1504
Anal. Chem. 1987, 59, 1504-1509
Simultaneous Determination of Flurbiprofen and Its Major Metabolite in Physiological Fluids Using Liquid Chromatography with Fluorescence Detection W. J. Adams,* B. E. Bothwell, W. M. Bothwell, G. J. VanGiessen, and D. G. Kaiser Drug Metabolism Research, The Upjohn Company, Kalamazoo, Michigan 49001
A highly speclflc and sensiUve hlgh-perfonnance llqukl chromatography method that utlllres fluorescence detectkrl was developed for the rapid and precise determination of Hurblprofen, a potent nonsterddal antlhdlammatory agent, and Its major metabolite, 4‘-hydrorytlwb@rokn, In 100 pL of serum or urine. The samples were prepared for chromatographlc analysts by deprotelnlrlng with acetonltrlle (1 mL) and bufferlng wHh 0.05 M potassium phosphate (pH 2.6, 2 mL). A structural analogue of flurblprofen, 2-(2-methoxy-4-blpheny1)proplonlc acld, was used as an Internal standard. AHquots of the supernatant (100 pL) were chromatographed on a Waters peondapak C-18 column uslng a mobile phase containing 0.05 M potassium phosphate (pH 2.6) and tetrahydrofuran (55/45 (v/v)) at a flow rate of 1.9 mL/mh. The fluorescence response (Aex 260 nm, A, 320 nm) was linear ( r 2 > 0.9998, n = 2 X 12) for concentrationsof Hurbiprofen and 4’-hydroxyHurblprofen from 100 ng/mL, the lower IhnIt of quantltatlon, up to 50 pg/mL. Flurblprofen and 4’-hydroxyflurblprofen concentratlons were determlned before and after alkaline hydrolysis. Recoveries from unhydrolyzed and hydrolyzed controls ranged from 97.4 f 1.2 to 105.5 f 5.9%. The Utlly of the methodology was confirmed by analysis of serum and urine speclmens from male volunteers recelvlng single 50-mg oral doses of flurblprofen.
Flurbiprofen [ (RS)-2-(2-fluoro-4-biphenyl)propionic acid; F, Figure 11 is a well-tolerated, orally effective nonsteroidal antiinflammatory drug for the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and mild to moderate pain (1-4). Single oral doses of flurbiprofen usually do not exceed 100 mg. The highly potent antiinflammatory activity of flurbiprofen derives, in part, from its ability to inhibit prostaglandin biosynthesis and other mediators responsible for the inflammatory process (5). Flurbiprofen has been marketed internationally since 1977 and is currently awaiting marketing approval in the United States as a filmcoated compressed tablet [ANSAID, The Upjohn Co.]. Drug disposition studies with flurbiprofen in animals and normal human volunteers (6-8) indicated that absorption was rapid and complete following oral administration. In humans, peak plasma concentrations ranging from 5 to 7 bg/mL were achieved 1 to 2 h after single-dose oral adminstration of 50 mg of micronized drug. Disappearance of flurbiprofen from systemic circulation was biphasic with the terminal disposition phase having a half-life of approximately 7 h. No metabolites of flurbiprofen were detected in plasma (6,8,9). More than 95% of the orally administered dose was excreted in urine within 24 h. Major metabolites reported to be excreted in urine were 4’-hydroxyflurbiprofen (4’-HF, Figure 1;40-47 %), 3’-hydroxy-4’-methoxyflurbiprofen (3’-H-4’-MOF, Figure 1; 20-30%), and intact flurbiprofen (F, Figure 1;20-25%) (6). A minor metabolite which accounted for only about 5% of the dose was 3’,4’-dihydroxyflurbiprofen (3’,4’-DHF, Figure
