Journal of Chromatography B (v.879, #27)

Tamoxifen is the agent of choice for the treatment of estrogen receptor-positive breast cancer. Tamoxifen is a substrate of P-glycoprotein (P-gp) and microsomal cytochrome P450 (CYP) 3A, and biochanin A (BCA) is an inhibitor of P-gp and CYP3A. Hence, it could be expected that BCA would affect the pharmacokinetics of tamoxifen. In the present study we have developed and validated a simple, sensitive and specific LC–ESI-MS/MS method for the simultaneous quantification of tamoxifen and its metabolite 4-hydroxytamoxifen with 100 μL rat plasma using centchroman as an internal standard (IS). Tamoxifen, 4-hydroxytamoxifen and IS were separated on a Supelco Discovery C18 (4.6 mm × 50 mm, 5.0 μm) column under isocratic condition using 0.01 M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) as a mobile phase. The mobile phase was delivered at a flow rate of 0.8 mL/min. The method was proved to be accurate and precise at linearity range of 0.78–200 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.89 to 8.54% and 3.97 to 10.26%, respectively; and intra- and inter-day assay accuracy was between 87.63 and 109.06% and 96 and 103.89%, respectively for both the analytes. The method was successfully applied to study the effect of oral co-administration of BCA (an isoflavone) on the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen in female rats. The coadministration of BCA caused no significant changes in the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen. However, the peak plasma concentration (C max) of 4-hydroxytamoxifen in BCA pretreated rats was significantly (P  < 0.05) lower than those from control group.
Keywords: Tamoxifen; 4-Hydroxytamoxifen; Rat plasma; Validation; LC–MS/MS; Pharmacokinetics;

In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe3+, were prepared by in situ chemical modification of fused silica capillary using two chemistries, polymer brush coating and surface functionalization. One column was based on a poly-(glycidyl methacrylate) brush (GMA) and the other on 3-glycidoxypropyltrimethoxysilane (GLYMO). Phosphopeptide enrichment on the open tubular columns was evaluated on an αS1, αS2 mixture and β casein peptides. The optimized enrichment protocol includes sample loading in a slightly acidic solution made with pure deionized water, a washing step with 10% acetonitrile, 0.1% formic acid, and an elution step with 50% acetonitrile, 0.1% phosphoric acid at pH 8.0. MALDI-TOF spectra generated from eluted fractions show several phosphorylated peptides. For example, 7 phosphorylated peptides of the αS1, αS2 casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe3+ IMAC polymer brush column elutes all phosphorylated peptides in one fraction independently of phosphorylation degree, whereas the GLYMO-IMAC polymer brush provides longer elution times for higher phosphorylation states. In particular, the pentaphosphorylated peptide was eluted after a 30 min elution versus 5 min for monophosphorylated species (isocratic gradient).
Keywords: Proteomics; Phosphoprotein; Phosphopeptide; Open tubular capillary; Polymer brush; Surface functionalization; IMAC enrichment;

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 μL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated “small volume” sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing “small volumes” was developed. The automated PPT method was achieved by extracting and processing 10 μL out of a minimum sample volume of 15 μL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton “small volume” method platform. Analyses of a 10 μL plasma aliquot from 15 μL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C18 Extend, 1.0 × 50 mm, 3.5 μm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90–107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0–1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1 mg/kg and oral administration at 2 mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T 1/2) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 μL of plasma from 15 μL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.
Keywords: PD 0332991; Automated micro-sample processing; Small volume quantitation; Microbore LC–MS/MS;

Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions by Kinya Iwatsuka; Shin-ichi Yasueda; Eiji Bando; Hiroyuki Fujii; Takashi Terada; Hiroya Okubo; Hiroki Iwamoto; Mitsuhiro Kinoshita; Kazuaki Kakehi (2866-2870).
Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62–78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.
Keywords: N-acetylneuraminic acid; Eye mucus; Ophthalmic solution; HPLC; LC/MS;

