Influence of the differentiation system on the analytical parameters for the spectrophotometric determination of clonazepam in urine

MICROCHEMICAL

JOURNAL

4,

249-257 (1991)

Influence of the Differentiation System on the Analytical Parameters for the Spectrophotometric Determination of Clonazepam in Urine F.RANDEZ-GIL,A. Department

of Analytical

Chemistry,

SALVADOR,AND M. DE LAGUARDIA' University of Valencia, Valencia, Spain

50, Dr. Moliner

St., 46100 Burjassot,

Received January 24, 1991; accepted May 2, 1991 Electronic and mathematical differentiation of the ultraviolet visible spectra were compared from the study of the analytical parameters of the direct determination of clonazepam in urine samples. Third-order derivative spectra were used. The mathematical diierentiation provides higher sensitivity, a lower limit of detection, and better accuracy and precision than the electronic one. The use of a diode array instrument provides lower sensitivity than that found using a conventional spectrometer; however, the limit of detection obtained in both cases is of the same order. e IWI Academic press. IX.

INTRODUCTION

Derivative spectrophotometry is, at present, a well-established technique (1-3) which permits improved qualitative information of ultraviolet-visible spectroscopy (4, 5), and provides quantitative information about the overlapping bands in the zero-order spectra of multicomponent samples (64). It has also been demonstrated that the use of the derivative spectra allows for the direct analysis of a compound in a complex matrix (Wl). Derivative spectra can be obtained optically (12), electronically, or mathematically. Among these methods the use of analog resistance-capacitance devices, which permit the derivative to be obtained as a function of time, and those which employ mathematical treatments of the digitalized signals are the most commonly used. Electronic differentiation is carried out simultaneously with spectrum recording and it depends strongly on the instrumental parameters employed to obtain the spectra such as the slitwidth, scan speed, and differentiation time (13, 14). Mathematical differentiation is carried out on the output signals and consists of determining the slope of the spectra at each wavelength. These parameters can be measured from the quotient between the increment of absorbance and a fixed wavelength interval. AA/AA, by manual calculation, but in general, a microcomputer is used to generate derivative spectra. A variety of algorithms are employed to obtain derivatives, the Savitsky-Golay being the most commonly used (15); recently it has been demonstrated that the Fourier transform algorithm offers the highest reproducibility (16). ’ To whom correspondence

should be addressed. 249

250

RANDEZ-GIL,

SALVADOR,

AND DE LA GUARDIA

At present the popularization of the diode array instruments has increased interest in the mathematical differentiation (17, Z8). In recent years a series of studies have been carried out to compare the different strategies for obtaining derivative spectra (24) and also to evaluate the instrumentation commercially available (19). The comparative studies have focused on the signal-to-noise ratio (13); however, it has been reported also that the precision and accuracy of the analysis by derivative ultraviolet spectrophotometry can be affected by the method used to interpret the data of electronic differentiation (20). In the present paper we have employed several analytical parameters, such as, sensitivity, limit of detection, precision, and accuracy to compare the electronic differentiation with the mathematical one and also the use of a conventional spectrometer with a diode array instrument. Clonazepam in urine, has been used as a test system. We have selected this benzodiazepine because it has a broad spectrum of activity against the various types of epilepsy (21) and is more potent than other benzodiazepines as well as phenytoin and the barbiturates in patients whose condition has proved resitant to those antiepileptic drugs. Therefore clonazepan determination is very interesting in clinic analysis. MATERIALS AND METHODS Apparatus A Shimadzu UV 240 double beam ultraviolet-visible spectrophotometer equipped with a l-cm quartz cell and an OPI 2 optional program, which permits the first four derivative spectra to be obtained both mathematically and electronically, was used. 0.1

A

“4 -0 m>” -0

-0.1 225

275

325 A (nm)

375

425

475

395

400

405

410

415

420

h (nm)

FIG. 1. Ultraviolet visible and derivative spectra of aqueous solutions of clonazepam and urine samples containing this drug. (A) Ultraviolet spectra of clonazepam in 0.1 M NaOH (-) and urine sample containing clonazepam (---). (B) Third-order derivative spectra of clonazepam in 0.1 M NaOH (-) and urine sample containing clonazepam (- - -).

