The aim of this study was to examine whether Xuesaitong, a multiherbal formulation for coronary heart disease, alters the pharmacokinetics of losartan. Adult male Sprague Dawley rats randomly received losartan (10 mg/kg) or losartan plus Xuesaitong (10 mg/kg) through an oral gavage (
Xuesaitong is a traditional Chinese medicine with multiple pharmacological activities. Major components of Xuesaitong are saponins from
As a nonpeptide angiotensin-II-receptor antagonist, losartan is the first of its kind in the market [
Metabolic drug interactions can occur when an herbal medicine and chemical pharmaceutical are coadministered such that the herbal medicine may alter the metabolism of the pharmaceutical via induction or obstruction of CYP450 isoforms [
Losartan (purity >98%) and irbesartan, the latter of which was used as an internal standard (IS; purity >98%), were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). EXP3174 (purity >98%) was obtained from Toronto Research Chemicals (Toronto, Canada). Figure
Structures of losartan (a), EXP3174 (b), and irbesartan (IS) (c).
Approval was obtained from the Animal Ethics Committee of the Second Military Medical University (Shanghai, China) for all of the animal experimental procedures. Male adult Sprague Dawley rats (180–220 g; Sino-British SIPPR/BK Lab, Shanghai, China) were housed at 50 ± 10% relative humidity and 22 ± 2°C. Water and standard-rate chow were available
Twelve rats (six per group) randomly received losartan (10 mg/kg), either with or without Xuesaitong pretreatment (10 mg/kg, 5 min earlier), through an oral gavage. Dosage calculations were performed according to the body-surface-area ratio between human subjects and rats. A pestle was used to grind losartan and Xuesaitong tablets into powder, which was then dissolved in 5 mL of water before being added to a 10 mL of 1.5% Tween 80 solution (as a solubilizer) for homogenization. Blood samples (0.2 mL) were obtained via the tail vein at 0 (predosing), 5, 10, 15, and 30 min and 1, 2, 3, 4, 6, 8, 12, 24, and 36 h after the treatment. Plasma samples were stored at −80°C before being analyzed.
We used a noncompartmental model to obtain the following pharmacokinetic parameters with Phoenix WinNonlin 7.0 pharmacokinetic software (Pharsight Corporation, Princeton, NJ, USA): the area under the concentration-time curve from time 0 to the last-measured concentration (AUC0–
An Agilent 1290 Infinity UPLC system (Agilent Technologies, USA) was used for chromatographic analysis and included a binary pump, a high-performance well-plate autosampler, an online vacuum degasser, and a temperature controller for the column. Samples were separated on a SHISEIDO MG-C18 column (100 mm × 3.0 mm, 3.0
The LC system was linked to an Agilent 6470 Triple Quad MS system (Agilent Technologies, USA) with an Agilent Jet Stream Technology (AJS) electrospray-source interface (ESI). The positive-ion mode was optimized for MS detection via multiple-reaction monitoring (MRM). Precursor and product ions had
Precursor and product ions of EXP3174 (a), irbesartan (IS) (b), and losartan (c).
Losartan (1.0 mg/mL) and EXP3174 (1.0 mg/mL) stock solutions were prepared using reference standards in methanol solution. The stock solution of IS was diluted to 100 ng/mL prior to use. Working standard solutions of losartan and EXP3174 were prepared by combining and diluting each stock solution with methanol. Additional standard working solutions were prepared by dilution with methanol. The working solutions were spiked into blank rat plasma at concentrations of 2–2000 ng/mL (2, 5, 10, 50, 100, 500, 1000, 1600, and 2000 ng/mL) to establish calibration standards for losartan and EXP3174. Quality control (QC) samples for validation were similarly set at 5, 100, and 1600 ng/mL. Standards and QC specimens were removed on every analysis day via a method identical to that used for actual samples, as detailed below.
Portions of 50
We evaluated method selectivity by examining six separate blank rat-plasma samples that we contrasted with the ones acquired by placing analytes into correlating blank plasma specimens with IS to observe interference. Calibration curves for losartan and EXP3174 comprised plots of the peak-area ratio of the analyte to IS against plasma concentration with a 1/
The accuracy and precision for both interday and intraday were determined with QC specimens for three consecutive days at three concentrations. Five replicates were used for each concentration. Each analyte concentration was quantified with a calibration curve on the same testing day.
Analyte-extraction recovery was performed by contrasting the peak areas acquired from blank plasma, in which analytes had been combined prior to removal, with areas from samples, in which they were combined following removal; this was performed five times. The matrix effect was examined by contrasting the solution in which the blank processed matrix had been combined with the solution at various QC concentrations; this procedure was performed five times.
To determine the stability of samples, three QC samples at high, medium, and low concentrations were examined under various conditions, including short-term storage stability at room temperature for 3 h, postpreparative stability with the autosampler for 24 h, three freeze-thaw cycles at −80°C, and long-term storage stability at −80°C for 30 days.
