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Rapid Liquid Chromatographic Assay for the Determination of Amiodarone and Its N-Deethyl Metabolite in Plasma, Urine, and Bile
Rapid Liquid Chromatographic Assay for the Determination of Amiodarone and Its N-Deethyl Metabolite in Plasma, Urine, and Bile SCOTT J. WEIR A N D CLARENCE T. UEDA' Received August 7, 1984,from the Department of Pharmaceutics, College of Pharmacy, The University of Nebraska Medical Center, Omaha, NE Accepted for publication November 16, 1984.
68705.
rapid high-performance liquid chromatographic assay was developed for the determination of amiodarone (1) and its N-deethyl metabolite (desethylamiodarone, 2) in plasma, urine, and bile. Analysis was performed on a Cle reversed-phase column and precolumn using a mobile phase consisting of methanol:water:58% ammonium hydroxide (94:4:2)delivered at a flow rate of 1.5mL/min. The eluant was monitored at 244 nm. Under these conditions, 1.2, and the internal standard eluted with retention times of 5.5, 4.6, and 6.8 min, respectively. Samples (100 eL) of plasma were prepared by precipitating the plasma proteins with acetonitrile containing the internal standard and injecting an aliquot of the supernatant directly onto the column. Samples (100 pL) of urine and bile were prepared for injection by acidifying the sample with concentrated HCI and then extracting the mixture with six volumes of 2,2dirnethoxyproprane. The recovery of 1 and 2 from plasma was virtually complete. The recovery from urine and bile was 80-90% for 1 and 6065% for 2. The limit of sensitivity of both compounds in plasma was 100 ng/mL. For urine and bile, the detection limits were 1 and 5 pg/mL, respectively. Over the plasma concentration range of 0.1-1 0.0 pg/mL, the within-day CV ranged from 1 to 10% for 1 and from 1 to 8% for 2. The between-day CV ranged from 2 to 12% and from 1 to 17% for 1 and 2, respectively. Of 44 drugs tested for potential assay interference, four interfered with the determination of 2 and one with the analysis of 1. The assay has been used for pharmacokinetic studies in rats and routine monitoring of the concentrationsof 1 and 2 in human plasma. Abstract 0A
principal advantages of the method are its applicability with urine and bile, use of small samples, simple sample preparation, and short analysis time. The clinical applicability of the method was demonstrated with plasma samples obtained from patients taking amiodarone.
1: R = CPHS
2:R = H 0 It
kBr
/ C3H7
3
Amiodarone, 2-hutyl-3-[3,5-diiodo-4-~-diethylaminoethoxyExperimental Section henzoyl]henzofuran ( l ) ,is a diiodinated henzofuran derivative structurally unique to antiarrhythmic agents. It is a type 111 Materials-Amiodarone, 2-butyl-3-[3,5-diiodo-4-/3-diethylantiarrhythmic marketed in Europe and South America and is aminoethoxybenzoyl]benzofuran ( l ) ,desethylamiodarone, 2butyl-3- [ 3,5-diiodo-4-~-ethylaminoethoxyhenzoyl]benzofuran currently under limited investigation in the United States. The drug is considered a "hroad-spectrum" antiarrhythmic, being ( 2 ) ,and the internal standard, 2-ethyl-3-[3,5-dihromo-4-y-dieffective in controlling supraventricular and ventricular arpropylaminopropoxybenzoyl]benzothiophene (3) were ohtained from Sanofi Centre de Recherches, Montpellier, France. rhythmias,' the difficult to treat reentry-type arrhythmias associated with Wolff-Parkinson-White syndrome,' and those Hydrochloric acid, ammonium hydroxide, and 2,2-dimethoxyproprane (Mallinckrodt, St. Louis, MO) were analytical grade; arrhythmias resistant to currently available antiarrhythmics methanol and acetonitrile (Burdick and Jackson Laboratories, such as verapamil, quinidine, lidocaine, propranolol, and pheMuskegon, MI) were HPLC grade quality. nytoin." Like many antiarrhythmics, the therapeutic plasma Human plasma collected in heparinized tubes and urine concentration range of amiodarone is narrow! Thus, the monitoring of plasma amiodarone concentrations is highly desiraspecimens were obtained from a normal, adult, male, drug-free ble. volunteer. Rat plasma, urine, and bile were obtained from adult, Prior to 1980, radioisotopic male Sprague-Dawley rats (Sasco, Omaha, NE). using 13'1- and I4Clabeled drug were used for the determination of blood and Chromatographic Procedures-The analysis of 1 and 2 tissue amiodarone concentrations. Since then, amiodarone has was performed with a CISreversed-phase column (10-pm parbeen assayed almost exclusively by HPLC, norma17-'l and ticles, 30 cm x 3.9 mm; Waters Associates, Milford, MA) and reversed-phase.I2-lfiAll of these HPLC methods, however, suffer precolumn (30-38-pm particles, 5 cm X 3.2 mm; Whatman, from one or more of the following limitations. Many9-I2'l5lack Clifton, NJ) using a mobile phase consisting of methathe ability to quantify the N-deethyl metabolite of amiodarone nol:water:58% ammonium hydroxide (94:4:2)delivered with a (desethylamiodarone, 2)," require multiple extractions in the Constametric I11 pump (Laboratory Data Control, Riviera or require long analysis work-up of the samples,'"~ll~l' Beach, FL) at a flow rate of 1.5 mL/min. The mobile phase times,9-12.14. I f Others have poor extraction efficien~ies',~~or was prepared fresh on the day of assay and deaerated by they cannot he used for the determination of 1 or 2 in urine sonication (Cole-Parmer Instrument Co., Chicago, IL). The and/or bile.' l 5 eluant was monitored at 244 nm with a Spectromonitor I11 This paper describes the assay of 1 and 2 in plasma, urine, variable-wavelength UV detector (Laboratory Data Control, and bile by reversed-phase HPLC with UV detection. The Riviera Beach, FL) and chart recorder (Houston Instruments, 460
/ Journal of Pharmaceutical Sciences Vol. 74,No. 4, April 1985
0022-3549/85/0400-0460$0 7.OOIO 0 1985, American PharmaceuticalAssociation
Austin, TX) over a detector sensitivity range of 0.01-0.50 AUFS. Sample volumes of 200 pL were injected onto the column with a manual HPLC injector (Rheodyne, Cotati, CA) fitted with a 200-pL loop. All assays were performed a t ambient temperature. Under these conditions, 2, 1, and 3 eluted with retention times of 4.6, 5.5, and 6.8 min, respectively. S t o c k Solutions-Stock solutions of 1 and 2 were prepared by dissolving 200 mg of drug in 100 mL of acetonitrile in glass volumetric flasks. Working standards of 1 and 2 were prepared by serially diluting the stock solutions with additional acetonitrile such that 50 pL of working standard solution, when added to 1 mL of plasma, produced plasma concentrations of 0.1, 0.25, 0.50, 0.75, 1.0, 2.5, 5.0, and 10 pg/mL, and to 1 mL of urine and bile, concentrations of 1.0, 2.5, 5.0, 7.5, 10, 25, 50, and 100 pg/mL. Solutions of the internal standard were prepared in acetonitrile a t concentrations of 1pg/mL for the assay of plasma and 10 pg/mL for urine and bile. All solutions were stored at 0-4°C in glass volumetric flasks. a 1.5-mL disposable polypropylene Assay of Plasma-To centrifuge tube (Sarstedt, Princeton, N J ) was added 100 pL of plasma and 200 pL of the 1-pg/mL internal standard solution. After mixing the tube with a vortex mixer (Scientific Products, McGaw Park, IL) for 30 s and centrifuging (Microfuge B; Beckman Instruments, Palo Alto, CA) a t 13,000 rpm (9500Xg) for 4 min, the supernatant was transferred to a new 1.5-mL polypropylene centrifuge tube. Two hundred microliters of the supernatant was injected into the HPLC. a 1.5-mL polypropylene Assay of U r i n e and Bile-To centrifuge tube were added 100 pL of urine or bile and 20 pL of concentrated hydrochloric acid. After mixing the tube with a vortex mixer for 30 s, 600 pL of 2,2-dimethoxypropane was added and the tube mixed again for an additional 30 s. The tube was then cooled to -10°C for 4 h. Following centrifugation a t 13,000 rpm (9500Xg) for 4 min, the supernatant was transferred to a new 1.5-mL