Determination of quinidine and dihydroquinidine in plasma by high

Mar 1, 1979 - Berry J. Kline, Vilma A. Turner, and William H. Barr. Anal. Chem. , 1979, 51 (3), ... Gary H. Smith , René H. Levy. Drug Intelligence &...
0 downloads 0 Views 415KB Size
ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979

us with analyzed patient serum samples and their results.

LITERATURE CITED

(13) H. Huang, J. W. Kuan, and G. G. Guilbault, Clin. Chem. ( Winston-Salem. N . C . ) , 21, 1605 (1975). (14) A. Noma and K. Nakayama, Clin. Chem. (Winston-Salem, N . C . ) , 22, 336 (1976). (15) F:Williams, A. Brunsman, J. Huntington, J. Johnson, and D. Newman, Amperometric-OxidaseEnzyme Probes for Biochemical Analysis", Yellow Springs Instrument Company., Inc., Technical paper, Yellow Springs, Ohio, March 1976, p 12. (16) Chem. Eng. News, January 5, 1976, p 19. (17) D. S. Papastathopoulos and G. A. Rechnitz, Anal. C k m . , 47, 1972 (1975). (18) W. D. Mason and C. L. Olson, Anal. Chem., 42, 488 (1970). (19) M. D. Smith and C. L. Olson, Anal. Chem., 46, 1544 (1974). (20) M. D. Smith and C. L. Olson, Anal. Chem., 47, 1074 (1975). (21i L. Meites and T. Meites, Anal. Chem., 20, 984 (1948). (22) M. Dixon and E. C. Webb, "Enzymes", 2nd ed., Academic Press, New York, 1964, p 338. 1231 H. H. Leffler, A m . J . Clin. Pathol.. 31, 310 (1959). \

D. B. Tonks, Clin. Biochem., 1, 12 (1967). R. J. Henry, D. C. Cannon, and J. W. Winkelman, Ed., "Clinical Chemistry Principles and Technics", Bio-Science Laboratories, 2nd ed., Harper & Row, New York, 1974. H. M. Flegg, Ann. Clln. Biochem., 10, 79 (1973). W. Richmond, Clin. Chem. (Winston Salem, N.C.), 19, 1350 (1973). C. C. Alhin. L. S . Poon, C. S. G. Chan. W. Richmond, and P. C. Fu, Clin. Chem. ( Winston-Salem, N . C . ) ,20, 470 (1974). P. N. Tarbutton and C. R. Gunter, Clin. Chem. (Winston-Salem, N . C . ) , 20, 724 (1974). D. L. Witte, D. A. Baren 11, and D. A. Wycoff, Ch.C k m . ( Winsfon-Salem. N . C . ) , 20, 1282 (1974). P. W. Wentz. R. E. Cross. and J. Savorv. Clln. Chem. I Winston-Salem. N . C . ) . 22. 188 11976). R. J. 'Morin, C l i i . C h i h . Acta, 71, 75 (1976). F. Zoppi and D. Fenili, Chir. C h m . ( Winston-Salem,N.C.).22,690 (1976). C. A. Robinson, Jr., L. M. Hall, and J. Vasiliades, Clin. Chem. (Winston-Salem, N.C.). 22, 1542 (1976). R. Haeckei and M. Perlick, J . in. C k m . c/in. Biochem., 14, 41 I (1976).

449

- I

"

RECEIVED for review April 12, 1978. Accepted December 18, 1978. This work was partially supported by the National Institue of General Medical Sciences Grant G M 15821.

Determination of Quinidine and Dihydroquinidine in Plasma by High Performance Liquid Chromatography Berry J. Kline," Vilma A. Turner, and William H. Barr Department of Pharmacy and Pharmaceutics, Medical College of Virginia, Virginia Common wealth University, Richmond, Virginia 23298