Figure
Chromatograms of (a) blank plasma; (b) blank plasma with losartan, EXP3174, and irbesartan (IS); and (c) rat plasma after oral administration. In the chromatograms, “1” denotes EXP3174, “2” denotes losartan, and “IS” denotes irbesartan.
The LLOQ for losartan and EXP3174 with S/N >10 was 0.5 ng/mL and 1.0 ng/mL, respectively. Calibration curves for losartan and EXP3174 were created by plotting the peak-area ratios of the analytes to IS against the plasma concentration with a 1/
Five separate runs on the same day for five consecutive days with three QC sample concentrations were performed for evaluating interday and intraday comparisons. The precision (relative standard deviation (RSD)) of this technique was <7% for losartan and EXP3174. The accuracy (relative error (RE)) was from −9.78% to 7.78% for losartan and −9.92% to 3.06% for EXP3174. The results are indicated in Table
Accuracy and precision of losartan and EXP3174 in rat plasma.
Compounds | Concentration (ng·mL−1) | Intraday ( |
Interday ( | ||
---|---|---|---|---|---|
RSD (%) | RE (%) | RSD (%) | RE (%) | ||
Losartan | 5 | 1.19 | −9.78 | 4.03 | −8.45 |
100 | 1.52 | 4.90 | 1.07 | 4.60 | |
1600 | 2.00 | 7.78 | 2.58 | 6.01 | |
EXP3174 | 5 | 5.89 | −9.92 | 6.46 | −8.77 |
100 | 2.22 | 2.65 | 4.68 | −0.53 | |
1600 | 3.62 | 3.06 | 4.30 | 0.83 |
RSD, relative standard deviation; RE, relative error.
The extraction recoveries for losartan and EXP3174 were >85%, and there were no significant variations among the three concentrations. The analyte-matrix effect ranged from 92.43 to 115.74%. The results are indicated in Table
Matrix effect and extraction recovery of losartan and EXP3174 in rat plasma (
Compounds | Concentration (ng·mL−1) | Matrix effect (%) | RSD (%) | Recovery (%) | RSD (%) |
---|---|---|---|---|---|
Losartan | 5 | 92.43 | 8.69 | 85.60 | 6.34 |
100 | 93.25 | 4.04 | 108.28 | 1.46 | |
1600 | 96.82 | 1.34 | 111.12 | 0.96 | |
EXP3174 | 5 | 101.98 | 6.27 | 85.19 | 2.71 |
100 | 115.74 | 7.75 | 101.25 | 2.38 | |
1600 | 111.36 | 2.59 | 109.79 | 2.51 |
RSD, relative standard deviation; RE, relative error.
We evaluated stability using a variety of conditions. Both losartan and EXP3174 remained stable at 4°C for 3 h at room temperature, for 24 h in the autosampler after three cycles of freeze-thawing, and after long-term storage for 30 days at −80°C. The concentration variation was <10%. The results are indicated in Table
Stability outcomes of losartan and EXP3174 in rat plasma (
Compounds | Concentration (ng·mL−1) | Storage at 25°C for 3 h | Storage at 4°C for 24 h | Three freeze-thaw cycles | Long-term storage at −80°C for 30 days | ||||
---|---|---|---|---|---|---|---|---|---|
Measured (ng·mL−1) | RE (%) | Measured (ng·mL−1) | RE (%) | Measured (ng·mL−1) | RE (%) | Measured (ng·mL−1) | RE (%) | ||
Losartan | 5 | 5.25 | 5.00 | 5.47 | 8.30 | 5.39 | 6.71 | 5.41 | 8.31 |
100 | 104.60 | 4.60 | 109.52 | 9.52 | 106.54 | 6.54 | 107.46 | 7.46 | |
1600 | 1633.48 | 2.09 | 1673.44 | 4.59 | 1576.38 | −1.48 | 1686.27 | 5.39 | |
EXP3174 | 5 | 5.37 | 7.40 | 5.49 | 7.76 | 5.44 | 7.78 | 5.46 | 7.15 |
100 | 102.33 | 2.33 | 107.57 | 7.57 | 97.22 | −2.78 | 94.40 | −5.60 | |
1600 | 1648.96 | 3.06 | 1653.63 | 3.35 | 1487.01 | −7.06 | 1492.90 | −6.94 |
RSD, relative standard deviation; RE, relative error.
No detectable carryover was noted. The accuracy was within ±15.0%, and the precision was under 15%, according to dilution-integrity experiments.
Concentration-time curves for losartan and EXP3174 in rat plasma (upon losartan treatment vs. losartan plus Xuesaitong treatment) are shown in Figure
Mean concentration-time curves upon losartan treatment vs. losartan plus Xuesaitong. (a) Losartan. (b) EXP3174. The error bars represent standard deviations (SDs).
