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Anal. Chem. 1907, 59, 1349-1351
Direct Determination of @-LactamAntibiotics by Circular Dichroism Neil Purdie* and Kathy A. Swallows Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-0447
Circular dkhrolsm spectra have been characterized for nlne penklllns and three cephalosporlns dissolved In aqueous pH 5.4 buffer solutions. Discrlmlnations among either the penlclHln or the cephalosporin homologues are not posslbie from these spectral data, but the distinction between the two groups Is easy. Dtrect determinatlons of penlclllln V present In prepared laboratory mixtures with lactose, starch, caffeine, and cephalothln and In pharmaceutlcai preparations were accomplished after a simple extractlon Into ailquots of the buffer. Correspondence between the experimental and theoretical resuits was better than 1% for the prepared samples and within a few percent for the pharmaceutical preparatlons.
The analytical determinaton of the penicillins is important for a number of reasons and has been the object of a very large number as well as a wide variety of studies. These have been conveniently reviewed in recent publications (1-3). In a summary of the methods it is appropriate to point out that a direct method of analysis has not been described. Determinations have usually required that some kind of prior chemical reaction or derivatization step be performed, and the products of these processes were used in the detection step. In this way a certain degree of selectivity is introduced by the exclusion of interfering compounds. Among the methods employed are titrimetry, colorimetry, polarography, and chromatography. Where speed in performing repetitive analyses was an important priority, such as in quality control applications, automatic and kinetic methods of analysis have been developed. Circular dichroism (CD) spectropolarimetry has emerged as an analytical detector with considerable selectivity which is based upon the fact that two prerequisites must be satisfied before an analyte is CD active, namely, optical activity and absorption. The degree of selectivity has been sufficient in a number of cases to determine an analyte without any chromatographic separation of the sample, or any other kind of workup, except a simple extraction into a recommended solvent system. Examples of single analytes successfully analyzed in this way are (-)-cocaine (4),heroin (5),and (+)-LSD (6). More complex systems containing more than one CDactive analyte have also been successfully determined, the most impressive among these being plant extracts (7). Pharmaceutical products have also been investigated, for example, demerol for meperidine (8) and seconal for secobarbital (9). The P-lactam drugs are known to be CD-active (10-12) and as such are potential candidates for their direct determination by CD. Accordingly we have characterized the CD spectra of 12 analogues in a variety of solvents and have chosen a slightly acid medium as the optimum solution for extraction and analysis.
EXPERIMENTAL SECTION The substances obtained for this study were amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin (Bristol Meyers), cephalexin, cephalothin, and cephapirin (Bristol Meyers and Sigma Chemical co.) or their sodium salts, and the 0003-2700/87/0359-1349$01.50/0
potassium salts of penicillin G and penicillin V (Sigma Chemical Co.). All were used without further purification. Pen-V tablets (250 mg) (Parke-Davis)were obtained from a local pharmacy. A sample of filtered and arbitrarily diluted penicillin (Pen-V) fermentation broth was provided to us by Eli-Lilly, Inc. Lactose, starch, and caffeine hydrobromide, used in the preparation of laboratory mixtures, were obtained from Fischer, Mallinckrodt, and Matheson Coleman and Bell, respectively. These are typical of the additives commonly encountered in pharmaceutical preparations. Their special interest in this case is that lactose and starch are both chiral but nonabsorbing, while caffeine absorbs radiation but is not chiral. None should interfere with the CD measurement but their presence might affect the signal quality. Starch was also included because it is insoluble in the buffer system chosen for the study. Centrifugation was required to separate any undissolved matter in the workup of the commercial preparations. As a test to see if any of the analyte is retained by adsorption, starch was added to stimulate this condition for the laboratory samples. The penicillin content in the laboratory samples was varied from approximately7% to 70% by weight. These were thoroughly mixed. To sample the pellets, the entire pill was first ground and then finely powdered by shaking in a Wig-LBug for approximately 2 min. Several samples of approximately 20 mg were chosen at random from each of the solid mixtures, extracted into 25-mL aliquots of the buffer solution, and centrifuged where necessary and the spectrum was measured. For the sample of fermentation broth 50-rL aliquots were diluted with 10 mL of buffer and the spectrum was run directly. Limited solubilities eliminated a number of potential organic solvents as the extracting medium. Aqueous solutions worked very well but strong acid and strong basic media were avoided to minimize hydrolysis problems. The signal quality was best for the solutes dissolved in a 5.4 buffer solution, spectra at pH 7.6 and 9.2 showing considerably more noise. CD measurements were made as before with a JASCO500A/DP-500N automatic spectropolarimeter/data processor combination (7). Instrument parameters were arranged to give the optimum signal-to-noise (S/N) ratio. The instrument was calibrated daily with a standard solution of androsterone in dioxane as recommended.
