Simultaneous Determination of Otilonium Bromide and Diazepam by First-Derivative Spectroscopy

CARLO MANNUCCI, JACOPO BERTINI’, ALDO COCCHINI, ANDREA PERICO, FRANCO SALVAGNINI, AND ANTONIO TRIOLO Received August 12, 1991, from the Analytical Research Department, A. Menarini lndustrie Fannaceutiche Riunite s.r.1.. Vi8 Sette Sanfi 3, 50131 Florence, Italy. Accepted for publication April 8, 1992.

Abatract 0 A rapid, simple assay procedure was developed for simul- taneous analysis of otilonium bromide, a smooth-muscle relaxant, and diazepam in tablets containing 20 mg of otilonium bromide and 2 mg of diazepam (20:2 tablets) or 40 mg of otilonium bromide and 2 mg of diazepam (40:2 tablets) by “zerocrossing” first-derivative spectroscopy. The tablets were dissolved in 0.01 N HCI, mixtures were centrifuged at 3500 rpm (2472 x g) for 5 min, and first-derivative spectra were recorded. The absolute values of the derivative were measured at 264 nm for determination of otilonium bromide and between 406 and 408 nm (380 nm for analysis of 40:2 tablets) for determination of diazepam. The method is linear, quantitative, and reproducible and can also be used for the tablet dissolution test. Ten tablets of the same batch were analyzed by the described method and by a high-performance liquid chromato- graphic method, and the results were in good agreement.

Otilonium bromide, a smooth-muscle relaxant devoid of the side effects of other antimuscarinic drugs,l is marketed either alone (as Spasmomen, Menarini, in Italy, Belgium, and Hong Kong; as Spasmoctyl, Menarini, in Spain; as Spasen, Firma, in Italy) or in combination with diazepam (as Spasmomen Somatico, Menarini, and as Spasen Somatico, Firma, in Italy). The pharmaceutical combination of otilonium bromide and diazepam is available in tablets at two different dosages: 20 mg of otilonium bromide with 2 mg of diazepam (20:2 tablets) and 40 mg of otilonium bromide with 2 mg of diazepam (40:2 tablets). A gas chromatographic-mass spectrometric method for the determination of otilonium bromide in serum has been reported.2 Otilonium bromide and diazepam in pharmaceu- tical preparations have also been determined by near- infrared spectroscopy.3

We report here a fast and specific method for the simulta- neous quantitative determination of otilonium bromide and diazepam in tablets a t different dosages by “zerocrossing” firstderivative spectroscopy, a technique recently developed and applied with success for quantitation of binary mixtures of pharmaceutical interest.4-11 A rapid, simultaneous mea- surement of active components in tablets by assay of individ- ual units is useful for determination of content uniformity, as required by the Italian official pharmacopeia.12 To determine the validity of the method, 10 tablets of the same batch were analyzed by this spectrophotometric method and by a high- performance liquid chromatographic (HPLC) method that had been performed and validated in our laboratory. The good agreement of results indicates the suitability of this UV spectrophotometric method. In addition, use of this spectro- photometric method leads to considerable savings in costs and time.

Experimental Section UV Analysis-A Hewlett-Packard 8452A diode-array spectropho-

tometer was used, and spectra were determined with a l-cm quartz cell from 200 to 500 nm. Standard spectra of the first derivatives of

absorbance8 of otilonium bromide and diazepam (in 0.01 N HCl solutions) were stored in the memory of the spectrophotometer.

HPLC Analys isThe liquid chromatograph (Hewlett-Packard 1090L) was equipped with a n autoinjector, a n autosampler, and a diode-array variable-wavelength detector (Hewlett-Packard 1040M Series 11) connected to a workstation (Hewlett-Packard 79994A) with a hard disk, printer, and plotter. The analytical column used was a 60 RP Select B (Merck; 5 pm; 250 x 4.0 mm), and the precolumn was a 60 RP Select B (Merck; 5 pm; 4 x 4 mm). The mobile phase was 0.5 M sodium acetate trihydrate buffer containing 5 mM l-heptanesulfo- nic acid monohydrate sodium salt mixed with methanol (30:70, vlv) and adjusted to pH 6.0 with glacial acetic acid. The mixture was stirred, filtered, and deaerated. A flow rate of 1 mumin was used.

Materials and Reagents-Water was deionized, and methanol and 1-heptanesulfonic acid monohydrate sodium salt were HPLC grade. Glacial acetic acid, sodium acetate trihydrate (99.5%), acetonitrile, and n-butylp-hydroxybenzoate (99%) were analytical grade and were used as received. Hydrochloric acid (3638%) was analytical grade. Otilonium bromide was synthesized by Research Laboratories, Chemistry Department, A. Menarini s.r.l., Florence, Italy. Diazepam was supplied by the manufacturer (lot 9506, Fabbrica Italiana Sintetici S.p.A., Alte di Montecchio Maggiore, Italy) and was char- acterized in our laboratories for use as a n analytical reference standard.

