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\begin{document}

\title{Determination of rosmarinic acid and its N-substituted analog A1 in rat
plasma by high-performance liquid chromatography-tandem mass
spectrometry and its application in pharmacokinetics}



\author[1]{Shujie Fu}%
\author[2]{Qinglang Zhang}%
\author[2]{Shiyu Zhang}%
\author[1]{Weizhe  Jiang}%
\author[2]{Minjie Jiang}%
\affil[1]{Guangxi Medical University}%
\affil[2]{GuangXi University of Chinese Medicine}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The researcher will introduce the way of effective liquid chromatography
tandem mass spectrometric method (HPLC-MS) which is quick, sensitive,
and alternative to determine the (E)-3-(3,4-dihydroxyphenyl)
-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A1) and rosmarinic
acid (RA) of the plasma in rats. The analyses were divided into a C18
column (1.9 \selectlanguage{greek}μ\selectlanguage{english}m, 2.1 mm \selectlanguage{ngerman}× 100 mm) with a security guard C18 column (5 \selectlanguage{greek}μ\selectlanguage{english}m,
2.1 mm \selectlanguage{ngerman}× 10 mm) and a triple-quadrupole mass spectrometry with an
electrospray ionization (ESI) ion-source generates ions. With
Pseudoephedrine hydrochloride being a standard, the sample pretreatment
is relevant to the one-step protein precipitation with isopropanol:
ethyl acetate (v/v, 20:80). This method presented a linear relationship
within ranges of the concentration of 5--750 ng/ml for RA and A1.
Relative standard deviations (RSD) in daily courses stood less than 15\%
and the relative errors (RE) registered within 15\%. The means adopted
in this research makes the RA's and A1's unambiguous quantification and
identification and in vivo possible. And this study can be the first one
to focus on determining the A1 and RA of rat plasma with administration
orally. The results served as a significant basis to evaluate the
medicine's applications in the clinic.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Determination of rosmarinic acid and its N-substituted analog A1
in rat plasma by high-performance liquid chromatography-tandem mass
spectrometry and its application in pharmacokinetics}

Shujie Fu\textsuperscript{1}, Qinglang Zhang\textsuperscript{2}, Shiyu
Zhang\textsuperscript{2}, Weizhe Jiang\textsuperscript{1}, Minjie
Jiang\textsuperscript{2*}

1 School of Pharmaceutical, Guangxi Medical University, Nanning 530021,
China, 2 Guangxi University of Chinese Medicine, Nanning 530001, China

* Corresponding author. Tel.: +86 13768509293; fax: +86 07713137535.

E-mail address: jiangminjietp@me.com (M. Jiang).

\textbf{Introduction}

Caffeic acid esters, rosmarinic acid (RA, Fig. 1a), exhibit different
biological properties, such as antiviral, antibacterial,
anti-inflammatory, and antioxidant properties{[}1,2{]}. It has been
recently noted that RA pharmacology is important{[}2--5{]}, but very few
papers have evaluated its pharmacokinetics and pharmacodynamics
{[}6--11{]}using high-performance liquid chromatography-tandem mass
spectrometry. In order to investigate the pharmacodynamic mechanisms of
RA in more depth, we synthesized many analogs based on RA, including
(E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic
acid (A1, Fig. 1b). Previous studies have shown that A1 has higher
pharmacological activity against tumors than RA{[}12{]}. Thus, in order
to simultaneously determine A1 and RA, pharmacokinetics (PK) must be
studied using a sensitive and precise method. Recently, researchers have
expressed concern about RA absorption and metabolism, but few papers
have used HPLC-MS to study the pharmacokinetics and pharmacodynamics of
RA. By avoiding the need to fully analyze the chromatograms, this method
guarantees high sensitivity and speedy quantification. The majority of
previous studies, however, only focused on administering one substance
at a time; thus, no one has conducted a simultaneous study on RA and
other compounds and their pharmacokinetics. The objective of this study
was to implement and validate a new method for estimating RA and A1 in
plasma samples collected from rats. Therefore, this method is very
useful for pharmacokinetics studies.

