Talanra, Vol 39, No. 9, pp. 1175-I 180, 1992 0039-9140/92 $5.00 + 0.00 Pnnted in Great Britain. All nghts reserved Copyright 0 1992 Pergamon Press Ltd

AUTOMATIC KINETIC DETERMINATION OF OXAZEPAM BY THE CONTINUOUS ADDITION OF

REAGENT TECHNIQUE

MANUEL CARMONA, MANUEL SILVA and DOLORES PEREZ-BENDITO

Department of Analytical Chemistry, Faculty of Sciences, University of Grdoba, 14004 Cbrdoba, Spain

(Received 22 October 1991. Revised 21 January 1992. Accepted 27 January 1992)

Summary-An automatic continuous-addition-of-reagent method for the routine determination of oxazepam based on the formation of a coupling product between diazotized 2-amine-5-chlorobenzophe- none (the hydrolysis product of oxazepam) and I-naphthol in a basic medium is proposed. The reaction is developed by continuously adding basic I-naphthol to a reaction vessel containing the other ingredients and is monitored spectrophotometrically at 530 nm. The results obtained show the proposed kinetic method to surpass the performance of its conventional equilibrium counterpart as it permits distinguishing the intact drug from its hydrolytic degradation product and is suitable for the determination of oxazepam in dosage forms and urine samples.

Benzodiazepines make a major class of sedative- hypnotic drugs of wide use in the treatment of anxiety and insomnia. They are also the most frequently encountered in cases of drug over- dose.’ The detection and rapid determination of benzodiazepines in various biological and non- biological specimens is therefore of paramount importance in a number of clinical, forensic and pharmaceutical applications. Oxazepam, a 1,4- benzodiazepine derivative of this type, is widely employed as an antidepressant.

Analytical methods currently available for the determination of benzodiazepines in various matrices include gas chromatography’ and high-performance liquid chromatography pro- cedure$ that take just a few minutes to com- plete. In addition, the thermal instability of some of these drugs-particularly oxazepam- complicates their gas chromatography and HPLC analyses for this compound often require careful selection of the mobile phase. As far as the determination of oxazepam is concerned, official pharmacopeias”,’ recommended a spec- trophotometric assay for assaying this drug in dosage form. The spectrophotometric methods used for this purpose are generally based on the acid hydrolysis of this compound, which is followed by ring opening, and the subsequent determination of the benzophenone thus ob- tained. In fact, oxazepam and other benzo- diazepines have been determined in binary mixtures by UV-Vis spectrophotometry using micellar media6 of by fourth-derivative spec-

trophotometric techniques after separation by HPLC.’ In other methods, benzophenone is diazotized with nitrous acid and then coupled with a chromogenic reagent such as N-(l- naphthyl)ethylenediamine, Bratton-Marshall reaction,8*9 or I-naphthol.“,”

A new method for the kinetic determination of oxazepam is presented in this paper. It is based on the rate of coupling between 1 -naphthol and diazotized benzophenone ob- tained from the acid hydrolysis of this drug. This automatic kinetic method is substantially simpler, faster and more sensitive than its equilibrium counterpart and relies on the con- tinuous-addition-of-reagent (CAR) technique.12 It is quite inexpensive to implement and lends itself readily to routine analyses for benzo- diazepines. It has been previously used in various application fields with good results.13

EXPERIMENTAL

Reagents

The reagents used were of analytical-reagent grade and distilled water and ethanol were used to prepare the solutions. The oxazepam solution (200 pg/ml) was prepared by dissolving 200.0 mg of the drug (Sigma Chemical Co.) in one litre of ethanol. More dilute solutions were prepared as needed. The coupling reagent sol- ution for the CAR procedure was prepared by mixing 5.0 ml of 3.47 x IO-‘M 1-naphthol in 2:3 v/v ethanol/water and 20 ml of 7.5M

1175

1176 MANUEL CARMONA et al.

0.6 - al : +j 0.4 -

z 0.2-

; ; ; 1 Time, set

Fig. 1. Typical response curve obtained in the CAR determi- nation of 1.5 pg/ml oxazepam by using the proposed

reaction. (For experimental conditions, see Procedure.)

sodium hydroxide in a 50-ml standard flask and diluting to the mark with 2 : 3 v/v ethanol/water. A 1.45 x 10-Zit4 sodium nitrite solution was also used.

Apparatus

All measurements were made by using a CAR system described elsewhere,‘* which consists essentially of an automatic burette (Mettler DL40), a fan stirrer, a photometric detector (Mettler DKl8), an immersion probe (Mettler DK181) for measuring the absorbance in the visible region and a data acquisition and pro- cessing unit consisting of a Hewlett-Packard 98561AE computer and a 16-bit Hewlett- Packard analog-to-digital converter provided with a program for the determination of the reaction rate written by the authors. A Radiometer PHM62 pH-meter was also used.

Procedures

Kinetic determination of oxazepam. A volume of standard oxazepam solution (9-220 pg) of up to 10 ml was mixed with the same volume of 6M hydrochloric acid in a lOO-ml reaction vessel and allowed to stand in a boiling water- bath for one hour. Once the solution was cool, 0.6 ml of 1.45 x IO-‘M sodium nitrite solution was added and the mixture was accurately di- luted to 60 ml with distilled water. The reaction was developed by continuously adding the coupling reagent solution at a rate of 35 ml/min with stirring at 200 rpm. The reaction was monitored at 530 nm and kinetic data (ab- sorbance VS. time) were collected at a rate of 30 msec per point and subsequently processed by the computer. The reaction rate was

measured in about 1 .O set and each sample was assayed in triplicate.

