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Zero-Crossing Derivative Spectrophotometry for the Determination of Mixtures of Cephaloridine and Cephalothin in Pure and Dosage Forms
Zero-Crossing Derivative Spectrophotometry for the Determination of Mixtures of Cephaloridine and Cephalothin in Pure and Dosage Forms BASILIOMORELLI Received October 26,1987, from the Universitrl degli Studi-Bari, Dipattimento di Chimica, Campus Universitario-4, Trav. 200 Re David, 707264ar1, Italy. Accepted for publication February 16, 1988. ~~
Abstract 0 First- and second-derivative spectrophotometry, with a zero-crossing technique of measurement, has been used for the quantitation of two-component mixtures of cephaloridine and cephalothin Na,
which are cephalosporins with closely overlapping spectral bands. Beer's Law is fcillowed for up to 28 and 36 luglmL of cephaloridine in the first-and second-derivative modes, respectively, and up to 36 N / m L of cephalothin Na in both modes. Detection limits at the 0.05 level of significancewere calculated to be 0.13 and 0.37MlmL of cephaloridine and cephalothin Na, respectively, in the first-derivativemode, and 0.25 and 0.29 N/mL, respectively, in the second-derivativg mode. The recovery of these antibiotics in mixtures of injectable dosage forms Is also reported.
The term derivative spectroscopy refers to a spectral measurement technique in which the slope of the specthm, that is, the rate of change of spectral intensity with wavelength, i s measured. There are many instances in biochemical, pharmaceutical, and environmental areas where the derivative technique may be useful. It is perfectly general in its application and can be used equally for spectroscopic, chromatographic, densitometric, etc., data.14 In previous papers, we showed the usefulness of derivative spectrophotometry for the simultaneous determination of ihorganic cations in mixtures by using 2-thiobarbituric acid &' and ally1 thiourea8 as the reagents. Derivative spectrophotometry represents an elegant approach to the problem of resolving spectral overlap in pharmaceutical analysis. It has been successfully used for the analysis of pharmaceutical dosage formsg and, more recently, for the determination of several drugs alone or in mixtures.10-12 In the course of systematic research of new procedures for the quantitation of antibiotics and substances of clinical and pharmaceutical interest,Is17 we described in a recent paper a sensitive method of derivative spectrophotometry fsr the determination of cephacetrile and ceftezole in mixture.'*
Continuing with this investigation, we developed a simple quantitative method for the fast determination of mixtures of cephaloridine and cephalothin, which are semisynthetic analogues of cephalosporin C with a wide antimicrobial spectrum of activity (see structures). These compounds are characterized by closely overlapping absorption spectra. The aim of the present work was to demonstrate the relative ease with which the proposed zero-crossing derivative methodology overcomes this difficulty, allowing the simultaneous determination of the two drugs without the need for prior separations. The method i d sensitive and yields accurate and reproducible results. It has been applied to laboratory mixtures of the pure drugs and to mixtures of injections used parenterally for the treatment of diseases of the genitourinary and respiratory apparatus, otorhinolaringologic and obstetric-gynecological infections, skin and venereal diseases, etc. It cannot be excluded that other techniques, such as chromatography, would also give good results. The technology and applications of HPLC have been developed at a fast rate during the past years, although the detector still remains the factor that sometimes limits ultimate sensitivity and flexibility. The use of the derivative technique for resolution enhancement in HPLC and GLC, by applying the derivative technique to the elution profile itself, was discussed by Fell.'g The increasing interest in derivative spectroscopy is such that the method has been patented again to give a device for low-noise derivatives up to the eighth order, although for resolution enhancement in the UV-VIS range, the most useful derivatives are up to the fourth ~ r d e rFor .~ these reasons, coupled with analysis time, ease of operations, and widespread availability of commercial instruments with derivative capability, it is probably of value to present this work as a n encouragement to others to apply this simple and versatile technique in routine practical analytical work.
Experimental Section M a t e r i a l s h r e drug samples of cephaloridine and cephalothin
Na were purchased from Sigma Chemical Company, St. Louis, MO.
Cephaloridine
COOH
Cephalothin 0022-3549/88~700-0615$0 I .OO/O 0 1988, American Pharmaceutical Association
Stock solutions (0.2 mg/mL) were freshly prepared in water. Injectable dosage forms of Keflin (Eli Lilly S.p.A., Italy) were labeled to contain 1g of cephalothin Na per vial, and Keflodin (Eli Lilly S.p.A., Italy) and Ceporin (by Glaxo S.p.A., Italy) were both labeled to contain 1 g of cephaloridine per vial. The apparatus used was a Perkin Elmer 555 double beam UV-VIS spectrophotometer with 1-cm quartz cells. Suitable settings were: 1nm slit width, 4-s response time, and 120-ndmin monochromator scan speed. Sample Preparation and Procedure-A few microlitres of the cephalosporin stock solutions were added to a 5-mL volumetric flask which was made to volume with distilled water. The derivative spectra of the mixture against water was then recorded. The absolute Journal of Pharmaceutical Sciences / 615 Vol. 77, No. 7, July 1988
values of the first derivative were measured at 232 and 235 nm for the determinationof cephaloridine and cephalothin Na, respectively, and the values of the second derivative were measured at 253 and 262 nm for cephaloridine and cephalothin Na, respectively.
