ORIGINAL ARTICLE
A Validated Fluorometric Method for the Rapid Determination of Pregabalin in Human Plasma Applied to Patients With Pain Nozomi Yoshikawa, BSc, Takafumi Naito, PhD, Tatsuya Yagi, PhD, and Junichi Kawakami, PhD
Background: Pregabalin has been used for the treatment of pain. A clinically accepted method applied to patients with pain has not been published for the determination of pregabalin in human plasma. This study developed a fluorometric ultrahigh-performance liquid chromatography (UHPLC) method to measure pregabalin concentration in patients with pain.
Methods: After plasma pretreatment involving protein precipitation, pregabalin and gabapentin as an internal standard were derivatized with 4-fluoro-7-nitrobenzofurazan (NBD-F) under the following reaction conditions: 1 minute, pH 10, and 608C. The UHPLC separation was performed using a 2.3-mm particle size octadecylsilyl column. The fluorescence detector was set at excitation and emission wavelengths of 470 and 530 nm, respectively. The predose blood samples were collected from 40 patients with pain who have been treated with 75 mg of pregabalin twice daily. Results: The chromatographic run time was 1.25 minutes. No interfering peaks were observed in the blank plasma at the retention times of NBD derivatives. The calibration curve of pregabalin was linear at a range of 0.05–10 mcg/mL (r . 0.999). The lower limit of quantification was 0.05 mcg/mL. The intraassay accuracy and precision were 98.3%–99.8% and within 4.3%, respectively. The inter-assay accuracy and precision were 103.2%–107.1% and within 4.1%, respectively. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL. Conclusions: This study provides a validated fluorometric UHPLC method with fast analytical performance for the determination of pregabalin in human plasma. The present method could be applied to patients with pain and be used for the clinical research or therapeutic drug monitoring of pregabalin. Key Words: pregabalin, NBD-F, UHPLC, human plasma, pain (Ther Drug Monit 2016;38:628–633)
Received for publication March 17, 2016; accepted July 18, 2016. From the Department of Hospital Pharmacy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. The authors declare no conflict of interest. Correspondence: Takafumi Naito, PhD, Department of Hospital Pharmacy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan (e-mail: [email protected]). Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
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INTRODUCTION
Pregabalin, g-aminobutyric acid analogue, has been used for the treatment of epilepsy, fibromyalgia, anxiety, and neuropathic pain. Among them, the increased frequency of treatment for pain, including cancer pain, has led to a more widespread clinical use of pregabalin.1–4 Pregabalin is completely absorbed and predominantly eliminated by renal excretion as unchanged drug. Pregabalin clearance is expected to depend on the glomerular filtration rate.5–8 In contrast, the interindividual variability in the response to pregabalin is dependent not only on renal function in clinical settings. To date, the target range of pregabalin concentration has not been determined for each indication as there is only limited amount of information on the relationship between the pregabalin concentration and drug response. In addition, clinically accepted method applied to patients with pain has never been published for the determination of pregabalin. Because pregabalin has no specific visible or ultraviolet absorption, the determination of pregabalin is commonly performed with fluorescent derivatization reagents, such as o-phthaladehyde (OPA),9,10 picryl sulfonic acid,11 fluorescamine,12,13 and 4-chloro-7-nitrobenzofurazan.14 These reagents react with the amino group of analytes. Earlier derivatization methods had complicated pretreatment procedures, such as longer reaction times and unstable derivatives. 4-Fluoro-7nitrobenzofurazan (NBD-F) itself is nonfluorescent and generates highly fluorescent stable derivatives.15 NBD-F possesses a shorter reaction time than other fluorescent reagents. A conventional high-performance liquid chromatography (HPLC) system was widely used for the determination of pregabalin in human plasma in earlier reports.9–14,16–18 Tandem mass spectrometry (MS/MS) or liquid chromatography-mass spectrometry (LC-MS) were also used as a tool for the determination of pregabalin.16–20 However, the MS methods are not commonly used for the measurement of plasma drug concentration in clinical settings due to various technical limitations and high operating cost. In recent years, an ultrahigh-performance liquid chromatography (UHPLC) method has been recognized for its rapid determination of drugs in a biological matrix. The sensitivity of UHPLC is 2–3 times higher than that of conventional HPLC.21 A UHPLC method using an octadecylsilyl (ODS) column with particles around 2 mm can achieve a short run time compared with a 5-mm particle size column, without decreasing sensitivity. The shorter run time results in not only reducing organic solvent volumes of mobile phases and the running cost but also ensuring high-throughput analysis. Ther Drug Monit Volume 38, Number 5, October 2016
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Ther Drug Monit Volume 38, Number 5, October 2016
A UHPLC method coupled to fluorescence detection for determination of pregabalin could not be found in our literature search conducted from the PubMed database, from its inception through December 2015, using the following terms: “pregabalin hplc” or “pregabalin liquid chromatograph*” or “pregabalin fluorometr*” or “pregabalin human plasma” or “pregabalin derivatization.” Furthermore, the plasma concentration of pregabalin in patients with pain has not yet been reported. The aim of this study was to develop a validated fluorometric method for the rapid determination of pregabalin in human plasma and to use it for the measurement of pregabalin concentration from patients with pain.
MATERIALS AND METHODS Chemicals and Solutions Pregabalin and gabapentin as an internal standard (IS) were obtained from Kemprotec Limited (Cumbria, United Kingdom) and Toronto Research Chemicals (North York, Canada), respectively. NBD-F was purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals and organic solvents were of analytical grade and commercially available. Stock solutions of pregabalin (100 mcg/mL) and IS (100 mcg/mL) were prepared with methanol. Calibrators of pregabalin and IS were obtained each time by dilution of stock solutions with methanol. NBD-F was dissolved each day in acetonitrile at a final concentration of 50 mmol/L. A reaction solution was composed of 50 mmol/L borate buffer at pH 10 containing 20 mmol/L ethylenediaminetetraacetic acid (EDTA) disodium salts. A terminal solution was a mixture of 1 mol/L hydrochloric acid solution and acetonitrile (50/50, vol/vol). All solutions were stored at 48C and prepared freshly when all the stock has been used.
