Anal. Chem. 1986, 58, 1896-1898
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AIDS FOR ANALYTICAL CHEMISTS Liquid Chromatographic Determination of Penicillins by Postcolumn Degradation wlth Sodium Hypochlorite J u n Haginaka* and J u n k o Wakai Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 4-16 Edagawa-cho, Nishinomiya, Hyogo 663, Japan In order to enhance selectivity and sensitivity, several high-performance liquid chromatographic (HPLC) methods combined with precolumn (1-6)and postcolumn (4,7-15) reactions have been developed for the assay of penicillins. In a previous paper (12),we reported an HPLC method for the determination of penicillins using postcolumn degradation with sodium hydroxide, mercury(I1) chloride, and ethylenediaminetetraacetic acid disodium salt in solution. The method was successively applied to the assay of ampicillin and its metabolites in urine (15). However, the procedure is at a disadvantage in that it requires the use of rnercury(II) chloride (which is toxic to humans and is an environmental pollutant) M. a t such a high concentration as 2 x I t is found that the treatment of a penicillin with sodium hypochlorite in alkaline solution yields a product(s) having an absorption maximum at 270-280 nm. This paper describes the reaction of penicillins with sodium hypochlorite in alkaline solutions in the absence and the presence of methanol and an HPLC method for the determination of penicillins using this degradation reaction as postcolumn derivatization. EXPERIMENTAL SECTION Drugs and Reagents. Potassium benzylpenicillin (PCG) was purchased from Sigma Chemical (St. Louis, MO). Disodium ticarcillin (TIPC), disodium carbenicillin (CBPC), ciclacillin (ACPC), and ampicillin (ABPC) trihydrate were kindly donated from Beecham Yakuhin (Tokyo, Japan), Takeda Chemical Industries (Osaka, Japan), and Meiji Seika Kaisha (Tokyo). The structures and abbreviations of penicillins used in this study are listed in Figure 1. (5R,GR)-Penicilloic acid of ABPC and the (5S,GR)-epimerwere prepared by the methods similar to those reported previously (16). ABPC piperazine2,5-&one was prepared according to the procedures described by Bundgaard and Larsen (17). Other chemicals of reagent grade were purchased from Nakarai Chemicals (Kyoto, Japan) and used without further purification. Deionized, glass-distilled water and distilled methanol were used to prepare the HF'LC eluents and the samples for the measurements of ultraviolet (UV) spectra. A 100 pg/mL stock solution of each penicillin was prepared with water. The stock solution was diluted with water, if necessary. Apparatus. The following HPLC system was used: two pumps, LC-5A (Shimadzu, Kyoto, Japan) and NP-DX-2 (Nihon Seimitsu Kagaku, Tokyo), for delivering the eluent and postcolumn reagent, respectively; a Model 7125 loop injector (Rheodyne, Cotati, CA) equipped with a 100-pL loop for the loading of the samples; and an SPD-6AV (Shimadzu, Kyoto) spectrophotometer equipped with an 8-pL cell for detection. A Model 228 spectrophotometer (Hitachi, Tokyo) was used for the measurements of UV spectra. Chromatography. Two chemically C18-bonded silica columns, 15 cm X 4.6 mm i.d., were used: column I, Develosil ODs-5 (5-pm particle size) (Nomura Chemicals, Seto, Japan), and column 11, Nucleosil 5CI8(5-pm particle size) (Macherey-Nagel,Diiren, West Germany). The mobile phases used were as follows: eluent A, 2 mM NaH2P04-2 mM NazHP04-methanol (l:l:l,v/v); eluent B, 15 mM tetrabutylammonium bromide3 mM NaH2P04-3 mM 0003-2700/86/0358-1896$01.50/0
