MS Method for Quantification of Liquiritigenin in Rat Plasma: Application to Pharmacokinetic Study of Liquiritin

Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60 

53

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Chinese Herbal Medicines (CHM)  ISSN 1674-6384

 

Journal homepage: www.tiprpress.com

E-mail: [email protected]  

Original article 

LC–MS/MS Method for Quantification of Liquiritigenin in Rat Plasma: Application to Pharmacokinetic Study of Liquiritin Shi-qi Dong1, Hui-rong Fan2, Quan-sheng Li2, Guang-li Wei2, Ya-zhuo Li2, Chang-xiao Liu1,2, Duan-yun Si2* 1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China 2. State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, China

ARTICLE INFO  Article history Received: May 4, 2015 Revised: June 20, 2015 Accepted: August 30, 2015 Available online:

  January 18, 2016 DOI: 10.1016/S1674-6384(16)60008-4 

  ABSTRACT    Objective A simple, sensitive, and rapid LC-MS/MS method has been established and

validated for the determination of liquiritigenin (LG) in rat plasma. Methods Naringenin was chosen as internal standard (IS). LG and IS were separated on a Diamonsil C18 analytical column with a mobile phase of methanol-10% methanol in water containing

0.5 mmol/L ammonium formate and 0.2% formic acid (55:45) at the isocratic flow rate of

0.6 mL/min for 10 min. The multiple reaction monitoring (MRM) was performed on a mass spectrometer in the negative ion mode with electro-spray ionization (ESI) source and the transition from precursor ion to product ion was m/z 255.0→119.0 for LG and

m/z 271.0→151.0 for IS, respectively. Results The linearity was acceptable in the range of 5-5000 ng/mL (r = 0.9973). The inter-day and intra-day accuracies were in the

ranges of −0.09%−3.25% and −5.02%−9.21%, respectively. The precision was in the

ranges of 3.60%−12.4% and 0.909%−6.89%, respectively. LG was stable in the course of

analysis and storage. Conclusion

The LC-MS/MS method was successfully applied to

the pharmacokinetic study for the first time in rats after ig and iv administration of liquiritin (LQ), a glycoside of LG, at pharmacologically effective levels.

Key words

LC-MS/MS; liquiritigenin; liquiritin; pharmacokinetics

© 2016 published by TIPR Press. All rights reserved.

1.    Introduction  Licorice roots or Glycyrrhizae Radix (RG) is one of the most commonly used Chinese materia medica (CMM), and has been widely used in the treatment of spleen invigorating, Qi replenishing, heat clearing, and toxic substance removing.

In modern medicine, licorice extract exerts its pepticulcer (Asl and Hosseinzadeh, 2008), neuroprotective effects (Fu and Li, 2012), antiviral activity (Wang et al, 2006), anti-inflammatory (Zhang and Shen, 2011), antitumor, and antibiosis effects (Wang et al, 2009). Licorice roots contain flavonoids and terpenoids (Hatano

                      *

Corresponding author: Si DY E-mail: [email protected]

Funds: “Twelfth Five-year Plan”-Major Technological Projects of “Creation of Major New Drug” (2012ZX09506-001); National 973 Program of China (2010CB933900); Tianjin Science and Technology Plan Project (10SYSYJC28600)

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Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60 

et al, 1989). Liquiritin (LQ) is the main flavonoid glycoside isolated from licorice roots and often used to treat injuries or swelling, due to its life-enhancing and detoxifying properties (Chen et al, 2009). In the study, LQ was found to have anti-myocardial ischemia activity which has not been explored so far, and now is being developed as a drug candidate to treat myocardial ischemia. In order to define the pharmacokinetic profile of LQ, an LC-MS/MS method for the determination of LQ in rat plasma needs to be established. However, preliminary study following ig administration of LQ to rats showed that the content of LQ in rat plasma was extremely low, while the content of liquiritigenin (LG, aglycone of LQ) after hydrolysis by

β-glycuronidase was far more than that of LQ, suggesting LQ may undergo hydrolysis in intestinal tract and phase II metabolism. According to literature report, flavonoids have been proved to be metabolized in liver and intestinal, and mainly hydrolyzed in intestinal into aglycone, and then conjugated with UDPGA to produce glucuronoconjugates, sulfoconjugates, and methylconjugates (Graf et al, 2006; Van Derwode et al, 2004; Gradolatto et al, 2004). Additionally, LG was one of the main metabolites of LQ and was known to be actually absorbed into the body (Kitagawa et al, 1998; Zuo et al, 2002). Therefore, the contents of LG hydrolyzed by β-glycuronidase may reflect dynamic exposure of LQ in vivo. The chemical structures of LQ and LG were shown in Figure 1.

