Simultaneous determination of ceftriaxone and streptomycin in mixture by ‘ratio-spectra’ 2nd derivative and ‘zero-crossing’ 3rd derivative spectrophotometry

Tahta, Vol. 41, No. 5, pp. 673-683, 1994 Copyright 0 1994 Else&r ScienceLtd Printed in Great Britain. All rights resewed 0039-9140/94 $7.00 + 0.00

SIMULTANEOUS DETERMINATION OF CEFTRIAXONE AND STREPTOMYCIN IN MIXTURE BY ‘RATIO-SPECTRA’ 2nd DERIVATIVE AND ‘ZERO-CROSSING’ 3rd DERIVATIVE SPECTROPHOTOMETRY BA~ILIO MOPELLI Universita’ degli Studi, Bari, Dipartimento di Chimica, Campus Universitario, Via E. Orabona, 4, 70126_Bari, Italy (Received 2 August 1993. Reoised 7 October 1993. Accepted 7 October 1993) Summar-Binary mixtures of antibiotics, ceftriaxone sulphate and streptomycin sodium, are assayed by ‘ratio-spectra’ 2nd derivative and ‘zero-crossing’ 3rd derivative spectrophotometry. Both procedures did not require any separation step and/or solving of equations. In the first method, calibration plots are linear up to 40 pg/ml of ceftriaxone at 225, 241.5, 255.5,255.5-241.5 and 225-241.5 nm (peak-to-peak), with r ranging from 0.9999 to 1.0000, and up to 30 rg/ml of streptomycin at 206 mn, r 0.9998. Detection limits, at P = 0.05 level of significance: ceftriaxone, from 0.24 to 0.47 pg/ml (at the various wavelengths), streptomycin, 0.42 rg/ml. By the second method, lines of regression are linear up to 40 pg/ml of ceftriaxone, at 227.8 and 241.7 nm (r, 0.9999 and 1.0000) and up to 35 pg/ml of streptomycin (r, 0.9999). Detection limits were calculated to be 0.35 and 0.15 fig/ml for ceftriaxone and 0.27 &ml for streptomycin. Both methods were successfully applied to laboratory mixtures and to mixtures of commercial injections for these drugs.

Derivative spectrophotometry offers a convenient solution to a number of well defined analytical problems, such as resolution of multicomponent systems, elimination of interference from sample turbidity and matrix background, and enhancement of spectral details.‘-’ Recently, there has been an evolution in UV-Vis spectrophotometry, by the application of computer control systems to the spectrophotometers, which have acted as a catalyst for the improvement of the traditional instruments, particularly in the introduction of microprocessors control, self-checking routines, rapid scanning, digital readout, visual display systems, etc. The results of this has been an improvement of sensitivity, precision and rapidity of data acquisition. Fundamental principles and applications of derivative spectrophotometry have been frequently and comprehensively reviewed.’ In the last years, derivative spectrophotometry is becoming increasing popular in the determinations of several drugs (alone or in mixture) in a pure form or in their formulations.‘k’3 From continuing investigations for determining substances of clinical interest,ikz3

we present and compare two different applications of derivative spectrophotometry for the simultaneous determination of ceftriaxone sulphate and streptomycin sodium in mixture, antibiotics with closely overlapping spectra. Ceftriaxone (Beta-lactam antibiotic) is a third generation cephalosporin characterized by a broad antibacterial spectrum and a resistance to Beta-lactamase-producing organisms. In addition to its antimicrobial activity (streptococci, staphylococci, pneumococci, et~.‘~), ceftriaxone exhibits a long elimination half-life and diffuses well into cerebrospinal fluid. These characteristics are of considerable clinical and, hence, analytical interest.25 Streptomycin (an aminoglucoside) is the second antibiotic, after penicillin-G, which was isolated from cultures of moulds. Because of its potential toxicity, its use in the human field, is today limited to serious affections, sometimes in associations with Beta-lactams or tetracyclines. However, it is still the main antibiotic for the therapy of tularaemia, Yersinia pestis, soft cancer (b. Ducrey), acute septicaemia and endocarditis,24 other than in veterinary field. 673

674

BASILIOMORELLI

We retain useful to propose reliable procedures to quantitate mixtures of these antibiotics. The methods are the “ratio-spectra” 2nd derivative and the “zero-crossing” 3rd derivative spectrophotometry. Both methods gave accurate results and applied favourably to either laboratory samples or commercial injections. An attempt to a comparison between the usefulness of the two methods was made. THEORY

