Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 405–410 Direct determination of verapamil in rat plasma by coupled column O.H. Jhee, J.W. Hong, A.S. Om, M.H. Lee, W.S. Lee, a Department of Food and Nutrition, College of Human Ecology and Institute of Biomedical Science, Hanyang University, Seoul 133-791, South Korea b Department of Pharmacology, College of Medicine and Institute of Biomedical Science, Hanyang University, Seoul 133-791, South Korea c Department of Internal Medicine, College of Medicine, Hanyang University, Seoul 133-791, South Korea d Scientific Affairs Department, Sanofi-Synthelabo Korea Co. Ltd., Seoul 135-932, South Korea e Department of Pathology and Lab medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA Received 6 August 2004; received in revised form 29 October 2004; accepted 1 November 2004 Abstract
This report describes an automated coupled column microbore-high-performance liquid chromatography (HPLC) with fluorescence de- tection for direct determination of verapamil in small volume of rat plasma. We used HPLC system consisting of three columns such asprecolumn, intermediate and analytical column and six-port switching valve and injected small volume of rat plasma to the system withoutsample preparation. An aliquot of sample was directly injected into Capcell Pak MF Ph precolumn for clean-up and enrichment, 35 mmCapcell Pak C18, intermediate column for concentration of compounds and 250 mm Capcell Pak C18 analytical column for separation ofcompounds and two mobile phases are used as mobile phase A (50 mM ammonium phosphate, pH 4.5) and B (50 mM ammonium phos-phate:acetonitrile = 70:30 v/v). Analysis of verapamil and internal standard, propranolol was performed with direct injection of 10 ␮l of ratplasma to the system and were eluted at 22 and 12 min, respectively, at a mobile phase flow rate of 0.5 (mobile phase A) and 0.15 ml/min(mobile phase B). The peaks of verapamil and internal standard were good shapes and well separated from any interfering endogenous peaksduring a total run time of 25 min. The calibration curve for verapamil showed good linearity (r2 = 0.9997) over the concentration range of0.01–2.50 ␮g/ml. The mean RSD (%) values of intra-day (n = 5) and inter-day (n = 5) variability of verapamil ranged from 1.96 to 9.06 and 0.62to 3.08%, respectively. The LOD and LOQ were 0.01 and 0.025 ␮g/ml, respectively, for verapamil using 10 ␮l of rat plasma. An automatedcoupled column microbore-HPLC method was successfully applied to a pharmacokinetic study after intravenous injection of 3 mg/kg ofverapamil to the normal and dimethylnitrosamine (DMN)-induced hepatofibrotic rats.
2004 Elsevier B.V. All rights reserved.
Keywords: Verapamil; Propranolol; Coupled column; Automated microbore-HPLC; Fluorescence detector; Rat plasma; DMN-induced hepatofibrotic rat 1. Introduction
of oral dosage of verapamil (40–180 and 120–140 mg in thecase of conventional tablets and slow releasing tablets, re- Verapamil (a synthetic paraverin derivate, which spectively) have been mostly used. Despite of 90% of oral belongs to phenylalkylamine class, is a calcium blocker absorption rate, low oral bioavailability (only 10–20%) of It has been used an important therapeutic agent for angina verapamil is attributed by extensive hepatic first-pass ef- pectoris, ischemic heart disease, hypertension and hyper- fect leading to a pharmacologically inactivation. During the trophic cardiomyopathy For clinical purpose two types metabolism it converted into norverapamil (N-demethylatedmetabolite), which exerts 20% pharmacological activity of verapamil, and other inactive metabolites In pharma- Corresponding author. Tel.: +82 2 2290 0652; fax: +82 2 2292 6686.
cokinetic study of verapamil therapeutic plasma levels were E-mail address: [email protected] (J.S. Kang).
0731-7085/$ – see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2004.11.003 O.H. Jhee et al. / Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 405–410 and HPLC/MS these techniques have limitationsincluding requirement of expensive instruments and spaciouslaboratory. In addition, gas liquid chromatography has beensuccessfully applied to analysis of verapamil and some of itsmetabolites in biological fluids But it is not appropri-ate to detect verapamil in micro-volume of plasma, becausethis method also requires at least 1 ml of plasma samples.
Furthermore, HPLC using either reversed-phase r nor-mal phase or ion-exchange column spectro-metric detection ve been described. In these meth-ods isolation of varapamil from biological fluids was per-formed by liquid–liquid extraction that is extraction from al-kaline sample into a volatile, water-immiscible, and organicphase followed by evaporation, subsequently reconstitutioninto mobile phase, or solvent compatible with mobile phaseHowever, the initial extraction is followed by a back-extraction into dilute aqueous acid that is then injected onto Fig. 1. Structures of verapamil (a) and propranolol (b); internal standard.