A

B Figure 1

Chemical structures of LQ (A) and LG (B)

LC–MS/MS method for determination of LG concentration in rat plasma in the range of 5–5000 ng/mL has been described. The method we reported is accurate and sensitive, on the basis of liquid extraction. This method was then successfully applied to characterizing the pharmacokinetics of LQ in rat following ig and iv administration.

2.    Materials and methods   

2.1    Chemicals and reagents 

after passing a mass calibration and sensitivity test using polypropylene glycol (PPG) standard (Applied Biosystems Company, USA) according to the user’s manual. The instrument was interfaced to a computer running Applied Biosystems Analyst version 1.5.2 software for the data acquisition and processing.

Both LQ (purity of 98.23%) and LG (purity of 100%) were kindly provided by Jilin Institute of Traditional Chinese Medicine (China). Naringenin used as internal standard (IS) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Methanol and ethyl acetate of HPLC-grade were purchased from Concord Co., Inc (Tianjin, China) and Formic acid for HPLC was from Tianjin Guangfu Fine Chemical Research Institute (China). Water was prepared in-house with the BM−40 Water Purification System from Zhongsheng Maoyuan Tech. Co., Ltd. (Beijing, China). All other chemicals were of analytical grade. Drug-free heparinized rat plasma was freshly collected from Wistar rats in our laboratory and stored at −20 oC before use.

Chromatographic separation was performed on a Diamonsil C18 column (Dikma Technologies, 150 mm × 4.6 mm, 5 µm) at an isocratic flow rate of 0.6 mL/min for 10 min with column temperature at 40 oC. The mobile phase was composed of methanol and 10% methanol in water containing 0.5 mmol/L ammonium formate and 0.2% formic acid (55:45). Autosampler temperature was maintained at 4 oC and the injection volume was set at 3 μL.

2.2    Instrumentation 

2.4    MS/MS conditions 

The analysis was performed on an LC-MS/MS system consisted of a binary LC−20 AD delivery pump, a DGU-20A3 vacuum degasser, a CTO−20A column oven, a SIL−20A auto-sampler, a CBM−20A system controller (Shimadzu, Japan) and an API 4000 QTRAP Mass Spectrometer (Applied Biosystems, USA). The LC system coupled with the mass spectrometer (mass range from 50 Da to 3000 Da) through an electro-spray ionization (ESI) source. The mass spectrometer was operated in negative ion mode

Analytes were detected by tandem mass spectrometry using multiple reaction monitoring (MRM) with a dwell time of 200 ms. The deprotonated ions [M − H]− for LG and IS were selected in the first quadrupole, and the collision energy was −40 eV for LG and −25 eV for IS which were adjusted to achieve maximum sensitivity for each ion transition. The transitions from precursor ion to product ion were m/z 255.0→119.0 for LG and m/z 271.0→151.0 for IS, respectively. MS parameters were optimized by syringe pump infusing of

2.3    Chromatographic conditions 

Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60  standard solution containing LG and IS. Optimized instrument settings were as follows: collision gas, 6.895 × 104 Pa; curtain gas, 6.895 × 104 Pa; sheath gas, 3.448 × 105 Pa; auxillary gas, 5.516 × 105 Pa; ion spray voltage, −3000 V; capillary temperature, 500 oC. The compound parameters, including entrance potential, declustering potential, and collision cell exit potential were −12 V, −60 V, and −10 V for LG and −12 V, −75 V, and −10 V for IS, respectively.