Ratio-spectra method

Blanc0 et aLz6 reported a linear regression graphical method for resolving binary mixtures, called multi-wavelength linear regression analysis (MLRA). As extention of this method, a new spectrophotometric procedure for resolving mixtures, named “ratio-spectra” 1st derivative, has been, recently, developed and its basic principles described.27-2g “Zero-crossing”

derivative method

The fundamental principles and convention have been announced in the works of O’Haver and Green’ and Fell et a1.,3o*31 as well as in our previous papers.4T’“20

EXPERIMENTAL SECTION

Reagents

Stock solutions (0.2 mg/ml in water) were prepared from pure samples of ceftriaxone sulphate and streptomycin sodium (Sigma Chem. Co., U.S.A.), C and S, from this point, for brevity. Injectable dosage forms of rocefin (H. Hoffmann-La Roche and Cle. S. A., Switzerland) and of streptomycin (Farmitalia-Carlo--Erba, Italy), labelled to contain, respectively, lg of C and lg of S per vial were tested. Apparatus

Perkin-Elmer Lambda 3A spectrophotometer, coupled with Epson PCe computer and Graph& Pen Plotter MP 4100. Suitable settings. Scan speed 60 nm/min, wavelength interval 0.5 nm. The computer calculates the derivatives using the Savitzky-Golay method;32 a value of Delta = 6 was found optimal (in both methods) in connection with both slit width and wavelength interval (Delta represents the width

of the boundaries over which the derivative is calculated3*). Procedure

Suitable volumes of C and S stock solutions, expected to contain up to 40 pg/ml of C and 30 (ratio-spectra method) or 35 pgglml (zero-crossing method) of S were mixed in a 5-ml calibrated flask and diluted to volume with distilled water. (a) Ratio-spectra method. Ratio-spectra for mixtures of C + S were suitably obtained by The concentration of C following the theory. 27-2g was proportional to the value of 2nd derivative of the ratio-spectra at 225 nm (a maximum), 241.5 nm (a minimum), 255.5 nm (a maximum), 255.5-241.5 and 225-241.5 nm (peak-to-peak). The concentration of S was proportional to the amplitude of the ratio-spectra 2nd derivative, at 206 nm. (b) Zero-crossing method. The 3rd derivative spectrum of mixtures of C + S was recorded against water and the values of derivatives were measured at 227.8 and 241.7 nm (zero-crossings of S) for the determination of C, and at 216.4 nm (zero-crossing of C) to determine S. Procedure for injections

The contents of each vial was dissolved in two separate 500-ml calibrated flasks and made to volume with distilled water. Suitable aliquots of the two antibiotics were mixed in 5-ml volumetric flasks and diluted to the mark with distilled water. Then the assay was completed as described above under procedure for both methods. The percentage recovery of the two drugs was computed from the corresponding regression equations of C and S. RESULTS AND DISCUSSION

Figure 1 shows the zero-order spectra of C (10 pg/ml) and S (30 pgg/ml) and their sum spectrum. The spectra of the two antibiotics closely overlap, hence we circumvented the problem by making use of derivative spectrophotometry. (a) Ratio-Spectra 2nd derivative method

In a preliminary investigation, various concentrations of C and S as divisors were tentatively assayed. A correct choice of the concentration of divisors is fundamental for several reasons.27-2g Among these, in the wavelength range where the absorbance of the standard spectrum used as

675

Simultaneous determination of ceftriaxone and streptomycin

divisor is zero or below the base-line the noise of ratio-spectra is greatly increased (also an high smoothing function may result insufficient). Hence, a certain overlap of spectra in the working wavelength region is actually desirable, to avoid an increase of the error.27 Then, if the concentration of divisor is increased or decreased, the resulting derivative values (hence, the slope of calibration graphs) are proportionately decreased or increased,27-2g with consequent variation of both sensitivity and linearity range. From several tests, the best results in terms of signal-to-noise ratio, sensitivity, repeatibility and range of validity of Beer’s law, followed using as divisors standard spectra of 10-l 1 pg/ml of S and 12-13.5 pug/ml of C, with minimal differences. We selected, for all subsequent measurements, standard

spectra of 10 pg/ml of S and 12 ,ug/ml of c. Because of the wrong results obtained with the 1st derivative of the ratio-spectra, an attempt was made to use the 2nd derivative. No problem arose with the last one, so that the 2nd derivative was preferred for a better resolution of the ratio-spectra and more accurate and precise results. Figure 2 shows a series of spectra of samples of S (from 12 to 30 pg/ml) divided by the standard spectrum of C (dashed lines, left ordinate) and a series of C spectra (from 10 to 30 pg/ml) divided by the standard spectrum of S (continuous lines, right ordinate). Because of a certain extent of the noise level of the ratio-spectra, we found useful a smoothing function of 7, on the basis of the Savitzky and Golay method.32

