Abstract

A sensitive and selective liquid chromatography mass spectrometry method was developed for the determination of rhynchophylline in rat plasma. After the addition of estazolam as the internal standard (IS), protein precipitation by acetonitrile was used for sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column (2.1 × 150 mm, 5 µm) with acetonitrile–0.1% formic acid as the mobile phase with gradient elution. The electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification by using target fragment ions m/z 385 for rhynchophylline and m/z 295 for the IS. Calibration plots were linear over the range of 5–500 ng/mL for rhynchophylline in plasma. The lower limit of quantification for rhynchophylline was 5 ng/mL. The mean recovery of rhynchophylline from plasma was in the range of 87.7–92.6%. The coefficients of variation of intra-day and inter-day precision were both less than 11%. This method is sensitive and selective enough to be used in pharmacokinetic research for the determination of rhynchophylline in rat plasma.

Introduction

China has an abundant resource of Uncaria rhynchophylla (Miq.) jacks. The hooks on this plant are used in the treatment of infantile convulsion, headache, dizziness, hypertension and stroke in traditional Chinese medicine and Japanese Kampo medicine (1). Oxindole alkaloids such as rhynchophylline (R) and isorhynchophylline (IR) are major components of U. rhynchophylla (2), responsible for the cardiocerebral vascular effects, including hypotension, vasodilation (3), anti-platelet aggregation (4) and protective effects against neuronal damage (5).

One analytical liquid chromatography mass spectrometry (LC–MS) method has been published for the determination of R in rat plasma (6). However, the analysis method was not fully verified; the selectivity, matrix effect and stability were missing. LC–tandem mass spectrometry (MS-MS) does not guarantee the effective elimination of interferences from endogenous impurities, but an easy and effective way to make this adjustment is to modify gradient conditions. In this paper, a sensitive and selective LC–MS method was developed and validated for the determination of R in rat plasma, using one-step protein precipitation with gradient elution. The LC–MS method was successfully applied to a pharmacokinetic study of R after oral administration to rats.

Experimental

Chemicals and reagents

R (purity > 98%) was purchased from Chengdu Mansite Pharmaceutical Co. (Chengdu, China). Estazolam (purity > 98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). High-performance liquid chromatography (HPLC) grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA).

Instrumentation and conditions

All analyses were performed with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump, a degasser, an autosampler, a thermostatted column compartment and a Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with an electrospray ion source and controlled by ChemStation software [Version B.01.03 (204); Agilent Technologies].

Chromatographic separation was achieved on an Agilent Zorbax SB-C18 (2.1 × 150 mm, 5 µm) column at 25°C, with acetonitrile–0.1% formic acid as the mobile phase. The flow rate was 0.4 mL/min. A gradient elution program was conducted for chromatographic separation with mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile) as follows: 0–4.0 min (10–80% B), 4.0–7.0 min (80–80% B), 7.0–8.0 min (80–10% B), 8.0–12.0 min (10–10% B).

Drying gas flow and nebulizer pressure were set at 7 L/min and 25 psi, respectively. Drying gas temperature and capillary voltage of the system were adjusted at 350°C and 3,500 V, respectively. LC–MS was performed with selected ion monitoring (SIM) mode using target ions at m/z 385 for R (Figure 1A) and m/z 295 for estazolam [internal standard (IS); Figure 1B], in the positive ion electrospray ionization (ESI) interface.

Figure 1.

Mass spectra in the positive mode: rhynchophylline (A); estazolam (IS) (B).

Figure 1.

Mass spectra in the positive mode: rhynchophylline (A); estazolam (IS) (B).

Calibration standards and quality control samples

The stock solution of R (1.0 mg/mL) was prepared in methanol–trichloromethane (70:30) and the stock solution of the IS (100 µg/mL) was prepared in methanol. Working solutions for calibration and controls were prepared from the stock solution by dilution using methanol. The 2.0 µg/mL working standard solution of the IS was prepared by dilution of the IS stock solution with methanol. All of the solutions were stored at 4°C and brought to room temperature before use.

R calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 5–500 ng/mL for R in rat plasma (concentrations of 5, 10, 20, 50, 100, 200 and 500 ng/mL). Quality control (QC) samples were prepared by the same method as the calibration standards at three different plasma concentrations (10, 100 and 500 ng/mL). The analytical standards and QC samples were stored at –20°C.

