2344
Anal. Chem. 1985, 57,2344-2346
Table 11. Response of TDI-Treated Silicone Coatings to Some Organic Interferants interferant
FS-1265
acetone methyl ethyl ketone toluene xylene (mixed)
790 590
responsea DC high vac
LS-420
360 710 730
2210 840 90
690
600
400 550
Concentration (ppm v/v) required to change response to 100 m b TDI bv 1 Hz.
water that adhered to the crystal face (coated or not) was a function of the relative humidity and not of the coating material. A stable base line could be realized only after this hydroscopic equilibrium was reached, usually in less than 10 min. This apparent stabilization occurred only once at the beginning of the day for a crystal that was not already in use. Since the measurement of TDI response is relative to a stable base line and the humidity of any given test area is reasonbly constant, water vapor did not appear to alter the TDI response over the range of 10-85% relative humidity. At high levels of moisture (85% and higher) the crystals tend to drift severely due to condensation. The silicone materials make rugged coating materials as evidenced by the long functional lifetime of the crystal detectors. Silicone-coated crystals were found to be stable indefinitely when not in use, while 5-6 weeks was a common lifetime for crystals in use (average 10 assays/day). Base line drift, caused primarily by coating materials flaking off the crystal due to piezoelectric vibrations, and normal crystal aging, averaged only about 6 Hz (0.1%)per day. Sensitivity remained nearly constant, for several hundred assays, although the time of response increased with the number of analyses
performed and the concentration of sample vapor adsorbing on the crystal. It can be concluded that various modified silicone compounds coated onto piezoelectric crystals can be analytically important as sensitive and selective detectors for toluene diisocyanate in air. Registry No. TDI, 26471-62-5; quartz, 14808-60-7.
LITERATURE CITED (1) Chem. Eng. New, 1983, 67,25. (2) Graham, Jeffery D. J . Chromatogr. Sci. 1980, 78,384. (3) Bagon, D. A,; Purnell, C. J. J . Chromatogr. 1980, 790 (l),175. (4) Sangoe, C. J . Liq. Chromatogr. 1979, 2 ( 6 ) ,763. (5) Hardy, H. L.; Walker, R. F. Analyst (London) 1979, 104 (1242),890. (6) Marcali, Kalman Anal. Chem. 1957. 29 (4).552. Posniak, Malgorzata Pr. Cent. Inst. Ochi. Pr. 1979, 29 (loo),35. Walker, R. F.; Pinches, M. A. Analyst (London)1979, 704 (1243),928. Ben-Efraim,David A. Chem. Cyanates Their Thio Derlv. 1977, 7, 191. Hlavay, J.; Guilbault, George G. Anal. Chem. 1977, 49, 1890. Scheide, Eugene P.; Warner, R . B. Am. Ind. Hyg. Assoc. J . 1978,
39, 745.
King, W. H., Jr. Anal. Chem. 1984, 36, 1735. King, W. H., Jr. Environ. Sci. Techno/. 1970, 4 , 1136. Janghorbani, M.; Freund, H. Anal. Chem. 1973, 45, 325. Cheney, J. L.; Homolya, J. B. Anal. Lett. 1975, 8 , 175. Karmarkar, K. H.; Guilbault, George G. Anal. Chim. Acta 1974, 71, 419.
Scheide, Eugene P.; Gullbault, George
G. Anal. Chem. 1972, 44,
1764.
Karmarkar, K. H.; Guilbault, George G. Anal. Chim. Acta 1975, 75, 111. Hlavay, J.; Guilbault, George G. Anal. Chem. 1978, 5 0 , 1044. Hlavay, J.; Gullbault, George G. Anal. Chem. 1978, 5 0 , 965. Webber, L. M.; Karmarkar, K. H.; Gullbault, George G. Anal. Chim. Acta 1978, 97,29. Sauerbrey, G. 2. Z . Phys. 1959, 155, 206. Sauerbrey, G. Z.2.Phys. 1964, 778,457. Bruins, Ed. “Silicone Technology”; Interscience: New York, 1970; p
75. Ozaki, S.Chem. Rev. 1972, 72,(5),457.
RECEIVED for review September 20, 1984. Resubmitted June 3, 1985. Accepted June 3, 1985.
