Simultaneous determination and tissue distribution studies of four phenolic acids in rat

tissue by UFLC-MS/MS after intravenous administration of salvianolic acid for

injection

Shuang Li 1+, Xiuman Xie 1+, Dongxiang Li 2, Zhiguo Yu 2, Ling Tong 2* and Yunli Zhao
1*

1
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang
110016, People’s Republic of China

2
State Key Laboratory of Core Technology in Innovative Chinese Medicine, Pharmaceutical
Analysis Institute, Tasly Academy, Tianjin 300410, People’s Republic of China

* Correspondence to: Yunli Zhao, School of Pharmacy, Shenyang Pharmaceutical University,

Shenyang Liaoning Province, 110016, People’s Republic China. E-mail: [email protected];
Ling Tong, Tasly R&D Institute, Tianjin Tasly Holding Group Co., Ltd., Tianjin, 300410,
People’s Republic of China. E-mail: [email protected].

+
These authors contributed equally to this work

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/bmc.4128

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Abbreviations used: UFLC-MS/MS, ultra-fast liquid chromatography tandem mass
spectrometric; SAFI, Salvianolic acid for injection; HPLC, high-performance liquid
chromatography; UHPLC, ultra-high-performance liquid chromatography;
UHPLC/Q-TOF-MS , ultra-high-performance liquid chromatography coupled with
quadrupole-time-of-flight mass spectrometry; Sal B, Salvianolic acid B; RA, rosmarinic acid;
LA, lithospermic acid; Sal D, salvianolic acid D; IS, Internal standard; ESI, electrospray
ionization; MRM, multiple reaction monitoring; QC, quality control; USFDA, United States
Food and Drug Administration; LLOQ, lower limit of quantification; CV, coefficient of
variance; RSD, relative standard deviation; SD, standard deviation; CE, collision energy; DP,
declustering potential; EP, entrance potential; CXP, cell exit potential; PP, protein
precipitation; LLE, liquid-liquid extraction; Cmax, maximum concentrations.

Abstract
A rapid, simple and sensitive ultra-fast liquid chromatography tandem mass spectrometric
method was developed and validated for simultaneous determination and tissue distribution
studies of rosmarinic acid, salvianolic acid D, lithospermic acid and salvianolic acid B in
rats after intravenous administration of salvianolic acid for injection. The tissue
homogenate samples were pretreated by protein precipitation with pre-cooled acetonitrile.
Chromatographic separation was achieved on a Waters Cortecs UPLC C18 column (1.6 μm,
2.1 × 100 mm) with a mobile phase composed of 0.1% formic acid-water and 0.1% formic
acid-acetonitrile. Analytes were detected by electrospray ionization mass spectrometry and
quantitated using multiple reaction monitoring. The method was fully validated. The
calibration curves for the four phenolic acids were linear in the given concentration ranges.
The precision (relative standard deviation) in the measurement of quality control samples
were less than 10% and the accuracy (relative error) were in the range of 0.28%-11.22%.
The reliable method was successfully applied to the tissue distribution studies of the four
phenolic acids. The results showed that rosmarinic acid, salvianolic acid D, lithospermic
acid and salvianolic acid B were rapidly distributed in tissues with the major amount found
in kidney, and little amount crossed the blood-brain barrier. The developed method and the
results can provide a basis for further studies.
Keywords
UFLC-MS/MS; tissue distribution; salvianolic acid for injection; intravenous administration

