and United States have shown the illicit abuse potential of these drugs and deaths have been observed. A simple, reliable and fully validated method was developed for the screening and quantification of BLF, GBP, and PGL in human post-mortem (PM) blood. The analytes and their deuterated analogues as internal standard were extracted from blood using a single addition acetonitrile protein precipitation reaction followed by analysis using triple-quadrupole liquid chromatography-tandem mass spectrometry (LC-MS/MS) with triggered dynamic multiple reaction monitoring mode (t-DMRM) for simultaneous quantification and confirmation. The assay was linear from 0.05 – 1.00 µg/mL for BLF and 0.5 – 50.0 µg/mL for GBP and PGL respectively with r2 values greater than 0.999 (n=9) for all analytes. Intra-day and inter-day imprecisions (n=80) were calculated using one-way ANOVA; no significant difference (P>0.99) was observed for all analytes over 8 non-consecutive days. The average recovery for all analytes was greater than 98.9 %. The limit of detection and limit of quantification was 0.05 µg/mL for BLF and 0.5 µg/mL for GBP and PGL. The method was highly selective with no interference from endogenous compounds or from 55 commonly encountered drugs in PM toxicology. To prove method applicability to Coroners PM cases, authentic PM blood samples were analysed.

Keywords: baclofen, gabapentin, pregabalin, post-mortem blood, Liquid chromatography-tandem mass spectrometry

Introduction

Baclofen (BLF), gabapentin (GBP) and pregabalin (PGL) are structural analogues of gama-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the mammalian central nervous system. Baclofen is prescribed for the alleviation of spasticity due to multiple sclerosis (1), but the General Medical Council (UK) has approved its off-label use for treating alcoholics. GBP and PGL are used for the treatment of epilepsy and neuropathic pain. Pregabalin is additionally used for generalized anxiety disorder, and GBP has off-label use for treatment of restless leg syndrome, substance dependence and migraine (2). In the UK, BLF has been prescribed since the 1970s, GBP since 1993 and PGL since 2004. From 2009-2015 in England there was a rise in prescriptions for BLF, GBP and PBL of 44%, 175% and 225% respectively (3, 4).

In Coroners post-mortem (PM) toxicology, the knowledge of compliance and non-compliance of medication by the deceased is important especially in cases where anti-epileptic drugs are involved. Baclofen, GBP and PGL are prescription drugs only; however governmental reports and case studies indicate a growing illicit market for the abuse of these drugs (5, 6). Baclofen, GBP and PGL misuse amongst addicts and people with no history of drug abuse have been observed throughout Europe (6-8). Gabapentin and PGL abuse has been strongly observed amongst the heroin addict population (6, 9) and overdoses have been reported in literature (10). Thus, these drugs should be routinely analysed for in Coroners PM toxicology, however due to the amphoteric nature of the drugs, they are not detected in a broad-spectrum screen for basic or acidic drugs. Therefore, a quick, sensitive and cost-efficient method is required for the screening and quantification of BLF, GBP and PGL in PM blood at therapeutic concentration and above. The therapeutic concentration for BLF ranges between 0.04 – 1.10 µg/mL in plasma (11) and 2 - 20 µg/mL for GBP in serum (12). The therapeutic reference range has not yet been identified for PGL however a group of patient receiving 600 mg/day showed a steady state concentration of 2.8 - 8.3 µg/mL(13).

There are many analytical methods available for the analysis of BLF, GBP and PGL in biological matrices. They have been analysed individually using high-pressure liquid-chromatography (HPLC): (13-16); gas chromatography-mass spectrometry (GC-MS) (17-19) and more recently liquid chromatography-tandem mass spectrometry (LC/MS-MS) has been employed (20-22). Several methods have been reported where GBP and PGL have been analysed together (23, 24) however to our knowledge only one quantitative method has been reported where all three drugs were analysed together in whole blood (22). Most methods use a form of protein precipitation (25, 26) or solid-phase extraction (11, 18). Most published HPLC methods require pre-column derivatisation as the drugs do not have a suitable chromophor for florescence UV detection (13, 14, 24). Many GC-MS analysis of these drugs also requires a form of derivatisation (17-19). Derivatisation is a time consuming process involving multiple steps of concentrating extracts before derivatisation, and in some instances requiring multiple reagents.

