Nicotine Pharmacokinetics in Rat Brain and Blood by Simultaneous

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Nicotine Pharmacokinetics in Rat Brain and Blood by Simultaneous Microdialysis, Stable-isotope Labeling and UHPLC-HRMS: Determination of Nicotine Metabolites Yan Xu, Qidong Zhang, Peng Li, Guangfeng Hong, Dingzhong Wang, Junhui Liu, Hao Zhou, Guobi Chai, Binbin Lu, Shengbao He, Wenjuan Zhang, Shihao Sun, Jianxun Zhang, and Jian Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05078 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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Analytical Chemistry

Nicotine Pharmacokinetics in Rat Brain and Blood by Simultaneous

Microdialysis,

Stable-isotope

Labeling

and

UHPLC-HRMS: Determination of Nicotine Metabolites Yan Xu,1 Qidong Zhang,2 Peng Li,2 Guangfeng Hong,2 Dingzhong Wang,2 Junhui Liu,2 Hao Zhou,3 Guobi Chai,2 Binbin Lu,2 Shengbao He,4 Wenjuan Zhang,2 Shihao Sun, 2 Jianxun Zhang,2 Jian Mao,2, * 1

School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China

2

Zhengzhou Tobacco Research Institute of China National Tobacco Company, Zhengzhou

450001, China 3

Technology Center, China Tobacco Henan Industrial Co., Ltd., Zhengzhou 450000, China

4

China National Tobacco Quality Supervision and Test Center, Zhengzhou 450001, China

*Corresponding

Author. E-mail: [email protected]

ABSTRACT The disposition and metabolism of nicotine in the brain is an important determinant of its exposure. We have developed a novel method for the dynamic determination of nicotine and its metabolites in rat brain and blood by simultaneous microdialysis sampling, stable-isotope labeling and ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) assaying. Microdialysis probes were inserted into both the right striatum and jugular vein of Sprague Dawley rats. The collections of dialystes after nicotine intraperitoneal injection were analyzed by the optimized UHPLC-HRMS. Nicotine-pyridyl-d4 was used as a metabolic tracer and several stable labeled isotopes were applied to calibrate the in vivo recoveries of retrodialysis. The quadrupole-Orbitrap HRMS provided reliable characterization of the nicotine derivatives with less than 3.5 ppm mass measurement accuracy. Good precision and accuracy were obtained for different analytes within the predefined limits of acceptability and the range of the standard curve. Nicotine and its 11 metabolites were identified in most microdialysis samples from the blood and brain tissue samples. Besides cotinine as the main metabolic product of nicotine, trans-3’-hydroxy-cotinine, nicotine-N-oxide and norcotinine were proved to be the second most abundant metabolites. The other seven nicotine products, including 4-oxo-4-(3-pyridyl)-butanoic acid, 4-hydroxy-4-(3-pyridyl)-butanoic acid, cotinine-N-oxide, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3’hydroxy-cotinine-O-glucuronide, which have not been determined previously in animal brain, were present in minor amounts. Pharmacokinetic profile of nicotine

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metabolites indicated that the metabolic characteristic of nicotine in the brain was relatively different from that in the blood. The present work would provide comprehensive evidence for clarifying the differences between nicotine metabolism in brain and peripheral system.

