For thousands of years, traditional Chinese medicine has been widely used in China to treat diseases. With the development of modern medicine, more and more people are trying to combine herbs and western medicines clinically to explore new medication options [
Levofloxacin (LVFX) is used to treat numerous bacterial infections belonging to the third-generation fluoroquinolone antibiotic family [
In vivo distribution studies are crucial in drug research as they can be performed to demonstrate the pharmacokinetic features of a drug [
An ACQUITY I-Class UPLC system with a conditional autosampler and an Acquity I-Class UPLC BEH C18 Column (2.1 mm × 100 mm, internal diameter 1.7
Mass spectrometric detection was performed with an XEVO TQS Triple-Quadrupole Tandem Mass Spectrometer (Waters Corp, Milford, MA, USA) equipped with an electrospray ionization (ESI) source. The mass spectrometer parameters were capillary voltage, 3.0 kV; capillary ionization voltage, 3 kV; ion source temperature, 120°C; spray gas and backflush gas, N2; desolvation gas flow rate, 650 L·h−1; desolvation gas temperature, 350°C. Multiple reaction monitoring (MRM) mode was used for quantification. The optimal parameters for the analyte and internal standard (IS, puerarin) in the MRM mode are listed in Table
Mass spectrum parameters of the analyte and puerarin (IS).
Component | Mass spectrum parameters | ||||
---|---|---|---|---|---|
Parent ( | Daughter ( | Cone (V) | Collision (V) | ESI | |
IS | 417.00 | 267.00 | 40 | 30 | + |
LVFX | 362.20 | 261.10 | 20 | 10 | + |
Sprague–Dawley rats (230 ± 20 g) were supplied by Changsha Tianqin Biotechnology Co. Ltd. (Changsha, China; certificate No. SCXK (Xiang) 2014-0010). All studies were approved by the Animal Ethics Committee at Guizhou Medical University and conducted in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals in China.
The stock solutions of LVFX were separately weighed and dissolved in methanol to obtain the final concentration of 1.002 mg/mL. An appropriate amount of puerarin was dissolved in methanol and diluted to obtain the IS solution (20 ng/mL). The stock solutions of LVFX were successively diluted to the following concentrations to generate the calibration curves: 5.01–15,030.00 ng/mL, 5.01–10,020.00 ng/mL, and 5.01–1002.00 ng/mL. Quality control (QC) samples containing 10.02, 100.20, and 5010.00 ng/mL of LVFX were prepared for the tissue distribution study, 10.02, 200.40, and 4008.00 ng/mL of LVFX for the urinary excretion study, and 10.02, 20.04, and 200.40 ng/mL of LVFX for the fecal excretion study. All stock and working solutions were stored at 4°C and brought to room temperature before use.
The tissues were cut on ice and mixed evenly. Each weighed tissue was homogenized in physiological saline (1 : 4, w/v) after thawing. The corresponding tissue homogenate with no drug was used as the blank homogenate.
One milliliter of tissue homogenate and the bladder homogenates were centrifuged (5000 rpm, 4°C, 8 min). Thereafter, 50
Feces were crushed with a grinder. Thereafter, 0.5 g of the feces was homogenized with physiological saline (1 : 8, w/v), vortexed for 2 min, sonicated for 10 min, and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was then collected in a centrifuge tube.
A 100
The specificity of the method was evaluated by analyzing a blank rat liver tissue homogenate, adding LVFX and IS to this blank, and collecting the tissue homogenate at 30 min after oral administration.
As described in Section
The precision and accuracy of the test were determined by analyzing the quality control samples of the five replicates at three concentration levels (low, medium, and high) on the same day (intraday) and three consecutive days (interday).
The extraction recovery of the analyte was evaluated by comparing the peak area ratio of the low concentration, medium concentration, and high concentration of the pretreated QC samples to the peak area ratio of the supernatant containing the same concentration of the standard solution. The matrix effect was evaluated by comparing the peak area of the analyte in the spiked sample postextraction, with the peak area of the analyte dissolved in the same concentration of methanol. Five replicate analyses were performed on the QC samples.
