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 Meeting, Academy Of part at the 35th 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
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product were prepared in the following manner. Three stock solutions combining either (A) 2.32 g/L cysteine hydrochloride (B) 30 g/L chymopapain, or (C) 2.5 g/L metabisulfite and 10 g/L EDTA were prepared by diluting the appropriate weights of each salt in 0.3% phosphoric acid. The actual artificial samples were prepared by adding 1 mL of the chymopapain stock, 0.5 mL of the other mixed inorganic stock, and 3.3 mL of the cysteine stock in a 5 mL volumetric flask and diluting to volume with 0.3% phosphoric acid. The working samples were diluted 3-50 mL prior to analysis. Chromatographic System. The chromatographic system used included a Perkin-Elmer Series 10 pump, a Waters Model 440 UV-visible spectrophotometric detector, Perkin-Elmer PCR-1 postcolumn reaction system and a Perkin-Elmer LC 600 autosampler. A 10-cm X 0.46-cm column packed with 5-pm CI8 resin Hypersil ODS (Alltech Associates, Deerfield, IL) was used to produce the separations. Detector wavelength was 412 nm, sample size was 10 pL and detector sensitivity was set at 0.4 absorbance units full scale (AUFS). The mobile phase was an aqueous mixture containing 10% acetonitrile,0.1% sodium octyl sulfate, and 0.3% phosphoric acid and was filtered and degassed by vacuum filtration through 0.2-pm polycarbonate media prior to use. The postcolumn reagent was a 0.5 M sodium citrate/sodium phosphate buffer at pH 7, which contained, by weight, 0.03% 5,5’-dithiobis(2-nitrobenzoic acid) and 0.025% EDTA. The postcolumn reagent was filtered through 0.45-1m membrane media prior to use. Flow rates for the mobile phase and postcolumn reagent were 1.0 and 0.5 mL/min, respectively. Assay Integrity. In order to confirm the purity of the cysteine response, the apparatus as described above was expanded t o include an additional ultraviolet detector (Perkin-Elmer LC85B) positioned between the column and the postcolumn reactor. This detector was used at a wavelength of 205 nm, and was thus sensitive to cysteine, its primary degradates, and the chymopapain. The rest of the apparatus as described above remained intact throughout the course of this experimentation. In addition to analyzing the sample solutions described above, samples continuing 1 g/L of the common cysteine degradates (cystine, sulfocysteine, and cysteic acid) were prepared by dissolving the appropriate weight of each reagent salt in 0.3% phosphoric acid and were analyzed with the dual detector system. Calibration. Calibration standards were prepared by dilution of a 2320 mg/L stock solution of cysteine hydrochloride by using 0.3% phosphoric acid as the diluent. Working standards containing approximated 20,40, and 60 ppm cysteine were prepared by diluting 2, 3.5, and 5 mL of the stock to a final volume of 200 mL. Standard response is determined as both peak area and peak height by using an in-house computer integrator. Calibration is accomplished by performing a least-squares linear regression curve fit on the standard detector response.
RESULTS AND DISCUSSION Aspects of the developed cysteine assay that were considered in this research included cysteine response integrity, method optimization, assay performance, and system stability. Considering the first of these, it is necessary that the assay be stability indicating, that is, the response assigned to cysteine must be due to this moiety alone. This issue of peak purity can be addressed by using the dual series detection system, where the first UV detector (A = 205 nm) functions as a universal detector capable of responding to all organic matrix components (formulation compounds and degradates) and the second detection system (reactor and second visible detector a t X = 412 nm) serves as the thiol selective proposed method. The system’s response to solutions containing chymopapain, cysteine-spiked chymopapain, and several cysteine-related oxidation products was determined and are summarized in Table I. Cystine and cysteic acid, the main cysteine oxidation products, have chromatographic behavior dramatically different from that of cysteine. In addition, they have no observable response with the DTNB under these assay conditions. Sulfocysteine, another potential degradate, has a slight DTNB response (approximately 0.004 times the response to a molar equivalent concentration of cysteine), but has re-
Table I. Chromatographic Behavior of Potential Cysteine/Oxygen Reaction Products” 205 nm retention time, cysteine cystine sulfocysteine cysteic acid
412 nm
min
k’
retention time, min
3.5 7.5 1.0 1.0
3.2
4.0 nr
8.4 0.2 0.2
1.5 nr
Onr = no response.