INTRODUCTION Nicotine (Nic) is the key component of mainstream smoke and is responsible for cigarette addiction.1-3 The occurrence of tobacco addiction or dependence originates in the continual neuropharmacological effects of Nic exposure, which is closely related to the amount and the body’s metabolic ability of Nic.4-7 Generally, the transformation process of Nic in liver mainly includes oxidation, N-demethylation and hydroxylation, which generates cotinine (Cot), nicotine-N-oxide (NNO), cotinine-N-oxide (CNO), nornicotine (NorNic), norcotinine (NorCot) and trans-3’-hydroxy-cotinine (OH-Cot).8-10 In addition, it has been proved that Nic could also be transformed to 4-oxo-4-(3-pyridyl)-butanoic acid (OxPyBut) and 4-hydroxy-4-(3-pyridyl)-butanoic acid (HyPyBut) in smokers.11 Moreover, these products as well as Nic may be further glucuronidated to form other metabolites such as nicotine-N-glucuronide (Nic-Gluc), cotinine-N-glucuronide (Cot-Gluc), trans-3’-hydroxy-cotinine-O-glucuronide (OH-Cot-Gluc) and so on.12 In fact, about 10 Nic metabolites can be well detected simultaneously, partly due to the limitation of analytical approaches and partly due to their trace levels in the body.13-19 Nic has easy access to the brain. However, the metabolic results in the peripheral system could not truly reflect its metabolic characteristics in the central nervous system (CNS).20 As some Nic metabolites have been shown to have pharmacological activity and may contribute to the neuropharmacological effects resulting from the Nic exposure, it is therefore necessary to consider the Nic biotransformation in the brain.21-23 Even though peripheral biotransformation of Nic has been studied extensively, its metabolism process in CNS has been given little attention because of the difficulty to obtain brain samples in vivo. In some previous studies, GC-MS, HPLC and LC-MS/MS methods were performed to detect the Nic and its metabolites in animal brain tissues, and Nic, Cot, NorNic, NorCot, NNO and OH-Cot could be quantified, but no information on other Nic metabolites was available.24-28 Other studies using high-performance liquid radiochromatographic analysis identified Nic, Cot, NorNic, NorCot, NNO and two other unknown minor products in brain.29,30 These developments provide useful initial insights; however, more effective studies are needed to understand how Nic metabolizes in the brain and the differences between the CNS and peripheral system. In addition, novel and efficient techniques are needed to investigate the


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metabolic process of Nic both in the brain and blood synchronously, which will reveal the Nic metabolic characteristics in a more realistic manner. In this study, we have employed simultaneous microdialysis coupled with UHPLC-HRMS using stable labeled isotope to firstly determine Nic and its 11 metabolites in rat brains, and further we compared the time-course of these metabolites both in the brain and the blood synchronously to elucidate their mechanistic similarities and differences.

EXPERIMENTALS The Supporting Information contains full details of the materials and methods for this study. Briefly, the SD rats were anesthetized with 2% pentobarbital sodium and the guide cannula for brain probes were implanted in the right striatum using the stereotaxic apparatus for orientation. After at least 24 h, under anesthesia, the blood microdialysis probes (CMA/20) were fixed in the jugular vein/right atrium, subsequently, the brain microdialysis probes (CMA/12) were inserted into the brain probe guide cannula. Finally, both of the blood and brain microdialysis probes were connected separately to the syringe pump (CMA/402). Then the filtered Ringer’s solution was perfused at a flow rate of 2.0 μL min-1 for the post-surgical balancing at least 120 min. After balancing, with the animal freely moving in the system (CMA/120), injection solutions containing 2.0 mg kg-1 Nic and (or not) 1.0 mg kg-1 Nic-d4 in saline (i. p.) was administered to the rats. Simultaneously, the sample collection was started with the brain and blood dialysates gathering every 15 min in separate tubes at 4 ℃ using the refrigerated fraction collector (CMA/140). The sampling amount of each collection was checked as about 30 μL and the microdialysis samples were analyzed directly by validated UHPLC-HRMS. A diagram of microdialysis devices and configuration in this experiment was shown as Figure S-1.

RESULTS AND DISCUSSION UHPLC-HRMS optimization. In term of the strong polarity of Nic and its metabolites, the HILIC chromatographic column was selected based on the previous studies.26,31 The flow phase eluting gradient was adjusted so that the target compounds were well separated within 10 min (Figure 1). Even though microdialysis samples were protein-free and could be directly loaded, the high salinity background (more than 150 mM) might lead to ion interference. To reduce this matrix effect, online desalination for 1.0 min elution “to waste” before automatically switching to MS detection was employed at the beginning of the gradient program. For the best ion response, MS parameters such as spray voltage, capillary temperature, vaporizer temperature, sheath gas, auxiliary gas and S-lens RF level were fine tuned by



manually. To optimize the mass resolution, 70000 full width at half maximum (FWHM) of full MS scan and 17500 FWHM of targeted-MS2 scan were used to obtain the required mass accuracy. The mass tolerance window of 5 ppm proved to be the most suitable, considering that this parameter played an important role on the selectivity.32 In addition, the NCEs for each analyte were also optimized corresponding to the signal intensity. Table 1 demonstrated the high level of confidence between the theoretical and experimental masses for each analyte with the mass measurement accuracy less than 3.5 ppm, indicating that the Q-Exactive HRMS could provide more reliable characterization of the compounds. Notably, the same theoretical mass for OH-Cot and CNO did not pose any problem for identification because of their distinct retention times and fragment ion masses. The main daughter ion of each analyte was chosen as selected reaction monitoring (SRM) transition for quantitative analysis.