Quantification using LC/MS-MS is becoming more common due to increased sensitivity and selectivity. Most published methods utilise structurally similar compounds to the analytes as an internal standards (IS) rather than using the deuterated analogue e.g. BLF–d4, GBP-d10, PGL-d6 (20, 21, 27). It is essential to use deuterated analogues when analysis is performed by LC/MS-MS as it is the only way to accurately compensate for potential matrix enhancement or suppression.

Many LC/MS-MS methods only monitor 1 product ion (25, 27, 28), whilst others monitor 2 (23, 29) or 3 (22) product ions for both quantification and confirmation. Although it is an acceptable approach in toxicology, only using 1 product ion for confirmation increases the chance of reporting a false positive result. Using full-scan monitoring, or selective ions monitoring in tandem with full-scan monitoring is possible with LC/MS-MS, however loss of sensitivity is generally observed. Alternatively analysis performed on an Agilent 6460 Triple Quadrupole mass spectrometer (TQ-MS) (Agilent Technologies, UK) in the triggered dynamic multiple reaction monitoring (t-DMRM) acquisition mode enables a product ion spectrum to be generated; it scans for up to 10 product ions per analyte without compromising sensitivity.

A simple, cost-efficient protein precipitation-LC/MS-MS method was developed and validated using deuterated IS and t-DMRM mode for the screening and quantification of BLF, GBP and PGL in human post-mortem blood. Authentic samples were analysed to demonstrate method applicability. To our knowledge, this is the first published method using t-DMRM mode to quantitate the three analytes together in PM blood.

Materials and methods

Chemicals

Baclofen (±) (0.5 mg/mL) GBP, PGL (1 mg/mL), GBP-d10 and PGL-d6 (0.1 mg/mL) were purchased from Sigma-Aldrich Company Ltd (Poole, UK). BLF–d4 (10 mg) was purchased from LGC Standards (Teddington, UK). Acetonitrile (LC/MS grade) and 90% formic acid (analytical grade) were purchased from VWR (Lutterworth, UK). Deionised water (15.0 MΩ.cm at 25°C) was produced in-house using PURELAB Option-R7/15 (ELGA-VEOLIA, UK) water purifier.

Instrumentation and chromatographic conditions

For chromatographic separation and analysis, a 1260 Infinity Binary high-performance liquid chromatography system coupled to a 6460 TQ-MS (Agilent Technologies, UK) was used. Mobile phase A was composed of deionised water: formic acid (0.1%) and mobile phase B contained acetonitrile: formic acid (0.1%). Chromatographic separation was performed at 40°C column temperature and a flow rate of 0.20 mL/min using a Poroshell 120 EC-C18 HPLC column (2.1 x 100 mm, 2.7 µm) (Agilent Technologies, UK). A complementary guard cartridge (EC-C18 2.1 x 5 mm, 2.7 µm) was connected to the chromatographic column. Electrospray ionisation (ESI) in the t-DMRM mode was used for analysis.

The injection volume was 1 µL. The sample run time was 8.10 min with a 5 min post-run time to re-equilibrate the column (total run time per sample: 13.10 min). To control the LC system and the TQ-MS, MassHunter Data Acquisition software (version B.06.00) was used. For data analysis QQQ Quantitative Analysis software (version B.06.00) was used. The LC gradient system parameters, TQ –MS parameters and the quantifier, qualifier and triggered ion transitions monitored by TQ-MS are listed in Table 1.

Preparation of standard solutions

Stock working solution containing BLF at 1.00 µg/mL, GBP and PGL at 50.0 µg/mL was prepared in deionised water. Stock solution of the deuterated IS containing BLF-d4 (0.30 µg/mL), GBP-d10 and PGL-d6 (0.50 µg/mL) was also prepared in deionised water. Quality control standards (QC) at a low and high concentration (BLF: 0.08, 0.80 µg/mL; GBP and PGL: 1.5, 40.0 µg/mL) were prepared in deionised water. All stock solutions and QCs were prepared independently and were stored at -20°C.