1.2000

\

o.sOo

0.7200

A32

0.4300

0.2400

0 .oooo 200.0

220.0

240.0

220.0

220.0

300.0

320.0

340 .o

NM

Fig. 1. Absorption spectra of (1) streptomycin sulphate (30 pg/ml), (2) cefiriaxone sodium (10 rg/ml) and (3) their sum spectrum. Reference, water.

BASIL10 MORm.LI

676

i .sooo

1.2000

0.2000

A22

0.2000

0.3000

0.0000

I

I

I

I

I

I

I

Fig. 2. Ratio-spectra for different concentrations of streptomycin sulphate (12, IS, 20,25 and 30 rg@l, dashed cwves I-5, divisor ceftriaxone sodium 12 pg/ml, left ordinate scale) and of ceftriaxone sodium (10,15,20,25 and 30 pg/ml, continuous curves l-5, divisor streptomycin sulphate 10 rg/ml, right ordinate scale).

In Fig. 3 are depicted the corresponding 2nd derivatives of the the ratio-spectra of Fig. 2 (a value of DELTA = 6 was found optimal). For calibration graph of S (dotted lines, left ordinate), the amplitude of the peak of 2nd derivative at 206 nm was selected; for calibration graphs of C (continuous lines, right ordinate) five derivative values were selected as optimum, i.e., 225, 241.5, 255.5 mn corresponding to two maxima and a minimum, and 255.5-241.5 and 225-241.5 nm (adjacent minimum and maximum). The absence of interactions between C and S at the selected wavelengths it is clearly evidenced. The little interaction at 241.5 nm have no influence on the results, as it is demonstrated by the intercept of the corresponding line of regression (see later Tables 1 and 2).

Hence the interference is only apparent, i.e. it is a graphic artifact, due to the extent of the difference between the amplitudes of left and right ordinate scales. The regression equations for C and S, calculated as described above, are assembled in Table 1 (under ratio-spectra section), together with correlation coefficients, variance’8*33 and detection limits18*33J4 at P = 0.05 level of significance. The lines of regression are linear up to 40 pg/ml C and 30 pg/ml S, with intercept near to zero. Both linearity of the lines of regression and negligible scatter of experimental points are evidenced by correlation coefficients and variances, The best results, in terms of correlation coefficient and detection limit, are obtained for C at 225 nm.

Simultaneous determination of ceftriaxone and streptomycin

Tests of significance of the intercepts of the regression lines, showed that the experimental intercepts, “a”, did not differ significantly from the theoretical value, zero. A simplified method of calculating the differences a - 0, followed of the quantities from the calculation t = a/&J m33 and their comparison with the tabulated data for the Student’s_t distribution (Su is a measure of the accuracy of the determination of a, which is calculated by making use of the law of the accumulation of errors18*33 ). The results of these calculations are reported in Table 2. The values calculated for t never exceed the 95% criterion, 2.31, which denotes that the intercepts of all regression lines (including those of “zero-crossing” method, described later) are not significantly different from zero, and hence the proposed methods are free from procedural errors, including those depend-

677

ing on the simultaneous presence of two components. To test accuracy and precision, five replicate determinations on various syntehtic mixtures of C + S were performed. For determining C, the stored spectra of the mixtures were divided by the standard spectrum of S (10 pg/ml). The ratio-spectra thus obtained were smoothed (level 7) and the 2nd derivatives (DELTA = 6) were recorded. The values of 2nd derivatives at the working wavelengths, previously reported, allowed to determine the concentration of C from the regression equations of Table 1. Analogously, for determining S, the spectra of the mixtures were first divided by the standard spectrum of C (12 pg/ml) and the resulting ratio-spectra smoothed (level 7) and derivated (DELTA = 6). The concentration of S was calculated, as above, from the derivative value and 0 A060

0.0500

0.0304

o.oz!w

Da

0.0152

-

-0.0600

-

Da

-

-0.3200

0.0038

nm

-0.ooao

Fig. 3. 2nd derivative spectra of the ratio-spectra of streptomycin, dotted lines, left ordinate and ceftriaxone, continuous lines right ordinate, shown in Fig. 2. The working wavelengths are indicated.