Figure 1. Selected ion monitoring chromatogram of the standard mixture at 50 ng mL-1.

Identification of Nic metabolites in CNS. According to the presence of pyridyl ring in all the Nic derivatives, nicotine-pyridyl-d4 (Nic-d4) was selected as a metabolic tracer in the present study. It was coupled with free Nic for the peripheral administration during the microdialysates collection. Analysis of the brain samples as extracted ion chromatogram provided 8 pairs of compounds with the same retention time, such as Nic and Nic-d4, Cot and Cot-d4, NorNic and NorNic-d4, NorCot and NorCot-d4, OH-Cot and OH-Cot-d4, NNO and NNO-d4, OxPyBut and OxPyBut-d4, HyPyBut and HyPyBut-d4, respectively (Figure S-2). Also, these Nic metabolites could be further identified by the SRM transitions and MS2 fragment ions (Figure S-3). Although the other d4-labled products were undetectable, the biotransformation products of CNO, Nic-Gluc1 (m/z 339.1544), Cot-Gluc and OH-Cot-Gluc were proved to exist in CNS according to the consistence with the standard MS spectrogram and MS2 fragment ions (Figure S-4). Another minor glucuronide metabolite nicotine-N-(4-deoxy-4,5-didehydro)-β-D-glucuronide (Nic-Gluc2, m/z 321.1445) probably did not appear in rat body (both in brain and blood samples), which was consistent with the previous study using human plasma after removal of a


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7-mg transdermal Nic patch.17 Above all, the results indicated that Nic and its 11metabolites could present in rat brain after peripheral Nic exposure. Table 1. Accurate mass and SRM parameters of the analytes. Analyte

Parent ion masses [M+H]+ Theoretical

Experimental

Mass error (ppm)

NCE (%)

Main daughter ion

Nic

163.1230

163.1227

−1.8

55

(m/z) a 130.0651; 132.0806

Nic-d4

167.1471

167.1478

3.5

55

134.0902 ; 136.1058

Cot

177.1022

177.1020

−1.1

60

80.0500; 98.0604

NorNic

149.1073

149.1072

−0.7

50

132.0807; 130.0651

NorNic-d4

153.1325

153.1323

−1.3

50

134.0902; 136.1058

NorCot

163.0866

163.0864

−1.2

65

80.0500

NorCot-d4

167.1117

167.1116

−0.6

65

84.0751

NNO

179.1179

179.1178

−0.6

50

132.0808; 130.0652

CNO

193.0972

193.0973

0.5

55

96.0448; 98.0604

OxPyBut

180.0655

180.0654

−0.6

45

134.0600; 162.0550

OxPyBut-d4

184.0906

184.0905

−0.5

45

138.0851; 166.0800

HyPyBut

182.0812

182.0810

−1.1

50

164.0703; 109.0524

OH-Cot Nic-Gluc1

193.0972 339.1551

193.0970 339.1544

−1.0 −2.1

65 20

80.0500; 134.0599 163.1229

Nic-Gluc2

321.1441

321.1445

1.2

20

163.1227

Cot-Gluc

353.1340

353.1348

2.3

15

177.1019

OH-Cot-Gluc

369.1292

369.1302

2.7

15

193.0970

a Quantification

ion transitions are in bold.