Preparation calibrators and quality control

Stock working solution was diluted with deionised water to prepare 7 calibration standards consisting of blank, 0, 0.05, 0.10, 0.20, 0.50 and 1.00 µg/mL for BLF, and 9 calibration standards consisting of blank, 0, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0 and 50.0 µg/mL for GBP and PGL in micro-centrifuge tubes (100 µL total). One hundred-microliters of QC standards (low and high in singlet) were pipetted into micro-centrifuge tubes.

Sample preparation

To the all calibrators and QCs (100 µL), 100 µL of human drug-free whole blood (DFB) was added. To the PM bloods (100 µL), 100 µL of deionised water was added. To all calibrators (excluding blank) and QCs, 200 µL of the internal standard solution was added. To the blank, 200 µL of deionised water was added. The samples were vortex mixed thoroughly and then micro-centrifuged for 2 minutes at 12000 rpm (8049.6 x g).

Protein precipitation

After centrifugation 100 µL of supernatant from each sample was pipetted into fresh micro-centrifuge tubes. Two-hundred microliters of acetonitrile was added to the aliquots followed by vortex mixing and centrifugation as previously mentioned.

Fifty-microliters of the sample supernatants were taken into LC micro-vials and diluted with 50 µL acetonitrile. The samples were injected (1 µL) onto the LC/MS-MS.

Method validation

For validation the following criteria were determined: specificity/selectivity, regression line weighting, linearity and range, limit of detection (LOD) and limit of quantification (LOQ), intra-day and inter-day accuracy and precision, stability, recoveries and assessment matrix effect. Validation was preformed according to United Kingdom and Ireland Association of Forensic Toxicologists (UKIAFT) guidelines (30). All analysis was carried out using human DFB to matrix match with PM blood.

Specificity /Selectivity

The specificity of the method was determined by extracting and analysing 8 DFB samples to establish the absence of chromatographic interference from blood endogenous compounds with the analytes of interest.

Authentic PM blood samples often contain other drugs in combination with the analytes of interest. To prove that no interference was occurring from other drugs that maybe present, a single drugs mix containing 52 frequently observed drugs in toxicological screening was extracted in blood using the analytical method (concentrations ranging from 0.05 – 125 µg/mL). The mix included amphetamines, benzodiazepines, cocaine, opiates, anti-depressants, anti-psychotics, drugs given during emergency treatment and over-the-counter drugs.

Regression model

Five replicates of each calibration standard were extracted and an average linear calibration line was generated for the analytes. Using the data obtained a residual graph was plotted to determine homoscedasticity of the analytes. To determine the type of weighting required, calibration curves (linear) using the average response from the standard point replicates (n=5) were prepared with no-weighting, 1/x and 1/x2 weighting for all analytes. The accuracy at each calibration point was calculated by comparing the theoretical concentration of each standard to the calculated concentration obtained from the different calibration curve type. The weighting type was selected based on the simplest model that adequately described the concentration–response relationship.

Linearity, limit of detection and quantification

Linearity was verified by analysing three full calibration lines on three non-consecutive days (n=9). For the calibration line to be acceptable, the lowest quantifiable value had to be within ± 20% of its theoretical value compared to its extracted values against the obtained calibration line and the rest of the calibration standards were required to be within ± 15%. The LOQ was defined as the lowest concentration that could be reproducibly detected within ± 20% error and a signal to noise ratio (S:N) greater than 10. The LOD was set as the lowest concentration that could be detected with the presence of the quantifier ion, qualifier ion and all triggered product ions at an acceptable ratio (± 20%) and a S:N > 3.

Accuracy and precision

For intra-day and inter-day analysis, 5 replicates of the low and high QCs were extracted with full calibration standards. This was repeated on eight non-consecutive days (total of 80 QCs analysed). One-way ANOVA statistical analysis was performed using the alpha p-value of 0.05 to determine intra-day and inter-day variance.

Stability

Three different stability studies were performed: stability of extracted sample, short and long term stability of analytes under different storage conditions and a freeze-thaw study.

For extracted sample stability, 2 full calibration lines of the analytes were extracted and analysed on day zero (day of extraction). The extracts were then left at ambient temperature and re-analysed on days 1, 3 and 6. The average percentage relative standard deviation (%RSD) was calculated comparing the results obtained for the calibration standards on day 0 to that obtained on days 1, 3 and 6 where the samples were left at ambient temperature.