1). Approximately 60-70% of the dose was excreted as conjugates. Quantitation of intact flurbiprofen in physiological fluids has been accomplished by using both gas (10-12) and liquid chromatographic (13,14) methods. Plasma, serum, and urine have been analyzed, with a lower limit of detection sensitivity in plasma of 50 ng/mL achieved using isothermal gas chromatographic conditions and electron capture detection (IO). More recently, liquid chromatographic methods were reported for the quantitation of flurbiprofen in dog (13)and human (14) serum. One of these methods utilized fluorescence detection and allowed quantitation at the 100 ng/mL level when 0.5 mL of serum was analyzed (14). Intact flurbiprofen and its metabolites have previously been determined by thin-layer chromatography (8) or gas chromatography with temperature programming (8) and isothermal chromatographic conditions (9). The sensitivity, precision, and accuracy of the thin-layer (8) and temperature-programmed gas chromatographic (8)methods were not reported. This report describes a simple yet highly specific and sensitive liquid chromatographic method that utilizes fluorescence detection for the rapid determination of flurbiprofen and its major metabolite, 4’-hydroxyflurbiprofen, in unhydrolyzed or alkaline hydrolyzed human serum or urine. Quantitation of as little as 100 ng/mL of flurbiprofen and 4‘-hydroxyflurbiprofen can be achieved when analyzing only 0.1 mL of serum or urine. The methodology was used to analyze serum and urine specimens from normal male volunteers receiving single 50-mg oral doses of flurbiprofen and the serum flurbiprofen concentrations were compared with those obtained by using a previously reported electron capture gas chromatographic method (10). EXPERIMENTAL PROCEDURE Reagents. Synthetic samples of flurbiprofenand the c o d i e d metabolites of flurbiprofen in man (Figure l),and the internal standard, (RS)-2-(2-methoxy-4-biphenyl)propionic acid, were supplied by the Pharmaceutical Research and Development Laboratories of The Upjohn Co. Distilled-in-glassspectroscopic grade solvents (Burdick and Jackson, Muskegon, MI) were used as received. Inorganic chemicals were AR grade and were prepared in deionized water. Apparatus. The high-performance liquid chromatograph used in this study was a modular component system consisting of a dual piston solvent pump (Model 6000A, Waters Associates, Milford, MA), an in-house designed and fabricated autoinjector (15) fitted with a 100-pL sample loop, a commercially prepared 30 cm X 3.9 mm i.d. reversed-phase column (pBondapak C-18, Waters Associates, Milford, MA), a dual monochromator spectrofluorometer equipped with a 28-pL flow cell (Model 650-10s, Perkin-Elmer, Norwalk, CT), and a dual-channel recorder (Model 585, Linear Instruments, Irvine, CA). The autoinjector was modified to provide for accurate injection of small sample volumes by replacing the vacuum sample aspirator with a precision motorized buret (Metrohm Model E412, Brinkman Instruments, Westbury, NY). A commercially prepared 4.0 cm X 4.6 mm
0003-2700/87/0359-1504$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987
-
CONJUQATION
C H O ?-& O !-H
/
4‘-HF (4047%)
&Loon \ E
\
I20-;5%l
I
no
i
CONJUGATION
3‘,4’ -DHF 15%)
I
CONJUGATION
1
CONJUGATION
Figure 1. Biotransformation of flurbiprofen in man (6, 8 ) . The abbreviated names (arbitrary) and percentage of the dose excreted in urine are shown lmmediiateiy below each structure: F, flurbiprofen, (RS).2~2-fluor&iphenyl)propionic acid; 4‘HF, 4’-hydroxyflurbiprofen, (RS)-2-(2-fluoro-4‘-hydroxy-4-biphenyl)propionic acid; 3’,4’-DHF, (RS)-2-(2-fluoro-3’,4’dihydroxy-4-biphenylbropionic acid: 3’-K4‘-MOF, 3‘-hydroxy-4’-methoxyfiurbiprofen, (RS)-2-(2-fiuoro-3’-hydroxy-4’methoxy-4biphenyl)propionic acid.