A rapid, specific and sensitive high performance liquid chromatographic method has been developed for the determination of quinidine (Q) and dihydroquinidine (DHQ) in human plasma. Plasma samples are extracted with a benzene-isoamyl alcohol mixture ( 1:1) followed by separation on a microparticulate reverse-phase column. Quinidine, dihydroquinidine, and their metabolltes are detected by fluorescence In acid medlum using post-column additlon of sulfuric acid. Plasma concentrations as low as 0.05 pg/mL can be measured with a relative standard deviation less than 10%. The complete assay procedure takes about 30 min. Recovery from plasma is at least 95 YO. A linear response is obtained in the concentration range of 0.05 to 10 pg/mL of quinidine in plasma. The minimum detectable quantity is 1 ng on column.

metabolites are being measured along with the quinidine in this method. They recommend washing the benzene layer with NaOH to eliminate interferences from less polar metabolites. Numerous chromatographic procedures have been developed for the determination of quinidine (7-19). Several of them (7-10) are specific but time-consuming TLC procedures which we did not consider suitable for routine clinical monitoring. Some of them ( 1 1 , 12) have been applied only to pharmaceutical preparations. Several others (13-15) did not separate quinidine from dihydroquinidine. The method presented here provides good separation of quinidine, dihydroquinidine, and their metabolites, is sensitive enough to determine low concentrations of quinidine and dihydroquinidine, and is rapid enough for routine clinical monitoring of plasma levels.

To study quinidine pharmacokinetics in human subjects, a specific method for the separation and determination of quinidine, dihydroquinidine, and their metabolites is needed. Quinidine may contain u p to 20% of a natural contaminant, dihydroquinidine, depending on the source ( I ) . Dihydroquinidine is an analogue of quinidine with similar if not greater antiarrhythmic effect ( 2 , 3 ) . Drayer et al. have reported that three metabolites of quinidine found in man are pharmacologically active in mice and rabbits ( 4 ) . Until this activity, or lack thereof, is verified in humans, plasma level determinations of quinidine for the purpose of therapeutic dosage adjustment should be specific with minimal contribution from dihydroquinidine or metabolites. The analytical method commonly used to determine quinidine in plasma is that of Cramer and Isaksson ( 5 ) . This method is based on extraction of alkalinized plasma samples with benzene, which excludes the more polar metabolites, and then back extraction into H2S04 for fluorimetric quantitation of t h e quinidine. However, Huynh-Ngoc and Sirios (6) have found t h a t some

Apparatus. A Waters Model ALC202 liquid chromatograph fitted with a Model 6000 constant flow pump, a 30 cm X 3.9 mm i.d. pBondapak C18column (Waters Associates, Milford, Mass.) and a Rheodyne Model 7120 syringe loading sample injector with a 175-pL sample loop was used. A 5 cm X 2 mm i.d. guard column packed with C18 Corasil (37-50 pm particle size) was added to the system to prevent clogging of the main column. A T-union (Altex, k200-22) was used to combine the column eluate with 2 N sulfuric acid supplied by a peristaltic pump (Manostat Cassette pump) through Teflon tubing (0.8-mm i.d.). The sulfuric acid and the column eluate were introduced into the T-union at a 180' angle so that the opposing flows created turbulence, thus eliminating the need for a long mixing coil. A second Teflon tube (75 cm X 0.8 mm id.) connected the T-union t o the detector flow cell. Acid-resistant flexible tubing (0.056-inch i.d.) was used to pump the sulfuric acid to the Teflon tubing. The detector was a filter fluorimeter (Aminco Fluoro-Monitor) equipped with a 70-pL flow cell, 350-nm excitation, and 450-nm emission filters (Turner, 27-60 and 2A). The areas under the chromatographic peaks were measured with a Spectra Physics Autolab Minigrator. Reagents. Water was single-distilled, and acetonitrile was 99 mol 90 pure (Fisher Scientific Co.). All other chemicals and