BASILIOMORELLI

618

Table 1. Statistical analysis of the determination of aextriaxone and streptomycin in mixture by “ratio-spectra” derivative and “zero-crossing” 3rd derivative spectrophotometry* Wavelength (nm)

Drug

Regression equations “Ratio-spectra”

s C C C C

206 225 241.5 255.5 255.51241.5 2251241.5

S C C

216.4 227.8 241.7

D2 D2 D2 D2 D2 D2

2nd

Correlation coefficient

Variance (4)

Detection limit olglml)

0.9998 ;Kz 09999 0.9999 0.9999

l.O8E-07 2.33E-06 2.32E-05 1.58E-06 3.84E-05 3.52E-05

0.42 0.24 0.40 0.47 0.42 0.32

0.9999 0.9999 1.0000

2.08E-09 1.73E-08 1.29E-09

0.27 0.35 0.15

2nd Derivative

= 1.39E-04 + 1.67E-03Cs = - 1.35B03 + 1.35E-02Cc = 3.17E-03 + 2.59E-02Cc = -3.62E-04 + 5.78E-03Cc = 2.72B03 + 3.17E-02Cc = 2.17E-03 + 3.94E-02Cc

‘Zero-crossing” 3rd Derivative D3 = 1.61E-03 + 3.63E-04Cs D3 = 7.69E-05 + 8.05E-04Cc D3 = - 5.98E-05 + 5.26E-04Cc

*S = Streptomycin sulphate; C = ceftriaxone sodium. Cs and Cc, concentrations of streptomycin sulphate and ceftriaxone sodium @g/ml). Number of standard specimens, n = 10; level of significance, P = 0.05

the corresponding line of regression at 206 nm. The experimental findings are shown in Table 3 (under “ratio-spectra” method). (b) “Zero-crossing”

3rd derivative method

Preliminary tests were carried-out to choice the derivative order and the types of measurements, i.e. “graphical” (peak-to-base-line, or peak-to-peak) or “zero-crossing,1*30,31 which gave the best results. 1st and 2nd derivative gave an insul-hcient resolution of the absorption spectra of mixtures and, consequently, unacceptable conditions. The was resolution power of 4th derivative very satisfactory; unfortunately, the noise was greatly increased, hence it required too high a level of smoothing, with consequent distortion and variation of shape of curves and location of peaks. On the contrary, no problem arose with the 3rd derivative, which was selected as optimum.

Table 2. Estimate of differences a - 0, where a is the intercept of the regressions lines Method “Ratio-spectra” 2nd derivative

“Zero-crossing” 3rd derivative

Wavelength (nm) 206 225 241.5 255.5 255.51241.5 2251241.5 216.4 227.8 241.7

Dmst S C C C C

C S

C C

a/Sa

0.67 1.62 1.20 0.53 0.80 0.67 2.10 1.04 2.01

*Theoretical value oft at P = 0.05 level of significance, 2.31 (number of degrees of freedom, f = n - 2 = 8). $S = Streptomycin sulphate; C = ceftriaxone sodium.

Graphical measurements gave calibration graphs with greater scatter of experimental points and/or intercept on y-axis quite different from zero. The zero-crossing method was considered more convenient in terms of rapidity and simplicity, and gave linear calibration curves with zero (or near to zero intercept, as expected). The 3rd derivative spectra of S, dotted curves 1 and 2, and C, continuous curves 3 and 4, (smoothing 7, DELTA 6) are shown in Fig. 4, with the zero-crossing wavelengths. Among these, 227.8 and 241.7 nm for determination of C and 216.4 nm for S, were selected as working wavelengths in that measurements of the absolute value of the total-derivative spectra taken at these wavelengths gave the best response to analyte concentration. Note that two spectra at different concentration for each one of the two drugs are reported, in order to evidence the repeatibility of the values of the zero-points. In Fig. 5 are presented typical sets of 3rd derivative spectra of laboratory mixtures of 5 pg/ml of C and increasing concentration of S, from 8 to 30 pg/ml (dotted curves 1 to 5, left ordinate) and of mixtures of 15 ~1g/ml of S plus increasing amounts of C, from 15 to 40 pgg/ml (continuous curves 1 to 5, right ordinate). All curves were recorded with a value of DELTA = 6 and a smoothing level of 7, which eliminated a little noise without distortion, variation of shape of curves or location of the characterizing wavelengths. It is interesting to observe that all curves converge to distinct isosbestic points, corresponding to zero-crossing wavelengths (indicated by arrows) of S and C, i.e. irrespective of the concentration of S and C, respectively.