The purpose of this study is to develop and validate a fully automated reverse-phase coupled column microbore- considerably variable from 20 to 500 ng/ml depending on HPLC method with fluorometric detector for measuring ver- drug form used This suggests that patients require fre- apamil in micro-volume (10 ␮l) without sample preparation.
quent adjustments of individual dosage of this drug. For anal- The procedures includes precolumn for clean-up, enrichment ysis of drugs and their metabolites in biological samples, an of compounds followed by intermediate column for sample efficient high-performance liquid chromatography (HPLC) concentration or extraction, finally transferred to the analytic combined with a various mode of detection, which is com- column for subsequent HPLC analysis by means of an au- plicate to use, has been used. Because of undesirable sen- tomated column-switching valve. To confirm the feasibility sitivity and selectivity, sample preparations including sam- of the method we tried to apply this method to assess the ple clean-up, and pre-concentrations are required prior to pharmacokinetic properties of verapamil in hepatofibrotic rat analysis Traditional sample preparation methods such as liquid–liquid extraction and solid-phase extraction (SPE)are time-consuming and imprecise. The case of liquid–liquidextraction is difficult to be automated unlike the SPE method 2. Experimental
that is extremely flexible except for requirement of complexof robotic devices. Also, both liquid–liquid extraction and SPE methods require large amounts of high-purity solventsn contrast using an automated one-line coupled column Dimethylnitrosamine, verapamil hydrochloride and pro- or column-switching devices coupled advanced separation pranolol hydrochloride (I.S., internal standard) were pur- media technologies are enable to perform automated clean- chased from Sigma–Aldrich Co. (St. Louis, MO, USA) and up and trace enrichment of analytes in biological samples purity of these materials was greater than 99.5%. Ammonium as well as enable to improve analytical process Vari- phosphate dibasic (Wako Pure Chemical Industries Ltd., ous analytical techniques have been introduced for quanti- Japan) and pentobarbital sodium salt (TCI, Tokyo, Japan) fying verapamil in biological fluids such as plasma, serum were analytical reagent grade. Acetonitrile (HPLC grade) and urine, as well as tissues To date, HPLC method was purchased from Merck Co. (Darmstadt, Germany). Water has been mainly employed to quantify the concentrations was deionised and purified using a Milli-Q system (Millipore, of verapamil in biological samples even though gas Bedford, MA, USA). Stock solutions of verapamil and pro- chromatography coupled nitrogen–phosphorous detector has pranolol containing 1.0 and 10.0 mg/ml of each products in been widely used for measuring trace levels of drugs mobile phase A were prepared and stored at −20 ◦C. Stock It has been reported that verapamil in biological fluids and standard solutions of verapamil were prepared at a concen- tissue homogenates was determined by a reproducible fluo- tration of 1 mg/ml in mobile phase and diluted at a concen- rometric method However, this method requires a very tration of 1 ␮g/ml using the same solvent; the resulting solu- large volume of sample (greater than 4 ml), complicated sam- tion, was stable for several weeks at 4 ◦C. Under these con- ple preparation and interfering detection by fluorescent in- ditions, the solution was stable for several weeks. Working active several metabolites. More sensitive and specific ap- standard solutions of verapamil were prepared by sequential proaches to measure plasma verapamil are mass spectrome- dilution by drug-free plasma at 10, 25, 50, 250, 500, 1000 try (MS) with isotope dilution (mass fragmentography) O.H. Jhee et al. / Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 405–410 2.2. HPLC apparatus and chromatographic conditions The coupled column HPLC system used was a NANOSPACE SI-1 microbore system equipped with a six-port switching valve unit (Shiseido Co., Tokyo, Japan). Thesystem is designed for semi-microcolumn LC by reducingany possible dead spaced volume in the entire system be-cause the dead volumes in the connections between columnsand any switching valves can negatively affect the separationefficiency. The column used for the sample clean-up step wasan MF-Ph precolumn, Capcell Pak (4.0 mm i.d. × 20 mm,Shiseido Co., Tokyo, Japan). A C-18 column, Capcell PakUG120 (2.0 mm i.d. × 35 mm, Shiseido Co., Tokyo, Japan)was used for the primary separation of compound fromplasma using the mobile phase A at 0.5 ml/min; an analyticalC-18 column, Capcell Pak UG120 (1.5 mm i.d. × 250 mm,Shiseido Co., Tokyo, Japan) was employed in the analysisof verapamil by using the mobile phase B at a flow rateof a 0.15 ml/min. The column temperature was maintainedconstant at 45 ◦C. The column effluent was monitored us- Fig. 2. Separation of verapamil and propranolol-spiked plasma on MF Ph-1 ing a fluorescence detector with an excitation wavelength of precolumn and determination of switching time (min) for valves ( 3.10, 3.85 and 4.85); conditions: mobile phase A, flow rate: 0.5 ml/min, 280 nm and an emission wavelength of 313 nm. Mobile phase A (50 mM ammonium phosphate, pH 4.5) and mobile phase B(50 mM ammonium phosphate:acetonitrile = 70:30 v/v) were A at flow rate of 0.5 ml/min. The analytical column was filtered and degassed through a 0.22 ␮m Magna-R filter (Whatman International Co., Maidstone, UK).