2.5    Preparation  of  calibration  curve  and  quality  control samples  Two stock solutions of LG were prepared from independent preparations. Working standard solutions (0.25, 0.5, 2.5, 10, 25, 125, and 250 µg/mL) were prepared by serial dilution of the stock solution with methanol. Low, medium and high concentration of QC solutions (0.5, 10, and 200 µg/mL) were prepared in the similar way. The stock solution of IS (0.1 mg/mL) was also prepared in methanol and then diluted to a final concentration of 2 µg/mL. All solutions were stored at 4 ◦C and used within one month after preparation. Calibration standards and QC samples were prepared by spiking 20 µL of either standard or QC solutions to 1 mL of blank plasma in rat. The final concentration of LG in calibration curves were 5, 10, 50, 200, 500, 2500, and 5000 ng/mL, and those in QC samples were 10, 200, and 4000 ng/mL. All of the spiked plasma samples were then treated according to sample preparation procedure. Both the calibration standard samples and QC samples were applied in the method validation and the pharmacokinetic study. All calibration and QC samples were prepared daily to avoid potential degradation or adsorption issues.

2.6    Plasma sample pretreatment  After adding 10 µL of acetic acid (0.56 mol/L), an aliquot (50 µL) of rat plasma was transferred to the glass tube and then 25 µL of β-glucuronidase (4000 units) was added. The mixture was vortexed for 30 s and then placed into water bath at 37 oC for 1 h. After hydrolysis, 50 µL of IS working solution (2 µg/mL) was then added into each mixture and mixed gently, and then 2.5 mL of ethyl acetate was added and the resulting solution was vortexed for 2 min, followed by centrifugation for 10 min at 4 oC at 3000 g. The organic layer (2 mL) was then transferred into another glass tube and evaporated to dryness with nitrogen at 40 oC. The residue was reconstituted in 100 µL of methanol-water (50:50) solution and centrifuged for 10 min at 4 oC at 1200 g, 3 µL was injected for LC-MS/MS analysis.

  2.7    Method validation  Guidance for Industry-Bioanalytical Method Validation implemented by FDA was used as guidance for validation described as follows. The specificity was defined as non-interference at retention time of LG and IS from the endogenous plasma

55

components and no cross-interference between LG and IS using the proposed extraction procedure and LC-MS/MS conditions. Blank plasma samples collected from six different rats were analyzed to investigate the potential interferences at the LC peak region for analytes and IS. Carryover was assessed by a blank sample analyzed following a ULOQ sample. Only if the peak area of LG in the blank sample was less than 20% of that in the LLOQ sample, carryover can be accepted. Linear calibration curves in rat plasma were generated by plotting the peak area ratio of LG to the IS versus the seven known plasma LG concentration over the range of 5–5000 ng/mL. Each calibration curve consisted of a blank sample, a zero sample and seven calibrator concentration. The calibration curves were fitted using a least square linear regression model y = ax + b, weighted by 1/x2 using the Analyst software. The deviations of these back-calculated concentration from calibration standard samples should be within ± 15 % of the theoretical value. The lower limit of quantification (LLOQ) in this experiment was determined by finding the lowest concentration with a signal to noise > 10 over six independent runs with accuracy and precision within 20% for every run. The intra- and inter-day precision and accuracy were evaluated by parallel analytical runs performed on three consecutive days. Six replicates of LG at four QC levels (5, 10, 200, and 4000 ng/mL) were analyzed. Each analytical run consisted of a matrix blank, a set of calibration standards, six replicate LLOQ samples, and a set of low, medium, and high concentration QC samples. Intra-day precision and accuracy were calculated using replicate (n = 6) determinations for each concentration of the spiked plasma sample during a single analytical run. Inter-day precision and accuracy were calculated using replicate (n = 18) determinations of each concentration made on three separate days. The precision was determined as the RSD (%) and the accuracy was expressed as a percentage of the nominal concentration (relative error). The criteria for acceptability should be within ± 15%, except for LLOQ, where it should not exceed ± 20% of accuracy as well as precision. The recovery and matrix effects of the method were determined by comparing the peak areas of LG at three concentration (10, 200, and 4000 ng/mL) and those of IS at 2 µg/mL with six replicates, including standards (a), standards spiked before extraction (b), and standards spiked after extraction (c). The recoveries of LG and IS were evaluated by comparing the peak area b with c calculated as equation (1). The matrix effects of LG and IS were assessed by comparing the peak area a with c calculated as equation (2). The experiments were performed at the three QC levels in six replicates. Recovery =