Standard Solution-The otilonium bromide standard solution was prepared by accurately weighing 20 mg of otilonium bromide refer- ence standard into a 25-mL volumetric flask (50 mL for the analysis of 40:2 tablets) and diluting to volume with 0.01 N hydrochloric acid. Then, 1.0 mL of this solution was pipetted into a 20-mL volumetric flask (10 mL for the analysis of 40:2 tablets) and diluted to volume with 0.01 N HCl. The diazepam standard solution was prepared by accurately weighing 2 mg of diazepam reference standard into a 200-mL volumetric flask and diluting to volume with 0.01 N hydro- chloric acid. Then, 4.0 mL of this solution was pipetted into a 10-mL volumetric flask (20 mL for the analysis of 40:2 tablets) and diluted to volume with 0.01 N HC1.

Sample Solution-An accurately weighed tablet was transferred to a 200-mL volumetric flask already containing 200 mL of 0.01 N HC1. The tablet was completely dissolved with the aid of magnetic s t imng (-10 min). An aliquot of this solution was then centrifuged a t 3500 rpm (2472 x g) for 5 min, and 10.0 mL (2.0 mL for the analysis of 40:2 tablets) of the clear supernatant was pipetted, transferred into a 25-mL volumetric flask (10 mL for the analysis of 4 0 2 tableta), and diluted to volume with 0.01 N HCl.

Proeedur-The firstderivative spectra of standards and sample solutions were recorded between 200 and 500 nm. The absolute values of the derivative were measured at 264 nm for determination of otilonium bromide and between 406 and 408 nm (380 nm for the analysis of 40:2 tablets) for determination of diazepam.

Results and Discussion Spectrophotometric Measurement-Reliable results

were obtained by first-derivative spectroscopy. The value of the first-derivative spectrum of diazepam is -0 at 264 nm; however, a t this wavelength it is possible to take derivative measurements of the mixture proportional to the otilonium bromide concentration. Similarly, the value of the first- derivative spectrum of otilonium bromide is -0 at 406408

0022-3549/92/1200- 1 175$02.50/0 8 1992, American Pharmaceutical Association

Journal of Pharmaceutical Sciences I 1175 Vol. 81, No. 72, December 1992

0.04571 264 n m n d

A

2: -.007024 \

200 2SO 300 350 IJRUELENGTH

400

B 0.00718-

d

486-488 ma 0.00322-

380 n m + Y =- - ---.++- c -7.SE-04 - a 3 - I w -.OM70 - n

-.00867 -

-.01263,. . . . , . . . . , . . . . , . . . . I . 200 250 300 350 400

URVELENGTH

Flgure 1-First-derivative spectra of (A) otilonium bromide (40.83 &mL) and (B) diazepam (2.12 Fg/mL) in 0.01 N HCI.

nm, whereas in the same range, a very good linearity between derivative measurements of the mixture and diazepam con- centration is present (Figure 1).

Because the value of the first-derivative spectrum of the excipients in the 20:2 tablets was >O at 380 nm (wavelength used for the analysis of diazepam in 40:2 tablets), it was necessary to choose a wavelength at which the first-derivative spectra of both excipients and otilonium bromide are -0 while maintaining a good proportionality between derivative mea- surements of the mixture and diazepam concentration. The first-derivative spectra of a placebo formulation and otilo- nium bromide solution showed that the best wavelength range to determine diazepam in 20:2 tablets was 406-408 nm. Tovalidate the present method for the analysis of 20:2 tablets, calibration graphs were prepared by measurements of the total first-derivative spectra of a mixture in 0.01 N HC1 of diazepam at 2.32 pg/mL with increasing amounts of otilo- nium bromide (10.21-30.62 pg/mL) and another one contain- ing otilonium bromide at 20.42 pg/mL with increasing amounts of diazepam (1.16-3.49 pg/mL). For the analysis of 40:2 tablets, calibration graphs were prepared by measure- ments of the total first-derivative spectra of a mixture in 0.01 N HC1 of diazepam at 2.32 pg/mL with increasing amounts of otilonium bromide (20.10-60.27 pg/mL) and another one containing otilonium bromide at 40.18 pg/mL with increasing amounts of diazepam (1.014.04 pg/mL).

Calibration Graphs and Statistical Analysis of Results- Determination coefficients, slopes, and intercepts calculated for mixtures of otilonium bromide and diazepam by the measurements described are given in Table I.

Precision and Accuracy-The reproducibility of repeated scans, that is, the precision of the system, was determined by

making six replicate scans of two solutions containing otilo- nium bromide and diazepam at concentration (pg/mL) ratios of 20.42~2.32 and 40.83:2.32 and determining the relative standard deviations (RSDs) of the first-derivative values. Mean first-derivative values of 0.553 and 1.108 with RSDs of 0.06 and 0.1%, respectively, were obtained for otilonium bromide; mean values of 0.0039 (406408 nm) and 0.0183 (380 nm) with RSDs of 2.7 and 0.5%, were obtained for diazepam.