Quality control samples were prepared at a low and high concentration (BLF: 0.20, 0.80 µg/mL; GBP and PGL: 5.0, 15 µg/mL) in DFB and were divided into tubes containing preservative (fluoride oxalate 1.1%) and tubes containing no preservatives. A full calibration line was extracted and analysed with triplicate sets of low and high QCs, preserved and un-preserved, on day 0 (day of sample preparation). The remaining QC samples (preserved and un-preserved) were stored at ambient temperature, fridge (5 °C) and freezer (-20 °C). Short and long term stability of the analytes was determined on days 3, 7, 14 and two months. On each analysis day a full calibration line was extracted with 3 replicates of each QC sample (low and high concentration, preserved and un-preserved). Stability was determined by calculating the percentage difference for the QC results obtained on day 0 to days 3,7,14 and two months.

To determine the stability of the analytes after 3 freeze-thaw cycles in preservative and non-preservative containing tubes, QC samples at low and high concentration left over from day 0 analysis (used for short and long term stability) were frozen, and underwent 3 freeze-thaw cycles and were analysed over 3 non-consecutive days with a full calibration line. Freeze-thaw stability was determined by calculating the percentage relative standard deviation the average QCs results obtained on day 0 to that obtained on each thawing day (6 low QC and 6 high QC: n=12).

Extraction efficiency

Low and high QCs (BLF: 0.20, 0.80 µg/mL; GBP and PGL at 10.0, 40.0 µg/mL) were prepared in DFB (five at each concentration) and extracted using the described method but replacing the IS with deionised water. Ten DFB samples were also extracted using the method stated above. Fifty-microliters of all extracted samples were placed to micro-vials and an external standard, containing BLF–d4, GBP-d10 and PGL-d6, was added. To the extracted QCs, 50 µL of acetonitrile was added to the micro-vials. To 5 of the drug-free extracts, 50 µL of acetonitrile containing the equivalent concentration of the extracted low QC was added. To the remaining 5 drug-free extracts, 50 µL of acetonitrile containing the equivalent concentration of the extracted high QC was added. All samples were then analysed. The percentage recovery was calculated by dividing the mean peak area ratio of the QC samples, fortified prior extraction, by the mean peak area ratio of the samples to which the analytes were added after extraction multiplied by 100.

Assessment of matrix effect

A matrix effect experiment was carried out to determine if any potential suppression or enhancement was occurring to the ion signals of the analytes due to other endogenous compounds present in the sample.

Eight different sources of DFB samples were extracted in duplicate (n=16), and 50µL extracts were placed into micro-vials. Into six empty micro-vials 50 µL of acetonitrile was added (no matrix). To all vials 50 µL of acetonitrile containing the equivalent concentration of extracted 0.50 µg/mL BLF and 25.0 µg/mL of GBP and PGL, was added. The average response of the analytes in the matrix containing samples was compared to the response of the analytes in the non-matrix containing sample.

Post-mortem samples

The Toxicology Unit performs routine toxicological analysis on PM samples submitted from Coroners’ cases from in and around London (UK). All cases that requested a general drugs screen were also screened for BLF, GBP or PGL in femoral blood; any positive cases were quantitated in duplicate (n=17). The samples were stored at 5 °C until analysis.

Results

Specificity /Selectivity

No interference was observed from the drug-free blood samples (n=8) analysed or from the 55 most commonly observed drugs in routine toxicological screening using the described method on the analytes or internal standards of interest.

Regression model

For the residual plot, it was observed that GBP and PGL were heteroscedastic and required a form of weighted calibration curve whilst BLF was homoscedastic and required no weighting. All calibration lines were linear. The weighting type required for GBP and PGL was determined as 1/x.

Linearity, limit of detection and quantification

BLF calibration lines (n=9) were linear from 0.05 – 1.00 µg/mL, and for GBP and PGL the lines (n=9) were linear from 0.5 – 50.0 µg/mL, with a correlation coefficient (r2) > 0.999 for all analytes. Acceptable % error (± 15 %) was obtained throughout the calibration range. The results are summarised in Table 2.