Sample preparation

Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 µL of the IS working solution (2.0 µg/mL) was added to 100 µL of the collected plasma sample, followed by the addition of 300 µL of acetonitrile. The tubes were vortex-mixed for 0.5 min. After centrifugation at 15,000 rpm for 10 min, the supernatant was collected, transferred into clean tubes and evaporated to dryness with a gentle stream of nitrogen gas at 40°C. The residues were dissolved in 150 µL of acetonitrile–water (50:50) and the supernatant (5 µL) was injected into the LC–MS system for analysis.

Method validation

Validation of the method was conducted following Food and Drug Administration (FDA) guidelines with respect to specificity, recovery, intra-day and inter-day precision, lower limit of detection (LOD), lower limit of quantification (LLOQ) and sample stability. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples.

The selectivity of the method was evaluated by analyzing blank rat plasma, blank plasma spiked R and the IS and a rat plasma sample.

Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of R to the IS were plotted against analyte concentrations and standard curves were fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration squared (1/x2) in the concentration range of 5–500 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves.

To evaluate the matrix effect, blank rat plasma samples were protein precipitated and spiked with the analyte at 10, 100 and 500 ng/mL. The corresponding peak areas were compared to those of neat standard solutions at equivalent concentrations, and this peak area ratio was defined as the matrix effect (ME). The ME of the IS was evaluated at the working concentration (200 ng/mL) in the same manner.

Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (10, 100 and 500 ng/mL) in three validation days. The precision was expressed by the coefficient of variation (CV).

The recovery values of R at three QC levels (n = 6) were determined by comparing the peak areas of the analytes in QC samples to which the analytes were added after protein precipitation at equivalent concentrations. The recovery of the IS was determined in a similar manner.

The stability values of R in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 10 and 500 ng/mL, which were exposed to different conditions. These results were compared with those obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. The freeze/thaw stability was evaluated after three complete freeze/thaw cycles (–20 to 25°C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at –20°C for 30 days. The stability of the IS (200 ng/mL) was evaluated in a similar manner.

Pharmacokinetic study

Male Sprague-Dawley rats (200–220 g), obtained from Laboratory Animal Center of Wenzhou Medical College (Wenzhou, China), were used to study the pharmacokinetics of R. All six rats were housed at Wenzhou Medical College Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College and were in accordance with the Guide for the Care and Use of Laboratory Animals. Animals were housed under controlled conditions [25 ± 1°C, relative humidity (RH) 55 ± 10%] with a natural light–dark cycle. They were allowed to adapt to the housing environment for at least one week before the study. Diet was prohibited for 12 h before the experiment, but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0, 0.08333, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6 and 8 h after oral administration of R (15 mg/kg). The samples were immediately centrifuged at 5,000 rpm for 5 min. The obtained plasma (100 µL) was stored at –20°C until analysis. The plasma concentration of R versus time data for each rat was analyzed by DAS software (Version 2.0; Wenzhou Medical College).

Results

Selectivity and ME

Figure 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with R and the IS and a plasma sample. No interfering endogenous substance was observed at the retention times of the analyte or the IS.

Figure 2.

Representative LC–MS chromatograms: of R (peak 1) and estazolam (peak 2): blank plasma (A); blank plasma spiked with R (10 ng/mL) and the IS (200 ng/mL) (B); rat plasma sample 3 h after oral administration of a single dosage of 15 mg/kg of R (32 ng/mL) (C).

Figure 2.

Representative LC–MS chromatograms: of R (peak 1) and estazolam (peak 2): blank plasma (A); blank plasma spiked with R (10 ng/mL) and the IS (200 ng/mL) (B); rat plasma sample 3 h after oral administration of a single dosage of 15 mg/kg of R (32 ng/mL) (C).

The MEs for R at concentrations of 10, 100 and 500 ng/mL were calculated to be 94.5 ± 8.6, 104.0 ± 5.1 and 92.6 ± 5.6% (n = 6), respectively. The ME for IS (200 ng/mL) was 100.9 ± 6.4% (n = 6). As a result, ME from plasma was negligible in this method.

Calibration curve and sensitivity

The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range of 5–500 ng/mL for R in rat plasma. A typical equation of the calibration curve was: y = 0.0146x + 0.0903, r = 0.9987, where y represents the ratios of the peak area of R to that of the IS and x represents the plasma concentration.

The LLOQ for the determination of R in plasma was 5 ng/mL. The precision and accuracy at LLOQ were 11.6 and 86.6%, respectively. The LOD, defined as a signal–noise ratio of 3, was 2 ng/mL for R in rat plasma.

Precision, accuracy and recovery

The precision of the method was determined by calculating the CV for QCs at three concentration levels over three validation days. Intra-day precision was 11% or less and inter-day precision was 7% or less at each QC level. The accuracy of the method ranged from 88.9 to 103.4% at each QC level. Mean recovery values of R were better than 87.2%. The recovery of the IS (200 ng/mL) was 93.6%.

