HPLC_ESI_MS法测定人血浆中依普利酮的浓度及其药代动力学研究_英文_

QIAN Wen-juan1, DING Li1*, WEN Ai-dong2*, GONG Bin1, LENG Ye1, YUN Chang-hong1, YANG Lin2 (1. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China; 2. Organization for

State Drug Clinical Trial, Xijing Hospital Affiliated to the Fourth Military Medical University, Xi’an 710032, China)

Abstract: A sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was established for the determination of eplerenone (EP) in human plasma. The plasma samples of EP were extracted with ethyl acetate and separated by HPLC on a reversed phase C18 column with a mobile phase of 10 mmol·L−1 ammonium acetate water solution-methanol (30∶70, v/v). EP was determined with electrospray ionization-mass spectrometry (ESI-MS) in the selected ion monitoring (SIM) mode. The calibration curves were linear over the range of 2−4 000 ng·mL−1 for EP. The lower limit of quantification was

2 ng·mL−1. The method has been successfully applied in the pharmacokinetic study of the EP tablets. The

main pharmacokinetic parameters of EP after oral administration of 25 mg, 50 mg, 100 mg were as follows, t1/2:

(4.9 ± 2.1), (4.7 ± 1.5), (5.9 ± 1.2) h; AUC0−∞: (4 402 ± 1 735), (8 150 ± 2 509), (13 783 ± 4 102) µg·h·L−1; and

MRT: (6.2 ± 2.1), (6.6 ± 1.3), and (7.2 ± 1.6) h. Parameters of EP after oral administration of multiple doses of

50 mg were as follows, t1/2: (6.1±1.7) h; AUC ss: (10 071±4 220) µg·h·L−1; MRT: (8.1±2.3) h; and DF: (3.2± 1.0).

Key words: eplerenone; HPLC-ESI-MS; pharmacokinetics

CLC number: R917 Document code: A Article ID: 0513-4870 (2009) 07-0771-07 HPLC-ESI-MS法测定人血浆中依普利酮的浓度及其药代动力学研究钱文娟1, 丁黎1*, 文爱东2*, 龚斌1, 冷晔1, 云昌宏1, 杨林2

(1. 中国药科大学药物分析教研室, 江苏南京 210009;

2. 中国人民第四军医大学西京医院国家药品临床试验机构, 陕西西安 710032)

摘要: 建立测定人血浆中依普利酮浓度的HPLC-ESI-MS法。依普利酮血浆样品采用乙酸乙酯提取, 色谱柱为反相色谱C18柱, 流动相为10 mmol·L−1醋酸铵水溶液-甲醇 (30∶70, v/v)。质谱离子源为电喷雾离子化 (ESI) 源, 选择性离子检测方式检测; 血浆样品在2～4 000 ng·mL−1内线性良好, 定量下限为2 ng·mL−1。本文采用该方法对健康受试者的人体药代动力学进行研究, 3个单剂量 (25 mg、50 mg和100 mg) 口服给药后的药代动力学参数分别为t1/2: (4.9 ± 2.1)、(4.7 ± 1.5)、(5.9 ± 1.2) h; AUC0−∞: (4 402 ± 1 735)、(8 150 ± 2 509)、(13 783 ± 4 102) µg·h·L−1; MRT: (6.2 ± 2.1)、(6.6 ± 1.3)、(7.2 ± 1.6) h; 多剂量口服给药50 mg后药代动力学参数为t1/2: (6.1 ± 1.7) h;

AUC ss : (10 071 ± 4 220) µg·h·L−1; MRT: (8.1 ± 2.3) h; DF: (3.2 ± 1.0)。

关键词: 依普利酮; HPLC-ESI-MS; 药代动力学

Introduction

Eplerenone (EP, Figure 1A), pregn-4-ene-7α,21- Received 2008-12-11.