The LOD and LOQ for BLF was 0.05 µg/mL whilst 0.5 µg/mL for GBP and PGL. At this concentration the quantifier, qualifier and all triggered product ions were present in an acceptable ratio (± 20%) and with an S:N >3. Figure 1 shows the quantifier and qualifier ion peaks generated and their product ion spectra at the analytes LOD.

Accuracy and imprecision

The analysis of variance (one-way ANOVA) showed there was no significant difference between intra-day and inter-day analysis of all three analytes at the low and high QC concentration; BLF: F, (7, 72) = 0.032, P = 0.99, GBP: F, (7, 72) = 0.0023, P = 1.00 and PGL: F, (7, 72) = 0.0029, P = 1.00.

Stability

Extracted samples left at ambient temperature were stable for 6 days with less than 5.5% RSD (n=10) when comparing day-zero’s to day-six results for all analytes. The criteria at the LOD concentration were satisfied for all analytes on each day of analysis.

All three analytes were stable when stored at ambient temperature, fridge (5 °C) and freezer (-20 °C) for up to two-months in both preservative and non-preservative containers. The maximum percentage difference, comparing day-zero’s results to days under storage, over the two-months were; BLF: -11.3%, GBP: -6.7%, PGL: +4.2%. These are within the acceptable error of 20% for forensic cases (as described in the UKIAFT guidelines).

The average %RSD (n=12) of the concentration obtained after 3 freeze-thaw cycles compared to the concentrations obtained on day 0 was; BLF in preserved blood: 1.5%, non-preserved blood: 3.7% , GBP in preserved blood: 1.4 %, non-preserved blood: 1.4 %, PGL in preserved blood: 0.4 %, non-preserved blood: 0.4 %. The results show that all three analytes are stable after 3 freeze-thaw cycles.

Data from the freeze-thaw cycles and the long and short-term stability study indicate there is no substantial difference between use of a preservative or non-preservative containing tubes for blood sample storage with a maximum difference of ± 4.1 SD between them.

Extraction efficiency

The average extraction efficiency for; BLF: 98.9 % (± 9.2 SD), GBP: 102.9 % (± 2.1 SD,), PGL: 104.8 % (± 2.9 SD) (n=10).

Assessment of matrix effect

The mean matrix effect observed for; BLF -2.2 % (± 4.9 % RSD), GBP: -1.0 % (± 1.9% RSD), PGL: +1.0 % (± 1.8 % RSD) (n= 16). Results indicate a small amount of matrix suppression for BLF and GBP and enhancement for PGL was occurring.

Method application on PM samples

No complications were experienced during the extraction or analysis of the PM samples. The results from 17 PM cases analysed are summarised in Table 3. The concentration range observed in PM for BLF was 0.08 – 102.00 µg/mL, for GBP 1.0 – 134 µg/mL and 2.0 - 540 µg/mL for PGL. Human drug-free blood was used for dilution in cases where the drug concentration was greater than the calibration range. In all cases, multiple drug consumption was observed (illicit drugs/prescription drugs). The previously highest reported level of PGL in PM whole blood was 226 µg/mL (10), we report two of the highest concentrations to date of 301 and 540 µg/mL.

Method suitability for screening

In addition to being used as a full quantitative analysis, the described method has been successfully used as a routine screening method for the three analytes. Extracted full calibration curves for each analyte were stored in a quantitative database using the MassHunter QQQ Quantitative Analysis software.

During the screening procedure, singlet case blood samples (diluted 1 in 2 with drug-free blood) were extracted alongside blank, low and high QC concentrations (BLF: 0.08, 0.80 µg/mL, GBP and PGL: 1.5, 40.0 µg/mL). The case blood data were then processed against the stored calibration lines. Negative cases were easily identified and for cases positive for the analytes, a semi-quantitative result was obtained. The semi-quantitative result can be used to report a therapeutic result, as long as the QCs are within ± 20% of their theoretical value when processed with the stored calibration curves. The semi-quantitative result can help determine the dilution factor required for analyte quantification. For this study all cases positive for the analytes were quantified (in duplicate) with a full set of fresh calibration standards.

