Development of a HPLC-MS/MS Method to Determine the 13 Elements of Semen Cuscutae and Application to a Pharmacokinetic Study in Rats

This study developed a method for simultaneous determination of 13 elements of Semen Cuscutae (quercitrin, quercetin, hyperoside, caffeic acid, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercitrin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, astragalin, and rutin) in rat plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the negative MRM mode. The analytes were analyzed with CORTECS®C18 column (4.6 × 150 mm, 2.7 μm) with mobile phases consisting of 0.1% formic acid in water (A) and acetonitrile (B). The intra- and interday precision of the target compounds were expressed as relative standard deviation (RSD) in the range of 0.5%–10.4%, and the accuracy of the target compounds was expressed as relative error (RE) not exceeding ±14.5% for all analytes. In the meantime, the extraction recovery of the target compounds in plasma samples ranged from 87.4% to 106.2% and matrix effect from 81.0% to 115.5%. The established method was successfully accomplished for the pharmacokinetic study of the analytes in rat plasma samples following oral administration of Semen Cuscutae extract, and the pharmacokinetic parameters of seven compounds were obtained.


Introduction
Semen Cuscutae (Longxuzi, Tusizi), the dry mature seed, belongs to Cuscuta australis R.Br. or Cuscuta chinensis Lam. of Convolvulaceae family [1,2]. It was first recorded in the "Shen Nong's Herbal" as an upper grade drug [1]. As a wellknown traditional Chinese medicine, Semen Cuscutae has numerous pharmacological functions, such as regulating the body's endocrine system, nourishing the liver and kidney, improving eyesight, and preventing miscarriage [3,4]. It has also been reported to have neuroprotective, hepatoprotective, antioxidative, osteoblastogenic, and immunomodulatory properties and to have positive effects on chronic prostatitis [5][6][7].
ere are many natural active ingredients in Semen Cuscutae, which includes flavonoids, lignans, polysaccharides, alkaloids, and other chemicals from Semen Cuscutae between normal and kidney-deficiency rats. e metabolism process in vivo of six flavonoids were clarified and showed prospective results in their study. However, it is insufficient to study the pharmacokinetics of only one kind of component, and other active ingredients from Semen Cuscutae need to be further investigated too.
As we know, study on the pharmacokinetics of traditional Chinese medicine can elucidate its material basis and explain the mechanism of drug absorption, distribution, metabolism, and excretion in vivo [14]. erefore, in our study, we simultaneously separated and determined 13 elements from Semen Cuscutae extract by a selective HPLC-MS/MS method and analyzed their pharmacokinetics data.
is research would contribute to the understanding of the metabolism of these elements, as well as the mechanism of action of Semen Cuscutae.

Analytical Standards and Reagents.
Methanol and acetonitrile (HPLC pure grade) were purchased from Fisher Co., Ltd. Formic acid was of chromatographic purity obtained from ROE Co., Ltd. Ultrapure water for the HPLC-MS/MS analysis was purified by Milli-Q water purification system (Millipore, Milford, MA, USA). Hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercitrin, cryptochlorogenic acid, isorhamnetin-3-Oglucoside, astragalin, and liquiritin (internal standards, IS) were obtained from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China). Quercitrin and quercetin were purchased from National Institutes for Food and Drug Control. Semen Cuscutae was purchased from Anguo, Hebei province. e structures of 13 compounds are displayed in Figure 1.

Instruments and Experimental Conditions.
e electrospray ionization (ESI) source was used to connect the HPLC-MS/MS system consisting of an Agilent 1200 high-performance liquid chromatography equipped with an Agilent 6430 series triple quadrupole mass spectrometer (Agilent Technologies, USA). e target compounds and IS were separated on the CORTECS ® C 18 column (4.6 × 150 mm, 2.7 μm) with mobile phases consisting of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. e gradient elution method as follows: 0-5 min, 25%-75% B; 5-7 min, 75%-95% B; 7-12 min, 95%-95% B. And the column balance procedure was 12-13 min, 95%-25% B; 13-17 min, 25%-25% B. e column temperature was maintained at 30°C and the injection volume set at 5 μL. e data obtained were processed using Mass Hunter workstation software (Agilent Technologies, USA).