EXPERIMENTAL

0003-2700/79/0351-0449$01.00/062 1979 American Chemical Society

450

ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979

organic solvents were reagent grade and were used as received. Quinidine sulfate dihydrate and dihydroquinidine were obtained as gifts from A. H. Robins Co. Although the batch of quinidine sulfate used contained approximately 5% dihydroquinidine and, likewise,the dihydroquinidine was contaminated with about 1.4% of quinidine, analytical results were not corrected for these because the same batches of each were used in all experiments. Correction of the actual amounts weighed and measured for these contaminants made no difference in recovery or precision data because of internal consistency. When patient samples are analyzed using standards with significant levels of contamination, this correction should be applied. Since quinidine and dihydroquinidine give equivalent responses in the chromatographic system (as will be discussed later), estimates of the contamination levels of these two compounds in each other may be obtained by chromatographic peak area normalization. A stock solution containing 2 mg/mL of quinidine was prepared by dissolving and diluting 24.2 mg of quinidine sulfate dihydrate to 10 mL with distilled water. A similar solution of dihydroquinidine, 1 mg/mL was prepared in methanol. A quinidine plasma stock solution (10 ,ug/mL) was prepared by the addition of 50 ,uL of quinidine stock solution (2 mg/mL) t o 10 mL of drug-free plasma. Quinidine standard solutions in the 5 to 0.05 pg/mL range were prepared by serial dilution of the 10 pg/mL quinidine stock solution with drug-free plasma. Procedure. Pipet 0.5 mL of plasma into a 5-mL stoppered test tube, alkalinize with 0.5 mL of 5N NaOH, add 2 mL of benzene-isoamyl alcohol (1:l mixture), mix gently by rocking for approximately 10 min on a mechanical mixer, and centrifuge at 2000 rpm for 10 min. Inject 175 pL of the organic layer into the HPLC column (full-loop injection). The concentration of quinidine in the plasma sample is determined from a calibration curve of quinidine peak area vs. quinidine concentration. Chromatographic Conditions. The mobile phase was prepared to contain 4% (v/v) of a 1% (w/v) aqueous (NH,)2C03 solution, 31% (v/v) of methanol, and 65% (v/v) of acetonitrile. It was degassed and filtered through a 0.45-pm membrane filter before use. The flow rate of the mobile phase was 3.5 mL/min (1000 psi). The sulfuric acid flow rate was approximately 0.45 mL/min.

RESULTS AND DISCUSSION F l u o r e s c e n t Detection. After development of the HPLC column-solvent system which separates quinidine from dihydroquinidine and their metabolites, it was obvious that ultraviolet photometric detection was not sensitive enough for quantitation of the low plasma levels of dihydroquinidine and its metabolites. Since Cramer and Isaksson ( 5 ) utilized the fluorescence of quinidine in its determination, it was anticipated that fluorescence detection might improve sensitivity. Although quinidine did not fluoresce in the HPLC mobile phase, addition of aqueous sulfuric acid restored the fluorescence characteristics of the quinidine. Therefore, post-column addition of aqueous sulfuric acid t o the column eluate was introduced by use of a peristaltic pump and a simple T-joint. Since the quality of the HPLC separation is determined by the flow rate of the mobile phase, the only other variables which could be manipulated to improve detectability were the concentration and the post-column addition rate of sulfuric acid. Fluorescence was optimized a t a sulfuric acid concentration of 2 N and a flow rate of approximately 0.45 mL/min. Since the mobile phase contains ammonium carbonate, the possibility of COPbubble formation during the addition of the aqueous acid was a concern because the bubbles could cause detector noise. However, this anticipated problem never appeared. E x t r a c t i o n . During the initial investigation, direcc injection of plasma into the HPLC column was attempted (141, but precipitated plasma proteins tended to clog the column. Several protein precipitation methods were then investigated. Precipitation of plasma proteins with trichloroacetic acid (TCA) yielded low recovery of quinidine. Other protein precipitants such as methanol and tungstic acid were tested,

