Quantitative study on a simple electrochemical dsDNA-pregabalin biosensor; multi-spectroscopic, molecular docking and modelling studies.

Pregabalin (PGB) is a γ-aminobutyric acid (GABA) alkylated analog prescribed to treat neuropathic pain, fibromyalgia, and postherpetic neuralgia. Using analytical, spectroscopic methods and molecular docking and molecular dynamics (MD) simulations, a detailed experimental and theoretical investigation was conducted into the binding process and interactions between PGB and double-stranded fish sperm deoxyribonucleic acid (dsDNA). It was evident from the collected experimental results that PGB binds with ds-DNA. PGB attaches to dsDNA via minor groove binding, as demonstrated by the results of electrochemical studies, UV-Vis absorption spectroscopy, and replacement study with ethidium bromide and Hoechst-32588. PGB's binding constant (Kb) with dsDNA, as determined by the Benesi-Hildebrand plot, is 2.41×104 ± 0.30 at 298 K. The fluorescence investigation indicates that PGB and dsDNA have a binding stoichiometry (n) of 1.21 ± 0.09. Molecular docking simulations were used in the research to computational determination of the interactions between PGB and dsDNA. The findings demonstrated that minor groove binding was the mechanism by which PGB interacted with dsDNA. Based on the electrochemically responsive PGB-dsDNA biosensor, we developed a technique for low-concentration detection of PGB utilizing differential pulse voltammetry (DPV). The voltammetric analysis of the peak current decrease in the deoxyadenosine oxidation signals resulting from the association between PGB and dsDNA enabled a sensitive estimation of PGB in pH 4.80 acetate buffer. The deoxyguanosine oxidation signals exhibited a linear relationship between 2 and 16 µM PGB. The values for the limit of detection (LOD) and limit of quantitation (LOQ) were 0.57 µM and 1.91 µM, respectively.

Review of the demographic factors and toxicology related to deaths due to pregabalin in Northern Ireland.

The potential for recreational abuse of the analgesic and antiepileptic drug pregabalin is now well established in the literature. The potential minimum lethal dose in post mortem cases is however less defined. All post mortem examinations in Northern Ireland where the cause of death was found to be due to pregabalin were examined for demographic and toxicological analysis. Deaths are generally seen in young men, especially 30-39-year-olds, many of whom have a history of substance misuse and are often prescribed pregabalin. Until recently, prescription rates have been on the rise regionally. The overall median post mortem peripheral blood concentration of pregabalin found in this study is 10.6 mg/L, however this rises when concurrent drugs and alcohol are considered and in cases where pregabalin is considered responsible for death alone (i.e. outside of multidrug toxicity). Pregabalin peripheral blood concentrations returned in this study suggest previously offered minimum lethal dosages may need to be revised downward.

A simple and easy non-derivatization gas chromatography-mass spectrometry method for simultaneous quantification of valproic acid, gabapentin, pregabalin, and vigabatrin in human plasma.

Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988-0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%-94.5%, 90.0%-90.9%, 90.0%-92.1%, and 88.0%-92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.

Forensic application and evaluation of a commercially available pregabalin immunoassay test in serum on an Olympus AU480.

Misuse of pregabalin and its forensic relevance is steadily increasing. The aim of this study was to evaluate the usability of the commercially available ARKTM Pregabalin II Assay (ARK Diagnostics) for serum analysis of forensic samples. Overall, 156 samples were tested by both the immunoassay and a validated liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method. Sensitivity was 100%, and specificity was 98.7% (n = 79 positive cases confirmed by LC-MS/MS in a range of 380-37,000 ng/mL). A good correlation (R2  = 0.73) could also be shown between quantitative immunoassay and LC-MS/MS results. In conclusion, the assay shows excellent reliability for screening of forensic serum samples.

Postmortem analysis of quetiapine and pregabalin in human bone.

In this study quetiapine and pregabalin were analyzed in human bones. A method previously developed for the determination of antidepressants in human bone was tested for the analysis of these two substances. Bones were pulverized and subjected to the extraction protocol, and after undergoing solid-phase extraction, samples were analyzed using gas chromatography-mass spectrometry. The assay was validated in the range 0.3-500 ng/mg, mean analytical recovery was 76.9% for quetiapine and 90.9% for pregabalin, matrix effect was 83% for quetiapine and 91% for pregabalin and process efficiency was 63.8% for quetiapine and 82.7% for pregabalin. The intra- and inter-day precision was below 3% in all cases and the intra- and inter-assay accuracy values were in almost all cases better than 12%. The validated method was then applied to bone samples from forensic cases. Drugs were detected in bone in 2 of the 3 blood positive cases. The approximate concentrations in bone were 40 ng/mg for pregabalin and 7 ng/mg for quetiapine. To our knowledge, this is the first time these substances were detected in bones. With this study the number of substances with a validated protocol to be used in human bones in case of necessity is expanded.

A repeated cross-sectional study of factors associated with pregabalin-positive poisoning deaths in Ireland.