Simultaneous determination of cefiriaxone and streptomycin

0.0040 D3 -0.0040

-0.0120

Fig. 4. 3rd derivative of streptomycin sulphate, dotted lines (1) 20 &ml and (2) 10 &ml; and Ceftriaxone sodium, continuous lines (3), 20 pg/ml and (4) 10 &ml. The arrows indicate the zero-crossing wavelengths. Reference, water.

The linear regression equations for mixtures of C and S are assembled in Table 1 (under “zero-crossing” section), together with the correlation coefficients, variances and detection limits, at level of significance of P = 0.05. Beer’s law is followed for concentrations up to 40 pg/ml and 35 pgg/ml of C and S, respectively. The best results in terms of correlation coefficients and detection limits were obtained for C at 241.7 nm. As in “ratio-spectra” method, an estimate of differences a-0 is reported in Table 2 (under “zero-crossing” section). Also in the present instance, the values calculated for t, did not exceed the 95% criterion of t = 2.31. To test accuracy and precision, five determinations on the same synthetic mixtures of C + S of the previous method were performed. The results reported in Table 3 (under “zero-crossing” section) confirm that accuracy and precision are very satisfactory. Assay of Ceftriaxone ture of injections

and Streptomycin

in mix-

Because of the difficulties encountered in obtaining pharmaceuticals products containing

both the antibiotics tested in mixture, we applied the two proposed methods to the recovery of C and S, by mixing commercial injectable dosage forms of C and S. The assay was performed as previously described in the Experimental Section, under “Procedure for injections”. The results of five replicate determinations of mixtures of Rocefin and Streptomycin vials are shown in Table 4. Both the procedures yield good recoveries and the minor differences observed may be considered acceptable. We consider a useful test to see if there was a statistically significant difference in the mean recoveries in mixtures of injections, carried-out “ratio-spectra” “zero-crossing” and by methods, i.e to verify the null hypothesis ~1l-c12 = 0 (p 1 and ,u2 are the mean recovery with the two methods). This problem can be solved with the help of t-criterion.33 In Table 5 are reported the calculated t-values at the 95% confidence level. Full values found for t do not exceed the theoretical t-value 2.3 1, therefore the null hypothesis is verified, by denoting no significant differences in the results achieved by the two methods.

BASILIOMORELLI

680 CONCLUSIONS

One of the purposes of the present work was to investigate if the 2nd derivative of ratio-spectra was suitable for resolving mixtures of compounds with overlapping spectra and to verify if it was possible to obtain better findings in respect to the ratio-spectra 1st derivative method in all cases in which the last one gives poor results or fails completely (it is useful to remember that the ratio-spectra method presents many limitations*“~. It is important to note that in previous works, derived from the

work of Blanc0 et al.,26 it is always used the 1st derivative, while nothing is reported about the capability of using 2nd or higher derivative orders. A novelty of this paper is the demonstration that 2nd derivative may be profitably utilized in the ratio-spectra method and, moreover, that it may be an advantageous alternative to 1st derivative. In fact, all preliminary tests made in the present work with 1st derivative (as reported in results and discussion), by testing also various concentrations of divisors, always gave wrong results in terms of scatter of experimental points

0.0028

-0.0022

-0.0071

D3

-0.0121

-0.0170

-0.0220

I

I

I

I

I

I

I

I

i

-0.0350

Fig. 5. Dotted curves, left ordinate: 3rd derivative spectra of binary mixtures with constant concentration of ceftriaxone sodium (5 pg/ml) and increasing concentration of streptomycin sulphate (8, 10, 20, 26 and 30 pg/ml, curves l-5) The working wavelength at 216.4 nm is marked. Continuous curves, right ordinate: 3rd derivative spectra of binary mixtures, with constant concentration of streptomycin sulphate (15 pg/ml) and increasing concentration of ceftriaxone sodium (15,25,30,36 and 39 fig/ml, curves l-5). The working wavelengths at 227.8 and 241.7 nm are marked. The arrows point-out the isosbestic points, coinciding with the zero-crossing wavelengths of streptomycin sulphate and ceftriaxone sodium, respectively. Reference, water.

