Talanta 131 (2015) 46–54

Contents lists available at ScienceDirect

Talanta journal homepage: www.elsevier.com/locate/talanta

Determination of acrylamide and glycidamide in various biological matrices by liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study Tae Hwan Kim a,1, Soyoung Shin b,1, Kyu Bong Kim c, Won Sik Seo d, Jeong Cheol Shin d, Jin Ho Choi d, Kwon-Yeon Weon d, Sang Hoon Joo d, Seok Won Jeong d, Beom Soo Shin d,n a

School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Korea Department of Pharmacy, Wonkwang University, Iksan, Jeonbuk, Korea School of Pharmacy, Dankook University, Cheonan-si, Chungnam, Korea d College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Gyeongbuk, Korea b c

art ic l e i nf o

a b s t r a c t

Article history: Received 5 May 2014 Received in revised form 14 July 2014 Accepted 15 July 2014 Available online 24 July 2014

Acrylamide (AA) is a heat-generated food toxicant formed when starchy foods are fried or baked. This study describes a simple and sensitive liquid chromatography–tandem mass spectrometry assay for the simultaneous quantification of AA and its active metabolite, glycidamide (GA) in rat plasma, urine, and 14 different tissues. The assay utilized a simple method of protein precipitation and achieved a lower limit of quantification of 5, 10 and 25 ng/mL of AA and 10, 20 and 100 ng/mL of GA for plasma, tissues and urine, respectively. The assay was fully validated to demonstrate the linearity, sensitivity, accuracy, precision, process recovery, and stability using matrix matched quality control samples. The mean intraand inter-day assay accuracy was 91.6–110% for AA and 92.0–109% for GA, and the mean intra- and interday assay precisions were r 10.9% for AA and r 8.60% for GA. The developed method was successfully applied to a pharmacokinetic study of AA and GA following intravenous and oral administration of AA in rats. Tissue distribution characteristics of AA and GA were also determined under steady-state conditions. & 2014 Elsevier B.V. All rights reserved.

Keywords: Acrylamide Glycidamide LC–MS/MS Pharmacokinetics Tissue distribution

1. Introduction Acrylamide (AA) is a white and highly water-soluble chemical that has been used in various fields such as oil, cosmetic, water treatment, and textile industries since mid-1950s [1]. AA has shown to cause a variety of tumors in experimental animals. The carcinogenicity of AA has been extensively studied and increased incidences of tumors in mammary gland, thyroid, and skin have been documented [2–4]. Based on the animal studies, AA is classified as a probable human carcinogen by the United States Environmental Protection Agency [1] and International Agency for Research on Cancer [5]. Various other toxicities have been reported, including neurotoxicity in animals [6,7] and humans [8–10], reproductive toxicity in rodents [11,12], and mutagenicity in somatic cells in vitro [13] and in vivo [14], as well as in germ cells in vivo [15].

n Correspondence to: College of Pharmacy,Catholic University of Daegu,13-13 Hayang-ro, Hayang-eup Gyeongsan-si, Gyeongbuk 712-702, Korea. Tel.: þ 82 53 850 3617; fax: þ 82 53 850 3602. E-mail address: [email protected] (B.S. Shin). 1 Authors contributed equally to this work.

http://dx.doi.org/10.1016/j.talanta.2014.07.042 0039-9140/& 2014 Elsevier B.V. All rights reserved.

In 2002, it has been reported that AA is formed by the Maillard reaction during cooking of carbohydrate-rich foods at high temperature [16,17]. Significant levels of AA were found in certain fried, baked, and deep-fried foods such as potato chips (318 ppb), ground coffee (205 ppm), crackers and snack (169 ppb), and bakery products (34 ppb) [16–19]. Since the hazardous effects of AA as an industrial chemical are well known, the discovery of AA in the daily diet renewed the interests in its potential health effects and raised considerable concern [20]. AA toxicities are known to be mediated by its epoxide metabolite, glycidamide (GA). GA is predominantly formed by CYP2E1 [21–24]. GA-induced genotoxicity and mutagenicity have been reported in rodents [14,25], rodent germ cells [15,26], and Salmonella typhimurium [27]. GA reacts with DNA to form DNA adducts with even higher affinity than AA [28–30], which appears to be the major cause of mutagenicity and carcinogenicity by AA exposure [5,31,32]. To assess the potential risks of AA exposure on human health, it is essential to determine AA and GA concentrations in biological fluids. While many analytical methods have been described for the determination of AA in drinking water [33–36] and food stuffs [37–39], few bioanalytical methods are available that can be used

T.H. Kim et al. / Talanta 131 (2015) 46–54

for in vivo pharmacokinetic or toxicokinetic studies. The first reported method to determine AA in biological samples was the radioactivity assay of labeled AA (2,3-14C AA) [40]. Later, assays utilizing high pressure liquid chromatography (HPLC) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) have been developed. These assays include an HPLC/UVD with protein precipitation for determination of AA in rat plasma [41], an LC–MS/MS assay with solid phase extraction (SPE) for determination of AA and GA in mouse serum [42] and an LC–MS/MS assay with protein precipitation to determine AA and GA in human placenta [43]. However, the HPLC/UVD and LC–MS/MS methods utilizing protein precipitation lack sufficient sensitivity (LLOQ ¼500 ng/mL) [41,43]. The only bioanalytical method which achieves sufficient sensitivity is the LC–MS/MS assay with SPE sample preparation (LLOQ ¼0.71 ng/mL for AA, 8.7 ng/mL for GA) [42]. To date, most of the pharmacokinetic studies of AA [31,32,44,45] rely on this LC–MS/MS assay with SPE which is complicated, time consuming, and less cost efficient. A simple and rapid assay which is adaptable to various biological samples with high sensitivity is not yet available. In the present study, a simple and sensitive LC–MS/MS method using single-step protein precipitation sample preparation was developed and validated for the simultaneous determination of AA and GA in biological matrices including plasma, urine, and various tissues. The method was applied to an in vivo study to characterize the absorption, tissue distribution and disposition pharmacokinetics of AA and GA after intravenous (i.v.) injection, i.v. infusion, and oral administration in rats.