reversed-phase guard column (5 pm RP-8, Brownlee Labs, Berkeley, CA) was used to protect the analyticalcolumn. Solvents were evaporated with an analytical evaporator (Organomation Associates, Shrewsbury, MA) and samples were mixed on a vortex mixer (Labline Instruments, Melrose Park, IL). Automated data acquisition and processing were accomplished with a Harris 500 minicomputer. Standards. Stock solutions containing approximately 100 wg/mL flurbiprofen, 4’-hydroxyflurbiprofen, and 3’-hydroxy-4’methoxflurbiprofen were prepared by diluting accurately weighed samples of the respective reference standards in methanol. Reference standard solutions containing approximately 50, 20, 10,5,2,1,0.5,0.2,0.1,0.05,0.02, and 0.01 pg/mL of the respective standards were prepared by making appropriate dilutions with methanol. A 1 pg/mL internal standard solution was prepared in acetonitrile. Fortified serum and urine controls containing flurbiprofen, 4’-hydroxyflurbiprofen, and 3’-hydroxy-4’-methoxyflurbiprofen at concentrations of 25.0,2.5, and 0.25 yg/mL, respectively,were prepared by transferring appropriate aliquots of the reference standard solutions to a volumetric flask, evaporating the solvents to dryness at 40 “C using a stream of filtered nitrogen, and diluting with blank control serum or urine. Sample Preparation. Calibration curve standards were prepared for processing by transferring 0.1-mL aliquots of the reference standard solutions to 16 X 125 mm culture tubes fitted with Teflon-lined caps and evaporating the solvent to dryness using a stream of filtered nitrogen. One-tenth-milliliter aliquots of blank control serum or urine were added to each tube and thoroughly mixed on a vortex mixer. Fortified controls and subject specimens were prepared for processing by transferring 0.1-mL aliquots to 16 X 125 mm culture tubes fitted with Teflon-lined caps. Unhydrolyzed samples were processed for quantitation of free (unconjugated) flurbiprofen and 4‘-hydroxyflurbiprofen by sequential addition with mixing of (1) 0.1 mL of 0.5 M sodium chloride, (2) 1 mL of internal standard solution, and (3) 2 mL of 0.05 M potassium phosphate (pH 2.6). The samples were centrifuged at 2000 rpm for 10 min prior to chromatographic analysis. Hydrolyzed samples were processed for quantitation of total (conjugated and unconjugated) flurbiprofen and 4‘-hydroxyflurbiprofen by sequential addition with mixing of (1) 0.050 mL of 1M sodium hydroxide and hydrolysis at ambient temperature for 20 min, (2) 0.050 mL of 1M hydrochloric acid to neutralize the sodium hydroxide, (3) 1mL of internal standard solution, and (4) 2 mL of 0.05 M potassium phosphate (pH 2.6). The samples were centrifuged at 2000 rpm for 10 min prior to chromatographic analysis. Chromatography. Aliquots of each sample (100 pL) were chtomatographed on a pBondapak (2-18 reversed-phase column at ambient laboratory temperature using a 0.05 M potassium phosphate/tetrahydrofuran (55/45 (v/v)) mobile phase at a flow
1505
rate of 1.9 mL/min. Column eluate was monitored at an emission wavelength of 320 nm, using an excitation wavelength of 260 nm and 20 nm excitaton and emission slit widths. Concentrations of flurbiprofen and 4’-hydroxyflurbiprofen in unknowns were calculated from peak height ratios using the through-origin slope calculated by unweighted linear regression analysis of calibration curve data. In Vivo Studies. Apparently healthy male volunteers between 19 and 28 years of age and weighing between 150 and 170 lbs participated in the study. The volunteers were fasted 12 h prior to and 2 h after oral administration of a single 50-mg dose of flurbiprofen as a film-coated compressed tablet (ANSAID, The Upjohn Co.). Blood specimens were obtained by venipuncture at predetermined times ranging from 0.5 to 24 h post drug administration. The blood was allowed to clot and the collected serum was stored frozen (-18 OC) until analysis. Urine specimens were collect2 at predetermined time intervals out to 48 h post drug administration and stored frozen (-18 “C) until analysis.