polypropylene tube and evaporated to dryness under nitrogen (N-Evap; Organomation Associates, Northborough, MA). Following reconstitution of the residue with 300 pL of lO-pg/mL internal standard solution, a 200-pL aliquot was injected into the HPLC. Standard Curves-Standard plasma, urine, and bile samples were prepared by adding 50 pL of the appropriate working standard solution to 1 mL of plasma, urine, or bile in a culture tube to produce concentrations of 0.1-10.0 pg/mL for plasma and 1-100 pg/mL for urine and bile. Samples (100 pL) of these standard samples were treated as described above and processed with the unknown samples. Standard curves were constructed by plotting the peak height ratios of 1 or 2 to the internal standard versus amiodarone or N-deethyl metabolite concentration with the equation of best-fit obtained by linear regression analysis. Recovery Studies-The recovery of 1 and 2 from plasma, urine, and bile was determined by comparing the peak height ratios of 1 and 2 to the internal standard obtained with the spiked plasma, urine, and bile samples to the ratios obtained with the injection of equivalent amounts of each compound dissolved in acetonitrile. Stability and Compatibility Studies-Plasma, urine, and bile samples spiked with 1 and 2 were stored in 1.5-mL polypropylene centrifuge tubes at -10°C and assayed a t various times to assess how long the specimens could be stored prior to assay. Additionally, to investigate the stability and/or compatibility of 1 and 2 in various types of blood collection tubes, after removing control specimens, 1-mL samples of 5%dextrose solution, whole blood, and plasma containing 10 pg/mL of 1 and 2 were placed in 12 x 75-mm blood collection tubes. The tubes were then inverted to allow the samples to come in contact with a glass-stopper, laboratory film (Parafilm; American Can Co., Greenwich, CT), and red, green, and lavender stoppers (Becton, Dickinson and Co, Rutherford, NJ) for 60
min a t ambient temperature. One-hundred-microliter aliquots of the control (0 min) and 60-min aqueous and plasma samples were assayed for 1and 2 as described above. I n t e r f e r e n c e Studies-A number of commonly prescribed drugs were tested to determine if they interfere with the assay for 1 and/or 2.
Results Representative chromatograms for the assay of 1 and 2 in plasma, urine, and bile are presented in Figs. 1-3. The retention times of 1, 2, and internal standard were 5.5,4.6, and 6.8 min, respectively. No interfering peaks were observed with the control plasma, urine, and bile blank samples. As shown in Fig. 4, the separation of 1 and 2 was dependent on the ammonium hydroxide concentration in the mobile phase. An ammonium hydroxide concentration of 2% v/v afforded near maximal separation of 1 from 2 (R, = 0.95). Increasing the mobile phase ammonium hydroxide concentration above 2.5% v/v produced no appreciable further enhancement in the resolution of the two compounds. On the other hand, at 3% v/v ammonium hydroxide, the mobile phase reached an apparent pH of -10.5 which with continued use could damage the column. The amount of water in the mobile phase had little influence on the separation of 1 , 2 ,and internal standard from each other. It did, however, affect the separation of the three peaks from the solvent front. Over the concentration ranges of 0.1-10 pg/mL for plasma and 1-100 pg/mL for urine and bile, the relationship between the peak height ratio of 1 or 2 to the internal standard and the concentration of 1 and 2 in plasma, urine, or bile was linear. The correlation coefficients for the plasma, urine, and bile standard curves were >0.99 (Table I). Assay sensitivity, defined as three times the quotient of the value of the y-intercept divided by the slope for the standard calibration curve, was 20 ng/mL for the detection of 1in plasma and 340 ng/mL for urine and bile. For 2, the assay sensitivities were 85 ng/mL for plasma and 2.2 pg/mL for bile and urine. At these concentrations, the coefficients of variation were 535%. An acceptable detection limit (515% CV) for both compounds in plasma was 100 ng/mL. For urine and bile, 1 and 2 could be assayed with a CV of 515% down to 1 and 5 pg/mL, respectively. As shown in Table 11, the precision of the assay for 1 and 2 in human and rat plasma over the concentration range of 0.110.0 pg/mL ranged from 1 to 10% and from 1 to 17% for the within- and between-day coefficients of variation, respectively. To test for assay specificity, aliquots of a human plasma sample obtained from a patient receiving 200 mg of 1 twice daily for several months were subjected to HPLC analysis with varying mobile phase characteristics. When different combinations of three mobile phase variables were investigated (acetonitrile substitution for methanol, varied percentages of acetonitrile mobile phase concentration, and apparent mobile phase pH varying from 3.5 to 9.5), only single peaks were seen for 1 and 2 and no indications of an underlying component were detected, e.g., peak shoulders. These observations suggested assay specificity for 1 and 2. Representative chromatograms of a patient plasma sample and rat bile specimen following amiodarone treatment are shown in Figs. 1 and 3, respectively. Forty-four drugs were tested for their potential to interfere with the assay of 1 and 2. The retention times of' these compounds are presented in Table 111. Of the compounds investigated, chloroquine, nortriptyline, thioridazine, and thiothixene interfered with the assay of 2 and desipramine with the analysis of 1. The recovery of 1 and 2 from spiked human and rat plasma samples is summarized in Table IV. Both compounds were recovered almost totally from human and rat plasma and with Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
I
461
RAT
HUMAN C
1 I
1
Figure 1-Chromatograms of blank human plasma (A), human plasma spiked with 0.5 pgpnL of I and 2 (B), plasma of a patient receiving 200 mg of amiodarone hydrochloride twice daily for several months (C), blank rat plasma (D), rat plasma spiked with 0.5 pglmL of 1 and 2 (E), and plasma of a rat given an intraperitoneal infusion of 15 mglkgld of amiodarone hydrochloride for 28 d (F). Peaks: (1) the deethyl metabolite; (2) amiodarone; (3) internal standard. Detector sensitivity: 0.01 AUFS; chart speed: 20 cmlh.
!
I
3 1
A
m
m
8 4
8
0
'r m
I
4
c
m
8 4 0
8 4 0
0
m
8 4 0
m
8 4 0
MINUTES good reproducibility. With plasma, the column life was a t least 500 injections if the guard column was cleaned and repacked after every 100-150 injections. The extraction efficiencies of 1 and 2 from urine and bile are given in Table V. The recovery of 1 and 2 from the two biological fluids when added in concentrations of 1-100 pg/mL was -80-90% for 1 and -6065% for 2 . In spite of the lower recoveries, the reproducibilities of the assay of 1 and 2 in urine and bile were similar to the plasma assay. Plasma, urine, and bile samples spiked with known concen trations of 1 and 2 were stored in disposable polypropylene tubes at -10°C to assess their stability characteristics on storage. When assayed over 25 d, no loss of either drug was observed. Further, the stock solutions of 1 and 2 stored in glass at 4°C were stable for at least 9 months. When 5% dextrose solutions containing 10 pg/mL of 1 and 2 were allowed to come in contact with laboratory film or red, green, and lavender rubber stopper tops for 60 min a t ambient
temperature, reductions in the concentrations of both drugs of 56, 58, 55, and 5396, respectively, were observed. In plasma, no changes in 1 or 2 concentration were seen after contact with any of the closures investigated. With whole blood, on the other hand, after 60 min of contact, the plasma concentrations of both compounds in these specimens increased 32, 16, 10, and 44% with the laboratory film and red, green, and lavender stopper tops, respectively.
