University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Faculty Publications and Other Works -- Veterinary Medicine -- Faculty Publications and Biomedical and Diagnostic Sciences Other Works 2020 Determination of Thiafentanil in Plasma Using LC–MS Sherry Cox University of Tennessee, Knoxville Joan Bergman University of Tennessee, Knoxville Matthew C. Allender University of Illinois Kimberlee Beckmen Alaska Department of Fish and Game William Lance Wildlife Pharmaceuticals Inc Follow this and additional works at: https://trace.tennessee.edu/utk_compmedpubs Recommended Citation Cox, Sherry; Bergman, Joan; Allender, Matthew C.; Beckmen, Kimberlee; and Lance, William, "Determination of Thiafentanil in Plasma Using LC–MS" (2020). Faculty Publications and Other Works -- Biomedical and Diagnostic Sciences. https://trace.tennessee.edu/utk_compmedpubs/151 This Article is brought to you for free and open access by the Veterinary Medicine -- Faculty Publications and Other Works at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Faculty Publications and Other Works -- Biomedical and Diagnostic Sciences by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Journal of Chromatographic Science, 2020, Vol. 58, No. 1, 1–4 doi: 10.1093/chromsci/bmz098 Advance Access Publication Date: 9 December 2019 Article Article Determination of Thiafentanil in Plasma Using LC–MS Downloaded from https://academic.oup.com/chromsci/article-abstract/58/1/1/5667452 by guest on 14 April 2020 Sherry Cox1, Joan Bergman1, Matthew C. Allender2, Kimberlee Beckmen3, and William Lance4 1Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN, USA 2Wildlife Epidemiology Laboratory, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana IL, USA 3Wildlife Health and Disease Surveillance Program, Division of Wildlife Conservation, Alaska Department of Fish and Game, 1300 College Road, Fairbanks AK, USA 4Wildlife Pharmaceuticals Inc., 1230 West Ash, Windsor, CO, USA Author to whom correspondence should be addressed. Email: [email protected] Abstract A new method of analysis has been developed and validated for the determination of thiafentanil in plasma. After protein precipitation, samples were separated on an XBridge BEH C18 column and quantified using mass spectrometry. The mobile phase was a mixture of water with 0.1% formic acid and acetonitrile with 0.1% formic acid (90:10). The standard curve ranged from 0.1 to 25 ng/mL. Intra- and Inter-assay variability for thiafentanil was less than 10%, and the average recovery was greater than 95%. The lower limit of quantification was 0.1 ng/mL. This is the first validated method for thiafentanil analysis in plasma. Introduction effects after apparent antagonism. Renarcotization is dangerous for free-ranging wildlife because prolonged struggling during recovery Opioid agonists are valuable for immobilization of many nondo- could cause several life-threatening problems, such as hyperthermia mestic species. Opioids such as etorphine and carfentanil are often and trauma (2). The shorter half-life also means that targeted animals preferred for their potency, fast action and ability to have their effects may be handled and secured quicker than with other opioids, prevent- reversed. However, this class of drug is associated with adverse side ing problems with trauma, overheating and escapes by free-ranging effects such as excitement, muscle rigidity, regurgitation, bradypnea, wildlife. abnormal blood pressure, hyperthermia and lactic acidosis (1). A literature search revealed no published methods for the deter- Thiafentanil is a potent opioid that is a synthetic fentanyl deriva- mination of thiafentanil in plasma. Therefore, the aim of this paper tive, structurally similar to sufentanil. It has a morphine-like anal- was to develop a simple, sensitive, specific and reliable method for gesic mode of action and produces rapid immobilization following determining thiafentanil concentrations using protein precipitation intramuscular injection. It is used for immobilization of captive minor and mass-spectrometry detection. species and free-ranging hoof stock and represents the next genera- tion of opioid immobilizing agents. The restraint and immobilization of nondomestic ungulates has been extremely problematic and there Experimental continues to be a need for agents that will immobilize these animals quickly and safely (2). When compared to other opioids, thiafentanil Instrumentation and reagents has a much shorter induction time, by as much as 50%, while The chromatography system consisted of an Acquity Arc system and retaining agonist activity (3). In addition, recovery times after antago- an Acquity QDa single-quadrupole mass detector (Waters, Milford, × nization are also shorter (4). Because it has a shorter half-life, there is MA). Separation occurred on an XBridge BEH C18 column (4.6 less incidence of renarcotization, which is the reoccurrence of