Sample Pretreatment and NBD-F Derivatization Fifty microliters of EDTA plasma samples (pooled or obtained from patients) and 150 mL of IS solution were mixed and centrifuged at 13,000g at 48C for 10 minutes. A 150-mL aliquot of the supernatant was evaporated to dryness using a centrifugal concentrator without heating. The residue was reconstituted with 100 mL of reaction solution and 25 mL of NBD-F solution in a thin wall polypropylene microtube (Quality Scientific Plastics, San Diego, CA). The mixture was incubated immediately at 608C for 1 minute using a thermal cycler (GeneAmp PCR System 2400; PerkinElmer Japan, Yokohama, Japan). After cooling to 48C, 50 mL of terminal solution was added and centrifuged at 13,000g at 48C for 10 minutes. The supernatant was injected into a UHPLC system. With respect to the optimization of NBD-F derivatization, its reaction was examined under the following conditions: pH value of the reaction solution (pH 7, 8, 9, 10, and 11), the concentration of NBD-F solution (25, 50, and 100 mmol/L), reaction time (0.5, 1, 2.5, and 5 minutes), and reaction temperature (40, 50, 60, 70, and 808C).
Rapid Determination of Pregabalin
controlled by LabSolutions software (version 5.73). The system comprised CBM-20A system controller, DGU-20 A3R mobile phase degassing system, LC-20ADXR pump, SIL-20ACXR automatic injector, CTO-20AC column heater, and RF-20AXS fluorescence detector. The isocratic mobile phase composed of acetonitrile, methanol, and 50 mmol/L phosphate buffer pH 2 (20/20/60, vol/vol/vol) and its flow rate was 1.3 mL/min. The injection volume of sample into the UHPLC system was 0.5 mL. The UHPLC separation was performed using a 2.3-mm particle size ODS column (TSKgel ODS-140HTP, 50 · 2.1 mm internal diameter; Tosoh, Tokyo, Japan) set to a temperature of 508C. The fluorescence detector with a 3-mL flow cell was set at excitation and emission wavelengths of 470 and 530 nm, respectively. The response time and flow cell temperature of the fluorescence detector were optimized at 0.05 seconds and 308C, respectively.
Calibration Curve and Quality Control Calibrators were prepared from drug-free pooled human plasma (Tennessee Blood Services, Memphis, TN), which was spiked to obtain 8 pregabalin concentration levels: 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mcg/mL. Quality control samples were prepared from independent stock solutions and spiked at pregabalin concentrations of 0.15, 3, and 8 mcg/mL in drugfree plasma. Intra- and inter-assays were repeated 6 times within the same day and over different days, respectively. Accuracy was obtained by dividing the measured value by the theoretical value. Precision was expressed as the relative standard deviation (RSD). The lower limit of quantification was defined as the concentration at which RSD did not exceed 20%.
Stability All stability studies of pregabalin and IS in human plasma were examined in triplicate by analyzing the low and high quality controls. The short-term stability was evaluated after 24 hours storage at 48C and 258C. The long-term stability was measured after 1 month storage at 2808C. The freeze/ thaw stability was assessed for 3 freeze/thaw cycles every 12 hours. The stability of derivatized samples was observed at hourly intervals by keeping the samples in the automatic injector at 48C for 4 hours. The stability of stock solutions was determined after 3 months storage at 48C.
Ethics This study was conducted according to the ethical principles stated in the Declaration of Helsinki. The protocol was approved by the Ethical Committee of Hamamatsu University School of Medicine. Each patient gave informed consent and was provided with information about the scientific aim of this study and was free to withdraw at any time at their discretion.
Patients
All chromatographic analyses were performed using a UHPLC system (UFLCXR; Shimadzu Corp, Kyoto, Japan)
The method described here was applied to patients with pain (n = 40) treated with pregabalin capsule (Lyrica; Pfizer, Tokyo, Japan) 75 mg twice daily for at least 2 weeks. All patients were inpatients admitted to the Hamamatsu University Hospital from January 2014 to October 2015. Noncompliant patients were excluded in this study. The enrolled
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Instrumentation and Chromatographic Conditions
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Yoshikawa et al
patients were not coadministered any enzyme inducers or inhibitors. The predose blood samples were collected in tubes containing EDTA disodium salts at close to 12 hours after last dose. The plasma samples were obtained by centrifugation of predose blood samples at 1670g at 48C for 10 minutes and stored at 2808C until plasma pretreatment and NBD-F derivatization.
RESULTS Optimization of NBD-F Derivatization Figure 1 shows the results of optimization of NBD-F derivatization for pregabalin and IS in human plasma. Stable and high fluorescence intensity of pregabalin-NBD and IS-NBD were obtained under the following conditions: reaction solution pH of 10, 50 mmol/L NBD-F solution, reaction temperature of 608C, and reaction time of 1 minute.
Chromatographic Separation The UHPLC chromatograms of pregabalin-NBD and IS-NBD in human plasma are shown in Figure 2. The chromatographic run time was 1.25 minutes. The pregabalin-NBD and IS-NBD were well separated and eluted at 0.75 and 0.90 minutes, respectively. No interfering peaks were observed in the blank plasma at the retention times of the NBD derivatives.
Calibration Curve, Accuracy, and Imprecision The calibration curve was linear over a range of 0.05– 10 mcg/mL. The correlation coefficient of the calibration curve was greater than 0.999 (y = 0.354x 2 0.021). Table 1 shows the results of intra- and inter-assays for pregabalin in human plasma. The intra-assay accuracy and precision were 98.3%–99.8% and within 4.3%, respectively, while the
inter-assay accuracy and precision were 103.2%–107.1% and within 4.1%, respectively.
Stability In all stability tests, the peak area of NBD derivatives was maintained at a level of at least 92% compared with that of freshly prepared samples, whose peak area was taken to be 100%. Pregabalin in human plasma was stable at 48C and 258C for 24 hours, at 2808C for 1 month, and after 3 freeze/thaw cycles every 12 hours. The fluorescence intensity of NBD derivatives was maintained at the initial intensity by keeping the samples in the automatic injector at 48C for at least 4 hours. The stock solutions of pregabalin and IS prepared with methanol were stable at 48C for at least 3 months.