Discussion A rapid HPLC assay method with simple sample preparation has been developed for the determination of 1 and 2 in several biological fluids. The method is specific for both compounds and has been found to be sensitive enough for therapeutic drug monitoring and pharmacokinetic studies. The assay represents RAT
w
RAT
B
A
I
3
m
m
8 4 0
m 8 4 0
m
8 4 0
rTT 8 4 0
MINUTES Figure 2-Chromatograms of blank human urine (A), human urine spiked with 5.0 pglmL of 1 and 2 (B), blank rat urine (C), and rat urine spiked with 5.0 pg/mL of 1 and 2 (0).Detector sensitivity: 0.7 AUFS. See Fig. 7 for key.
462
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
a 4
J m
I
8 4 0
rn 8 4 0
MINUTES Figure 3-Chromatograms of blank rat bile (A), rat bile spiked with 5.0 pglmL of 1 and 2 (B), and bile obtained from a rat in the first hour after an intravenous bolus dose of 50 mglkg of amiodarone hydrochloride (C). Detector sensitivity: 0.7 AUFS. See Fig. 7 for key.
0.8
Table It-lntraday and lnterday Reproducibilityof the Assay of Amiodarone (1) and Its Deethyl Metabolite 2 in Human and Rat Plasma
4
Coefficient of Variation, o/o Amount
Intraday"
Added,
8
U
PLglmL
0.4
o.2 0.0 0
2
1
~
0.1 0.5 1.o 5.0 10.0
4
3
'n
% NH4OH ( v ~ v )
Figure 4-Resolution (RJ between 7 and 2 as a function of mobile phase ammonium hydroxide 58% content. Chromatographic conditions as described in the Experimental Section.
an improvement over existing HPLC procedures7-'" in several important aspects. With the method described herein, amiodarone and its N deethyl m e t a b ~ l i t e 'can ~ be quantified in plasma, urine, and bile with good precision and reproducibility. Many of the existing HPLC procedures"" are unable to separate 1 from 2 , and with the they are not applicable to urine and/or bile. other^'^','^,'^ do not employ an internal standard, thereby reducing the precision and accuracy of the assay.IRAs shown in Figs. 1-3, there is good resolution between 1 and 2 with the present method. The HPLC analysis time is more rapid when compared with other assays. With a retention time for the internal standard of 6.8 min and the elution of 1 and 2 before the internal standard, a sample can be assayed in 8 min (Figs. 1-3). In contrast, other procedures require analysis times of at least 12 min per ample.^-^".'^"^ With most procedure^,^^"^'^ 1 and/or 2 is extracted from plasma or serum with an organic solvent prior to HPLC analysis. In this laboratory, the recoveries of both compounds were poor using those methods that employ hexane or ether as the extracting ~ o l v e n t . ~ .Other ~ . " procedures'0~1~~'4'1F, were time consuming, requiring multiple extractions or lengthy mixing steps. With the present method, simple precipitation of plasma proteins with acetonitrile is all that is required to prepare a plasma (or serum) sample for HPLC injection. The preparation time is <5 min, and the recovery of 1 and 2 is virtually complete. For urine and bile, simple treatment with acetonitrile was unsatisfactory for two reasons. Amiodarone is present in urine in low con~entrations.'"~~ With some specimens, the addition of acetonitrile diluted the samples below the detection limits of the assay. More important, endogenous substances were present in both specimens which interfered with the analysis of 1 and 2. Therefore, an extraction procedure was sought that would concentrate the analytes and at the same time, remove the interfering endogenous substances. Table I-Calibration Curves for Amiodarone (1) and Its Deethyl Metabolite 2 in Plasma, Urine, and Bile
Sample Plasma Human Rat Urine Human Rat Bile Rat
Calibration Range, ua/mL 0.1-10.0
Slope
Intercept
1
2
1.09 1.02
1.38 1.34
1
0.01 0.01
2 0.04 0.06
r 1
Human
2
0.997 0.998 0.991 0.992
1-100
0.096 0.110 0.012 0.081 0.994 0.996 0,111 0.126 0.011 0.094 0.996 0.996
7-100
0.115 0.128 0.039 0.119 0.991 0.989
= 5.
lnterdayb Rat
Human
1
2
1
2
10.3 6.1 1.6 4.2 1.0
7.6 3.2 1.2 5.0 2.6
6.2
6.1
0.9
0.8
2.2
3.2
1
12.2 9.1 5.0 7.9 1.9
Rat
2
1
2
16.5 9.7 1.0 7.9 2.6
9.0
10.5
5.6
5.6
3.5
3.9
b n = 6.
Bousquet et a1." reported the use of 2,2-dimethoxypropane as a dehydrating agent to remove water from aqueous samples. Under acidic conditions, water reacts with 2,2-dimethoxypropane to form 1 mol of acetone and 2 mol of methanol. The reaction is endothermic, which aids in the precipitation of Table Ill-Drugs
Tested for Potential Assay Interference
Compound Acetaminophen Acetazolamide Amiodarone (1) Amitriptyline
Ampicillin Aspirin
Caffeine Chlorcyclizine Chloroquine Chlorpromazine Cyclizine Cyproheptadine Deethyl metabolite of amiodarone 2 Desipramine Diphen hydramine Haloperidol Heparin H ydralazine Hydrocortisone Hydroxyzine lmipramine Internal Standard 3 Lidocaine Lorazepam Loxapine Meclizine Mesoridazine Methapyrilene Nortriptyline Pentobarbital Perphenazine Phenobarbital Phenoxybenzamine Phenytoin Procainamide Procaine Propranolol Propylthiouracil Pyrilamine Quinidine Quinine Theophylline Thioridazine Thiothixene Tranylcypromine Trifluoperazine ValDroic Acid
Retention Time, min 2.2 1.5 5.5 3.4 1.4 1.7 2.4 3.1 4.6 3.9 2.9 3.3 4.6 5.6 2.8 1.9 2.3 2.7 2.1 2.6 3.7 6.8 2.5 2.9 2.1 3.4 3.4 2.7 4.7 1.7 2.8 1.7 2.2 1.5 2.5 2.7 2.7 1.4 2.8 3.1 3.1 2.4 4.2 4.9 3.3 3.4 2.8
Journal of Pharmaceutical Sciences I 463 Vol. 74, No. 4, April 1985
Table IV-Recoverv
of Amiodarone (1) and Its Deethvl Metabolite 2 from SDiked Samoles of Human and Rat Plasma"
Recovery, %
Amount Humanb
Added,
a
Ratc
m/mL
1
2
0.1 0.5 1.o 5.0 10.0
91.8 k 8.4 (9.2) 91.4 t 2.7 (3.0) 101.3 rf: 1.4(1.4) 100.1 f 3.8 (3.8) 98.2 +- 0.9 (0.9)
92.5 f 6.2 (6.7) 88.5 f 2.5 (2.8) 102.2 f 1.1 (1.1) 105.3 f 4.8 (4.6) 105.1 f 1.3 (1.2)
Mean f SD (% CV). n
Table V-Recovery
= 6.
n
=
1
2
* 3.7 (3.9)
91.3 f 5.4 (5.9)
112.2 f 0.8 (0.7)
102.3 f 1.1 (1.1)
100.1 f 2.1 (2.1)
104.5 f 2.4 (2.2)
93.7
5.