2. Experimental 2.1. Materials AA, acrylamide-D3 standard solution (500 mg/L, internal standard, IS) and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). GA was purchased from Toronto Research Chemicals (Toronto, ON, Canada). Acetonitrile, methanol, and distilled water (all HPLC grade) were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA).

2.2. Preparation of standard solutions Primary stock solutions of AA and GA were prepared by dissolving 10 mg each of AA and GA separately in 10 mL of distilled water. The working standard solutions were prepared by further dilution of the primary stock solutions with acetonitrile. The acrylamide-D3 standard solution (100 μL) was diluted to 10 mL with acetonitrile.

47

2.3. Calibration standards and quality control (QC) samples All calibration curves consisted of at least seven calibrator concentrations, a blank sample (without IS) and a zero sample (with IS). The calibrator and QC samples were prepared by spiking 5 μL of the working standard solution to 45 μL of the blank rat biological matrices. Various blank biological samples were obtained from at least 10 rats by collecting blood, urine, and tissues of liver, kidney, spleen, lung, heart, testis, stomach, small intestine, large intestine, fat, skin, muscle, brain, and thyroid. Tissue samples were homogenized in isotonic saline. Four different levels of low QC, medium QC, high QC, and lower limit of quantification (LLOQ) samples were prepared for AA and GA in each biological matrix. Different calibration ranges were constructed depending on the biological matrices of plasma, urine, and tissue samples. The calibration standards and QC samples for AA and GA in different biological matrices are presented in Table 1. The QC samples were prepared once, and aliquots (50 μL each) were stored at –20 1C until analysis. 2.4. Sample preparation The working standard solutions of IS (500 ng/mL for plasma and tissue samples, and 2000 ng/mL for urine samples) were added to 50 μL of the biological samples. Blank acetonitrile (50 μL for plasma and tissue samples and 650 μL for urine samples) was used as the protein precipitating solvent. The mixture was mixed on a vortex mixer for 1 min and centrifuged at 16,060g for 10 min (Biofuge Fresco; Kendro, Osterode, Germany). For all tissue samples except thyroid, the supernatant (75 μL) was diluted with the same volume of acetonitrile and centrifuged at 16,060g for 10 min. Finally, 75 μL of the diluted supernatant was mixed with 25 μL of distilled water, and a portion (7.5 μL) was injected into the LC–MS/MS. 2.5. LC/MS/MS instruments and conditions A model 1200 HPLC coupled with a model 6430 LC–MS/MS system (Agilent, Santa Clara, CA, USA) was used for sample analysis. Samples were separated on a dC18 column (150  2.10 mm2 i.d., 3 μm; Atlantis, Milford, MA, USA) with a Security Guard column (Phenomenex, Torrence, CA, USA). The mobile phase was a mixture of acetonitrile and 0.05% of formic acid (10:90 v/v). The flow rate was 0.1 mL/min, and the column oven temperature was 30 1C for all samples. The electro spray ionization source was operated in a positive mode and samples were detected in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms per MRM channel. Gas temperature, gas flow rate, and nebulizer gas pressure was set at 300 1C, 10 L/min, and 20 psi, respectively. The MRM transition of precursor to product ion pairs were m/z 71.9-55.0 for AA, m/z

Table 1 Concentrations of calibrators and QC samples for acrylamide and glycidamide in various biological samples. Compound

Matrix

Concentration (ng/mL) Calibrator

QC samples

Acrylamide

Plasma Urine All tissuesn

5, 10, 50, 100, 500, 1000, 2000, and 5000 25, 50, 100, 500, 1000, 2000, 5000, and 10,000 10, 20, 50, 100, 500, 1000, 2000, and 5000

5, 20, 500, and 4500 25, 300, 1000, and 9000 10, 40, 500, and 4500

Glycidamide

Plasma Urine All tissuesn

10, 50, 100, 500, 1000, 2000, and 5000 100, 150, 500, 1000, 2000, 5000, and 10,000 20, 50, 100, 500, 1000, 2000, and 5000

10, 20, 500, and 4500 100, 300, 1000, and 9000 20, 40, 500, and 4500

n

The lowest concentration in the calibrator and QC samples for thyroid was 5 ng/mL for acrylamide and 10 ng/mL for glycidamide."

48

T.H. Kim et al. / Talanta 131 (2015) 46–54

87.9-44.2 for GA, and m/z 75.0-58.0 for acrylamide-D3. The fragmentor voltage was set at 90 V for AA and acrylamide-D3 and 35 V for GA. The collision energy was set at 2 V for AA and acrylamide-D3 and 5 V for GA. Data acquisition was performed with MassHunter Quantitative Analysis (Agilent). 2.6. Assay validation 2.6.1. Specificity, linearity and sensitivity Specificity was assessed by analyzing blank matrix and the blank matrix spiked with AA, GA, and IS. The linearity of the method was evaluated over the concentration ranges described for different biological matrices (Table 1). All calibration curves were constructed by the weighted regression method (1/x) of the peak area ratios of AA and GA to the internal standard vs. theoretical concentrations. The LLOQ was defined as the lowest concentrations of AA and GA of the calibration curves that yielded a signalto-noise (S/N) ratio 420 with acceptable accuracy and precision (r15%). 2.6.2. Accuracy and precision Intra- and inter-day accuracy and precision were determined by assaying five replicates of LLOQ, low QC, medium QC, and high QC samples in each matrix each day for 5 consecutive days. Acceptable criteria for accuracy and precision were within 715% relative error from the nominal values and within 715% relative standard deviation except at LLOQ, where it should not deviate by more than 20%. 2.6.3. Recovery Process recoveries were evaluated by comparing the LC–MS/MS responses of reference and test samples using five replicates of the low and high QC samples. Reference samples and test samples were prepared by dissolving AA, GA, and IS in distilled water and biological matrix, respectively. The sample preparation procedures were the same as described. The recovery was calculated by dividing the analyte peak area of the test sample by that of the reference sample. 2.6.4. Stability The stability of AA and GA was examined under four different conditions using five replicates of low and high matrix-matched QC samples. The short term stability was determined by analyzing low and high QC samples left at room temperature for 4 h. The long-term stability was determined by analyzing low and high QC samples left at –20 1C for 2 weeks. The auto sampler stability was determined by analyzing low and high QC samples left in the auto sampler at 4 1C for 24 h. Lastly, the freeze and thaw stability was determined by analyzing QC samples subject to three freeze-thaw cycles. All stability data are expressed as the percentage of the mean calculated vs. actual concentrations.

