ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
A SIMPLE AND RAPID LC-MS/MS METHOD FOR THE DETERMINATION OF ENALAPRIL IN HUMAN PLASMA FOR PHARMACOKINETIC AND BIOEQUIVALENCE STUDIES IN KOREAN HEALTHY VOLUNTEERS
UNDER FASTING CONDITIONS
© 2014 C. N. Kang*, H. J. Kim**, Y. S. Park**, ***, S. H. Kim**, ***, H. K. Park****, *****,
H. S. Hwang*****, J. S. Kang**, ***
*Department of Orthopaedic Surgery, College of Medicine, Hanyang University Seoul 133-791 South Korea **Division of Molecular Therapeutics Development, Hanyang Biomedical Research Institute
Seoul 133-791 South Korea ***Department of Pharmacology & Clinical Pharmacology Lab, College of Medicine, Hanyang University
Seoul 133-791 South Korea ****Department of Medical Education & Family Medicine, College of Medicine, Hanyang University
Seoul 133-791 South Korea *****Department of Family Medicine, College of Medicine, Hanyang University Seoul 133-791, South Korea Received 17.01.2012; in final form 04.03.2013
A simple and rapid liquid chromatography-tandem mass spectrometry method for Enalapril in human plasma was developed and applied to pharmacokinetic and bioequivalence test for 2 formulations of Enalapril (10 mg) capsules in healthy korean volunteers under fasting state. The analytes were extracted from plasma by simple protein precipitation by acetonitrile, separated on YMC C8 column using methanol—10 mM ammonium formate (80 : 20, v/v) as the mobile phase, and detected by tandem mass spectrometry with Turbo IonSpray interface operating in the positive ion mode for Enalapril and Glibenclamide (IS) in MRM mode. The ionization was optimized using electro-spray ionization (ESI) (+) and selectivity was achieved by MS/MS analysis, m/z 376.447 ^ 234.1 for Enalapril and m/z 494.1 ^ 369.1 for IS. The assay exhibited good linearity in the concentration ranges of 1.0 ~ 300 ng/mL for Enalapril in human plasma with lower limit of quantification (LLOQ) of 1.0 ng/mL. The chromatographic run time was approximately 2.0 min. No endogenous compounds were found to interfere with the analysis. The accuracy and precision were acceptable for concentrations over the standard ranges. The method was successfully applied to pharmacokinetic (PK) and bioequivalence (BE) studies by determination of Enalapril in the blood sample taken up to 12 h after oral administration of two Enalapril (10 mg) formulations and results from PK analysis suggested that the 2 types of 10 mg Enalapril tablets should be considered to be bioequivalent for both the extent and rate of absorption in normal volunteers.
Keywords: LC-MS/MS method, Enalapril, pharmacokinetics, bioequivalence test.
DOI: 10.7868/S0044450214050041
Enalapril (N- [( 1 ,^)-1-(ethoxycarbonyl)-3-phenyl-propyl]-Z-alanyl-Z-proline) maleate is an effective prodrug, which is deesterified to an active diacid form enalaprilat for the treatment of renovascular hypertension and congestive heart failure [1]. The active metabolite, enalaprilat, is an inhibitor of angiotensin converting enzyme (ACE) that leads to decreased vasopressor activity [2]. Following oral administration of Enalapril in healthy subjects, absorption is rapid and bioavailability is about 60 to 70% [3, 4]. The therapeutic daily dose ofEnalapril is 10—20 mg and the time for
maximum concentration of Enalapril in the blood is about 1 h; it disappears from circulation below level of 10 ng/mL after 4 h of drug dose. The terminal half-life of Enalapril and active metabolite, enelaprilat, is approximately 2 h and 30—35 h, respectively [5, 6]. Furthermore, 7max of active metabolite, enalaprilat, is about 3—4 h after administration [7—9]. Therefore, Enalapril and enalaprilat are often determined simultaneously in biological fluids [10]. Most of the pharmacokinetic (PK) studies [1, 11] were based on determining Enalapril in plasma by converting it to enalap-
rilat by alkaline hydrolysis and then assaying the latter form by a fluorometric [12], radio-enzymatic [13], ACE inhibition assay, or radioimmunoassay [14]. Modern methods, which include chemiluminescence using flow injection [15], GC-MS [6], LC-MS/MS [16-18], LC-MS [19] and LC-ESI-MS [20], have made it possible to directly measure Enalapril and enalaprilat.
Recently, these methods have been developed for simultaneous measurement of Enalapril and enalaprilat in human plasma to apply to PK and BE studies. However, simultaneous measurement of the two substances by these methods has disadvantages for BE study, as the terminal half-life of enalaprilat is about 30—35 h after an oral dose of Enalapril and the period of drug monitoring lasts about 72 h [18]. Recently, most ofLC-MS/MS and LC-MS methods were used in the simultaneous determination of Enalapril and enalaprilat. However, the long time for analysis (>2.5 min) and large volume of plasma sample (>0.2 mL), or low extraction recovery may be weak point for high throughput, speed and sensitivity in the PK or BE studies. Because the elimination half-life of enalaprilat is very long, and long monitoring time is reluctant and inconvenient to volunteers and researcher, monitoring of only pro-drug, Enalapril, is sufficient in routine BE studies of Enalapril.