of Amiodarone (1) and Its Deethyl Metabolite 2 from Spiked Samples of Human and/or Rat Urine and Bile"
Recovery, O% Amount Added,
Urine
1 10 100 a
Rat Bile
~~~
NlmL
Human
Rat
.~
1
2
1
2
1
2
82.4 i 6.0 (7.3) 93.3 & 1.3 (1.4) 85.6 k 2.9 (3.4)
64.8 F 10.7 (16.5) 62.0 & 4.7 (7.6) 59.8 1.7 (2.8)
87.9 f 12.2 (13.9) 75.1 f 1.7 (2.2) 91.9 + 1.1 (1.2)
50.6 f 7.4 (14.6) 65.0 f 1.8 (2.8) 68.9 f 0.9 (1.3)
87.8 k 6.6 (7.5) 84.3 f 7.2 (8.5) 92.0 k 4.9 (5.3)
66.2 f 11.9 (18.0) 64.5 k 4.3 (6.7) 65.2 f 1.4 (2.1)
Mean k SD ( O h CV), n
=
*
5
endogenous substances. After centrifugat,ion, the acetone and methanol mixture that is formed is easily removed and evaporated. After reconstitution of the residue with an appropriate solvent, the sample is ready for HPLC analysis. In the present assay, 2,2-dimethoxypropane was used to determine 1 and 2 in urine and bile. The extraction procedure, which was adapted from the work of Holcslaw," effectively eliminated the Hank interferences in the two fluids (Figs. 2 and 3). Other reports that describe the extraction of 1 from urine" I(i,I') and bile"' did not present recovery or reproducibility data. Therefore, it was not possible to compare the results of this assay for urine and bile with the previous methods. Plomp et al.1'3obtained good recovery of 1 and 2 from urine using disposable extraction columns and multiple extractions. With the present procedure, the recoveries of both compounds are good and the preparation of a urine sample for HPLC analysis is simpler and less expensive. In urine samples that were available for this work from humans and rats treated with amiodarone hydrochloride, no detectable levels of' 1 or 2 were observed. Andreasen e t al.15 and Harris et al."' noted that the concentrations of 1 in the urine after intravenous arid oral administrations were low. These observations suggested that perhaps the levels of 1 and 2 in the urine specimens tested may have been below the sensitivity of the assay. If this was the case, these findings suggest that renal excretion of 1 and 2 is not a primary mechanism of elimination for either of these drug species. With measurable concent rations of 2 in the plasma, perhaps this metabolite undergoes further metabolism or is excreted by a nonrenal mechanism. These possibilities will require further investigations. Blood collection tubes have been shown to affect the plasma concentrations of propranolo122and quinidine.'" In this study, dextrose solution the concentrations of 1 and 2 were cantly reduced after coming in contact with laboratory film and rubber stoppers. A similar effect was not seen, however, with the two compounds in plasma. Lalloz et aLZ4recently report,ed that 1 was bound extensively and with high affinity t o serum proteins. The hound plasma drug fraction was reported to be 96%, with the primary amiodarone binding site exhibiting an aff'initv constant of5.6 x 106L/mol. The unusually strong and ext.ensive plasma binding characteristics of 1 would explain the absence of any changes in the plasma concentrations of both compounds following contact with the same 464 f Journal of PharmaceuticalSciences Vol. 74, No. 4, April 1985
tube closures since the availability of the two drugs for interactions with the closures is reduced in plasma. In whole blood, the erythrocyte concentrations of 1 and 2 are sizeable.2s These findings may explain the increases in the plasma concentrations of both compounds after the blood specimens from whence they came contacted the laboratory film and rubber stopper tops. The increases may have been due to hemolysis of the erythrocytes, thus liberating additional drug into the plasma. Alternatively, contact of the erythrocytes with the closures may have caused a redistribution of both compounds from the red cells to the plasma. In this study, no visual evidence of hemolysis was observed in any of the plasma samples. Because of these findings, care is taken in this laboratory to prevent the blood specimen from coming in contact with the rubber stopper during the collection of blood for plasma assays. Additionally, the plasma is separated from the red blood cells immediately after the blood specimen is collected. The method described has been used to monitor steady-state plasma concentrations of 1 and 2 in patients receiving amiodarone for their dysrhythmias. At similar dosages, the observed concentrations of 1 and 2 were in agreement with those reported by other investigators4 and/or were in the desired therapeutic plasma concentration range of 1-2.5 pg/mL.26 For example, the concentrations of 1 and 2 in the patient plasma specimen shown in Fig. 1 were 1.2 and 1.0 pg/mL, respectively.
References and Notes 1. Rosenbaum, M. B.; Chiale, P. A,; Halpern, M. S.; Nau, G. J . ; Przybylski, J.; Levi, R. J.; Lazzari, ?J. 0.; Elizari, M. V. Am. J . Cardiol. 1976. 38. 934-944. 2. Rosenbaum, M. B.; Chiale, P. A.; Ryba, D.; Elizari, M. V. A m . J . Cardiol. 1974,34, 215-223. 3. Wheeler, P. J.; Puritz, R.; Ingram, D. V.; Chamberlain, D. A.
Postgrad. Med. J . 1979, 5Fi, 1-9. 4. Haffajee, C. I.; Love, J . C.; Alpert, J . S.; Asdourian, G. K.; Sloan, K. C. Am. Heart J . 1983. 106. 935-943. 5. Broekhuysen, J.; Laruel,' R.; 'Sion R. Arch. Int. Pharmacodyn. 1969, 177, 340-359. 6. Ravin, L. J.; Shami, E. G.; Intoccia, A.; Rattie, E.; Joseph, G . J . Pharm. Sci. 1969, 58, 1242-1245. 7. Storey, G. C. A.; Holt, D. W. J . Chromatogr. 1982, 245, 377-380. 8. Mostow, N. D.; Noon, D. L.: Mvers. C. M.; Rakita. L.: Blumer. J. L. J Chromatogr. 1983, 227, 289-237. 9. Lesko, L. J.; Marion, A,; Canada, A. T.; Haffajee, C. J Pharm. Sci 1981, 70, 1366-1368. 10. Debbas, N. M G.; Du Cailar, C.; Sassine, A,; Derancourt, d.;
DeMaille, J. G.; Puech, P. Eur. J . Clin. Invest. 1983, 13, 123-127. 11. Cervelli, J. A,; Kerkay, J.; Pearson, K. H. Anal. Lett. 1981, 14, 137-153. 12. Riva, E.; Gerna, M.; Latini, R.; Giani, P.; Volpi, A.; Maggioni A. J . Cardiovasc. Pharmacol. 1982,4, 264-269. 13. Plomp, T. A,; Engels, M.; Robles de Medina, E. 0.; Maes, R. A. A. J . Chromatogr. 1983,273, 379-392. 14. Brien, J. F.; Jimmo, S.; Armstrong, P. W. Can. J. Physiol. Pharmacol. 1983, 61, 245-248. 15. Andreasen, F.; Agerbaek, H.; Bjerregaard, P.; Gotzsche, H. Eur. J. Clin. Pharmacol. 1981, 19, 293-299. 16. Gobatto, S.; Padrini, R.; Candelpergher, G.; Bettero, A.; Cargnelli, G.; Ferrari, M. Int. J. Clin. Pharmacol. Res. 1982, 2, 279-281. 17. Flanagan, R. J.; Storey, G. C. A.; Holt, D. W.; Farmer, P. B. J. Pharm. Pharmacol. 1982, 34, 638-643. 18. Johnson, E. L.; Stevenson, R. “Basic Liquid Chromatography”; Varian Associates: Palo Alto, 1978; pp 238-239. 19. Harris, L.; Hind, C. R. K.; McKenna, W. J.; Savage, C.; Krikler, S. J.; Storey, G. C. A,; Holt, D. W. Postgrad. Med. J . 1983,59, 440442. 20. Bousquet, W. F.; Christian, J. E.; Knevel, A. M.; Spahr, J. L. Anal. Biochem. 1962, 3, 519-520.
21. Holcslaw, T. L. Masters Thesis, Purdue University, 1972. 22. Cotham, R. H.; Shand, D. Clin. Pharmacol. Ther. 1975, 18, 535538. 23. Ooi, D. S.; Poznanski, W. J.; Smith, F. M. Clin. Biochem. 1980, 13, 297-300. 24. Lalloz, M. R. A,; Byfield, P. G. H.; Greenwood, R. M.; Himsworth, R. L. J . Pharm. Pharmacol. 1983,36, 366-372. 25. Weir, S. J.; Ueda, C. T., unpublished results. 26. Latini, R.; Tognoni, G.; Kates, R. E. Clin.Pharmacokinet. 1984, 9, 136-156.