oral gavage (n ¼4) at a dose of 2 mg/kg. Rats were fasted 12 h prior to the AA dose. Approximately 0.2 mL of the jugular venous blood samples were collected at 5, 10, 15, and 30 min, 1, 2, 4, 6, 8, 10, 12, 14, and 15 h post-dose. Plasma samples were harvested by centrifugation of the blood samples at 16,060g for 5 min. Urine samples were collected for over 24 h. Tissue distribution of AA and GA were examined under steadystate conditions after i.v. infusion of AA. The rats were anesthetized by intraperitoneal injection of Zoletil 50s (20 mg/kg; Virbac Laboratories, Carros, France) and surgically cannulated with polyethylene (PE) tubing (0.58 mm i.d. and 0.96 mm o.d.; Natume, Tokyo, Japan) in the right jugular vein for blood sampling and femoral vein for i.v. injection and infusion. After 2 days of recovery, AA dissolved in distilled water was administered by i.v. injection as a loading bolus dose (0.236 mg/kg) and i.v. infusion (2 μg/min/kg) for 12 h. The loading dose was calculated as the volume of distribution (0.589 L/kg) multiplied by the target steady-state plasma concentration (400 ng/mL), while the infusion rate was calculated as the clearance (5.13 mL/min/kg) multiplied by the target steady-state plasma concentration (400 ng/mL). The volume of distribution and clearance of AA were obtained from the i.v. injection study. Blood samples were collected at 1, 2, 4, 6, 8, 10, and 12 h during infusion and centrifuged at 16,060 g for 5 min. At the end of the infusion, rats were sacrificed with diethyl ether and the tissues of liver, kidney, spleen, lung, heart, testis, stomach, small intestine, large intestine, fat, skin, muscle, brain, and thyroid were excised and immediately homogenized in normal saline (T10 basic; IKA, Wilmington, DE, USA). All samples were stored at  20 1C until analysis. 2.8. Data analyses The plasma concentration–time data were analyzed by a noncompartmental method using the WinNonlin 2.1 nonlinear least squares regression program (Pharsight, Cary, NC, USA) to obtain pharmacokinetic parameters. The terminal elimination half-life (t1/2) was calculated as 0.693/λz. The area under the serum concentration–time curve from time zero to the last observation time point (AUCall) was calculated using the trapezoidal rule and AUC to infinity time (AUCinf) was obtained by adding Cn/λz to AUCall. The systemic clearance (CL) was calculated as dose/AUC. Apparent volume of distribution during the terminal phase (Vz) and steady state volume of distribution (Vss) were calculated as CL/ λz and CL  MRT, respectively. The peak serum concentration (Cmax) and the time to reach Cmax (Tmax) were read directly from the observations. The fraction of dose excreted into urine (Fe,urine) was calculated by dividing the amount excreted into urine (Ae,urine) by dose. The absolute bioavailability (F) after oral dosing was calculated as F¼(Doseiv  AUCoral)/(Doseoral  AUCiv). The tissue-toplasma partition coefficients (Kp) were expressed as the tissue-toplasma concentration ratios. Obtained data were statistically tested by unpaired t-test between the two means for the unpaired data. The statistical significance level was set at po 0.05.

2.7. In vivo pharmacokinetic study Male Sprague-Dawley rats (8–10 weeks, 230–290 g; Hyochang Science, Daegu, Korea) were kept in plastic cages with free access to standard diet (Cargill Agri Purina, Seong-nam, Korea) and water. The animals were maintained at a temperature of 237 2 1C with a 12 h light–dark cycle and relative humidity of 50 710%. The animal study was approved by the Ethics Committee for the Treatment of Laboratory Animals at Catholic University of Daegu (IACUC 2012-07) and conducted following standard operating procedures. AA was dissolved in distilled water at a concentration of 1 mg/mL and administered by penile vein injection (n ¼4) and

3. Results and discussion 3.1. Optimization of sample preparation and chromatography The present assay employed a simple protein precipitation procedure for the determination of AA and GA in biological samples. Protein precipitation has advantages over other sample preparation methods such as SPE [42], liquid–liquid extraction [46], and column switching [47] in that it is rapid, simple, and may be adaptable to high-throughput screening. It also allows a high process recovery by minimizing the chances to loss of analytes

T.H. Kim et al. / Talanta 131 (2015) 46–54

Fig. 1. Representative MRM chromatograms of acrylamide, glycidamide and acrylamide-D3 (IS) obtained at LLOQ in plasma, urine and thyroid.

49

50

T.H. Kim et al. / Talanta 131 (2015) 46–54

during the sample clean-up process. In contrast, protein precipitation may be more susceptible to endogenous interferences or matrix effects because the efficacy of removing endogenous

interference may be lower than other complicate methods. Concerning AA and GA, which both have a low molecular weight and high hydrophilicity, polar endogenous components may possibly

Table 2 Intra- and inter-day accuracy and precision of acrylamide and its active metabolite, glycidamide in various biological matrices (n ¼5). Matrix

Acrylamide Conc (ng/mL)

Plasma

Urine

Liver

Kidney

Spleen

Lung

Heart

Testis

Stomach

Small intestine

Large intestine

Fat

Skin

Muscle

Brain

Thyroid

5 20 500 4500 25 300 1000 9000 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 10 40 500 4500 5 40 500 4500

Glycidamide

Intra-day (%)

Inter-day (%)

Conc (ng/mL)

