An LC–MS/MS method for the determination of salidroside and its metabolite p-tyrosol in rat liver tissues

Abstract

Context: Salidroside and its metabolite p-tyrosol are two major phenols in the genus Rhodiola L. (Crassulaceae). They have been confirmed to possess various pharmacological properties and are used for the prophylaxis and therapeutics of many diseases. Several analytical methods have been developed for the determination of the two compounds in plant materials and biological plasma matrices. However, these methods are not optimal for biological samples containing complex organic interferences, such as liver and brain tissues.

Objective: This study aimed to further develop and validate a simple and specific LC–MS/MS method for the determination of salidroside and its metabolite p-tyrosol in rat liver tissues using paracetamol as the internal standard (IS).

Materials and methods: Salidroside and p-tyrosol with the IS paracetamol and liver tissues were used as model compounds and biological samples. Samples were processed by protein precipitation (PP) with methanol, the supernatant was dried under nitrogen and the residue was reconstituted in a mobile phase that consisted of a mixture of acetonitrile and water (1:9, v/v). Salidroside and p-tyrosol were detected in negative mode under multiple reaction monitoring (MRM) by a triple quadrupole tandem mass spectrometer coupled with electrospray ionization.

Results: Standard curves were linear over the concentration range of 50–2000 ng/mL with correlation coefficients of 0.995 or better for both salidroside and p-tyrosol. The intra- and inter-day accuracy for salidroside ranged between 104.90 and 112.73% with a precision of 3.51–14.27%. For p-tyrosol, the intra- and inter-day accuracy was between 92.38 and 100.59%, and the precision was 8.54% or less. The stability data showed that no significant degradation occurred under the experimental conditions. The recoveries were 111.44, 108.10, and 102.00% for salidroside at concentrations of 50, 500 and 2000 ng/mL, respectively, and were 105.44, 105.50, and 113.04% for tyrosol at concentrations of 50, 500 and 2000 ng/mL, respectively. The matrix effects were 83.85–92.45% for salidroside and 85.61–92.49% for p-tyrosol at three QC levels. This method was successfully applied to a liver tissue distribution study of salidroside and its metabolite p-tyrosol in rats.

Discussion and conclusion: This newly established method is validated as simple, reliable and accurate. It can be used as a valid analytical method for the intrinsic quality control of biological matrices, especially tissue samples.

• LC–MS/MS
• method validation
• p-tyrosol
• salidroside
• tissue distribution

Introduction

Rhodiola rosea L. is a popular medicinal plant in the Crassulaceae family found in mountains at high altitudes. It has been used in the traditional Tibetan medicine system as an adaptogen to enhance the body's resistance to fatigue. The plant displays a range of pharmacological properties, including anti-inflammation, antihypoxia, antioxidative, anti-aging, anticancer and hepatoprotection activities (Díaz Lanza et al., 2001; Kanupriya et al., 2005; Kelly 2001; Kučinskaitė et al., 2004; Nan et al., 2003; Zhang et al., 2007, 2009). Phenolic glycosides are a group of glycosides in which a sugar is bound to the phenolic compounds via a glycosidic bond (Tahvanainen et al., 1985). Salidroside (Figure 1a), a natural phenol glycoside, is extracted from the root of Rhodiola rosea as one of its main active ingredients and is responsible for all the documented pharmacological effects of this medicinal plant (Chen et al., 2008). The neuroprotective effects of salidroside against hydrogen peroxide (H₂O₂)-induced apoptosis (Zhang et al., 2007) could markedly attenuate H₂O₂-induced cell viability loss and apoptotic cell death in a dose-dependent manner. The mechanisms by which salidroside protects neuronal cells from oxidative stress include the induction of several antioxidant enzymes (thioredoxin, heme oxygenase-1 and peroxiredoxin-I), the downregulation of the pro-apoptotic gene Bax and the upregulation of the anti-apoptotic genes Bcl-2 and Bcl-X(L) (Wu et al., 2008). p-Tyrosol (Figure 1b, 4-(2′-hydroxyethyl) phenol), the aglycone of salidroside, is another major active constituent in Rhodiola (Mao et al., 2007a). p-Tyrosol relieves mental and physical fatigue, enhances memory and favorably influences the overall physical state. Commercially, p-tyrosol is known as an intermediate product in the synthesis of β-choline, some cardiovascular drugs and biologically active polymers (Storozhok et al., 2002). Clinical data indicated that p-tyrosol may counteract the reactive oxygen metabolite that mediates cellular damage and related diseases by improving in vivo antioxidant defenses (Giovannini et al., 1999).