Clinical Application The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL (Figure 3). All plasma samples used in this study were measurable within the calibration curve.
DISCUSSION This study developed a validated UHPLC method using NBD-F for determination of pregabalin in human plasma. The reaction time for derivatizing pregabalin with NBD-F and the chromatographic run time were 1 and 1.25 minutes, respectively. The linear calibration curve for pregabalin in human plasma was obtained at a range of 0.05–10 mcg/mL. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL. The present method could be helpful for evaluating the plasma exposure of pregabalin in clinical settings. The conditions of NBD-F derivatization were optimized at reaction solution pH of 10 and 50 mmol/L NBD-F
FIGURE 1. Optimization of NBD-F derivatization for pregabalin and gabapentin as an IS in human plasma: A, pH value of the reaction solution, B, concentration of NBD-F solution, C, reaction temperature, and D, reaction time. All peak areas of pregabalin-NBD (filled circles) and IS-NBD (filled triangles) are presented as the mean (n = 3). Reaction conditions: A, 50 mmol/L NBD-F, 1 minute, 608C. B, pH 10, 1 minute, 608C. C, 50 mmol/L NBD-F, pH 10, 608C. D, 50 mmol/L NBD-F, pH 10, 1 minute. A, RSD of pregabalin-NBD peak area was 0.86% (pH 10) and 2.5% (pH 11), and RSD of ISNBD peak area was 0.73% (pH 10) and 0.76% (pH 11). B, RSD of pregabalin-NBD peak area was 6.3% (25 mmol/L) and 0.86% (50 mmol/L), and RSD of IS-NBD peak area was 2.5% (25 mmol/L) and 1.0% (50 mmol/L).
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Ther Drug Monit Volume 38, Number 5, October 2016
Rapid Determination of Pregabalin
FIGURE 2. Fluorometric UHPLC chromatograms of pregabalin-NBD (1) and gabapentin as an IS-NBD (2) in human plasma. A, Blank human plasma, not including IS. B, Blank human plasma spiked with pregabalin and IS. C, Human plasma from a patient with pain.
solution to obtain stable and high fluorescence intensity of NBD derivatives. With reaction solution pH of 11 and 25 mmol/L NBD-F solution, higher RSD values were observed than at pH 10 and 50 mmol/L for the peak area. NBD-F reacts with the amino group of pregabalin under basic conditions, and its reaction terminates under acidic conditions.15 The terminal solution containing 1 mol/L hydrochloric acid solution causes strong acidity and stabilizes NBD derivatives. Our previous study demonstrated that NBD derivatives were unstable when 250 mmol/L hydrochloric acid solution was added for the termination.22 The reaction time was 1 minute for NBD-F derivatization of pregabalin. The stability of NBD derivatives was verified at
48C for up to 24 hours in the automatic injector. The fluorescence intensity of NBD derivatives declined with a reaction time of more than 1 minute. The increase in background noise by heating for more than 1 minute implies that NBD-F reacts with endogenous plasma proteins, such as albumin and globulin. Vermeij and Edelbroek9 used OPA for derivatization of pregabalin and its reaction time was 10 minutes. OPA derivatives are unstable, and samples need to be injected immediately into an HPLC system after sample preparation. Martinc et al14 described an HPLC method using 4-chloro-7-nitrobenzofurazan, which takes 15 minutes for derivatization of pregabalin. Earlier studies required complicated sample preparation involving longer reaction time and produced unstable derivatives that are not suitable
TABLE 1. Analytical Performance for Determination of Pregabalin in Human Plasma Intra-assay (n = 6) Theoretical Value, mcg/mL 0.05 (LLOQ) 0.15 (low QC) 3 (middle QC) 8 (high QC)
Inter-assay (n = 6)
Mean 6 SD, mcg/mL
Accuracy, %
RSD, %
6 6 6 6
102.7 98.3 98.7 99.8
4.6 4.3 1.3 1.8
0.052 0.148 2.96 7.98
0.004 0.006 0.04 0.14
Mean 6 SD, mcg/mL
Accuracy, %
RSD, %
6 6 6 6
101.3 103.2 104.3 107.1
7.8 3.7 2.3 4.1
0.051 0.154 3.13 8.18
0.003 0.005 0.04 0.34
LLOQ, lower limit of quantification; QC, quality control; SD, standard deviation; RSD, relative standard deviation.
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Yoshikawa et al
FIGURE 3. Predose plasma concentration of pregabalin in patients with pain. All patients were treated with 75 mg of pregabalin twice daily. The pregabalin concentration ranged from 0.14 to 8.5 mcg/mL.
for clinical settings. The method described here using NBD-F has a reaction time that is 10–15 times shorter and more stable derivatives than other derivatization reagents. With a 3-mL flow cell volume, the resolution performance of the detector increases compared with that with a conventional flow cell volume. The response time of the fluorescence detector was optimized at 0.05 seconds for the best peak detection in our method. Optimization of the mobile phase including phosphate buffer pH 2 was performed to obtain the best peak sharpness and chromatographic separation. The injection volume of sample into the UHPLC system was 0.5 mL. The lower limit of quantification of our method was 0.05 mcg/mL. Our method could detect 25 pg of pregabalin and has the sensitivity similar to that of MS/MS or LC-MS detection.16–20 The smaller injection volume of sample contributes to the durability of the column. More than 500 chromatographic runs could be performed with one ODS column without any deterioration of the chromatographic separation performance. The present method has sufficient sensitivity and analytical performance for high-throughput analysis and/or routine analysis of pregabalin in clinical settings. The run time of the present UHPLC method was 1.25 minutes. Martinc et al14 described a conventional HPLC method for determination of pregabalin that requires a total run time of more than 15 minutes. A longer run time was also reported for the determination of pregabalin in several studies that used a conventional HPLC.9–13,16–18 A UHPLC method using an ODS column with particles around 2 mm can achieve not only a shorter run time but also higher injection cycle times. A shorter run time is suitable for routine monitoring and is able to reduce the running cost due to a reduction in the amount of organic solvents.