Rapid and Convenient Spectrophotometric Method for Determination of Clavulanic Acid’ Arie L. Gutman,* Vered Ribon, and Jean Pierre Leblanc2 Department of Chemistry, Technion-Israel
Institute of Technology, Haifa 32000, Israel
A convenient and sensltlve spectrophotometrlc method for the determlnatlon of the cllnlcally Important clavulanlc acld In blologlcal fluids has been developed. The method Is based on the property of clavulanlc acld tQ lnhlblt P-lactamase facilitated degradatlon of penlcllllns. I t makes use of the fact that at 240 nm the molar absorptlon coefficient of penlcillln G is greater than that of Its correspondlng degradatlon product, peniclllolc acid.
Clavulanic acid, produced by Streptomyces clavuligerus
0&k--J*,,,
2
clavulanic acid
( I ) , exhibits only weak antibacterial activity but is most im-
Dedicated to Professor David Ginsburg on the occasion of his 65th birthday. 2Present address: Inst. Chemie et Phys. Ind., Lyon, France.
portant for its capability to inhibit P-lactamases, which hydrolyze the cyclic amide bond of P-lactams converting them into ring-opened antibiotically inactive compounds (2) (eq 1).
As a powerful inhibitor of P-lactamases, clavulanic acid provides a means for removing this mechanism of bacterial resistance, thus extending the spectrum of antibacterial activity of various P-lactam antibiotics. Although first reported only in 1976, it is already used clinically in a formulation with amoxoceline against a large variety of gram-positive and gram-negative P-lactamase producing bacteria, which are otherwise resistant to P-lactam antibiotics. In the course of research on the biosynthesis of clavulanic acid, we required a rapid and reliable method for its quantitative determination in aqueous solutions in general and in particular in samples of culture broths of S. clauuligerus A.T.C.C. 27064 (3). To our knowledge the only reported method involves a tedious multistep assay and is not suitable
0003-2700/85/0357-2344$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985 2345
for determinations of small quantities of clavulanic acid (4, 5). It is based on the inhibition of enzymic degradation of penicillin to penicilloic acid with the results expressed as concentration of clavulanic acid giving 50% of the enzyme inhibition (Ibo).It therefore requires in each case the examination of several dilutions of the clavulanic acid containing solution. Furthermore, the amount of enzymic degradation is determined indirectly by the hydroxylamine assay for penicillin. The aim of this study was to develop a more convenient one-step spectrophotometric assay, whose sensitivity would allow detection and determination of clavulanic acid in aqueous solutions in the microgram per milliliter range.
EXPERIMENTAL SECTION Reagents. All chemicals used were C. P. grade commercial preparations. Crystalline sodium penicillin G was obtained from Rafa Laboratories, Israel. Crystalline lithium clavulinate was obtained from Beecham Pharmaceuticals Research Division, England. Crystalline potassium clavulinate was obtained from Glaxo Group Research, England. The j3-lactamases used was bacto-penase penicillinase 2000 L.U. mL-', purchased from Difco Laboratories (catalog no. 0345-61, control 715750). Enzymatic reactions were carried out in 0.1 M phosphate buffer, pH 7, prepared with double distilled water. Fresh solutions of sodium penicillin G and of salts of clavulanic acid were prepared daily in the same buffer and kept in a thermostat at 30 f 0.2 OC. Instrumentation. All spectra and kinetic measurements were carried out on a Perkin-Elmer Model 554 UV-vis spectrophotometer equipped with a thermostated cell holder, maintained at a temperature of 30.0 0.2 "C. The enzymatic reaction was carried out in a 3-mL quartz cell with a 1-cm light path and its progress was monitored by recording the absorbance of the reaction solution to ultraviolet light at 240 nm against a blank containing the buffer solution. The decrease in absorbance correspond to the enzymatic degradation of sodium penicillin G. Enzymatic Reaction. To the thermostated cell at 30.0 f 0.2 "C was added 970 pL of pH 7.00 phosphate buffer and 30 p L of bacto-penase penicillinase and the enzymatic reaction was started by addition of 1 mL of the stock solution of sodium penicillin G (0.85 X M). The cell was covered, its contents were thoroughly mixed by inversion for 5 s, and the cell was placed in the cell holder of the spectrophotometer. Recording of the decrease in absorbance at 240 nm began 15 s after addition of penicillin and was continued until the absorbance did not change for 5 min (AJ. Extrapolation of the time dependent curve to the beginning of incubation gave the A. absorbance at the beginning of incubation. tl was calculated as the time required for the absorbance to reach Allzhalf way between A, and A, (see Figure 1). Preparation of t h e Calibration Curve for Lithium Clavulinate. The stock solution of lithium clavulinate (232 pg mL-'; 1.13 X M) was made up in pH 7.00 phosphate buffer. To the thermostated cell at 30.0 h 0.2 "C was added (970 - x ) pL of pH 7.00 phosphate buffer, 30 pL of bacto-penase penicillinase, and X wL of lithium clavulinate (a, x = 2 pL; b, x = 4 pL; c, x = 6 p L ; d, x = 8 pL; e, x = 10 pL; f, x = 15 pL; g, x = 65 pL). The contents of the cell were mixed and preincubated for 5 min. The enzymatic reaction was started by addition of 1mL of the stock M). The contents solution of sodium penicillin G (0.85 X of the cell were again thoroughly mixed and recording of the decrease in absorbance at 240 nm began 15 s after addition of penicillin (Figure 1). In each case t l j z was calculated as described above. Assay of a n Unknown Solution. The assay is carried out as described above for the preparation of the calibration curve, except that instead of the stock solution of lithium clavulinate an aliquot of the unknown solution is taken.