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1. Introduction
Salvia miltiorrhiza is a very common traditional Chinese medicine that has been extensively
applied for many years to treat coronary heart disease, cerebrovascular disease, hepatitis,
hepatocirrhosis and chronic renal failure, either alone or in combination with other Chinese
plant-based medicines (Zhang et al., 2005; Zhao et al., 2004; Su et al., 2015). The chemical
constituents of Salvia miltiorrhiza include both lipophilic and hydrophilic components (Ling
et al., 1999; Li et al., 2009). The hydrophilic components, which are mostly phenolic acids
(salvianolic acids) are responsible for the therapeutic effects of the extract of Salvia
miltiorrhiza (Zheng et al., 1989; Xu et al., 2007). Previous studies demonstrated that phenolic
acids have the pharmacological activities of anti thrombus, improve blood circulation, protect
myocardial ischemia and hypoxia, and promote tissue repair (Liu et al., 2012; Li et al., 2003;
Chen et al., 2015).
Salvianolic acid for injection (SAFI) is a kind of powder for injection that prepared by
phenolic acids extract from Salvia miltiorrhiza and mannitol. The active chemical
composition in SAFI is very complicated. Salvianolic acid B (Sal B, 59%), rosmarinic acid
(RA, 3.3%), lithospermic acid (LA, 2.9%) and salvianolic acid D (Sal D, 2.3%) comprise the
main active ingredients of SAFI (Xu et al., 2015). SAFI shows a significant efficacy in
protecting against cerebral infarction and ischemia/reperfusion-induced brain injury owing to
its antioxidant activity (Tang et al., 2008; Bai et al., 2016; Liu et al., 2017).
Tissue distribution study is of great importance for the better understanding of the
biological effects and safety of drug. Up to now, several analytical methods have been
reported for SAFI determination, such as high-performance liquid chromatography (HPLC)
method for quantitative determination of SAFI (Tang et al., 2014), ultra-high-performance
liquid chromatography (UHPLC) method for fingerprint and qualitative analysis (Xu et al.,
2016), ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight
mass spectrometry (UHPLC/Q-TOF-MS) method for characterization of metabolites of SAFI
(Miao et al., 2016), and an ultra-fast liquid chromatography tandem mass spectrometric
(UFLC-MS/MS) method for quantitative determination of SAFI in rat plasma (Xie et al.,
2015). However, no method has been published for tissue distribution study of these

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compounds after intravenous administration of SAFI. Therefore, the objective of this study
was to develop and validate an UFLC-MS/MS method for the determination of the four main
active ingredients (RA, Sal D, LA and Sal B) and to investigate their tissue distribution in rats,
to provide evidence for the safety and effectiveness of drug clinical application.

2. Material and methods

2.1. Chemical and reagents
SAFI was provided by Tasly Pharmaceutical Co., Ltd. (Tianjin, China), RA, Sal D, LA and
Sal B were obtained from Tianjin Yifang science and technology Co., Lt., Chloramphenicol
(Internal standard, IS) was obtained from the National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China). The purity of all of the standards
was > 98% by HPLC analysis. LC-MS grade acetonitrile and methanol were purchased from
Omni (North Carolina, USA). HPLC grade formic acid (purity > 95.0%) was purchased from
Sigma (Saint Louis, Mo, USA). High purity deionized water was prepared by a Milli-Q
system (Bedford, MA, USA). Other reagents were analytical grade.
2.2. Instruments and UFLC-MS/MS conditions
Chromatographic analysis was performed on a Shimadzu UFLC system equipped with an
LC-30AD binary pump, a DGU-20 degasser, an SIL-30AC autosampler and a CTO-30AC
column oven (Shimadzu Corp, Tokyo, Japan). Chromatographic separation was achieved on a
Waters Cortecs UPLC C18 (1.6 μm, 2.1 × 100 mm) column with a guard cartridge at
temperature of 30°C. The mobile phase was composed of 0.1% formic acid-water (A) and
0.1% formic acid-acetonitrile (B) with a gradient elution as follows: 0-2 min, 95% A;
2.01-5.0 min, 67% A; 5.01-5.5 min, 95% A; 5.51-7.5 min, 50% A; 7.51-8 min, 95% A. The
flow rate was set at 0.4 mL/min and the injection volume was 3 μL.

A Q-Trap® 5500 triple quadruple mass spectrometry equipped with an electrospray

ionization (ESI) source was employed in the negative ion mode (Applied Biosystems Sciex,
CA, USA). Quantification was performed in multiple reaction monitoring (MRM) mode. MS
parameters were set as follows: ion-spray voltage, -4500 V; ion source temperature, 550°C;
nebulizer gas (gas 1, nitrogen) and heater gas (gas 2, nitrogen), 55 psi; curtain gas, 25 psi.