Figure 1. Chemical structures of (a) salidroside, (b) p-tyrosol and (c) paracetamol (internal standard, IS).

Several analytical methods have been developed for the determination of salidroside in plant materials and biological plasma matrices, including liquid chromatography with ultraviolet (UV) detection (HPLC–UV) (Mao et al., 2007b), liquid chromatography with mass spectrometry (LC–MS) (Yu et al., 2008; Zhang et al., 2008) and liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) (Chang et al., 2007; Li et al., 2006). The simultaneous determination of salidroside and p-tyrosol in Rhodiola was established by HPLC (Mao et al., 2007a; Wang et al., 2006). In our previous study, p-tyrosol was identified as the deglycosylated metabolite of salidroside in plasma samples after intravenous (i.v.) administration at a dose of 50 mg/kg, but was not detectable after intragastric gavage (i.g.) administration through using HPLC–photodiode array detection (PDA) and LC–MS/MS analysis (Guo et al., 2012b). Many methods, such as HPLC with UV detection, fluorescence detection (FLD), mass spectrometry (MS) and gas chromatography–mass spectrometry (GC–MS), have been developed for the determination of compounds in rat tissue samples and successfully applied to pharmacokinetic and tissue distribution studies. The sample preparation methods used for method development included PP, liquid–liquid extraction (LLE) and solid-phase extraction (SPE) (Guo et al., 2012a; Liang et al., 2013; Jia et al., 2013; Wang et al., 2007, 2009; Zheng et al., 2012). However, until now, few studies in the literature have measured salidroside and p-tyrosol in biological tissues, such as the liver and brain, due to their complexity.

In the present study, we further developed and validated a simple and specific LC–MS/MS method for the determination of salidroside and its metabolite p-tyrosol in rat liver tissues using paracetamol (structure shown in Figure 1c) as the internal standard (IS). The method has been successfully applied to a tissue distribution study in rat liver tissues after i.v. administration of 50 mg/kg salidroside.

Materials and methods

Chemicals and reagents

Salidroside was purchased from the National Institutes for Food and Drug Control (Beijing, China). Paracetamol, the IS (purity≥98%), was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). HPLC-grade acetonitrile was obtained from Sigma (St. Louis, MO). Ultra-pure water was produced by a Millipore Milli-Q system (Billerica, MA). All other reagents or solvents used were commercially available and of reagent grade. Blank rat liver tissues were collected from healthy male Wistar rats weighing 200 ± 20 g (Laboratory Animal Center of Jilin University, Changchun, Jilin Province, China).

Healthy male Wistar rats (Imprinting Control Region (ICR), 200 ± 20 g) were obtained from the Laboratory Animal Center of Jilin University (Changchun, Jilin Province, China). Animal handling procedures were in accordance with the standard operating procedures approved by the institutional animal care and use committee. All rats were dosed following an overnight fasting (except for water).

Chromatographic conditions

Chromatographic conditions were carried out according to our previously published method (Guo et al., 2012b) with minor modifications. The quantitative analysis of salidroside and p-tyrosol was performed using a LC–MS/MS system composed of an Alliance 2695 HPLC and a Quattro-Micro^TM mass spectrometer (Waters Co., Milford, MA). A C18 mass column (3.5 µm, 50 × 3.0 mm, xTerra, Kings Beach, CA) was used for separation and was equilibrated and eluted with an isocratic mixture of acetonitrile and water (1/9, v/v) at a flow rate of 0.3 mL/min. The injection volume was 20 µL. The total run time was 4.5 min. The retention time of salidroside, p-tyrosol and the IS were 1.98, 2.85 and 1.86 min, respectively. Each wash cycle consisted of 200 μL of strong wash solvent (acetonitrile/H₂O, 9/1, v/v) and 600 μL of weak wash solvent (acetonitrile/H₂O, 1/9, v/v). The analytes were monitored in MRM mode using negative electrospray ionization (ESI). The precursor/product transitions (m/z) were 299.0→118.8 for salidroside, 137.0→118.9 for p-tyrosol and 150.1→106.9 for paracetamol, the IS. Major fragmentation pathways were observed for deprotonated salidroside, p-tyrosol and the IS (M–H)⁻ ions and the corresponding mass spectra are shown in Figure 2. The ESI–MS/MS detection was performed under the following conditions: extractor 2.00 V, RF Lens 0.1 V, desolvation gas 500 L/h, cone gas 50 L/h, source temperature 120 °C and desolvation temperature 350 °C, and the corresponding cone voltage and collision energy are shown in Table 1. Waters MassLynx 4.0 software (Waters Corporation, Milford, MA) was used for system control and data acquisition.