Simultaneous determination of ceftriaxone and streptomycin

681

Table 3. Replicate determinations on laboratory mixtures of ceftriaxone and streptomycin by “ratio-spectra” 2nd derivative and “xero-crossing” 3rd derivative methods* 3rd Derivative “zero-crossing method”

2nd Derivative “ratio-spectra method Foundt

Nominal value Ratio

s OlglW

C

c/s

206 S

225 C

241.5 C

255.5 C

255.51 241.5 C

2251 241.5 C

216.4 S

227.8 C

241.7 C

20

2.00

40.00

1.98 *0.03 (1.52)

40.02 kO.06 (0.15)

39.96 kO.06 (0.15)

39.80 f0.06 (0.15)

40.05 *0.05 (0.12)

39.90 f0.04 (0.10)

2.03 f 0.03 (1.48)

39.75 *0.04 (0.10)

39.80 kO.02 (0.05)

1

20.00

20.00

20.09 *0.05 (0.25)

20.18 40.09 (0.45)

19.95 *0.05 (0.25)

20.20 *0.07 (0.35)

20.08 f0.06 (0.30

20.09 f 0.02 (0.10)

20.02 *0.03 (0.15)

20.10 +0.04 (0.20)

20.07 kO.02 (0.10)

0.60

25.00

15.00

24.95 *0.05 (0.20)

15.10 *0.05 (0.33)

15.05 + 0.03 (0.20)

14.98 kO.05 (0.33)

15.01 +0.02 (0.13)

15.07 kO.04 (0.27)

25.20 kO.04 (0.16)

14.99 kO.05 (0.33)

14.96 *0.03 (0.20)

0.53

8.00

5.00

8.05 f 0.05 (0.62)

4.98 &0.04 (0.80)

5.01 f 0.02 (0.40)

5.10 +0.04 (0.78)

*?z2 (0.40)

5.05 kO.03 (0.59)

8.07 f 0.05 (0.62)

5.09 +0.04 (0.79)

4.99 kO.02 (0.40)

30.09 +0.04 (0.13)

1.99 io.03 (1.51)

1.99 *0.01 (0.50)

2.05 *0.03 (1.46)

1.99 *0.01 (0.50)

2.00 f 0.02 (1.00)

30.10 *0.01 (0.33)

2.02 kO.01 (0.49)

2.02 *0.01 (0.49)

0.07

30.00

2.00

lS = Streptomycin sulphate; C = ceftriaxone sodium. tMean f standard deviation @g/ml) for five determinations,

with RSD(%) in parentheses.

and/or intercepts and slope of lines of regression (consequently, high values of detection limits), narrow range of validity of Beer’s law and, obviously, unacceptable accuracy and precision in the assay of synthetic mixtures and bad recovery in mixtures of injections. As concerns the choice of ceftriaxone and streptomycin, we emphasize that this mixture has been principally selected for the following reasons: to test the 2nd derivative ratio-spectra by means of compounds with strictly overlapping spectra; for its importance in therapeutic field; as a consequence of the toxic effects of streptomycin especially to children, old patients and patients with renal failure, a particularly sensitive and accurate quality control of vials is recommended. Although the mixture studied

was not easily available, i.e. with the two antibiotics formulated together, we emphasize that streptomycin and ceftriaxone (or other beta-lactam antibiotics) are administered simultaneously in serious diseases, such as, for example, acute endocarditis,“*3’ for the synergic effect developed by the two drugs. In particular, ceftriaxone for its action on the cell-wall, allows to obviate to the impermeability of Streptococci-D from streptomycin. The mixture of vials prepared by us corresponds very well to the mixture which is administered in therapy, hence it may be considered as a “real” sample. Furthermore, for the toxicity of streptomycin24 remarked above, it is advisable to scrupulously monitor the quantities administered, hence we retain very useful the procedures described

Table 4. Recovery of ceftriaxone and streptomycin in mixtures of injections by “ratio-spectra” “zero-crossing” 3rd derivative methods* Recovery, %t 2nd Derivative “ratio-spectra” method

Mixture

206

Rocefin Streptomycin

101.6 *0.5

225

241.5

102.1 *0.5

102.0 *0.5

Wavelength, mn 255.51 255.5 241.5 102.0 +0.5

101.5 *0.4

2nd derivative and

3rd Derivative “zero-crossing” method 2251 241.5

216.4

102.1 *0.3

227.8

241.7

102.1 *0.5

102.0 kO.3

101.8 *0.5

*Rocefin (F. Hoffmann-La Roche and Cle S.A., Switzerland) and Streptomycin (Farmitalia-Carlo Erba S.p.A., Italy) powder for injections, labelled to contain 1 g ceftriaxone and 1 g streptomycin for vial, respectively. tMean and standard deviation for five determinations, given as percentage of the label claim.