2. Experimental section

2.1. Reagents and chemicals

RA (batch number 111871) with a purity above 99\% and pseudoephedrine
hydrochloride (PPD, batch number 171237-201510) were obtained from the
National Institute for the Control of Pharmaceutical and Biological
Products (Beijing, China) (Fig. 1c) and used as standards. A1 (99.8\%
purity) was synthesized at the School of Pharmaceutical, Guangxi Medical
University (Nanning, China). Ethyl acetate was obtained from Dikma
Technologies, Inc. (Richmond Hill, ON, Canada). The methanol and
acetonitrile used were obtained from Sigma-Aldrich Chemicals (St. Louis,
MO, USA). Deionized water was purified with an Alpha-Q water
purification system and then filtered with 0.20-\selectlanguage{greek}μ\selectlanguage{english}m membranes.

\emph{2.2. Instruments and conditions}

A Dionex UltiMate 3000 HPLC system with a binary pump, an on-line
degasser, etc. was employed for analysis. Separation was carried out by
chromatography at 40 \selectlanguage{ngerman}°C using a Hypersil GOLD C18 column (100 mm x 2.1
mm, 1.9 mm particle size) and C18 guard column. Acetonitrile-0.1\%
formic acid (25:75, v/v) was introduced at a flow rate of 0.2 ml/min.
Moreover, 2 \selectlanguage{greek}μ\selectlanguage{english}l aliquots were added to the HPLC system for analysis.

In order to analyze MS data, a Thermo Scientific TSQ Quantum Access MAX
triple stage quadrupole mass spectrometer was operated in negative
ionization mode with an electrospray ionization source (ESI).The typical
parameters of the ion source were as follows: sheath gas pressure
(N\textsubscript{2}): 20 units; spray voltage: 3500 V; collision gas
pressure (Ar): 1.5 mTorr; and scan width: 0.002; selected-reaction
monitoring (SRM) with monitoring ion pairs was used to analyze the
samples at m/z 359 - 161 for RA, 358 - 161 for A1, and 164 - 146 for the
internal standard (IS). Every channel had a scanning dwell time of 0.1
s. Xcalibur 2.2 software was used in the centroid mode to acquire and
process data.

\emph{2.3. Preparation of standards and quality control samples}

By dissolving 10 mg of A1 or RA in 100 ml of methanol, the standard
stock solution was prepared. Then, individual solutions were diluted
with methanol to achieve ideal concentrations and used as standard
solutions. We also dissolved and diluted PPD (10.0 mg) in methanol to
obtain a stock solution with a concentration of 1.0 mg/ml, which was
subsequently diluted to obtain an IS working solution at a concentration
of 5.0 \selectlanguage{greek}μ\selectlanguage{english}g/ml. These solutions were kept at 4 \selectlanguage{ngerman}°C and subsequently exposed
to normal temperature prior to usage. One hundred microliters of blank
plasma was spiked with 50 \selectlanguage{greek}μ\selectlanguage{english}l of appropriate RA and A1 standard solutions
to obtain RA or A1 concentrations of 5, 25, 50, 100, 250, 500 and 750
ng/ml, and calibration standards were used every day. In a similar
manner, quality control (QC) samples were prepared at low, medium, and
high concentrations to obtain samples containing 10, 100, and 500 ng/ml
RA and A1, respectively.

\emph{2.4. Plasma sample preparation}

The mixtures were put into 1.5-ml microcentrifuge tubes containing 20 ml
of standard solution and 100 ml of plasma. Then, 1000 \selectlanguage{greek}μ\selectlanguage{english}l of
isopropanol:ethyl acetate (20:80, v/v) was injected into the tubes and
vortexed for 60 s. The tubes were subsequently centrifuged at 8049.6\selectlanguage{ngerman}×g
for 10 min. Another tube was used for the organic layer, which was
subsequently evaporated to dryness. After dissolving the residue in 100
\selectlanguage{greek}μ\selectlanguage{english}l of mobile phase, 2 \selectlanguage{greek}μ\selectlanguage{english}l of the extract was injected into the HPLC-MS/MS
system. SYKG2003-0005 is the license number of the Animal Protection and
Utilization Committee at Guangxi Medical University that approved this
study.