Figure 2. Product ion mass spectra of [M−H]⁻. (a) Salidroside ([M−H]⁻, m/z 299.0), (b) p-tyrosol ([M−H]⁻, m/z 137.0), and (c) the IS (paracetamol) ([M−H]⁻, m/z 150.1).

Table 1. ESI–MS/MS parameters for salidroside, p-tyrosol and IS.

Analyte	Precursor ion (m/z)	Daughter ion (m/z)	Dwell time (s)	Cone voltage (V)	Collision energy (eV)
Salidroside	299.0	118.8^a, 179.0	0.2	28	14
p-Tyrosol	137.0	105.8, 118.9^a	0.2	35	16
IS	150.1	106.9^a	0.2	35	20

^aIon for quantification.

Preparation of liver homogenate

After collecting blood using cardiac puncture, the rats were dissected and the liver tissues were excised. The blood was flushed off from the liver tissues with phosphate buffer solution (pH = 7.4). The tissues were gently blotted with absorbent paper, weighed and sliced coarsely with scissors. They were then added to phosphate buffer solution (pH = 7.4) (50% tissue w/v) and homogenized in a homogenizer. After being transferred to a centrifuge tube, the liver tissue homogenate was stored at −20 °C until analysis.

Preparation of standard and quality control samples

Stock solutions of salidroside, p-tyrosol and the IS (paracetamol) were prepared at 200 μg/mL in acetonitrile–water (1:9, v/v), then further diluted using acetonitrile–water (1:9, v/v), giving a series of working solutions. All solutions were stored at −20 °C until use.

Calibration curves were prepared by spiking 20 μL of the appropriate working solution with 100 μL of blank rat liver tissue homogenate. The effective concentrations were 50, 100, 200, 500, 1000, 1500 and 2000 ng/mL for both salidroside and p-tyrosol. The quality control (QC) samples were prepared in a pool as a single batch at concentrations of 50, 500 and 2000 ng/mL and then divided into aliquots and stored at −20 °C until use. The IS working solution of 200 ng/mL was diluted from the stock solution. The spiked rat liver tissue homogenate samples (standards and QCs) were treated following the sample processing procedure on each analytical batch along with the unknown samples.

Sample processing

A liver tissue homogenate sample (100 μL) (blank liver tissue homogenate, spiked liver tissue homogenate or liver tissue distribution study sample) and 20 µL of IS working solution were pipetted into a 1.5-mL polypropylene tube, then 280 µL of methanol was added followed by vortex mixing for 1 and 10 min of ultrasonic incubation. After centrifuging at 45 000 g for 5 min, the clear supernatant was transferred to a new tube and evaporated to dryness under a nitrogen stream. The residue was reconstituted in 200 µL of the mobile phase. After centrifuging at 45 000 g for 10 min, an aliquot of 20 µL was injected into the LC–MS/MS system.

When the concentration of salidroside in a liver tissue homogenate sample was over the range of the calibration curve, appropriate dilutions were applied to the sample with blank rat liver tissue homogenate before sample processing.

Method validation

The method was evaluated for specificity, precision, accuracy, extraction efficiency, matrix effects and stability based on the FDA guidelines for bioanalytical method validation (Food and Drug Administration, 2001).

The specificity and selectivity of the method were assessed by comparing the chromatograms of six different batches of blank liver tissue homogenate samples. Peak areas of endogenous and exogenous compounds co-eluting with the analytes should not exceed 20% of the peak area of the lower limit of quantitation (LLOQ) standard and less than 5% of the response of the IS. The deviations of the nominal concentrations for the LLOQ samples should be within ± 20%.