Semen Cuscutae Extract Preparation. 3 kg of Semen
Quality control (QC) samples included low, middle, and high concentrations, prepared with the appropriate mixed stock solutions and blank plasma sample to meet the desired concentration. All the solutions were stored at − 4°C.
2.5. Treatment of Plasma. 20 μL of methanol, 20 μL of IS (liquiritin, 200 ng/mL) and 20 μL of formic acid were added to 100 μL of plasma sample and then vortex-mixed. e mixture was extracted with 800 μL of acetonitrile by vortexing for 3 min. After centrifugation at 14000 rpm for 10 min, the supernatant was transferred to a clean glass tube and dried with nitrogen. e residue was reconstituted in 100 μL of methanol and then centrifuged at 14000 rpm for 10 min. 5 μL of supernatant was injected into the LC-MS/ MS system for analysis.
2.6. Method Validation. Specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, extraction recovery, matrix effect, and stability for the method were validated based on the guidelines published by regulatory authorities [15].

Specificity.
e specificity was investigated by analyzing the chromatography of blank plasma samples from six different rats to determine whether the endogenous substances in the sample would affect the quantitative analysis of each component.

Linearity and LLOQ.
e linearity was achieved by spiking rat plasma with the mixed standard solution and IS in a series of concentrations. e calibration curves were constructed with peak-area ratio of analyte to IS (y) against concentration of the calibration standard (x), with 1/x 2 as the weighing factor. LLOQ was evaluated according to the lowest concentration of standard curve at which the signalto-noise ratio (S : N) was about 10 : 1.

Matrix Effect and Extraction Recovery.
e extraction recovery was assessed by comparing the peak areas obtained from pretreatment procedures samples with those from postextracted spiked samples. e matrix effect of the 13 analytes was determined by comparing the peak area obtained from postextracted spiked samples to that from pure standards solutions. All the extraction recovery and matrix effect experiments were evaluated in six replicates with three concentration levels.

Stability.
e stability of the 13 analytes was evaluated by QC samples in different processing and storage conditions, including short-term stability (putting the analytes in ambient temperature for 4 h and storing analytes in autosampler for 12 h after treatment), long-term stability (storing the analytes at − 70°C for 21 days) and subjecting analytes to three freeze-thaw cycles. All stability experiments were tested in six parallels with low, middle, and high concentrations.

Pharmacokinetic Studies.
Six Sprague-Dawley rats (SPF, 200 ± 10 g, male) were purchased from HFK Laboratory Animal Technology Co., Ltd. e rats were housed in a standard laboratory condition (12 h dark-light cycle; temperature was 25°C ± 2°C and humidity was kept 50 ± 5%) and fed standard dry pellet diet and water for one week for acclimatization. Before the experiments, the rats were in fasting state for 12 h, with free access to water. e Semen Cuscutae extracts were dissolved in 0.5% carboxymethyl cellulose-sodium, prepared into suspension and gavage to the rats at a dose of 13 g/kg. Approximately 200 μL of rat blood samples was collected from the orbital venous plexus at 0, 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h after oral administration into 1.5 mL heparinized centrifuge tube. After centrifugation at 7000 rpm for 10 min immediately, the supernatant was collected into a new centrifuge tube and stored at − 70°C until analysis.

Method Development.
In order to improve sensitivity and shorten analysis time, we tested different columns and various mobile phase systems. Comparing different columns such as CORTECS ® C 18 column (4.6 mm × 150 mm, 2.7 μm), Xbridge ™ C 18 column (2.1 mm × 150 mm, 3.5 μm), and Xbridge ™ C 18 column (4.6 mm × 50 mm, 2.5 μm), and various mobile phase systems such as acetonitrile-water or 0.1% formic acid in water and methanol-water or 0.1% formic acid in water, we found that the sensitivity and signal response of compounds analyzed with CORTECS ® C 18 column (4.6 mm × 150 mm, 2.7 μm) and acetonitrile-water containing 0.1% formic acid were more satisfactory.  Figure 2 suggested no interfering peaks from the endogenous matrix in rat blood sample.

Linearity and Sensitivity.
e data of the linear equation, correlation coefficients, linearity ranges, and LLOQ of all target compounds are listed in Table 2. e correlation coefficients of all analytes were greater than 0.9903, indicating that the 13 analytes in the plasma sample had good linearity in the corresponding concentration range. LLOQ with S/N ratio >10 ranged from 1-2 ng/mL of the 13 analytes, showing that the above method developed is suitable for quantitative pharmacokinetic studies.