RESULTS A N D DISCUSSION Chromatography and Detection Conditions. The virtues of reversed-phase liquid chromatography for the analysis of drugs and metabolites in physiological fluids have been emphasized by Karger and Giese (16). It is particularly advantageous to use reversed-phase liquid chromatography for the analysis of polar metabolites when concentrations and detector sensitivity are high enough to preclude the need for labor-intensive sample extraction. In those situations, sample preparation is usually accomplished by precipitation of proteins with a suitable organic solvent or inorganic precipitation reagent and the resulting supernatant injected directly into the chromatographic system following centrifugation. Flurbiprofen and its metabolites are well suited for reversed-phase chromatography because therapeutic doses of the drug are relatively high and the strong fluorophore possessed by these compounds allows high sensitivity. Initially, a method utilizing paired-ion reversed-phase chromatography was developed which successfully resolved all the known metabolites of F (Figure 1). In this method, 0.1 mL of internal standard reagent was added directly to 0.1 mL of serum or urine followed by precipitation with 0.4 mL of acetonitrile. The clear supernatant was injected directly onto a reversed-phase column (yBondapak C-18) using a mobile phase containing 45% methanol and 55% aqueous solution of 0.05 M (NH4)2HP04and 0.004 M tetrabutylammonium phosphate a t a flow rate of 1.9 mL/min. In this assay, the lower limits of detection of F and 4’-HF were 1.0 and 0.1 yg/mL, respectively. Subsequently, the present method was developed which provided greater sensitivity and reduced analysis time. The detection of all four drug-related materials simultaneously at optimum sensitivity is not possible without reproducible excitation and emission wavelength programming or using multiple detectors, as is clear from the excitation and emission spectra shown in Figures 2 and 3. The wavelengths used in the present assay were selected to provide optimum response for the parent compound and the major metabolite, 4’-HF, with less response for the 3’-H-4’-MOF and extremely low response for the 3’,4’-DHF. Lower limits of quantitation (SIN 1 6) of F, 4’-HF, 3’-H-4’-MOF, and 3’,4’-DHF were 100, 100,2000, and 6000 ng/mL, respectively, under the described analytical conditions. Use of these wavelengths to maximize the sensitivity of F and 4’-HF was possible since early studies showed that, under optimum conditions, undetectable levels of 3’-H-4’-MOF and 3’,4’-DHF were present in serum specimens of male volunteers administered 50-mg doses of flurbiprofen. Furthermore, little if any 3’-H-4’-MOF and 3’,4’DHF were present in urine from the same subjects. By use of excitation and emission wavelengths at which 3’,4’-DHF gave essentially no response, a spectroscopic resolution of the 3’-H-4’-MOF and 3’,4/-DHF was achieved, which under the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987
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4’-HF
4’-HF
F 312nm
IS
I] 220
zw
’
’> 3w
I
340
380
420
500 220 280 WAVELENGTH (nm)
460
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/
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.
.
”
340
380
4 s
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5w
spectra of fiurbiprofen
(F) and 4’-hydroxyflurbiprofen (4’-HF). 3 ’ - H , 4’-MOF
F
i I 12
RETENTION TIME (rnin)
Flgure 4. Chromatogram of authentic standards of flurbiprofen (F), 4’-hydroxyflwbiprofen(4’-HF), 3’-hydroxy-4’-methoxyflwbiprofen (3’H-4’-MOF), 3’,4‘-dihydroxyflurbiprofen (3‘,4’-DHF), and the internal standard (IS). The 3‘-H-4’-MOF and 3‘,4’-DHF coeiute under the
described chromatographic conditions. WAVELENGTH (nm)
Figure 3. Excitation (---) and emission (-) spectra of 3’-hydroxy-4’-methoxyflurbiprofen (3’-H-4’-MOF)and 3’,4’dihydroxyflurbi-
profen (3‘,4’-DHF). described chromatographic conditions coelute (Figure 4). Because as much as 20-30% of the dose of F administered had previously been reported (8) to be excreted in urine as 3’-H-4’-MOF, calibration standards for this metabolite were prepared and analyzed during these studies. Typical chromatograms of predose serum and urine and serum and urine from patients administered single 50-mg doses of F, before and after alkaline hydrolysis, are shown in Figures 5 and 6, respectively. No peaks were observed in normal human serum or urine which interferred with F, 4’-HF, or the internal standard under these analytical conditions. In urine a very small peak, which eluted very close to 3’-H-4’MOF and 3’,4’-DHF, was occasionally present in unhydrolyzed predose as well as postdose samples. Examination of the chromatogram of unhydrolyzed urine (Figure 6) indicates the presence of several drug-related peaks that elute prior to the known metabolites of flurbiprofen. It is probable that these compounds are the reported conjugates of flurbiprofen or its metabolites (6, 8). Assay Precision and Accuracy. Linear regression analysis of calibration curve data indicated no significant deviations from linearity for F, 4’-HF, or 3’-H-4’-MOF for concentrations up to the highest concentration used, 50 pg/mL. Correlation coefficients (r2)were better than 0.9998 for calibration curves prepared in serum and urine, with and without alkaline hydrolysis, and analyzed on three different assay days. The intercepts were not significantly different