Accuracy

Precision

Accuracy

Precision

105 99.8 96.0 106 102 103 103 102 105 95.3 94.2 104 107 94.7 92.6 101 97.8 99.8 102 106 101 96.7 94.1 101 107 99.8 94.6 106 105 95.5 97.3 105 105 101 103 105 99.6 99.2 99.6 101 100 91.7 94.7 96.2 102 100 102 97.9 102 98.9 94.1 105 110 97.9 91.6 101 104 92.6 93.9 106 98.1 93.9 93.7 105

3.67 3.83 2.30 3.04 4.28 3.85 2.00 0.86 4.55 4.68 4.58 2.01 3.29 4.55 4.86 2.63 7.92 7.45 5.52 3.71 7.05 6.21 2.84 1.64 3.21 3.86 1.92 2.68 4.41 6.28 1.55 1.70 4.89 4.77 4.65 2.70 6.52 3.40 4.03 4.69 4.05 1.31 3.82 3.05 10.9 3.90 6.57 3.38 6.24 1.36 3.44 3.10 4.90 5.12 1.01 1.74 5.77 1.49 2.15 0.93 4.34 2.51 1.02 1.24

106 95.7 97.0 103 103 103 102 96.8 97.0 99.2 98.3 106 103 96.5 95.7 103 102 98.6 99.7 108 109 99.3 99.9 102 105 97.0 93.7 107 105 96.9 97.5 105 105 96.8 99.4 98.2 99.0 98.0 98.9 101 99.4 95.0 96.1 99.5 107 97.8 94.1 101 98.3 96.6 99.3 105 102 93.7 92.7 101 103 96.5 99.4 106 99.8 97.3 97.0 102

1.34 2.70 1.52 1.36 5.68 3.66 1.95 3.62 9.03 6.76 4.26 2.50 5.53 3.27 4.79 4.16 4.95 4.41 5.82 1.46 4.07 4.57 5.04 2.38 5.58 5.63 2.24 2.99 4.52 3.57 6.57 2.40 4.62 4.18 7.28 4.96 5.76 5.05 5.24 6.00 6.81 5.49 8.14 5.38 6.51 3.30 4.99 3.38 4.35 5.10 4.80 2.51 6.87 3.47 1.61 4.23 5.69 4.57 7.63 1.45 5.46 3.37 5.69 1.03

10 20 500 4500 100 300 1000 9000 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 20 40 500 4500 10 40 500 4500

Intra-day (%)

Inter-day (%)

Accuracy

Precision

Accuracy

Precision

103 101 98.3 106 102 97.5 97.9 103 105 100 96 103 92.0 93.7 94.9 100 102 99.3 98.9 104 105 102 94.6 99.5 97.5 96.5 99.1 103 104 98.5 93.1 104 102 96.2 101 108 103 95.0 99.5 106 97.4 97.1 94.8 104 97.7 101 106 108 101 97.0 102 101 105 101 97.2 101 101 94.0 101 105 107 98.5 95.4 104

4.21 4.65 3.74 2.12 5.66 4.56 3.74 1.44 5.36 5.29 3.51 1.61 3.93 4.82 3.02 1.84 7.00 6.56 5.11 1.04 4.55 4.93 3.43 2.56 4.89 4.19 5.59 3.79 3.68 1.47 1.95 1.24 5.44 2.56 1.85 1.24 4.86 4.81 6.92 3.33 5.35 4.74 1.56 2.92 7.45 5.57 4.63 2.43 3.86 5.25 3.29 2.01 3.13 3.44 2.14 2.03 7.74 2.98 4.21 3.28 3.54 5.03 3.74 2.83

101 102 98.2 100 98.2 98.8 98.2 99.5 104 102 101 104 96.0 95.4 93.2 99.8 101 96.5 95.3 100 103 103 94.0 101 94.3 98.1 98.1 106 105 97.9 94.7 102 98.7 95.1 97.8 102 104 99.1 100 106 100 103 98.1 103 103 98.1 99.9 105 104 98.2 97.0 105 103 97.8 102 105 103 97.9 104 109 100 98.1 97.4 102

6.96 5.75 3.76 7.63 7.90 5.40 4.71 3.65 4.14 5.55 4.37 2.75 8.58 5.14 3.60 1.76 7.17 5.76 4.54 2.74 5.89 3.30 3.11 1.79 3.38 4.29 3.33 3.21 4.79 3.97 2.42 1.86 5.44 3.80 4.61 6.91 6.86 4.28 6.35 2.94 7.87 5.07 3.93 1.42 6.63 5.85 3.04 4.68 8.12 5.29 1.50 3.30 5.41 7.52 4.03 7.92 5.53 5.07 2.90 1.55 7.79 3.61 2.30 1.53

T.H. Kim et al. / Talanta 131 (2015) 46–54

be co-eluted leading to low resolution or matrix effect. Thus, separation of the target analytes from endogenous compounds is challenging during assay development. To separate the target analytes from polar endogenous components and achieve a sufficient retention, highly aqueous compositions of the mobile phase (490%, 0.05% formic acid) were initially adapted with a high resolution Kinetex C18 column. However, the recovery of AA and GA was 4150% for plasma and o 20% for urine with high variability, suggesting the possibility of signal enhancement or suppression caused by a matrix effect. The peak shape of the IS was split and endogenous interferences peaks were also observed. Other columns specially designed for the separation of polar analytes under reverse phase conditions (e.g., Luna CN column, Atlantis HILIC column, and Atlantis C18 column) were then tested. The CN column resulted in unsatisfactory recovery (o 10%) and the HILIC column resulted in highly unstable baselines. Acceptable separation and recovery were achieved using the Atlantis C18 column. With the mobile phase composition of acetonitrile and 0.05% formic acid in water (10:90 v/v), AA, GA, and IS peaks were well separated from endogenous interferences in all tested biological samples (Fig. 1). To optimize the sample preparation, methanol and acetonitrile were evaluated as the protein precipitating solvents with different dilution factors. Acetonitrile showed stable baselines with less endogenous interferences and was selected as the protein precipitation agent. The dilution factor was also optimized by analyzing peak intensities with different volumes of the protein precipitating solvent. Optimal recoveries were obtained with 3-, 15-, and 6-fold dilution for plasma, urine, and tissue samples, respectively. Representative MRM chromatograms of AA, GA, and IS obtained by analyzing blank biological matrices and the matrices spiked with each compound yielding concentrations of LLOQ are shown in Fig. 1. The retention time for AA, GA, and IS was 5.4, 5.0, and 5.4 min, respectively. Examination of blank sample, zero sample, and other calibrators showed no interfering peaks at the retention times corresponding to the target analytes. 3.2. Method validation 3.2.1. Linearity, sensitivity, precision, and accuracy Calibration ranges were determined depending on the analytes and matrices. The calibration curves were linear over AA concentration ranges of 5–5000 ng/mL for plasma and thyroid,