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Data acquisition and integration were performed using the Analyst 1.6.2 software (Applied
Biosystems Sciex, CA, USA).
2.3. Preparation of standards, internal standard and quality control (QC) samples
Stock solutions of RA, Sal D, LA, Sal B (2.0 mg/mL) and IS (0.05 mg/mL) were prepared in
methanol, respectively. A series of working solutions ranging from 5.0 to 5000 ng/mL for RA,
Sal D, LA and 10.0 to 10000 ng/mL for Sal B were prepared by diluting stock solutions with
methanol, respectively. IS working solution (1.0 μg/mL) was made by diluting the stock
solution with methanol. All the solutions were stored at -20 °C before use.
Calibration standards were prepared by spiking working solutions into blank rat tissue
homogenate to yield final tissue homogenate concentrations of 5, 10, 25, 50, 100, 250, 1000,
5000 ng/mL for RA, Sal D, LA and 10, 20, 50, 200, 1000, 2000, 10000 ng/mL for Sal B,
respectively. QC samples in tissue homogenate concentration of 10, 100, 4000 ng/mL for RA,
Sal D, LA and 20, 200, 8000 ng/mL for Sal B were acquired by the same procedures as above.
Standard calibration samples and QC samples were stored at -20 °C until analysis.
2.4. Sample preparation
Tissue homogenate samples were thawed to room temperature before used. 100 μL methanol
and 20 μL IS solution (1.0 μg/mL) were added into 100 μL tissue homogenate samples. The
mixture was vortex mixed for 30 s, followed by the addition of 180 μL pre-cooled acetonitrile
containing 5% fomic acid to each tube for protein precipitation. The mixture was centrifuged
for 7 min at 13,000 rpm after vortex mixed for 5 min. Then the supernatant was transferred to
a 1.5 mL eppendorf tube and centrifuged for another 5 min. Finally, 3 μL of the supernatant
was injected into the UFLC-MS/MS system for analysis.

2.5. Method validation

The developed UFLC-MS/MS method was fully validated according to the United States
Food and Drug Administration (USFDA) (Food and Drug Administration, 2013) and
supplemented by European Medicines Agency guidelines on bioanalytical method validation
(European Medicines Agency, 2011).

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a. Selectivity was proved using at least 6 individual sources of the appropriate blank matrix,
which were individually analysed and evaluated for interference. It was ensured at the
lower limit of quantification (LLOQ).
b. Linearity of each calibration curve was established by plotting the peak area ratio (y) of
analytes to IS versus the analyte concentration (x) by least-squares linear regression with
a weighted factor (1/x2). The LLOQ was the lowest concentration of analyte in a sample
which can be quantified reliably, with an acceptable accuracy and precision. The
coefficient of variance (CV) values of accuracy and precision should not exceed 20% for
LLOQ.
c. Accuracy and precision (intra- and inter-day) were determined by analyzing 6 replicates
of low QC, medium QC and high QC samples in rat tissue homogenate for three
consecutive days. The criteria for acceptability of the data included accuracy within ±15%
deviation from the nominal values and precision of within ±15% relative standard
deviation (RSD).
d. Extraction recovery of an analyte in an assay was the detector response obtained from an
amount of the analyte added to and extracted from the biological matrix, compared to the
detector response obtained for the true concentration of the pure authentic standard.
e. Matrix effect was investigated using at least 6 blank matrix from individual donors, by
calculating the ratio of the peak area in the presence of matrix (measured by analysing
blank matrix spiked after extraction with analyte) to the peak area in absence of matrix
(pure solution of the analyte) at low, medium, and high concentrations.
f. The analyte stability was investigated at three QC levels under several different
conditions, each of six replicates. Stability studies included freeze-thaw (three cycles)
stability, short-term stability (25 °C for 24 hours), and long-term sample storage (−20 °C
for 20 days).
g. Dilution integrity was performed five-fold of the high QC concentration. Six replicates of
the concentration (20000 ng/mL for RA, Sal D, LA and 40000 ng/mL for Sal B) were
prepared and diluted to 4000 ng/mL for RA, Sal D, LA and 8000 ng/mL for Sal B and
then analyzed against the fresh calibration curve.