To evaluate the linearity, calibration curves were prepared and analyzed for salidroside and p-tyrosol in triplicate on six consecutive days. A linear least-square regression analysis was conducted to determine the slope, intercept, and regression correlation coefficient (r²). The linear regression correlation coefficients were greater than 0.995 in all validation runs and exhibited good linearity over the concentration range of 50–2000 ng/mL for both salidroside and p-tyrosol. Liver tissue samples were quantified by comparing the peak area ratio of salidroside or p-tyrosol to that of the IS. Standard curves are in the form of y = A + Bx, where y represents the concentration of the analyte and x represents the ratio of the analyte peak area compared to that of IS. The acceptance criterion for each back-calculated standard concentration was ± 15% deviation from the nominal value except at the LLOQ.

The LLOQ was defined as the lowest concentration on the calibration curve at which the signal-to-noise (S/N) ratio was no less than 10 with a precision less than or equal to 20% and an accuracy within ± 20%. The LLOQ was determined using six replicates on three consecutive days.

The precision and accuracy of the assay were obtained by comparing the predicted concentration (obtained from the calibration curve) to the actual concentration of salidroside and p-tyrosol spiked in blank liver tissue homogenate samples at three QC levels (50, 500 and 2000 ng/mL) on different validation days. The accuracy was expressed by (mean observed concentration)/(spiked concentration) × 100%, and the precision was expressed by the relative standard deviation (RSD%). The concentration of each sample was determined using the calibration curve analyzed on the same day. Intra-day precision and accuracy were determined by analyzing QC samples at three concentrations (50, 500 and 2000 ng/mL) on the same day (n = 6), and inter-day precision and accuracy were assayed on six consecutive days (n = 6).

Stability

The stability of the standard solutions was tested at room temperature for 4 h and upon refrigeration (4 °C) for 30 days. The stability of the analytes was examined by keeping replicates of the salidroside and p-tyrosol QC samples in the autosampler tray for 24 h and in a freezer at −20 °C for 30 days; the freeze–thaw stability was obtained over three freeze–thaw cycles by thawing at room temperature for 2–3 h and then refreezing at −20 °C for 12–24 h. For each concentration and each storage condition, six replicates were analyzed in one analytical batch. The concentration of the analytes after each storage period was related to the initial concentration, which was determined when the sample was originally prepared and processed.

Recovery

The extraction recoveries of salidroside and p-tyrosol were determined at three QC levels (50, 500 and 2000 ng/ml). Recoveries were calculated by comparing the analyte/IS peak area ratios of each analyte in spiked liver tissue homogenate samples with those of the analytes in the matrices by spiking extracted analyte-free liver tissue homogenate samples prior to chromatography.

Matrix effects

The matrix effects were investigated using the post-extraction spike method in the present study. The peak area (A) of a standard analyte in spiked blank liver tissue homogenate was compared with the corresponding peak area (B) obtained by directly injecting the standard analytes into the mobile phase at concentrations of 50, 500 and 2000 ng/mL for both salidroside and p-tyrosol in triplicates. The peak area ratio of A/B (as a percentage) was used as a quantitative measure of the matrix effects (Yang et al., 2011).

Liver tissue distribution studies in rats

For the tissue distribution study, rats were intravenously administered salidroside (in saline) through the vena caudalis at a dose of 50 mg per kg body weight (i.v. 50 mg/kg). After collecting blood by cardiac puncture, six rats were dissected and the liver tissues were excised at 0.17, 0.33, 0.5, 1.0, 2.0, and 4.0 h after administration. The liver tissues were treated and extracted using the procedure described above. The six rats were counted as six repetitions.

Statistical analysis

A non-compartmental pharmacokinetic analysis using the Kinetica^TM software package (version 5.0, Thermo Fisher Scientific Inc., Franklin, MA) was performed to determine key parameters including the elimination half-life (T_1/2), the mean residence time (MRT), clearance (CL), the apparent volume of the plasma compartment (VSS), the area under the concentration–time curve from 0 to 4 h (AUC_0–4) and the area under the concentration–time curve from time zero to infinity (AUC_0–∞).