The stability of the analytes was determined using low, medium, and high concentrations (
The dilution integrity of the test was determined by analyzing the high-concentration samples in the ultralinear range (urine: 1,002.00
For the tissue distribution study, Sprague–Dawley rats were randomly divided into two groups, with 24 rats in each group. Group one was administered 42 mg·kg−1 LVFX while group two was administered 1.86 g·kg−1
For the urinary and fecal study, Sprague–Dawley rats were randomly divided into two groups (6 rats/group). Group one was administered 42 mg·kg−1 LVFX while group two was administered 1.86 g·kg−1
Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the statistical software package, Statistical Product and Service Solutions (SPSS 11.5, SPSS Inc., Chicago, IL, USA). Factorial analysis of variance was used for comparison between groups. A
Figure
Typical chromatograms. (a) Blank tissue homogenate; (b) blank tissue homogenate spiked with LVFX and IS; (c) rat tissue homogenate collected at 30 min after oral administration of LVFX. (1) LVFX; (2) puerarin.
As described in Section
Calibration curves, linear ranges, correlation coefficients, and LLOQ of LVFX in rat tissues and urine and fecal samples.
Biosamples | Calibration curves | Linear ranges (ng/mL) | LLOQ (ng/mL) | |
---|---|---|---|---|
Heart | 0.9966 | 5.01–15,030.00 | 5.01 | |
Liver | 0.9951 | 5.01–15,030.00 | 5.01 | |
Spleen | 0.9967 | 5.01–15,030.00 | 5.01 | |
Lung | 0.9953 | 5.01–15,030.00 | 5.01 | |
Kidney | 0.9961 | 5.01–15,030.00 | 5.01 | |
Stomach | 0.9957 | 5.01–15,030.00 | 5.01 | |
Intestines | 0.9967 | 5.01–15,030.00 | 5.01 | |
Bladder | 0.9954 | 5.01–15,030.00 | 5.01 | |
Urine | 0.9998 | 5.01–10,020.00 | 5.01 | |
Feces | 0.9996 | 5.01–1002.00 | 5.01 |
The precision and accuracy of LVFX are shown in Table
Precision and accuracy of LVFX in rat liver tissues, urine, and feces (
Biosamples | Spiked concentration (ng/mL) | Intraday | Interday | ||||
---|---|---|---|---|---|---|---|
Calculated concentration (ng/mL) | Precision (RSD, %) | Accuracy (%) | Calculated concentration (ng/mL) | Precision (RSD, %) | Accuracy (%) | ||
LVFX in liver tissue | 10.02 | 9.33 ± 1.03 | 11.03 | 93.07 | 9.18 ± 0.55 | 5.94 | 91.59 |
100.20 | 104.61 ± 3.89 | 3.72 | 104.40 | 103.86 ± 4.09 | 3.94 | 103.65 | |
5010.00 | 4815.20 ± 158.68 | 3.30 | 96.11 | 4803.63 ± 111.96 | 2.33 | 95.88 | |
LVFX in urine | 10.02 | 9.49 ± 0.85 | 8.99 | 94.67 | 10.46 ± 0.51 | 4.85 | 104.35 |
200.40 | 209.45 ± 10.49 | 5.01 | 104.52 | 207.55 ± 12.98 | 6.25 | 103.57 | |
4008.00 | 4183.48 ± 102.27 | 2.44 | 104.39 | 3875.60 ± 97.53 | 2.52 | 96.70 | |
LVFX in feces | 10.02 | 9.54 ± 0.86 | 9.03 | 95.17 | 10.54 ± 0.73 | 6.90 | 105.16 |
20.04 | 20.56 ± 1.16 | 5.66 | 102.58 | 19.54 ± 0.91 | 4.64 | 97.53 | |
200.40 | 215.78 ± 13.68 | 6.34 | 107.67 | 190.05 ± 20.49 | 10.78 | 94.83 |
The mean extraction recovery and the matrix effect of LVFX are shown in Table
Recovery and matrix effect of LVFX in rat liver tissues, urine, and feces (
Biosamples | Spiked concentration (ng/mL) | Extraction recovery | Matrix effect | ||
---|---|---|---|---|---|
Mean ± SD | RSD (%) | Mean ± SD | RSD (%) | ||
LVFX in liver tissue | 10.02 | 90.84 ± 9.21 | 10.14 | 93.09 ± 6.72 | 7.22 |
100.20 | 89.11 ± 6.24 | 7.00 | 103.03 ± 2.50 | 2.43 | |
