Chemicals and reagents
Epimedin C (purity ≥ 98%), used as the dosage administration, was purchased from Cdmust Biology Technology Ltd. (Chengdu, China). Lamivudine (internal standard, IS, purity > 98%), adenosine (purity > 99%), guanosine (purity > 98%), and cytidine (purity > 98%) were purchased from Tokyo Chemical Industry (Shanghai) (Shanghai, China). Uridine (purity 99%), m6A (purity 97%), and m5C (purity 98%) were purchased from J&K Chemical (Shanghai, China). nuclease P1 and alkaline phosphatase were purchased from Takara Biotechnology (Dalian, China). Methanol (HPLC grade) and Formic acid (chromatographic grade) were obtained from Fisher Scientific (Shanghai, China). Ultra-purified water was used throughout this study and was prepared using a Milli-Q purification system (Millipore, Milford, MA, USA). All of the other chemicals and reagents were of analytical grade.
Animals
Male Balb/c mice (6–8 weeks old and weighing 18.0–22.0 g) were obtained from Beijing HFK bioscience Co., Ltd. (Beijing, China). Mice were kept in cages under controlled conditions of 22 ± 0.5 ◦C, 50 ± 2.0% RH and maintained with free access to standard laboratory food and water for one week before experiments.
Establishment of the Epimedin C-induced hepatitis model and experimental groups
Animals were randomly divided into three groups (n = 7 each): the normal control group, the Epimedin C (10 mg/kg) group, and the Epimedin C (40 mg/kg) group. Epimedin C were completely dissolved in 0.9% saline before passing through a 0.22 µm cell strainer. The Epimedin C groups were intragastrically administered single doses of 10 and 40 mg/kg body weight of Epimedin C per day, and the normal control group were given the same volume of saline. After four weeks of intragastric administration, all mice were sacrificed, and blood samples and liver tissues were collected. The procedures for the present study were approved by the Guide for the Care set by the National Institutes of Health.
Assessment of liver injury
Serum alanine transaminase (ALT) and aspartate transaminase (AST) contents in serum were analyzed using colorimetric tests (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). Liver tissues fixed in 4% paraformaldehyde were embedded in paraffin using a tissue procedure, and 4-µm-thick slices were cut and stained with hematoxylin and eosin (H&E) reagent. Photomicrographs were observed with a light microscope to evaluate liver injury.
LC-MS/MS instruments and conditions
The LC was performed using an ExionLCTM analytical (UPLC) system (AB Sciex, USA). Chromatographic separation was carried out on a Kinetex® 2.6 µm Polar C18 100A LC column (100 mm × 2.1 mm i.d.). The flow rate was 0.3 mL/min. The mobile phase included ultra-purified water containing 0.1% formic acid (solvent A) and methanol (solvent B) in a linear gradient. The gradient program was as follows: 0 to 0.5 min, 95% A; 0.5 to 3 min, 95 to 30% A; 3 to 4 min, 30% A; 4 to 4.1 min, 30 to 95% A; 4.1 to 6 min, 95% A. The injection volume was 10 µL and the total run time was 8 min. The temperature of the autosampler was set at 4 ◦C, and the column temperature was maintained at 40 ◦C. MS/MS analysis was carried out on a Qtrap 6500 mass spectrometer (AB Sciex, Redwood City, CA, USA) equipped with Turbo Ionspray interface operating in positive ESI mode. The instrument was operated with an ion spray voltage of 4.5 kV and a heater gas temperature of 500 ◦C. Mass-dependent parameters (declustering potential, entrance potential, collision energy, and collision cell exit potential) were set to the optimal values obtained by automated optimization. Data acquisition was achieved by multiple reaction monitoring (MRM). The precursor-product ion pair and the optimal values of mass parameters are listed in Table 1. Positive ion mode was used and the dwell time was set at 100 ms. Data acquisition was generated and processed using the Analyst 1.6.2 software (AB Sciex).
Table 1
Multiple reaction monitoring transitions and optimized mass parameters for the analytes
Analytes
|
Precursor ion
(m/z)
|
Product ion
(m/z)
|
DP
(V)
|
EP
(V)
|
CE
(V)
|
CXP
(V)
|
Adenosine (A)
|
268.1
|
136.1
|
30
|
10
|
22
|
10
|
Uridine (U)
|
245.0
|
113.1
|
12
|
10
|
12
|
10
|
Cytidine (C)
|
244.1
|
112.0
|
30
|
10
|
13
|
10
|
Guanosine (G)
|
284.2
|
152.1
|
30
|
10
|
15
|
10
|
N6-methyladenosine (m6A)
|
282.2
|
150.2
|
30
|
10
|
24
|
10
|
N5-methylcytidine (m5C)
|
258.2
|
126.1
|
30
|
10
|
15
|
10
|
Lamivudine (IS)
|
230.2
|
112.0
|
30
|
10
|
14
|
10
|
DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: collision cell exit potential |
Preparation of calibration standards and QC samples
Stock solutions for calibration and quality control (QC) were accurately weighed and dissolved in dimethylsulfoxide (2% of the total volume) before adding an appropriate volume of methanol to final concentration of 1 mg/mL. Working solutions were prepared by serially diluting the stock solutions with water, and then the corresponding working solutions were mixed to prepare mixed working solutions with concentration in the ranges of 160-80000 pg/mL for A, C, m5C and m6A, 0.8-400 ng/mL for G, and 8-4000 ng/mL for U. The stock solution (1 mg/mL) of the IS was dissolved in water to 4 ng/mL containing 0.4% formic acid. All solutions were kept at -20 ◦C and brought to room temperature before use. The calibration standards were prepared by spiking 10 μL of the corresponding working solutions mentioned above into 30 μL of mixtures of nuclease P1 (0.1 U) and alkaline phosphatase (2 U) to yield concentrations of 40, 120, 500, 1000, 2000, 4000, 8000 and 20000 pg/mL for A, C, m5C and m6A, 0.2, 0.6, 2.5, 5, 10, 20, 40 and 100 ng/mL for G , and 2, 6, 25, 50, 100, 200 400 and 1000 ng/mL for U. The QC samples were prepared in the same way as the calibration samples at three concentrations 120, 1600 and 16000 pg/mL for A, C, m5C and m6A, 0.6, 8, 80 ng/mL for G and 6, 80 and 800 ng/mL for U.