Precision and Accuracy.
In this experiment, all results of the intra-and interday precision and accuracy were analyzed at three different concentration levels, including low, medium, and high concentrations in six replicates, as displayed in Table 3. Intra-and interday RSD for the analytes ranged from 0.5%-10.4% and 0.8-7.9%, respectively. RE of accuracy did not exceed ±14.5% for all analytes. e results implied that this method is reliable and accurate for the study of the above target compounds in rat plasma.

Extraction Recovery and Matrix Effect.
e results of extraction recovery and matrix effect are given in Table 4. e extraction recoveries of the analytes in rat plasma sample at three concentration levels ranged from 87.4% to 106.2%. e matrix effects of the target compounds were in the range of 81.0-115.5%. e data showed that the process of the experiment is efficient and there was no significant matrix effect observed for the plasma sample tested.

Stability.
Stability data of the 13 analytes in different conditions are listed in Table 5. All analytes subjected to different processing and storage conditions had an acceptance criterion in the range of 1.1%-11.1% for QC sample at three concentration levels.
e results suggested that the analytes had a satisfactory stability for storage and analytical process.

Pharmacokinetic Application.
e validated method was successfully applied to a pharmacokinetic study of orally administered Semen Cuscutae extract (13.0 g/kg) in rats with the determination of 13 active ingredients of Semen Cuscutae in rat plasma. e corresponding pharmacokinetic parameters are listed in Table 6. Mean plasma concentration-time profiles of target compounds were illustrated in Figure 3.
In our experiment, 6 ingredients, namely, quercitrin, quercetin, apigenin, kaempferol, luteolin, and cryptochlorogenic acid were found to be of low content in vivo, and their concentrations after 15 minutes of intragastric administration were lower than the LLOQ. erefore, complete pharmacokinetic curves of the above 6 compounds were not obtained. In the previous studies, researchers have explored the pharmacokinetics rules of kaempferol and quercetin [16,17]. Compared with our study, the dose of oral administration was significantly higher than ours, and caused by the low oral bioavailability of them, we could just detect them at a few time points, and a whole mean plasma concentration-time curve could not be obtained. Hence, their pharmacokinetic parameters were not further discussed.
e T max of hyperoside, chlorogenic acid, isoquercitrin, isorhamnetin-3-O-glucoside, and astragalin was less than 1 h. ese results showed that the 5 ingredients were absorbed quickly in vivo. e elimination half-life (t 1/2 ) of caffeic acid and rutin was more than 7.5 h, suggesting that caffeic acid and rutin are present in the body for a longer time, and may exert continuous therapeutic action, enhancing clinical efficacy. Similar pharmacokinetic trends have been reported in previous studies [18,19]. In addition to this, the t 1/2 of hyperoside, astragalin, isoquercitrin, chlorogenic acid, and sorhamnetin-3-O-glucoside was less than 1.5 h, indicating that the above compounds were Evidence-Based Complementary and Alternative Medicine 5  (1). Quercetin (2). Hyperoside (3). Caffeic acid (4). Rutin (5). Chlorogenic Acid (6). Luteolin (7). Apigenin (8). Kaempferol (9). Isoquercitrin (10). Cryptochlorogenic Acid (11). Isorhamnetin-3-O-glucoside (12). Astragalin (13). IS (14).  Evidence-Based Complementary and Alternative Medicine       In the previous study of our research group, we have established a HPLC-MS/MS method to explore the pharmacokinetic rule of rutin from mulberry leaves [16]. e results were different from this study. In this research, we found that rutin had an isomeride from Semen Cuscutae extract, that is, the isomeride with a high concentration. However, its plasma concentration was low. So, we guessed this isomeride may transform into rutin after oral administration in vivo. us, in the pharmacokinetic application, C max of rutin is higher than that of other components.

Conclusions
A validated and selective method of HPLC-MS/MS for simultaneous quantification of 13 compounds of Semen Cuscutae was established in this study, and the pharmacokinetics of Semen Cuscutae extract in rats was investigated. We found that the content of quercitrin, quercetin, apigenin, kaempferol, luteolin, and cryptochlorogenic acid were at a lower level in vivo after oral administration, which could only be detected at a few time points. Meanwhile, other compounds except caffeic acid and rutin had shorter elimination half-life. e pharmacokinetic parameters indicate the metabolism rate of these elements and may provide references for further research of Semen Cuscutae.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.