than zero (p > 0.05) for any of the curves, indicating insignificant interference from endogenous serum components. An estimate of the interassay reproducibility and precision was obtained by comparing the percent relative standard deviations (% RSD) of the slopes for F, 4’-HF and 3’-H-4’-MOF in serum and urine, hydrolyzed and unhydrolyzed. Relative standard deviations were in all cases less than 6.3%. Analytical precision and accuracy were established by adding known quantities of F, 4’-HF, and 3’-H-4’-MOF, at three different levels, to serum and urine and analyzing aliquots on three different assay days. Mean recoveries of F, 4’-HF, and 3’-H-4’-MOF from unhydrolyzed and hydrolyzed serum and urine of 102.4 f 1.15, 99.0 f 1.00, and 101.6 f 1.75 % , respectively, indicated the methodology was highly accurate as well as precise under all analytical conditions. Applicability of the Methodology. The utility of the analytical methodology was demonstrated by analyzing serum and urine specimens collected from four normal male volunteers each administered a single oral dose of 50 mg of flurbiprofen. Low but significant levels of 4’-HF were observed for the first time in systemic circulation (6, 8, 9). Levels of F and 4’-HF in serum after alkaline hydrolysis were the same as without hydrolysis, indicating insignificant levels of conjugated F or 4’-HF in systemic circulation. Peak plasma concentrations of F ranged from 7.6 to 16.2 pg/mL in the four volunteers. Plasma concentrations of 4’-HF were 10-20 times less than simultaneous F concentrations. Semilogarithmic plots of serum concentration-time profiles of F and 4’-HF for a typical volunteer are shown in Figure 7 . Visual examination of F serum concentration-time profiles indicated that disappearance of the drug from systemic circulation was biphasic. Although limited data were available, log linear regression
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987 2 hr
0 hr
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1507 4 hr
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12 RETENTION TIME (min)
RETENTION TIME (min)
4
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0 hr
2 hr
0 hr
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8
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4
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0
4
a
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RETENTION TIME (mln)
Flgum 6. Chromatogam of u n w e d (top)and alkaline hydrolyzed (bottom)predose urine (0 h) and urine collected 4 h postdose (4 h) from a subject that received a 50-mg oral dose of flurbiprofen. High levels of flurbiprofen (F) and 4‘4-1ydroxyfiurbiprofen(4’-HF) were found in the 4-h urine. Internal standard (IS) was added to the 4-h specimens.
F or 4’-HF remaining to be excreted in urine ( U , - Ut) for a typical volunteer is shown in Figure 9. Terminal disposition half-lives calculated for F by log linear regression from the urinary excretion data were in good agreement with those calculated by log linear regression from serum concentration-time data. The terminal disposition half-lives calculated for 4’-HF by log linear regression from urinary excretion data were slightly longer than those calculated for F. It has been reported that 20-30% of the administered dose of F is excreted in urine as the glucuronide of 3’-H-4’-MOF, and approximately 5% as 3’,4’-DHF, 70-80% of which was conjugated (6,8). Much lower levels of these metabolites were excreted in the present study. In order to explore this difference further, a more accurate measurement of these components was made in urine since both give poor response under the reported detection conditions. The detector wavelengths were changed to 295 nm (excitation) and 355 nm (emission), which provides optimum sensitivity for these metabolites. The efficiency of alkaline hydrolysis (NaOH) was also compared to hydrolysis with glucuronidase, glusulase, and acid hydrolysis (HC1). The levels of F and 4’-HF after alkaline hydrolysis were higher than those after enzyme or acid hydrolysis, based on fluorescence response, indicating more complete hydrolysis in alkali. In the case of 3’-H-4’-MOF and 3’,4’-DHF, acid and enzyme hydrolysis produced variable interfering peaks which prevented direct comparison of peak heights. On the basis of the results obtained for F and 4’-HF using the various hydrolysis procedures, it was concluded that the alkaline
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987
1508 10.0
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5.0 a
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= 6.17 hrs tli2 0 = 5.76 hrs
I
4 6 12 16 20 TIME AFTER DRUG ADMINISTRATION (hn)
24
Figure 7. Semilogarithmic plots of serum flurbiprofen (0)and 4’-hydroxyflurbiprofen (m)concentrations in healthy male volunteers. Terminal disposition half-lives calculated by log linear regression analysis are shown for each compound.