I1

l

l

0

2

l

2

l

l

l

4 6 810 MINUTES

Figure 1. Chromatogram of human plasma spiked with quinidine ( l ) , 10 pg/mL containing dihydroquinidine (2) at the natural contaminant level ( - 0.5 pglmL). The arrows indicate the relative sensitivity of the fluorimetric detector at those points also with unsatisfactory results. Since direct injection after protein precipitation did not appear promising, several solvent extraction methods were then investigated. However the main disadvantage with them was a lengthy solvent evaporation step, and/or adsorption of quinidine by the glassware. T h e latter could be reduced by adding a small amount of isoamyl alcohol, but that increased the evaporation time. T h e highly fluorescent characteristics of quinidine and its affinity for the benzene-isoamyl alcohol solvent system made it possible t o avoid the evaporation step by direct injection of the extract onto the column. The analysis can then be performed in 30 min. T h e advantage of this solvent system over others investigated is that it extracts quinidine, dihydroquinidine, and their metabolites (8) since it was desired to determine all of these in this laboratory. Identification and quantitation of the metabolites by this method are currently under investigation. Q u a n t i t a t i o n . Because the internal standard method is preferred in quantitative chromatography, initial investigations were directed a t finding a compound which would serve as an internal standard in this procedure. However, the severe constraints placed upon an internal standard by the analytical sk-stem drastically restricted the choice of compounds. For example, the internal standard must (1)be extracted efficiently from plasma under the same conditions as quinidine in this procedure; (2) elute from the HPLC column with a retention time different from t h a t of quinidine, dihydroquinidine, and their metabolites; and (3) have the same fluorescence characteristics as quinidine in the column eluate-sulfuric acid mixture. Dibucaine met conditions 1 and 3, but interfered with one of the metabolites. Thiopropazate met conditions 2 and 3, but was poorly extracted from plasma in this procedure. Numerous other compounds were investigated, but none of them conformed to the constraints of the analytical system. Therefore, external standardization was adopted after it was proved that acceptable precision was obtainable with that technique in this procedure. Figure 1 is a chromatogram of a 10 pg/mL standard plasma solution of quinidine with dihydroquinidine a t the natural contaminant level ( - 5 % in this batch). Although it is not necessary to increase detector sensitivity when determining dihydroquinidine by electronic integration, i t was increased for purposes of clarity in this figure. Figure 2 shows a chromatogram for the plasma from a patient who was dosed

ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979 3

Table 111. Reproducibility for the Determination of Quinidine and Dihydroquinidine in Plasma drug Q" Q Q DHQ mean concn, 1.82 0.10 0.051 0.91 cLg/mL no. of 20 10 15 6 determinations std. dev. 0.067 0.007 0.0032 0.045 rel. std. dev., % 3.7 7.3 6.3 4.9 a Q: Quinidine. DHQ: Dihydroquinidine.

I1

I

2

! 0

4

8

1

2

MiNUTES

Figure 2. Chromatogram of plasma from a patient dosed with quinidine sulfate. Peaks represent 3-hydroxyquinidine (a quinidine metabolite) (1). as yet unidentified metabolites of quinidine or dihydroquinidine (2, 4, and 5), quinidine, 3.8 wg/mL (3), and dihydroquinidine (6)

Table I. Relative Recovery of Dihydroquinidine from Plasma Using Quinidine Standards concn added, pg/mL concn found, pg/mL mean recovery, %

0.246 0.233 94.7

0.493 0.496

100

0.983 0.973 98.7

4.93 5.23 106

Table 11. Absolute Recovery of Quinidine and Dihydroquinidine from Plasma drug concn added, pg/mL concn found, pg/mL % recovered a

Q: Quinidine.

451

&"

0.1

0.095 95.0

Q

10.0 9.73 97.0

DHQ

DHQ

0.107 107

1.04 104

0.1

1.0

DHQ: Dihydroquinidine.

with quinidine sulfate. This figure illustrates the separation of metabolites and the parent compounds. Calibration Curve. A calibration curve was constructed with quinidine concentrations of 10, 5, 2,0.1, and 0.05 pg/mL in drug-free plasma. A linear curve of peak area vs. quinidine concentration was obtained with a correlation coefficient of 0.999. T h e same curve can be used to measure the concentration of dihydroquinidine in plasma since it was found that the peak areas for equal concentrations of quinidine and dihydroquinidine are the same. This was determined by measuring the relative recovery of dihydroquinidine from plasma spiked a t 0.25 to 5 Fg/mL, using quinidine as the standard. As can be seen in Table I, the recovery is within experimental error of 100% a t all levels. Recovery. T h e absolute recoveries of quinidine and dihydroquinidine were determined by comparing chromatographic peak areas from extracted plasma solutions with areas generated by direct injection of aqueous or methanolic solutions of the two drugs. As shown in Table 11, absolute recoveries are within experimental error of 100%. Recoveries were significantly lower when 0.05 mL of 5 N NaOH was used for alkalinization. Further experiments indicated that the recovery was improved when plasma samples were diluted with an equal volume of water. Therefore, 0.5 mL of 0.5 N NaOH was used to alkalinize and dilute plasma samples. Since the absolute recoveries approximate 10070,it should be possible to substitute water for drug-free plasma in the