The accuracy and the utility of this method were demon- strated by the assay of 40:2 and 20:2 tablets (Figure 2). The amount of each component in 10 individual tablets of the same batch was determined by this method, and the results were compared with those obtained by an HPLC method that had been validated in our laboratory. The results (Table 11) are in good agreement, as shown by analysis of variance (Table 111).

Conclusions A simple W method for the analysis of a multicomponent

tablet formulation containing otilonium bromide and diaze- pam was developed. This method has many advantages over analyses that require a separation technique, such as HPLC or gas chromatography; it is very reproducible and easy to perform in analytical laboratories with a diode-array spec- trophotometer or an equivalent apparatus with the capability of derivative spectra. With the opportunity to interface a dissolution test apparatus with a diode-array spectrophotom- eter, it is possible to automate the dissolution procedure and to determine simultaneously and rapidly the active compo- nents and reduce costs without sacrificing precision and accuracy.

11 76 I Journal of Pharmaceutical Sciences Vol. 81, No. 12, December 1992

2

UAUELENGTH

0.05549~ B d

380 nn w = - c -.01005- a =t - - w -.M282- 0

-.m -

200 250 300 UAUELENGTH

Flgum 2-Firstderivative spectra of (A) 20:2 and (B) 40:2 tablets in 0.01 N HCI.

Table CRegresslon Analysls of Resulta of Assay by FIrst-Derhratlve UV Spectroscopy

Drug pb Intercept Slope Tablet Composition.

20:2 Otilonium bromide 0.998 4.00 x 1.15 x

40:2 Otilonium bromlde 1.OOO 2.81 x lo-' 1.37 x Diazepam

Diazepam

0.984 -3.33 x 10-5 1.29 x 10-4

1.000 -4.49 x 10-5 1.28 x 10-4

Otilonium bromide:diazepam ratio. Correlation coeffiaent squared.

Tabk ICComparlson of Results of Assay by FIrst-Derhratlve UV Spectroscopy and by HPLC

Diazepam, mg/tablet Otilonium Bromide, mg/tablet

Tablet 20:2 Tablet 40:2 Tablet 20:2 Tablet 402 Tablet

W HPLC UV HPLC UV HPLC UV HPLC

1 2 3 4 5 6 7 8 9 10

Mean RSD, %

21.03 20.17 18.99 19.38 19.34 19.01 19.79 19.36 20.41 19.45 19.69 3.3

21.25 38.97 20.14 41.92 19.36 37.71 19.14 39.62 19.51 38.65 19.59 39.45 19.90 39.06 18.98 38.77 20.04 38.84 19.92 39.65 19.78 39.26 3.3 2.8

39.95 1.98 39.78 2.01 38.97 1.92 39.30 1.99 39.70 1.96 39.88 2.00 39.58 2.08 39.50 1.92 39.65 1.95 39.94 1.94 39.62 1.98 0.8 2.5

1.94 2.1 0 1.88 1.92 1.86 1.95 1.97 1.85 1.91 1.96 1.93 3.7

2.02 1.99 1.99 2.10 1.98 1.99 1.94 2.00 1.92 2.13 2.01 3.2

1.95 1.94 1.95 1.92 2.01 1.99 1.97 1.94 1.93 2.07 1.97 2.3

Tabk Ill-Comparlson of Results of Composlte Tablets Assay ObtalneU by Flrst-Derlvatlve UV Spectroscopy and by HPLC.

F Test Tablet Compositionb P

Otilonium bromide 20:2 0.48 0.8283 40:2 0.88 0.3608

Diazepam 20:2 2.25 0.1511 40:2 1.66 0.2146

Analysis of variance. Otilonium br0mide:diazepam ratio.

1.

2.

3.

4. 5. 6. 7. 8. 9. 10. 11. 12.

References and Notes Ghelardoni, M.; Pisanti, N.; Volterra, G. J. Med. Chern. 1973,16, 1063-1065. Signorini, C.; Tosoni, 9.; Ballerini, R.; Chinol, M.; Mannucci, C. Drug Exp. Clin. Res. 1984, 10, 273-276. Corti, P.; Dreasei, E.; Corbini, G.; Ballerini, R.; Gravina, S. Pharrn. Acta Helv. 1990, 65, 189-193. Morelli, B. J. Pharm. Sci. 1988,12, 1042. Morelli, B. Anal. Lett. 1988,21, 43. Morelli, B. J. Pharm. Bwmed. Anal. 1988, 6, 199. Morelli, B. Anal. Lett. 1988,21, 759. Morelli, B. Analyst 1988, 113, 1077. Morelli, B. J. Phann. Sci. 1988, 7,615. OHaver, T. C.; Green, G. L. Anal. Chern. 1976,48, 312. O'Haver, T. C. Clin. Chem. 1979,25, 1548. Farmaco a Ufiicrle (F. U.) IX, vol. I; Istituto Poligrafico e Zecca: Rome, l&; pp 415-416.

Journal of Pharmaceutical Sciences I 1 t 77 Vol. 81, No. 12, December 1992