BASILIOMORELLI

682

Table 5. Comparison of the mean recovery in mixture of injections by “ratio-spectra” 2nd derivative and “zero-crossing” 3rd derivative spectrophotometry, with the help of the r-criterion Injection Ro&in

Streptomycin

Method Ratio-spectra Zero-crossing Ratio-spectra Zero-crossing Ratio-spectra Zero-crossing Ratio-spectra Zero-crossing Ratio-spectra Zero-crossing Ratio-spectra Zero-crossing

Wavelengths (m) 225 227.8 241.5 227.8 255.5 227.8 255.5/241.5 227.8 2251241.5 227.8 206 216.4

Wavelengths

t -talc.

@ml

r-talc.

0.06

225 241.7 241.5 241.7 255.5 241.7 255.512415 241.7 2251241.5 241.7

0.49

0.45 0.15 1.99 0.04

0.15 0.24 2.25 0.65

0.43

*Number of degrees of freedom f = n I +x2-2 = 8 (n 1 = n2 = 5, number of samples); theoretical value of t at P = 0.05 level of significance, 2.3 1.

which allow an accurate and precise assay of mixture before inoculation. However, this is not the only case of literature of mixtures of drugs formulated separately.7*‘~‘5~‘8~27-2p*3G37 Furthermore, from 50 to 80% of S and 60 to 65% of C administered are excreted unaltered in the urine,“*3* hence, in our opinion, the methods could be easily applied to the analysis of urine. The analysis of biological fluids was outside the scope of this work, and also difficulties in obtaining these samples from local hospitals (for the lack of patients submitted to the particular therapy specified above) prevented this type of investigation from being undertaken. However, the results presented above may be an incentive to other workers to apply such methods to the analysis of urine. This task should be quite simple, even for the high concentrations of antibiotics excreted.3Q Furthermore, the clinical efficacy of the therapy can be enhanced by the ability to determine concentrations accurately and rapidly, thereby ensuring that bactericidal levels are achieved and maintained and so to enable dosage adjustments to be made. Apart from this, another purpose of this work was to compare two different applications of derivative spectrophotometry, by putting in evidence the respective advantages and/or drawbacks. From an analysis of the results obtained with the two methods do not appear to have substantial differences. Both, “ratio-spectra” 2nd derivative and “zero-crossing” 3rd derivative give reliable findings with good accuracy and precision. A certain superiority of the “zero-crossing” over the “ratio-spectra”, results from an

observation of detection limits in Table 1, and of the SD and RSD(%) values in Table 3, generally smaller in the “zero-crossing” than in “ratio-spectra” method. Vice versa, in the estimate of differences a-0 (Table 2), on average, better results were found by the “ratio-spectra” method. Apart from this, the main advantage of the “ratio-spectra” method may be the chance of doing measurements in correspondence of peaks, hence a potential greater sensitivity and accuracy. On the contrary, the main disadvantages of the “zero-crossing” method are, as it is well known, the risk of small drifts of the working wavelengths (even if this risk was practically eliminated with the recent spectrophotometers) and the circumstance that the working wavelengths generally do not fall in correspondence of peaks of the derivative spectrum. This may be particularly dangerous when the slope of the spectrum is very high with consequent loss of accuracy and precision (even if a suitable enlarging of x-axis scale minimizes well this effect), and/or the working wavelength is in proximity of the base of the spectrum (less sensitivity). Fortunately, in the present case, the above circumstances did not occur. Finally, the “zero-crossing” derivative is a more rapid, simple and universal analytical method for solving multi-component systems, than the “ratio-spectra”. In fact, the last one requires an accurate investigation about the influence of the variables, as previously described. There are mixtures which cannot be assayed by this method*’ as personally verified with binary-samples of other antibiotics.

Simultaneous determination of ceRriazone and streptomycin

In addition to this, a greater number of measures is required, hence the method is more time spending. In our judgment, the “ratio-spectrg” may b

an interesting

and B&d

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