Assay performance data are presented in Table I. The previously discussed results demonstrate that the values are within the acceptable range and that the method is accurate and precise.

Table I

Precision, Accuracy and Recovery for R of QC Samples in Rat Plasma (n = 6)

Concentration (ng/mL) CV (%)
 
Accuracy (%)
 
Recovery (%) 
Intra-day Inter-day Intra-day Inter-day 
10 10.3 6.3 88.9 ± 8.3 92.1 ± 9.3 87.2 ± 6.3 
100 5.1 3.3 103.4 ± 3.8 95.9 ± 4.9 88.2 ± 6.9 
500 3.7 4.7 94.2 ± 9.2 100.1 ± 6.7 92.6 ± 4.9 
Concentration (ng/mL) CV (%)
 
Accuracy (%)
 
Recovery (%) 
Intra-day Inter-day Intra-day Inter-day 
10 10.3 6.3 88.9 ± 8.3 92.1 ± 9.3 87.2 ± 6.3 
100 5.1 3.3 103.4 ± 3.8 95.9 ± 4.9 88.2 ± 6.9 
500 3.7 4.7 94.2 ± 9.2 100.1 ± 6.7 92.6 ± 4.9 

Stability

The stability results showed that R spiked into rat plasma was stable for 2 h at room temperature, for 30 days at –80°C and during three freeze–thaw cycles. The stability of R extracts in the sample solvent on the autosampler was also observed over a 24 h period. The results of stability experiments are listed in Table II.

Table II

Summary of Stability of R and IS under Various Storage Conditions (n = 3)

Condition Concentration (ng/mL)
 
CV (%) Accuracy (%) 
Added Found 
Ambient, 2 h 10 9.9 ± 0.3 3.5 98.7 
500 506.5 ± 11.6 2.3 101.3 
IS (200) 195.9 ± 4.7 2.4 97.9 
–20°C, 30 days 10 10.7 ± 0.9 8.9 106.5 
500 480.4 ± 31.2 6.5 96.1 
IS (200) 188.8 ± 12.8 6.8 94.4 
3 Freeze/thaw 10 9.5 ± 0.9 9.8 95.4 
500 536.6 ± 29.0 5.4 107.3 
IS (200) 206.6 ± 13.0 6.3 103.3 
Autosampler ambient, 24 h 10 9.8 ± 0.2 2.3 97.6 
500 485.2 ± 6.3 1.3 97.0 
IS (200) 202.7 ± 4.9 2.4 101.3 
Condition Concentration (ng/mL)
 
CV (%) Accuracy (%) 
Added Found 
Ambient, 2 h 10 9.9 ± 0.3 3.5 98.7 
500 506.5 ± 11.6 2.3 101.3 
IS (200) 195.9 ± 4.7 2.4 97.9 
–20°C, 30 days 10 10.7 ± 0.9 8.9 106.5 
500 480.4 ± 31.2 6.5 96.1 
IS (200) 188.8 ± 12.8 6.8 94.4 
3 Freeze/thaw 10 9.5 ± 0.9 9.8 95.4 
500 536.6 ± 29.0 5.4 107.3 
IS (200) 206.6 ± 13.0 6.3 103.3 
Autosampler ambient, 24 h 10 9.8 ± 0.2 2.3 97.6 
500 485.2 ± 6.3 1.3 97.0 
IS (200) 202.7 ± 4.9 2.4 101.3 

Robustness

Five chromatographic conditions were tested and the retention times and areas of R and IS were achieved in every trial; the results of the robustness of the chromatographic method were satisfactory, as shown in Table III [relative standard deviation (RSD) < 10%].

Table III

Robustness Results of the Chromatographic Method

Condition Retention time (min)
 
Area
 
IS IS 
25°C, 80% B 5.1 6.1 286,670 90,194 
27°C, 80% B 5.1 6.1 289,242 88,706 
23°C, 80% B 5.1 6.1 285,182 90,253 
25°C, 83% B 5.1 6.1 282,322 80,679 
25°C, 77% B 5.1 6.1 272,670 74,660 
RSD (%)   2.3 8.2 
Condition Retention time (min)
 
Area
 
IS IS 
25°C, 80% B 5.1 6.1 286,670 90,194 
27°C, 80% B 5.1 6.1 289,242 88,706 
23°C, 80% B 5.1 6.1 285,182 90,253 
25°C, 83% B 5.1 6.1 282,322 80,679 
25°C, 77% B 5.1 6.1 272,670 74,660 
RSD (%)   2.3 8.2 

Application

The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after administration of a single 15 mg/kg oral dose of R is shown in Figure 3. The primary pharmacokinetic parameters from a two-compartment model analysis are summarized in Table IV.