Results and Discussion Spectrophotometric M e a s u r e m e n t e I n Figure 1 we show the absorption (zero-order)spectra of the following: (a) cephaloridine (12 pgimL) with a maximum a t 239 nm; (b) cephalothin Na (12 pg/mL) with a maximum a t 236 nm; and ( c ) a mixture of cephaloridine and cephalothin Na (12 pg/mL each) with a maximum at 237 nm. The large overlap of the spectral bands of the two components prevents the formation, from the total zero-order spectrum, of any spectral feature utilizable for analytical purposes. We circumvented this problem by making use of the first- and second-derivative spectra of the mixture. Peak-to-peak and baseline measurements (generally referred to as graphical measurements) and zero-crossing measurements,'a are the most common techniques used to prepare analytical working curves. In practice, that measurement which exhibits the best linear response to analyte concentration, which gives a zero intercept on the ordinate axis of the calibration graph, and which is least affected by the concentration of any other component is selected. In the present instance, preliminary experiments showed the difficulty of a correct use of graphical measurements. This was probably due to the closeness of the overlapping zero-order spectra of cephaloridine and cephalothin with the subsequent poor resolution of both the first- and secondderivative spectra of the mixtures. Hence, the zero-crossing technique was preferred. This involves the measurement of the absolute value of the total derivative spectrum at an abscissa value corresponding to the zero-crossing wavelengths of the derivative spectra of the individual components. Measurements made at the zero-crossingof the derivative spectrum of one of the two components would be a function only of concentration of the other component.
The first- and second-derivative spectra of cephaloridine and cephalothin Na (12 pg/mL in all instances) are shown in Figure 2. In the first-derivative mode, the zero-crossings of cephaloridine occur a t 216 and 235 nm, while those of cephalothin occur a t 210 and 232 nm. In the second-derivative mode, 222 and 262 nm are the zero-crossings wavelengths of cephaloridine and 218, 240, 253, and 272 nm are those of cephalothin. Among these wavelengths, we selected (see below) 232 and 235 nm as optimal for determination, by the first-derivative mode, of cephaloridine and cephalothin, respectively; 253 and 262 nm were chosen for the quantitation of cephaloridine and cephalothin, respectively, by the second-derivative mode. At the other wavelengths, worse results were obtained: the calibration curves were poorly linear, the scattering of experimental points was somewhat unacceptable, and/or the intercept on the y-axis was significantly different from zero. First-Derivative Mode-In Figure 3(a) we present a series of first-derivative spectra of mixtures of 10 pg/mL of cephalothin Na plus increasing quantities of cephaloridine (from 4 to 28 pg/mL). In Figure 3(b) is a series offirst-derivative spectra +0.060;
/ -\
rossing s izofe ro1st- cderivatives)
-0.060'
I
I
1
1
+0.008
,\
-0.0081 200
250
300
i(nm)
Flgure 1-Absorption spectra of: (a) cephaloridine (12 pg/mL); (b) cephalothin Na (12 pg/mL); and (c) mixture of cephaloridine and cephalothin Na (12 pg/mL each). The reference was water. 616 i Journal of Pharmaceutical Sciences Vol. 77, No. 7,July 1988
1
1
I
I
220
240
260
280
1 (nm)
Flgure 2-( a) First- and (b) second-derivative spectra of cephaloridine (dashed lines) and cephalothin Na (continuous lines). The cephalosporin concentrations were 12 p g h L each, and the reference was wafer.
+
+0.016
0.22, 3
A
C
%
I
I
I
I
I
I
220
240
260
280
-0.016
I
W
I
I
I
I
a + U
3
-0.20
1
I
I
l(nm)
Figure 3-First-derivative spectra of mixtures of cephaloridine and cephalothin Na. (a) Cephalothin Na concentration, 10 pg/mL; cephaloridine concentrations, 4, 20, and 28 pg/mL in curves 1, 2, and 3, respectively. (b:, Cephaloridine Na concentration, 13 pg/mL; cephalothin Na concentrations, 4, 12, and 36 pg/fnL in curves 1, 2, and 3, respectively. The reference was water.
of mixtures of 13 pg/mL of cephaloridine plus increasing amounts of cephalothin Na (from 4 to 36 pglmL). Denoting the height of the first derivative a t 232 nm (zerocrossing wavelength of cephalothin) by h l and the height at 235 nm (zero-crossingwavelength of cephaloridine) by h2, we found that hX and h2 were proportional to cephaloridine and cephalothin concentrations, respectively. The variation of h l and h2 was not affected by the presence of cephalothin and cephaloridine, respectively (i.e., there is a mutual independence between the two cephalosporins for any ratio of concentrations, in the full range investigated). It is important to point out that, as expected, all curves in Figures 3(a) and (b) converge to an abscissa value (235 and 232 nm, respectively) corresponding to the zero-crossing wavelengths of the two cephalosporins. Second-Derivative Mode-In Figure 4(a) is reported a series of second-derivative spectra of mixtures of 10 pg/mL of cephalothin Na and increasing concentrations of cephaloridine (from 12 to 32 pglmL). In Figure 4(b) are the secondderivative spectra of mixtures of 13 pglmL of cephaloridine
and increasing amounts of cephalothin Na (from 4 to 36 pgt mL). The heights a t 253 nm (zero-crossingwavelength of cephalothin), h3, and at 262 nm (zero-crossing wavelength of cephaloridine), h4, were proportional to cephaloridine and cephalothin Na concentrations, respectively. As in the firstderivative spectra, the curves converge to the zero-crossing wavelengths of 262 nm [Figure 4(a)l and 253 nm [Figure 4(b)] for cephaloridine and cephalothin Na, respectively. Calibration Graphs and Statistical Analysis-The above technique allowed us to obtain the linear regression equations for mixtures of cephaloridine and cephalothin Na. These equations are displayed in Table I together with correlation coefficients, variance, and detection limits a t p = 0.05 level of significance and for n = 10 standard specimens. The ordinate values, H, of the lines of regression were obtained from the h l , h2, and h3, and h4 (mm) measurements and standardized as follows:""18 H = recorder divisions (h mm) x scale expansiord100 mm full scale. Beer's Law rigorously holds up to 28 and 36 pg/mL of cephaloridine in the first- and second-derivative modes, reJournal ofPharmaceutical Sciences / 617 Vol. 77, No. 7, July 1988
Table 1-Statistical Analysis of the Determination of Cephaioridine and Cephalothln in Mixtures by First- and Second-Derivative Spectrophotometry" Antibiotic Cephaloridine Cephalothin Na Cephaloridine Cephaiothin Na
Derivative Mode
nm
Regression Equation
Correlation Coefficient
Variance
Detection Limit,
(St)
W/mL
First First
232
Second Second
0.9999 0.9999 0.9999 0.9999
6.88E-09 1.04E-07
0.13 0.37
253
H1 = -5.73E-05 + 1.37E-03C H 2 = 6.71 E-06 + 1.86E-03~ H3 = - 1.84E-05 + 4.20E-04c H4 = -2.45E-05 + 2.59E-04C
2.40E-09
0.25
1.27E-09
0.29
'Number of standard specimens: n
A,
235 262 =
10; level of significance: p
=
0.05.