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2.6. Tissue distribution study
Thirty male Wistar rats (250 ± 10 g) were obtained from Vital River Lab Animal Technology
Co., Ltd. (Beijing, China), and housed with a 12 h light/12 h night cycle at ambient
temperature (25 °C) and 60 % relative humidity. All animal experiments were based on the
guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals,
and were approved by the Animal Ethics Committee of Tianjin Tasly academy. The approval
number of rats is TSL-IACUC-2015-014. They were acclimated to the laboratory for one
week and fasted overnight with free access to water before experiments. They were randomly
divided into 5 groups (6 in each group) and intravenous administration of SAFI at dose of 56
mg/kg. SAFI was dissolved in normal saline. The rats were killed by cervical vertebra
dislocation 0.033, 0.166, 0.5, 1, 2 h after dosing, respectively, then tissue samples (heart, liver,
spleen, lung, kidney and brain) were rapidly obtained and placed in normal saline solution to
remove blood. All these samples were weighed and homogenized in normal saline (two-fold
tissue weight) at 0 °C. Tissue homogenate was centrifuged at 13,000 rpm for 15 min and the
supernatant was stored at -80 °C until use.
2.7. Statistical analysis
The pharmacokinetic parameters were calculated by DAS 3.0 (Chinese Pharmacological
Society) using non-compartmental analysis. All the data were expressed as mean ± standard
deviation (SD).

3. Results and discussion

3.1. Optimization of UFLC-MS/MS method
MS detection was performed in the negative ion mode, which was more sensitive than the
positive one for phenolic acids. MS parameters of collision energy (CE), declustering
potential (DP), entrance potential (EP) and cell exit potential (CXP) were optimized by
infusing standard solutions individually to achieve maximum responses of precursor and
product ions. The results are shown in Table 1. The precursor and product ions mass spectra
of RA, Sal D, LA, Sal B and IS and their proposed fragmentation pathways are shown in Fig
1.
The composition of mobile phase has a significant influence on chromatographic behavior

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and appropriate ionization. Acetonitrile was chosen as the organic phase for higher responses.
Because the analytes in this study were acidic, adding acid in mobile phase can improve the
peak shape and retention for acidic samples. Therefore, two additives (formic acid and acetic
acid) were investigated. Finally, optimum chromatographic peak shapes were observed after
adding 0.1% formic acid to both aqueous phase and organic phase.
3.2. Selection of internal standard
Chloramphenicol was chosen as the preferred internal standard as it showed suitable retention
time, good peak shape and high response in the chromatographic conditions and extraction
procedure. Previous studies also used chloramphenicol as the internal standard when
detecting phenolic acids (Xu et al., 2007; Miao et al., 2016).
3.3. Selection of tissue for method validation
Considering that using each tissue for method validation was time-consuming, mixed tissue
was investigated in this study. The results showed that the recovery and matrix effect of
analytes in mixed tissue homogenate could meet the requirements of biological sample
analysis. Furthermore, the RSD values for retention time of four analytes in mixed tissue
homogenate sample were calculated and the results were within 0.46%.
3.4. Development of sample preparation procedure
During the development of extraction methods, protein precipitation (PP) and liquid-liquid
extraction (LLE) were explored. Due to the various steps and time-consuming of LLE, PP
was applied for sample preparation in this study. However, we found that recovery of the four
phenolic acids could not meet the requirements of biological sample measurement, what’s
more, phenolic acids are often unstable and easily oxidized, lead to the unstable measurement
results. Based on previous research, adding acid could improve the recovery and stability of
phenolic acids (Xie et al., 2016). Therefore, formic acid and hydrochloric acid were
investigated. Finally, adding 5% formic acid in protein precipitation solution was found to be
an optimal choice for yielding the highest recovery.
3.5. Method validation
3.5.1. Selectivity
Typical chromatograms obtained from a blank mixed tissue homogenate sample, a spiked