Obesity is currently epidemic in many countries worldwide. In the young adult, obesity often accompanies hyperlipemia, which is strongly related to the occurrence and development of obesity-related chronic diseases such as diabetes mellitus, hypertension and cardiovascular disease. This study investigated the differences in metabolomic profiling between obese (with hyperlipemia, n  = 30) and normal-weight (n  = 30) young men. Anthropometric parameters and conventional metabolites were measured. There were no significant differences between obese and normal-weight young men in age, height and fasting plasma glucose level. Obese young men showed increased weight, body mass index, fat mass, systolic blood pressure, and triglyeride, total cholesterol and insulin levels, and lower levels of testosterone. The endogenous metabolite profile of urine was investigated by UPLC/Q-TOF MS (ultra performance liquid chromatography and Q-TOF mass spectrometry) with electrospray ionization (ESI). Partial least squares (PLS) enabled clusters to be visualized. Eight urine principal metabolites contributing to the clusters were identified; these included increased l-prolyl-l-proline, leucyl-phenylalanine, and decanoylcarnitine in positive ESI mode (m/z 213.1267, 279.1715 and 316.2459, respectively) and N-acetylornithine, 17-hydroxypregnenolone sulfate, 11β-hydroxyprogesterone, 5a-dihydrotestosterone sulfate and glucosylgalactosyl hydroxylysine in negative ESI mode (m/z 173.0931, 411.1883, 331.185, 369.1751 and 485.1875, respectively). These metabolite changes in obese men suggested early changes of metabolism in young-male obesity with hyperlipemia. The study may further aid the clinical prevention and treatment of obesity and related chronic disease.
Keywords: Metabolic profiles; Young obese men; Hyperlipemia; UPLC/Q-TOF MS;

Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody by Diya Ren; Jian Zhang; Ross Pritchett; Hongbin Liu; Jennifer Kyauk; Jun Luo; Ashraf Amanullah (2877-2884).
Sequence variants, also known as unintended amino acid substitutions in the protein primary structure, are one of the critical quality attributes needed to be monitored during process development of monoclonal antibodies (mAbs). Here we report on analytical methods for detection and identification of a sequence variant in an IgG1 mAb expressed in Chinese hamster ovary (CHO) cells. The presence of the sequence variant was detected by an imaged capillary isoelectric focusing (ICIEF) assay, showing a new basic species in mAb charge variant profile. The new basic variant was fractionated and enriched by ion-exchange chromatography, analyzed by reduced light and heavy chain mass determination, and characterized by HPLC–UV/MS/MS of tryptic and endoproteinase Lys-C peptide maps. A Serine to Arginine sequence variant was identified at the heavy chain 441 position (S441R), and confirmed by using synthetic peptides. The relative level of the S441R variant was estimated to be in the range of 0.3–0.6% for several mAb batches analyzed via extracted ion chromatogram (EIC). This work demonstrates the effectiveness of using integrated analytical methods to detect and identify protein heterogeneity and the importance of monitoring product quality during mAb bioprocess development.
Keywords: Monoclonal antibody; Sequence variant; HPLC–UV/MS/MS; Peptide mapping; Imaged capillary isoelectric focusing;

The alkaloids from Piper longum L. showed protective effects on Parkinson's disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC–ESI–MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25 °C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1–201.1 for piperine, m/z 274.0–201.1 for piperlonguminine, and m/z 472.4–436.4 for the IS. The calibration curves were both linear (r  > 0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze–thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L.
Keywords: Piper longum L.; Piperine; Piperlonguminine; UFLC–ESI–MS/MS; Pharmacokinetics;

Molecularly imprinted microspheres as SPE sorbent for selective extraction of four Sudan dyes in catsup products by Fengxia Qiao; Yuru Geng; Changqing He; Yupei Wu; Pengyu Pan (2891-2896).
A highly selective molecularly imprinted solid-phase extraction (MISPE) coupled with high performance liquid chromatography (HPLC) ultraviolet–visible detection was developed for the simultaneous isolation and determination of four Sudan dyes (I, II, III and IV) in catsup products. The novel molecularly imprinted microspheres (MIM) were synthesized by aqueous suspension polymerization using phenylamine and naphthol as template, which showed high affinity to Sudan dyes in aqueous solution. In order to develop a selective extraction protocol for simultaneous determination the four Sudan dyes from catsup products, the molecular recognition properties of MIM as a SPE sorbent were evaluated. Under the optimized condition, good linearity was obtained from 0.01 to 2.5 μg g−1 (r 2  ≥ 0.9990) with the relative standard deviations of less than 3.4%. This proposed MISPE-HPLC procedure eliminated the effect of template leakage on quantitative analysis and could be applied to direct determination of four Sudan dyes in complicated food samples.
Keywords: Molecularly imprinted solid-phase extraction; Imprinted microspheres; Sudan dyes; Catsup products;

A headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography–mass spectrometry (GC–MS) method was developed to determine a type of terpenoid named as cantharidin in the false blister beetles, family Oedemeridae. The experimental parameters for HS-SPME method were optimized. Six commercial fibers for HS-SPME method development were tested and the divinylbenzene/carboxene/polydimethylsiloxane fiber was selected to provide the best detection of analyzed compound. The calibration curve showed linearity in the range of 0.1–50 μg mL−1, correlation coefficient (R 2  = 0.992), limit of detection (0.01 ng mL−1) and quantitation (0.04 ng mL−1) were obtained for the proposed method. The relative standard deviations of intra-day and inter-day assays were 7.8 and 3.4%, respectively. The recovery values, obtained after spiking the beetle samples by three concentration levels of standard solution, were higher than 87%. The results indicated the successful application of the proposed method on the analysis of cantharidin from the false blister beetles.
Keywords: Solid-phase microextraction; Cantharidin; False blister beetle; Oedemeridae;

An LC–MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys by Qingfang Meng; Zhihang Chen; Jinjing Che; Yiping Lu; Huanzhang Wang; Chengqi Shan; Yunan Hou; Hongjiao Shi; Yangjun Zhang; Junying Wei; Xiaohong Qian; Hongwei Peng; Yuanguo Cheng (2902-2908).
A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10–5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51–7.70% with accuracy of 95.1–104.2%. The average recovery of cTP6 for three concentration levels was 59.6–64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6–8 h after intra-muscular injection. The half-life was 2.24–2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 μg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 μg/kg for 7 consecutive days in rhesus monkeys.
Keywords: Cyclic thymic hexapeptide; Thymopentin; Quantification; LC–MS/MS; Pharmacokinetics;

Pharmacokinetic studies and postmortem toxicological investigations require a validated analytical technique to quantify drugs on a large number of matrices. Three-step liquid/liquid extraction with online derivatization (silylation) ahead of analysis by gas chromatography–tandem mass spectrometry was developed and validated on rabbit specimens in order to quantify citalopram and 4 benzodiazepines (diazepam, nordazepam, oxazepam and temazepam) in 11 biological matrices (blood, urine, bile, vitreous humor, liver, kidney, skeletal muscle, brain, adipose tissue, bone marrow (BM) and lung). Since the 11 biological matrices came from the same animal species, full validation was performed on 1 matrix, bone marrow (considered the most complex), while the other 10 underwent partial validation. Due to non-negligible matrix effects, calibration curves were performed on each matrix. Within-day and between-day precision (less than 12.0% and 12.6%, respectively) and accuracy (from 88.9% to 106.4%) were acceptable on BM at both low and high concentrations. Assessment on the other matrices confirmed accuracy and within-day precision (less than 12%, and generally between 85.1% and 114.5%, respectively). The lower limit of quantification of the method was 1 ng/g for nordazepam, 5 ng/g for citalopram and 10 ng/g for oxazepam, diazepam and temazepam. The combination of 3-step extraction and MS/MS detection provided good selectivity in all matrices, including the most lipid-rich. Application to real-case samples showed that the method was sensitive enough to describe distribution patterns in an animal experiment, and specific enough to detect molecules in highly putrefied samples from human postmortem cases.
Keywords: Benzodiazepine; Validation; Citalopram; GC–MS/MS; Postmortem; Tissues;

Full-scan high resolution accurate mass spectrometry (HRMS) in regulated bioanalysis: LC–HRMS for the quantitation of prednisone and prednisolone in human plasma by Eliza N. Fung; Yuan-qing Xia; Anne-Francoise Aubry; Jianing Zeng; Timothy Olah; Mohammed Jemal (2919-2927).
A liquid chromatography–full scan high resolution accurate mass spectrometry (LC–HRMS) method for quantifying prednisone and prednisolone in human plasma using a quadrupole time-of-flight mass spectrometer (Q-TOF) was developed. Plasma samples were extracted using a liquid–liquid extraction procedure. Full scan data were acquired in the TOF only mode and extracted ion chromatograms were generated post-acquisition with the exact masses of the analytes. The calibration range was 5–2500 ng/mL, with a Lower Limit of Quantitation (LLOQ) of 5 ng/mL. The assay accuracy was between 98.4% and 106.3%. The between-run (inter-day) and within-run (intra-day) precision were within 1.7% and 2.9%, respectively. The matrix effect was between 0.98 and 1.10 for the six different lots of human plasma evaluated. Pooled incurred samples were analyzed by the method and the results matched those obtained from an LC–MS/MS method. In addition, qualitative information on phospholipids, and other endogenous components were also extracted from the full-scan data acquired.
Keywords: High resolution accurate mass spectrometry; Prednisone and prednisolone; Regulated bioanalysis; LC–MS/MS; LC–HRMS;

Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis by Takehiro Oyama; Masahiro Yodohsi; Ayako Yamane; Kazuaki Kakehi; Takao Hayakawa; Shigeo Suzuki (2928-2934).
Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N 4-(N-acetyl-β-d-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α1-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3 h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100 fmol and 4 fmol, respectively.
Keywords: Glycoprotein; N-linked oligosaccharides; High-performance liquid chromatography; Capillary electrophoresis with laser-induced fluorometric detection;

► Hydrophobic interaction on phenyl-sepharose works at 80% saturation ammonium sulfate. ► Bovine PEBP-1 and Ubiquitin are readily isolated by this technique. ► Two isoforms of PEBP-1 are evidenced by mass spectrometry and Western blotting.Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2 M). This soluble fraction (S80) was directly loaded onto a phenyl sepharose column equilibrated at the same ammonium sulfate concentration (3.2 M). A stepwise elution of the retained material at 1.0, 0.5, 0.2, 0.1 M ammonium sulfate in ammonium hydrogen carbonate was performed and then with ammonium hydrogen carbonate alone and finally with 50% ethylene glycol. All fractions were analyzed by SDS–PAGE and Western blotting and the fractions containing PEBP-1 was further fractionated by size exclusion chromatography on a HR75 Superdex column permitting the isolation of ubiquitin in addition to PEBP-1 as demonstrated by Western blotting and mass spectrometry. This study shows the feasibility of hydrophobic interaction chromatography (HIC) on phenyl sepharose at a very high ammonium sulfate concentration (3.2 M; 80% saturation) to efficiently purify the proteins that are still soluble in these extreme conditions.
Keywords: Phosphatidyl-ethanolamine-binding protein; Hydrophobic interaction chromatography; Raf-1 kinase inhibitory protein; Hippocampal cholinergic neurostimulating peptide precursor protein; Isoform;

HPLC-UV method development for fentanyl determination in rat plasma and its application to elucidate pharmacokinetic behavior after i.p. administration to rats by Ahmed A. Almousa; Rie Ikeda; Mitsuhiro Wada; Naotoka Kuroda; Ruri-Kikura Hanajiri; Kenichiro Nakashima (2941-2944).
A simple, rapid and validated high performance liquid chromatography method with UV detection for the quantification of an opioid agonist, fentanyl (FEN), in rat plasma was developed. The assay procedure involved chromatographic separation using a ZIC-HILIC SeQUANT column (250 mm × 4.6 mm, i.d., 5 μm) and a mobile phase of acetonitrile and acetate buffer (pH 3.4, 20 mM) of ratio (=65:35, v/v) at a flow rate of 1.2 mL/min and detection wavelength of 201 nm. Plasma sample (100 μL) pretreatment was based on simple deprotienization by acetonitrile spiked with clonidine as an internal standard (I.S.) of 20 ng/mL followed by extraction with tert-butyl methyl ether and centrifugation. The organic layer was evaporated under N2 gas and reconstituted with 100 μL of acetate buffer (pH 3.4, 20 mM), and 50-μL portions of reconstituted sample were injected onto the column. Sample analysis including sample pretreatment was achieved within 35 min. Calibration curve was linear (r  ≥ 0.998) from 5 to 100 ng/mL. Both intra- and inter-day assay precisions that are presented through RSD were lower than 12.6% for intra-day and lower than 12.0% for inter-day assessment. Limit of detection was 0.8 ng/mL at S/N of 3. This method was omitting the use of expensive solid phase extraction and time consuming liquid extraction procedures. Moreover, the present method was successfully applied to study pharmacokinetic parameters of FEN after intraperitoneal administration to male Wistar rat. Pharmacokinetic parameters estimated by using moment analysis were T 1/2 198.3 ± 44.7 min, T max 28.3 ± 2.9 min and AUC0–180 15.6 ± 2.9 (×102) ng min/mL.
Keywords: Fentanyl; High performance liquid chromatography; Deproteinization; UV detection; Rat plasma; Pharmacokinetics;