RESULTS AND DISCUSSION The basic structures of the penicillin and the cephalosporin homologues are given in Figure 1,the fundamental differences being associated with the identities of the R and R1substituents which are listed in Table I for reference. All the analytes absorb in the UV range of the spectrum, Figure 2, which is the typical range for an aromatic chromophore, so the CD spectra were measured from 320 to 220 nm, Figure 3. Considerably more use can be made of CD for analytical purposes based on the comparative appearances of the UV vs. CD spectra. CD spectra for the penicillins are qualitatively indistinguishable from one another showing a strong positive Cotton band maximizing at 230 i 2 nm, Figure 3a. A similar spectral correspondence is observed for the three cephalosporins which show two maxima. The first band is positive &h a maximum around 260 i 2 nm, associated with the ring unsaturation, and is separated from a second negative band with a maximum at 230 2 nm by a Cross Over Point of zero ellipticity a t approximately 243 nm, Figure 3b. Discrimination between the groups is easy because of these
*
0 1987 American Chemical Society
1350
ANALYTICAL CHEMISTRY, VOL. 59, NO. 9, MAY 1, 1987
Table I. Molecular Structures and Molar Ellipticities for Penicillins and Cephalosporins
name (a)
(b)
Figure 1. Molecular structures of (a) the penicillins and (b) the cephalosporins.
220 I
I
240 I
I
I
I
+4?0
h
amoxicillin
2-amino(p-hydroxyphenyl)ampicillin 2-amino-2-phenylcloxacillin 3-(o-chlorophenyl)-Zdicloxacillin 3-(2,6-dichlorophenyl)-Zmethicillin 2,6-dimethoxyphenylnafcillin 2-ethoxynaphthalenyl3-phenyl-Zoxacillin benzylPen-G benzoxyPen-V CHB cephalexin 2-amino-2-phenylcephalothin 242-thienyl)cephapirin
2-(4-pyridylthio)-
Figure 2. UV absorption curves for (a) P e n 4 and (b) cephalothin in pH 5.4 buffer.
OM (nm)
R1
R"
+398 (230) +431 (230) +333 (230) +323 (230) +265 (230)
+237 (230)
+482 (230) +394 (230) +363 (230) +395 (260) -632 (230) -CH20COCH3 +452 (260) -600 (230) -CH20COCH3- +501 (260) -674 (230)
"The symbol Z is used to represent
N//
' 0
3
Table 11. Determinations of In-House and Commercial Lactam Mixtures
mixture
s1
I
i
s2
s3
h
v
-300
-600
s4 s5
S6 PC1 PC2 PC3
FlQwe 3. CD spectra for (a) Pen-V and (b) cephalothin in pH 5.4 buffer.