5010.00 | 89.60 ± 4.69 | 5.24 | 97.64 ± 6.08 | 6.23 | |
LVFX in urine | 10.02 | 94.07 ± 7.88 | 8.38 | 93.02 ± 4.09 | 4.39 |
200.40 | 91.84 ± 10.21 | 11.11 | 89.06 ± 6.53 | 7.33 | |
4008.00 | 93.12 ± 5.71 | 6.14 | 103.18 ± 9.78 | 9.48 | |
LVFX in feces | 10.02 | 93.94 ± 8.39 | 8.93 | 89.96 ± 4.86 | 5.41 |
20.04 | 91.36 ± 7.60 | 8.31 | 93.00 ± 5.36 | 5.76 | |
200.40 | 101.13 ± 7.14 | 7.06 | 92.67 ± 9.76 | 10.53 |
Table
Stability of LVFX under different storage conditions (
Biosamples | Spiked concentration (ng/mL) | Calculated concentration (ng/mL) | ||
---|---|---|---|---|
Room temperature | Cold storage | Three freeze-thaw cycles | ||
LVFX in liver tissue | 10.02 | 9.36 ± 0.44 | 9.28 ± 0.85 | 9.60 ± 0.94 |
100.20 | 102.33 ± 8.31 | 102.92 ± 10.23 | 108.10 ± 3.75 | |
5010.00 | 4887.83 ± 105.12 | 4738.62 ± 157.74 | 4798.11 ± 159.01 | |
LVFX in urine | 10.02 | 8.98 ± 0.97 | 9.22 ± 0.99 | 10.50 ± 0.75 |
200.40 | 207.47 ± 10.63 | 212.35 ± 11.46 | 197.20 ± 10.17 | |
4008.00 | 3869.70 ± 143.60 | 3888.71 ± 161.29 | 3833.85 ± 91.54 | |
LVFX in feces | 10.02 | 9.02 ± 0.84 | 9.74 ± 0.67 | 9.33 ± 0.82 |
20.04 | 20.96 ± 1.66 | 18.75 ± 1.19 | 21.44 ± 0.83 | |
200.40 | 192.05 ± 17.68 | 191.49 ± 18.34 | 196.81 ± 13.40 |
The dilution integrity of LVFX in urine and fecal are shown in Table
Dilution integrity of LVFX in urine and fecal samples (
Biosamples | Mean ± SD | RSD (%) |
---|---|---|
LVFX in urine | 965.35 ± 39.82 | 4.12 |
LVFX in feces | 100.51 ± 9.00 | 8.96 |
The content of LVFX in the heart, liver, spleen, lung, kidney, stomach, intestine, and bladder is shown in Figure
The content of LVFX in rat tissue homogenate of single and coadministration group at four different time points (mean ± SD). Compared with single group:
Figure
Urinary and fecal cumulative excretion ratio of LVFX after oral administration to rats (
In the present study, we established a rapid, simple, and sensitive UPLC-MS/MS method. Thereafter, we opted to apply this method to determine the tissue distribution and excretion changes when LVFX was combined with
After drugs enter the blood circulation, they are distributed to the tissues of the body and blood. Understanding the characteristics of the tissue distribution of a drug will enable the identification of its target organs and the prediction of its pharmacological effect, which are significant for expanding its clinical use [
Generally, multiple mechanisms may be responsible for the HDI of a specific drug. Traditional Chinese medicine mainly causes pharmacokinetic interactions by inhibiting or inducing drug-metabolizing enzymes and transporters, which play a decisive role in the absorption, distribution, metabolism, and excretion of drugs [
LVFX excretion in urine and feces is shown in Figure
Herein, we developed an LC–MS/MS method to determine LVFX in rat biological samples after the combined oral administration of the
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was supported by the National Natural Science Foundation of China (grant no. 81760675/U1812403); Guizhou Science and Technology Department (grant nos. [2019] 2777/5660, [2018]4006, and [2017]5601); Guiyang Science and Technology Bureau (grant no. [2017]30-29); and Guizhou Education Department (no. KY[2017]073).