spectively, and up to 36 Fg/mL of cephalothin Na in both modes. At higher concentrations, we observed a progressive degradation of the shape of derivative spectra (i.e., a n increase of the background noise, with subsequent increase of the scattering of the experimental points around the corresponding calibration curves) because of the difficulty in taking correct heights measurements. This may appear to be in contrast to the not very high values of absorbance of the total zero-order spectra a t the concentrations and wavelengths used; in fact, the mathematical derivative operation would give erroneous results when the absorption spectra of the mixture are outside the range of measurement of the instrument. However, the development of a derivative spectrophotometric assay requires a great number of instrumental variables to be controlled, depending on the derivative module employed. Presumably, the observed degradation is an artifact of the electronic analogue RC device utilized, which computes the derivative with respect to time, as the spectrum is scanned a t constant speed. The linearity of calibration graphs in the useful concentration range and the negligible scatter of experimental points is clearly evidenced by the values of correlation coefficients and variances. Tests of significance of the experimental intercepts (a of lines of regression H = a + bc) showed that these did not differ significantly from the expected value of zero. A simplified method of estimating the differences a - 0 is based on the calculation of the quantities t = a/s,20 and their comparison with the tabular data for the t distribution (Table 11). The values calculated for t do not exceed the 95%criterion (t, = 2.311, which indicates that the intercepts of all lines of regression are not significantly different from zero. Hence, the proposed methods for mixtures of cephalosporins are free from procedural errors, depending on the concentration of one of the two components. However, a more rigorous approach requires the construction of a joint confidence region for the possible values of slope and intercept, because of the strong correlation which exists between the two parameters; this has the further advantage of allowing for the evaluation of the calibration graphs as a whole. The 95%joint confidence regions for slopes and intercepts of equations of regression reported in Table I, constructed by the method of Mandel and Linning,20.21are displayed in
Table ii-Estimate of Differences a Type of Measurement First Derivative ( h l ) First Derivative (h2) Second Derivative (h3) Second Derivative (h4)
Figure 5: they are bounded by a n ellipse having the point of best fit as the center (i.e., coordinates a and b). The question as to whether the calculated intercepts are not significantly different from zero is answered by determining whether the ellipses in Figure 5 contain points for which the intercept is zero. Such points lie on a vertical line through abscissa = 0, and it is evident that they fall well inside the ellipses, confirming the previous conclusions. In Figure 6 are the histograms of the absolute error, S , , ~ O in the determination of a given concentration, calculated by means of statistical analysis of the regression equations
l(C EPHALORlDlME 1st de 1.1
,EPHALOTHlN - 1 s t der.)
~
7-c
1.83 Y
n
0, v)
- 2 . 0 -1.0
-r - - - 7
-
I
-1.0
-0.5
I
I
0 *1.0
x10-4 I(CEPHALORIOINE-2nd d.)
- 0'
Antibiotic Cephaloridine Cephaiothin Na Cephaloridine Cephalothin Na
bb 1.04
2.31
x~o-'
0
*0.5
XlO"
0.034
0.62 1.14
INTERCEPT
a
Figure +Joint confidence regions, at p = 0.05 level of significance, for
f = 8.
slope and intercept of regression equations of cephaloridine and cephalothin Na by first- and second-derivative methods.
Experimental value of intercept of lines of regression. bTheoretical value of tat p = 0.05 level of significance; no. of degrees of freedom:
618 /Journal of Pharmaceutical Sciences Vol. 77, No. 7,July 1988
reported in Table I. The absolute error, sc, is defined by the relationship:*O
Recovery of Cephaloridine and Cephalothin in Pharmaceuticals-Because of the difficulty encountered in obtaining pharmaceuticals containing both the cephalosporins tested, we applied the proposed method t o the recovery of these antibiotics in mixtures of commercial injectable dosage forms. Working mixtures were prepared by dissolving the required amounts of pharmaceuticals in water. The assay was completed as described in the Experimental Section, and the results of five replicate determinations of mixtures of Keflin (cephalothin Na) with Ceporin and Keflodin (cephaloridine), respectively, are shown in Table IV. Satisfactory results were obtained: the method developed yields good recoveries and the minor differences observed may be considered acceptable.