51

25–10,000 ng/mL for urine, and 10–5000 ng/mL for all tissues except thyroid, and over GA concentrations ranges of 10– 5000 ng/mL for plasma and thyroid, 100–10,000 ng/mL for urine, and 20–5000 ng/mL for all tissues except thyroid, with correlation coefficients 40.999. At LLOQ of each analyte in various matrices, the signal-to-noise ratio was 4 20 (range 41.0–145), and accuracy and precision were acceptable. While the LLOQ for AA in the rat plasma is higher than the LLOQ from the reported assay which was determined in the mouse serum [42], the LLOQ for GA is comparable. Table 2 shows the intra- and inter-day accuracy and precision determined at the LLOQ, low QC, medium QC, and high QC matrix matched samples with five replicates each day on 5 consecutive days. The intra- and inter-day accuracies were 91.6–110% for AA and 92.0–109% for GA. The precision of AA and GA was r 10.9% and r8.58%, respectively. 3.2.2. Recovery and matrix effect The recoveries of AA and GA at low and high QC samples and IS in each biological matrix are presented in Table 3. The process recovery was calculated by comparing the peak area of the analyte obtained from the standard solution spiked in matrices followed by protein precipitation process to that of the analyte obtained from the matrix free solvent. Since no extraction step was involved, the process recovery directly reflected the matrix effect. The process recovery of AA in all tested matrices ranged from 37.1 72.0% (small intestine) to 77.3 73.2% (fat). The highest process recovery of GA was observed for kidney (68.7 73.6%) and the lowest recovery was observed for urine (39.9 70.3%). For IS, the recovery was highest for fat (75.5 72.9%) and lowest for thyroid (44.9 71.6%). Even if the recovery of target compounds was not high mainly due to the matrix effects, the process recoveries were consistent and reproducible in all tested batches (Table 3) with minimal variation (CV 1.3–8.9%). 3.2.3. Stability The stability tests were conducted by using low and high matrix matched QC samples. Table 4 summarizes the results of the auto sampler stability, freeze–thaw stability, short-term, and long-term stability of AA and GA in each biological matrix as the mean percentages of the calculated vs. theoretical concentrations. No significant deviations were observed compared to the theoretical concentrations, indicating that AA and GA were stable under all tested conditions.

Table 3 Process recovery of acrylamide, glycidamide, and the internal standard (acrylamide-D3) in rat biological matrices (n¼ 5, mean 7 S.D.). Matrix

Plasma Urine Liver Kidney Spleen Lung Heart Testis Stomach Small intestine Large intestine Brain Fat Thyroid Skin Muscle n

Acrylamide

Glycidamide

Acrylamide-D3 (IS)

Low QC

High QC

Low QC

High QC

500 ng/mL

63.3 71.3 46.5 70.9 45.0 71.0 66.4 71.2 56.6 70.9 42.7 71.2 56.6 71.1 55.9 70.9 64.2 71.3 37.1 72.0 56.3 70.8 47.9 72.5 74.7 72.0 44.4 71.2 72.5 73.4 62.8 71.3

60.8 7 0.8 42.6 7 2.0 44.9 7 1.0 69.4 7 3.9 56.3 7 0.5 44.3 7 0.7 54.4 7 0.2 57.0 7 0.6 56.17 1.9 39.6 7 3.1 56.17 0.7 42.7 7 0.8 77.3 7 3.2 46.6 7 0.7 69.5 7 0.4 59.3 7 0.6

59.3 7 3.1 45.4 7 1.5 47.0 7 2.2 68.77 3.6 57.9 7 2.5 50.7 7 2.6 59.3 7 3.5 58.7 7 3.1 55.4 7 2.3 49.87 2.0 56.6 7 1.7 47.4 7 2.3 60.3 7 0.8 52.4 7 2.5 63.3 7 2.6 65.2 7 2.6

56.6 7 0.5 39.9 7 0.3 46.0 7 0.6 65.9 7 0.7 56.2 7 0.6 43.5 7 0.6 59.9 7 0.4 59.6 7 0.3 55.8 7 0.8 48.77 0.6 60.5 7 0.5 43.5 7 0.4 60.9 7 0.5 57.4 7 0.5 63.9 7 0.8 64.17 0.6

58.7 7 3.2 46.0 7 0.9* 45.6 7 1.4 70.37 3.2 64.07 3.0 45.17 2.0 58.2 7 2.8 61.9 7 2.5 63.4 7 2.0 51.6 7 1.8 59.17 1.0 46.17 1.8 75.5 7 2.9 44.9 7 1.6 74.17 2.5 64.57 1.7

The process recovery of acrylamide-D3 (IS) in urine samples was determined at 2000 ng/mL.