Method validation. Although the internal standard method is generally preferred approach, the procedure is complicated by the addition of internal standards. In order to realize the direct loading test for microdialysis samples, the external standard method was selected to carry out the quantitative analysis. Good linearity with all the correlation coefficients (R2) > 0.9954 was acquired, as calculated with the peak area of selected SRM transitions (Y) to the concentration of analyte (X, ng mL-1). Generally, the LODs were below 0.081 ng mL-1 for all the analytes and the LOQs were from 0.05 to 0.266 ng mL-1. The intra-day precision ranged from 0.4 % to 9.2 % and the accuracy was 1.2 % to 8.8 %, respectively. As listed in Table S-1, the present method showed good linear correlation, high sensitivity, and was suitable for the trace analysis of the microdialysis samples. In vivo recovery of retrodialysis. The relative recovery of an analyte from the microdialysis probe (Rdial) is crucial for obtaining the apparent extracellular concentration (Ca).33,34 In the case of Nic microdialysis, the measurement of in vivo recovery can be achieved by retrodialysis method before the experiment while the Ca of Nic and its metabolites are known to be zero.35 The perfusate (Cperf) solution containing Nic metabolites at three different QC levels (1, 10, 100 ng mL-1) and the dialysate (Cdial) concentrations of the analytes were determined at different time.



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Since the retrodialysis of stable labeled isotope has been developed a better standard to infuse and give the equal Rdial,36 some of the analytes in the experiments were used as their stable-isotope labeled form including Nic-d4, NorNic-d4, NorCot-d4 and OxPyBut-d4. The results from Figure S-5 showed that, although stable-isotope labeled retrodialysis could monitor dynamic changes in Rdial, no significant variations of the Rdial of Nic-d4 in rat brain and blood were found under the transient conditions. Therefore, the in vivo Rdial of Nic metabolites were determined at three different time (0.5 h, 2 h, and 4 h after the probe balancing, respectively) using the three QC samples. The average recoveries of Nic metabolites through the microdialysis probes were between 11.3 % and 18.1 % in the brain, while ranged from 26.1 % to 32.8 % in the blood (Table 2). Table 2. In vivo microdialysates recoveries from rat blood and brain (n=9). Rdial in blooda

Rdial in braina

Nic-d4

30.6 ± 2.8

15.5 ± 2.9

Cot

32.8 ± 3.1

18.1 ± 2.7

NorNic-d4

29.4 ± 2.9

11.9 ± 1.9

NorCot-d4

31.7 ± 2.7

14.2 ± 3.5

NNO

29.6 ± 3.2

11.3 ± 2.8

CNO

30.2 ± 2.2

11.8 ± 2.5

OH-Cot

29.8 ± 2.6

17.1 ± 2.7

OxPyBut-d4

28.1 ± 3.6

16.2 ± 3.2

HyPyBut

27.6 ± 2.8

17.6 ± 2.7

Nic-Gluc

26.1 ± 3.2

16.3 ± 2.1

Cot-Gluc

27.2 ± 2.1

14.4 ± 2.5

OH-Cot-Gluc

26.9 ± 2.4

13.0 ± 2.6

Analytes

a Results

shown represent mean±S.D..

Pharmacokinetic studies of Nic metabolites in rat brain and blood. We previously studied the pharmacokinetic profiles of Nic and its 9 metabolites in rat blood using microdialysis coupled with HPLC-ESI/MRM.31 Here, results proved that subsequent to the Nic exposure, Nic and its 11 metabolites could be detected not only in blood dialysates, but also in brain samples under the experimental conditions. The determined microdialysate concentrations (Cm) were converted to the Ca according to the equation: Ca=Cm/Rdial. The metabolic time-course curves for Nic and its metabolites were made by the mean Ca levels in the brain and blood after the injection of Nic (Figure 2). The pharmacokinetic profiles were obtained in Table 3. Unlike intravenous injection, where the plasma Nic decreases directly from its peak, the concentration of Nic increases rapidly and then decreases gradually after intraperitoneal injection. Actually, all the Nic metabolites suggested an increase phase followed by an elimination phase. To some extent, the observed behavioral changes