Area b Area c

Matrix effect =

Area c Area a

(1)

(2)

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Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60 

The stability of LG was assessed at the concentration of 10 and 4000 ng/mL with six replicates for each concentration level, which involved sample storage, preparation, and analysis, including stability of three cycles of freeze/thaw (−80 oC/20 oC), stability of analyte in the auto-sampler at 4oC for 24 h, stability of plasma samples at room temperature for 6 h, long-term stability of plasma samples at −80 oC for 1 and 3 months. The concentration obtained from all stability studies were compared with the spiked concentration, and the percentage concentration deviation was calculated. The analytes were considered stable in rat plasma when the concentration difference was less than 15 %. Dilution integrity was carried out on higher concentration (above ULOQ), which may be encountered during real rat samples analysis. Dilution integrity experiment was performed at 40 μg/mL (10-fold of high quality control). Six replicate samples were diluted to 1/10 (4000 ng/mL) by spiking blank plasma of rats and their concentration were calculated by applying the dilution factor of 10 against calibration curves.

2.8    Animal experiment  Wistar rats [6-week age and weighing (220 ± 20) g] were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were housed in an environmentally controlled room (temperature: (25 ± 2) oC, humidity: (50 ± 20)% with a 12h light/dark cycles under filtered, pathogen-free air with food and water available ad libitum for one week before use. The protocols for animal studies were carried out by the Guidelines of the Animal Care and Use committee of Tianjin Institute of Pharmaceutical Research, which was approved by Tianjin Municipal Science and Technology Commission (Tianjin, China). All animals were fasted overnight with water allowed ad libitum. Twelve rats were divided into two groups randomly (bisexual each half), one group was given a single dose of LQ at 30 mg/kg by oral using an animal feeding needle after overnight fasting (~12 h); The other group was iv given LQ at a dosage of 30 mg/kg. Animals were fasted for the first 3 h and had free access to water during the process of experiment. Blood (300 µL) was collected from the orbital venous plexus at 0.167, 0.5, 1, 2, 3, 4, 6, 9, 12, 17, 24, 30, and 48 h after ig administration. Similarly, rat blood (300 µL) was collected at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 9, 12, 17, 24, 30, and 48 h after iv administration. All samples were centrifuged at 4000/g for 5 min immediately and separated plasma samples were stored at -80 oC until analysis.

3.    Results and discussion   

3.1    Method optimization  Optimization of MS was involved in tuning of MS parameters in positive and negative ionization modes using a 1000 ng/mL tuning solution, and both LG and IS could be

ionized under either positive ESI (ESI+) or negative ESI (ESI−) modes, but signal intensity was much higher at ESI− than ESI+, therefore, the ESI− mode was used for quantification. Mass spectrometric parameters were optimized so as to achieve maximum abundance of product ions. LG and IS predominantly produced singly charged precursor [M − H]− ions at m/z of 255.0 and 271.0, respectively in Q1 MS full scan spectra. Fragmentation was initiated using sufficient nitrogen for collision induced dissociation and the most abundant ions found in the product ion mass spectra were m/z 119.0 and 151.0 at −40 eV and −25 eV collision energy for LG and IS, respectively. The chemical structures and product ion mass spectra of [M − H]− ions of LG and IS were shown in Figure 2. An IS is necessary for the determination of analytes in biological samples and several possible compounds were explored, including wogonin, hesperetin, and naringenin. Wogonin was initially abandoned for its unacceptable retention time, and hesperetin was also not appropriate due to its low response. Finally, naringenin, with a similar structure to LG, was selected in terms of chemical structure as well as good chromatographic behavior and higher response. Chromatographic optimization includes column type and mobile phase composition. Different types of analytical columns were tested and the analytes were well retained on the C18 column at 40 oC. Feasibility of various mixture (s) of solvents such as acetonitrile and methanol using different buffers, for instance ammonium acetate, ammonium formate, and formic acid, along with altered flow rates (in the range of 0.4−1.0 mL/min) were tested to get symmetric peak shape, selectivity and sensitivity for LG and IS, and also to reduce matrix effect. Methanol as an organic solvent showed better sensitivity compared to acetonitrile. The addition of formic acid and ammonium formate remarkably enhanced the sensitivity and improved the peak shape. The optimized mobile phase was composed of methanol and water (55:45) containing 0.5 mmol/L ammonium formate and 10% methanol supplemented with 0.2% formic acid. After optimization, the chromatographic retention times of LG and IS were at 5.8 and 8.2 min, respectively.