Pharmacokinetic parameters of losartan and EXP3174 in rats following oral administration of two pretreatments (A: losartan alone; B: losartan plus Xuesaitong).
Parameter | Losartan | EXP3174 | ||
---|---|---|---|---|
Losartan alone (A) | Losartan plus Xuesaitong (B) | Losartan alone (A) | Losartan plus Xuesaitong (B) | |
|
6.35 ± 2.10 | 4.26 ± 1.51 |
8.22 ± 1.41 | 6.29 ± 1.38 |
|
0.13 ± 0.05 | 1.06 ± 1.04 |
6.83 ± 4.12 | 11.33 ± 1.63 |
|
1.60 ± 0.59 | 1.50 ± 0.49 | 1.61 ± 0.61 | 1.60 ± 0.54 |
AUC0– |
12.92 ± 5.01 | 14.17 ± 4.21 | 27.02 ± 10.49 | 26.40 ± 10.08 |
AUC0–inf ( |
13.54 ± 5.54 | 14.28 ± 4.23 | 29.40 ± 11.85 | 27.64 ± 10.88 |
AUMC0– |
101.66 ± 26.75 | 120.88 ± 27.41 | 324.38 ± 65.68 | 320.31 ± 58.17 |
AUMC0–inf ( |
101.15 ± 26.13 | 127.30 ± 25.52 | 440.49 ± 34.34 | 361.21 ± 36.31 |
MRT0– |
8.41 ± 1.80 | 7.76 ± 0.88 | 12.42 ± 1.48 | 11.98 ± 1.27 |
MRT0–inf (h) | 9.86 ± 2.80 | 8.03 ± 1.00 | 15.09 ± 2.72 | 13.31 ± 2.08 |
|
7.20 ± 2.41 | 4.41 ± 1.61 |
4.52 ± 1.66 | 3.62 ± 1.21 |
CL (mL·h−1) | 0.84 ± 0.32 | 0.77 ± 0.31 | 0.39 ± 0.16 | 0.42 ± 0.21 |
To optimize the pretreatment, we chose the simple method of protein precipitation and studied the effects of precipitation of methanol and acetonitrile as well as the influence of volume ratio on the effects of precipitation. The results showed that methanol protein deposition using the same volume as the plasma yielded the most optimal results. The extraction efficiency surpassed 90% with methanol as the extraction solution. The direct precipitation method also revealed convenience with the low matrix effect, indicating that it was an excellent precipitating agent.
To determine the suitable retention time and reaction of losartan and EXP3174 in rat plasma, water, methanol, formic acid, and acetonitrile were examined as mobile phases. Solvent A (0.1% formic acid) and solvent B (acetonitrile; 50 : 50, v/v) after optimization were determined to improve the ionization efficiency and led to an elevated intensity compared to water for each constituent evaluated. To devise a precise and sensitive LC-MS/MS method, we conducted quantitative analysis in the MRM mode because of its high sensitivity and selectivity. Precursor and product ions had
According to the results shown in Table
Interactions between losartan and Xuesaitong could occur during absorption, metabolism, distribution, and/or excretion. A previous study indicated that tanshinone IIA and salvianolic acid B (two components of danshen) have distinct effects on the metabolism of losartan. Tanshinone IIA inhibits losartan metabolism via CYP3A4 and CYP2C9; salvianolic acid B, in contrast, induces losartan metabolism via CYP3A4 and CYP2C9 [
In this study, we developed a UPLC-MS/MS technique with high sensitivity and accuracy to measure losartan and EXP3174 in rat plasma. We used this technique successfully for the pharmacokinetic evaluation of losartan and EXP3174 following losartan and Xuesaitong oral administration. Our results showed that Xuesaitong affected the pharmacokinetics of losartan following coadministration. We recommend an increased dosage of losartan in patients who also receive Xuesaitong.
Agilent Jet Stream Technology
The area under the concentration-time curve
The area under the first-moment plasma concentration-time curve
The clearance rate
The maximum plasma concentration
The apparent volume of distribution
Electrospray-source interface
Internal standard
Lower limit of quantification
Multiple-reaction monitoring
The mean residence time
Quality control
Relative error
Relative standard deviation
Standard deviation
The elimination half-life
The time to reach the maximum concentration
Ultraperformance liquid chromatography-tandem mass spectrometry.
The raw/processed data required to reproduce these findings are available from the corresponding author upon request.
The authors have no conflicts of interest related to this work.
Weina Ma and Lei Lv contributed equally to this work.
This project was supported by the fund of Shanghai Baoshan Luodian Hospital (grant no. 19-A-8) and the fund of project introduction at Shanghai University of Traditional Chinese Medicine-Gaoyuan Gaofeng Clinical Medicine Grant and Shanghai Municipal Commission of Health and Family Planning (grant no.201540265). We thank Accdon (