Results and discussion

Method development

The liquid chromatographic conditions published in our previous paper were used in the present study and gave acceptable resolutions for both salidroside and p-tyrosol in liver tissues. In the present method, LC–MS/MS in multiple reaction monitoring (MRM) mode was performed using the transitions described in Table 1 to obtain high specificity and low noise. Paracetamol was chosen for quantification as the IS due to its similarity with the analytes in structure, chromatographic and mass spectrographic behavior and stability (Zhang et al., 2008).

Different methods for sample preparation were investigated including LLE with various organic solvents, such as n-butanol and ethyl acetate, and SPE, but they were all restrained by low or irreproducible recovery and a time-consuming procedure. Protein precipitation using 3-fold volumes of methanol was adopted because of the fast and simple processing procedure and good reproducibility.

Method validation

Specificity

The mass chromatograms of a blank liver tissue homogenate sample, a liver tissue homogenate sample spiked with salidroside (500 ng/mL), p-tyrosol (500 ng/mL) or the IS (200 ng/mL) and a liver tissue homogenate sample obtained after 0.17 h after i.v. administration of salidroside (i.v. 50 mg/kg) are shown in Figure 3. There was no endogenous interference in the retention times of the analytes and IS in blank liver tissue homogenate and good resolution and selectivity between the analytes and the IS under the optimal conditions were observed. The specificity was verified by comparing the retention time of salidroside, p-tyrosol and the IS in QC samples (n = 6). The differences were less than 1%.

Figure 3. MRM chromatograms of salidroside, p-tyrosol and the IS in (a) a blank rat liver tissue homogenate sample, (b) a blank rat liver tissue sample spiked with salidroside (500 ngmL⁻¹), p-tyrosol (500 ngmL⁻¹) and the IS (200 ngmL⁻¹), (c) a rat liver tissue homogenate sample collected 0.17 h after i.v. administration of salidroside (50 mgkg^-1) with the IS (200 ngmL⁻¹).

Calibration curve, linearity, LLOQ and LOD

The calibration curves exhibited good linearity over the concentration range of 50–2000 ng/mL for both salidroside and p-tyrosol. The typical regression equations were y₁ = 0.0002 x₁ + 0.0137 (r²= 0.9959) and y₂ = 0.0004 x₂ − 0.0022 (r²= 0.9951), where y represents the peak area ratios of the analytes to IS, and x represents the concentration of the analytes, and r² is the coefficient of correlation, which statistically confirmed the linearity of the method.

The LLOQ for both salidroside and p-tyrosol was defined as 50 ng/mL based on S/N = 10. The LOD was estimated to be below 20 ng/mL based on S/N = 3. Based on our preliminary experiments, 50 ng/mL for salidroside and p-tyrosol could meet the needs in the tissue distribution studies and was selected as the lower concentration on the calibration curves.

Accuracy and precision

The intra-day accuracy of salidroside ranged between 104.90 and 110.10% with a precision of 5.18–12.79%. The inter-day accuracy of salidroside ranged between 102.51 and 112.73% with a precision of 3.51–14.27%. For p-tyrosol, the intra-day precision was 10.44% or less, and the accuracy was between 96.32 and 99.56%; the inter-day precision was 8.54% or less, and the accuracy was between 92.38 and 100.59%. All the results were within the acceptable limits (± 15%) and suggested that the method was accurate and precise for the simultaneous analysis of salidroside and p-tyrosol in rat liver samples (Table 2).

Table 2. Accuracy and precision for the determination of salidroside and p-tyrosol in rat liver tissues (n = 6).

		Intra-day			Inter-day
Analyte	Concentration (ng/mL)	Mean ± SD (ng/mL)	Precision (%)	Accuracy (%)	Mean ± SD (ng/mL)	Precision (%)	Accuracy (%)
Salidroside	50	52.45 ± 6.71	12.79	104.90	51.26 ± 5.11	9.97	102.51
	500	550.51 ± 28.53	5.18	110.10	563.64 ± 19.79	3.51	112.73
	2000	2126.53 ± 235.05	11.05	106.33	2121.73 ± 302.85	14.27	106.09
p-Tyrosol	50	49.16 ± 5.13	10.44	98.33	46.19 ± 3.85	8.54	92.38
	500	481.62 ± 31.32	6.80	96.32	467.05 ± 30.99	6.64	93.41
	2000	1991.17 ± 50.72	2.55	99.56	2011.83 ± 12.92	0.64	100.59