*Corresponding author Tel / Fax: 86-25-832712,

E-mail: dinglihg@sina.com, adwen@fmmu.edu.cn dicarboxylic acid, 9,11α-epoxy-17α-hydroxy-3-oxo, γ- lactone, methyl ester, is the first selective aldosterone receptor antagonist (SARA) approved for the treatment of hypertension and left ventricular dysfunction after acute myocardial infarction[1]. The pharmacokineticprofile of EP in Chinese volunteers has not been reported yet. To evaluate the pharmacokinetics of EP tablets in humans, a sensitive method for the determination of EP in human plasma is required. A SPE-LC-MS/MS method[2] for the determination of EP and its hydrolyzed metabolite with a lower limit of quantification (LLOQ) of 10 ng·mL−1 in human plasma for EP has been reported. The hydrolyzed metabolite was an open lactone-ring form of EP[3], plasma concen-trations of which were much lower than EP concentra-tions, and the area under the plasma-concentration time curve (AUC) for the metabolite was only 4% of the AUC of EP[4], further more, the ring-opened form of EP and other metabolites were all inactive[4,5]. So in the pilot pharmacokinetic study in our laboratory, only EP was determined. Meantime our study showed that the concentration levels of EP in many plasma samples obtained on the terminal elimination phase were below 10 ng·mL−1, especially when the low dose of 25 mg EP was given. In this paper, a more simple and sensitive HPLC-ESI-MS method with the LLOQ of 2 ng·mL−1 EP in human plasma was established. The validated method was used to evaluate the pharmacokinetics of EP in healthy Chinese volunteers. This paper reported, for the first time, the pharmacokinetics of EP in healthy Chinese volunteers after oral administration of the three single-doses of 25, 50, or 100 mg and the multiple-dose of 50 mg of EP.

Figure 1Chemical structures of EP (A) and the IS (B)

Materials and methods

Chemicals and reagents EP (99.7% purity), and the internal standard (IS) Δ9,11-eplerenone (99.3% purity, see Figure 1B) were both obtained from Changzhou Siyao Pharmaceutical Co., Ltd. (Jiangsu, China). The test formulation was EP tablets (each tablet containing 25 mg EP) provided by Changzhou Siyao Pharmaceutical Co., Ltd. (Jiangsu, China). Methanol was of HPLC grade (Merck KgaA, Germany). Ethyl acetate and ammonium acetate were of analytic grade purity and purchased from Nanjing Chemical Regent Co., Ltd. (Nanjing, China). Distilled water, prepared from demineralized water, was used throughout the study.

Instruments The HPLC-ESI-MS method was performed using an Agilent Technologies Series 1100 LC/MSD SL system (Agilent Technologies, Palo Alto, CA), which included an Agilent 1100 G1312A binary pump, vacuum degasser (model G1322A), G1316A injection temperature controlled column compartment, Agilent 1100 autosampler (model G1313A), and an Agilent 1100 MSD single quadrupole mass spectrometer equipped with an electrospray source (model G1956B).

A Hanban Lichrospher-C18 column (250 mm × 4.6 mm ID, 5 μm, Haban Sci & Tech Co., Ltd., China) was used for the separation. The signal acquisition, peak integration and concentration determination were performed by using the ChemStation software (10.02A) supplied by Agilent Technologies.

Preparation of standard solutions The stock solutions of EP were prepared at the concentration level

of 1.0 mg·mL−1 in methanol. The stock solution of EP was diluted with methanol to prepare the working solutions at the concentration levels of 100 μg·mL−1, 10 μg·mL−1, 1.0 μg·mL−1, and 100 ng·mL−1. The stock solution of IS was also prepared in methanol. All of the solutions were stored at −20 ℃.

Sample preparation Aliquot of 0.5 mL plasma sample and 50 µL IS (5 μg·mL−1) were both placed into a 10 mL centrifuge tube and vortex-mixed for 10 s, then the plasma sample was extracted with 3 mL ethyl acetate by vortex mixing for 3 min. After being centrifuged for 8 min, the ethyl acetate phase was separated and evaporated to dryness under a stream of nitrogen in a water bath at 37 ℃. The residue was reconstituted with aliquot of 120 µL mobile phase, and

a 5 µL aliquot of the reconstituted solution was injected onto the HPLC-ESI-MS for analysis.

