Establishment of a UPLC-MS/MS Method for Studying the Effect of Salt-Processing on Tissue Distribution of Twelve Major Bioactive Components of Qing'e Pills in Rats

Qing'e pills is clinically used for treating osteoporosis in postmenopausal women in China. Eucommiae Cortex and Psoraleae Fructus are the main herbs of Qing'e pills and are both required to be salt-processed. In order to study the influence of salt-processing on the tissue distribution of Qing'e pills, a UPLC-MS/MS method was established for studying the tissue distribution of 12 main bioactive ingredients of Qing'e pills in rats. The linear relationships of the 12 compounds in each tissue were good. The method was fully validated for its selectivity, accuracy, precision, stability, matrix effect, and extraction recovery. Then, the validated method was successfully applied for simultaneous determination of the 12 chemical components in Qing'e pills in tissues for the first time. Areas under the curve (AUC) results showed that, except for pinoresinol diglucoside, psoralen, and isopsoralen, the distribution of the other components was increased in the kidney, uterus, ovary, and testes. Relative targeting efficiency (RTE) results showed that all 12 chemical components targeted the kidney and sexual organs. The results indicated that the Eucommiae Cortex and Psoraleae Fructus after salt-processing could significantly increase the distribution of components to the kidney and generative organs.


Introduction
Qing'e pills is included in the 2015 edition of the Chinese Pharmacopoeia, which has the effect of strengthening the kidney and waist [1]. It is clinically used for the treatment of osteoporosis in postmenopausal women. e causes of osteoporosis in postmenopausal women are mainly ascribed to estrogen deficiency, endocrine dysfunction, and metabolic disorder. Moreover, the postmenopausal osteoporosis is characterized by an imbalance in bone formation and bone resorption. Because bone resorption function is superior to bone formation, an imbalance occurs in bone remodeling. Qing'e pills consist of Eucommiae Cortex (salt-processed), Psoraleae Fructus (salt-processed), Juglandis Semen (fried), and Allii Sativi Bulbus [1], of which Eucommiae Cortex and

UPLC-MS/MS Instruments and Analytical Conditions.
e UPLC-MS/MS system consisted of a Shimadzu UPLC system, which was equipped with a LC-10 ATvp binary pump (Shimadzu Corporation UFLC XR, Kyoto, Japan) and a 5,500 triple quadrupole mass spectrometer. e latter was equipped with an electrospray ionization (ESI) source (AB SCIEX, Foster City, CA, USA). e separation of the analytes was achieved on a Waters BEH-C 18 column (100 mm × 2.1 mm, 1.7 μm).
e chromatographic conditions in negative and positive ionization modes were as follows: injection volume of 2 μL; column temperature at 40°C; and flow rate at 0.3 mL/min. e mobile phase was composed of 0.1% formic acid aqueous solution (A)-acetonitrile (B). e gradient elution procedures in the negative ionization mode were as follows: 0-1 min, 5% B; 1-1. e optimized parameters were as follows: ion source temperature (TEM), 550°C; curtain gas (CUR), 35 psi; ion source gas 1 (GAS1), 55 psi; ion source gas 2 (GAS2), 55 psi; and ion spray voltage (IS), 5,500 V. e multiple reaction monitoring (MRM) was chosen for the quantification of the components. e precursor ions, product ions, declustering potential (DP), and collision energy (CE) for each analyte and IS in negative and positive ionization modes are shown in Table 1.

Preparation of Crude and Salt-Processed Qing'e Pills
Extracts. Crude (500.0 g) and salt-processed (500.0 g) Qing'e pills were soaked in 4,000 mL 95% ethanol and pure water, heated, and refluxed twice for 1 h. e extracts were combined and condensed to 500 mL separately to yield crude and salt-processed Qing'e pills extracts. 1 mL of concentrated liquid was equivalent to 1 g of Qing'e pills. Final solutions were stored at 4°C before use.

Preparation of Calibration Standards and Quality Control
Samples.
e stock solutions of geniposidic acid, pinoresinol diglucoside, psoralenoside, psoralen, isopsoralen, isobavachin, neobavaisoflavone, bavachin, bavachalcone, isobavachalcone, and bavachinin were prepared by dissolving accurately the weighed reference substance in methanol at a concentration of 1 mg/mL. Psoralidin was prepared at a concentration of 0.5 mg/mL. e stock solutions were diluted into serial standard solutions. e stock solution of IS, including rhein (negative ionization mode) and scoparone (positive ionization mode), were prepared by dissolving rhein (2.5 mg) and scoparone (5.0 mg) in methanol at concentrations of 50 μg/mL and 1 mg/mL, respectively. Working solutions of IS were prepared by serial dilution of the stock solutions with methanol at a concentration of 500 ng/mL for rhein and 100 ng/mL for scoparone.