Acknowledgments The authors acknowledge the gifts of amiodarone, its deethyl metabolite, and internal standard from Dr. C . Lafille of Sanofi Centre de Recherches, Montpellier, France and the assistance of Mrs. Elizabeth A. McCafferty of the New York Office in acquiring these materials. We are grateful to Dr. John Kugler for providing the patient plasma and urine samples and to Mrs. Marilyn Kircher for preparing the manuscript.
Journal of PharmaceuticalSciences Vol. 74, No. 4, April 1985
1
465
68705.
rapid high-performance liquid chromatographic assay was developed for the determination of amiodarone (1) and its N-deethyl metabolite (desethylamiodarone, 2) in plasma, urine, and bile. Analysis was performed on a Cle reversed-phase column and precolumn using a mobile phase consisting of methanol:water:58% ammonium hydroxide (94:4:2)delivered at a flow rate of 1.5mL/min. The eluant was monitored at 244 nm. Under these conditions, 1.2, and the internal standard eluted with retention times of 5.5, 4.6, and 6.8 min, respectively. Samples (100 eL) of plasma were prepared by precipitating the plasma proteins with acetonitrile containing the internal standard and injecting an aliquot of the supernatant directly onto the column. Samples (100 pL) of urine and bile were prepared for injection by acidifying the sample with concentrated HCI and then extracting the mixture with six volumes of 2,2dirnethoxyproprane. The recovery of 1 and 2 from plasma was virtually complete. The recovery from urine and bile was 80-90% for 1 and 6065% for 2. The limit of sensitivity of both compounds in plasma was 100 ng/mL. For urine and bile, the detection limits were 1 and 5 pg/mL, respectively. Over the plasma concentration range of 0.1-1 0.0 pg/mL, the within-day CV ranged from 1 to 10% for 1 and from 1 to 8% for 2. The between-day CV ranged from 2 to 12% and from 1 to 17% for 1 and 2, respectively. Of 44 drugs tested for potential assay interference, four interfered with the determination of 2 and one with the analysis of 1. The assay has been used for pharmacokinetic studies in rats and routine monitoring of the concentrationsof 1 and 2 in human plasma. Abstract 0A
principal advantages of the method are its applicability with urine and bile, use of small samples, simple sample preparation, and short analysis time. The clinical applicability of the method was demonstrated with plasma samples obtained from patients taking amiodarone.
1: R = CPHS
2:R = H 0 It
kBr
/ C3H7
3
Amiodarone, 2-hutyl-3-[3,5-diiodo-4-~-diethylaminoethoxyExperimental Section henzoyl]henzofuran ( l ) ,is a diiodinated henzofuran derivative structurally unique to antiarrhythmic agents. It is a type 111 Materials-Amiodarone, 2-butyl-3-[3,5-diiodo-4-/3-diethylantiarrhythmic marketed in Europe and South America and is aminoethoxybenzoyl]benzofuran ( l ) ,desethylamiodarone, 2butyl-3- [ 3,5-diiodo-4-~-ethylaminoethoxyhenzoyl]benzofuran currently under limited investigation in the United States. The drug is considered a "hroad-spectrum" antiarrhythmic, being ( 2 ) ,and the internal standard, 2-ethyl-3-[3,5-dihromo-4-y-dieffective in controlling supraventricular and ventricular arpropylaminopropoxybenzoyl]benzothiophene (3) were ohtained from Sanofi Centre de Recherches, Montpellier, France. rhythmias,' the difficult to treat reentry-type arrhythmias associated with Wolff-Parkinson-White syndrome,' and those Hydrochloric acid, ammonium hydroxide, and 2,2-dimethoxyproprane (Mallinckrodt, St. Louis, MO) were analytical grade; arrhythmias resistant to currently available antiarrhythmics methanol and acetonitrile (Burdick and Jackson Laboratories, such as verapamil, quinidine, lidocaine, propranolol, and pheMuskegon, MI) were HPLC grade quality. nytoin." Like many antiarrhythmics, the therapeutic plasma Human plasma collected in heparinized tubes and urine concentration range of amiodarone is narrow! Thus, the monitoring of plasma amiodarone concentrations is highly desiraspecimens were obtained from a normal, adult, male, drug-free ble. volunteer. Rat plasma, urine, and bile were obtained from adult, Prior to 1980, radioisotopic male Sprague-Dawley rats (Sasco, Omaha, NE). using 13'1- and I4Clabeled drug were used for the determination of blood and Chromatographic Procedures-The analysis of 1 and 2 tissue amiodarone concentrations. Since then, amiodarone has was performed with a CISreversed-phase column (10-pm parbeen assayed almost exclusively by HPLC, norma17-'l and ticles, 30 cm x 3.9 mm; Waters Associates, Milford, MA) and reversed-phase.I2-lfiAll of these HPLC methods, however, suffer precolumn (30-38-pm particles, 5 cm X 3.2 mm; Whatman, from one or more of the following limitations. Many9-I2'l5lack Clifton, NJ) using a mobile phase consisting of methathe ability to quantify the N-deethyl metabolite of amiodarone nol:water:58% ammonium hydroxide (94:4:2)delivered with a (desethylamiodarone, 2)," require multiple extractions in the Constametric I11 pump (Laboratory Data Control, Riviera or require long analysis work-up of the samples,'"~ll~l' Beach, FL) at a flow rate of 1.5 mL/min. The mobile phase times,9-12.14. I f Others have poor extraction efficien~ies',~~or was prepared fresh on the day of assay and deaerated by they cannot he used for the determination of 1 or 2 in urine sonication (Cole-Parmer Instrument Co., Chicago, IL). The and/or bile.' l 5 eluant was monitored at 244 nm with a Spectromonitor I11 This paper describes the assay of 1 and 2 in plasma, urine, variable-wavelength UV detector (Laboratory Data Control, and bile by reversed-phase HPLC with UV detection. The Riviera Beach, FL) and chart recorder (Houston Instruments, 460
/ Journal of Pharmaceutical Sciences Vol. 74,No. 4, April 1985
0022-3549/85/0400-0460$0 7.OOIO 0 1985, American PharmaceuticalAssociation
Austin, TX) over a detector sensitivity range of 0.01-0.50 AUFS. Sample volumes of 200 pL were injected onto the column with a manual HPLC injector (Rheodyne, Cotati, CA) fitted with a 200-pL loop. All assays were performed a t ambient temperature. Under these conditions, 2, 1, and 3 eluted with retention times of 4.6, 5.5, and 6.8 min, respectively. S t o c k Solutions-Stock solutions of 1 and 2 were prepared by dissolving 200 mg of drug in 100 mL of acetonitrile in glass volumetric flasks. Working standards of 1 and 2 were prepared by serially diluting the stock solutions with additional acetonitrile such that 50 pL of working standard solution, when added to 1 mL of plasma, produced plasma concentrations of 0.1, 0.25, 0.50, 0.75, 1.0, 2.5, 5.0, and 10 pg/mL, and to 1 mL of urine and bile, concentrations of 1.0, 2.5, 5.0, 7.5, 10, 25, 50, and 100 pg/mL. Solutions of the internal standard were prepared in acetonitrile a t concentrations of 1pg/mL for the assay of plasma and 10 pg/mL for urine and bile. All solutions were stored at 0-4°C in glass volumetric flasks. a 1.5-mL disposable polypropylene Assay of Plasma-To centrifuge tube (Sarstedt, Princeton, N J ) was added 100 pL of plasma and 200 pL of the 1-pg/mL internal standard solution. After mixing the tube with a vortex mixer (Scientific Products, McGaw Park, IL) for 30 s and centrifuging (Microfuge B; Beckman Instruments, Palo Alto, CA) a t 13,000 rpm (9500Xg) for 4 min, the supernatant was transferred to a new 1.5-mL polypropylene centrifuge tube. Two hundred microliters of the supernatant was injected into the HPLC. a 1.5-mL polypropylene Assay of U r i n e and Bile-To centrifuge tube were added 100 pL of urine or bile and 20 pL of concentrated hydrochloric acid. After mixing the tube with a vortex mixer for 30 s, 600 pL of 2,2-dimethoxypropane was added and the tube mixed again for an additional 30 s. The tube was then cooled to -10°C for 4 h. Following centrifugation a t 13,000 rpm (9500Xg) for 4 min, the supernatant was transferred to a new 1.5-mL polypropylene tube and evaporated to dryness under nitrogen (N-Evap; Organomation Associates, Northborough, MA). Following reconstitution of the residue with 300 pL of lO-pg/mL internal standard solution, a 200-pL aliquot was injected into the HPLC. Standard Curves-Standard plasma, urine, and bile samples were prepared by adding 50 pL of the appropriate working standard solution to 1 mL of plasma, urine, or bile in a culture tube to produce concentrations of 0.1-10.0 pg/mL for plasma and 1-100 pg/mL for urine and bile. Samples (100 pL) of these standard samples were treated as described above and processed with the unknown samples. Standard curves were constructed by plotting the peak height ratios of 1 or 2 to the internal standard versus amiodarone or N-deethyl metabolite concentration with the equation of best-fit obtained by linear regression analysis. Recovery Studies-The recovery of 1 and 2 from plasma, urine, and bile was determined by comparing the peak height ratios of 1 and 2 to the internal standard obtained with the spiked plasma, urine, and bile samples to the ratios obtained with the injection of equivalent amounts of each compound dissolved in acetonitrile. Stability and Compatibility Studies-Plasma, urine, and bile samples spiked with 1 and 2 were stored in 1.5-mL polypropylene centrifuge tubes at -10°C and assayed a t various times to assess how long the specimens could be stored prior to assay. Additionally, to investigate the stability and/or compatibility of 1 and 2 in various types of blood collection tubes, after removing control specimens, 1-mL samples of 5%dextrose solution, whole blood, and plasma containing 10 pg/mL of 1 and 2 were placed in 12 x 75-mm blood collection tubes. The tubes were then inverted to allow the samples to come in contact with a glass-stopper, laboratory film (Parafilm; American Can Co., Greenwich, CT), and red, green, and lavender stoppers (Becton, Dickinson and Co, Rutherford, NJ) for 60
min a t ambient temperature. One-hundred-microliter aliquots of the control (0 min) and 60-min aqueous and plasma samples were assayed for 1and 2 as described above. I n t e r f e r e n c e Studies-A number of commonly prescribed drugs were tested to determine if they interfere with the assay for 1 and/or 2.