where so = VZ(Y - Y,,,c)2/n - 2; Y is the experimental value of ordinate; Ycalc is the ordinate value calculated from the regression, equation; b is the angular coefficient of line of regression, and Z and P are the average concentration and ordinate values, respectively, for n standard specimens. From an inspection of the graphs in Figure 6, we see that the error is minimal at -16 pg/mL of cephaloridine and cephalothin Na in the first-derivative mode, and at -18 pg/mL in the second-derivative mode. In Figure 7 are the histograms of confidence limitsz0for the Conclusions determination of cephaloridine and cephalothin Na, in mixture, at p := 0.05 level of significance. The curves are plotted The above findings substantiate the usefulness of derivafrom calibration data in a particular way;crs.13-16.17-18.21-24 tive spectrophotometry for the assay of mixtures of cephalorithat is, as uncertainty (%) on concentration (relative error), dine and cephalothin Na, either in the pure or injectable tpsc/c,against the concentration of cephaloridine and cephaform. Both first- and second-derivative spectrophotometry lothin Na, respectively. These graphs can give useful inforgive reproducible and accurate results; statistical analysis of mation about the degree of precision that may be expected in the experimental data demonstrates that there are no subthe full range of concentrations investigated. Accuracy and stantial differences between the two techniques. precision were tested by five successive determinations on Although the analyte mixture studied was not easily found laboratory mixtures of the pure drugs (Table 111). in commercial pharmaceutical dosage forms, in our opinion
n
A
I
CEPHRLORIDINE
CEPHALOTHIN
tlST DERIV.1
tlST DERIV.)
0
v,
pg
m1-’
I
A CEPHRLORIOINE
CEPHRLOTHIN
12ND DERIV.1
(2ND DERIV.1
”
U
N n
cn
pg Figure 6-Histograms spectrophotometry.
p g m 1-1
m 1-’
pg m1.l
of the absolute error in the determination of cephaloridine and cephalothin Na by first- and second-derivative
Journal of Pharmaceutical Sciences / 61 9 Vol. 77, NO. 7, July 1988
_
r
Y
4
CEPHRLOTHIN
IlST D E R I V . 1
CEPHALOTHIN
[ZND DERIV.)
s U
\ '?
LI
pg
ml-I
Figure 7-Histograms of the variation of confidence limits, at p = 0.05 level of significance, in the form of uncertaintypercent on the concentration (relative error) of cephaloridine and cephalothin Na by first- and second-derivativespectrophotometry.
Table Ill-Replicate Determinations on Synthetic Mixtures of Cephaloridlne and Cephalothin Nominal Value, rdml
easurement
BC
Ab
First Derivative (hl and h2) Second Derivative (h3 and h4) a Mean of
Mean Value, pg/mLa
A
Standard Deviation
Coefficient of \lariatinn
O-/
B
A
B
A
B
0.040
0.39
0.22
0.050
0.37
0.20
9.0
18.0
8.99
18.01
0.035
15.0
25.0
15.03
24.99
0.055
five determinations. Cephaloridine. Cephalothin Na.
Table IV-Recovery
of Cephaloridine and Cephalothin in Mixtures of Injections'
Type of Measurement
Mixture
Mean Recovery, %
First Derivative (hl and h2) Second Derivative (h3 and h4)
Ceporin Keflodin Ceporin Keflodin
100.02 2 100.03 ? 100.04 2 100.03 2
0.035 0.041 0.055 0.052
Mixture Keflin Keflin Keflin Keflin
Mean Recovery, yob 99.99 2 100.01 2 99.99 2 99.99 2
0.048 0.050 0.058 0.057 ~~
Keflin powder for injections (Eli Lilly S.p.A. Italy), with 1 g of cephalothin Ndinjection; Ceporin (Glaxo S.p.A. Italy) and Keflodin(E1iLilly S.p.A. Italy) powder for injections, both with 1 g of cephaloridinehnjection.bMean of five determinations ? standard deviation; assay as percentageof label claim. a
620 /Journal of Pharmaceutical Sciences Vol. 77, No. 7, July 7988
the proposed methods eould be also applied to real samples, such as biological fluids.
References and Notes 1. O'Haver, T.C.;Green, G. L. Anal. Chem. 1976,48,312. 2. OHaver, T.C.Anal. Chern. 1979,51,91A. 3. OHaver, T.C. Clin. Chern.1979,25,1548. 4. Fell, A. F.; Smith, G. Anal. Proc. 1982,19,28. 5. Morelli, B. Anal st 1982,107,282. 6. Morelli, €3. Anarst 1983,108,870. 7. Morelli, B. Anarst 1983,108,1506. 8. Morelli, B. AnatLett. 1985,18(A19), 2453. 9. Fell, A. F.Proc. Anal. Diu.Chem. Soc. 1978,15,260. 10. Korany, M. A.; Wanby, A.M.; Mandour, S. Anal. Lett. 1984, 17(B12), 1373. 11. El-Yazby, F. A.; Barary, M. H. Anal. Lett. 1985,18fB51, 629. 12. Mohamed, M.E. Anal. Lett. 1986,19(11-12), 1323. 13. Morelli, B.; Peluso, P. Anal. Lett. 1985,18fB9), 1113.
14. Morelli, B.; Peluso, P. Anal. Lett. 1985,18(B15), 1865. 15. Morelli. B. Anal. Lett. 1987.20(1). 141. 16. Morelli: B. J . Pharrn.Biorned. Anal. 1987,5, 577. 17. Morelli. B.; Mariani, M.; Gesmundo, M. Anal. Lett. 1987,20f91, 1429. 18. Morelli, B. Anal. Lett 1988,21f11, 43. 19. Fell, A. F.Anal. Proc. 1980,17,512. 20. Nalimov, V.V. The Application of Mathematical Statistics to Chemical Analysis; Pergamon: Oxford, 1963;pp 167-189. 21. Mandel, J.; Linnin , F J. Anal. Chern. 1957,29,743. 22. Morelli, B.;Pelusos. Anal. Lett. 1986,19f5-61,503. 23. Morelli, B.Anal st 1986,111, 1289. 24. Morelli, B.A n a i s t 1987,112, 1395.