Na2HP04-methanol (2:2:2:5, v/v); and eluent C, 15 mM sodium heptylsulfonate-3 mM NaHZPO4-27 mM H,P04-methanol (3:3:35, v/v). Column I and eluent A were used for the separation of PCG, ABPC, and ACPC, column I and eluent B, CBPC and TIPC, and column I1 and eluent C, ABPC and its metabolites. The flow rates were maintained at 0.8 mL/min. The postcolumn reagent used was 0.5 M sodium hydroxide plus 0.02% sodium hypochlorite solution, whose flow rate was maintained at 0.2 mL/min. Teflon tubing (1-2 m X 0.5 mm i.d.) was used as the postcolumn reactor. The reagent stream and eluent were mixed in a Diflon tee-piece (each angle, 120'). Detection was performed at 280-284 nm for penicillins and 270 nm for ABPC and its metabolites. All separations and postcolumn reactions were carried out at ambient temperature. UV SpectralMeasurements. A 1-mL aliquot of PCG solution (100 pg/mL) was mixed with a mixture of 3 mL of HzO and 1 mL of 0.5 M sodium hydroxide plus 0.02% sodium hypochlorite solution. A 1-mL aliquot of PCG solution (100 pg/mL) was mixed with a mixture of 2 mL of H20, 1 mL of methanol, and 1mL of 0.5 M sodium hydroxide plus 0.02% sodium hypochlorite solution. The UV spectra of the above reaction solutions between 310 and 250 nm were recorded at reaction times of 0 (immediately after initiation of the reaction), 1, 2, 3, and 4 min (Figure 2). RESULTS AND DISCUSSION Degradation of PCG with Sodium Hypochlorite. Figure 2, parts A and B, shows the time-dependent UV spectral changes of PCG reacted with sodium hypochlorite in alkaline solutions in the absence and the presence of methanol at ambient temperature. In the absence of methanol, the maximum absorbance a t about 270 nm was attained at 1min after initiation of the reaction (A). In the presence of methanol, the maximum absorbance at about 280 nm was attained just after initiation of the reaction and was twice that in the absence of methanol (B). The results in Figure 2 indicate that the reaction in the presence of methanol provides a faster and higher UV response than that in the absence. Similar results were also obtained for the reaction of other penicillins. It is well-known (18, 19) that imino acids (e.g., proline, hydroxyproline) undergo oxidative decarboxylation to primary amines, when treated with sodium hypochlorite. In a previous paper (12),we reported that penicillins were degraded in alkaline solutions to the corresponding penicilloates and methyl penicilloates in the absence and the presence of methanol, respectively. In the above-mentioned degradation reactions, a penicillin may be degraded to the penicilloate (which is an imino acid) and further undergo oxidative decarboxylation to a compound(s) having A, at about 27C-280 nm. The structural study of the reaction product is now being investigated. Postcolumn Reaction Conditions. The postcolumn reaction conditions (pH, reagent concentration, time) were examined to study their effects on the UV absorbance produced by the reaction of penicillins with sodium hypochlorite. Since the system containing methanol in the reactioin solution was 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
1897
Table I. Precision of the Assay of Penicillins" assay
PCG
ABPC
ACPC
CBPC
TIPC
l b
2b 36
4.95 f 3.03 5.13 f 1.81 4.92 f 4.47
4.93 h 2.06 5.02 f 1.01 4.98 f 3.39
4.95 f 1.56 5.03 f 2.87 5.07 f 3.04
4.92 f 2.53 5.25 f 1.18 5.06 f 3.13
5.00 f 1.91 5.03 f 2.94 5.08 f 2.38
betweemrune
5.00 f 2.27
4.98 f 0.91
5.02 f 1.22
5.08 f 3.26
5.04 f 0.80
'The concentration of each compound was 5 pg/mL. bMean f relative standard deviation of five replicates (%). 'Mean f relative standard deviation of three replicates (%). H
A
benzylpenicillin (PCG)
i
@CH2-
ampicillin ( A B PC)
-
ciclacillln ( ACPC)
"2
0
O ( N H 2
10
20
~kne (mln)
carbenicillin (C BPC)
0
10
20
Time (min)
Flgure 3. Separation of penicillins: (A) ABPC (l), ACPC (2), and PCG
(3); (B) (1OR)- and (lOS)-epimers of TIPC (1) and (10R)- and
COOH
(10S)epimers of CBPC (2). Each concentration was 25 pg/mL. Injection volume was 20 gL. Detection was performed at 280 nm and at a sensitivity of 0.016 AUFS. Reactlon coil length was 1 m. For other conditions, see the Experimental Section.