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were closely related to the metabolic process of Nic. Rats were found to be hyperactive for ambulation and distance traveled shortly after Nic treatment, then began to tremble in short time followed with sedation, polypnea and quick heart rate. With the clearance of Nic and the generation of other metabolites, the rats breathed normally and resumed their activities gradually. Both brain and blood levels of Nic peaked above 1000 ng mL-1 within 45 min and declined rapidly. By 360 min, Nic was almost completely cleared from rat body. Moreover, Nic was relatively predominately diffused into brain tissue with the higher Cmax and bigger AUC. After 75 min of post-injection, the close concentrations of Nic between brain and blood indicated that Nic was equilibrated between the peripheral compartment and CNS in rat body. The mean elimination half-life of Nic in rat blood was 69.5 min, which was close to Kyerematen’s study and our previous study.31,37 Meanwhile, the rate of Nic clearCance in rat brain was relatively slower with different t1/2, MRT and CL/F comparing to those in blood. These results were different from Chang’s study with a single peripheral dose of 2.0 mg kg-1 nicotine (i. v.),25 the differences may be due to the inconsistency of measure time and test method. As the primary metabolite of Nic, Cot was proved to be the major product in rat body over the time course studied with their AUC showing that the Nic-Cot equilibrium favored Cot. Unlike the previous findings that Cot remained at a consistent level in rat blood and did not appear in brain dialysates,25 Cot was found to be able to pass the blood-brain barrier (BBB) and was eliminated from the central nervous system relatively slowly. Cot is regarded as the best biomarker in smokers because it is present at a much higher concentration than Nic and persists longer in the body with a plasma half-life of about 16-20 h.38 In the present study, Cot had a brain half-life of about 285 min and a plasma half-life of about 240 min in rat body, which is close to 333 min in rat brain in Ghosheh’s study and 291 to 318 min in rat blood in previous studies.21,31,37 Nic (blood) Nic (brain)

1500 1000 500

1500

Concentration (ng/mL)


2000

Cot (blood) Cot (brain)

1200 900 600 300 0

0 0

60

120

180

240

300

0

360

60

120

180

240

300

360

Time (min)

80

NorNic (blood) NorNic (brain)

60 40 20


Time (min) Concentration (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 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 48 49 50 51 52 53 54 55 56 57 58 59 60


180

NorCot (blood) NorCot (brain)

150 120 90 60 30 0

0 0

60

120

180

Time (min)

240

0 300Paragon 360 Plus Environment ACS

60

120

180

Time (min)

240

300

360


80

OxPyBut (blood) OxPyBut (brain)

60 40 20

300 200 100

60

120

180

240

300

Time (min)

400

360

NNO (blood) NNO (brain)

300 200 100

0



HyPyBut (blood) HyPyBut (brain)

0 0

60

120

180

Time (min)

240

300

80

360

CNO (blood) CNO (brain)

60 40 20 0

0 60

120

180

240

300

Time (min)

800

OH-Cot (blood) OH-Cot (brain)

600

0

360

400 200


0


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400

0

60

120

180

Time (min)

150

240

300

360

OH-Cot-Gluc (blood) OH-Cot-Gluc (brain)

120 90 60 30 0

0 0

60

120

180

240

300

0

360

60

Nic-Gluc (blood) Nic-Gluc (brain)

60 40 20 0


80

120

180

240

300

360

Time (min)

Time (min) Concentration (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 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 48 49 50 51 52 53 54 55 56 57 58 59 60



40

Cot-Gluc (blood) Cot-Gluc (brain)

30 20 10 0

0

60

120

180

Time (min)

240

300

360

0

60

120

180

Time (min)

240

300

Figure 2. Time-course curves of Nic and its metabolites in rat brain (triangles) and blood (circles) after administration of Nic (2.0 mg kg-1, i. p.); each point is a mean±S. D. from four rats.

The second most abundant metabolite in rat body was OH-Cot, and the time-concentration profiles for OH-Cot were comparable with that for Cot. OH-Cot had mean half-lives of 274.5 min and 314 min in rat blood and brain respectively, indicating that it presented relatively longer residence time than Cot in rat body. The concentration of OH-Cot in the brain was lower than in the blood, which might originate from that the expression of the responsible metabolic enzymes in the brain tissue was less abundant than in the liver. The metabolic process from Nic to Cot and


360

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Cot to OH-Cot is believed to be mediated by the same cytochrome P450 enzyme.9 In many smoking behavior studies, although the fractional clearance of Nic to Cot and the plasma ratio of OH-Cot to Cot can both be shown to be indicators of CYP2A6 enzymatic activity, the ratio of OH-Cot to Cot has been widely used as a biomarker of the rate of CYP2A6-mediated nicotine metabolism.39-41 Table 3. Estimated pharmacokinetic parameters for Nic and its metabolites from rat brain and blood treated with single injection of Nic (values from Figure 2 were used). AUC0-360 min (μg min mL-1)