tention very different from cysteine. These results confirm that this postcolumn reaction system is indeed selective for thiols (specifically cysteine) in the presence of other thiol degradation products. T o this point, however, this discussion has not addressed the possibility of the chymopapain material containing impurities (peptide fragments), which contain a thiol moiety. To consider this possibility, six different lots of chymopapain materials were used to generate chymopapain blank and cysteine containing chymopapain formulations that were analyzed with the dual detector system. Figure 1 shows the nature of the typical chromatographic response. In lA, one notes that there are several peaks apparent in the chymopapain blank monitored with the UV detector. However, no chymopapain-related response was observed in any of the six material batches when the thiol-selective detector was monitored. Cysteine-spiked chymopapain samples (Figure 1B) show good response in both detection systems. The UV system, however, clearly shows the presence of low-concentration contaminants that elute near or coelute with cysteine. These contaminants produce no response, and thus pose no problem, in the thiol selective system. Sodium bisulfite, also present in the DISCASE formulation, will also react with DTNB to produce the same colored product, but, as seen in Figure 2 , it is well separated from cysteine chromatographically. Since the thiol selective detection involves postcolumn derivatization, one must be concerned with the kinetics of this reaction, that is, is the postcolumn reaction complete by the time the analyte containing portion of the mixed mobile phaselreagent solution reaches the detector? With the reactor system used herein, residence time between solution mixing and entrance to the detector is approximately 40 s. This residence time can only be increased by increasing the amount of tubing between the mixing and detection components. This is an inappropriate strategy, because it produces a worsening i n the chromatographic behavior due to increased band broadening. Thus, in order to develop a reproducible assay, one must insure that the reaction has gone to completion prior to detection in this 40-s interval. While increasing the reaction temperature can produce this goal, utilization of this approach requires specialized equipment and close control. Another approach is to modify the chemistry of the mixed solution; in this study both DTNB concentration and pH of the reagent solution were considered. As shown in Figure 3, both these variables have a significant effect on the completeness of the postcolumn reaction. The optimized assay conditions include a reaction solution that contains 300 mg/L DTNB (as a compromise between reaction efficiency and reagent waste) and has a pH of 7 (so as to give high reaction efficiency and provide relative freedom from and insensitivity to minor pH fluctuations). An additional part of the optimization process was to consider the chromatographic behavior of cysteine. Since the separation is accomplished via ion pairing and cysteine has both anionic and cationic functionalities, pH of the mobile phase is expected to have a profound effect on its behavior.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987 t
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A
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tol
t , , , , ,
1 2 3 4 5 Analysis Time, min.
Figure 2. Chromatographic behavior of cysteine and sodium bisulfite, thiol selective detection at 412 nm.
20
6.2 6.5 6.8 7.0 Post-Column Reagent pH
,
;
;
;
I
; 5 6 Analysis Time, min
,
I
7
0
B
T
tol
Cysteine Response in PCR/Visible System
DTNB Content in Post-Column Reagent, mg/L
\
Figure 3. Optimization of postcolumn reaction conditions. The postcolumn reagent was investigated for DTNB concentration and pH effects. I n the pH study, the reagent was 0.5 M citrate and pH 6.5. A 1 mL reactlon/delay coil was used In the derivatization; thus at a combined (mobile phase and reagent) flow rate of 1.5 mL/min the reaction time was approximately 4 0 s.
q\
Cysteine Response in UV System
\
2
1
15
8
z
$
5
G 20
25
3.0
35
Mobik Phase pH
Figure 4. Cysteine chromatographic performance: effect of mobilephase pH.
t I
I
1
2
I
I
I
I
I
I
3
4
5
6
7
8
Analysis time, min
Figue 1. (A) Dual series analysis of dhymopapain blank sample. The lower trace represents UV detection at 205 nm while the upper trace utilizes thiol-selective postcolumn derivatization with detection at 4 12 nm. (B) Dual series analysis of chymopapain spiked with cysteine. The lower trace represents UV detection at 205 nm whlle the upper trace utilizes thloCseiective postcolumn derivatizatlon with detection at 4 12 nm.