BACKGROUND: The increasing use of pregabalin and the presence of pregabalin in poisoning deaths, particularly with opioids, highlight it as a potential drug of abuse. In this study we examined factors associated with pregabalin-positive poisoning deaths (PPPD) between 2013 and 2016 in Ireland. METHODS: Data were extracted from the National Drug-Related Deaths Index (NDRDI). Analysis included univariate and multivariate logistic regression to estimate unadjusted and adjusted odds ratios (OR) and 95 % confidence intervals (CI) for factors associated with PPPD (primary outcome) by logistic regression models for the total sample and stratified by gender. RESULTS: Pregabalin was present on 240 (16 %) toxicology reports of 1489 poisoning deaths; significantly rising from 15 (4.5 %) in 2013 to 94 (26 %) in 2016. Women (AOR 2.69, 95 % CI: 1.95-3.70), opioid misuse (AOR 1.74, 95 % CI: 1.17-2.59), in receipt of treatment for problem drug use (AOR 1.95, 95 % CI: 1.33-2.86) and year of death (2016 vs 2013) (AOR 7.95, 95 % CI: 4.58-13.79) were associated with increased odds of PPPD. Alcohol dependence was associated with reduced odds of PPPD (AOR 0.59, 95 % CI: 0.41-0.85). For men, opioid misuse, in receipt of treatment for problem drug use, and year of death were associated with increased odds of PPPD, while alcohol dependence was associated with reduced odds of PPPD. For women, in receipt of treatment for problem drug use and year of death were associated with increased odds of PPPD. CONCLUSIONS: Enhanced training to prescribers and treatment providers on the potential risks associated with pregabalin, particularly among people who use drugs, is required.

Misuse and Mortality Related to Gabapentin and Pregabalin are Being Under-Estimated: A Two-Year Post-Mortem Population Study.

Due to the rise in their misuse and associated mortality, the UK government is reclassifying gabapentin (GBP) and pregabalin (PGL) to Class C controlled drugs from April 2019. However, it is impossible to gauge the extent of their use with current post-mortem toxicological screening, where GBP and PGL are only screened for if they are mentioned in the case documents. This study determines the prevalence of GBP and PGL, the potential extent of their under-reporting and poly-drug use in a post-mortem population. Between 1 January 2016 and 31 December 2017, 3,750 deceased from Coroners' cases in London and South East England underwent a routine drugs screen and a specific screen for GBP and PGL. The prevalence of both drugs was determined in the cohort and the subcategories of heroin users and non-heroin-users. The prevalence of both drugs was compared to tramadol (Class C drug). Case documents were reviewed to investigate the under-reporting of GBP and PGL and poly-drug use. Of 3,750 samples analyzed, 118 (3.1%) were positive for GBP, 229 (6.1%) for PGL and 120 (3.2%) were positive for tramadol. If routine analysis without additional screening of GBP and PGL had been performed in this cohort, GBP would have been under-reported by 57.6% (P < 0.0001) and PGL by 53.7% (P < 0.0001) in deaths. The most common drug group observed with GBP and PGL was non-heroin-related opioids at 60.2% and 64.6%, respectively. In total 354 deceased (9.4%) were heroin users. GBP was positive in 23 (6.5%) of these cases and PGL was positive in 69 (19.5%). The prevalence of PGL in heroin users (19.5%) was 4.1 times greater than in non-heroin users (4.7%) (P < 0.0001). GBP and PGL are being significantly under reported in fatalities. Both drugs are extensively used with opioids. The prevalence of PGL in heroin users is highly significant.

Saliva versus Plasma Therapeutic Drug Monitoring of Pregabalin in Jordanian Patients.

The objective is using saliva instead of plasma for pregabalin therapeutic drug monitoring (TDM) since saliva reflects the free non-protein bound drug concentration, simple and noninvasive sampling, cheaper and does not require the expertise of drawing blood. Forty four patients participated in this study, two samples of saliva and another two of blood were taken from each patient; first sample of both saliva and blood is the trough sample and was taken just before the first dose of the day and second sample is the peak sample and was taken 1 h after taking the first dose of the day. Descriptive statistics and t-testing after log transformation were done using Excel, p-value=0.05 was adopted for significant difference. Optimized effective intestinal permeability of pregabalin was estimated by PK-Sim program version 7. This study for the first time revealed that pregabalin is excreted in saliva and classified as class 1 based on Salivary Excretion Classification System (SECS). A good correlation of 0.71-0.83 between Cmin and Cmax of plasma and saliva pregabalin was observed respectively which indicate that saliva sampling is a good alternative matrix for pregabalin TDM. C/D-ratios were calculated to demonstrate pharmacokinetic variability of Pregabalin; the results showed that C/D-ratio was higher in women, elderly and in those patients who had Scr.≥0.9 mg/dl. Proposed pregabalin therapeutic ranges are 0.7 to 1.84 µg/ml in plasma and 0.055 to 0.145 µg/ml in saliva, for neuropathic pain, diabetic neuropathy and disc prolapse patients.

Assessment of Pregabalin Use by Hair Testing.