CL/F (mL min-1)

t1/2 (min)

MRT (min)

tmax (min)

Brain

Blood

Brain

Blood

Brain

Blood

Brain

Blood

Brain

Blood

Nic

124.6

97

15.0

20.0

97.2

69.5

132.1

116.3

45

Cot

290.8

263

4.0

5.0

285.3

240.3

460.2

385.7

NorNic

12.9

8.3

115.0

188.0

138.4

190.5

288.0

256.9

NorCot

29

34.5

30.0

25.0

317.5

390.7

545.1

NNO CNO

44.6 11.7

74.5 10.2

32.0 78.0

15.0 80.0

183.5 304.9

261.1 437.9

286.1 505.7

OxPyBut

13.4

13.4

101.1

112.0

187.7

164.2

HyPyBut

19.2

16.7

82.0

109.0

168.8

OH-Cot

82. 4

133.9

12.0

7.0

314.0

OH-Got-Gluc

19.3

18.2

57.0

51.0

Nic-Gluc

8.1

11. 5

107.0

Cot-Gluc

4.2

6.5

183.0

Cmax (ng mL-1) Brain

Blood

45

1639.6

1134.8

105

120

1206.7

1116.4

90

45

50.8

46.3

620.7

240

150

104.2

147.7

433.6 665.0

105 90

120 105

259.5 40.5

282.8 45.3

322.6

278.7

60

75

51.3

60.6

124.6

217.6

139.5

30

30

334.0

191.8

274.5

480.0

481.4

150

150

318.7

561.0

314.6

335.8

468.0

546.5

105

180

98.8

79.0

72.0

388.2

398.0

603.2

627.8

90

75

37.6

45.6

119.0

424.5

434.7

673.2

676.3

90

90

18.0

26.5

Another pair of important metabolites is NorNic and NorCot, which are indicative of the extent of N-demethylation of Nic and its major proximate metabolite, Cot. The low concentrations of NorNic in the blood indicated that peripheral biotransformation of Nic to NorNic was a minor pathway. However, the relatively high levels of NorNic found in the brain dialysates suggested that NorNic could be formed via oxidative N-demethylation of Nic locally.30 NorNic was found to have a half-life of 138.4 min in the brain and a half-life of 190.5 min in the blood (present study), which were comparable to its reported half-life of 166 min in the brain and 198 min in the plasma.21,42 The pharmacologically active NorNic was shown to evoke a concentration-dependent increase and had a similar potency with Nic in dopamine release studies.22,43,44 Compared with Nic, NorNic is also a major alkaloidal component of tobacco and has a significantly longer half-life. Thus, it is likely that alkaloidal and metabolically formed NorNic will accumulate in the brain and play a contributory role in the neuropharmacological effects of Nic as a consequence of tobacco use.21 Compared with NorNic, NorCot had relatively higher levels and longer residence times in rat body. NorCot has been reported as a central nervous system Nic metabolite and its pharmacological activity is unknown. Notably, the different pharmacokinetic profiles of NorCot in rat brain and blood may originate from the different biotransformation process, because the results in Crooks’ study indicated