SPECTROPHOTOMETRIC

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OF CLONAZEPAM

251

A Hewlett-Packard 8452 A diode array spectrophotometer equipped with a I-cm quartz cell and a Vectra ES/12 calculus program, which provides the first nine derivatives of the normal spectra mathematically, was used. Reagents

NaOH (0.1 M) aqueous standards containing 2,4,8,12,16, and 20 mg liter-’ clonazepam were prepared from a 100mg liter- ’ stock solution (0.1 M NaOH) of the drug, obtained from the Toxicology Department of the University of Valencia. 0.1

3E-2

A

B ,'i !

r-l

--I

G

G \ QO n -0

-20 -w

i \J

4

-0.1

1 420

I

400

410

-3E-2

410

J

420

h (nm)

h b-4 8E-3

-SE-3

I

400

C

+ 420

400 A (nm)

FIG. 2. Third-order electronic derivative spectra of clonazepam obtained with differentiation slitwidth of (A) 1 nm, (B) 2 mn, (C) 4 nm. (-) Spectrum of a 16 mg liter-’ standard solution of clonazepam. (- - -) Spiked sample of urine containing 16 mg liter-’ of clonazepam.

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RANDEZ-GIL,

SALVADOR,

AND DE LA GUARDIA

Spiked samples were prepared by adding known amounts of clonazepam to diluted urine samples, making the diluted samples 0.1 M in NaOH. General Procedure The calibration curve of clonazepam was obtained using the third-order derivative spectra, because in that order more precise and accurate results were obtained in the direct determination of this benzodiazepine in biological fluids (22). From the calibration curve obtained, both electronically and mathematically with the conventional spectrometer and mathematically with the diode array, the corresponding sensitivity values were established from the slope and the limit of detection determined from the variation of the blank readings. Accuracy and precision were determined from the analysis of synthetic samples and evaluated from the recovery percentage and the variation coefficient, respectively. RESULTS AND DISCUSSION The Use of a Conventional

Spectrometer

The analytical parameters of the determination of clonazepam in urine have been established using both the electronic and the mathematical differentiation with the OPI 2 UV 240 Shimadzu spectrophotometer. Figure 1 indicates the zeroand the third-order absorbance spectra of Clonazepam. As can be seen, the use of the derivative spectra permits comparable values for standards and spiked samples with the same concentration of drug. Electronic Differentiation The characteristics of each derivative spectra, obtained via electronic differentiation, depend on the slitwidth, the differentiation time, and the scan speed. However, the program used with the spectrophotometer does not permit change in the differentiation time, which is automatically fixed according to the scan speed employed. The effects of the slitwidth and the scan speed on the direct analysis of clonaTABLE 1 Effect of the Slitwidth on the Analytical Parameters of the Determination of Clonazepam in Urine, by Electronic Differentiation Analytical parameters Slithwidth (nm)

Sensitivity

R.P.

V.C.

LOD

(nm)

A

@pm-9

(%I

(%)

@pm)

1 2 4

418.8 418.0 416.2

-4.0764 x 10-3 -1.0709 x 10-j -2.7884 x 1O-4

112 115 118

4.4 1.8 2.2

1.0 0.7 0.7

Note. Sensitivity was evaluated from the slope of the regression line in derivative absorbance units for ppm of drug. R.P., recovery percentage in the analysis of spiked samples containing 16 ppm of drug. V.C., variation coefficient of 10 readings of three independent analyses. LOD, limit of detection for a probability level of 95% (K = 3).

SPECTROPHOTOMETRIC

DETERMINATION

253

OF CLONAZEPAM

zepam in urine were studied in order to obtain the experimental conditions at which higher sensitivity and better precision could be found. Eflect of the slitwidth. Figure 2 shows the third-order derivative spectra of a standard solution containing 16 mg liter - ’ of clonazepam and a spiked sample of urine with the same concentration. It can be seen that a change in the slitwidth from 1 nm to 2 and 4 nm provides a smoothing of the spectra with a strong reduction in the sensitivity obtained for the d3AldA3 experimental values. On the other hand an appreciable wavelength shift can be observed, as has been indicated previously by other authors (14). 3E-2

3E-2

-3E-2

I

400

410

1 420

-3E-2

400

A (nm>

410

420

A (nm) 3E-2

3E-2

I

-3E-2 )

410

h (nm>

’ 420

-3E-2

-

--

4ocl

I

410

I

420

A (nm>

FIG. 3. Effect of the scan speed on the clonazepam derivative spectra. (A) Fast, (B) medium, (C) slow, (D) accumulative. In all cases a register speed of 4 nmkm and a slitwidth of 2 nm was employed.

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SALVADOR,

AND DE LA GUARDIA

The effect of this instrumental parameter on the analytical features of the direct analysis of clonazepam in urine at the third-order derivative spectra has been evaluated (see Table 1) at the absorbance wavelengths at which the measurements for samples and standards were more coincident. Data in Table 1 evidences that the use of higher slitwidth provides lower sensitivity but higher stability. The variation coefftcient of 10 readings corresponding to each one of three independent analyses of a sample is lower for the higher slitwidth and so the stability of the blank provides a lower limit of detection in this case. The accuracy is poor at increasing values of the slitwidth assayed. 0.2

\

405

h (nm>

410

415

420

h (nm)

0.1

-0.1 395

I 400

I 405

410

I 415

I 420

h (nm) FIG. 4. Effect of the differentiation slitwidth on the third-order mathematical derivative spectra of clonazepam. (A) 1 nm, (B) 2 nm, (C) 4 nm. (-) Spectra of standard solutions of clonazepam. (- - -) Spectra of spiked mine samples.