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mixed tissue homogenate sample with analytes (at LLOQ) and IS, and a kidney homogenate
sample after intravenous administration of SAFI are presented in Fig 2. The retention time of
RA, Sal D, LA, Sal B and IS in mixd tissue homogenate sample were 3.94, 3.66, 3.93, 4.15
and 4.64 min, respectively. No interference of endogenous peaks exhibited in blank tissue
sample, which indicated the high selectivity of the UFLC-MS/MS method.
3.5.2. Linearity and LLOQ
The calibration curves showed good linearity over the concentration ranging from 5.0-5000
ng/mL for RA, Sal D, LA and 10.0-10000 ng/mL for Sal B. Typical linearity regression data
for the analytes in rat tissue homogenate sample are listed in Table 2. The LLOQ of RA, Sal
D, LA and Sal B were 5.0, 5.0, 5.0, 10.0 ng/mL, respectively. The RSD values of all analytes
for precision were less than 9.30%, and the RE values for accuracy were less than 3.40%,
which meet the requirement of a bioanalytical method according to the Guideline of USFDA.
3.5.3. Accuracy and precision
The intra- and inter-day precision and accuracy obtained by QC samples are listed in Table 3.
The RSD values of all analytes for precision were less than 9.96% and the RE values for
accuracy were less than 11.22%. The results indicated that the method was reliable and
reproducible enough for further tissue distribution study.
3.5.4. Extraction recovery and matrix effect
As showed in Table 3, matrix effect of the four analytes were at the range of
85.42%-100.87%, extraction recovery of all analytes were ranged from 85.42%-114.26%,
which demonstrated that the interference in rat tissue homogenate could be negligible.
3.5.5. Stability
The results of short-term stability, long-term stability and freeze-thaw stability are presented
in Table 4, which were well within the acceptable limit. The results suggested that the four
analytes were stable under all tested conditions.
3.5.6. Dilution integrity
The dilution integrity data are listed in Table 5, the RE values for accuracy for analyzing
diluting QC samples (1/5 dilution) were within 13.67%, and RSD values for precision were
within 10.30%. The results suggested that tissue homogenate samples with a higher

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concentration of analytes than ULOQs could be quantified reliably by appropriate dilution.
3.6. Tissue distribution study
3.6.1. Selection of potential active compounds for tissue distribution
Although SAFI contains a variety of components, only a few components were detected in
tissue samples in pre-experiments, including RA, Sal D, LA and Sal B, which comprise
67.5% of the total amount of SAFI. Other compounds with low content in this injection, such
as protocatechuic aldehyde, caffeic acid, were only detected in the beginning few minutes.
Therefore, RA, Sal D, LA and Sal B were chosen as detection indicators for tissue
distribution study of SAFI.
3.6.2. Selection of sampling time-points
In pre-experiment, we found that the concentration of analytes in tissues after 2 hours were
generally lower than the LLOQ except Sal B. Furthermore, RA concentrations were below
the LLOQ in spleen and brain at 10 minutes. Therefore, the time of tissue sampling was set to
2 hours, and the time-points were set at 0.033, 0.166, 0.5, 1, and 2 h.
3.6.3. Tissue distribution of SAFI
The tissue distribution of RA, Sal D, LA and Sal B after intravenous administration of SAFI
at dose of 56 mg/kg in rats is presented in Fig 3. The main pharmacokinetic parameters are
listed in Table 6. The four phenolic acids were quickly distributed among tissues at 0.033h
after intravenous administration and reached the maximum concentrations (Cmax) at the same
time, indicating that SAFI has a fast onset time in vivo.
The amount of RA was in the order of kidney > liver > lung > heart > spleen > brain. In
spleen and brain, RA was only detected in the beginning 0.033h after dose, and the
concentration in heart was below the LLOQ after 1 hour. The Cmax level in kidney was
significantly higher than other tissues, and cleared rapidly, which might result from RA was
excreted mainly from kidney into urine (Xu et al., 2007).
The content of Sal D were generally low in tissues with the highest level in kidney,
followed by lung, liver, heart, spleen, and the lowest level in brain. Sal D was cleared slowly
in these tissues except kidney, suggesting that Sal D has higher affinity to kidney.
Remarkably, in earlier pharmacokinetics study (Xie et al., 2017), a large apparent volume of