Chromatographic conditions and mass spectro-metric conditions The mobile phase was methanol-

10 mmol·L−1 ammonium acetate aq. (70∶30, v/v) at

a flow rate of 1.0 mL·min−1. The column temperature was maintained at 30 ℃. The HPLC-ESI-MS was carried out using nitrogen to assist nebulization. The quadrupole mass spectrometer equipped with an ESI source was set with the drying gas (N2) flow of 10 L·min−1, nebulizer pressure of 276 kPa, drying gas temperature of 350 ℃, capillary voltage of 4.0 kV and the positive ion mode. The fragmentor voltage was 100 V. The ESI-MS was performed in the selected-ionQIAN Wen-juan, et al: Establishment of HPLC-ESI-MS method for the determination of eplerenone in human

plasma and its pharmacokinetics·773·

monitoring (SIM) mode using the target ions [M+H]+

at m/z 415.3 for EP and m/z 399.3 for IS. Figure 2 showed the typical full-scan ESI mass spectrum of EP and the IS.

Preparation of calibration curves and quality control samples The calibration standards of EP were prepared by spiking appropriate amounts of the standard solutions in 0.5 mL blank plasma, obtained from healthy volunteers. The standard curves were prepared in the range of 2−4 000 ng·mL−1 for EP at concentration levels of 2, 10, 50, 200, 500, 1000, 2 000,

3 000 and

4 000 ng·mL−1. The calibration curves were prepared and assayed along with the quality control (QC) samples. The QC samples for EP were prepared

in 0.5 mL blank plasma at concentration levels of 5, 300 and 3 600 ng·mL−1.

Assay validation

Selectivity The selectivity of the method was checked by comparing the chromatograms of the blank plasma samples from six different batches with the corresponding spiked plasma samples. Each blank plasma sample was tested using the proposed extraction procedure and the HPLC-ESI-MS conditions to ensure

no interference of the analysands from the plasma.

Linearity The calibration standards of nine EP concentration levels at 2, 10, 50, 200, 500, 1 000, 2 000,

3 000 and

4 000 ng·mL−1 were extracted and assayed.

To evaluate the linearity, plasma calibration curves were prepared and assayed on five different days. The calibration curve was constructed by plotting the peak- area ratios of the EP to the internal standard versus the concentrations of the EP, using weighed least squares linear regression (1/C2).

Quality control samples and lower limit of quanti-fication The calibration curve was prepared and assayed along with quality control (QC) samples and each batch of clinical plasma samples. The concentra-tions of quality control samples for EP at low, medium and high levels were 5, 300 and 3 600 ng·mL−1, respec-tively. The assay precision was calculated using RSD (%) and the accuracy is defined as the relative deviation

in the experimental calculated value (E) of a standard from that of its true value (T) expressed as a percentage (RE%). It was calculated using the formula: RE (%) = (E−T)/T×100. The LLOQ was established using five samples independent of standards.

Extraction recovery and matrix effect The extraction recovery of EP was evaluated by analyzing five replicates at concentration levels of 5, 300 and 3 600 ng·mL−1. Recovery was calculated by comparison

of the peak areas of analysands extracted from plasma samples with those of injected standards.

The matrix effect (ME) was examined by comparing

the peak area of the analysands between two different sets of samples. In set 1, EP and IS were added to the reconstructed solutions of the blank plasma samples and assayed by HPLC-MS, and the obtained peak area

of the two analysands was defined as A. In set 2, EP and IS were resolved in the mobile phase and assayed

by HPLC-MS, and the obtained peak area of analysands were defined as B. The ME values were calculated by using the formula: ME (%) = A / B × 100. The matrix effect on EP was evaluated at low, middle and high concentration levels. The matrix effect of the IS was also evaluated.

