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. 2016 Jan 15;54(5):689–696. doi: 10.1093/chromsci/bmv227

A Comparative Pharmacokinetic Study of Myrislignan by UHPLC–MS After Oral Administration of a Monomer and Myristica fragrans Extract to Rats

Zhe Zhu 1,†, Shu Yang 2,†, Waiou Zhao 3, Rui Li 1,*, Chengliang Zhao 4
PMCID: PMC4890437  PMID: 26774114

Abstract

An ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC–MS) method was developed and validated to quantify myrislignan in rat plasma using podophyllotoxin as an internal standard (IS). The chromatographic separation of myrislignan and IS was performed on a 3.0 µm Hypersil C18 column (50 mm × 4.6 mm) with methanol and water containing 0.1% acetic acid (80:20, v/v) as the mobile phase at a flow rate of 0.3 mL/min. An electrospray ionization was used in the positive selective-ion monitoring mode for the target ions at m/z 397 and m/z 437 for the quantification of myrislignan and IS. The total run time was 3.6 min for each run. The calibration curve was linear over the range of 0.75–300 ng/mL (r > 0.995) with the lower limit of quantitation at 0.75 ng/mL. Intra- and interday precision was below 11.49%, and the mean accuracy ranged from −9.75 to 7.45%. The proposed method was successfully applied to evaluate the pharmacokinetic properties of myrislignan after oral administration of the myrislignan monomer and Myristica fragrans extract in rats. Statistical analyses indicate that the pharmacokinetic properties of myrislignan in rats have significant differences between two groups.

Introduction

Semen Myristicae, also known as “nutmeg”, is derived from the seeds of Myristica fragrans (1). This medicine has been used for the treatment of asthma, rheumatism, muscle spasm, atherosclerosis, decreased appetite and diarrhea (2, 3). In addition, increasing number of studies about Semen Myristicae are reported because of its newly discovered anti-angiogenic (4), anti-inflammatory (5), antifungal (6, 7), antihelmintic (8), anti-obesity (9), antioxidant (10, 11), anti-tumor (12), anxiolytic (13), anti-parasitic (14, 15), hypolipidemic (16), hepatoprotective (17), neuroprotective (18), immunomodulatory and radioprotective (19) effects. The M. fragrans extract can inhibit the expression of COX-2 and cAMP response element binding protein, as well as p38 MAPK phosphorylation in murine microglial BV2 cells induced by lipopolysaccharide (LPS) (20, 21), which suggests a protective effect on murine microglial BV2 cell.

M. fragrans contains a variety of ingredients such as dihydroguaiaretic acid, elemicin, myristica acid, myristicin and lignan compounds (12, 22). Lignans are the main effective components isolated from M. fragrans. Myrislignan (Figure 1) is a typical neolignan isolated from M. fragrans, and it has been intensively studied by many scientists because of its antifeeding and antifungal properties (23, 24), as well as other health benefits, such as neoplasm and vascular smooth muscle contraction inhibition (25, 26). More recently, Jin et al. reported that myrislignan exerted a significant anti-inflammatory effect in LPS-stimulated macrophage cells by inhibiting the NF-κB signaling pathway activation (27).

Figure 1.

Figure 1.

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The chemical structures of myrislignan and podophyllotoxin (the internal standard).

Currently, information related to the in vivo pharmacokinetic study of myrislignan is limited (28, 29). To the best of our knowledge, only two HPLC methods have been reported regarding its quantification. Moreover, one of the methods has a lower limit of quantification (LLOQ) of 500 ng/mL in rat plasma and the other method has an LLOQ of 100 ng/g in rat tissues (28, 29). However, these methods are not satisfactory for the pharmacokinetic study due to poor selectivity, insufficient sensitivity and long chromatographic run time. Hence, a more sensitive quantification method is needed to better understand the myrislignan pharmacokinetics in vivo. In this study, a simple and sensitive ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC–MS) method was developed for the determination of myrislignan in rat plasma. The method was successfully applied to a comparative pharmacokinetic study of myrislignan after oral administration of myrislignan alone and M. fragrans extract to rats. To our knowledge, no analytical method has been developed and validated for the comparative pharmacokinetic study of myrislignan.