*
RESULTS AND DISCUSSION We report a convenient direct spectrophotometric assay for accurate (experimental error *lo%) determination of clamlanic acid in aqueous solutions. The method is simple, sensitive, and suitable even for very dilute solutions of clavulinate (the limit of detection of the assay is in the area of 1 pg mL-l).
t
I 0
'I; ' I
i
2
~
3
I! 4
~
5
I 6
I
7
~
8
~
9
i
i
IO
t1
~
I2
~
13
I
I
14
15
~
16
~
17
! 18
I 19llminh)
Figure 1. Effect of added lithium clavulinate on the enzymatic decomposition of penicillin G, as determined by decrease in absorbance
at 240 nm (see Experimental Section for incubation details): A o , absorbance at the beginning of decomposition; A m absorbance at the end of decomposition; t 1,2, time required for the decomposition of half of the penicillin. I t is based on the ability of clavulanic acid to inhibit @-lactamase facilitated degradation of penicillin and makes use of the fact that at 240 nm the molar absorption coefficient of sodium penicillin G is greater than that of its corresponding degradation product, penicilloic acid ( A Q = ~ 0.6 ~ X lo3 M-' cm-') (6). I n our assay the clavulinate containing solution is preincubated with the @-lactamasea t 30 "C in 0.1 M phosphate buffer, pH 7, in a quartz spectrophotometric cell. After 5 min a solution of sodium penicillin G in the same buffer is added, the contents of the cell are mixed, and the absorbance as 240 nm is recorded. The gradual decrease in absorbance at 240 nm corresponds to the enzymatic conversion of penicillin into the corresponding penicilloic acid (as in eq 1). Figure 1summarizes the kinetic profile of several incubation experiments carried out with identical concentration of enzyme and penicillin but varying concentrations of lithium clavulinate. The sensitivity of the method is emphasized by the fact that small changes in concentration of cladinate significantly affect the rate of decomposition of penicillin. The decomposition rate could be expressed by measuring the slope of the time-dependent continuous absorption curve. We found, however, that it is most convenient to measure the time, required for the decomposition of half of the penicillin present, Le., tllz (the point at which the absorbance reaches Al12half way between the absorbance a t the beginning of decomposition, Ao, and the absorbance a t the end of decomposition, Am). As the activity of the enzyme solution could vary from batch to batch and is generally decreasing with time, for standardization of the method, it is best to operate with figures showing the relative increase in t l l z ,compared with that of the standard experiment (without any clavulanic acid), rather than with absolute tljz values. As the kinetics of the inhibition process is very complex, the dependence of the reaction rate, as expressed by the increase in tljz,on the concentration of added clavulinate could not be described by a linear function. However, since the focus of this study was to develop a method for quantitative determination of clavulanic acid, a suitable, although not linear, calibration curve was obtained by plotting the percent of increase in t l j zagainst the concentration of added lithium clavulinate (Figure 2). An excellent correlation was obtained when potassium clavulinate was used instead of lithium clavulinate. The calibration curve makes it possible to estimate the concentration of clavulanic acid in aqueous solutions. All that needs to be done is (a) determination of t l j zof the enzymatic degradation of penicillin without any added clavulanic acid,
2346
Anal. Chem. 1985, 57,2346-2349
In conclusion, a quantitative determination of clavulanic acid in different biological solutions can be carried out with a very high degree of sensitivity and accuracy. The analysis is rapid and it requires a minimum of manipulations and preparation. Since relative values of increase in t l / *are used, the accuracy of the assay is not affected by the activity of the enzyme. Indeed, within experimental error (&lo%),identical calibration curves were obtained when the quantity and/or activity of the enzyme was varied widely. It should, however, be borne in mind that the assay is based on detection of /3-lactamase inhibitory activity and it could not be used for determination of clavulinate in the presence of another 0lactamase inhibitor.