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distribution for Sal D was found after intravenous administration of SAFI, inferring that that
Sal D should have high concentration in tissues, while it was inconsistent with present result.
It might be related to metabolites of Sal D exist mainly in bile (Miao et al., 2016). Therefore,
further studies on distribution in bile and other tissues of Sal D are necessary to investigate its
distribution position.
LA was widely distributed in the six tissues studied. The amount was in the descending
order of kidney > lung > heart > liver > spleen > brain. The high-concentration was found in
kidney, and content in heart, liver, spleen and lung were close. While in previous report (Li et
al., 2007), content of LA in kidney was less than heart and lung after injection of depside salts
from Salvia miltiorrhiza, this might explained by other compounds in SAFI preparation may
have influence on LA distribution in tissues.
As the main ingredient of SAFI, Sal B possessed the highest concentrations among the four
phenolic acids in all tissues studied. The amount of Sal B in tissues was in the order of
kidney > liver > lung > heart > spleen > brain. Sal B was found to be cleared rapidly after
0.166h especially in liver. According to previous research, this may because that Sal B was
metabolized mainly by liver, and rapidly excreted into bile as four methylated metabolites (Li
et al., 2007; Zhang et al., 2004).
The amounts of the four phenolic acids were low in brain tissue, which indicated that the
four compounds could not effectively across the blood-brain-barrier. Previous studies also
demonstrated that Sal B could not cross the blood-brain-barrier by a blood-brain-barrier
model in vivo (Hu, 2014). Although only a few amount of them could enter the brain by
intact, their metabolites such as caffeic acid or other compounds also have the therapeutic
potential for cerebrovascular diseases (Zhang et al., 2005; Baba et al., 2005), and might more
easily cross the blood-brain-barrier (Miao et al., 2016). In addition, the elimination
half-lives for the four phenolic acids in tissues (expect brain) were less than 2 h, and
concentrations were generally low in all tissues 2h after intravenous administration,
indicating there were no long-term accumulation in these tissues.

4. Conclusions
The fully validated method was successfully applied to the tissue distribution studies of the

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four components of SAFI in rats. The results showed that the four phenolic acids were rapidly
distributed in tissues and reach the Cmax at 0.033 h in all tissues studied. The highest
concentrations of the four analytes were found in kidney, while the lowest were found in
brain, which indicated that the four analytes could not efficiently across the
blood-brain-barrier. The method and results of tissue distribution in this paper could lay a
solid foundation for future studies of SAFI.

Acknowledgments
This work was supported by National Key Special Project of Science and Technology for
Innovation Drug of China (no. 2013ZX09402202).

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Baba, S., Osakabe, N., Natsume, M., Yasuda, A., & Muto, Y. (2005). Absorption, metabolism,
degradation and urinary excretion of rosmarinic acid after intake of Perilla frutescens
extract in humans. European Journal of Nutrition, 44, 1-9.

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Tables
Table 1. Optimized MRM parameters of rosmarinic acid, salvianolic acid D, lithospermic
acid, salvianolic acid B and chloramphenicol (IS).
Q1Mass Q3Mass Declustering Entrance Collision Cell exit
Analyte
(Da) (Da) potential potential energy potential

Rosmarinic acid 359.0 161.0 -100 -10 -23 -16

Salvianolic acid D 417.2 197.1 -33 -3 -27 -20

Lithospermic acid 537.1 493.2 -90 -11 -14 -16

Salvianolic acid B 717.2 519.1 -40 -10 -27 -18

Chloramphenicol 321.1 152.1 -125 -10 -23 -16

Table 2. Linear regression data of UFLC-MS/MS method for analysis of rosmarinic acid,
salvianolic acid D, lithospermic acid and salvianolic acid B in rat tissue (n=6).
Analyte Slope (× 10-4) Intercept (× 10-2) R Range (ng/ml)

Rosmarinic acid 6.66 1.06 0.9982 5-5000

Salvianolic acid D 3.75 0.64 0.9938 5-5000

Lithospermic acid 0.85 0.55 0.9931 5-5000

Salvianolic acid B 1.94 0.33 0.9992 10-10000

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Table 3. Precision, accuracy, matrix effect and recovery of rosmarinic acid, salvianolic acid D, lithospermic acid and salvianolic acid B in rat
tissue (n=6).