\emph{2.5. Method validation}

We evaluated the selectivity by comparing the chromatograms of six
batches of blank rat plasma with spiked rat plasma. The linearity of six
calibration curves was determined through a weighted
(1/x\textsuperscript{2}) analysis. By analyzing three samples (n = 6) of
low-, medium-, and high-concentration QCs on different days, we
determined intraday and interday precisions (relative standard
deviation, RSD) and accuracy(relative error, re). The matrix effect was
studied by comparing the peak areas of the analytes in low-concentration
and high-concentration postextraction spiked blank plasma samples with
those of the corresponding standard solutions. In order to determine the
recovery rate, six extracted samples were compared to six extracted
samples with low, medium, and high QC concentrations. Stability was
evaluated by comparing the repeatability of low and high QC samples (n =
6) during storage and processing. A freeze-thaw stability test was
performed after three freeze-thaw cycles. By analyzing the QC samples at
4 \selectlanguage{ngerman}°C in one day, the stability of the samples during postpreparation was
assessed. For long-term and short-term stability evaluations, six
aliquots of QC samples were stored at 20 °C for two weeks and at ambient
temperature for four hours.

2.6. Pharmacokinetic (PK) study in rats

Male Sprague--Dawley rats weighing 250-300 g were selected for the PK
study. All animal experiments were conducted according to the guidelines
of the institutions and with the approval of the Use and Care of Animals
of Guangxi Medical University. Twelve rats were individually treated
with RA and A1 aqueous solutions at 10 mg/kg. We collected blood samples
(0.5 ml) at 0, 5, 15, 30, 45, 60, 90, 120, 240, 360, 480, and 720 min
following oral administration of the drug. These samples were stored in
heparinized tubes. After centrifugation at 8049.6×g and 4 °C for 10 min,
plasma was collected and frozen at \selectlanguage{english}-20 degC until analysis. Using TopFit
2.0 software, pharmacokinetic parameters were assessed in a
noncompartmental model. The elimination half-life (t\textsubscript{1/2})
is 0.693 k\textsubscript{e}, where k\textsubscript{e} is the elimination
rate constant, which is calculated by fitting the mean data of the
plasma concentration curve at 4 end points with a logarithmic linear
regression equation using the least squares method. Based on the
observed data, we calculated the maximum drug plasma concentration
(Cmax) and time to reach Cmax (Tmax). The area under the plasma
concentration-time curve from zero to the final measurable sample time
(AUC\textsubscript{0--t}) was calculated using the linear trapezoidal
rule.

\textbf{3. Results and discussion}

\emph{3.1. Chromatography and mass spectrometry}

We optimized the chromatographic conditions by using various proportions
of methanol in water to achieve optimal peak separation and peak shape
with little peak tailing in a short analysis time, but very poor
chromatography was achieved. Acetonitrile and 0.1\% formic acid in water
(25:75, v/v) were employed. The final chromatographic conditions showed
that PPD behaved similarly to RA in terms of retention and ionization,
and was adopted as the IS.

Methanol was first tested as a pretreatment method for protein
precipitation. However, methanol caused poor efficiency in extracting A1
in this study. Hence, we introduced isopropanol:ethyl acetate (50:50,
v/v) to increase the efficiency of sample extraction, which provided
cleaner extracts and higher IS recoveries. By adjusting the ratio of
isopropanol and ethyl acetate (20:80, v/v) to extracts, the recovery
rates were further increased without compromising extract cleanliness.