Acknowledgments Thanks are due to Dr. Mariateresa Gesmundo for scientific collaboration and for help in performing part of the experimental work.
Journal of Pharmaceutical Sciences / 621 Vol. 77, No. 7, July 1988
Abstract 0 First- and second-derivative spectrophotometry, with a zero-crossing technique of measurement, has been used for the quantitation of two-component mixtures of cephaloridine and cephalothin Na,
which are cephalosporins with closely overlapping spectral bands. Beer's Law is fcillowed for up to 28 and 36 luglmL of cephaloridine in the first-and second-derivative modes, respectively, and up to 36 N / m L of cephalothin Na in both modes. Detection limits at the 0.05 level of significancewere calculated to be 0.13 and 0.37MlmL of cephaloridine and cephalothin Na, respectively, in the first-derivativemode, and 0.25 and 0.29 N/mL, respectively, in the second-derivativg mode. The recovery of these antibiotics in mixtures of injectable dosage forms Is also reported.
The term derivative spectroscopy refers to a spectral measurement technique in which the slope of the specthm, that is, the rate of change of spectral intensity with wavelength, i s measured. There are many instances in biochemical, pharmaceutical, and environmental areas where the derivative technique may be useful. It is perfectly general in its application and can be used equally for spectroscopic, chromatographic, densitometric, etc., data.14 In previous papers, we showed the usefulness of derivative spectrophotometry for the simultaneous determination of ihorganic cations in mixtures by using 2-thiobarbituric acid &' and ally1 thiourea8 as the reagents. Derivative spectrophotometry represents an elegant approach to the problem of resolving spectral overlap in pharmaceutical analysis. It has been successfully used for the analysis of pharmaceutical dosage formsg and, more recently, for the determination of several drugs alone or in mixtures.10-12 In the course of systematic research of new procedures for the quantitation of antibiotics and substances of clinical and pharmaceutical interest,Is17 we described in a recent paper a sensitive method of derivative spectrophotometry fsr the determination of cephacetrile and ceftezole in mixture.'*
Continuing with this investigation, we developed a simple quantitative method for the fast determination of mixtures of cephaloridine and cephalothin, which are semisynthetic analogues of cephalosporin C with a wide antimicrobial spectrum of activity (see structures). These compounds are characterized by closely overlapping absorption spectra. The aim of the present work was to demonstrate the relative ease with which the proposed zero-crossing derivative methodology overcomes this difficulty, allowing the simultaneous determination of the two drugs without the need for prior separations. The method i d sensitive and yields accurate and reproducible results. It has been applied to laboratory mixtures of the pure drugs and to mixtures of injections used parenterally for the treatment of diseases of the genitourinary and respiratory apparatus, otorhinolaringologic and obstetric-gynecological infections, skin and venereal diseases, etc. It cannot be excluded that other techniques, such as chromatography, would also give good results. The technology and applications of HPLC have been developed at a fast rate during the past years, although the detector still remains the factor that sometimes limits ultimate sensitivity and flexibility. The use of the derivative technique for resolution enhancement in HPLC and GLC, by applying the derivative technique to the elution profile itself, was discussed by Fell.'g The increasing interest in derivative spectroscopy is such that the method has been patented again to give a device for low-noise derivatives up to the eighth order, although for resolution enhancement in the UV-VIS range, the most useful derivatives are up to the fourth ~ r d e rFor .~ these reasons, coupled with analysis time, ease of operations, and widespread availability of commercial instruments with derivative capability, it is probably of value to present this work as a n encouragement to others to apply this simple and versatile technique in routine practical analytical work.
Experimental Section M a t e r i a l s h r e drug samples of cephaloridine and cephalothin
Na were purchased from Sigma Chemical Company, St. Louis, MO.
Cephaloridine
COOH
Cephalothin 0022-3549/88~700-0615$0 I .OO/O 0 1988, American Pharmaceutical Association
Stock solutions (0.2 mg/mL) were freshly prepared in water. Injectable dosage forms of Keflin (Eli Lilly S.p.A., Italy) were labeled to contain 1g of cephalothin Na per vial, and Keflodin (Eli Lilly S.p.A., Italy) and Ceporin (by Glaxo S.p.A., Italy) were both labeled to contain 1 g of cephaloridine per vial. The apparatus used was a Perkin Elmer 555 double beam UV-VIS spectrophotometer with 1-cm quartz cells. Suitable settings were: 1nm slit width, 4-s response time, and 120-ndmin monochromator scan speed. Sample Preparation and Procedure-A few microlitres of the cephalosporin stock solutions were added to a 5-mL volumetric flask which was made to volume with distilled water. The derivative spectra of the mixture against water was then recorded. The absolute Journal of Pharmaceutical Sciences / 615 Vol. 77, No. 7, July 1988
values of the first derivative were measured at 232 and 235 nm for the determinationof cephaloridine and cephalothin Na, respectively, and the values of the second derivative were measured at 253 and 262 nm for cephaloridine and cephalothin Na, respectively.