QC Accuracy Precision (RSD %) Matrix effect Recovery

Analyte
level (RE %) Intra-day Inter-day (Mean ± SD %) (Mean ± SD %)

Low QC 8.00 8.36 6.74 99.32 ± 14.30 99.50 ± 11.32

Rosmarinic acid Medium QC 11.22 7.50 1.80 92.83 ± 2.17 102.8 ± 8.78
High QC 3.21 6.74 0.89 98.13 ± 8.51 96.99 ± 5.98
Low QC 5.45 6.28 3.96 98.11 ± 11.62 101.4 ± 14.26
Salvianolic acid D Medium QC 0.28 6.33 6.26 85.42 ± 4.73 98.31 ± 6.29
High QC 5.79 6.75 0.95 94.63 ± 9.92 93.67 ± 9.60
Low QC 9.17 5.83 7.89 92.96 ± 9.46 102.1 ± 11.07
Lithospermic acid Medium QC 10.70 3.46 1.15 86.07 ± 6.41 103.5 ± 4.48
High QC 6.67 9.96 5.16 91.56 ± 10.61 88.76 ± 7.07
Low QC 0.75 8.62 1.04 100.9 ± 12.85 100.6 ± 13.82
Salvianolic acid B Medium QC 8.33 4.23 2.29 86.33 ± 4.68 113.4 ± 5.84
High QC 1.15 6.57 7.42 86.05 ± 5.74 114.3 ± 4.59

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Table 4. Stability of rosmarinic acid, salvianolic acid D, lithospermic acid and salvianolic acid B in rat tissue (n=6).

QC 20 °C for 20 days Room temperature for 24 hours Freeze–thaw (three cycles)

Analyte
level RE (%) RSD (%) RE (%) RSD (%) RE (%) RSD (%)
Low QC 1.27 13.18 1.98 13.29 5.37 12.80
Rosmarinic acid Medium QC 1.10 3.61 1.13 7.67 1.13 7.67
High QC 9.17 6.70 8.04 5.55 9.08 7.63
Low QC 4.97 9.36 4.15 6.39 2.05 10.19
Salvianolic acid D Medium QC 3.82 3.18 9.50 6.83 10.17 6.53
High QC 9.46 3.33 8.04 8.54 6.04 7.19
Low QC 0.47 10.22 6.78 6.36 3.78 6.44
Lithospermic acid Medium QC 2.12 6.49 5.68 2.33 7.60 5.13
High QC 10.38 4.52 6.63 5.74 8.92 8.34
Low QC 7.92 7.43 0.67 6.76 2.42 6.44
Salvianolic acid B Medium QC 0.58 7.62 2.33 7.09 2.33 5.13
High QC 8.31 4.29 12.21 7.20 4.98 8.34

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Table 5. Dilution integrity data of rosmarinic acid, salvianolic acid D, lithospermic acid and salvianolic acid B in rat tissue (n=6).

Analyte Concentration (mean ± SD) Accuracy (RE%) Precision (RSD %)

Rosmarinic acid 4547 ± 439.6 13.67 9.67

Salvianolic acid D 4263 ± 192.5 6.58 4.52

Lithospermic acid 4188 ± 268.6 4.71 6.41

Salvianolic acid B 7937 ± 817.4 0.79 10.30

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Table 6a. Main pharmacokinetic parameters of rosmarinic acid after intravenous administration in rat tissues (mean ± SD; n=6).
Parameters Heart Liver Spleen Lung Kidney Brain
AUC0-∞ (ng h/mL) － 93.39 ± 59.19 － 81.87 ± 23.15 1246.2 ± 42.87 －
t1/2(h) － 1.79 ± 2.26 － 0.85 ± 0.79 0.24 ± 0.21 －
Tmax (h) 0.033 0.033 0.033 0.033 0.033 0.033
Cmax (ng/mL) 149.91 ± 23.63 175.61 ± 24.03 68.46 ± 17.24 198.04 ± 45.46 9353.9 ± 311.32 5.12 ± 0.91