Results Representative chromatograms for the assay of 1 and 2 in plasma, urine, and bile are presented in Figs. 1-3. The retention times of 1, 2, and internal standard were 5.5,4.6, and 6.8 min, respectively. No interfering peaks were observed with the control plasma, urine, and bile blank samples. As shown in Fig. 4, the separation of 1 and 2 was dependent on the ammonium hydroxide concentration in the mobile phase. An ammonium hydroxide concentration of 2% v/v afforded near maximal separation of 1 from 2 (R, = 0.95). Increasing the mobile phase ammonium hydroxide concentration above 2.5% v/v produced no appreciable further enhancement in the resolution of the two compounds. On the other hand, at 3% v/v ammonium hydroxide, the mobile phase reached an apparent pH of -10.5 which with continued use could damage the column. The amount of water in the mobile phase had little influence on the separation of 1 , 2 ,and internal standard from each other. It did, however, affect the separation of the three peaks from the solvent front. Over the concentration ranges of 0.1-10 pg/mL for plasma and 1-100 pg/mL for urine and bile, the relationship between the peak height ratio of 1 or 2 to the internal standard and the concentration of 1 and 2 in plasma, urine, or bile was linear. The correlation coefficients for the plasma, urine, and bile standard curves were >0.99 (Table I). Assay sensitivity, defined as three times the quotient of the value of the y-intercept divided by the slope for the standard calibration curve, was 20 ng/mL for the detection of 1in plasma and 340 ng/mL for urine and bile. For 2, the assay sensitivities were 85 ng/mL for plasma and 2.2 pg/mL for bile and urine. At these concentrations, the coefficients of variation were 535%. An acceptable detection limit (515% CV) for both compounds in plasma was 100 ng/mL. For urine and bile, 1 and 2 could be assayed with a CV of 515% down to 1 and 5 pg/mL, respectively. As shown in Table 11, the precision of the assay for 1 and 2 in human and rat plasma over the concentration range of 0.110.0 pg/mL ranged from 1 to 10% and from 1 to 17% for the within- and between-day coefficients of variation, respectively. To test for assay specificity, aliquots of a human plasma sample obtained from a patient receiving 200 mg of 1 twice daily for several months were subjected to HPLC analysis with varying mobile phase characteristics. When different combinations of three mobile phase variables were investigated (acetonitrile substitution for methanol, varied percentages of acetonitrile mobile phase concentration, and apparent mobile phase pH varying from 3.5 to 9.5), only single peaks were seen for 1 and 2 and no indications of an underlying component were detected, e.g., peak shoulders. These observations suggested assay specificity for 1 and 2. Representative chromatograms of a patient plasma sample and rat bile specimen following amiodarone treatment are shown in Figs. 1 and 3, respectively. Forty-four drugs were tested for their potential to interfere with the assay of 1 and 2. The retention times of' these compounds are presented in Table 111. Of the compounds investigated, chloroquine, nortriptyline, thioridazine, and thiothixene interfered with the assay of 2 and desipramine with the analysis of 1. The recovery of 1 and 2 from spiked human and rat plasma samples is summarized in Table IV. Both compounds were recovered almost totally from human and rat plasma and with Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
I
461
RAT
HUMAN C
1 I
1
Figure 1-Chromatograms of blank human plasma (A), human plasma spiked with 0.5 pgpnL of I and 2 (B), plasma of a patient receiving 200 mg of amiodarone hydrochloride twice daily for several months (C), blank rat plasma (D), rat plasma spiked with 0.5 pglmL of 1 and 2 (E), and plasma of a rat given an intraperitoneal infusion of 15 mglkgld of amiodarone hydrochloride for 28 d (F). Peaks: (1) the deethyl metabolite; (2) amiodarone; (3) internal standard. Detector sensitivity: 0.01 AUFS; chart speed: 20 cmlh.
!
I
3 1
A
m
m
8 4
8
0
'r m
I
4
c
m
8 4 0
8 4 0
0
m
8 4 0
m
8 4 0
MINUTES good reproducibility. With plasma, the column life was a t least 500 injections if the guard column was cleaned and repacked after every 100-150 injections. The extraction efficiencies of 1 and 2 from urine and bile are given in Table V. The recovery of 1 and 2 from the two biological fluids when added in concentrations of 1-100 pg/mL was -80-90% for 1 and -6065% for 2 . In spite of the lower recoveries, the reproducibilities of the assay of 1 and 2 in urine and bile were similar to the plasma assay. Plasma, urine, and bile samples spiked with known concen trations of 1 and 2 were stored in disposable polypropylene tubes at -10°C to assess their stability characteristics on storage. When assayed over 25 d, no loss of either drug was observed. Further, the stock solutions of 1 and 2 stored in glass at 4°C were stable for at least 9 months. When 5% dextrose solutions containing 10 pg/mL of 1 and 2 were allowed to come in contact with laboratory film or red, green, and lavender rubber stopper tops for 60 min a t ambient
temperature, reductions in the concentrations of both drugs of 56, 58, 55, and 5396, respectively, were observed. In plasma, no changes in 1 or 2 concentration were seen after contact with any of the closures investigated. With whole blood, on the other hand, after 60 min of contact, the plasma concentrations of both compounds in these specimens increased 32, 16, 10, and 44% with the laboratory film and red, green, and lavender stopper tops, respectively.
Discussion A rapid HPLC assay method with simple sample preparation has been developed for the determination of 1 and 2 in several biological fluids. The method is specific for both compounds and has been found to be sensitive enough for therapeutic drug monitoring and pharmacokinetic studies. The assay represents RAT
w
RAT
B
A
I
3
m
m
8 4 0
m 8 4 0
m
8 4 0
rTT 8 4 0
MINUTES Figure 2-Chromatograms of blank human urine (A), human urine spiked with 5.0 pglmL of 1 and 2 (B), blank rat urine (C), and rat urine spiked with 5.0 pg/mL of 1 and 2 (0).Detector sensitivity: 0.7 AUFS. See Fig. 7 for key.