Clinical study design and pharmacokinetic analysis

V olunteers and study design Twenty healthy Chinese volunteers (ten males and ten females), aged 33.2 ± 3.5 years, were recruited. All volunteers gave their written consent and the study protocols were approved by the relevant Ethical Review Committee, in accordance with the principles of the Declaration of Helsinki. The study was carried out with a randomized sequence, open-label, parallel design. The eligible volunteers were twenty healthy volunteers who were randomly divided into two groups (ⅠandⅡ), each group had five males and five females. The group Ⅰ

was assigned to receive a single 25 mg dose first, and then after a 1-week washout period, they were given a single 100 mg dose of EP. The group Ⅱ was given a single 50 mg dose first (day 1), collected blood samples

and then given multiple dose on the consecutive six days (from day 3 to day 8, once 50 mg per day). The drugs were administered with 250 mL of water at 7∶30

am after a 12 hour overnight fast. Standard meals were provided 4 h post-dosage. For the group Ⅰ, the venous blood samples of 5 mL were collected pre-dosage (0 h) and at 1, 1.5, 2, 3, 4, 6, 9, 12, 15, 24 and 30 h post-dosage, respectively. For the group

Ⅱ, the venous blood samples of 5 mL were collected

pre-dosage (0 h) and at 1, 1.5, 2, 3, 4, 6, 9, 12, 15, 24 and 30 h post-dosage on day 1 and day 2 for the single-dosage pharmacokinetic study, then the venous blood samples of 5 mL were collected at 0 h (pre- dosage) on day 5, 6, 7 and 8, and at 1, 1.5, 2, 3, 4, 6, 9, 12, 15, 24 and 30 h post-dosage on day 8 for the multiple-dose pharmacokinetic study. All blood·774·药学学报Acta Pharmaceutica Sinica 2009, 44 (7): 771−777

samples were collected in heparinized tubes, and the plasma were separated immediately by centrifugation at 1 500 ×g for 10 min and stored at −20 ℃ until analysis.

Pharmacokinetic analysis and statistical analysis of eplerenone Pharmacokinetic analysis was performed by using the drug and statistics (DAS) 2.0 pharmacoki-netic program authorized by the Chinese Pharmacology Society (Beijing, China). Data obtained from the volunteers were subjected to non-compartmental pharmacokinetic analysis. All the values of the pharmacokinetic parameters were expressed as mean ± standard deviation (SD). In the study, linear regression and one-way analysis of variance (ANOVA) were used to study the linear pharmacokinetic characteristics of EP and whether EP accumulation existed after multiple dosing, respectively.

Results

1 Selectivity, calibration curve and sensitivity

Figure 2 showed the typical full-scan ESI mass spectra of EP and the IS. Selectivity was assessed by comparing the chromatograms of six different batches of blank human plasma with the corresponding spiked plasma. Figure 3 showed the typical chromatograms of a blank plasma sample, a spiked plasma sample with EP at the LLOQ of 2 ng·mL−1 and IS, a spiked plasma sample of 4 000 ng·mL−1 EP and the IS, and a plasma sample from a healthy volunteer of the study. There was no significant interference from endogenous sub-stances observed at the retention times of the analysands.

For EP, good linearity was showed over the range of 2−4 000 ng·mL−1, the typical calibration curve for EP had a slope of 0.001333, an intercept of 0.000 552 0 and r > 0.998. Calibration curves were prepared and analyzed with each run of clinical samples and QC samples. The LLOQ of the method was 2 ng·mL−1, at which the RSD was 6.9% (n = 5) and the RE % was less than 20%.

2 Assay precision and accuracy

Table 1 summarizes the intra- and inter-batch precision and accuracy of the method. The precision was calculated by using one-way ANOVA. The results demonstrated that the method was accurate and precise. Table 1 Accuracy and precision for the analysis of EP in human plasma (in prestudy validation, three batches, five replicates per run)

Added C

/ng·mL−1

Found C

/ng·mL−1

Intra-batch

RSD/%

Inter-batch

RSD/%

RE/%

5.200 5.287 5.8 14.9 1.7

312.0 345.9 4.8 6.1 10.9

3 74

4 3 630 1.6 4.6 −3.1

RE: Relative error

3 Extraction recovery and stability

For the determination, ethyl acetate was chosen as

the extraction solvent for its higher extraction efficiency

of the two target compounds. It does not only eliminate

the interference of endogenous substances, but also meet the requirement of sensitivity for the method. The values of the recovery at three evaluated concentration levels of 5, 300 and 3 600 ng·mL−1 were 73.7% ± 5.4%, 75.5% ± 2.2% and 73.6% ± 4.1% (n = 5), respectively.