DHQ

4.96 6

0.185 3.7

preparation of standard solutions, although this has not been tested experimentally. Reproducibility. Drug-free plasma spiked with quinidine and dihydroquinidine a t three different concentrations was analyzed over a period of several days. As shown in Table 111, the relative standard deviation is less than 10% a t all levels. Sensitivity. Quinidine and dihydroquinidine concentrations of 0.05 wg/mL are easily measurable by this method. It is possible to increase the detector sensitivity or the sample volume so t h a t as little as 1 ng of quinidine or dihydroquinidine on column can be detected. Interferences. No attempt was made to screen a large number of drug compounds which might be coadministered with quinidine and interfere with this determination. I t became obvious during the search for an internal standard that the constraints placed upon other drugs (and their metabolites) by this analytical system would limit the number of compounds which could interfere. Because of these constraints, the method is considered to be quite specific. However, the potential for interference by other compounds should be recognized. T o investigate the possibility that metabolites were eluting with the quinidine or dihydroquinidine, a sample of urine from a patient dosed with quinidine sulfate was analyzed by this procedure, and the eluate fractions containing the quinidine and dihydroquinidine were collected. T h e solvent was evaporated, and the residues were analyzed by mass spectrometry. The spectra obtained were identical to those of pure quinidine and dihydroquinidine. LITERATURE CITED (1) "The United States Pharmacopeia", XIX rev., Mack Publishing Co.. Easton, Pa.. 1975 D 435. (2) T. Lewis, Am. J. Med. Sci., 163, 781 (1922). (3) F. Alexander, H. Gold, L. N. Katz, R. L. Levy. R. Scott, and P. D. White, J . Pharmacal. ~ x p Tber., . 90, 191 (1947). (4) D. E Drayer, C. E. Cook, and M. M. Reidenberg. Clin. Res., 24, 623-A 119761. (5) G . Cramer and B. Isaksson, Scand. J. Clin. Lab. Invest., 15,553 (1963). (6) T. Huynh-Ngoc and G. Sirois, J , Pharm. Sci., 66, 591 (1977). (7) C. T. Ueda, D. S. Hirschfeld, M. M. Scheinman. M. Rowland, B. J. Williamson, and B. S. Dzindzio, Clin. fbarmacol. Tber., 19, 30 (1976). (8) G. Hartel and A. Korhonen, J. Chromatogr.,37, 70 (1968). (9) B. Wesley-Hadziia and A. M. Mattocks, J . Cbromatcgr.. 144, 223 (1967). 10) J. M. Steyn and H. K. L. Hundt, J. Chromatogr., 111. 463 (1975). 11) E. Smith, S.Barkan, 6. Ross, M. Maienthal, and J. Levine, J. Pharm. Sci., 62, 1151 (1973). 12) N. J. Pound and R. W. Sears, Can. J. Pbarm. Sci., 10, 122 (1975). 13) J. L. Valentine, P. Driscoll, E. L. Hamburg. and E. D. Thompson, J. pharm. Sci.. 65. 96 11976). 14) K. A. Conrad,' B. L.'Molk, and C. A. Chidsey, Circulation, 55, 1 (1977). 15) J. L. Powers and W. Sadee, Clin. Cbem. (Winston-Salen, N.C.). 24, 299 (1978). (16) B. A. Persson, and P. 0. Lagerstron, J. Chromatogr., 122, 305 (1976). (17) M. A. Moulin and H. Kinsun, Lin. Chim. Acta. 7 5 , 491 (1977). (18) K. K. Midha and C. Charette, J . Pharm. Sci., 63, 1244 (1974). (19) R. G. Achari, J. L. Baldridge, T. R. Koziol, and L. Yu, J. Cbromatogr. Sci., 16, 271 (1978).

RECEIVED for review June 26, 1978.

1978. Accepted December 8,