U K H
ticarcillin ( T I P C )
Flgure 1. Structure and abbreviation of penicillins. A
B 0.9-
A
0.4.-
-0.11
:
-0.1
250
300
Wavelongth (nm)
250
300
Wavelength (nm)
Flgure 2. UV absorption spectra of PCG reacted with sodium hypochlorite in alkaline solution in the absence (A) and presence (6)of methanol. Reaction tlmes are as follows: (1) 0 min (immedlately after initiation of reaction), (2) 1 min, (3)2 min, (4)3 min, (5)4 min.
preferred for the present purpose as described above, the reaction conditions were examined in the presence of methanol using the flow injection analysis. The carrier solutiion (eluent A for PCG, ABPC, and ACPC and eluent B for CBPC and TIPC) and postcolumn reagent were delivered at flow rates of 0.8 and 0.2 mL/min. The concentration of sodium hydroxide was changed from 0.1 to 0.75 M a t a sodium hypo-
chlorite concentration of 0.02%. The maximum UV response was obtained a t a sodium hydroxide concentration of 0.5 M or more for PCG, ABPC, and ACPC and 0.25 M or more for CBPC and TIPC. At a sodium hydroxide concentration of 0.5 M, the concentration of sodium hypochlorite was varied from 0.005 to 0.04%. The maximum UV response was obtained at a sodium hypochlorite concentration of 0.02% or more for all penicillins tested. The effect of reaction time was examined by changing the reaction coil length between 0.5 and 4 m (0.5 mm i.d.). The maximum UV response was obtained at a coil length of 1m (corresponding to 12 s of the residence time) for ABPC, CBPC, and TIPC and 2 m for PCG and ACPC. Detection wavelength was examined by changing the wavelength of HPLC detector. The optimal detection wavelengths were 280 nm for PCG, ABPC, and TIPC, 282 nm for CBPC, and 284 nm for ACPC. Thus, the postcolumn reaction conditions were established as described in the Experimental Section. Separation of Penicillins. Under the optimal conditions, the HPLC separation and detection of penicillins were carried out as shown in Figure 3. The separation of PCG, ABPC, and ACPC was attained on a reversed-phase C18 column by using eluent A (A), while the separation of CBPC and TIPC was attained by using eluent B (which contains a cationic ion pairing agent) (B). Since the standard TIPC and CBPC were a mixture of (108)- and (lOS)-epimers,they showed two peaks on a chromatogram. The analytical standard curves of eight points (0.2-100 gg/mL) of penicillins constructed by plotting peak height against concentration were linear with a correlation coefficient of 0.999 or above and passed through the origin. The method permits detection of 100 ng/mL fur PCG, ABPC, and ACPC and 200 ng/mL for CBPC and TIPC with a 50-gL injection at the signal-to-noise ratio of 3. Table I shows the within- and between-run precisions. At a concentration of 5 pg/mL, within- and between-run pre-
1808
Anal. Chem. 1988, 58, 1898-1900
are metabolites of ABPC in human urine (20). Recently, it has been reported that the piperazine-2,5-dioine (3) is excreted in rat (21) and human (22) urine after dose of ABPC is given. Figure 4 shows the structures of these metabolites, and Figure 5 shows a chromatogram of ABPC, 1,2, and 3 detected by the proposed method. The limits of detection of 1,2, and 3 are as low as 0.5 pg/mL with a 50-pL injection. These results reveal that the method is able to detect not only a penicillin but also a metabolite having a thiazolidine ring.
1
ACKNOWLEDGMENT We are grateful to T. Uno and H. Yasuda, Mukogawa Women's University, for their interest and support. W S t W NO. 1,32746-94-4;2,71774-55-5; 3,94595-14-9;PCG, 61-33-6; ABPC, 69-53-4; ACPC, 3485-14-1; CBPC, 4697-36-3; TIPC, 34787-01-4; penicillin, 1406-05-9; sodium hypochlorite, 7681-52-9.