462
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
a 4
J m
I
8 4 0
rn 8 4 0
MINUTES Figure 3-Chromatograms of blank rat bile (A), rat bile spiked with 5.0 pglmL of 1 and 2 (B), and bile obtained from a rat in the first hour after an intravenous bolus dose of 50 mglkg of amiodarone hydrochloride (C). Detector sensitivity: 0.7 AUFS. See Fig. 7 for key.
0.8
Table It-lntraday and lnterday Reproducibilityof the Assay of Amiodarone (1) and Its Deethyl Metabolite 2 in Human and Rat Plasma
4
Coefficient of Variation, o/o Amount
Intraday"
Added,
8
U
PLglmL
0.4
o.2 0.0 0
2
1
~
0.1 0.5 1.o 5.0 10.0
4
3
'n
% NH4OH ( v ~ v )
Figure 4-Resolution (RJ between 7 and 2 as a function of mobile phase ammonium hydroxide 58% content. Chromatographic conditions as described in the Experimental Section.
an improvement over existing HPLC procedures7-'" in several important aspects. With the method described herein, amiodarone and its N deethyl m e t a b ~ l i t e 'can ~ be quantified in plasma, urine, and bile with good precision and reproducibility. Many of the existing HPLC procedures"" are unable to separate 1 from 2 , and with the they are not applicable to urine and/or bile. other^'^','^,'^ do not employ an internal standard, thereby reducing the precision and accuracy of the assay.IRAs shown in Figs. 1-3, there is good resolution between 1 and 2 with the present method. The HPLC analysis time is more rapid when compared with other assays. With a retention time for the internal standard of 6.8 min and the elution of 1 and 2 before the internal standard, a sample can be assayed in 8 min (Figs. 1-3). In contrast, other procedures require analysis times of at least 12 min per ample.^-^".'^"^ With most procedure^,^^"^'^ 1 and/or 2 is extracted from plasma or serum with an organic solvent prior to HPLC analysis. In this laboratory, the recoveries of both compounds were poor using those methods that employ hexane or ether as the extracting ~ o l v e n t . ~ .Other ~ . " procedures'0~1~~'4'1F, were time consuming, requiring multiple extractions or lengthy mixing steps. With the present method, simple precipitation of plasma proteins with acetonitrile is all that is required to prepare a plasma (or serum) sample for HPLC injection. The preparation time is <5 min, and the recovery of 1 and 2 is virtually complete. For urine and bile, simple treatment with acetonitrile was unsatisfactory for two reasons. Amiodarone is present in urine in low con~entrations.'"~~ With some specimens, the addition of acetonitrile diluted the samples below the detection limits of the assay. More important, endogenous substances were present in both specimens which interfered with the analysis of 1 and 2. Therefore, an extraction procedure was sought that would concentrate the analytes and at the same time, remove the interfering endogenous substances. Table I-Calibration Curves for Amiodarone (1) and Its Deethyl Metabolite 2 in Plasma, Urine, and Bile
Sample Plasma Human Rat Urine Human Rat Bile Rat
Calibration Range, ua/mL 0.1-10.0
Slope
Intercept
1
2
1.09 1.02
1.38 1.34
1
0.01 0.01
2 0.04 0.06
r 1
Human
2
0.997 0.998 0.991 0.992
1-100
0.096 0.110 0.012 0.081 0.994 0.996 0,111 0.126 0.011 0.094 0.996 0.996
7-100
0.115 0.128 0.039 0.119 0.991 0.989
= 5.
lnterdayb Rat
Human
1
2
1
2
10.3 6.1 1.6 4.2 1.0
7.6 3.2 1.2 5.0 2.6
6.2
6.1
0.9
0.8
2.2
3.2
1
12.2 9.1 5.0 7.9 1.9
Rat
2
1
2
16.5 9.7 1.0 7.9 2.6
9.0
10.5
5.6
5.6
3.5
3.9
b n = 6.
Bousquet et a1." reported the use of 2,2-dimethoxypropane as a dehydrating agent to remove water from aqueous samples. Under acidic conditions, water reacts with 2,2-dimethoxypropane to form 1 mol of acetone and 2 mol of methanol. The reaction is endothermic, which aids in the precipitation of Table Ill-Drugs
Tested for Potential Assay Interference
Compound Acetaminophen Acetazolamide Amiodarone (1) Amitriptyline
Ampicillin Aspirin
Caffeine Chlorcyclizine Chloroquine Chlorpromazine Cyclizine Cyproheptadine Deethyl metabolite of amiodarone 2 Desipramine Diphen hydramine Haloperidol Heparin H ydralazine Hydrocortisone Hydroxyzine lmipramine Internal Standard 3 Lidocaine Lorazepam Loxapine Meclizine Mesoridazine Methapyrilene Nortriptyline Pentobarbital Perphenazine Phenobarbital Phenoxybenzamine Phenytoin Procainamide Procaine Propranolol Propylthiouracil Pyrilamine Quinidine Quinine Theophylline Thioridazine Thiothixene Tranylcypromine Trifluoperazine ValDroic Acid
Retention Time, min 2.2 1.5 5.5 3.4 1.4 1.7 2.4 3.1 4.6 3.9 2.9 3.3 4.6 5.6 2.8 1.9 2.3 2.7 2.1 2.6 3.7 6.8 2.5 2.9 2.1 3.4 3.4 2.7 4.7 1.7 2.8 1.7 2.2 1.5 2.5 2.7 2.7 1.4 2.8 3.1 3.1 2.4 4.2 4.9 3.3 3.4 2.8
Journal of Pharmaceutical Sciences I 463 Vol. 74, No. 4, April 1985
Table IV-Recoverv
of Amiodarone (1) and Its Deethvl Metabolite 2 from SDiked Samoles of Human and Rat Plasma"
Recovery, %
Amount Humanb
Added,
a
Ratc
m/mL
1
2
0.1 0.5 1.o 5.0 10.0
91.8 k 8.4 (9.2) 91.4 t 2.7 (3.0) 101.3 rf: 1.4(1.4) 100.1 f 3.8 (3.8) 98.2 +- 0.9 (0.9)
92.5 f 6.2 (6.7) 88.5 f 2.5 (2.8) 102.2 f 1.1 (1.1) 105.3 f 4.8 (4.6) 105.1 f 1.3 (1.2)
Mean f SD (% CV). n
Table V-Recovery
= 6.
n
=
1
2
* 3.7 (3.9)
91.3 f 5.4 (5.9)
112.2 f 0.8 (0.7)
102.3 f 1.1 (1.1)
100.1 f 2.1 (2.1)
104.5 f 2.4 (2.2)
93.7
5.