To determine the stability of the stored calibration curves, QCs at a low and high concentration were extracted, analysed and calculated using the stored calibration curve over several weeks. The stored calibration curves have been determined to be stable for at least 7 weeks, with the average % error of the QCs of less than 5% and % RSD ≤12.0% (n=14).

To further determine the accuracy of the concentration obtained for the analytes during the screen (using the stored calibration curve) they were compared to the concentration obtained when the samples were extracted with a full set of calibration standards at the same time. The concentrations were compared by calculating the % RSD screen and quantification results; BLF: 6.0% (n=5), GBP: 10.3% (n=15), PGL: 5.8% (n=15). The results indicate the screen provides a good estimate of the analytes true concentration.

Discussion

An efficient and cost-effective screening and quantification method has been developed for the analysis of BLF, GBP and PGL in PM blood using protein precipitation and LC/MS-MS.

Most published methods using HPLC and GC/MS for the analysis of BLF, GBP and PGL require time consuming extraction procedure involves of concentration of samples followed by derivatisation (13, 14, 19). The described method is highly efficient with a simple extraction procedure and does not require concentrating or derivatisation. Extraction can be performed with reagents that are readily available in most laboratories.

There are many LC/MS-MS methods for the analysis of BLF, GBP and PGL; however, many methods only monitor 1 to 3 product ions for both quantification and confirmation. The developed method utilises t-DMRM with scanning mode of the instrument set to dynamic–MRM (d-MRM). The t-DMRM mode reduces the number of concurrent MRMs monitored and keeps the cycle and dwell times at optimal for the analytes; this is done by monitoring the 2 primary products ions (quantifier and qualifier ion) only at their specific Rt window. At a pre-set analyte threshold a secondary product ion spectrum is generated (up to 8 additional ions) which can be matched with a spectral library for analyte confirmation. Being able to simultaneously quantitate and confirm the analytes in a single run without loss of sensitivity maximises the time and cost efficiency of the method.

Many published LC-MS/MS methods use structurally similar compounds to the analytes as an internal standard (25-27) or just one deuterated analogue internal standard, to analyse multiple analytes (23). In the described method BLF-d4, GBP-d10 and PGL-d6 were used as the internal standards. Although LC-MS/MS is a very sensitive and selective technique, in complex samples such as post-mortem blood, matrix effects can be observed. As all post-mortem blood samples are different and are at variable states of decomposition (viscous and oleaginous) matrix effects are more likely to be prominent and difficult to correct for if non – deuterated analogues are used as an internal standards. Using deuterated analogues as internal standards is the most effective way to compensate for potential matrix effects and preventing erroneous results being reported.

During the literature search, it was observed that many authors performed short and long-term stability studies on the analytes in biological matrices (13, 27, 31); however, no comparisons have been made between stability of the analytes in preservative and non-preservative containing tubes. In some instances the authors do not clarify what containers the biological samples were stored in for the stability studies (20, 25) whilst others preformed stability studies on drugs without biological matrix (31). Many mortuaries do not store PM blood samples in tubes containing preservatives. It is important to know if special storage is required to prevent degradation of the analytes from occurring. Comparing stability between analytes stored in preservative and non-preservative containing tubes is the only way to answer this. The short and long-term stability studies presented here analysed human blood stored in both preservative and non-preservative containing tubes; no significant degradation or differences were observed between the storage conditions for the three analytes.

To our knowledge only one other quantitative LC-MS/MS method has been published (by Sørensen et al. 2014) which analysed for BLF, GBP and PGL together in PM blood and monitors 3 ions per analyte (22). Although the sample volume in the Sørensen method and the method described here are the same (100 µL) and both methods are a form of protein precipitation, Sørensen uses 1500 µL of ACN per sample, compared to only 250 µL in the method described here, and additionally requires methanol. The Sørensen method also requires an ultrafiltration step and expensive Amicon Ultra filter units are required. The method described here is cost-effective, requires only a single solvent, standard micro-centrifuge tubes.