Table IV

Primary Pharmacokinetic Parameters after Oral Administration of Single Dosage of 15 mg/kg of R to Six Rats

Pharmacokinetic parameters* Mean (± SD) 
t1/2 (h) 1.72 ± 0.34 
Cmax 131.67 ± 26.87 
tmax (h) 0.56 ± 0.13 
MRT(0–t) (h) 1.86 ± 0.11 
MRT(0–∞) (h) 2.12 ± 0.13 
CL (L/h) 51.21 ± 10.61 
AUC(0–t) (h/ng/mL) 294.03 ± 64.49 
AUC(0–∞) (h/ng/mL) 302.54 ± 63.09 
Pharmacokinetic parameters* Mean (± SD) 
t1/2 (h) 1.72 ± 0.34 
Cmax 131.67 ± 26.87 
tmax (h) 0.56 ± 0.13 
MRT(0–t) (h) 1.86 ± 0.11 
MRT(0–∞) (h) 2.12 ± 0.13 
CL (L/h) 51.21 ± 10.61 
AUC(0–t) (h/ng/mL) 294.03 ± 64.49 
AUC(0–∞) (h/ng/mL) 302.54 ± 63.09 

*t1/2: half-life; Cmax: maximum plasma concentration; tmax: time to reach maximum plasma concentration; CL: plasma clearance; MRT: average dwell time; AUC: area under the plasma concentration–time curve.

Figure 3.

Mean plasma concentration time profile after oral administration of a single dosage of 15 mg/kg R to 6 rats.

Figure 3.

Mean plasma concentration time profile after oral administration of a single dosage of 15 mg/kg R to 6 rats.

Discussion

The mobile phase played a critical role in achieving good chromatographic behavior (including peak symmetry and short analysis time) and appropriate ionization. Various combinations of acetonitrile, methanol, water and formic acid in water with changed contents of each component were investigated and compared to identify the optimal mobile phase. Acetonitrile was chosen as the organic phase because it provided sharper peak shapes than methanol. Formic acid added to the water improved the sensitivity, therefore 0.1% formic acid in water was chosen as the aqueous phase. Gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid avoided MEs for the analyte and IS, and provided proper retention times and better peak symmetry than isocratic elution (7–9). A flow rate of 0.4 mL/min produced good peak shapes and permitted a run time of 12 min.

The high selectivity of LC–MS does not guarantee the effective elimination of interferences from endogenous impurities. An operational strategy is to modify the chromatographic conditions to shift the retention time of the target analytes far away from the area affected by signal suppression or enhancement (10). In this study, an easy and effective way to make this adjustment was to modify gradient conditions. The MEs for R in rat plasma were measured to be 92.6–104.0%, which showed that the signal of R in rat plasma was not suppressed or enhanced.

In the study by Wang et al. (6), the chromatogram in Figure 3 shows considerable peak overlap between the IS and R; basically, the authors depend on the MS to extract the quantitative signals. The choice of IS and LC conditions in the current study appear to be better, with no peak overlap, as shown in Figure 2. The pharmacokinetic time profile (Figure 3) shows a maximum of approximately 0.6 h, whereas that of the Wang et al. study shows a maximum of approximately 4–5 h (6). The animals used in the study by Wang et al. were Male Wistar rats, whereas those in this study were Sprague-Dawley rats, which may cause the differences in pharmacokinetic data.

An efficient cleanup for biological samples to remove protein and potential interferences prior to LC–MS analysis was an important point in this study. The simple and effective protein precipitation was employed in this work. Acetonitrile was chosen as the protein precipitation solvent because it exhibited better effects than methanol, trichloroacetic acid (10%) or perchloric acid (6%), which provided acceptable recovery values.

Conclusion

A sensitive and specific LC–MS method was developed and validated for the determination of R in rat plasma over the concentration range of 5–500 ng/mL. Protein precipitation by acetonitrile was used for sample preparation, which is much simpler than the solid-phase extraction or liquid–liquid extraction reported in the literature (6). The LC–MS method was successfully applied to a pharmacokinetic study of R after oral administration of a single dosage of 15 mg/kg to rats.

Acknowledgments

The authors acknowledge financial support from the Youth Talent Program Foundation of The First Affiliated Hospital of Wenzhou Medical College (qnyc010), the key academic subject (clinical Chinese pharmacy) of the Twelfth Five Year Program of State Administration of Traditional Chinese Medicine.

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