A more accurate response for the two analytes was achieved using the ESI
source when compared with the response using the APCI source. In full
scan precursor ion spectra, the most abundant ions of A1, RA, and IS
should be deprotonated molecules with m/z 358, 359, and 164,
respectively (Fig. 2). Parameters such as the ESI source temperature and
flow rate of the desolvation gas have been enhanced to obtain the
maximum response of deprotonated molecules. To obtain the maximum
response of the compound fragments, collision-induced decomposition
(CID) and the SRM collision gas pressure and energy were selected
through the precursor \selectlanguage{english}- product ion transition. The product ion spectra
revealed abundant ions in fragments at m/z 161 for RA and A1 and 146 for
the IS. The characterization of the analyte fragments is shown in Fig.
3.

\emph{3.2. Method validation}

\emph{3.2.1. Specificity}

RA, A1 and IS chromatograms obtained from processed blank plasma samples
were examined to determine the degree of interference by endogenous
plasma components. Fig. 4(a)--(c) shows typical chromatograms of blank
plasma, plasma spiked with IS and plasma samples with A1 and RA. The
retention times of PPD (IS), RA, and A1 were 1.15, 1.38, and 1.53
minutes, respectively, with no endogenous interference or matrix
effects.

\emph{3.2.2. Linearity and LLOQ}

RA and A1 calibration curves showed good linearity over the
concentration range of 5-750 ng/ml. The typical calibration plot
equations and their correlation coefficients were calculated as follows:
RA: \emph{y} = 2.01 x 10\selectlanguage{english}\textsuperscript{-3} \emph{x} + 1.12 x
10\selectlanguage{english}\textsuperscript{-1} (\emph{r\textsuperscript{2}} = 0.9987);
A1:\emph{y} = 2.99 x 10\selectlanguage{english}\textsuperscript{-4} \emph{x} + 3.80 x
10\selectlanguage{english}\textsuperscript{-3} (\emph{r\textsuperscript{2}} = 0.9995). In the
equation \emph{y} = \emph{ax} + \emph{b} , \emph{x} is the concentration
of the analyte in the serum, and \emph{y} is the peak area ratio of the
analytes to the IS. The lower limit of quantitation (LLOQ) of RA and A1
is 5 ng/ml, and the coefficients are 8.14\% and 7.32\%, respectively.
The signal-to-noise ratios of RA and A1 (SN) are 97 and 102,
respectively.

\emph{3.2.3. Precision and accuracy}

The intraday accuracy, interday accuracy and precision were obtained by
measuring the QC samples at 3 concentration levels, as described in
Section 2. The intraday accuracy of RA detection was \selectlanguage{english}-0.21-1.32\%, with
precision below 2.39\%, and that of A1 detection was \selectlanguage{english}-1.03-2.01\%, with
precision below 3.51\%. The interday accuracy of RA detection was
\selectlanguage{english}-2.53-0.42\%, with precision below 1.32\%, and that of A1 detection was
\selectlanguage{english}-0.91-3.01\%, with precision below 4.05\%. Table 1 shows that the
method's overall reproducibility was acceptable.

\emph{3.2.4. Extraction recovery and ionization}

The mean extraction recoveries obtained for 6 replicates of the QC
samples at different concentrations were 89.52 +- 4.35\% (precision,
9.2\%), 87.23 +- 5.17\% (8.2\%), and 90.41 +- 3.65\% (8.2\%) for RA, 85.46
+- 4.19\% (6.60\%), 88.23 +- 4.37\% (7.60\%), and 84.98 +- 5.14\% (7.60\%)
for A1 and 92.18 +- 6.16\% (6.60\%) for the IS. These data were
calculated with precision.

In terms of ionization, the peak area ratios of the two target compounds
and IS after spiking the evaporated plasma samples at three
concentration levels were 96.8-103.2\% and 95.3-103.6\% of the
concentration levels of the neat standard solutions of RA and A1,
respectively, which suggests that the method was free from matrix
effects.

\emph{3.2.5. Stability}

As shown in Table 2, RA and A1 stabilities in the processed samples have
been assessed after freeze-thaw cycles and long-term cold storage at \selectlanguage{english}-20
degC for 14 days. The results show that the three analytes can be kept
stable for 24 h under the conditions of the autosampler after
preparation, 14 days under refrigeration, and through 3 freeze-thaw
cycles in plasma samples. Since the concentrations of RA and A1 have not
significantly changed, the RA and A1 in plasma should be tested within a
specified time under specified storage conditions.