  3.2    Sample preparation  Sample pretreatment plays a key role in determination of drugs in biological matrix, and a liquid-liquid extraction was used in this analysis. Several organic solvents including ethyl acetate and methyl-t-butyl ether were previously used to extract LG in plasma samples; Ethyl acetate was found to be optimal, which can produce a relative clean chromatogram for blank plasma samples, the best recovery, and the least matrix effect. Rat plasma after ig administration at a dose of 30 mg/kg was collected and applied to optimizing the parameters of enzymatic hydrolysis, including the choice of the amount of enzymes, incubation periods, and pH values of incubations. The results indicated that the concentration of LG increased during the first 45 min and then remained constant

Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60  MS2 (255.00) CE (−30):1.852 to 1.190 min from Sample 32 119.0

A

Max. 9. 5e5 cps.

m/z 135

100

57

−H+

m/z 91

m/z 255

Rel. int /%

80 60

m/z 119

135.3 40 91.3

20

255.0

0 60 80 100 120 140 160 180 200 220 240 260 280 300 MS2 (271.00) CE (−30):0.469 min from Sample 4 151.0

B

Max. 2.8e7 cps. m/z 151

100

m/z 107 80

−H+ m/z 255

119.0

60 Rel. int /%

107.0 m/z 109

40

271.0 20 0

  60

80 100 120 140 160 180 200 220 240 260 280 300 m/z

Figure 2

Da

Chemical structures and product ion mass spectra of [M − H]− for LG (A) and IS (B)

for 2 h, went up sharply for 1000 enzymatic units, and then kept constant over 5000 units. When the concentration of acetic acid was 0.56 mol/L, the concentration of LG was the highest. Therefore, the hydrolytic conditions for sample treatment were as follows: the incubation time over 1 h, enzymatic units 4000 units, and the concentration of acetic acid 0.56 mol/L.

3.3    Method validation  Representative chromatograms of LG and IS were shown in Figure 3, including blank plasma sample (Figure 3A), blank plasma spiked with LG at 5 ng/mL (Figure 3B), and rat plasma sample obtained at 2 h after ig administration (Figure 3C). No interfering peaks were observed at retention time of LG and IS. A blank sample was analyzed, followed by ULOQ to evaluate the carryover. The result showed that the carryover of the method was acceptable because the peak area of LG obtained from the blank plasma sample was far less than 20% of that obtained from the LLOQ sample. The plasma calibration curve was constructed by concentration of the seven calibrators over the range of 5−5000 ng/mL. A typical standard curve for LG was y = 0.234x + 0.00106 (r = 0.9987) with a weighting factor of 1/x2.

Ratio of LG to IS was calculated, and x represented the corresponding plasma concentration of LG. The accuracy and precision of LG at three concentration (10, 200, and 4000 ng/mL) were summarized in Table 1. The intra-day accuracy and precision of QC sample for LG were in the range −5.02%−9.21% and 0.909%−6.89%, respectively. The inter-day accuracy and precision were in the range −0.09%−3.25% and 3.60%−12.4%, respectively. The results indicated that the LC/MS/MS method had the good accuracy and excellent precision. The matrix effect can be regarded as the ion suppression or enhancement of the analyte (Peters and Remane, 2012). The extraction procedure and chromatographic condition were modified to decrease or even eliminate the unfavorable influence of the matrix effect (Earla et al, 2012). In this study, the matrix values were (105 ± 2.80)%, (101 ± 3.19)% and (99.2 ± 2.56)% for LG and (100 ± 4.36)% for the IS. The results showed that there was no matrix effect for LG and the IS in the method. Under the given set of operating condition, the results indicated that the extraction recoveries of LG and IS were acceptable. The detailed values of recoveries in rat plasma were (102 ± 6.74)%, (97.5 ± 2.17)% and (99.3 ± 1.03)% for LG and (105 ± 2.73)% for IS. The stability of QC samples for LG was studied under