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The method presented here has acceptable accuracy and precision according to the guidance published by the US Food and Drug Administration.23 The calibration curve of pregabalin was linear at a range of 0.05–10 mcg/mL. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL (including cancer pain ranged from 0.14 to 4.8 mcg/mL). In an earlier clinical study, the plasma concentration of pregabalin in patients with epilepsy ranged from 0.2 to 8.1 mcg/mL.24 The therapeutic dose of pregabalin for pain is similar to that for epilepsy or anxiety.24–26 The present method could be applied to patients with pain and be used for the clinical research or routine monitoring of pregabalin at therapeutic dose. The clinical applicability of our method remains to be confirmed for other indications, such as epilepsy or anxiety. This method has a few limitations that need to be considered. First, the suitability of the present method was evaluated only by the predose plasma concentration as all blood samples were obtained at trough. In a pharmacokinetic study in patients administered 300 mg of pregabalin twice daily, the mean peak concentration was approximately 8 mcg/mL.27 Our calibration curve ranged from 0.05 to 10 mcg/mL. Thus, the method described here could be applied to the measurement of pregabalin concentration either at peak or at trough. Second, the present method cannot be used in patients who receive gabapentin as gabapentin is used as an IS. Combination therapy with pregabalin and gabapentin is not common for the treatment of seizures or pain. Therefore, the method we have developed may be applicable in most clinical situations. Third, a fluorescence detector and system controller which are tolerant to high-speed data acquisition were required in the present method. The pump pressure value in our UHPLC method was around 300 kgf/cm2. A conventional HPLC system could be used if the separation column, flow cell volume, and a response time of detection satisfy our conditions.
CONCLUSIONS
A validated fluorometric method for the determination of pregabalin with fast analytical performance was developed. The present method could be applied to patients with pain and be helpful for evaluating the plasma exposure of pregabalin in clinical settings. REFERENCES 1. Michael IB, Barry L, Chantal van L, et al. Pregabalin for the management of neuropathic pain in adults with cancer: a systematic review of the literature. Pain Med. 2013;14:1681–1688. 2. Fallon M, Hoskin PJ, Colvin LA, et al. Randomized double-blind trial of pregabalin versus placebo in conjunction with palliative radiotherapy for cancer-induced bone pain. J Clin Oncol. 2016;34:550–556. 3. Dou Z, Jiang Z, Zhong J. Efficacy and safety of pregabalin in patients with neuropathic cancer pain undergoing morphine therapy. Asia Pac J Clin Oncol. In press, 2016. 4. Clark K, Quinn SJ, Doogue M, et al. Routine prescribing of gabapentin or pregabalin in supportive and palliative care: what are the comparative performances of the medications in a palliative care population? Support Care Cancer. 2015;23:2517–2520. 5. Bockbrader HN, Radulovic LL, Posvar EL, et al. Clinical pharmacokinetics of pregabalin in healthy volunteers. J Clin Pharmacol. 2010;50: 941–950.
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6. Randinitis EJ, Posvar EL, Alvey CW, et al. Pharmacokinetics of pregabalin in subjects with various degrees of renal function. J Clin Pharmacol. 2003;43:277–283. 7. Shneker BF, McAuley JW. Pregabalin: a new neuromodulator with broad therapeutic indications. Ann Pharmacother. 2005;39:2029–2037. 8. Huckle R. Pregabalin (Pfizer). Curr Opin Investig Drugs. 2004;5:82–89. 9. Vermeij TC, Edelbroek PM. Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2004; 810:297–303. 10. Dousa M, Gibala P, Lemr K. Liquid chromatographic separation of pregabalin and its possible impurities with fluorescence detection after postcolumn derivatization with o-phtaldialdehyde. J Pharm Biomed Anal. 2010;53:717–722. 11. Berry D, Millington C. Analysis of pregabalin at therapeutic concentrations in human plasma/serum by reversed-phase HPLC. Ther Drug Monit. 2005;27:451–456. 12. Shaalan RA. Spectrofluorimetric and spectrophotometric determination of pregabalin in capsules and urine samples. Int J Biomed Sci. 2010;6: 260–267. 13. Martinc B, Grabnar I, Mrhar A, et al. Rapid high-performance liquid chromatography method for determination of pregabalin in a pharmaceutical dosage form following derivatization with fluorescamine. J AOAC Int. 2010;93:1069–1076. 14. Martinc B, Roskar R, Grabnar I, et al. Simultaneous determination of gabapentin, pregabalin, vigabatrin, and topiramate in plasma by HPLC with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;962:82–88. 15. Uchiyama S, Santa T, Okiyama N, et al. Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton. Biomed Chromatogr. 2001;15:295–318. 16. Priez-Barallon C, Carlier J, Boyer B, et al. Quantification of pregabalin using hydrophilic interaction HPLC-high-resolution MS in postmortem human samples: eighteen case reports. J Anal Toxicol. 2014;38:143–148.
17. Nirogi R, Kandikere V, Mudigonda K, et al. Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:3899–3906. 18. Sørensen LK, Hasselstrøm JB. Determination of therapeutic g-aminobutyric acid analogs in forensic whole blood by hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry. J Anal Toxicol. 2014;38:177–183. 19. Dahl SR, Olsen KM, Strand DH. Determination of gammahydroxybutyrate (GHB), beta-hydroxybutyrate (BHB), pregabalin, 1,4butane-diol (1,4BD) and gamma-butyrolactone (GBL) in whole blood and urine samples by UPLC-MSMS. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;885–886:37–42. 20. Chahbouni A, Sinjewel A, Burger J, et al. Rapid quantification of gabapentin, pregabalin, and vigabatrin in human serum by ultraperformancel liquid chromatography with mass-spectrometric detection. Ther Drug Monit. 2013;35:48–53. 21. Cielecka-Piontek J, Zalewski P, Jelinska A, et al. UHPLC: the greening face of liquid chromatography. Chromatographia. 2013;76:1429–1437. 22. Yagi T, Naito T, Mino Y, et al. Rapid and validated fluorometric HPLC method for determination of gabapentin in human plasma and urine for clinical application. J Clin Pharm Ther. 2012;37:89–94. 23. Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation 2001. Available at: http://www.fda.gov/cder/guidance. Accessed March 15, 2016. 24. May TW, Rambeck B, Neb R, et al. Serum concentrations of pregabalin in patients with epilepsy: the influence of dose, age, and comedication. Ther Drug Monit. 2007;29:789–794. 25. Montgomery S. Pregabalin for the treatment of generalised anxiety disorder. Expert Opin Pharmacother. 2006;7:2139–2154. 26. Schulze-Bonhage A. Pharmacokinetic and pharmacodynamic profile of pregabalin and its role in the treatment of epilepsy. Expert Opin Drug Metab Toxicol. 2013;9:105–115. 27. Ben-Menachem E. Pregabalin pharmacology and its relevance to clinical practice. Epilepsia. 2004;45(suppl 6):13–18.