Journal of Analytical Methods in Chemistry 3
Calibration samples were prepared by using 90 μL of blank tissue, 10 μL of standard serial solution, and 10 μL of IS (50 ng/mL rhein and 100 ng/mL scoparone) to make the equivalent concentration of 1, 5, 10, 50, 250, 500, and 1,000 ng/m L of various tissue samples. Quality control (QC) solutions of the 12 compounds were prepared by adding standard solution with specified concentration into blank tissue, the samples with low, medium, and high concentrations according to the operation under "2.2." All solutions were stored at 4°C before use.

Method Validation.
Method validation was performed according to FDA's Guidance for Industry on Bioanalytical Method Validation [20].

Specificity.
e specificity of the method was investigated by comparing chromatograms of blank tissue homogenate samples, blank tissue homogenate samples spiked with standard solution and IS, and treated tissue homogenate samples.

Linearity and Quantification.
Various concentrations of 12 active ingredient calibration standard solutions with IS rhein (50 ng/m L, negative ionization ion mode) and scoparone (100 ng/m L, positive ionization ion mode) were added to blank tissue treated as tissue samples and assayed by using UPLC-MS/MS. e calibration curve was established via the 1/x 2 weighted linear least squares regression model. LLOQ had the lowest concentrations with signal-tonoise ratio ≥10, evaluated by analyzing samples in six replicates. e lower limit of detection (LLOD) was defined as the amount that could be detected with a signal-to-noise ratio ≥3.

Precision and Accuracy.
Accuracy and precision of the method were determined by repeated analyses of QC and LLOQ samples. e intraday precision and accuracy of the method were assessed by determining QC samples six times within a single day, while the intraday precision and accuracy were estimated by determining QC samples over three consecutive days.

Recovery and Matrix Effects.
e extract recovery was calculated by comparing the peak areas of extracted QC samples with peak areas of 12 active ingredients reference standard solutions. Matrix effects of the method were determined by comparing peak areas of blank tissue extracts spiked with standard samples with peak areas of neat standard solution.

Stability.
e stability of analytes in tissues was evaluated by measuring three concentrations of the QC samples (n � 6) under different conditions. e short-term stability was investigated by exposing the QC samples at 25°C for 4 h. e long-term stability was assessed after storing the QC samples at −20°C for 30 days. Freezingthawing stability was determined after QC samples underwent three freezing-thawing cycles by freezing at −20°C and thawing at 37°C in a waterbath.

Tissue Distribution Study.
A total of 84 Sprague-Dawley rats, half male and half female, were randomly divided into two groups (crude and salt-processed Qing'e pills groups). e rats were fed for a week and fasted for 12 h before the experiment.
is experiment has been approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine, license Number: 201903A011.
ese two groups of rats were orally administered raw and salt-processed extracts at the same dose of 1.2 mL/200 g of body weight. e rats were sacrificed at 10, 30, 90, 180, 360, 480, and 720 min (for each time point, 6 rats were sacrificed for each group, half male and half female). e heart, liver, spleen, lung, kidney, ovary, uterus, and testicular tissue samples were collected from the rats, the surface blood was washed with 0.9% normal saline solution, and the tissues were dried with filter paper. Different organ tissues were weighed, and two volumes of iced normal saline solution were added to obtain the homogenates, which were stored at −20°C for further analysis. Each tissue homogenate (90 μL) was placed in a 1.5 mL centrifuge tube, and 10 μL of the internal standard solution was added. Acetonitrile (300 μL) was added, vortexed for 5 min, and centrifuged at 11308.75 ×g for 5 min. e supernatants were transferred into the 1.5 mL Eppendorf tube, and after centrifugal concentration, 100 μL methanol was added and centrifuged at 11308.75 ×g for 5 min before being vortexed for 5 min. Finally, 80 μL of the supernatants was used for UPLC-MS/MS.

Targeting Efficiency Evaluation.
e purpose of saltprocessing of Qing'e pills in TCM theory is somewhat similar to modern drug target-delivery theory. AUC and RTE were utilized to investigate the effect of salt-processing on tissue distribution of Qing'e pills compounds. e relevant parameters were calculated according to previously described equations [21,22]: AUC sum � AUC heart + AUC liver + AUC spleen + AUC lung + AUC kidney + AUC ovary(testis) + AUC uterus(male rat is 0) . (1) In these equations, AUC sum involves the sum of AUC of all tissues in salt-processed and crude groups, respectively. e calculation formula is as the following equation:

Method Optimization of UHPLC-MS/MS Conditions
3.1.1. Specificity. As shown in Figure 2, the peak shapes measured under experimental conditions did not affect each other, and the endogenous substances in the heart, liver, spleen, lung, kidney, ovary, uterus, and testes did not interfere with the peaks of the detected components in each group.

Linearity and Sensitivity.
Under UPLC-MS/MS conditions, the equations for the calibration curves, correlation coefficient, linear range, and lower limit of quantification (LLOQ) of 12 bioactive ingredients are shown in Table 2. e results showed that the linear relationships of the 12 compounds in the linear range of the methanol solution and each tissue were good, and the ranges of concentrations and limits were suitable for the determination of Qing'e pills extract content and tissue distribution.