BACKGROUND: The detection and analysis of drugs in hair has progressively emerged as a consequence of the enhanced sensitivity of analytical techniques used in forensic toxicology; a greater advantage in using this matrix respect to classical ones (i.e., urine and blood) is an easier and noninvasive sample collection, even when the careful supervision of law-enforcement officers is required to avoid the risk that the sample may be adulterated or replaced. Moreover, according to the length of the hair, the history of drug exposure can be retrospectively monitored from few weeks up to months or years since sample collection. OBJECTIVE: Given the potential negative effects of pregabalin, an antiepileptic and analgesic drug with a high risk of misuse and abuse, the laboratory was asked to test for the drug in hair. METHOD: A new ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry was developed. The method involves incubation of 25 mg of cut hair in acetonitrile for 2 h in an ultrasonic bath and separation on an Acquity HSS C18 column (150 × 2.1 mm × 1.8 µm) maintained at 50°C in a thermostatically controlled oven. A gradient elution was performed. RESULTS: The method was fully validated according to international standards. The limit of quantitation of the test was 10 pg/mg. Five authentic cases of pregabalin in hair segments were tested using the method and the results were in the range 17-1487 pg/mg. CONCLUSION: This new method was found suitable to monitor both patients under pregabalin therapy and dependent subjects.

GC-MS/MS detects potential pregabalin abuse in susceptible subjects' hair.

Pregabalin, a GABA analogue, binds to the alpha 2 delta subunit of voltage-dependent calcium channels. It is recognised as efficacious in pathologies such as epilepsy, neuropathic pain, and anxiety disorders. Since pregabalin prescriptions have increased worldwide, reports of its abuse have been accumulating, mainly in patients with opioid abuse disorders. The present study investigated potential pregabalin abuse by means of hair analysis, a matrix that provides valuable retrospective information. Half of the pool of 280 susceptible patients had been occasional drug users and were being monitored for driving licence renewals. The other 140 patients had a history of opiate dependency and were monitored to assess compliance with methadone therapy. In view of determining pregabalin in hair samples, it was extracted in methanol, successfully derivatised to give the ethyl chloroformate derivative, and finally pregabalin was analysed by gas chromatography-tandem mass spectrometry. Selectivity, linearity, limit of detection, limit of quantification, recovery, intra- and inter-day precision, and accuracy of the quantification procedure were appraised. Pregabalin limits of detection and quantification were 30 pg/mg and 50 pg/mg, respectively. We found 10.7% of hair samples from methadone patients and 4.29% from occasional drug users were positive to pregabalin without medical prescription. The mean pregabalin concentration in hair was higher than in consumers with medical indications (1.45 ng/mg vs 0.74 ng/mg). These results suggest that pregabalin possesses a significant abuse potential particularly among individuals attending opiate dependence services and that pregabalin abuse is a serious emerging issue, which should be carefully monitored.

Stacking-cyclodextrin-microchip electrokinetic chromatographic determination of gabapentinoid drugs in pharmaceutical and biological matrices.

A facile, rapid, and highly sensitive microchip-based electrokinetic chromatographic method was developed for the simultaneous analysis of two gabapentinoid drugs, gabapentin (GPN) and pregabalin (PGN). Both drugs were first reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) via nucleophilic substitution reactions to yield highly fluorescent products with λex/em 470/540nm. Analyses of both fluorescently labeled compounds were achieved within 200s in a poly(methyl methacrylate) (PMMA) microchip with a 30mm separation channel. Optimum separation was achieved using a borate buffer (pH 9.0) solution containing methylcellulose and ß-cyclodextrin (ß-CD) as buffer additives. Methylcellulose acted as a dynamic coating to prevent adsorption of the studied compounds on the inner surfaces of the microchannels, while ß-CD acted as a pseudo-stationary phase to improve the separation efficiency between the labeled drugs with high resolution (Rs>7). The fluorescence intensities of the labeled drugs were measured using a light emitting diode-induced fluorescence detector at 540nm after excitation at 470nm. The sensitivity of the method was enhanced 14- and 17-fold for PGN and GPN, respectively by field-amplified stacking relative to traditional pinched injection so that it could quantify 10ngmL-1 for both analytes, with a detection limit lower than 3ngmL-1. The developed method was efficiently applied to analyze PGN and GPN in their pharmaceutical dosage forms and in biological fluids. The extraction recoveries of the studied drugs from plasma and urine samples were more than 89% with%RSD values lower than 6.2.

Gabapentin, Pregabalin and Vigabatrin Quantification in Human Serum by GC-MS After Hexyl Chloroformate Derivatization.

A simple, sensitive and robust method for simultaneous determination of antiepileptic drugs (gabapentin, pregabalin and vigabatrin) in human serum using GC-MS was developed and validated for clinical toxicology purposes. This method employs an emerging class of derivatization agents - alkyl chloroformates allowing the efficient and rapid derivatization of both the amino and carboxylic groups of the tested antiepileptic drugs within seconds. The derivatization protocol was optimized using the Design of Experiment statistical methodology, and the entire sample preparation requires less than 5 min. Linear calibration curves were obtained in the concentration range from 0.5 to 50.0 mg/L, with adequate accuracy (97.9-109.3%) and precision (<12.1%). The method was successfully applied to quantification of selected γ-aminobutyric acid analogs in the serum of patients in both therapeutic and toxic concentration ranges.