Recovery and matrix effects

The recoveries of salidroside at concentrations of 50, 500 and 2000 ng/mL were 111.44, 108.10 and 102.00%, respectively, and the recoveries of p-tyrosol were 105.44, 105.50, and 113.04%, respectively. The results suggested that the method was consistent and reproducible for both analytes (Table 3). The recoveries of the IS (200 ng/mL), salidroside (500 ng/mL) and p-tyrosol (500 ng/mL) were also calculated by comparing the peak areas with those for the neat solution without extraction. The value for IS was 66.42 ± 0.82% (n = 6), which was consistent with those of salidroside (52.13 ± 2.27%) and p-tyrosol (55.76 ± 3.48%).

Table 3. Recovery and matrix effects of salidroside and p-tyrosol in rat liver tissues (n = 3).

		Recovery		Matrix effects
Analyte	Concentration (ng/mL)	Mean ± SD	RSD (%)	Mean ± SD	RSD (%)
Salidroside	50	111.44 ± 10.26	9.20	92.45 ± 3.27	3.54
	500	108.10 ± 3.48	3.22	83.85 ± 3.61	4.32
	2000	102.00 ± 9.03	8.85	87.63 ± 6.77	7.72
p-Tyrosol	50	105.44 ± 1.99	1.88	85.61 ± 8.71	10.17
	500	105.50 ± 3.63	3.44	87.13 ± 1.78	2.04
	2000	113.04 ± 15.43	13.65	92.49 ± 3.27	3.53

By using protein precipitation for the sample preparation, salts and endogenous tissue material may cause ion suppression or enhancement of the signal, which might make it greater than that from the samples obtained by SPE and LLE (Wen et al., 2008). As shown in Table 3, the matrix effects were 83.85–92.45% for salidroside and 85.61–92.49% for p-tyrosol. The results were within the acceptable limit, indicating that ion suppression or enhancement in liver tissue matrices would be negligible in this study (Table 3).

Stability

The stability of salidroside and p-tyrosol was evaluated as described in the Method section. The results are shown in Tables 4 and 5 and indicated that no significant degradation occurred under all storage conditions.

Table 4. Stability of salidroside (n = 6).

Experimental condition	Added, C (ngċmL^–1)	Found, C ± S.D. (ngċmL^–1)	RSD (%)	Accuracy (%)
Standard solution 4 h at RT	50	52.04 ± 1.89	3.63	104.08
	500	517.76 ± 32.17	6.21	103.55
	2000	2041.93 ± 12.53	0.61	102.10
Standard solution 30 days at 4 °C	50	49.81 ± 4.78	0.59	99.62
	500	498.05 ± 40.72	8.18	99.61
	2000	1989.01 ± 69.32	3.48	99.45
QC samples Autosampler 24 h at RT	50	56.71 ± 4.47	7.88	113.42
	500	548.72 ± 39.38	7.18	109.74
	2000	2039.90 ± 180.52	8.85	102.00
QC samples 30 Days storage at − 20 °C	50	52.33 ± 7.74	14.79	104.66
	500	549.37 ± 32.81	5.97	109.87
	2000	2105.08 ± 12.62	0.59	105.25
QC samples 3 Freeze–thaw cycles	50	53.69 ± 7.06	13.14	107.38
	500	551.85 ± 32.76	5.94	110.37
	2000	2038.27 ± 147.43	7.23	101.91

Table 5. Stability of p-tyrosol (n = 6).

Experimental condition	Added, C (ngċmL^–1)	Found, C ± S.D. (ngċmL^–1)	RSD (%)	Accuracy (%)
Standard solution 4 h at RT	50	51.53 ± 1.09	2.11	103.06
	500	490.79 ± 34.55	7.04	98.16
	2000	2012.36 ± 13.24	0.66	100.62
Standard solution 30 days at 4 °C	50	52.55 ± 2.73	5.19	105.09
	500	486.26 ± 33.06	6.80	97.25
	2000	1980.65 ± 68.08	3.44	99.03
QC samples Autosampler 24 h at RT	50	52.55 ± 2.73	5.19	105.09
	500	486.26 ± 33.06	6.80	97.25
	2000	1980.65 ± 68.08	3.44	99.03
QC samples 30 days storage at − 20 °C	50	47.55 ± 4.21	8.86	95.10
	500	479.74 ± 35.83	7.47	95.95
	2000	2013.44 ± 11.03	0.55	100.67
QC samples 3 Freeze–thaw cycles	50	50.05 ± 5.46	10.91	100.10
	500	490.38 ± 28.22	5.76	98.08
	2000	1984.78 ± 56.20	2.83	99.24