Precision and Accuracy.
e precision and accuracy of the assay were evaluated using QC samples at low, medium, and high concentrations. e results for kidneys are shown in Table 3. e accuracy and precision of the method were within the specified range, meeting the relevant requirements for biological sample determination.

Extract Recovery and Matrix Effect.
e extract recoveries and matrix effects in kidney are presented in Table 4. e extract recoveries of QC samples at low, medium, and high concentrations were 81.59-90.84%, while the matrix effects of QC samples were 90.85-96.38%. e RSD of extract recoveries was less than 7.28%, and the matrix effects were less than 9.39%. e results indicated that the method was suitable for the treatment of kidney tissue samples in this experiment.

Stability.
e results of stability in the kidney tissue under different storage conditions are presented in Table 5.
e samples were all stable, and the storage conditions did not affect the determination of the chemical components in the experimental tissue samples.

Discussion.
Except for pinoresinol diglucoside, psoralen, and isopsoralen, the tissue distribution of other nine ingredients were increased after salt-processing in general. e distribution of psoralenoside, bavachin, isobavachin, bavachalcone, isobavachalcone, neobavaisoflavone, and bavachinin into the kidney increased. e distribution of geniposidic acid in the liver increased after salt-processing. e distribution of psoralidin into the spleen increased after salt-processing. Since the difference between the salt-processed group and the crude group was deducted from the in vitro content determination, the increase in the distribution of these nine components in the organs after salt-processing was not related to the concentration difference. e increased distribution of most components in the organs of the salt-processed group may be related to the high osmotic pressure caused by salt [23]. e decreased distribution of pinoresinol diglucoside and psoralen in organs may be due to the transformation of salt and other chemicals in vivo.
RTE showed that all twelve chemical components targeted the kidney or sexual organs (uterus, ovary, and testes) to some extent. After salt-processing, the components targeting the kidney were psoralen, isopsoralen, bavachin, isobavachin, bavachalcone, isobavachalcone, neobavaisoflavone, and psoralidin. e components targeting the uterus, ovary, and testes were geniposidic acid, pinoresinol diglucoside, psoralen, isopsoralen, psoralenoside, bavachin, isobavachin, bavachalcone, isobavachalcone, neobavaisoflavone, psoralidin, and bavachinin. Bavachalcone, isobavachin, bavachinin, psoralenoside, neobavaisoflavone, and psoralen targeted the kidney and sexual organs the most. e results of AUC and RTE indicated that salt-processing in Qing'e pills not only can enhance the dissolution of the effective ingredients but also increase the targeting of drugs into the kidney and sexual organs, which may be the potential reason for Qing'e pills to play the role of tonifying the liver and kidney and strengthening muscles and bones.
One of the main functions of TCM salt-processing was to tonify the kidney, and from the perspective of TCM syndrome differentiation and treatment, the main bone of the kidney gave birth to marrow, which was the innate basis, so salt-processing plays an important role in the treatment of osteoporosis [24,25]. e bioactive ingredients in Qing'e pills have certain pharmacological activities. Bavachalcone can inhibit osteoclastogenesis by interfering with the ERK and Akt signaling pathways and the induction of c-Fos and NFATc1 during differentiation [26]. Isobavachin, bavachinin, psoralenoside, neobavaisoflavone, and psoralen may be associated with isopentenyl and estrogen-like activity in the treatment of osteoporosis [3,[27][28][29]. Pinoresinol diglucoside, bavachin, and isobavachalcone targeted the lung. Geniposidic acid, pinoresinol diglucoside, isobavachin, and bavachalcone targeted the liver. e results indicated that salt-processing could strengthen the estrogen-like activity of salt. is enhances Qing'e pills's antiosteoporosis effect.

Conclusion
A sensitive and accurate UPLC-MS/MS for simultaneous determination of 12 bioactive components of Qing'e pills in different tissues of rats. is study investigated the effects of salt-processing on the tissue distribution of 12 main bioactive ingredients. After salt-processing, the tissue distribution of bavachalcone, isobavachin, bavachinin, psoralenoside, neobavaisoflavone, and psoralen were increased in the kidney and sexual organs. e result provided a basis for clinical usage of Qing'e pills.

TEM:
Ion source temperature CUR: Curtain gas GAS: Ion source gas MRM: Multiple reactions monitoring DP: Declustering potential CE: Collision energy AUC: Area under the curve ESI: Information-dependent acquisition IS: Internal standard LLOD: e lower limit of detection LLOQ: e lower limit of quantitation QC: Quality control RTE: Relative targeting efficiency SD: Sprague-Dawley TCM: Traditional Chinese medicine UHPLC-MS/ MS: Ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Data Availability
e main data used to support the findings of this study are included within the article. e methodological data for organs other than kidney's data used to support the findings Journal of Analytical Methods in Chemistry 13 of this study are included within the supplementary information file.

Disclosure
Jingxia Hou and Shangyang Lin contributed equally to the project and are considered as the co-first authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.