Experimental

Chemicals and reagents

Myrislignan (99.0% purity) and podophyllotoxin (internal standard, IS; 98.6% purity) were obtained from Sichuan Standard Center of Traditional Chinese Medicine (Chengdu, China). Methanol and glacial acetic acid were of chromatographic grade and supplied by Tedia Corporation (Fairfield, USA). Ultrapure water used for the animal experiments was prepared from a Milli-Q purification system (Millipore Corp., Bedford, MA, USA).

Preparation of M. fragrans extract

For preparation of the extract, the dried powder of M. fragrans (20 g) was extracted twice by refluxing the sample with 200 mL ethanol–water (90:10, v/v) for 1.5 h (30). Extraction solutions were combined and filtered; the extract was subsequently evaporated to dryness by using a rotary evaporator. The dried powders (1.31 g) of the M. fragrans extract and pure myrislignan were, respectively, dissolved in a 0.5% carboxymethyl cellulose sodium aqueous solution before use.

Preparation of working, calibration and quality control standards

The stock solutions of myrislignan and IS were prepared by dissolving the drug in methanol at a concentration of 120 and 200 µg/mL, respectively. Standard working solutions were prepared from the myrislignan stock solution by dilution using methanol. Calibration standards were prepared by a 1:10 dilution of the corresponding standard working solutions with blank rat plasma to achieve final concentrations of 0.75, 3.00, 7.50, 30.0, 75.0, 150 and 300 ng/mL. Quality control (QC) samples were similarly prepared at 2.25 ng/mL (LQC, low quality control), 15.0 ng/mL (MQC, medium quality control) and 270 ng/mL (HQC, high quality control). The IS stock solution was further diluted with methanol to make a working solution (50 ng/mL). All solutions were stored at 4°C in the dark prior to use.

Sample preparation

An aliquot of 50 µL of each plasma sample was mixed with 50 µL of the IS working solution (50 ng/mL). Methanol (300 µL) was then added for precipitation. After vortexing for 5 min and centrifuging at 11,000 rpm for 10 min, 350 µL of the supernatant was transferred to a new tube and evaporated to dryness at 45°C under a gentle nitrogen stream. The residue was then reconstituted in 100 µL of the mobile phase. After centrifuging at 11,000 rpm for 5 min, an aliquot of 2 µL was injected into the UHPLC–MS system for analysis.

UHPLC–MS analysis

Plasma samples were analyzed by an UHPLC–MS method. The system was composed of an UltiMate 3000 UHPLC system (Thermo Scientific, San Jose, USA) with a Hypersil C18 column (50 mm × 4.6 mm, i.d. 3.0 µm; Thermo Scientific, Waltham, MA, USA) coupled to a TSQ QUANTUM ULTRA mass spectrometer (Thermo Scientific, San Jose, USA) with an electrospray ionization (ESI) source. The mobile phase was composed of methanol and water containing 0.1% acetic acid (80:20, v/v) delivered at 0.3 mL/min. The eluate from the analytical column was directed to waste for the first 1.5 min via a divert valve and then introduced into the mass spectrometer. For the determination of myrislignan and IS, the positive ion mode was selected, along with the following conditions: ESI spray voltage, 3.5 kV; capillary temperature, 300°C; sheath gas, 35 Arb; auxiliary gas, 10 Arb. The mass spectrometer was operated in selective-ion monitoring (SIM) mode, with monitoring of the target precursor ions at m/z 397 for myrislignan and m/z 437 for IS (Figure 2).

Figure 2.

Figure 2.

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The full-scan precursor ion spectra of (A) myrislignan and (B) internal standard.

Method validation

A thorough and complete method validation of myrislignan in rat plasma was performed in terms of selectivity, sensitivity, linearity, matrix effect, accuracy, precision, recovery and stability according to the Food and Drug Administration (FDA) Guidelines.

Selectivity

The selectivity was evaluated by comparing the chromatograms of six different sources of blank rat plasma with those of corresponding plasma samples spiked with myrislignan and IS to exclude the interference of endogenous substances.

Linearity and LLOQ

The linearity was investigated by analyzing the myrislignan standards (concentration range of 0.75–300 ng/mL) in plasma. The calibration curves were constructed through weighted (1/x2) least-square linear regression of the determined peak areas as a function of the nominal concentrations. The LLOQ was defined as the lowest concentration of myrislignan on the calibration curve with a precision lower than 20% and deviation from the nominal concentration within ±20% by six replicate analyses.