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TABLE 2 Effect of the Slitwidth on the Analytical Parameters of the Determination of Clonazepam in Urine Samples by Mathematical Differentiation Analytical parameters Slithwidth (nm)

A (nm)

Sensitivity fmm-‘1

R.P. (%)

V.C. m

LOD (pw)

2 4

416.0 417.5

-2.9452 x lO-3 - 1.7329 x IO-’

99 100

1.6 1.1

0.2 0.08

Note. Sensitivity is indicated for the slope of the calibration line in derivative units for ppm of drug. R.P., recovery percentage in the analysis of a sample containing 16 ppm. V.C., variation coefficient of 10 measurements of three independent analyses of a sample. LOD, limit of detection for a probability level 95% (K = 3).

Efict of the scan speed. The scan speed does not affect the shape and size of the bands in the derivative spectra. (See Fig. 3) Results found do not agree with those obtained by Gill and colleagues (M), but this could be due to the special characteristics of the program employed. Mathematical Differentiation For the mathematical differentiation with the OP12 the only parameter which could affect the derivative spectra is the slitwidth. Figure 4 indicates that the use of a 1-nm slitwidth does not permit the characteristic third-order derivative spectra of the clonazepam solutions to be obtained. On the other hand, a lower sensitivity and a higher stability corresponds to higher slitwidth. Table 2 summarizes the analytical parameters corresponding to the analysis of Clonazepam, using the mathematical differentiation with a slitwidth of 2 and 4 nm at the third-order derivative spectra. The accuracy obtained with the mathematical differentiation is very good in both cases, being better for larger slitwidth, and the limit of detection in the more stable conditions is 80 ng liter-‘. Comparison between the Mathematical and the Electronic DiSferentiation Under the best experimental conditions, previously obtained, the analytical parameters of the spectrophotometric derivative determination were established. TABLE 3 Comparison between the Analytical Parameters of the Derivative Spectrophotometric Determination of Clonazepam by Mathematical and Electronic Differentiation Analytical parameters Sensitivity

R.P.

V.C.

LOD

Derivative

(nm)

A

@pm ‘1

(%I

ca

(mm)

Mathematical Electronic

417.5 416.2

-1.73 x lo-3 -2.79 x lO-4

100 118

1.1 2.2

0.08 0.7

Note. Sensitivity is indicated for the slope of the calibration line in derivative units for ppm of drug. R.P., recovery percentage in the analysis of a sample containing 16 ppm. V.C., variation coefftcient of 10 measurements of three independent analyses of a sample. LOD, limit of detection for a probability level of 95% (K = 3).

256

RANDEZ-GIL,

SALVADOR,

-0.1 395

400

405

AND DE LA GUARDIA

I

I

,

410

415

420

X (nm)

-1.5-4 420

390 h

b-4

FIG. 5. Derivative spectra corresponding to different standards of cionazepam used to obtain the calibration curve. Using a conventional spectrometer and a slitwidth of 4 nm (A) and those obtained using a diode array detector (B).

Table 3 indicates that the highest sensitivity, the best accuracy and precision, and the lowest limit of detection correspond to the use of the mathematical differentiation, using a 4-nm slitwidth. The Use of a Diode Array The configuration of the Hewlett-Packard 8452A diode array does not permit modification of the differentiation parameters. The best analytical features correspond to 408 nm for the determination of clonazepam in the derivative spectra. Under these conditions, a sensitivity of 2.95 X 10m6ppm-‘, a recovery percentage of 101%, a variation coefficient of 0.7%, and a limit of detection of 150 ppb were obtained.

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Comparison between the Results Obtained Using a Conventional Spectrometer and a Diode Array Figure 5 shows the calibration curves obtained for the third-derivative spectrophotometric determination of Clonazepam using the two apparatus assayed. From the comparison of the figures of merit, obtained in both cases, it can be concluded that, using the mathematical differentiation, accurate and precise results were obtained with both instruments. The sensivity of the diode array is lower than that of the conventional spectrometer (See Table 3). However, this is due only to the use of an arbitrary scale, and, so, the limit of detection in both cases is of the same order. The diode array instrumentation permits the first nine-order derivative spectra of the samples to be obtained very easily by a mathematical procedure and so the use of these apparatus is very appropiate for an accurate direct derivative spectrometric analysis of clonazepam in urine samples with limits of detection of the order of 150 ppb. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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