PC4
spectral dissimilarities. The same basic features are observed in alcoholic solvents and as a function of p H for aqueous solutions. The spectra in Figure 3 are for the drugs dissolved in an aqueous solution of pH 5.4, the condition that produced the best S / N ratio. The spectra are not quantitatively identical however, and a calibration cullre would be a necessary prerequisite before any specific analyte could be determined. Curves were obtained for ampicillin, amoxicillin, Pen-V, and cephalothin. For the others, values for the molar ellipticity, OM at the maxima were calculated for only one concentration in order to obtain a rough idea of the relative intensities, Table I. OM is defined in this work as the ellipticity of a one molar solution and values reported are 100 times smaller than molecular ellipticities contained in earlier work (11, 12). OM is the CD equivalent of the molar absorption coefficient in absorption spectrophotometry, and the Beer-Lambert law applies equally well to CD data. In the simplest case, where interference is not a serious problem, determinations can be made from data taken a t a single wavelength and as such the CD method can be automated for rapid repetitive analyses. The selectivity inherent to the method is the single important advantage of CD over potentially competitive methods. Obviously the groups of P-lactams could theoretically be distinguished by measuring the optical rotation at 230 nm, where the signs of the rotations are of opposite sense. One would have to be certain however that all possible optically active interferences had been successfully separated prior to making the measurement. One could just as easily presume that the distinction and determination could be done from absorption
T1 broth
lactam Pen-V Pen-V Pen-V Pen-V Pen-V Pen-V Pen-V cephalothin Pen-V cephalothin Pen-V cephalothin Pen-V cephalothin Pen-V Pen-V
additive
prepared, '3
lactose
23.9 40.5 caffeine 72.3 starch 7.1 starch 29.6 starch 63.5 lactose 1.28 0.63 lactose 2.22 lactose
4.79
caffeine 6.47 lactose
measd, % 23.6 39.5 72.2
6.3 28.2
63.8 1.53
0.65 2.53 4.74 7.19
5.58
5.46
3.50
4.11
1.68 63.3 f 0.9 (250 mg) see text (7.94 + 0.1 m d 8.07 f 0.12 1.80
?
measurements, Figure 2, but once again the total separation of the lactam from other absorbing species would have to be confirmed. Neither of these restrictions alone is limiting to the determination of a CD-active analyte. This is apparent from the spectra we have obtained, Figure 3, and the determinations we have performed on several laboratory in-house blinds, Sl-S6,PC1-PC4, and the samples of penicillin V in the commercially available products, T1,and broth, Table 11. The presence of the strongly absorbing caffeine in the mixtures affects the spectra only in the reduction of the SIN ratio. Whenever the nondrug residual material is completely soluble, the correspondence between the calculated and experimental results is uniformly excellent. Some indication of possible adsorption on undissolved starch is evident from the decrease in the correspondence with the increasing percentage of the insoluble material in the mixture. Reproducibility is excellent for the in-house mixtures. As might be expected it is poorer for the Pen-V tablets which are presumably prepared from aliquots taken from a much larger and less uniform sample. In fact considerable variation was observed in the total weights of the tablets. If the Pen-V content is reported as a percentage of the total weight, rather than compared with the nominal 250-mg prescription amount, then the correspondence between samples is much better (kO.9%,). A similar correspondence was observed for the determination of Pen-V in the fermentation broth. The composition
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Anal. Chem. 1907, 59, 1351-1356
I
ellipticity (millideg.)
t y220 I
I ’
40
n
@)
240 I
’
h
I
Figure 4. CD spectra for (a)the Pen-V fermentation broth and (b) the P e n 4 standard at a similar concentration in pH 5.4 buffer.
of the broth reported by Lilly was 7.94 mg/mL Pen-V and C0.1 mg/mL p-hydroxy-Pen-V (as the potassium salts), 1.01 mg/mL phenoxyacetic acid and 0.12 mg/mL p-phenoxyacetic acid (as the sodium salts), inorganic salts of alkali and alkaline-earth metals plus ammonium and trace heavy metal cations, and various unknown organic compounds typically found in fermentation broths. The organism and several large proteins were removed in the filtration step. Of these ingredients the only known CD-active molecules were the penicillins, and the extent of the matrix distortion in the CD spectrum of the standard is a slight asymmetry on the low wavelength end of the band with little loss in S / N quality, Figure 4. Since we are unable to distinguish between the penicillins, our value of 8.07 mg/mL is representative of the total penicillin calculated as if it were all Pen-V, which assumes that OM is the same for both, and within 1% it appears to be an acceptable assumption. In-house mixtures of Pen-V and cephalothin, PC1-PC4, Table 11, were determined without a separation step. The spectral analysis is relatively straightforward when cephalosporin is in large excess since it is essentially the only contributor to the positive band whose maximum is at 260 nm. For the other mixtures however the determinations were done by using a simple curve fitting program in which weighted contributions from the standard curves were added in order to simulate the experimental curve (7). Once again the
agreement between the prepared and calculated data is excellent. In summary therefore the present work is a further endorsement of the analytical capabilities of CD in the direct determination of pharmaceutically important substances. Assuming that every method employed for the determination of a lactam would involve the same extraction procedure, the relative time taken for detection by CD is very short, i.e., a matter of minutes, which can be improved by automation. In addition there is no cause for modifying the analyte by derivatization or for the addition of an internal standard.