of Amiodarone (1) and Its Deethyl Metabolite 2 from Spiked Samples of Human and/or Rat Urine and Bile"
Recovery, O% Amount Added,
Urine
1 10 100 a
Rat Bile
~~~
NlmL
Human
Rat
.~
1
2
1
2
1
2
82.4 i 6.0 (7.3) 93.3 & 1.3 (1.4) 85.6 k 2.9 (3.4)
64.8 F 10.7 (16.5) 62.0 & 4.7 (7.6) 59.8 1.7 (2.8)
87.9 f 12.2 (13.9) 75.1 f 1.7 (2.2) 91.9 + 1.1 (1.2)
50.6 f 7.4 (14.6) 65.0 f 1.8 (2.8) 68.9 f 0.9 (1.3)
87.8 k 6.6 (7.5) 84.3 f 7.2 (8.5) 92.0 k 4.9 (5.3)
66.2 f 11.9 (18.0) 64.5 k 4.3 (6.7) 65.2 f 1.4 (2.1)
Mean k SD ( O h CV), n
=
*
5
endogenous substances. After centrifugat,ion, the acetone and methanol mixture that is formed is easily removed and evaporated. After reconstitution of the residue with an appropriate solvent, the sample is ready for HPLC analysis. In the present assay, 2,2-dimethoxypropane was used to determine 1 and 2 in urine and bile. The extraction procedure, which was adapted from the work of Holcslaw," effectively eliminated the Hank interferences in the two fluids (Figs. 2 and 3). Other reports that describe the extraction of 1 from urine" I(i,I') and bile"' did not present recovery or reproducibility data. Therefore, it was not possible to compare the results of this assay for urine and bile with the previous methods. Plomp et al.1'3obtained good recovery of 1 and 2 from urine using disposable extraction columns and multiple extractions. With the present procedure, the recoveries of both compounds are good and the preparation of a urine sample for HPLC analysis is simpler and less expensive. In urine samples that were available for this work from humans and rats treated with amiodarone hydrochloride, no detectable levels of' 1 or 2 were observed. Andreasen e t al.15 and Harris et al."' noted that the concentrations of 1 in the urine after intravenous arid oral administrations were low. These observations suggested that perhaps the levels of 1 and 2 in the urine specimens tested may have been below the sensitivity of the assay. If this was the case, these findings suggest that renal excretion of 1 and 2 is not a primary mechanism of elimination for either of these drug species. With measurable concent rations of 2 in the plasma, perhaps this metabolite undergoes further metabolism or is excreted by a nonrenal mechanism. These possibilities will require further investigations. Blood collection tubes have been shown to affect the plasma concentrations of propranolo122and quinidine.'" In this study, dextrose solution the concentrations of 1 and 2 were cantly reduced after coming in contact with laboratory film and rubber stoppers. A similar effect was not seen, however, with the two compounds in plasma. Lalloz et aLZ4recently report,ed that 1 was bound extensively and with high affinity t o serum proteins. The hound plasma drug fraction was reported to be 96%, with the primary amiodarone binding site exhibiting an aff'initv constant of5.6 x 106L/mol. The unusually strong and ext.ensive plasma binding characteristics of 1 would explain the absence of any changes in the plasma concentrations of both compounds following contact with the same 464 f Journal of PharmaceuticalSciences Vol. 74, No. 4, April 1985
tube closures since the availability of the two drugs for interactions with the closures is reduced in plasma. In whole blood, the erythrocyte concentrations of 1 and 2 are sizeable.2s These findings may explain the increases in the plasma concentrations of both compounds after the blood specimens from whence they came contacted the laboratory film and rubber stopper tops. The increases may have been due to hemolysis of the erythrocytes, thus liberating additional drug into the plasma. Alternatively, contact of the erythrocytes with the closures may have caused a redistribution of both compounds from the red cells to the plasma. In this study, no visual evidence of hemolysis was observed in any of the plasma samples. Because of these findings, care is taken in this laboratory to prevent the blood specimen from coming in contact with the rubber stopper during the collection of blood for plasma assays. Additionally, the plasma is separated from the red blood cells immediately after the blood specimen is collected. The method described has been used to monitor steady-state plasma concentrations of 1 and 2 in patients receiving amiodarone for their dysrhythmias. At similar dosages, the observed concentrations of 1 and 2 were in agreement with those reported by other investigators4 and/or were in the desired therapeutic plasma concentration range of 1-2.5 pg/mL.26 For example, the concentrations of 1 and 2 in the patient plasma specimen shown in Fig. 1 were 1.2 and 1.0 pg/mL, respectively.
References and Notes 1. Rosenbaum, M. B.; Chiale, P. A,; Halpern, M. S.; Nau, G. J . ; Przybylski, J.; Levi, R. J.; Lazzari, ?J. 0.; Elizari, M. V. Am. J . Cardiol. 1976. 38. 934-944. 2. Rosenbaum, M. B.; Chiale, P. A.; Ryba, D.; Elizari, M. V. A m . J . Cardiol. 1974,34, 215-223. 3. Wheeler, P. J.; Puritz, R.; Ingram, D. V.; Chamberlain, D. A.
Postgrad. Med. J . 1979, 5Fi, 1-9. 4. Haffajee, C. I.; Love, J . C.; Alpert, J . S.; Asdourian, G. K.; Sloan, K. C. Am. Heart J . 1983. 106. 935-943. 5. Broekhuysen, J.; Laruel,' R.; 'Sion R. Arch. Int. Pharmacodyn. 1969, 177, 340-359. 6. Ravin, L. J.; Shami, E. G.; Intoccia, A.; Rattie, E.; Joseph, G . J . Pharm. Sci. 1969, 58, 1242-1245. 7. Storey, G. C. A.; Holt, D. W. J . Chromatogr. 1982, 245, 377-380. 8. Mostow, N. D.; Noon, D. L.: Mvers. C. M.; Rakita. L.: Blumer. J. L. J Chromatogr. 1983, 227, 289-237. 9. Lesko, L. J.; Marion, A,; Canada, A. T.; Haffajee, C. J Pharm. Sci 1981, 70, 1366-1368. 10. Debbas, N. M G.; Du Cailar, C.; Sassine, A,; Derancourt, d.;
DeMaille, J. G.; Puech, P. Eur. J . Clin. Invest. 1983, 13, 123-127. 11. Cervelli, J. A,; Kerkay, J.; Pearson, K. H. Anal. Lett. 1981, 14, 137-153. 12. Riva, E.; Gerna, M.; Latini, R.; Giani, P.; Volpi, A.; Maggioni A. J . Cardiovasc. Pharmacol. 1982,4, 264-269. 13. Plomp, T. A,; Engels, M.; Robles de Medina, E. 0.; Maes, R. A. A. J . Chromatogr. 1983,273, 379-392. 14. Brien, J. F.; Jimmo, S.; Armstrong, P. W. Can. J. Physiol. Pharmacol. 1983, 61, 245-248. 15. Andreasen, F.; Agerbaek, H.; Bjerregaard, P.; Gotzsche, H. Eur. J. Clin. Pharmacol. 1981, 19, 293-299. 16. Gobatto, S.; Padrini, R.; Candelpergher, G.; Bettero, A.; Cargnelli, G.; Ferrari, M. Int. J. Clin. Pharmacol. Res. 1982, 2, 279-281. 17. Flanagan, R. J.; Storey, G. C. A.; Holt, D. W.; Farmer, P. B. J. Pharm. Pharmacol. 1982, 34, 638-643. 18. Johnson, E. L.; Stevenson, R. “Basic Liquid Chromatography”; Varian Associates: Palo Alto, 1978; pp 238-239. 19. Harris, L.; Hind, C. R. K.; McKenna, W. J.; Savage, C.; Krikler, S. J.; Storey, G. C. A,; Holt, D. W. Postgrad. Med. J . 1983,59, 440442. 20. Bousquet, W. F.; Christian, J. E.; Knevel, A. M.; Spahr, J. L. Anal. Biochem. 1962, 3, 519-520.
21. Holcslaw, T. L. Masters Thesis, Purdue University, 1972. 22. Cotham, R. H.; Shand, D. Clin. Pharmacol. Ther. 1975, 18, 535538. 23. Ooi, D. S.; Poznanski, W. J.; Smith, F. M. Clin. Biochem. 1980, 13, 297-300. 24. Lalloz, M. R. A,; Byfield, P. G. H.; Greenwood, R. M.; Himsworth, R. L. J . Pharm. Pharmacol. 1983,36, 366-372. 25. Weir, S. J.; Ueda, C. T., unpublished results. 26. Latini, R.; Tognoni, G.; Kates, R. E. Clin.Pharmacokinet. 1984, 9, 136-156.
Acknowledgments The authors acknowledge the gifts of amiodarone, its deethyl metabolite, and internal standard from Dr. C . Lafille of Sanofi Centre de Recherches, Montpellier, France and the assistance of Mrs. Elizabeth A. McCafferty of the New York Office in acquiring these materials. We are grateful to Dr. John Kugler for providing the patient plasma and urine samples and to Mrs. Marilyn Kircher for preparing the manuscript.
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