Accuracy and precision

The accuracy and precision were measured by determining six replicates of three levels of QC samples (2.25, 15.0 and 270 ng/mL) on three consecutive days. Intraday accuracy and precision were evaluated via analysis of the samples in six replicates in the same day while the interday accuracy and precision were evaluated via repeating analysis over three consecutive days. The accuracy and precision were expressed in terms of relative error (RE) and relative standard deviation (RSD), respectively. The intra- and interday precision should not exceed 15%, and accuracy should be within ±15% for QC samples.

Extraction recovery and matrix effect

The extraction recovery was investigated by comparing the peak areas of the analyte obtained from the extracted plasma samples with those from the blank plasma extracts spiked with standard solution. The matrix effect was evaluated by comparing the responses of the analyte dissolved in the blank extracted rat plasma samples with those of the corresponding concentrations prepared in the reconstitution solution.

Stability

The stability of myrislignan in plasma was carried out under various conditions using three levels of QC samples. Long-term stability was studied by analyzing samples stored at −20°C for 45 days. Freeze–thaw stability were examined by analyzing samples after three freeze–thaw cycles (−20°C to 25°C). Short-term stability was assessed by analyzing samples at room temperature for 4 h. Postpreparative stability was investigated by analyzing samples kept in an autosampler at 4°C for 12 h. Samples were considered stable if the assay values were within the acceptable limits of accuracy (i.e., ±15% deviation) and precision (i.e., 15% RSD) in the tested samples versus control at the sample nominal concentrations (31, 32).

Application to a pharmacokinetic study in rats

Male Wistar rats weighing 220±20 g were purchased from the Laboratory Animal Center of Jilin University and kept in the controlled environment conditions (temperature 24 ± 2°C, humidity 50 ± 10%, 12 h dark/light cycle) with free access to food and water for 7 days. All the experimental protocols were approved by the Animal Ethics Committee of Jilin University (approval no: 20150213RR, approved in 13th Feb, 2015; Changchun, China). Rats were fasted for 12 h before the experiment and were randomly divided into two groups (n = 6 per group). One group received an oral dose of 18.3 mg/kg myrislignan monomer, and the other group received an oral dose of 0.33 g/kg M. fragrans extract, containing myrislignan 18.3 mg/kg. Blood samples (0.3 mL) were collected into heparinized centrifuge tubes from the orbital sinus venous plexus at 0, 0.08, 0.17, 0.33, 0.50, 0.67, 1, 1.5, 2, 3, 4 and 5 h post-dose. After centrifuging at 3,000 rpm for 10 min, the plasma samples were obtained and frozen at −20°C until analysis.

The pharmacokinetic parameters, peak plasma concentration (Cmax) and the time to Cmax (Tmax), elimination half-life (t1/2z), systemic clearance (CLz/F) and the area under the concentration–time curve from time zero to the last time point (AUC0–t) and to infinity (AUC0–∞) were calculated for each subject by the DAS 2.0 software (Chinese Pharmacology Society, China). Comparison of pharmacokinetic parameters between two groups was performed using the Student's t-test with statistical significance set at a P-value of <0.05 (33–35). Statistical analyses were performed using the SPSS 19.0 Software Package (Statistical Package for the Social Science; Yorktown Heights, NY, USA). All data are expressed as the mean ± standard deviation (SD).

Results

Assay validation

Selectivity

Figure 3 shows the representative chromatograms of blank plasma, a plasma sample at the LLOQ level, and a real plasma sample obtained at 1 h after oral administration of the monomer and M. fragrans extract. The assay was free of interference from endogenous substances in plasma at the retention times of myrislignan and IS.

Figure 3.

Figure 3.

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Typical SIM chromatograms of (A) blank rat plasma, (B) blank rat plasma spiked with myrislignan at the LLOQ of 0.75 ng/mL and internal standard (50 ng/mL), (C) plasma sample obtained at 1 h after oral administration of myrislignan and (D) plasma sample obtained at 1 h after oral administration of M. fragrans extract. Channel 1, myrislignan; Channel 2, IS.

Extraction recovery and matrix effect

The mean extraction recovery of myrislignan from rat plasma was 90.16 ± 5.03% at three QC levels, and the average extraction recovery of the IS was 91.55 ± 7.51%. The mean matrix effect of myrislignan at three QC levels was 96.57 ± 4.12%. The matrix effect for IS was 95.39 ± 2.80%. The results proved that there was no significant interference on the analyte's assay from the plasma matrix, while the extraction efficiency was consistent and reproducible.