Clearly, as shown in Figure 4, as pH is decreased (and approaches the pKa of cysteine carboxylate of 1.7) (30),the cysteine retention increases. A viable explanation for this behavior is that the protonation of the carboxylate site enhances the ion pair formation. One notes, however, that utilization of a pH 2 mobile phase could conceivably accelerate column degradation; thus, in this application, the use of a small CIS saturator column placed prior to the injector is recommended. In pharmaceutical applications, assay precision and accuracy are of primary importance, especially in terms of stability testing and product monitoring. The assay as described herein
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987 1--100%
*
1%-
I
I
Cysteine Recovery %
Flguro 5. Distribution of recoveries of cysteine in DISCASE samples. Cysteine concentrations represented 80%, loo%, and 120% of the product code while chymopapain concentrations represented 100% and 125% of the same code.
is characterized by a high degree of accuracy and precision. In terms of accuracy, recoveries of cysteine from 48 freshly prepared samples of DISCASE for Injection, using six different chymopapain lots and three different chromatographic columns, with cysteine concentrations at 80%, 100%, and 120% of the product code, are shown in Figure 5. Of the recoveries, 85% fell in the range 100 f 1%,97% in the range 100 f 1.5% and all fell in the range of 100 f 2%. The average precision, determined on 27 sets of samples and represented as the percent relative standard deviation (RSD) of triplicate analyses of samples, was 0.4%. None of the solutions assayed exhibited a precision worse than 0.8% RSD. Sensitivity is also of concern in this application. Since the original DISCASE for Injection product contains approximately 5 mg/mL chymopapain and 0.7 mg/mL cysteine after reconstitution, a typical sample after preparation will have a cysteine concentration of approximately 40 ppm. The limit of quantitation (lox peak to peak noise) obtained by using the postcolumn reaction system is in the sub parts per million range; thus these concentrations are easily accessible. Calibration of both peak heights and peak areas in the standard range used herein (20-60 ppm) is characteriaed by a high degree of linearity with correlation coefficients of the regression fit typically being 0.999 or better. As defined in this manuscript, system stability has three components; reagent, standard, and sample stability. Postcolumn reagent stability for periods in excess of 3 days has been confirmed by an unchanging percent reaction completion measured with the visible detector. Comparison of freshly prepared and stored (room temperature) standards, indicate that even after 4 days all standards fell within the 100 f 0.5% recovery range. The pH 2.1 phosphoric acid media is apparently effective in retarding any oxidative losses. It was observed, however, that the artificial DISCASE samples were much less stable with roughly a 2% loss of response being observed per day. The stability of these artificial samples was directly related to the chymopapain lot used for their preparations. It is thus suggested that in actual application the DISCASE sample be assayed within 8-12 h of reconstitution. Because the DISCASE matrix is moderately complex, it was anticipated that the quantitation of cysteine in other pharmaceutical matrices could be accomplished by the documented method. Recovery studies, performed in matrices that included 0.9% sodium chloride, 5% dextrose, lactated Ringer's Irrigating Solution, and a nutritional mixture containing 8.5% total amino acids,
confirmed that the method exhibits no bias in these types of samples. The proposed methodology is capable of quantitating analytes (other than cysteine) that fulfill two requirements: (1) they must show some affinity for the column packing, and (2) they must react with the postcolumn reagent. Species of pharmaceutical interest that fulfill these requirements and have been characterized with the documented system include n-acetylcysteine, homocysteine, mercaptoethanol and mercaptoacetic acid. It is noted that the technique is suitable for the quantitation of sulfite (see Figure 3), however, the response to this analyte is only poorly reproducible. The observed variation in sulfite response is attributed to trace-metal catalyzed oxidation of this species, which occurs in the chromatographic system. It has been observed that addition of EDTA to the mobile phase improves the precision of the sulfite response. Registry No. Cysteine, 52-90-4; DISCASE, 9001-09-6.