I
8 16 24 32 40 TIME AFTER DRUG ADMINISTRATION (hrs)
I
46
Figure 9. Amount of flurbiprofen (0)and 4‘-hydroxyflurbiprofen (m) remaining to be excreted In urine. Terminal disposition half-lives for both components were calculated, by log linear regression analysis.
on the peak. Double peaks and shoulders were not seen in the calibration standards or in the predose specimens. These results suggest that less, and perhaps much less, than 8.6% of a single 50-mg dose of F is excreted in urine as 3’-H-4’-MOF. Szpuner et al. (17) also found that only 6.75% of the administered dose of F was excreted as 3’-H-4’-MOF. In conclusion, the analytical methodology described in this paper allows the specific and sensitive determination of flurbiprofen and 4’-hydroxyflurbiprofen in human serum, plasma, or urine with minimal sample preparation required. Serum concentrationsof flurbiprofen determined by using this method were highly correlated with results obtained using the gas chromatographic method of Kaiser, et al. (IO). The method is suitable for bioavailability and bioequivalence studies, therapeutic drug monitoring, and forensic toxicology.
loor
t
Figure 8. Mean cumulative urinary excretion profiles and associated standard deviations (n = 4) for flurblpofen (F), 4‘-hydroxyflurblprofen (4‘-HF), and the summation of F and 4’-HF in healthy male volunteers.
ACKNOWLEDGMENT The design and conduct of the clinical phase of this study by C. D. Brooks and A. Wirbel are gratefully acknowledged. We thank L. Plagens and J. Katz for preparation of the manuscript. Registry No. F, 51543-38-5; 4’-HF, 80685-20-7.
hydrolysis procedure was most appropriate. By use of alkaline hydrolysis and optimum detection conditions, it was found that total levels of 3’-H-4’-MOF could not be greater than 8.6% of the F dose. Since 3’-H-4’-MOF and 3’,4’-DHF coelute under the chromatographic conditions used, a part of the observed response could be due to 3’,4’-DHF under the conditions used in these experiments. Furthermore, the chromatographic peaks either were double peaks or had a shoulder
(1) Lee, P.; Anderson, J. A.; Miller, J.; Webb, J.; Buchanan, W. W. J . Rheumatol. 1976. 3 . 283. (2) Sturrock, R. D.; &rt; F. D. Ann. Rheum. Dis. 1974, 3 3 , 129. (3) Kay, B. C u r . Med. Res. @in. 1975, 3(Suppl. 4). 49. (4) Muckle, D. S. Rheum. Rehab. 1977, 16, 5 8 . (5) Flower, R. J.; Moncada, S.; Vane, J. R. In Pharmacological Basis of Theraputics; Goodman, A., Goodman. L. S., Gilman, A,. Eds.; Macmlllan: New York, 1980; Chapter 29. (6) Adams, S. S.;Crampton, E. L.; Nlcholson, J. S.;Risdall, P. C. Znt. Congr. Rheum., 13th 1973, 173.
TIME AFTER DRUG ADMINISTRATION (hrs)
LITERATURE CITED
Anal. Chem. 1987, 59, 1509-1512 (7) Ishll, Y.; Sakai, Y.; Matsuda, T.; Nozu, K.; Inoue, M.; Tono, M.; Ofuji, T. Oyo Yakwll975, IO, 645. (6) Risdall. P. C.; Adams, S. S.; Crampton, E. L.; Merchant, 6.Xenoblotica 1.. 0.7.~ . .,8.9.. ,8 . (9) Kawahara, K.; Matsumura, Y.; Ishii, Y.; Ofuji, T. Oyo Yakuri 1975, IO, 653. (10) Kaiser, D. G.; Shaw, S. R.; VanGlessen, G. J. J . Pharm. Sci. 1974, 63. 567. (11) Adams, S. S.; McCullough, K. F.; Nicholson, J. S. Arznelm. Forscb. 1975, 25, 1766. (12) Funaki, H.; Matsuda, T.; Shindo, T. J. Kyoto Prefect. U n h . Med. 1976, 85, 661. (13) Snider, 6.G.; Beaubien, L. J.; Sears, D. J.; Rahn, P. D. J. pharm. Sci. 1901, 70. 1347.
1509
(14) Albert, K. S.; Glllesple, W. R.; Raatm, A.; Garry, M. J. fharm. Scl. 1904, 73, 1623. (15) Beyer, W. F.; Gleason, D. D. J. fharm. Sci. 1975, 6 4 , 1557. (16) Karaer.. 6.L.: Giese. R. W. Anal. Chem. - . . 1978. ... . 50. .., 1048A. . .. -. .. (17j Szpunar, G. J.; Albert, K. S.; Bole, 0 . G.; Dreyfus, J. N.; Lockwood, G. F.; Wagner, J. G., submitted for publication in Clln. fharm. Therap.