Linearity and LLOQ

The calibration curve for myrislignan was linear over the concentration range of 0.75–300 ng/mL by using weighted (1/x2) least-square linear regression analysis with a weighing factor of 1/x2. The typical equation for the calibration curves for myrislignan was y = 5.602x + 3.458 × 10–1 (r = 0.9962), where y represents the peak area ratio of myrislignan/IS and x represents the concentration of analyte in spiked plasma samples. The LLOQ for myrislignan was established at 0.75 ng/mL, at which the precision (%RSD) and accuracy (%RE) were 1.03 and 7.50%, respectively.

Accuracy and precision

The intra- and interday precision and accuracy for six replicates of QC samples are summarized in Table I. The intraday accuracy (%RE) for myrislignan ranged from −7.82 to 7.45% with the precision (%RSD) from 3.72 to 6.37%, and the interday accuracy for myrislignan ranged from −9.75 to 2.34% with the precision (%RSD) from 1.63 to 11.49%. The results demonstrated that this assay method was accurate, reliable and reproducible.

Table I.

Intraday and Interday Accuracy and Precision for Determination of Myrislignan in Rat Plasma

Nominal concentration (ng/mL) Intraday (n=6)
 Interday (n = 18)
Determined concentration
(mean ± SD, ng/mL)		 % RSD % RE Determined concentration
(mean ± SD, ng/mL)		 % RSD % RE
2.25 (LQC) 2.07 ± 0.13 6.37 −7.82 2.30 ± 0.04 1.63 2.34
15.0 (MQC) 16.12 ± 0.75 4.65 7.45 15.28 ± 1.76 11.49 1.87
270 (HQC) 280.18 ± 10.42 3.72 3.77 243.67 ± 24.30 9.97 −9.75
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Stability

The stability of QC samples was performed at three concentrations (2.25, 15.0 and 270 ng/mL) under various conditions during sample collection and processing, and the results are shown in Table II. The results of stability tests indicated that myrislignan is stable under various conditions (%RE: −11.30 to 9.00%, %RSD < 10.24%).

Table II.

The Stability Results for Determination of Myrislignan in Rat Plasma (n = 6)

Storage conditions Nominal concentration
(ng/mL)		 Determined concentration
(mean ± SD, ng/mL)		 % RSD % RE
Long-term stability (−20°C for 45 days) 2.25 (LQC) 2.45 ± 0.15 6.01 9.00
15.0 (MQC) 13.30 ± 0.60 4.51 −11.30
270 (HQC) 272.05 ± 4.51 1.66 0.76
Three freeze–thaw cycles (−20°C to 25°C) 2.25 (LQC) 2.03 ± 0.20 9.71 −9.69
15.0 (MQC) 14.78 ± 0.99 6.71 −1.44
270 (HQC) 272.66 ± 20.40 7.48 0.99
Short-term stability (room temperature for 4 h) 2.25 (LQC)  2.10 ± 0.01 0.55 −6.86
15.0 (MQC) 15.62 ± 1.31 8.38 4.15
270 (HQC) 295.67 ± 13.02 4.40 9.51
Postpreparation stability (in the autosampler at 4°C for 12 h) 2.25 (LQC) 2.25 ± 0.13 5.91 0.07
15.0 (MQC) 16.00 ± 0.17 1.07 6.66
270 (HQC) 288.94 ± 29.58 10.24 7.01
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Pharmacokinetic study

The plasma concentrations of myrislignan were determined after oral administration of the monomer and M. fragrans extract to rats (Figure 4). In addition, the estimated pharmacokinetic parameters are presented in Table III. As shown in Table III, after oral administration of 18.3 mg/kg monomer, myrislignan was absorbed and reached a Cmax of 102.44 ± 16.51 ng/mL. The Cmax of myrislignan from M. fragrans extract (0.33 g/kg body weight, containing 18.3 mg/kg of myrislignan) was 55.67 ± 13.32 ng/mL. The AUC0–t of myrislignan was 156.11 ± 39.54 µg h/L in the monomer group, whereas the AUC0–t of myrislignan in the M. fragrans extract group was 51.59 ± 20.30 µg h/L. These data indicate that the Cmax and AUC0–t values of myrislignan from the extract group were lower than those from the monomer group at the same dosage. However, the CLz/F value from the extract group was higher than that of the monomer group.

Figure 4.

Figure 4.

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Mean plasma concentration–time curves of myrislignan in rat plasma after oral administration of the monomer and M. fragrans extract (n = 6).