• Step 3 (4.85–25.0 min, The compounds trapped in the top of intermediate column were transferred to the analytical column and analyzed by fluorescence de-tector at an excitation wavelength of 280 nm and an emis- A diagram of the different column-switching positions and time sequences of column-switching procedure in the HPLCsystem is shown in vely.
• Step 1 (0–2.35 and 3.10–3.83 min, The calibration curve and linearity for verapamil were in- Plasma sample (10 ␮l) was introduced onto precolumn vestigated by diluting aliquots of the working standards with where plasma proteins, verapamil and internal standard normal rat plasma to obtain concentrations of 0.01, 0.025, were separated using mobile phase A at flow rate of 0.10, 0.25, 0.5, and 1.0 ␮g/ml of verapamil and 400 ng/ml of 0.5 ml/min. The intermediate column and analytical col- propranolol to drug-free rat plasma and the mixtures diluted umn were equilibrated using mobile phase B at a flow rate as same volume by mobile phase A. After dilution the sam- ples was centrifuged at 13 000 × g for 10 min to spin down • Step 2 (2.35–3.10 and 3.85–4.85 min, the precipitate, the supernatant loaded into autosampler and When the valve status was changed to B, target drug- 10 ␮l were injected into the HPLC system. For the analy- containing fraction separated in precolumn was focused sis, aliquots of 0.4 ml of plasma were supplemented with on to the top an intermediate column using mobile phase 40 ␮l of a propranolol (10.0 ␮g/ml) and subsequently pro- Table 1Time sequences of column-switching procedure in analytical process Plasma sample introduced into precolumn where proteins and compoundswere separated and sample fraction without analytes into the waste bypassintermediate column, analytical column and detector The fraction of analytes were introduced and trapped in the top ofintermediate column The compounds trapped in the intermediate column were transferred andanalyzed in the analytical column Mobile phase A: 50 mM ammonium phosphate, pH 4.5; mobile phase B: 50 mM ammonium phosphate (pH 4.5):acetonitrile = 70:30 v/v.
O.H. Jhee et al. / Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 405–410 cessed following the method described above. The calibration ples in the small volume without any loss in sensitivity and curves were obtained by plotting the ratio of the peak areas chromatographic efficiency obtained by semi-microcolumns of verapamil to internal standard against the concentrations Coupled column devices have been proved to sim- of verapamil spiked into drug-free rat plasma. Intra-day re- plify the HPLC analysis of drugs in biological samples, by producibility was evaluated by analyzing sets of drug-free rat facilitating the total automation of the chromatographic pro- plasma samples, spiked with four concentrations of verapamil cess, then increasing the speed and work capacity In hydrochloride in the range 0.025–2.50 ␮g/ml at five differ- this study, Capcell Pak MF Ph-1 precolumn was appropri- ent time periods. The assessment of inter-day reproducibility ate to remove proteins and concentrate verapamil and pro- was based on the analysis of the same spiked plasma samples pranolol from plasma. To determine an appropriate time for on five consecutive days. The limit of detection (LOD) for column-switching, the retention behavior of verapamil and verapamil was determined as the concentration of drug giv- propranolol in plasma on the Capcell Pak MF Ph-1 precol- ing a signal to noise ratio greater than 3. The lower limit of umn was evaluated using mobile phase A and shown in quantitation (LOQ) was determined as the minimum con- The constructed coupled column microbore-HPLC with flu- centration that can be accurately and precisely quantified orescence detector and time sequences of column procedure is illustrated in respectively. HPLC chromatogramsobtained from blank plasma, plasma spiked with verapamil 2.5. Application to animal studies (1.0 ␮g/ml) and propranolol (0.5 ␮g/ml) and plasma col-lected from DMN-induced hepatofibrotic rat at 120 min af- Pharmacokinetics studies were carried out using male ter single intravenous injection of 30 mg/kg of verapamil are Sprague-Dawley rats (BW, 200–250 g, Joongang Animal Co., shown in To update, an increasing number of HPLC Seoul, Korea) divided into groups of five rats each and fasted methods with on-line sample clean-up by solid-phase extrac- overnight for 12 h prior to an experiment. The hepatofibrosis tion using coupled column devices have been developed. The was induced by DMN according to George et al. method principle of coupled column technique for sample clean-up Experimental rats received the 1% DMN (1.0 ml/kgi.p.) by injection for three consecutive days per week fora period of 4 weeks and control rats were treated with thesame amount of saline. After anesthesia with pentobarbi-tal sodium (30 mg/kg i.p.), the rats were surgically exposedthe left femoral artery, of the rats was surgically exposedand cannulated with catheter, intravascular catheterisation(PE-50) for blood sampling purposes. Verapamil (3 mg/kgi.v.) was administered through the tail vein. Blood sampleswere drawn from the femoral artery at 10, 30, 60, 120 and180 min after verapamil administration into polyethylene testtubes in each rat, and immediately centrifuged at 1700 × gfor 15 min. The clear plasma layer was transferred to cleantest tubes and stored at −70 ◦C for verapamil analysis ineach plasma samples. For the analysis, aliquots of 0.4 ml ofplasma were supplemented with 40 ␮l of internal standardsolution (propranolol, 10.0 ␮g/ml) and the mixtures dilutedas same volume by mobile phase A. After dilution the sam-ples was centrifuged at 13 000 × g for 10 min to spin downthe precipitate, the supernatant loaded into autosampler and10 ␮l were injected into the HPLC system. Plasma levelsof verapamil were determined employing the developed au-tomated coupled column microbore-HPLC system with flu-orescence detector. Calculation of pharmacokinetic param-eters was done using pharmacological calculation systems(Pharm/PCS) version 4.0 (Springer-Verlag, New York, USA,1986).