The stability was studied under a variety of storage and handling conditions at QC levels. The results showed that no significant degradation of the analysands occurred at ambient temperature for 6 h. The sample

of EP in autosampler was stable for at least 9 h and there was no significant degradation during the three freeze-thaw cycles for EP plasma samples. EP in plasma at −20 ℃was stable for three months at least.

4 Matrix effect

The matrix effect of the method was evaluated at low, middle and high concentration levels. Five

Figure 2 Mass spectra of the positive ions of EP(A) and the IS (B) at 100 V fragmentor voltageQIAN Wen-juan, et al: Establishment of HPLC-ESI-MS method for the determination of eplerenone in human

plasma and its pharmacokinetics·775·

Figure 3Typical SIM mass chromatograms of blank plasma (A), LLOQ for EP (2 ng·mL−1) and the IS in plasma (B), plasma spiked with EP (4 000 ng·mL−1) and the IS (C), plasma obtained from a volunteer at 1 h after oral administration of EP tablets, the plasma concentration of EP was estimated to be 1069 ng·mL−1 (D)

samples at each concentration level of the analysands

were analyzed. The blank plasma samples used in this

study were from five different batches of human blank

plasma. The ME value of the internal standard was

also evaluated. If the ME values exceed the range

of 85%−115%, an exogenous matrix effect could be

implied. As shown in Table 2, the results obtained

were within the acceptable limit, and indicated that no

matrix effect of the analysands was observed in this study.

Table 2Matrix effect data for EP and the IS in the five different

batches of human plasma (n = 5, mean ± SD)

Sample Added C

/ng·mL−1

A B

Matrix

effect/%

EP 5.2 32 629 ± 1 354 31 731 ± 1 960 102.8 312 1 939 684 ± 56 368 1 811 680 ± 126 472 107.1

3 74

4 19 600 526 ± 934 30618 0 137 ± 100 834 108.4 IS 528.

5 4 027 990 ± 254 061 3 850 495 ± 212 713 104.65 Pharmacokinetic study

The developed method was applied to determine the plasma concentration-time profiles of EP in healthy volunteers after being given the single oral dose of 25, 50, or 100 mg of EP, and after a multiple dose of 50 mg, once daily for 6 days.

5.1 Single-dose administration The mean plasma concentration-time profiles of EP after the single oral dose of 25, 50 or 100 mg are shown in Figure 4, and the pharmacokinetic parameters are summarized in Table 3. The ANOVA analyses showed that there were no significant differences in t max, t l/2 and MRT of EP within the three different doses. Over the dosage range of 25 to 100 mg, the obtained values of C max and AUC0−∞ were all increasing proportionally to the dose, the C max - dose calibration curve had a slope of 15.54, an intercept of 35

6.0 and r > 0.99, the AUC0−∞- dose calibration curve had a slope of 123.3, an intercept·776·药学学报Acta Pharmaceutica Sinica 2009, 44 (7): 771−777

of 1586 and r>0.99, the MRT and t1/2 values did not increase following the dose escalation (Table 3). Therefore, the linear pharmacokinetics was found for EP in healthy Chinese volunteers after a single oral dose administration over the dose range of 25 to 100 mg.

5.2 Multiple-dose administration The mean plasma concentration-time profiles of EP after multiple oral doses (50 mg once daily for 6 days) are shown in Figure 4. Mean plasma concentrations of EP (n = 10) on day 5 to 8 before dosing were 6

6.84, 6

7.16, 71.90 and 76.46 ng·mL−1, respectively, indicating that plasma concentration reached steady state after ANOVA analysis. The pharmacokinetic parameters of EP, such as area under the curve at steady state (AUC ss), maximum steady-state plasma concentration (C ssmax), minimum steady-state plasma concentration (C ssmin), average steady-state plasma concentration (C ssav), and fluctuation percentage (DF), are summarized in Table 3. After 6 daily doses of 50 mg EP, the main pharmacoki-netic parameters (C max, t1/2, t max, MRT, AUC) were not significantly different from those obtained in the single dose treatment according to the results of the ANOVA (Table 4).