Results and Discussion Spectrophotometric M e a s u r e m e n t e I n Figure 1 we show the absorption (zero-order)spectra of the following: (a) cephaloridine (12 pgimL) with a maximum a t 239 nm; (b) cephalothin Na (12 pg/mL) with a maximum a t 236 nm; and ( c ) a mixture of cephaloridine and cephalothin Na (12 pg/mL each) with a maximum at 237 nm. The large overlap of the spectral bands of the two components prevents the formation, from the total zero-order spectrum, of any spectral feature utilizable for analytical purposes. We circumvented this problem by making use of the first- and second-derivative spectra of the mixture. Peak-to-peak and baseline measurements (generally referred to as graphical measurements) and zero-crossing measurements,'a are the most common techniques used to prepare analytical working curves. In practice, that measurement which exhibits the best linear response to analyte concentration, which gives a zero intercept on the ordinate axis of the calibration graph, and which is least affected by the concentration of any other component is selected. In the present instance, preliminary experiments showed the difficulty of a correct use of graphical measurements. This was probably due to the closeness of the overlapping zero-order spectra of cephaloridine and cephalothin with the subsequent poor resolution of both the first- and secondderivative spectra of the mixtures. Hence, the zero-crossing technique was preferred. This involves the measurement of the absolute value of the total derivative spectrum at an abscissa value corresponding to the zero-crossing wavelengths of the derivative spectra of the individual components. Measurements made at the zero-crossingof the derivative spectrum of one of the two components would be a function only of concentration of the other component.
The first- and second-derivative spectra of cephaloridine and cephalothin Na (12 pg/mL in all instances) are shown in Figure 2. In the first-derivative mode, the zero-crossings of cephaloridine occur a t 216 and 235 nm, while those of cephalothin occur a t 210 and 232 nm. In the second-derivative mode, 222 and 262 nm are the zero-crossings wavelengths of cephaloridine and 218, 240, 253, and 272 nm are those of cephalothin. Among these wavelengths, we selected (see below) 232 and 235 nm as optimal for determination, by the first-derivative mode, of cephaloridine and cephalothin, respectively; 253 and 262 nm were chosen for the quantitation of cephaloridine and cephalothin, respectively, by the second-derivative mode. At the other wavelengths, worse results were obtained: the calibration curves were poorly linear, the scattering of experimental points was somewhat unacceptable, and/or the intercept on the y-axis was significantly different from zero. First-Derivative Mode-In Figure 3(a) we present a series of first-derivative spectra of mixtures of 10 pg/mL of cephalothin Na plus increasing quantities of cephaloridine (from 4 to 28 pg/mL). In Figure 3(b) is a series offirst-derivative spectra +0.060;
/ -\
rossing s izofe ro1st- cderivatives)
-0.060'
I
I
1
1
+0.008
,\
-0.0081 200
250
300
i(nm)
Flgure 1-Absorption spectra of: (a) cephaloridine (12 pg/mL); (b) cephalothin Na (12 pg/mL); and (c) mixture of cephaloridine and cephalothin Na (12 pg/mL each). The reference was water. 616 i Journal of Pharmaceutical Sciences Vol. 77, No. 7,July 1988
1
1
I
I
220
240
260
280
1 (nm)
Flgure 2-( a) First- and (b) second-derivative spectra of cephaloridine (dashed lines) and cephalothin Na (continuous lines). The cephalosporin concentrations were 12 p g h L each, and the reference was wafer.
+
+0.016
0.22, 3
A
C
%
I
I
I
I
I
I
220
240
260
280
-0.016
I
W
I
I
I
I
a + U
3
-0.20
1
I
I
l(nm)
Figure 3-First-derivative spectra of mixtures of cephaloridine and cephalothin Na. (a) Cephalothin Na concentration, 10 pg/mL; cephaloridine concentrations, 4, 20, and 28 pg/mL in curves 1, 2, and 3, respectively. (b:, Cephaloridine Na concentration, 13 pg/mL; cephalothin Na concentrations, 4, 12, and 36 pg/fnL in curves 1, 2, and 3, respectively. The reference was water.
of mixtures of 13 pg/mL of cephaloridine plus increasing amounts of cephalothin Na (from 4 to 36 pglmL). Denoting the height of the first derivative a t 232 nm (zerocrossing wavelength of cephalothin) by h l and the height at 235 nm (zero-crossingwavelength of cephaloridine) by h2, we found that hX and h2 were proportional to cephaloridine and cephalothin concentrations, respectively. The variation of h l and h2 was not affected by the presence of cephalothin and cephaloridine, respectively (i.e., there is a mutual independence between the two cephalosporins for any ratio of concentrations, in the full range investigated). It is important to point out that, as expected, all curves in Figures 3(a) and (b) converge to an abscissa value (235 and 232 nm, respectively) corresponding to the zero-crossing wavelengths of the two cephalosporins. Second-Derivative Mode-In Figure 4(a) is reported a series of second-derivative spectra of mixtures of 10 pg/mL of cephalothin Na and increasing concentrations of cephaloridine (from 12 to 32 pglmL). In Figure 4(b) are the secondderivative spectra of mixtures of 13 pglmL of cephaloridine
and increasing amounts of cephalothin Na (from 4 to 36 pgt mL). The heights a t 253 nm (zero-crossingwavelength of cephalothin), h3, and at 262 nm (zero-crossing wavelength of cephaloridine), h4, were proportional to cephaloridine and cephalothin Na concentrations, respectively. As in the firstderivative spectra, the curves converge to the zero-crossing wavelengths of 262 nm [Figure 4(a)l and 253 nm [Figure 4(b)] for cephaloridine and cephalothin Na, respectively. Calibration Graphs and Statistical Analysis-The above technique allowed us to obtain the linear regression equations for mixtures of cephaloridine and cephalothin Na. These equations are displayed in Table I together with correlation coefficients, variance, and detection limits a t p = 0.05 level of significance and for n = 10 standard specimens. The ordinate values, H, of the lines of regression were obtained from the h l , h2, and h3, and h4 (mm) measurements and standardized as follows:""18 H = recorder divisions (h mm) x scale expansiord100 mm full scale. Beer's Law rigorously holds up to 28 and 36 pg/mL of cephaloridine in the first- and second-derivative modes, reJournal ofPharmaceutical Sciences / 617 Vol. 77, No. 7, July 1988
Table 1-Statistical Analysis of the Determination of Cephaioridine and Cephalothln in Mixtures by First- and Second-Derivative Spectrophotometry" Antibiotic Cephaloridine Cephalothin Na Cephaloridine Cephaiothin Na
Derivative Mode
nm
Regression Equation
Correlation Coefficient
Variance
Detection Limit,
(St)
W/mL
First First
232
Second Second
0.9999 0.9999 0.9999 0.9999
6.88E-09 1.04E-07
0.13 0.37
253
H1 = -5.73E-05 + 1.37E-03C H 2 = 6.71 E-06 + 1.86E-03~ H3 = - 1.84E-05 + 4.20E-04c H4 = -2.45E-05 + 2.59E-04C
2.40E-09
0.25
1.27E-09
0.29
'Number of standard specimens: n
A,
235 262 =
10; level of significance: p
=
0.05.