opioid 50 mm, 3.5 μm) preceded by a 3.5 μm BEH C18 guard column © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1 2 Cox et al. Method validation The method was validated according to the Guidelines for Bioanalyt- ical Method Validation published by the Food and Drug Administra- tion (5). Validation of the method was carried out using QC samples. All of the QC samples and calibration curves were prepared in a plasma matrix. The validation process looked at accuracy, precision, selectivity, sensitivity, reproducibility and stability. Selectivity Selectivity was determined by injecting blank plasma from six differ- ent deers to confirm no interfering peaks around the retention time of both thiafentanil and fentanyl, the internal standard (IS). Figure 1. Structures of thiafentanil and fentanyl. Downloaded from https://academic.oup.com/chromsci/article-abstract/58/1/1/5667452 by guest on 14 April 2020 Calibration curve, linearity and quality control samples × (3.9 5 mm). The mobile phase was a mixture of (A) 0.1% formic The final concentrations for the calibration standard curve were 0.1, acid in water and (B) 0.1% formic acid in acetonitrile (90:10, v/v). 0.25, 0.5, 1, 2.5, 5, 10 and 25 ng/mL. The calibration curve was The mixture was pumped at a starting ratio of 90% A and 10% B and constructed by using the ratio of the peak area of the analyte divided then changes to 10% A and 90% B over 4 minutes and then returns to by the peak area of the internal standard versus the concentration initial conditions over 3 minutes. The flow rate was 0.6 mL/min and ◦ and obtained on five different days. Linearity was assessed by linear the column temperature was 30 C. The compounds were detected regression analysis and expressed as the coefficient of determination by positive selected ion recording. The scan rate was 5 points/second, (r2). The standard deviations (SD) of the slope, intercepts and regres- gain 10, capillary voltage 0.8 kV,cone voltage for thiafentanil 8, cone ◦ sion coefficient were calculated. The QC samples were prepared in a voltage for fentanyl 15, ion source temperature 150 C and probe ◦ similar manner as the calibration standards at four different levels temperature 600 C. Nitrogen was used as the nebulizing gas and 0.3, 0.75, 3.5 and 17.5 ng/mL. The acceptance criterion for each maintained at 100 psi. Thiafentanil was detected at 417 m/z and back-calculated standard was 15% deviation from the nominal value fentanyl was detected at 337 m/z. except lower limit of quantification (LLOQ), which was set at 20%. Thiafentanil (Figure 1) (Wildlife Pharmaceuticals Inc., Windsor, CO) was 98% pure. Fentanyl (Figure 1), which was the internal standard (99% purity) was purchased from US Pharmacopeia Accuracy, precision and recovery (Rockville, MD). All other mass spectrometry grade chemicals and The precision and accuracy of the assay were determined using QC solvents were purchased from Fisher Scientific (Pittsburg, PA). Water samples of known thiafentanil concentrations (0.3, 0.75, 3.5 and (18.2 megaohm) was obtained from a Barnstead Nanopure Infinity 17.5 ng/mL), which were processed freshly each validation day. Five (Dubuque, IA) ultrapure water system. replicates of each QC were analyzed during the same day and on five different days, and the intra- and inter-assay means, SD and coefficient of variation were calculated. Recoveries were calculated as Preparation of calibration standards the measured concentrations divided by the expected concentrations Five milligrams of thiafentanil or fentanyl were weighed and dis- and expressed as a percentage (5). The tailing factor was calculated = solved in methanol to produce stock concentrations of 100 μg/mL. by As W0.05/2f,whereW0.05 is the width of the peak at 5% height Dilutions of the stock standards were prepared in methanol to and f is the distance from the peak maximum to the leading edge of produce 0.01, 0.1 and 1 μg/mL working stock solutions. Standards the peak height from the baseline (6). were aliquoted into 2 mL vials to prevent cross contamination and ◦ evaporation. All solutions were stored at 4 C. By comparing standard areas over time, it was determined that solutions were stable for a Results minimum of 8 months. Selectivity For preparation of calibration standards and quality control Endogenous components from the plasma did not interfere with the samples, appropriate volumes of stock solutions were placed in 13 elution of the compounds of interest. Six different blank plasmas were × 100 mm glass tubes and evaporated with nitrogen then untreated used in the pre-validation process. Figure 2 shows chromatograms of plasma was added. a (A) blank plasma, (B) a 0.5 ng/mL spiked plasma standard and (C) a plasma sample from a deer 30 minutes after a 0.07 mg/kg dose via nonmetal dart administration. Retention times were 4.32 minutes for Sample preparation thiafentanil and 4.56 minutes for fentanyl.