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A Validated Fluorometric Method for the Rapid Determination of Pregabalin in Human Plasma Applied to Patients With Pain Nozomi Yoshikawa, BSc, Takafumi Naito, PhD, Tatsuya Yagi, PhD, and Junichi Kawakami, PhD
Background: Pregabalin has been used for the treatment of pain. A clinically accepted method applied to patients with pain has not been published for the determination of pregabalin in human plasma. This study developed a fluorometric ultrahigh-performance liquid chromatography (UHPLC) method to measure pregabalin concentration in patients with pain.
Methods: After plasma pretreatment involving protein precipitation, pregabalin and gabapentin as an internal standard were derivatized with 4-fluoro-7-nitrobenzofurazan (NBD-F) under the following reaction conditions: 1 minute, pH 10, and 608C. The UHPLC separation was performed using a 2.3-mm particle size octadecylsilyl column. The fluorescence detector was set at excitation and emission wavelengths of 470 and 530 nm, respectively. The predose blood samples were collected from 40 patients with pain who have been treated with 75 mg of pregabalin twice daily. Results: The chromatographic run time was 1.25 minutes. No interfering peaks were observed in the blank plasma at the retention times of NBD derivatives. The calibration curve of pregabalin was linear at a range of 0.05–10 mcg/mL (r . 0.999). The lower limit of quantification was 0.05 mcg/mL. The intraassay accuracy and precision were 98.3%–99.8% and within 4.3%, respectively. The inter-assay accuracy and precision were 103.2%–107.1% and within 4.1%, respectively. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL. Conclusions: This study provides a validated fluorometric UHPLC method with fast analytical performance for the determination of pregabalin in human plasma. The present method could be applied to patients with pain and be used for the clinical research or therapeutic drug monitoring of pregabalin. Key Words: pregabalin, NBD-F, UHPLC, human plasma, pain (Ther Drug Monit 2016;38:628–633)
Received for publication March 17, 2016; accepted July 18, 2016. From the Department of Hospital Pharmacy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. The authors declare no conflict of interest. Correspondence: Takafumi Naito, PhD, Department of Hospital Pharmacy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan (e-mail: [email protected]). Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
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INTRODUCTION
Pregabalin, g-aminobutyric acid analogue, has been used for the treatment of epilepsy, fibromyalgia, anxiety, and neuropathic pain. Among them, the increased frequency of treatment for pain, including cancer pain, has led to a more widespread clinical use of pregabalin.1–4 Pregabalin is completely absorbed and predominantly eliminated by renal excretion as unchanged drug. Pregabalin clearance is expected to depend on the glomerular filtration rate.5–8 In contrast, the interindividual variability in the response to pregabalin is dependent not only on renal function in clinical settings. To date, the target range of pregabalin concentration has not been determined for each indication as there is only limited amount of information on the relationship between the pregabalin concentration and drug response. In addition, clinically accepted method applied to patients with pain has never been published for the determination of pregabalin. Because pregabalin has no specific visible or ultraviolet absorption, the determination of pregabalin is commonly performed with fluorescent derivatization reagents, such as o-phthaladehyde (OPA),9,10 picryl sulfonic acid,11 fluorescamine,12,13 and 4-chloro-7-nitrobenzofurazan.14 These reagents react with the amino group of analytes. Earlier derivatization methods had complicated pretreatment procedures, such as longer reaction times and unstable derivatives. 4-Fluoro-7nitrobenzofurazan (NBD-F) itself is nonfluorescent and generates highly fluorescent stable derivatives.15 NBD-F possesses a shorter reaction time than other fluorescent reagents. A conventional high-performance liquid chromatography (HPLC) system was widely used for the determination of pregabalin in human plasma in earlier reports.9–14,16–18 Tandem mass spectrometry (MS/MS) or liquid chromatography-mass spectrometry (LC-MS) were also used as a tool for the determination of pregabalin.16–20 However, the MS methods are not commonly used for the measurement of plasma drug concentration in clinical settings due to various technical limitations and high operating cost. In recent years, an ultrahigh-performance liquid chromatography (UHPLC) method has been recognized for its rapid determination of drugs in a biological matrix. The sensitivity of UHPLC is 2–3 times higher than that of conventional HPLC.21 A UHPLC method using an octadecylsilyl (ODS) column with particles around 2 mm can achieve a short run time compared with a 5-mm particle size column, without decreasing sensitivity. The shorter run time results in not only reducing organic solvent volumes of mobile phases and the running cost but also ensuring high-throughput analysis. Ther Drug Monit Volume 38, Number 5, October 2016
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Ther Drug Monit Volume 38, Number 5, October 2016
A UHPLC method coupled to fluorescence detection for determination of pregabalin could not be found in our literature search conducted from the PubMed database, from its inception through December 2015, using the following terms: “pregabalin hplc” or “pregabalin liquid chromatograph*” or “pregabalin fluorometr*” or “pregabalin human plasma” or “pregabalin derivatization.” Furthermore, the plasma concentration of pregabalin in patients with pain has not yet been reported. The aim of this study was to develop a validated fluorometric method for the rapid determination of pregabalin in human plasma and to use it for the measurement of pregabalin concentration from patients with pain.