2
LITERATURE CITED
4. Stnrctue of (5RW)genidlkic acid (l), the (5S,6R)-epimer (2) and piperazlne-2,5dlone (3). 1
4
1
h3 b
(1) Lam, S.; aushka, E. J . L i s . Chromatcar. 1978, 1 , 33-41. (2) Rogers, M. E.; Adlard, M. W.; Saunderc G.; Hoit, G. J . Llq. Chromat00r. 1983. 6 . 2019-2031. (3) MGazakl, K.; Ohtanl, K.; Sunada, K.; Arita, T. J . Chromatogr. 1983, 276, 478-482. (4) Rogers, M. E.; Adlard, M. W.; Saunders, G.; Holt, G. J . Chromatogr. 1984. 297, 385-391. (5) Haginaka, J.; Wakai, J. Amlyst(London) 1985, 110, 1185-1188. (6) Haglnaka. J.; Wakal, J. Analyst (London) 1985, 110, 1277-1281. (7) Lw, T. L.; D'arconte, L; Brooks, M. A. J . Pharm. Sci. 1979, 6 8 , 454-458. (8) Westerlund, 0.; Cerlqvlst, J.; Theodorsen, A. Acta Pharm. Sue. 1979, 16, 187-214. (9) Cadqvist, J.; Westerlund. D. J . Chromatogr. 1979, 164, 373-381. (IO) Rogers, M. E.; Adlard, M. W.; Saunders, G.; Holt, G. J . Chromatogr. 1088, 257, 91-100. (11) Buchberger, W.; Wlnsauer, K.; Nachtmann. F. Fresenius' 2. Anal. Chem. 1988, 315, 525-527. (12) Haaalnaka, J.: Wakai, J. Anal. Chem. 1985. 5 7 . 1568-1571. (13) Kok, W. Th.; Halvax, J. J.; Voogt, W. H.; Brinkman, U. A. Th.; Frei, R. W. Anal. Chem. 1085.. 5 7 ,~ . 2580-2583. (14) Carlqvist, J.; Westerlund, D. J . Chromatogr. 1985, 344, 285-296. (15) Haglnaka, J.; Wakal, J.. submitted for publication in J . Pharm. Phar~
macol .
0
10
20
30
Tlmo (mln) Flgure 5. Separation of ABPC (4), 1 (3), 2 (2), and 3 (1). Each concentratkn was 25 pg/rnL. Injection volume was 20 &. Detection was petfumed at 270 nm and at a SenSitMty of 0.016 AUFS. Fleadon coil length was 1 m. For other conditions, see the Experimental Section.
cisions (relative standard deviation) were 1.0-4.5% and 0.9-3.3%. Detection of ABPC and Its Metabolites. It was known that (5R,GR)-penicilloic acid (1)and the (5S,6R)-epimer (2)
(16) Uno, T.; Masada. M.; Yamaoka, K.; Nakagawa, T. Chem. Pharm. BuU. 1981, 2 9 , 1957-1968. (17) Bundgaard, H.; Larsen, C. I n t . J . Pharm. 1979, 3 , 1-11. (18) Bohbn, P.; Meibt, M. Anal. Blochem. 1970, 9 4 , 313-321. (19) Ishida, Y.; Fujita, T.; Asai, K. J . Chromatogr. 1981, 204, 143-148. (20) Bird, A. E.;Cutmore, E. A.; Jennlngs, K . R.; Marshall, A. C. J . Pharm. PhErrnz3COl. 1983, 35, 138-143. (21) Everett, J. R.; Jennlngs, K. R.; Woodnutt, J.; Buckingham, M. J. J . Chem. Soc., Chem. Commun. 1984, 894-895. (22) Haginaka. J.; Wakai, J. J . Pharm. Pharmacol. 1988, 38, 225-226.
RECEIVED for review January 2, 1986.
Resubmitted March
14, 1986. Accepted April 1, 1986.
Spectruphatometric System tor Klnetic Absorbance Measurements in Two-Phase Enzyme Immunoassays Vadiraja V. Murthy,* Lawrence Freundlich, and Arthur Karmen Department of Laboratory Medicine, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, The Bronx, New York 10461 Many microimmuncmsay methods currently in favor in the clinical laboratory involve measurement of the activity of an *Address all correspondence to this author at Hospital of the Albert Einstein College of Medicine, Room 3-33, 1600 Tenbroeck Ave., The Bronx, NY 10461. 0003-2700/86/035&1898$01.50/0
enzyme that is immobilized to the surface of a plastic or glass bead by an antibody-antigen reaction (1,2). These methods are preferable to radioimmunoassays for their relative ease of operation, freedom from exposure to radioactivity, and cost-effectiveness. In a typical enzyme immunoassay, a "sandwich" consisting 0 1986 American Chemical Society