\emph{3.3. Pharmacokinetic studies}

With oral administration of 10 mg/kg RA and A1 to 12 individual
Sprague--Dawley rats, this method was validated and proven to be
suitable for use in PK studies. The concentration-time data of A1 and RA
corresponded to the two-compartment pharmacokinetic model. Fig. 5 shows
the mean plasma concentration-time curve (mean +- SD) of RA and A1 after
administration of a dose. In spite of dosage differences, these RA
values did not differ significantly from those reported by Yang et
al.{[}13{]} and Noguchi-Shinohara et al.{[}14{]}. By replacing the O
atom with an N atom, no significant difference was observed between the
times to reach the maximum A1 and RA plasma concentrations; however,
C\textsubscript{max} increased from 209.3 +- 9.4 to 255.0 +- 20.8 min, and
the area under the plasma concentration-time curve from 0 min to the
time of the last measurable concentration (AUC\textsubscript{0--t}) was
twice that of RA, 16,859.2 +- 1,174.1 ng/ml min, amounting to 36,677.5 +-
4,493.6 ng/ml min. At the terminal phase, the drug elimination half-life
(t\textsubscript{1/2}) also increased from 108.4 +- 13.1 to 366.3 +- 12.2
min. Obviously, from the results, A1 has a higher oral bioavailability
than RA, which may indicate a direction for the structural modification
of such substances. The mechanisms of A1 need to be clarified by
additional studies.

\textbf{4. Discussion}

The current research hotspot is to modify the structure of RA to obtain
drugs with superior efficacy. We prepared N-substituted RA compounds and
completed a preliminary pharmacodynamic evaluation. To clarify the
mechanisms of A1, pharmacokinetic studies of A1 and RA are required. In
this study, a sensitive, simple and rapid HPLC--MS/MS method was adopted
to simultaneously analyze A1 and RA in a single-dose study on rat
plasma. It has been successfully applied to the PK study of RA and A1 in
rats. This result may provide a reference for the modification of the RA
structure.

\textbf{Author Contributions}

Conceived and designed the experiments: SF, WZ and MJ. Performed the
experiments: MJ, SF, QZ and SZ. Analyzed the data: MJ. Contributed
reagents/materials/analysis tools: MJ, SF and QZ. Wrote the paper: MJ.

\textbf{Funding} : This study was supported by the Natural Science
Foundation of Guangxi Province 2020GXNSFBA297042 and Youth Fund Projects
of Guangxi University of Traditional Chinese Medicine, Grant/Award
Number: YSL17023 and 2016QN010. The funders had no role in the study
design, data collection and analysis, decision to publish, or
preparation of the manuscript.

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\textbf{Fig. 1.} Chemical structures of RA (A), A1 (B) and PPD (C).

\textbf{Fig. 2.} Precursor ion and product ion spectra of RA (a), A1 (b)
and PPD (c).

\textbf{Fig. 3.} Characterization of the analyte fragments. (a) m/z 359
\selectlanguage{english}- 161 for RA; (b) m/z 358 \selectlanguage{english}- 161 for A1; and (c) m/z 164 \selectlanguage{english}- 146 for PPD.

\textbf{Fig. 4.} Representative SRM chromatograms of PPD (IS, I), RA
(II) and A1 (III) in rat plasma: (a) a blank rat plasma sample; (b) a
blank rat plasma sample spiked with PPD (100 ng/ml), RA (10 ng/ml), and
A1 (10 ng/ml); (C) a rat plasma sample 30 min after an oral dose of RA
and A1 was administered at 10 mg/kg (calculated as RA) to a
Sprague--Dawley rat.

\textbf{Fig. 5.} Mean plasma concentration--time profiles of RA and A1
administered to 12 Sprague--Dawley rats (mean +- SD).

\textbf{Hosted file}

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