LITERATURE CITED (1) Nakarnura, H.; Tamura, 2. Anal. Chem. 1981,53,2190-2193. (2) Allison, L. A.; Keddington, J.; Shoup, R. E. J . Liq. Chromatogr. 1983, 6 ,1785-1798. (3) De Master, E. G.; Shirota, F. N.; Redfern, B.; Goon, D. J. W.; Nagasawa, H. T. J . Chromatogr. 1984,308,63-91. (4) MacDonald, J. L.; Krueger. M. W. J . Assoc. Off. Anal. Chem. 1985, 68,826-829. ( 5 ) Malloy, M. H.; Rassin, D. K.; Gaull, G. E. Anal. Biochem. 1981, 173, 407-415. (6) Boyne, A. F.; Ellrnan, G. L. Anal. Biochem. 1972, 46,639-644. (7) Novak, T. J.; Pleva, S. H.; Epstein, J. Anal. Chem. 1980, 52, 1851-1854. (8) HunsBker, 0.B. Jr.; Schenk. G. H. Talanta 1983, 30,475-480. (9) Ohmori, S.; Ikeda, M.; Hattoni, H.; Iwase, C. J . Clin. Chem. Clin. Biochem. 1983,27, 851-857. (10) SaStry, S.;Satyanarayuna, P.; Tummura, M. K. Analyst (London) 1985, 170, 189-191. (11) United States Pharmacopeia, 21st ed.; The United States Pharmcopeial Convention, Inc.: Rockville, MD, 1984;pp 268. (12) Meckenzle, S. L.; Flnlayson, A. J. J . Chromatogr. 1980, 187, 239-243. (13) Moodie, I. M.; Walsh, D. L.; Burger, J. A. J . Chromatogr. 1983,267, 146-152. (14) Flelta, D. H.; Arsanious, H. N. Fresenius' Z . Anal. Chem. 1985,320, 753-756. (15) Prosod, B. N.; Kawak, G. B.; Padalikar. S. V.; Joglekar, V. D. Sci. Cult. 1980, 46,275-280. (16) Malyslk, G.; Soczewinski, E.; Wolski, T. Farm. Pol. 1984, 40, 593-596. (17) Nlshiyama, J.; Kunlnorl, T. Anal. Chem. 1984, 138,95-96. (le) Mopper, K.; Deirnas, D. Anal. Chem. 1984,56, 2557-2560. (19) Perrett, D.; Rudge, S. R. J . Chromatogr. 1984,294,380-384. (20) Werkhoven-Goewie, C. E.; Neissen, W. M. A.; Brinkman, U. A. Th.; Frei, R. W. J . Chromatogr. 1981,203, 165-172. (21) Cooper, J. D. H.; Turnell, D. C. J . Chromatogr. 1982,227, 158-161. (22)Toyo'oka, T.; Imai, K. J . Chromatogr. 1983,282, 495-500. (23) Kakehi, K.; Konishi, T.; Sagimoto, I.; Honda, S . J . Chromatogr. 1984, 378,367-372. (24) Sybilska, D.; Przanyski, M.; Mysior, B.; Sarnochocka, K. J . Chromatogr. 1984, 283, 453-456. (25) Graser, T. A.; Godel, H. G. Anal. Biochem. 1985, 757,142-152. (26) Komuro, C.; Ono, K.; Shibamoto, Y.; Nishidai, T.; Takahashi, M.; Abe, M. J . Chrometogr. 1985,338,209-212. (27) Toyo'oka, T.; Watanabe, Y. Anal. Biochem. 1983, 128, 471-473. (28) Toyo'oka, T.; Imai. K. Anaksr (London) 1984, 709,1003-1007. (29) Nakashima, H.; Akimoto, H.; Nishida, K.; Nakatsuji, S.; Akiyama, S. Talanta 1985, 32, 167-169. (30) Segel. I. H. Biochemlcal Calculations, 2nd ed.: Wiley: New York, 1976.
RECEIVED for review June 27,1986. Resubmitted January 26, 1987. Accepted February 19,1987. DISCASE for Injection is currently a product of Boots Pharmaceuticals, Inc. (Shreveport, LA).