Table III.

Main Pharmacokinetic Parameters of Myrislignan in Rats After Oral Administration of the Monomer and M. fragrans Extract (n = 6, mean ± SD)

Parameters Monomer group Extract group
Tmax (h) 0.867 ± 0.182 0.400 ± 0.149**
Cmax (ng/mL) 102.44 ± 16.51 55.67 ± 13.32**
t1/2z (h) 0.704 ± 0.081 0.594 ± 0.108
AUC0–t (µg h/L) 156.11 ± 39.54 51.59 ± 20.30**
AUC0–∞ (µg h/L) 158.09 ± 40.40 52.43 ± 20.39**
MRT0–t (h) 1.40 ± 0.23 0.894 ± 0.224**
MRT0–∞ (h) 1.45 ± 0.24 0.954 ± 0.222**
CLz/F (L/kg/h) 121.23 ± 27.09 409.38 ± 205.30*
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Values are mean ± SD.

*P < 0.05, **P < 0.01 compared with the level of myrislignan at the same dose.

Discussion

Mass spectrometric conditions

To optimize the MS conditions, the positive and negative modes were both tested. Myrislignan and IS could form a stable adduct ion ([M+Na]+) in the positive full-scan mode (Figure 2). However, the response of the two analytes in the negative mode was extremely weak without detection of an adduct ion. Therefore, we finally selected the [M+Na]+ in the positive full-scan mode as the precursor ions of myrislignan and IS. Nevertheless, neither of them could form the distinct product ions in MS/MS mode because the triple quadrupole mass spectrometer could not provide enough energy to obtain fragments for the analytes. Finally, the analyte quantification was performed using the SIM mode for data acquisition; in addition, the selected ions were m/z 397 [M+Na]+ for myrislignan and m/z 437 [M+Na]+ for IS.

Chromatographic conditions

Acetonitrile and methanol were first considered as optional elution solvents. Methanol showed stronger MS intensity and better peak shape for myrislignan and IS. The peak responses of the analyte decreased to 30% when the organic phase ratio decreased to 20%. Therefore, 80% of organic phase was finally applied. The addition of acetic acid to the mobile phase could greatly improve the ionization efficiency and the peak shape of myrislignan and IS. When the ratio of acetic acid in the mobile phase changed, the peak response showed no significant change. Therefore, an isocratic mobile phase that comprises methanol and water containing 0.1% acetic acid (80:20, v/v) was selected. Among various commercially available C18 columns tested, a Hypersil C18 column (50 mm × 4.6 mm, i.d. 3.0 µm) was found to show the most satisfactory chromatography. Furthermore, the 50 mm column length could reduce the analysis time.

Selection of IS

An ideal IS for an LC–MS assay is a deuterated form of the compound analyzed. However, the deuterated myrislignan is not generally available, and thus a compound structurally or chemically similar to myrislignan was considered in this study. To find a compound that could mirror the analyte and serve as a suitable IS, we tested several compounds. Podophyllotoxin was proved to be the most appropriate IS for this study due to its similar extraction efficiency, and chromatographic and MS behaviors with those of myrislignan. No significant interference in the SIM channels at the relevant retention times was observed during the study.

Pharmacokinetic parameters comparison

The pharmacokinetic parameters of myrislignan in the extract group, such as the Cmax, AUC0–t, AUC0–∞, Tmax, MRT0–t, MRT0–∞ and CLz/F, statistically differed from those in the monomer group (P < 0.01; P < 0.05). However, the t1/2z value showed no significant differences between the two groups. Notably, the significant differences in the Cmax, AUC0–t and AUC0–∞ suggested that other co-existing ingredients in the extract may have effects on reducing the myrislignan plasma concentrations by inhibiting its absorption. The mechanism that accounts for the difference in the pharmacokinetic behavior between the monomer and extract groups is unclear. However, the compound–compound interactions in the extract may present a main possible explanation. Further investigation on the compound–compound interactions of this herbal medicine is necessary.

Conclusion

A novel, simple and sensitive UHPLC–MS method was developed for the quantification of myrislignan in rat plasma. The developed method can offer sufficient selectivity, accuracy and precision. The method was successfully applied to the pharmacokinetic evaluation of oral administration of the monomer and M. fragrans extract using the rat as an animal model. Statistical analyses indicate that the pharmacokinetic properties of myrislignan in rats have significant differences between two groups.

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