Application to liver tissue distribution

The validated method was applied to quantify salidroside and p-tyrosol in the liver tissue samples obtained from male Wistar rats that were i.v. administered a single dose of 50 mg/kg salidroside. The results are presented in Figure 4. The concentration of salidroside was significantly higher than that of p-tyrosol at 0.17, 0.33, 0.5 and 1 h after administration and the two compounds were both reduced gradually over time. After 4 h, the concentration of salidroside and p-tyrosol decreased below the LLOQ. It is already known that salidroside is hydrolyzed to p-tyrosol in rat plasma after the i.v. administration of salidroside. Based on the estimation method and results described in this paper, a considerable amount of salidroside appeared to pass into the rat liver tissues after the i.v. administration, and the polyphenol was extensively metabolized to its aglycone in the rat livers. However, its aglycone, p-tyrosol also underwent a rapid and widespread distribution in liver tissues within the time course examined.

Figure 4. Mean tissue concentration–time histogram of salidroside and p-tyrosol in rat liver tissue after the i.v. administration of 50 mg/kg salidroside.

A summary of the pharmacokinetics parameters of salidroside and p-tyrosol in rat liver tissues is shown in Table 6. p-Tyrosol was eliminated in the liver (T_1/2 = 0.92 ± 0.03 h) approximately two times slower than salidroside (0.54 ± 0.06 h), The AUC_0–4 h and AUC_0–∞ of salidroside in liver homogenate samples were 4183.93 ± 905.58 h ng/mL and 4231.01 ± 899.11 h ng/mL, respectively, and were much higher than those of p-tyrosol. The higher MRT and CL values of p-tyrosol suggested that p-tyrosol was eliminated more slowly than salidroside in liver tissues. These differences in their pharmacokinetics parameters might be attributed to their chemical properties. Structurally, salidroside is the aglycone p-tyrosol attached to a p-glucopyranose through glycosidic linkage, which makes it as more water-soluble and consequently leads to a more rapid elimination than its aglycone (Bartholome et al., 2010). The deconjugation of flavonoid glucuronides could lead to prolonged circulation and enhanced bioactivity in in vitro studies (Bartholome et al., 2010; Day et al., 2000). The different values of T_1/2, MRT and CL between salidroside and p-tyrosol observed in the present study have extended previous findings that might also benefit from the bioactivity of salidroside. Thus, further research should include both the salidroside and the metabolite p-tyrosol.

Table 6. Pharmacokinetic parameters of salidroside and p-tyrosol in rat liver tissues (n = 6).

Parameters	Salidroside (mean ± SD)	p-Tyrosol (mean ± SD)
T1/2 (h)	0.54 ± 0.06	0.92 ± 0.03
AUC0–4h (h ng mL^-1)	4183.93 ± 905.58	962.64 ± 92.88
AUC0–∞ (h ng mL^-1)	4231.01 ± 899.11	1025.72 ± 68.85
MRT (h)	0.52 ± 0.05	0.99 ± 0.26
Cl (mL h^-1)	2.02 ± 0.38	9.06 ± 2.66
Vss (mL)	1.60 ± 0.39	12.15 ± 3.99

Conclusion

A simple and specific LC–MS/MS method for the determination of salidroside and its metabolite p-tyrosol was developed and validated using paracetamol as the IS and rat liver tissues as model compounds and biological matrices. This method was convenient for the quantification of salidroside and p-tyrosol in rat liver tissue homogenate samples. In addition, this method was successfully used to obtain the liver tissue distribution profiles of salidroside and p-tyrosol after i.v. administration of 50 mg/kg salidroside and can be used for future distribution studies of salidroside and p-tyrosol in other biological matrices, especially tissue samples.

Declaration of interest

The authors report no conflicts of interest.