RNA isolation from liver tissues and Enzymatic digestion of the mRNA
100 mg of liver tissue was completely disrupted and homogenized into 1 mL TRIzol reagent. Then, the total RNA of liver tissue was isolated according to the manufacturer’s instructions. After analyzed by a NanoDrop One (Thermo Scientific), the Dynabeads® mRNA Purification Kit (Ambion) was used to enrich mRNA. The contaminant DNA was removed from the mRNA samples by using DNase, and the concentration was analyzed by a Qubit 3.0 Fluorometer (Invitrogen).
Referring to the methods reported, enzymatic digestion of the mRNA was performed[16]. The mixture sample (30 µL) for m6A and m5C testing included 100 ng mRNA, 1 µL of nuclease P1 (0.1 U/µL, Takara), and 2 µL of alkaline phosphatase (calf intestine, 1 U/µL, Takara). The mixture sample (30 µL) for A, U, C and G testing included 0.1 ng mRNA, 1 µL of nuclease P1 (0.1 U/µL, Takara), and 2 µL of alkaline phosphatase (calf intestine, 1 U/µL, Takara). After fully vortexed, both high and low concentration mixtures were incubated at 37 ◦C for 12 h. Then, 10 µL IS solution (IS, lamivudine, 4 ng/mL) containing 0.4% formic acid was added. The mixtures were vortexed for 15 s and transferred into ultrafiltration tubes (MW cutoff of 3 kDa, Pall Corporation), and centrifuged at 4 ◦C, 14000 × g for 15 min. The filtrate was added to an autosampler vial, then 10 µL of the filtrate was used for UPLC-MS/MS analysis.
Method validation
The analytical methodology was under the guidelines set by the United States Food and Drug Administration [23] and the Chinese Pharmacopoeia Commission [24].
The selectivity was evaluated by comparing chromatograms of mRNA-free blank enzymolysis matrix, blank enzymolysis matrix with all analytes, and a liver mRNA enzymolysis sample containing IS from a mouse after Epimedin C treatment. The absence of peaks at retention times of seven analytes indicated no interference in the test samples.
After detection of the upper limit of the quantification (ULOQ) samples, blank samples were injected to evaluate the carryover effects. The response peak of any analytes must be < 10% of the lower limit of the quantification (LLOQ) samples.
The linearity was investigated by plotting the peak-area ratios of the analytes (A, U, C, G, m6A and m5C) to the IS versus the concentrations of the calibration standards. The calibration equations were fitted using a weighed least-squares linear regression analysis (weighing factor of 1/x2). The accuracy, expressed as the mean relative error (RE, %), should be ≤ 20% for LLOQ and ≤ 15% for the other seven concentrations of the calibration standards.
To assess the precision and accuracy of the method, five replicates of QC (at three concentration levels) and LLOQ were prepared and analyzed within three validation days. Both the accuracy (RE, %) and intra- and inter- precision (RSD, %) for LLOQ should be less than or equal to 20%. The accuracy and precision for the QC levels should be within ± 15%.
To determine whether matrix components affected the ion suppression or enhancement in the method, the matrix effect (ME) was assessed by comparing the corresponding peak area responses of enzymolysis matrix with all analytes and the blank samples in which the enzymolysis matrix was replaced with water. In this method, the variability values of the MEs (RSD, %) should be less than 15%.
To evaluate the stability of the analytes in the enzymolysis matrix during sample preparation and storage, the low and high QC concentration levels in different storage conditions were detected. The storage conditions included room temperature for 6 h, three freeze-thaw cycles, autosampler at ambient temperature for 20 h, and freezing at -20 ◦C for 30 days. The analytes were stable when 85–115% of the initial concentration was retained.
The dilution integrity was assessed by testing the solution which was diluted 100-fold with blank enzymolysis matrix before ultra-filtration from highly concentrated samples above the upper limit of standard curves. The accuracy and precision should be within ± 15%.
Data analysis
The data were presented as the arithmetic mean ± SD. Statistical analyses were performed using SPSS software for Windows. Statistical significance was assessed by unpaired two-tailed Student’s t-test between two samples. p value < 0.05 was considered statistically significant.