that NorCot detected in brain were most likely generated from 5’-C-oxidation of brain NorNic, rather than from N-demethylation of Cot as occurs peripherally.30 N-Oxidation is also a primary route of Nic metabolism, which is generally regarded as non-toxic pathway. In human, about 4-7 % of the Nic absorbed by smokers is being transformed via this direction.9 [14C]nicotine-N-oxide was detected by high pressure liquid radiochromatography in mouse brain following i. p. administration of [14C-N-methyl]nicotine,45 suggesting the presence of NNO in the brain after peripheral Nic administration. Here, we found that NNO and CNO were present in rat brain and blood simultaneously. The abundant amount of NNO in rat blood represented the active oxidation of the pyrrolidine ring for Nic occured peripherally, and the large AUC also indicated that NNO was one of the major metabolites of Nic. However, CNO was observed modestly with the long residence time in rat body. The detected half-life of CNO was 437.9 min in plasma and 505.7 min in the brain, respectively, which is comparable with 7.9 to 8.2 h in rat in Kyerematen’s study.37 OxPyBut and HyPyBut have been shown to arise directly from 2’-hydroxylation of Nic transformation and these metabolites were excreted in smoker’s urine accounting for about 10-15 % of total Nic.46 The exact ion chromatograms for OxPyBut (m/z 180.0654) and HyPyBut (m/z 182.0810) demonstrated their presence in rat brain and blood. Especially, this is the first report of these two compounds as Nic metabolites in rat body. Unlike OxPyBut as a minor metabolite, HyPyBut reached peak concentrations of 334.0 ng mL-1 in the brain and 191.8 ng mL-1 in the blood and declined rapidly. The HyPyBut t1/2 was 124.6 min in blood and 168.8 min in brain, and its elimination rate was comparable with that for Nic. Another pathway of Nic biotransformation is glucuronidation. In smokers, Nic, Cot and OH-Cot undergo conjugation reaction and their glucuronide conjugates are excreted in the urine.47 The high correlation in the extent of N-glucuronide formation between Nic and Cot within individuals indicated that the same enzyme was involved in N-glucuronidation of both agents.38 However, whether a different enzyme involved in conjugation of OH-Cot than in conjugation of Nic and Cot is unclear. Some studies showed that low-level or unmeasurable Nic glucuronidation activity was detected in animal microsomes and Cot glucuronidation was below limit of quantification for all the animal species.9 Current data suggest that Nic-Gluc and Cot-Gluc could be present in very minor amounts and almost nonquantifiable at 15 min. OH-Cot-Gluc was not observed at 15 min in the brain, but peaked between 105 min and 180 min with a maximal concentration of near 100 ng mL-1, and was still detectable at 6 h postinjection both in the brain and blood. Similar to the products of other metabolic pathways, these three metabolites had longer residence times and half-lives than those of Nic.


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In all, Nic and its 11 metabolites were simultaneously determined in the rat brain and blood. Among the metabolites studied, Cot was the main metabolite, OH-Cot, NNO and NorCot were the second most abundant products, and the other seven metabolites were present in minor but detectable amounts. Since the metabolites except Cot are unknown to be able to cross the BBB,29 it might be unclear which part of the metabolites measured in rat brain originates from local Nic transformation and which part comes from the uptake of peripheral metabolites into the brain. If these metabolites can pass through the BBB, they should be equilibrated between the peripheral compartment and CNS eventually like Nic, and the difference in the concentration of each substance between the blood and brain would vary slightly. However, the distinct metabolic profiles and pharmacokinetic data between rat brain and blood indicated that some Nic metabolites in the brain, such as Cot, NorNic, NorCot, NNO, HyPyBut, OH-Cot and OH-Cot-Gluc, might be generated partly from Nic in brain locally. On the other hand, given that the P450 enzymes responsible for Nic metabolism vary in species, there may be considerable variation between rodent species and human in the activity and stereospecificity of Nic metabolism, as well as in the relative amounts of Nic metabolites produced. This should be taken into account when consider the implication of the current work and its relevance to human smoking.

CONCLUSION In summary, we have developed an effective strategy on simultaneous microdialysis, stable-isotope labeling and UHPLC-HRMS for the identification and dynamic determination of Nic metabolites in vivo. This approach not only provides a sensitive, selective and rapid method for the simultaneous determination of Nic and its biotransformation products in complex biological fluids, but also continuously monitored the dynamic changes of unbound Nic during the metabolic process in freely-moving rats. As far as we know, this is the first study to detect Nic and its 11 metabolites in rat blood and brain tissue, and no previous studies have reported the analysis of central generations from all of the known pathways of Nic metabolism, including the important 2’-hydroxylation and glucuronidation. Our present work indicated that Nic and its 11 metabolites could be well quantified and pharmacokinetically profiled, and the metabolic characteristic of Nic in the brain was relatively different from that in the blood. The ability to measure these metabolites dynamically will be useful to study the mechanism of Nic metabolism and any differences between the brain and the peripheral system.

ACKNOWLEDGEMENTS



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We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant No. 21307163, 21175153), Scientific and Technological Project of Henan Province (Grant No. 22170009) and the assistance of Mr. Qidong Zhang in producing the pictures.

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TOC only

Dialytes collection

UHPLC-HRMS determination Nic/Nic-d4 administration

Nic metabolites


Nicotine Pharmacokinetics in Rat Brain and Blood by Simultaneous

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