The extent of accumulation was estimated as accumulation index (R) using the following three equations[6]:

R1 = C ssmax / C max

R2 = AUC ss / AUC0−τ

R3 = 1 / (1 − e−kτ)

Where τ was dosing interval (24 h) and k was elimination

rate constant of EP after multiple doses. The values of

R1, R2 and R3 were 1.10 ± 0.14, 1.26 ± 0.20 and 1.07 ±

0.04, respectively.

Table 4 The ANOVA of EP pharmacokinetic parameters in healthy Chinese volunteers after the single oral dose of 50 mg EP

and the multiple oral doses of EP (50 mg once daily for 6 days) Parameter d.f.a Mean

square F ratio P

C max 1 68

708 0.821

9

0.38

AUC 1 18 386 277 1.527 0.23

t1/2 1 10.76 4.024

0.06

MRT 1 10.21 2.868 0.11

t max 1 0.703

1 2.073 0.17 Degrees of freedom

Table 3Pharmacokinetic parameters of EP in healthy Chinese volunteers after the single oral doses of 25, 50 or 100 mg of EP and the multiple oral dose administration of EP, 50 mg once daily for 6 days. (n = 10, x± s)

Single dose Multiple dose

Parameter

25 mg 50 mg 100 mg 50 mg

C max /ng·mL−1703.5 ± 122.8 1 187 ± 236 1 882 ± 310 1 305 ± 334a

t max /h0.9 ± 0.4 1.8 ± 0.6 1.4 ± 0.3 1.4 ± 0.6

MRT /h 6.2 ± 2.1 6.6 ± 1.3 7.2 ± 1.6 8.1 ± 2.3

t1/2 /h 4.9 ± 2.1 4.7 ± 1.5 5.9 ± 1.2 6.1 ± 1.7

AUC0−30 h /µg·h·L−1 4 292 ± 1 587 8 001 ± 2 335 13 402 ± 3 732 10 071 ± 4 220b

AUC0−∞ /µg·h·L−1 4 402 ± 1 735 8 150 ± 2 509 13 783 ± 4 102 −

C ssmin /ng·mL−1−−− 76.46

±

77.56

C ssav /ng·mL−1−−− 419.6

±

175.8 DF −−− 3.2

±

1.0

a C

ssmax

; b AUC ss

Figure 4 Mean EP plasma concentration-time profiles in Chinese volunteers (n = 10) after administrations of the single oral doses of 25, 50 and 100 mg EP (A), the multiple oral dose of 50 mg EP once daily for 6 days (B)

QIAN Wen-juan, et al: Establishment of HPLC-ESI-MS method for the determination of eplerenone in human

plasma and its pharmacokinetics·777·

Discussion

Several experiments show that the use of an appropriate ratio of ammonium acetate buffer solution

in the mobile phase may improve the chromatographic peak shapes[7, 8]. So, the different concentrations of ammonium acetate buffer solution at levels of 10, 20 and 30 mmol·L−1 were tested in the mobile phase. The test results showed that the 10 mmol·L−1 ammonium acetate buffer was sufficient enough to improve the chromatographic peak shapes of EP and the IS, and result in the symmetric chromatography peaks of the analysands.

Several solvents were tested for the extraction. Finally, ethyl acetate was chosen as the extraction solvent that can produce a clean chromatogram for a blank plasma sample and yield the highest recovery for EP from the plasma.

The study showed that the t1/2, t max and MRT values of EP did not change with the increasing of the dose, but the AUC (including AUC0−30 h and AUC0−∞) and C max values increased following the increasing of the EP dose. Linear pharmacokinetics was found at dose range from 25 to 100 mg. The multiple-dose pharmacokinetic parameters of EP were consistent with those obtained from the single-dose administration. These suggested that there was no significant EP accumulation in the multiple-dose treatment of 50 mg per day for six days. The values of t1/2, t max and C max

of EP in Chinese volunteers have no difference with those values reported in Europeans[3], but the AUC value of EP in Chinese is a little higher than that in Europeans after a single dose of 100 mg EP.

Conclusion

The assay achieved high sensitivity and specificity for the determination of EP in human plasma. The simple and sensitive method was shown to be suitable for the pharmacokinetic study of eplerenone in human subjects and the pharmacokinetic profile of eplerenone in healthy Chinese volunteers was presented.

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