58

Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60  A

XIC of –MRM (3 paris): 255.000/119.000 ID: LG from sample 107 5.03 Intensity /cps

400 200 0

  1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Intensity /cps

XIC of –MRM (3 paris): 271.000/151.000 ID: YPS from sample 107 5.01 1200 1000 500 0

  1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Intensity /cps

XIC of –MRM (3 paris): 255.000/119.000 ID: LQ from sample 19 5.84 1500

B

1000 500 0 1.0

2.0

3.0

4.0

5.0

6.0

7.0

XIC of –MRM (3 paris): 255.000/119.000 ID: YPS from sample 19

9.0

8.16

2.0e5 Intensity /cps

8.0

1.0e5 0.0 1.0 4.9e5

2.0

3.0

4.0

5.0

6.0

7.0

8.0

XIC of –MRM (3 paris): 255.000/119.000 ID: YPS from sample 19 5.85

9.0

 

C

Intensity /cps

4.0e5 2.0e5 0.0

  1.0

2.0

3.0

4.0

5.0

6.0

7.0

XIC of –MRM (3 paris): 271.000/151.000 ID: YPS from sample 20

9.0

8.15

2.0e5

Intensity /cps

8.0

1.0e5 0.0 1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

t / min Figure 3

Representative chromatograms of LG (5.84 min) and IS (8.15 min)

(A) Blank plasma of rats sample; (B) Blank plasma sample spiked with 5 ng/mL of LG (LOQ) and 2 µg/mL of IS; (C) A plasma sample collected at 2 h after an oral dose of 30 mg/kg LQ.

Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60  Table 1

59

Accuracy and precision of LC-MS/MS analysis for determining LG in plasma of rats Accuracy / %

Spiked / (ng·mL−1)

Precision / %

Intra-day Day 1

Day 2

Intra-day

Inter-day

Day 3

Day 1

Day 2

Inter-day

Day 3

10

−5.02

0.0667

2.250

−0.09

5.730

6.89

6.13

9.22

200

1.42

4.4200

3.250

3.00

4.760

2.78

1.43

3.60

4000

9.21

−0.2920

0.667

3.25

0.909

1.93

1.73

12.40

different possible conditions, which included stability of three cycles of freeze/thaw (−80 oC /22 oC), stability of analyte in the auto-sampler at 4 oC for 24 h, stability of plasma samples at room temperature for 6 h, long-term stability of plasma samples at −80 oC for 1 and 4 months and stability of LG in the stock solutions at 4 oC for 1 month. The results, which were listed in Table 2, demonstrated that the analyte displayed excellent stability under all experiment conditions. The relative standard Table 2

deviation (RSD) range from 0.704% to 4.92%, and the relative error (RE) was in the range of −0.250%−13.2%. Dilution integrity was carried out at six replicates by 10-fold dilution with blank plasma. The accuracy of diluted QCs was 1.42% (RE); While precision values ranged from 3.06% (RSD) for LG. The results suggested that samples whose concentration were greater than the upper limit of calibration curve could be reanalyzed by10-fold dilution.