ACKNOWLEDGMENT We wish to thank Anthony J. Tietz of Eli Lilly, Inc., Indianapolis, IN, for providing us with the sample of penicillin broth. Registry No. Amoxicillin, 26787-78-0; ampicillin, 69-53-4; cloxacillin, 61-72-3; dicloxacillin, 3116-76-5; methicillin, 61-32-5; nafcillin, 147-52-4; oxacillin, 66-79-5; cephalexin, 15686-71-2; cephalothin, 153-61-7;cephapirin, 21593-23-7;penicillin G, 61-33-6; penicillin V, 87-08-1; penicillin, 1406-05-9; cephalosporin, 11111-12-9.
LITERATURE CITED (1) Hamilton Miller, J. M. T.; Smith, J. T.; Knox, R. J . Pharm. Pharmacol. 1963, 15, 81. (2) Hughes, D. W.; Vilim, A.; Wilson, W. L. Can. J . Pharm. Sci. 1978, 1 1 , 97. (3) Fairbrother. J. E. pharm. J . 1977, 218, 509. (4) Bowen, J. M.; Purdie. N. Anal. Chem. 1981, 5 3 , 2237. (5) Bowen, J. M.; Crone, T. A.; Kennedy, R. K.; Purdie, N. Anal. Chem. 1982, 5 4 , 66. (6) Bowen, J. M.; McMorrow, H. A.; Purdie, N. J . Forensic Sci. 1982, 27, 822. (7) Han, S. M.; Purdie, N. Anal. Chem. 1986, 5 8 , 113. (8) Han, S. M.; Purdie, N. Anal. Chem. 1984, 5 6 , 2822. (9) Han, S. M.; Purdie, N. Anal. Chem. 1984, 5 6 , 2825. (10) Nagarajan, R. I n Cephalosporins and Penicillins: Chemistry and Biology; Flynn, E. H., Ed.; Academic: New York, 1972; Chapter 15. (11) Richardson, F. S.; Yeh, C.-Y.; Troxell, T. C.; Boyd, D. B. Tetrahedron 1977, 33, 711. (12) Busson. R.; Roets, E.; Vanderhaeghe, H. J . Org. Chem. 1978, 43, 434.
RECEIVED for review November 12,1986. Accepted January 30, 1987.
Do Optical Sensors Really Measure pH? JiFi Janata Center for Sensor Technology, University of Utah, Salt Lake City, Utah 84112
The problems that are inherent in the optlcal determlnation of pH and of other Ion actlvltles are present also in the optrodes. Furthermore, because of the compllcated relationship between the bulk and the surface pH, these problems are amplified in optlcai sensors. While neither optrodes nor pH electrodes can measure pH, the themodynamk compromks that have to be made In electrochemical esthnatlon of pH are less severe then those that have to be made when using optlcal sensors. Furthermore, the flexlblllty in design of the ilquld junction suitable for any practlcal measuring situation makes electrochemical sensors preferable to optical ones.
A large number of papers dealing with optical sensors has been published recently, among them articles describing 0003-2700/87/0359-1351$01.50/0
various optical pH probes (optrodes or optodes) figure prominently (1-6). The main reasons cited for development of optical sensors are the lack of necessity of a reference electrode and electrical safety. Because it is the most ubiquitous species encountered in chemical reactions, hydrogen ion occupies a very special place in chemistry and biology. From the analytical point of view it is also special because it is almost always quantified in terms of pH-the negative logarithm of its activity:
pH = -log
UH+
(1)
It is a well-know and accepted fact that for thermodynamic reasons single ion activities cannot be measured (e.g. ref 7) and that all experimental techniques used for measurement of pH and other “ion activities” always contain some thermodynamic compromises. This issue has been long recognized 0 1987 American Chemical Society