ACKNOWLEDGMENT
025
050 0 7 5 100 125 1.50 pg o f lithium clavulinate/ ml
1.75
200
225
Figure 2. Calibration curve for lithium clavulinate as a function of percent increase in t ,,2. Each point represents a single incubation experiment: 0, no added lithium clavulinate; a, 0.233pg mL-’; b, 0.465 pg mL-’; c, 0.697 pg mL-’; d, 0.930 pg mL-‘; f, 1.743 pg mL-’; g, 7.553pg mi--’.
(b) determination of the tljzof the reaction carried out under identical conditions, but with addition of an aliquot of the unknown clavulinate containing solution, and (c) calculation of the relative percent increase in t l / , and correlation using the calibration curve. The accuracy of the assay decreases at high concentration of clavulinate; thus, if the increase in tllz is greater than 8070,a smaller aliquot should be taken. With this method any clavulinate containing solution could be accurately assayed within 1-2 h with minimal preparative work.
We thank Beecham Pharmaceuticals and the Glaxo Research Group for providing samples of lithium and potassium clavulinate. Registry No. Clavulanic acid, 58001-44-8; p-lactamase, 9073-60-3;penicillin G, 61-33-6.
LITERATURE CITED (1) Howarth, T. T.; Brown, A. G.; King, T. J. J . Cbem. SOC.,Cbem. Commun. 1976, 266-267. (2) Morin, R. B., Gorman, M., Ed. “Chemistry and Biology of P-Lactam Antibiotics”; Academic Press: New York, 1982; Vol. 3. (3) Higgens, 0.E.; Kastner, R. F. Int. J . Syst. Bacterial. 1971, 21, 326-331. (4) Reading, C.; Hepburn, P. Biocbem. J . 1979, 797, 67-76. (5) U. S. Patent 4 110 165. (6) Samunl, A. Anal. Biocbem. 1975, 63, 17-26.
RECEIVED for review March 4,1985. Accepted May 21,1985. This work was supported by the “Bat Sheva de Rothschild Fund for Encouragement of Research”.
Measurement of Creatine Kinase Isoenzyme MB in Serum with Immunoseparation and Electrochemical Detection Takashi Toyoda, Shia S. Kuan, and George G. Guilbault* Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148
A seml-on-llne method has been developed for rapid, slmple, accurate, and convenlent determination of CK-MB Isoenzyme in serum. An immunoreactor is used to separate the CK-MB Isoenzyme, whlch Is in turn determined electrochemically by coupllng the NADH produced In the prlmary reaction to the ferrlcyanide-diaphorase reaction. The reactor Is very stable and the assay procedure Is easy to manlpulate, thus resulting in excellent precision and recovery compared wlth the most frequently used electrophoresls procedure. After sllght modification, this method can be Incorporated into a continuous flow analytical system.
The determination of creatine kinase (CK) isoenzyme MB (CK-MB) is becoming increasing important in the diagnosis of acute myocardial infarction. Several separation techniques are now employed to measure the specific isoenzyme CK-MB:
electrophoresis (1-4), chromatography (5-7), and immunological methods (8-11). The most commonly used electrophoresis procedure is laborious and tedious. Ion exchange column chromatography uses more simplified instrumentation than electrophoresis; however, complete separation of each isoenzyme is difficult to effect. Immunochemical methods recently developed can be classified into two categories, namely, the immunoprecipitation test and the immunoinhibition test. The former can more accurately measure the activity of pure subunit isoenzymes, for example, CK-MM or CK-BB using anti-CK-BB or antiCK-MM. But this test requires a second antibody and centrifugation for separation; therefore, it is more complex and time-consuming. In the immunoinhibition test, the CK-M subunit is blocked by goat-anti-human CK-M-IgG (termed Inh-CK-M-antibody) and the activity of CK-B remaining is assayed. This test does not require separation and hence is more rapid, simple, and easy to automate. However, the
0 1985 American Chemical Society 0003-2700/85/0357-2346$01.50/0