MATERIALS AND METHODS Chemicals and Solutions Pregabalin and gabapentin as an internal standard (IS) were obtained from Kemprotec Limited (Cumbria, United Kingdom) and Toronto Research Chemicals (North York, Canada), respectively. NBD-F was purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals and organic solvents were of analytical grade and commercially available. Stock solutions of pregabalin (100 mcg/mL) and IS (100 mcg/mL) were prepared with methanol. Calibrators of pregabalin and IS were obtained each time by dilution of stock solutions with methanol. NBD-F was dissolved each day in acetonitrile at a final concentration of 50 mmol/L. A reaction solution was composed of 50 mmol/L borate buffer at pH 10 containing 20 mmol/L ethylenediaminetetraacetic acid (EDTA) disodium salts. A terminal solution was a mixture of 1 mol/L hydrochloric acid solution and acetonitrile (50/50, vol/vol). All solutions were stored at 48C and prepared freshly when all the stock has been used.
Sample Pretreatment and NBD-F Derivatization Fifty microliters of EDTA plasma samples (pooled or obtained from patients) and 150 mL of IS solution were mixed and centrifuged at 13,000g at 48C for 10 minutes. A 150-mL aliquot of the supernatant was evaporated to dryness using a centrifugal concentrator without heating. The residue was reconstituted with 100 mL of reaction solution and 25 mL of NBD-F solution in a thin wall polypropylene microtube (Quality Scientific Plastics, San Diego, CA). The mixture was incubated immediately at 608C for 1 minute using a thermal cycler (GeneAmp PCR System 2400; PerkinElmer Japan, Yokohama, Japan). After cooling to 48C, 50 mL of terminal solution was added and centrifuged at 13,000g at 48C for 10 minutes. The supernatant was injected into a UHPLC system. With respect to the optimization of NBD-F derivatization, its reaction was examined under the following conditions: pH value of the reaction solution (pH 7, 8, 9, 10, and 11), the concentration of NBD-F solution (25, 50, and 100 mmol/L), reaction time (0.5, 1, 2.5, and 5 minutes), and reaction temperature (40, 50, 60, 70, and 808C).
Rapid Determination of Pregabalin
controlled by LabSolutions software (version 5.73). The system comprised CBM-20A system controller, DGU-20 A3R mobile phase degassing system, LC-20ADXR pump, SIL-20ACXR automatic injector, CTO-20AC column heater, and RF-20AXS fluorescence detector. The isocratic mobile phase composed of acetonitrile, methanol, and 50 mmol/L phosphate buffer pH 2 (20/20/60, vol/vol/vol) and its flow rate was 1.3 mL/min. The injection volume of sample into the UHPLC system was 0.5 mL. The UHPLC separation was performed using a 2.3-mm particle size ODS column (TSKgel ODS-140HTP, 50 · 2.1 mm internal diameter; Tosoh, Tokyo, Japan) set to a temperature of 508C. The fluorescence detector with a 3-mL flow cell was set at excitation and emission wavelengths of 470 and 530 nm, respectively. The response time and flow cell temperature of the fluorescence detector were optimized at 0.05 seconds and 308C, respectively.
Calibration Curve and Quality Control Calibrators were prepared from drug-free pooled human plasma (Tennessee Blood Services, Memphis, TN), which was spiked to obtain 8 pregabalin concentration levels: 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mcg/mL. Quality control samples were prepared from independent stock solutions and spiked at pregabalin concentrations of 0.15, 3, and 8 mcg/mL in drugfree plasma. Intra- and inter-assays were repeated 6 times within the same day and over different days, respectively. Accuracy was obtained by dividing the measured value by the theoretical value. Precision was expressed as the relative standard deviation (RSD). The lower limit of quantification was defined as the concentration at which RSD did not exceed 20%.
Stability All stability studies of pregabalin and IS in human plasma were examined in triplicate by analyzing the low and high quality controls. The short-term stability was evaluated after 24 hours storage at 48C and 258C. The long-term stability was measured after 1 month storage at 2808C. The freeze/ thaw stability was assessed for 3 freeze/thaw cycles every 12 hours. The stability of derivatized samples was observed at hourly intervals by keeping the samples in the automatic injector at 48C for 4 hours. The stability of stock solutions was determined after 3 months storage at 48C.
Ethics This study was conducted according to the ethical principles stated in the Declaration of Helsinki. The protocol was approved by the Ethical Committee of Hamamatsu University School of Medicine. Each patient gave informed consent and was provided with information about the scientific aim of this study and was free to withdraw at any time at their discretion.
Patients
All chromatographic analyses were performed using a UHPLC system (UFLCXR; Shimadzu Corp, Kyoto, Japan)
The method described here was applied to patients with pain (n = 40) treated with pregabalin capsule (Lyrica; Pfizer, Tokyo, Japan) 75 mg twice daily for at least 2 weeks. All patients were inpatients admitted to the Hamamatsu University Hospital from January 2014 to October 2015. Noncompliant patients were excluded in this study. The enrolled
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Instrumentation and Chromatographic Conditions
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Ther Drug Monit Volume 38, Number 5, October 2016
Yoshikawa et al
patients were not coadministered any enzyme inducers or inhibitors. The predose blood samples were collected in tubes containing EDTA disodium salts at close to 12 hours after last dose. The plasma samples were obtained by centrifugation of predose blood samples at 1670g at 48C for 10 minutes and stored at 2808C until plasma pretreatment and NBD-F derivatization.
RESULTS Optimization of NBD-F Derivatization Figure 1 shows the results of optimization of NBD-F derivatization for pregabalin and IS in human plasma. Stable and high fluorescence intensity of pregabalin-NBD and IS-NBD were obtained under the following conditions: reaction solution pH of 10, 50 mmol/L NBD-F solution, reaction temperature of 608C, and reaction time of 1 minute.