52

T.H. Kim et al. / Talanta 131 (2015) 46–54

Table 4 Stability of acrylamide and glycidamide in various biological matrices (n¼ 5, mean 7 S.D.). Matrix

Auto sampler

Freeze-thaw

Short-term

Long-term

Low QC

High QC

Low QC

High QC

Low QC

High QC

Low QC

Acrylamide Plasma Urine Liver Kidney Spleen Lung Heart Testis Stomach Small Intestine Large intestine Fat Skin Muscle Brain Thyroid

98.5 7 2.9 99.2 7 2.5 1027 3 1017 3 95.2 7 4.1 1037 2 1037 3 93.8 7 1.9 99.2 7 3.1 92.6 7 2.1 1057 7 99.7 7 1.3 98.17 5.4 97.2 7 4.2 98.7 7 1.6 92.9 7 2.8

97.3 74.4 101 71 99.5 75.9 99.1 73.7 100 74 96.1 73.8 95.9 71.7 102 75 105 72 102 71 99.1 71.0 104 74 101 74 102 74 103 72 98.5 74.7

95.5 71.0 97.6 72.1 102 72 99.3 72.3 94.5 74.3 104 72 96.3 73.0 96.0 74.1 98.4 75.6 98.3 71.5 101 77 100 72 94.3 73.7 98.2 74.3 96.9 73.8 93.8 72.0

1017 6 99.7 7 1.6 1017 5 98.2 7 3.3 97.4 7 3.9 95.4 7 2.6 94.9 7 2.4 99.3 7 4.2 104 7 1 1037 1 99.7 7 1.6 1027 1 98.0 7 3.8 1037 5 1037 1 104 7 3

1017 2 1017 2 1037 4 1027 4 96.3 7 2.8 1057 2 104 7 2 90.4 7 2.7 96.7 7 4.7 94.0 7 3.2 1017 2 1007 2 97.8 7 5.3 95.3 7 3.8 96.9 7 1.8 93.0 7 3.3

95.17 1.1 1037 1 1017 6 97.2 7 3.9 104 7 2 1027 4 95.7 7 2.3 99.2 7 5.6 104 7 3 1037 3 99.2 7 1.4 1027 3 104 7 2 1017 3 1027 3 1017 6

1007 3 98.7 7 2.6 1017 3 1017 3 98.5 7 4.3 1037 2 98.7 7 0.7 92.17 1.3 95.5 7 3.8 95.3 7 3.3 1027 6 1007 1 95.8 7 3.0 95.4 7 4.5 98.8 7 2.0 92.6 7 2.1

96.2 7 1.3 102 7 1 96.9 7 2.7 1007 5 95.9 7 3.6 1047 6 96.6 7 2.3 102 7 4 102 7 2 1017 0 99.2 7 2.8 1047 4 105 7 5 102 7 3 1017 1 102 7 4

Glycidamide Plasma Urine Liver Kidney Spleen Lung Heart Testis Stomach Small Intestine Large intestine Fat Skin Muscle Brain Thyroid

1017 6 97.7 7 7.0 94.3 7 1.3 1017 3 97.2 7 3.1 1017 5 1027 3 98.2 7 5.1 98.8 7 5.2 99.5 7 4.0 1037 6 99.2 7 7.6 98.4 7 7.1 1037 5 97.2 7 3.8 1047 7

103 73 96.4 74.8 101 75 107 71 98.3 75.9 100 77 95.3 77.1 101 77 105 74 99.5 75.0 101 72 106 75 106 74 108 72 105 72 107 73

99.7 73.3 96.2 74.1 94.4 72.3 101 73 92.7 72.7 98.4 73.7 101 73 105 75 96.6 74.3 100 73 10776 100 76 97.1 74.9 103 74 98.2 75.4 102 77

1027 2 94.3 7 1.9 1037 5 106 7 2 96.7 7 5.9 95.5 7 1.2 90.8 7 1.1 1057 5 1027 1 99.3 7 4.6 104 7 3 106 7 4 1087 3 1087 2 1057 2 1097 4

104 7 6 1007 6 95.0 7 1.5 1017 5 97.9 7 3.2 1017 5 104 7 5 95.5 7 1.4 97.3 7 5.1 96.3 7 4.9 96.7 7 4.7 94.7 7 3.7 1027 6 1037 5 99.4 7 7.2 98.9 7 7.5

1077 2 97.7 7 4.1 97.9 7 5.7 106 7 2 1057 3 1037 5 1057 4 98.9 7 6.5 1077 3 1017 5 1007 2 106 7 3 1017 3 1057 1 104 7 3 1057 5

1037 5 98.0 7 5.2 98.2 7 4.2 1017 3 98.5 7 3.4 99.9 7 3.8 1037 3 1037 3 96.2 7 3.9 93.8 7 4.5 1007 4 97.7 7 4.3 98.4 7 7.8 93.7 7 3.5 98.3 7 6.6 99.0 7 4.5

105 7 2 99.7 7 1.6 1017 3 1077 1 92.7 7 3.6 108 7 3 91.4 7 2.6 105 7 2 105 7 1 93.9 7 1.5 1007 2 1117 2 1047 1 105 7 4 1067 1 1067 4

3.3. Application to pharmacokinetic study 3.3.1. Pharmacokinetics of AA and GA The developed method was applied to monitor the plasma concentration-time profiles and the urinary excretion of AA and its metabolite GA in rats after i.v. injection and oral administration of AA. The pharmacokinetics of AA is known to be linear over the dose of 1.0–100 mg/kg in rats [40]. In our study, the administered dose for both i.v. injection and oral administration were 2 mg/kg which is within the dose range of the linear pharmacokinetics in rats. The average plasma concentration-time profiles are shown in Fig. 2 and the obtained pharmacokinetic parameters of AA and GA are summarized in Table 5. After i.v. injection, plasma levels of AA declined in a mono-exponential manner. AA was converted to GA, which also declined in a mono-exponential manner. While the elimination half-lives (t1/2,λz) of AA and GA were independent of the administration routes, the t1/2,λz of GA was significantly longer than that of AA for both routes of administration (p o0.05, Table 5). Orally administered AA was rapidly and extensively absorbed; the time to reach the maximum concentration (Tmax) of AA was 0.50 70.00 h and the oral bioavailability was 80.5 76.3%. Following AA dose at 2.0 mg/kg in this study, the AUCall, which is based on the last observed plasma concentration, was over 99.6% and 93.8% of the extrapolated AUCinf for AA and GA, respectively. This data indicate that the assay was sensitive enough to evaluate the full pharmacokinetic profiles of AA and GA. Interestingly, metabolic conversion of AA to GA was more rapid and extensive after oral administration than after i.v. injection. The Tmax of GA was significantly faster after oral administration than after i.v. injection (2.50 7 1.00 vs. 6.00 70.00 h, po 0.05).