Conclusion

A simple, efficient and highly sensitive method has been validated for the screening and quantification of BLF, GBP and PGL in post-mortem blood using protein precipitation and LC-MS/MS in the t-DMRM mode. The screening method is cost effective and provides an accurate estimation of the analytes prior to quantification and can be used alone to report a therapeutic result without the need for further quantitation. To our knowledge, this is the first report comparing short and long-term stability of BLF, GBP and PGL in whole blood in preservative and non-preservative containing tubes and the first method to analyse BLF, GBP and PGL together in PM blood using t-DMRM mode.

Reference List:

1. Jones, R.F., Burke, D., Marosszeky, J., Gillies, J. (1970) A new agent for the control of spasticity. Journal of Neurology, Neurosurgery & Psychiatry, 33, 464-468.

2. Chiappini, S., Schifano, F. (2016) A Decade of Gabapentinoid Misuse: An Analysis of the European Medicines Agency’s ‘Suspected Adverse Drug Reactions’ Database. CNS Drugs, 30, 1-8.

3. (2010) Prescription Cost Analysis England 2009. Health & Social Care Information Centre. (accessed 22 June 2016)

4. (2016) Prescription Cost Analysis England 2015. Health & Social Care Information Centre (accessed 22 June 2016)

5. Kapil, V., Green, J.L., Le Lait, M.C., Wood, D.M., Dargan, P.I.(2014) Misuse of the γ-aminobutyric acid analogues baclofen, gabapentin and pregabalin in the UK. British Journal of Clinical Pharmacology, 78, 190-191.

6. (2013) Advice for prescribers on the risk of the misuse of pregabalin and gabapentin. Public Health England.

(accessed 22 June 2016)

7. Yates, C., Dines, A.M., Wood, D.M., Hovda, K.E., Heyerdahl, F., Giraudon, I., et al. (2015) Emergency Department presentations following recreational use of baclofen, gabapentin and pregabalin: A Euro-DEN case series. Clin Toxicol, 53, 372-373.

8. Wood, D.M., Besharat, A.C., Dargan, P.I., Martinez, E.M., Green, J.L. (2015) Pregabalin, gabapentin and baclofen: Sources of drug acquisition for non-medical use in an online national survey in the UK. Clin Toxicol, 53, 376-377.

9. Grosshans, M., Lemenager, T., Vollmert, C., Kaemmerer, N., Schreiner, R., Mutschler, J., et al. (2013) Pregabalin abuse among opiate addicted patients. European Journal of Clinical Pharmacology, 69, 2021-2025.

10. Eastwood, J.A., Davison, E. (2016) Pregabalin concentrations in post-mortem blood—A two year study. Forensic Science International, 266, 197-201.

11. Nahar, L.K., Cordero, R.E., Nut,t D., Lingford-Hughes, A., Turton, S., Durant, C., et al. (2016) Validated Method for the Quantification of Baclofen in Human Plasma Using Solid-Phase Extraction and Liquid Chromatography–Tandem Mass Spectrometry. Journal of Analytical Toxicology, 40, 117-123.

12.Patsalos, P.N., Berry, D.J., Bourgeois, B.F., Cloyd, J.C., Glauser, T.A., Johannessen, S.I., et al. (2008) Antiepileptic drugs—best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia, 49, 1239-1276.

13. Berry, D., Millington, C. (2005) Analysis of pregabalin at therapeutic concentrations in human plasma/serum by reversed-phase HPLC. Thererapeutic Drug Monit., 27, 451-456.

14. Wuis, E.W., Dirks, R.J.M., Vree, T.B., Vanderkleyn, E. (1985) High-performance liquid chromatographic analysis of baclofen in plasma and urine of man after precolumn extraction and derivatization with o-phthaldialdehyde. Journal of Chromatography, 337, 341-350.

15. Harrison, P., Tonkin, A., McLean, A. (1985) Determination of 4-amino-3-(p-chlorophenyl) butyric acid (baclofen) in plasma by high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications, 339, 424-428.

16. Sagirli, O., Çetin, S.M., Önal, A. (2006) Determination of gabapentin in human plasma and urine by high-performance liquid chromatography with UV–Vis detection. Journal of Pharmaceutical and Biomedical Analysis, 42, 618-624.