Chromatographic Separation The UHPLC chromatograms of pregabalin-NBD and IS-NBD in human plasma are shown in Figure 2. The chromatographic run time was 1.25 minutes. The pregabalin-NBD and IS-NBD were well separated and eluted at 0.75 and 0.90 minutes, respectively. No interfering peaks were observed in the blank plasma at the retention times of the NBD derivatives.
Calibration Curve, Accuracy, and Imprecision The calibration curve was linear over a range of 0.05– 10 mcg/mL. The correlation coefficient of the calibration curve was greater than 0.999 (y = 0.354x 2 0.021). Table 1 shows the results of intra- and inter-assays for pregabalin in human plasma. The intra-assay accuracy and precision were 98.3%–99.8% and within 4.3%, respectively, while the
inter-assay accuracy and precision were 103.2%–107.1% and within 4.1%, respectively.
Stability In all stability tests, the peak area of NBD derivatives was maintained at a level of at least 92% compared with that of freshly prepared samples, whose peak area was taken to be 100%. Pregabalin in human plasma was stable at 48C and 258C for 24 hours, at 2808C for 1 month, and after 3 freeze/thaw cycles every 12 hours. The fluorescence intensity of NBD derivatives was maintained at the initial intensity by keeping the samples in the automatic injector at 48C for at least 4 hours. The stock solutions of pregabalin and IS prepared with methanol were stable at 48C for at least 3 months.
Clinical Application The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL (Figure 3). All plasma samples used in this study were measurable within the calibration curve.
DISCUSSION This study developed a validated UHPLC method using NBD-F for determination of pregabalin in human plasma. The reaction time for derivatizing pregabalin with NBD-F and the chromatographic run time were 1 and 1.25 minutes, respectively. The linear calibration curve for pregabalin in human plasma was obtained at a range of 0.05–10 mcg/mL. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL. The present method could be helpful for evaluating the plasma exposure of pregabalin in clinical settings. The conditions of NBD-F derivatization were optimized at reaction solution pH of 10 and 50 mmol/L NBD-F
FIGURE 1. Optimization of NBD-F derivatization for pregabalin and gabapentin as an IS in human plasma: A, pH value of the reaction solution, B, concentration of NBD-F solution, C, reaction temperature, and D, reaction time. All peak areas of pregabalin-NBD (filled circles) and IS-NBD (filled triangles) are presented as the mean (n = 3). Reaction conditions: A, 50 mmol/L NBD-F, 1 minute, 608C. B, pH 10, 1 minute, 608C. C, 50 mmol/L NBD-F, pH 10, 608C. D, 50 mmol/L NBD-F, pH 10, 1 minute. A, RSD of pregabalin-NBD peak area was 0.86% (pH 10) and 2.5% (pH 11), and RSD of ISNBD peak area was 0.73% (pH 10) and 0.76% (pH 11). B, RSD of pregabalin-NBD peak area was 6.3% (25 mmol/L) and 0.86% (50 mmol/L), and RSD of IS-NBD peak area was 2.5% (25 mmol/L) and 1.0% (50 mmol/L).
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Ther Drug Monit Volume 38, Number 5, October 2016
Rapid Determination of Pregabalin
FIGURE 2. Fluorometric UHPLC chromatograms of pregabalin-NBD (1) and gabapentin as an IS-NBD (2) in human plasma. A, Blank human plasma, not including IS. B, Blank human plasma spiked with pregabalin and IS. C, Human plasma from a patient with pain.
solution to obtain stable and high fluorescence intensity of NBD derivatives. With reaction solution pH of 11 and 25 mmol/L NBD-F solution, higher RSD values were observed than at pH 10 and 50 mmol/L for the peak area. NBD-F reacts with the amino group of pregabalin under basic conditions, and its reaction terminates under acidic conditions.15 The terminal solution containing 1 mol/L hydrochloric acid solution causes strong acidity and stabilizes NBD derivatives. Our previous study demonstrated that NBD derivatives were unstable when 250 mmol/L hydrochloric acid solution was added for the termination.22 The reaction time was 1 minute for NBD-F derivatization of pregabalin. The stability of NBD derivatives was verified at
48C for up to 24 hours in the automatic injector. The fluorescence intensity of NBD derivatives declined with a reaction time of more than 1 minute. The increase in background noise by heating for more than 1 minute implies that NBD-F reacts with endogenous plasma proteins, such as albumin and globulin. Vermeij and Edelbroek9 used OPA for derivatization of pregabalin and its reaction time was 10 minutes. OPA derivatives are unstable, and samples need to be injected immediately into an HPLC system after sample preparation. Martinc et al14 described an HPLC method using 4-chloro-7-nitrobenzofurazan, which takes 15 minutes for derivatization of pregabalin. Earlier studies required complicated sample preparation involving longer reaction time and produced unstable derivatives that are not suitable
TABLE 1. Analytical Performance for Determination of Pregabalin in Human Plasma Intra-assay (n = 6) Theoretical Value, mcg/mL 0.05 (LLOQ) 0.15 (low QC) 3 (middle QC) 8 (high QC)
Inter-assay (n = 6)
Mean 6 SD, mcg/mL
Accuracy, %
RSD, %
6 6 6 6
102.7 98.3 98.7 99.8
4.6 4.3 1.3 1.8
0.052 0.148 2.96 7.98
0.004 0.006 0.04 0.14
Mean 6 SD, mcg/mL
Accuracy, %
RSD, %
6 6 6 6
101.3 103.2 104.3 107.1
7.8 3.7 2.3 4.1
0.051 0.154 3.13 8.18
0.003 0.005 0.04 0.34
LLOQ, lower limit of quantification; QC, quality control; SD, standard deviation; RSD, relative standard deviation.