spectively, and up to 36 Fg/mL of cephalothin Na in both modes. At higher concentrations, we observed a progressive degradation of the shape of derivative spectra (i.e., a n increase of the background noise, with subsequent increase of the scattering of the experimental points around the corresponding calibration curves) because of the difficulty in taking correct heights measurements. This may appear to be in contrast to the not very high values of absorbance of the total zero-order spectra a t the concentrations and wavelengths used; in fact, the mathematical derivative operation would give erroneous results when the absorption spectra of the mixture are outside the range of measurement of the instrument. However, the development of a derivative spectrophotometric assay requires a great number of instrumental variables to be controlled, depending on the derivative module employed. Presumably, the observed degradation is an artifact of the electronic analogue RC device utilized, which computes the derivative with respect to time, as the spectrum is scanned a t constant speed. The linearity of calibration graphs in the useful concentration range and the negligible scatter of experimental points is clearly evidenced by the values of correlation coefficients and variances. Tests of significance of the experimental intercepts (a of lines of regression H = a + bc) showed that these did not differ significantly from the expected value of zero. A simplified method of estimating the differences a - 0 is based on the calculation of the quantities t = a/s,20 and their comparison with the tabular data for the t distribution (Table 11). The values calculated for t do not exceed the 95%criterion (t, = 2.311, which indicates that the intercepts of all lines of regression are not significantly different from zero. Hence, the proposed methods for mixtures of cephalosporins are free from procedural errors, depending on the concentration of one of the two components. However, a more rigorous approach requires the construction of a joint confidence region for the possible values of slope and intercept, because of the strong correlation which exists between the two parameters; this has the further advantage of allowing for the evaluation of the calibration graphs as a whole. The 95%joint confidence regions for slopes and intercepts of equations of regression reported in Table I, constructed by the method of Mandel and Linning,20.21are displayed in
Table ii-Estimate of Differences a Type of Measurement First Derivative ( h l ) First Derivative (h2) Second Derivative (h3) Second Derivative (h4)
Figure 5: they are bounded by a n ellipse having the point of best fit as the center (i.e., coordinates a and b). The question as to whether the calculated intercepts are not significantly different from zero is answered by determining whether the ellipses in Figure 5 contain points for which the intercept is zero. Such points lie on a vertical line through abscissa = 0, and it is evident that they fall well inside the ellipses, confirming the previous conclusions. In Figure 6 are the histograms of the absolute error, S , , ~ O in the determination of a given concentration, calculated by means of statistical analysis of the regression equations
l(C EPHALORlDlME 1st de 1.1
,EPHALOTHlN - 1 s t der.)
~
7-c
1.83 Y
n
0, v)
- 2 . 0 -1.0
-r - - - 7
-
I
-1.0
-0.5
I
I
0 *1.0
x10-4 I(CEPHALORIOINE-2nd d.)
- 0'
Antibiotic Cephaloridine Cephaiothin Na Cephaloridine Cephalothin Na
bb 1.04
2.31
x~o-'
0
*0.5
XlO"
0.034
0.62 1.14
INTERCEPT
a
Figure +Joint confidence regions, at p = 0.05 level of significance, for
f = 8.
slope and intercept of regression equations of cephaloridine and cephalothin Na by first- and second-derivative methods.
Experimental value of intercept of lines of regression. bTheoretical value of tat p = 0.05 level of significance; no. of degrees of freedom:
618 /Journal of Pharmaceutical Sciences Vol. 77, No. 7,July 1988
reported in Table I. The absolute error, sc, is defined by the relationship:*O
Recovery of Cephaloridine and Cephalothin in Pharmaceuticals-Because of the difficulty encountered in obtaining pharmaceuticals containing both the cephalosporins tested, we applied the proposed method t o the recovery of these antibiotics in mixtures of commercial injectable dosage forms. Working mixtures were prepared by dissolving the required amounts of pharmaceuticals in water. The assay was completed as described in the Experimental Section, and the results of five replicate determinations of mixtures of Keflin (cephalothin Na) with Ceporin and Keflodin (cephaloridine), respectively, are shown in Table IV. Satisfactory results were obtained: the method developed yields good recoveries and the minor differences observed may be considered acceptable.