17. De Giovanni, N., D’Aloja, E. (2001) Death due to baclofen and dipyrone ingestion. Forensic Science International, 123, 26-32.

18. Kushnir, M., Crossett, J., Brown, P., Urry, F. (1999) Analysis of gabapentin in serum and plasma by solid-phase extraction and gas chromatography-mass spectrometry for therapeutic drug monitoring. Journal of Analytical Toxicology, 23, 1-6.

19. Mudiam, M.K.R., Chauhan, A., Jain, R., Ch, R., Fatima, G., Malhotra, E., et al. (2012) Development, validation and comparison of two microextraction techniques for the rapid and sensitive determination of pregabalin in urine and pharmaceutical formulations after ethyl chloroformate derivatization followed by gas chromatography–mass spectrometric analysis. Journal of Pharmaceutical and Biomedical Analysis, 70, 310-319.

20. Ramakrishna, N., Vishwottam, K., Koteshwara, M., Manoj, S., Santosh, M., Chidambara, J., et al. (2006) Rapid quantification of gabapentin in human plasma by liquid chromatography/tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 40, 360-368.

21. Shah, G.R., Ghosh, C., Thaker, B.T. (2010) Determination of pregabalin in human plasma by electrospray ionisation tandem mass spectroscopy. Journal of Advanced Pharmaceutical Technology & Research, 1, 354.

22. Sørensen, L.K., Hasselstrøm, J.B. (2014) Determination of Therapeutic γ-Aminobutyric Acid Analogs in Forensic Whole Blood by Hydrophilic Interaction Liquid Chromatography–Electrospray Tandem Mass Spectrometry. Journal of Analytical Toxicology, 38, 177-183.

23. Deeb, S., McKeown, D.A., Torrance, H.J., Wylie, F.M., Logan, B.K., Scott, K.S. (2014) Simultaneous Analysis of 22 Antiepileptic Drugs in Postmortem Blood, Serum and Plasma Using LC-MS-MS with a Focus on Their Role in Forensic Cases. Journal of Analytical Toxicology, 38, 485-494.

24. Martinc, B., Roskar, R., Grabnar, I., Vovk, T. (2014) Simultaneous determination of gabapentin, pregabalin, vigabatrin, and topiramate in plasma by HPLC with fluorescence detection. Journal of Chromatography B, 962, 82-8.

25. Kim, K.B., Seo, K.A., Kim, S.E., Bae, S.K., Kim, D.H., Shin, J.G. (2011) Simple and accurate quantitative analysis of ten antiepileptic drugs in human plasma by liquid chromatography/tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 56, 771-777.

26. Amini, M., Rouini, M.R., Asad-Paskeh, A., Shafiee, A. (2010) A new pre-column derivatization method for determination of gabapentin in human serum by HPLC using UV detection. Journal of Chromatographic Science, 48, 358-561.

27. Priez-Barallon, C., Carlier, J., Boyer, B., Benslima, M., Fanton, L., Mazoyer, C., et al. (2014) Quantification of pregabalin using hydrophilic interaction HPLC-high-resolution MS in postmortem human samples: eighteen case reports. Journal of Analytical Toxicology, 38, 143-148.

28. Flärdh, M., Jacobson, B.M. (1999) Sensitive method for the determination of baclofen in plasma by means of solid-phase extraction and liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 846, 169-173.

29. Kostić, N., Dotsikas, Y., Jović, N., Stevanović, G., Malenović, A., Medenica, M. (2015) Quantitation of pregabalin in dried blood spots and dried plasma spots by validated LC–MS/MS methods. Journal of Pharmaceutical and Biomedical Analysis, 109, 79-84.

30. Cooper, G.A., Paterson, S., Osselton, M.D. (2010) The United Kingdom and Ireland Association of Forensic Toxicologists: forensic toxicology laboratory guidelines (2010). Science & Justice, 50, 166-176.

31. Karinen, R., Vindenes, V., Hasvold, I., Olsen, K.M., Christophersen, A.S., Oiestad, E. (2015) Determination of a selection of anti-epileptic drugs and two active metabolites in whole blood by reversed phase UPLC-MS/MS and some examples of application of the method in forensic toxicology cases. Drug Testing and Analysis, 7, 634-644.

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download