This paper presents a simple and rapid LC-MS/MS method that described small volume of plasma (0.1 mL) and total run-time sparing (<2.0 min), more simple protein precipitation with acetonitrile and validated LC-MS/MS method for measuring Enalapril in human plasma to perform PK and BE studies after an oral dose of two capsules of a Enalapril (10 mg) formulations in 22 healthy volunteers.
EXPERIMENTAL
Chemicals and reagents. Enalapril maleate (CAS 76095-16-4) was purchased from Sigma-Ald-rich Co. (St. Louis, MO, USA) and Glibenclamide (internal standard, CAS 1023-21-8) was purchased from Wako Co. (Tokyo, Japan). HPLC grade acetonitrile, methanol, and ammonium formate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Other commercially available agents and solvents used were of analytical grade. The water was purified using a Milli-Q system (Millipore Co. USA). A reference drug (Lemipril™, Choongwae Pharm Co. South Korea) and test drug (Malepril™, Hanseo Pharm Co. South Korea), each containing 10 mg Enalapril maleate per tablet, were used in this study.
Preparation of standard and quality control (QC) samples. Standard stock solutions of Enalapril and IS were prepared in methanol at a final concentration of 1.0 mg/mL and stored at —20°C. A set of six nonzero calibration standards, ranging from 1.0 to 300 ng/mL, was prepared by spiking drug-free human plasma with
an appropriate amount of Enalapril. Quality control (QC) samples at four concentration levels [1.0 ng/mL (LLOQ), 10 ng/mL (low), 100 ng/mL (medium), and 300 ng/mL (high)] were prepared in a manner similar to the calibration standards. Blank human plasma was tested before spiking to ensure that no endogenous interference was found around retention times of Enala-pril and IS.
Plasma sample preparation. A 0.1 mL aliquot of human plasma was transferred to a screw cap 10-mL glass test tube with 20 |L of IS working solution (10 |g/mL) and 0.5 mL of acetonitrile. The mixture was vortex-mixed for 1 min. After centrifuging at 12000 rpm for 10 min, 0.3 mL of the upper organic layer was then evaporated to dryness under nitrogen in a TurboVap evaporator. The residues were reconstituted in 0.2 mL of mobile phase consisting of methanol and 10 mM ammonium formate (50 : 50, v/v) and centrifuged at 12000 rpm for 5 min. Then, 5 |L of the upper layer of reconstituted sample were directly injected into the LC-MS/MS system.
LC-MS/MS instrumentation and operation conditions. The LC system used for quantification of ana-lytes was an Agilent 1100 series (Agilent Technologies, Inc., Palo Alto, CA) chromatograph system equipped with an Agilent 1100 Autosampler (HTC PAL system, CTC analytics AG, Switzerland) and Peak Simple LC Data System (Lab Alliance Co., State College, PA, USA) with Analyst 1.4 software (Applied Biosystems). Mass spectrometric analysis was performed using an API 2000™ MS system (Applied Biosystems, Foster City, CA, USA) that was equipped with a TurboIonSpray ion source. ESI (5000.0 V) was performed in the positive ion mode with nitrogen as the nebulizing, turbo spray and curtain gas. This system was set to MRM (multiple reaction monitoring) mode for mass spec-trometric analysis and detection. The analytical column was an YMC hydrosphere C8 column (2.0 x 50 mm i.d., 3 |m; Waters Co., Milford, MA, USA). The mobile phase consisted of methanol and 10 mM ammonium formate (80 : 20, v/v). The flow rate was 0.2 mL/min and the injection volume was 5 | L. The dwell time per transition was set at 150 ms, and the source temperature was set at 350°C with ultra-high purity nitrogen as the curtain gas (50.0 L/min) and collision gas (5.0 L/min). A full-scan positive ion spectrum showed that the precursor ions were the protonated molecules, [M + H]+, of m/z 376.447 for Enalapril and m/z 494.1 for IS. After collision-induced dissociation, the most abundant ion in the product ion mass spectrum was at m/z 376.447 ^ 234.1 for Enalapril at a collision energy of 27.0 eV and m/z 494.1 ^ 369.1 for IS at a collision energy of 27.0 eV for identification and quantification in MRM. The strongest fragment of each compound was selected and used as the Q3 ion to be monitored. Unit resolution was used for both Q1 and Q3 mass detection. The ion source parameters were set as follows: curtain gas = 50 psi, collision gas 5 psi, ion spray voltage 5000 V, temperature 350°C, ion source (GS1)
50 psi and ion source (GS2) 50 psi. No significant interferences at the retention times of Enalapril or IS were observed in the mass chromatograms of blank plasma under the aforementioned LC-MS/MS conditions.
Method validation. Calibration curves were based on peak area ratios of Enalapril to IS for seven calibration standards over the range of 1.0—300 ng/mL for Enalapril in human plasma analyzed in duplicate. The LLOQ was d
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