High QC

The metabolite-to-parent compound area under the curve (AUC) ratio (AUCGA/AUCAA) was higher after oral administration than after i.v. injection (0.64 vs. 0.12). This observation suggests that orally administered AA is subject to a significant pre-systemic first-pass metabolism. Urinary excretion of AA and GA was minimal (o 1% of the administered dose).

3.3.2. Tissue distribution of acrylamide and glycidamide The developed assay was applied to characterize the tissue distribution characteristics of AA and GA in rats under steady-state conditions. Since AA is frequently found in the daily diet, humans may be exposed to AA continuously. Thus, it may be useful to characterize the tissue distribution of AA under steady-state conditions. The plasma concentration-time profiles of AA and GA during i.v. infusion and the steady-state tissue concentrations are shown in Fig. 3 and Table 6. Plasma concentrations of AA and GA reached their steady-states within 2 h after initiation of infusion. The observed steady-state plasma AA concentrations (mean 347 745 ng/mL) appeared to be comparable to the target concentration of 400 ng/mL. The average steady-state plasma GA concentration was lower (1137 37 ng/mL) than the parent compound. The steady-state tissue concentrations and the tissue-to-plasma partition coefficients (Kp) for AA and GA are presented in Fig. 3 and summarized in Table 6. This is the first report on the steady-state distribution of AA and its metabolite GA in a total of 14 tissues, including spleen, lung, heart, stomach, intestine, fat, and thyroid. Consistent with the hydrophilic nature of AA and its small volume of distribution, AA concentrations in tissues were lower than the plasma concentrations. The highest concentration of AA was

T.H. Kim et al. / Talanta 131 (2015) 46–54

53

Fig. 2. Average plasma concentration–time profiles of acrylamide and glycidamide after (A) intravenous injection and (B) oral administration of acrylamide at a dose of 2 mg/kg in rats (n¼ 4 each). Each point represents the mean 7 S.D.

Table 5 Pharmacokinetic parameters of acrylamide and glycidamide following i.v. injection and oral administration of acrylamide at a dose of 2 mg/kg to rats (mean 7 S.D.). Compound

Parameters

Route of administration i.v. (n ¼4)

p.o. (n¼4)

Acrylamide

t1/2 (h) Tmax (h) C0 or Cmax (ng/mL) AUCall (ng  h/mL) AUCinf (ng  h/mL) Vz or Vz/F (L/kg) CL or CL/F (mL/min/kg) Vss (L/kg) F (%) Ae,urine (μg) Fe,urine (%)

1.337 0.06 NA 27607 170 6490 7 250 65007 250 0.589 7 0.038 5.13 70.19 0.642 70.031 NA 5.217 2.36 0.961 70.495

1.32 7 0.08 0.50 7 0.00 1780 7 300 5220 7 410 52407 410 0.7307 0.039 6.40 7 0.51 NA 80.5 7 6.3 1.517 0.63 0.299 7 0.131

Glycidamide

t1/2 (h) Tmax (h) Cmax (ng/mL) AUCall (ng ·h/mL) AUCinf (ng ·h/mL) Ae,urine (µg)

2.317 0.33n 6.007 0.00 1057 41 7757 349 826 7329 4.61 71.60

1.93 7 0.28n 2.50 7 1.001;# 5417 32 3310 7300 3350 7310 7,28 7 3.21

n

po 0.05 vs. acrylamide. # p o 0.05 vs. i.v.

Fig. 3. (A) Average plasma concentration–time profiles of acrylamide and glycidamide during intravenous infusion of acrylamide, (B) the steady-state concentrations of acrylamide and glycidamide in various tissues and (C) their steady-state tissue-to-plasma partition coefficients in rats (n¼ 4).

observed in heart followed by thyroid, stomach, brain, spleen, skin, muscle, kidney, lung, liver, testis, fat, large intestine, and small intestine. GA concentration was the highest in thyroid followed by

54

T.H. Kim et al. / Talanta 131 (2015) 46–54

Table 6 Steady-state tissue concentrations and tissue-to-plasma partition coefficients (Kp) of acrylamide and glycidamide in rats (n¼ 4, mean 7 S.D.). Matrix

Plasma Liver Kidney Spleen Lung Heart Testis Stomach Small intestine Large intestine Fat Skin Muscle Brain Thyroid

Acrylamide

Glycidamide

Concentration (ng/g)

Kp

Concentration (ng/g)

Kp

3477 45 1157 43 1337 60 1577 38 1337 54 1967 50 1037 68 1697 127 42.9 7 26.8 64.77 46.1 78.2 7 10.8 1577 30 1567 48 1657 30 1727 45

NA 0.3247 0.093 0.380 7 0.153 0.456 7 0.112 0.381 7 0.136 0.564 70.130 0.295 7 0.188 0.4777 0.339 0.1247 0.073 0.1877 0.128 0.231 70.046 0.459 7 0.125 0.450 7 0.125 0.476 70.076 0.506 70.154

1137 37 BLOQ 65.6 7 5.3 93.3 7 30.4 74.4 7 26.5 1107 26 BLOQ 72.2 7 15.7 BLOQ 63.4 7 21.7 74.5 7 11.8 88.6 7 27.0 99.5 7 39.6 93.3 7 25.8 152 757

NA NA 0.661 70.281 0.956 7 0.575 0.5707 0.286 1.09 7 0.50 NA 0.7147 0.305 NA 0.6777 0.334 0.562 7 0.090 0.8317 0.274 0.905 7 0.245 0.840 7 0.082 1.20 7 0.42

heart, spleen, muscle, brain, skin, stomach, large intestine, kidney, lung, and fat. Distribution of GA into liver and small intestine was minimal. Although tissue concentrations of GA were mostly lower than AA concentrations, relatively high concentrations of GA were observed in thyroid, fat and large intestine, which were comparable with AA concentrations. It was of particular interest to observe the significant distribution of AA and GA in representative target tissues of carcinogenicity and neurotoxicity, such as thyroid, brain, and skin.