where so = VZ(Y - Y,,,c)2/n - 2; Y is the experimental value of ordinate; Ycalc is the ordinate value calculated from the regression, equation; b is the angular coefficient of line of regression, and Z and P are the average concentration and ordinate values, respectively, for n standard specimens. From an inspection of the graphs in Figure 6, we see that the error is minimal at -16 pg/mL of cephaloridine and cephalothin Na in the first-derivative mode, and at -18 pg/mL in the second-derivative mode. In Figure 7 are the histograms of confidence limitsz0for the Conclusions determination of cephaloridine and cephalothin Na, in mixture, at p := 0.05 level of significance. The curves are plotted The above findings substantiate the usefulness of derivafrom calibration data in a particular way;crs.13-16.17-18.21-24 tive spectrophotometry for the assay of mixtures of cephalorithat is, as uncertainty (%) on concentration (relative error), dine and cephalothin Na, either in the pure or injectable tpsc/c,against the concentration of cephaloridine and cephaform. Both first- and second-derivative spectrophotometry lothin Na, respectively. These graphs can give useful inforgive reproducible and accurate results; statistical analysis of mation about the degree of precision that may be expected in the experimental data demonstrates that there are no subthe full range of concentrations investigated. Accuracy and stantial differences between the two techniques. precision were tested by five successive determinations on Although the analyte mixture studied was not easily found laboratory mixtures of the pure drugs (Table 111). in commercial pharmaceutical dosage forms, in our opinion
n
A
I
CEPHRLORIDINE
CEPHALOTHIN
tlST DERIV.1
tlST DERIV.)
0
v,
pg
m1-’
I
A CEPHRLORIOINE
CEPHRLOTHIN
12ND DERIV.1
(2ND DERIV.1
”
U
N n
cn
pg Figure 6-Histograms spectrophotometry.
p g m 1-1
m 1-’
pg m1.l
of the absolute error in the determination of cephaloridine and cephalothin Na by first- and second-derivative
Journal of Pharmaceutical Sciences / 61 9 Vol. 77, NO. 7, July 1988
_
r
Y
4
CEPHRLOTHIN
IlST D E R I V . 1
CEPHALOTHIN
[ZND DERIV.)
s U
\ '?
LI
pg
ml-I
Figure 7-Histograms of the variation of confidence limits, at p = 0.05 level of significance, in the form of uncertaintypercent on the concentration (relative error) of cephaloridine and cephalothin Na by first- and second-derivativespectrophotometry.
Table Ill-Replicate Determinations on Synthetic Mixtures of Cephaloridlne and Cephalothin Nominal Value, rdml
easurement
BC
Ab
First Derivative (hl and h2) Second Derivative (h3 and h4) a Mean of
Mean Value, pg/mLa
A
Standard Deviation
Coefficient of \lariatinn
O-/
B
A
B
A
B
0.040
0.39
0.22
0.050
0.37
0.20
9.0
18.0
8.99
18.01
0.035
15.0
25.0
15.03
24.99
0.055
five determinations. Cephaloridine. Cephalothin Na.
Table IV-Recovery
of Cephaloridine and Cephalothin in Mixtures of Injections'
Type of Measurement
Mixture
Mean Recovery, %
First Derivative (hl and h2) Second Derivative (h3 and h4)
Ceporin Keflodin Ceporin Keflodin
100.02 2 100.03 ? 100.04 2 100.03 2
0.035 0.041 0.055 0.052
Mixture Keflin Keflin Keflin Keflin
Mean Recovery, yob 99.99 2 100.01 2 99.99 2 99.99 2
0.048 0.050 0.058 0.057 ~~
Keflin powder for injections (Eli Lilly S.p.A. Italy), with 1 g of cephalothin Ndinjection; Ceporin (Glaxo S.p.A. Italy) and Keflodin(E1iLilly S.p.A. Italy) powder for injections, both with 1 g of cephaloridinehnjection.bMean of five determinations ? standard deviation; assay as percentageof label claim. a
620 /Journal of Pharmaceutical Sciences Vol. 77, No. 7, July 7988
the proposed methods eould be also applied to real samples, such as biological fluids.
References and Notes 1. O'Haver, T.C.;Green, G. L. Anal. Chem. 1976,48,312. 2. OHaver, T.C.Anal. Chern. 1979,51,91A. 3. OHaver, T.C. Clin. Chern.1979,25,1548. 4. Fell, A. F.; Smith, G. Anal. Proc. 1982,19,28. 5. Morelli, B. Anal st 1982,107,282. 6. Morelli, €3. Anarst 1983,108,870. 7. Morelli, B. Anarst 1983,108,1506. 8. Morelli, B. AnatLett. 1985,18(A19), 2453. 9. Fell, A. F.Proc. Anal. Diu.Chem. Soc. 1978,15,260. 10. Korany, M. A.; Wanby, A.M.; Mandour, S. Anal. Lett. 1984, 17(B12), 1373. 11. El-Yazby, F. A.; Barary, M. H. Anal. Lett. 1985,18fB51, 629. 12. Mohamed, M.E. Anal. Lett. 1986,19(11-12), 1323. 13. Morelli, B.; Peluso, P. Anal. Lett. 1985,18fB9), 1113.
14. Morelli, B.; Peluso, P. Anal. Lett. 1985,18(B15), 1865. 15. Morelli. B. Anal. Lett. 1987.20(1). 141. 16. Morelli: B. J . Pharrn.Biorned. Anal. 1987,5, 577. 17. Morelli. B.; Mariani, M.; Gesmundo, M. Anal. Lett. 1987,20f91, 1429. 18. Morelli, B. Anal. Lett 1988,21f11, 43. 19. Fell, A. F.Anal. Proc. 1980,17,512. 20. Nalimov, V.V. The Application of Mathematical Statistics to Chemical Analysis; Pergamon: Oxford, 1963;pp 167-189. 21. Mandel, J.; Linnin , F J. Anal. Chern. 1957,29,743. 22. Morelli, B.;Pelusos. Anal. Lett. 1986,19f5-61,503. 23. Morelli, B.Anal st 1986,111, 1289. 24. Morelli, B.A n a i s t 1987,112, 1395.
Acknowledgments Thanks are due to Dr. Mariateresa Gesmundo for scientific collaboration and for help in performing part of the experimental work.
Journal of Pharmaceutical Sciences / 621 Vol. 77, No. 7, July 1988