Table 6b. Main pharmacokinetic parameters of salvianolic acid D after intravenous administration in rat tissues (mean ± SD; n=6).
Parameters Heart Liver Spleen Lung Kidney Brain
AUC0-∞ (ng h/mL) 33.70 ± 4.76 82.04 ± 10.44 25.82 ± 2.69 161.29 ± 28.19 202.35 ± 30.46 219.87 ± 85.60
t1/2(h) 0.78 ± 0.29 0.88 ± 0.21 1.03 ± 0.16 2.20 ± 0.58 1.00 ± 0.45 8.33 ± 3.53
Tmax (h) 0.033 0.055 ± 0.054 0.033 0.055 ± 0.054 0.033 0.70 ± 0.47
Cmax (ng/mL) 32.20 ± 2.40 59.90 ± 3.55 33.88 ± 4.09 59.78 ± 8.97 358.63 ± 80.36 34.67 ± 25.50

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Table 6c. Main pharmacokinetic parameters of lithospermic acid after intravenous administration in rat tissues (mean ± SD; n=6).
Parameters Heart Liver Spleen Lung Kidney Brain
AUC0-∞ (ng h/mL) 356.57 ± 119.20 86.15 ± 21.48 53.92 ± 7.15 358.44 ± 24.49 605.95 ± 196.13 72.50 ± 17.17
t1/2(h) 0.99 ± 0.47 1.28 ± 0.81 0.84 ± 0.22 0.94 ± 0.37 1.28 ± 0.92 2.16 ± 0.78
Tmax (h) 0.033 0.033 0.033 0.033 0.033 0.22 ± 0.39
Cmax (ng/mL) 329.30 ± 29.57 236.53 ± 21.34 149.02 ± 40.09 321.25 ± 79.61 950.96 ± 101.09 26.34 ± 5.52

Table 6d. Main pharmacokinetic parameters of salvianolic acid B after intravenous administration in rat tissues (mean ± SD; n=6).
Parameters Heart Liver Spleen Lung Kidney Brain
AUC0-∞ (ug h/L) 482.71 ± 20.72 1083.6 ± 168.13 － 1183.5 ± 330.22 4443.56 ± 533.01 210.71 ± 101.58
t1/2(h) 0.46 ± 0.30 1.08 ± 1.45 － 0.49 ± 0.008 1.12 ± 0.11 3.28 ± 1.83
Tmax (h) 0.033 0.033 0.033 0.033 0.033 0.033
Cmax (ng/mL) 2279.3 ± 233.06 7785.3 ± 966.95 1704.6 ± 340.66 4349.4 ± 1631.5 26058 ± 2803.5 88.44 ± 20.96
—: Not determined

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Fig 1 Precursor and product ions mass spectra and proposed fragmentation pathways of
rosmarinic acid (RA), salvianolic acid D (Sal D), lithospermic acid (LA), salvianolic acid B
(Sal B) and chloramphenicol (IS).

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Fig 2 Representative MRM chromatograms of rosmarinic acid (RA), salvianolic acid D
(Sal D), lithospermic acid (LA), salvianolic acid B (Sal B) and chloramphenicol (IS) in rat
tissue: blank tissue homogenate sample (A); mixed tissue homogenate sample spiked with
analytes (at LLOQ) and IS (1 μg/mL) (B); 1 h post dose kidney homogenate sample after
intravenous administration of SAFI (56 mg/kg) (C).

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Fig 3 Tissue distribution of rosmarinic acid (A), salvianolic acid D (B), lithospermic acid
(C) and salvianolic acid B (D) after intravenous administration of SAFI at dose of 56
mg/kg in rats.

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