--
~
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.
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RECEIVED for review November 10,1986. Accepted February 24, 1987. The work reported in this paper was presented in part at the 35th Meeting, Academy Of Pharmaceutical Sciences, Miami Beach, FL, 1983.
Determination of Cysteine in Pharmaceuticals via Liquid Chromatography with Postcolumn Derivatization Dennis R. Jenke* and David S. Brown Travenol Laboratories, Inc., 6301 Lincoln Avenue, Morton Grove, Illinois 60053
A hlgh-performance llquld chromatographic assay has been developed for cysteine in pharrnaceutlcal solutions and has been applled to the characterlzatkn of DISCASE for InJectlon samples. SeparaUon Is accomplished by uslng the Ion-palrlng agent sodlum octyl sulfate on IO-cm, 5 - ~ mparticle slre C,,-type columns. The column effluent Is contlnuously mixed with a pH buffered solution of 5,5'-dlthlobls[2-nitrobenzolc acM] (DTNB) M o r e entering the detector. The reaction b e tween cystelne (a thlol) and DTNB Is rapld and liberates a yellow chromophore monitored wlth vlslble llght at 412 nm. The method dlscrlminates against cysteine degradation products and chymopapaln raw material. Thls system was tested by measurlng cystelne recoverles In DISCASE for Injectlon samples made from flve dlfferent lots of chymopapain using three chromatographlc columns. Performance Is characterlred by high degrees of accuracy (100 f 2 % ) and precision (less than 0.8 % relatlve standard devlatlon). Reagents and standards are stable for at least 3 days but samples can, In some cases, begln to lose cysteine wlthln 24 h of sample reconstltutlon.
for Injection preparation as preservatives. Cysteine acts in two ways. Cysteine scavenges oxygen in the lyophilized storage form, and, more importantly, acts as a reducing agent to preserve the integrity of thiols in the chymopapain molecule. EDTA and sodium bisulfite are also added as stabilizers and activators. Because chymopapain is purified from crude extracts, one would expect batch variability in low-level contaminants. To circumvent coelution interference problems, a thiol-selective postcolumn derivatization/detection system was developed. The column effluent is combined and mixed with a reagent solution containing buffered sodium citrate (pH control) and the derivatizing reagent 5,5'-dithiobis[2-nitrobenzoicacid] (DTNB). Cysteine and DTNB react to form an intensely colored chromophore:
-0
The important role that thiol compounds in general and cysteine in particular play in pharmaceutical formulations places a premium on an analytical methodology that is highly sensitive and selective for this species. While many methods have been reported for the determination of cysteine, including ion exchange chromatography (1-4); spectophotometry (5-1 I); gas (12-14),thin-layer (15,16),and high-performance liquid chromatographies (17-26);polarography (11);and fluorometry (27-29);these methodologies generally suffer from sensitivity, specificity, or productivity limitations. Specificity is particularly critical in most pharmaceutical applications, where the formulations may include a large number (and quantity) of structurally similar species, their degradation products, and matrix components (pH buffers, preservatives). This situation is encountered in the characterization of DISCASE for Injection samples for cysteine content. DISCASE for Injection, a stabilized, sterile preparation of the proteolytic enzyme chymopapain, is used therapeutically to relieve herniated disc conditions. Cysteine, ethylenediaminetetraacetic acid (EDTA), and sodium bisulfite are present in the DISCASE
NO2
Cysteine
DTNB
Mixed Disulfide
Chromophore (A 41 2 nm, E = 14000)
While DTNB is thiol-selective (among possible protein functional groups), it also reacts with sodium bisulfite to yield the same chromophore. Hence, the chromatographic system must be capable of separating sodium bisulfite and cysteine. This report summarizes the capabilites, performance, and optimization of the developed method.
EXPERIMENTAL SECTION Materials. Solvents and reagents used in this study were all commercial analytical grade. DISCASE (chymopapain) for Injection (Travenol Laboratories, Inc., Deerfield, IL) samples were prepared for analysis by reconstituting the contents of individual vials in 5 mL 0.3% phosphoric acid followed by dilution (3-50 mL) in the same diluent. Artificial formulations simulating this
0003-2700/87/0359-1509$01.50/00 1987 American Chemical Society