[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]

4. Conclusions [20]

A specific and sensitive LC–MS/MS assay was developed and validated for the simultaneous determination of AA and its active metabolite GA in various biological matrices of rat plasma, urine, and 14 different tissues. The method was successfully applied to determine the oral bioavailability in rats after i.v. and oral administration of AA. In particular, tissue distribution characteristics of AA and GA were evaluated under steady-state conditions. The developed assay may be useful in risk assessment studies of AA and GA. Acknowledgment This work was supported by Grant 12162MFDS722 from the Korea Food and Drug Administration and National Research Foundation of Korea (NRF) Grant No. 2012R1A2A2A02044997 and 22A2013000073 (BK21 Plus). References [1] U.S. Environmental Protection Agency (EPA), EPA 749-F-94-005a: Chemical Summary for Acrylamide, 1994. [2] M.A. Friedman, L.H. Dulak, M.A. Stedham, Fundam. Appl. Toxicol. 27 (1) (1995) 95–105. [3] K.A. Johnson, et al., Toxicol. Appl. Pharmacol. 85 (2) (1986) 154–168. [4] R.J. Bull, M. Robinson, J.A. Stober, Cancer Lett. 24 (2) (1984) 209–212. [5] International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 1994. p. 389.

[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

[36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47]

P.S. Spencer, H.H. Schaumburg, Can. J. Neurol. Sci. 1 (2) (1974) 143–150. P.S. Spencer, H.H. Schaumburg, Can. J. Neurol. Sci. 1 (3) (1974) 152–169. C.J. Calleman, et al., Environ. Health Perspect 99 (1993) 221–223. L.O. Goffeng, et al., Neurotoxicol. Teratol. 30 (3) (2008) 186–194. J.W. Donovan, T. Pearson, Vet. Hum. Toxicol. 29 (1987) 462 (Abstract). K.L. Dearfield, et al., Mutat. Res. 330 (1–2) (1995) 71–99. R.W. Tyl, M.A. Friedman, Reprod. Toxicol. 17 (1) (2003) 1–13. A. Besaratinia, G.P. Pfeifer, J. Natl. Cancer Inst. 95 (12) (2003) 889–896. M.G. Manjanatha, et al., Environ. Mol. Mutagen 47 (1) (2006) 6–17. B.I. Ghanayem, et al., Biol. Reprod. 72 (1) (2005) 157–163. D.S. Mottram, B.L. Wedzicha, A.T. Dodson, Nature 419 (6906) (2002) 448–449. E. Tareke, et al., J. Agric. Food Chem. 50 (17) (2002) 4998–5006. J. Rosen, K.E. Hellenas, Analyst 127 (7) (2002) 880–882. U.S. Environmental Protection Agency (EPA), EPA/635/R-07/009F: Toxicological Review of Acrylamide, 2010. WHO, Joint FAO/WHO Consultation on Health Implication of Acrylamide in Food, 2002, 33 pp. E. Bergmark, C.J. Calleman, L.G. Costa, Toxicol. Appl. Pharmacol. 111 (2) (1991) 352–363. E. Bergmark, et al., Toxicol. Appl. Pharmacol. 120 (1) (1993) 45–54. T.R. Fennell, et al., Toxicol. Sci. 85 (1) (2005) 447–459. S.C. Sumner, et al., Chem. Res. Toxicol. 12 (11) (1999) 1110–1116. B. Paulsson, et al., Mutat. Res. 535 (1) (2003) 15–24. W.M. Generoso, et al., Mutat. Res. 371 (3–4) (1996) 175–183. K. Hashimoto, H. Tanii, Mutat. Res. 158 (3) (1985) 129–133. J.J. Solomon, et al., Cancer Res. 45 (8) (1985) 3465–3470. G. Gamboa da Costa, et al., Chem. Res. Toxicol. 16 (10) (2003) 1328–1337. D.R. Doerge, et al., Mutat. Res. 580 (1–2) (2005) 131–141. D.R. Doerge, et al., Toxicol. Appl. Pharmacol. 202 (3) (2005) 258–267. D.R. Doerge, et al., Toxicol. Appl. Pharmacol. 208 (3) (2005) 199–209. S. Cavalli, S. Polesello, G. Saccani, J. Chromatogr. A 1039 (1-2) (2004) 155–159. A. Hashimoto, Analyst 101 (1209) (1976) 932–938. U.S. Environmental Protection Agency (EPA), EPA Method 8316: Acrylamide, Acrylonitrile and Acrolein by High Performance Liquid Chromatogaphy (HPLC), 1994. U.S. Environmental Protection Agency (EPA), EPA Method 8032A: Acrylamide by Gas Chromatography, 1996. L.S. Bologna, et al., J. Chromatogr. Sci. 37 (7) (1999) 240–244. K. Takata, T. Okamoto, J. Environ. Chem. 1 (3) (1991) 559–565. Y. Zhang, et al., J. Chromatogr. A 1142 (2) (2007) 194–198. M.J. Miller, D.E. Carter, I.G. Sipes, Toxicol. Appl. Pharmacol. 63 (1) (1982) 36–44. D.S. Barber, et al., J. Chromatogr. B. Biomed. Sci. Appl. 758 (2) (2001) 289–293. N.C. Twaddle, et al., Cancer Lett. 207 (1) (2004) 9–17. K. Annola, et al., J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 876 (2) (2008) 191–197. E.K. Kopp, W. Dekant, Toxicol. Appl. Pharmacol. 235 (2) (2009) 135–142. D.R. Doerge, et al., Toxicol. Lett. 169 (1) (2007) 34–42. F. Sorgel, et al., Chemotherapy 48 (6) (2002) 267–274. E.K. Kopp, et al., J. Agric. Food Chem. 56 (21) (2008) 9828–9834.