Stability of LG in rat plasma under different storage conditions ( x ± s , n = 6 )

Storage conditions Stability for 6 h at room temperature

Concentration levels / (ng·mL−1) Skiped

Measured 4140

4527

4125 ± 36.7 10.10 ± 0.498

10

3562

4000

In this study, pharmacokinetics of LQ in rats after a single iv or ig administration was characterized. Due to its special mechanism of absorption and metabolism which remained unclear to date, determination of its total aglycone LG may represent the LQ profiling in vivo. Therefore, after the LC-MS/MS method was fully validated, the concentration of LG were successfully determined in plasma of rats. The method was sensitive to determine LG in rat plasma up to 48 h. Those samples, which concentration were over the upper calibrator, were diluted ten-fold with blank plasma of rats and reanalyzed. The major pharmacokinetic parameters of LG calculated, including t1/2, Cmax, Tmax, AUC0-t, AUC0-∞, Vd, CL, and MRT were shown in Table 3. The results indicated that the individual variation was significant because of the larger standard deviation (SD) values of most pharmacokinetic parameters. The oral bioavailability calculated by (AUCig./dose)/ (AUCiv./dose) of LG in rats was about 80.6%, and this basic pharmacokinetic evaluation prompts us to believe that it is positive for further development of LQ as a new drug for anti-myocardial ischemia. The mean plasma concentration-

± 70.0

9.98 ± 0.330

10

4.    Pharmacokinetic study 

± 53.2

11.10 ± 0.308

10

4000 Stability for freeze storage for 4 months (−80 oC)

± 96.5

4640

4000 Stability for freeze storage for 1 month (−80 oC)

RE / %

10.30 ± 0.493

10 4000

Stability for three freeze-thaw cycles (−80 oC /20 oC）

Accuracy

RSD / %

10.40 ± 0.445

10 4000

Stability for extracted samples in autosample for 24 h

Precision

± 94.5

4.270

4.22

2.330

3.50

4.800

2.77

0.704

13.20

2.760

11.30

1.550

13.20

3.310

-0.25

0.891

3.13

4.920

1.10

2.650

−11.00

time profiles of LG with a single iv and ig administration to six rats in each group were illustrated in Figure 4. Table

3

Pharmacokinetic

parameter

after

iv

and

ig

administration of LG (30 mg/kg) to rats Pharmacokinetic parameters

iv (30 mg·kg−1)

Units

C5min

ng·mL−1

Cmax

ng·mL−1

ig (30 mg·kg−1)

22 267 ± 2180 −

− 2870 ± 1166

Tmax

h

−

2.08 ± 1.11

AUC0-48h

h·ng·mL−1

30 391 ± 12 800

24 483 ± 12 764

AUC0-∞

h·ng·mL−1

30 750 ± 13 068

24 634 ± 12 765

MRT

h

Vd

mL·kg−1

CL

4.31 ± 2.01

6.53 ± 1.32

8248 ± 4813

11 692 ± 13 390

mL·h−1·kg−1

1159 ± 534

1516 ± 720

T1/2

h

4.98 ± 1.55

4.65 ± 3.47

F

%

80.6

C5min: The peak plasma concentration at 5 min; Cmax: The peak plasma concentration; Tmax: Time to reach Cmax; T1/2: Elimination half-life; AUC0-48h: Area under the curve the concentration-time curve from 0 to 48 h; AUC0-∞: Area under from 0 to infinity; CL: Clearance; Vd: Volume

of

Bioavailability.

distribution;

MRT:

Mean

residence

time;

F:

60

Dong SQ et al. Chinese Herbal Medicines, 2016, 8(1): 53-60  100 000

Concentration / (ng·mL−1)

10 000

ig (30 mg·kg−1) iv (30 mg·kg−1)

1000 100 10 1 0

10

20

30

40

50

60

t/h Figure 4

Plasma concentration logarithm-profile of LG after drug administration (ig 30 mg·kg−1 and iv 30 mg·kg−1) in rats

5.    Conclusion  A simple, sensitive, and rapid LC-MS/MS method for the quantification of LG is developed and fully validated in rat plasma for the first time. The method is accurate, sensitive, and has an advantage of wide linear range. After enzymatic hydrolysis converting the conjugated glycoside to the free forms, the method could be successfully applied to pharmacokinetic study of LQ after ig and iv administration to rats. This is the first report to develop and validate an LC–MS/MS method for ig determination of LG in plasma of rats and the first attempt to evaluate pharmacokinetics of LQ.

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