3. Results and discussion
Fig. 3. HPLC chromatograms of: (A) blank plasma, (B) blank plasma spikedwith propranolol (0.5 ␮g/Ml) and verapamil (1.0 ␮g/Ml), and (C) plasmasample at 2 h after intravenous injection of 30 mg/kg of verapamil and spiked Coupled column technique is a useful sample preparations with propranolol (0.5 ␮g/Ml) to a DMN-induced hepatofibrotic rat (I: pro- system that can directly analyze complex biological sam- O.H. Jhee et al. / Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 405–410 Table 2Precision and accuracy of coupled column microbore-HPLC method of determination of verapamil in rat plasma a Mean ± S.D. (n = 5): concentration were calculated from linear regression equation.
b Precision = (S.D./mean) × 100.
c A.C. (accuracy, %) = (measured amount/spiked amount) × 100.
is to trap the fraction of the sample that contains the ana- inter-day reproducibility, the same set of spiked plasma was lytes in the precolumn. The other compounds of the biolog- assayed on three consecutive days. The results are shown ical matrix are eluted to waste, whereas the cut-off effluent in The intra-day precision (RSD) was ranged from containing the analytes is diverted to the intermediate or an- 1.96 to 9.06%. The inter-day precision (RSD) was ranged alytical column, where they are separated for identification from 0.62 to 3.08%. The mean accuracy was 103.3% with and/or quantitation Zone cutting technique probably is RSD of 3.19%. The difference between the measured and the one of the most useful and versatile of the entire coupled spiked concentration were not more than 13% at any QC con- column techniques Therefore, we used two zones cut-ting techniques for propranolol and verapamil fraction inour system that is shown in The fractions of vera-pamil and propranolol isolated from precolumn by fractioncutting techniques were focused in the top of intermediatecolumn to obtain well-separated sharp peaks with retentiontime of 22 and 12 min in the final separation on analyti-cal column and the obtained chromatograms were shown inThe total analysis process was completed within ap-proximately 25 min. A calibration curve for verapamil quan-titation in plasma was obtained by plotting peak area ra-tios at seven concentrations in the range of 0.01–2.50 ␮g/ml,in the presence of 0.5 ␮g/ml of propranolol. The relation-ship between peak areas ratio and the concentrations showedgood linearity (y(ratio) = 0.0027 × (concentration) + 0.0504,r2 = 0.9997). To evaluate the precision and accuracy of ourmethod, repeated analysis of verapamil-spiked plasma sam-ples (n = 5) was carried out. The intra-day reproducibility wasevaluated by comparing the peak area ratios obtained in five Fig. 4. Plasma mean (±S.D.) concentration–time profiles of verapamil fol- different time periods for five sets of plasma serially spiked lowing a single intravenous injection of 30 mg/kg of verapamil in control with verapamil at 0.025, 0.05, 0.5 and 2.5 ␮g/ml. To assess and DMN-induced hepatofibrotic rats (n = 10).
Table 3Pharmacokinetic parameters (mean ± S.D., n = 10) of verapamil in control and DMN-induced hepatofibrotic rats Statistical significance was calculated by Student’s t-test; NS: not significant; AUC: area under the curve; Cmax: maximum concentration; Tmax: time to reach topeak serum concentration; MRT: mean residence time; Vdss: steady-state volume of distribution; CLp: plasma clearance; t1/2): terminal elimination half-life;ke: elimination rate constant.
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