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FIGURE 3. Predose plasma concentration of pregabalin in patients with pain. All patients were treated with 75 mg of pregabalin twice daily. The pregabalin concentration ranged from 0.14 to 8.5 mcg/mL.
for clinical settings. The method described here using NBD-F has a reaction time that is 10–15 times shorter and more stable derivatives than other derivatization reagents. With a 3-mL flow cell volume, the resolution performance of the detector increases compared with that with a conventional flow cell volume. The response time of the fluorescence detector was optimized at 0.05 seconds for the best peak detection in our method. Optimization of the mobile phase including phosphate buffer pH 2 was performed to obtain the best peak sharpness and chromatographic separation. The injection volume of sample into the UHPLC system was 0.5 mL. The lower limit of quantification of our method was 0.05 mcg/mL. Our method could detect 25 pg of pregabalin and has the sensitivity similar to that of MS/MS or LC-MS detection.16–20 The smaller injection volume of sample contributes to the durability of the column. More than 500 chromatographic runs could be performed with one ODS column without any deterioration of the chromatographic separation performance. The present method has sufficient sensitivity and analytical performance for high-throughput analysis and/or routine analysis of pregabalin in clinical settings. The run time of the present UHPLC method was 1.25 minutes. Martinc et al14 described a conventional HPLC method for determination of pregabalin that requires a total run time of more than 15 minutes. A longer run time was also reported for the determination of pregabalin in several studies that used a conventional HPLC.9–13,16–18 A UHPLC method using an ODS column with particles around 2 mm can achieve not only a shorter run time but also higher injection cycle times. A shorter run time is suitable for routine monitoring and is able to reduce the running cost due to a reduction in the amount of organic solvents.
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The method presented here has acceptable accuracy and precision according to the guidance published by the US Food and Drug Administration.23 The calibration curve of pregabalin was linear at a range of 0.05–10 mcg/mL. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL (including cancer pain ranged from 0.14 to 4.8 mcg/mL). In an earlier clinical study, the plasma concentration of pregabalin in patients with epilepsy ranged from 0.2 to 8.1 mcg/mL.24 The therapeutic dose of pregabalin for pain is similar to that for epilepsy or anxiety.24–26 The present method could be applied to patients with pain and be used for the clinical research or routine monitoring of pregabalin at therapeutic dose. The clinical applicability of our method remains to be confirmed for other indications, such as epilepsy or anxiety. This method has a few limitations that need to be considered. First, the suitability of the present method was evaluated only by the predose plasma concentration as all blood samples were obtained at trough. In a pharmacokinetic study in patients administered 300 mg of pregabalin twice daily, the mean peak concentration was approximately 8 mcg/mL.27 Our calibration curve ranged from 0.05 to 10 mcg/mL. Thus, the method described here could be applied to the measurement of pregabalin concentration either at peak or at trough. Second, the present method cannot be used in patients who receive gabapentin as gabapentin is used as an IS. Combination therapy with pregabalin and gabapentin is not common for the treatment of seizures or pain. Therefore, the method we have developed may be applicable in most clinical situations. Third, a fluorescence detector and system controller which are tolerant to high-speed data acquisition were required in the present method. The pump pressure value in our UHPLC method was around 300 kgf/cm2. A conventional HPLC system could be used if the separation column, flow cell volume, and a response time of detection satisfy our conditions.
CONCLUSIONS
A validated fluorometric method for the determination of pregabalin with fast analytical performance was developed. The present method could be applied to patients with pain and be helpful for evaluating the plasma exposure of pregabalin in clinical settings. REFERENCES 1. Michael IB, Barry L, Chantal van L, et al. Pregabalin for the management of neuropathic pain in adults with cancer: a systematic review of the literature. Pain Med. 2013;14:1681–1688. 2. Fallon M, Hoskin PJ, Colvin LA, et al. Randomized double-blind trial of pregabalin versus placebo in conjunction with palliative radiotherapy for cancer-induced bone pain. J Clin Oncol. 2016;34:550–556. 3. Dou Z, Jiang Z, Zhong J. Efficacy and safety of pregabalin in patients with neuropathic cancer pain undergoing morphine therapy. Asia Pac J Clin Oncol. In press, 2016. 4. Clark K, Quinn SJ, Doogue M, et al. Routine prescribing of gabapentin or pregabalin in supportive and palliative care: what are the comparative performances of the medications in a palliative care population? Support Care Cancer. 2015;23:2517–2520. 5. Bockbrader HN, Radulovic LL, Posvar EL, et al. Clinical pharmacokinetics of pregabalin in healthy volunteers. J Clin Pharmacol. 2010;50: 941–950.
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Rapid Determination of Pregabalin
6. Randinitis EJ, Posvar EL, Alvey CW, et al. Pharmacokinetics of pregabalin in subjects with various degrees of renal function. J Clin Pharmacol. 2003;43:277–283. 7. Shneker BF, McAuley JW. Pregabalin: a new neuromodulator with broad therapeutic indications. Ann Pharmacother. 2005;39:2029–2037. 8. Huckle R. Pregabalin (Pfizer). Curr Opin Investig Drugs. 2004;5:82–89. 9. Vermeij TC, Edelbroek PM. Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2004; 810:297–303. 10. Dousa M, Gibala P, Lemr K. Liquid chromatographic separation of pregabalin and its possible impurities with fluorescence detection after postcolumn derivatization with o-phtaldialdehyde. J Pharm Biomed Anal. 2010;53:717–722. 11. Berry D, Millington C. Analysis of pregabalin at therapeutic concentrations in human plasma/serum by reversed-phase HPLC. Ther Drug Monit. 2005;27:451–456. 12. Shaalan RA. Spectrofluorimetric and spectrophotometric determination of pregabalin in capsules and urine samples. Int J Biomed Sci. 2010;6: 260–267. 13. Martinc B, Grabnar I, Mrhar A, et al. Rapid high-performance liquid chromatography method for determination of pregabalin in a pharmaceutical dosage form following derivatization with fluorescamine. J AOAC Int. 2010;93:1069–1076. 14. Martinc B, Roskar R, Grabnar I, et al. Simultaneous determination of gabapentin, pregabalin, vigabatrin, and topiramate in plasma by HPLC with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;962:82–88. 15. Uchiyama S, Santa T, Okiyama N, et al. Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton. Biomed Chromatogr. 2001;15:295–318. 16. Priez-Barallon C, Carlier J, Boyer B, et al. Quantification of pregabalin using hydrophilic interaction HPLC-high-resolution MS in postmortem human samples: eighteen case reports. J Anal Toxicol. 2014;38:143–148.
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