Preparation and analysis of pharmaceutical and clinical samples

Pharmaceutical samples. To prepare the commercial pharmaceutical samples for analy- ses, a given number of tablets or capsules was ground to a fine powder. The accurately weighed sample was then stirred with 50 ml of ethanol (one portion of 20 ml and two portions of 15 ml) until dissolution was complete. If a residue was formed, it was filtered off and washed with portions of ethanol, and the filtrate and washings were hydrolysed as described above, the final solution being diluted to lOO-ml in a standard flask containing ethanol. In all cases appropri- ate aliquots of these solutions were analysed for oxazepam by the above-described pro- cedure.

Extraction of human urine samples. After col- lection, the human urine samples (approximate volume 25 ml) were spiked with various known amounts of oxazepam (50 to 450 pg) from the stock solution, pH adjusted to ca. 10 with sodium hydroxide and filtered. Next, each filtrate was shaken with 25 ml of methylene chloride in a lOO-ml separating funnel for 5 min. The resulting organic extract was evaporated to almost dryness and the resulting residue was dissolved in 30 ml of ethanol and hydrolysed as described above. The remaining solution was diluted with distilled water in a 50-ml standard flask and 20-ml aliquots were used to analyse for oxazepam by using the above-described procedure.

RESULTS AND DISCUSSION

Acidic solutions of diazotized 2-amine-5- chlorobenzophenone (the hydrolysis product of oxazepam) react with basic I-naphthol added from the autoburette of the CAR system to yield a red coupling product. Since this reaction is time-dependent and very fast, oxazepam must be determined by the continuous-addition-of- reagent technique by monitoring the reaction rate spectrophotometrically at 530 nm (absorp- tion maximum of the coupling product formed). A typical absorbance us. time plot for an oxa- zepam concentration of 1.5 pg/ml is shown in Fig. 1.

As stated in the literature, the acid hydrolysis of benzodiazepines to benzophenones is carried

Automatic kinetic determination of oxazepam 1177

- 0.4 Q, E; a

a)

-5.0 -4.0 -3.0

Log [Nitrite]

b)

1.0 2.0 3.0

PH

Fig. 2. Elect of {a) the sodium nitrite concentration and (b) pH on the kinetic determination of 1.5 pg/ml oxazepam. (For experimental conditions, see Procedure.)

out with hydrochloric acid at high tem- peratures.4’4-1p In our method, oxazepam is quantitatively (99.5%) hydrolysed to 2-amine-5- chlorobenzophenone with 6M hydrochloric acid at 100” in a boiling water-bath for 1 hr. Thus, a kinetic study of the influence on the rate of the coupling reaction was performed in order to develop a kinetic method for the determination of the drug.

Chemical and instrumen~l variables were optimized in order to maximize the sensitivity and precision of the proposed kinetic method. In short, stock Z-amine-5-chlorobenzophenone solutions were used and each variable was changed in turn while keeping all other con-

stant. In this case, a 1.0~pg/ml ~nzophenone solution was equivalent to one containing 1.24 p g/ml oxazepam.

The influence of the sodium nitrite concen- tration was assessed over a wide range (2.42 x 10-6-2.90 x 10m4M). The absorbance US. time curves obtained show an increase in the reaction rate with increase in the diazotizing agent concentration up to 4.84 x 10-5M, above which it remains virtually constant. This depen- dency shown in Fig. 2(a) (a log-log plot) arises from the wide concentration range studied. A concentration of 1.45 x 10e4M (0.6 ml of a 1.45 x lo-‘M solution), which lay on the plateau, was selected. The influence of pH is closely related to the former variable since the true diazotizing agent for benzophenone is

T

:

0.36

oi

2 0.26

a)

I 3 I

1.0 3.0 5.0

[~a0ii] , M

0.32 ai E

9” $ 0.24

fc

0.08

b)

654 3 2

1

I I I I

2 4 6 8

Time, set

Fig. 3. (a) influence of the sodium hydroxide ~n~ntration on the reaction rate. (b) Absorbance vs. time curves recorded at different NaOH concentrations: 1, 0.75; 2, 1.125; 3, 1.50; 4, 2.25; 5, 3.0 and 6,

3.75-5.25M. Oxazepam concentration, 1.5 #g/ml. (For ex~~mental conditions, see Procedure.)

1178 MANUEL CARMONA et al.

at “a -0.4 a

- 3.5 -3.0 -2.5

Log [l-Naphthol]

Fig. 4. Variation of the reaction rate as a function of the I-naphthol concentration. Oxazepam concentration, 1.5

fig/ml, (For experimental conditions, see Procedure.)

nitrous acid. Thus, the effect of pH was studied between 0.5 and 3.0 [Fig. 2(b)]. The plot of the reaction rate US. pH shows maximum depen- dency at about pH 1.25, above which it de- creases. Taking into account this dependency, a pH of 1.30 (nitrous acid is not dissociated) was selected for use in the kinetic determination of oxazepam. At this pH, not only the reaction rate, but also the final absorbance was maximal.

The effect of the sodium hydroxide concen- tration in the reagent solution added from the autoburette was also tested in the range 0.75-5.25M; as shown in Fig. 3(a), the reaction rate increases sharply with an increase in this variable up to 1.5M, above which it remains virtually constant. Continuous addition of this chemical to the reaction vessel has two distinct effects: first, it neutralizes the acid present in the reaction vessel, which is required for the diazo- tizing reaction to develop; then it provides the basic medium needed for the coupling reaction with l-naphthol. Therefore, the kinetic curves obtained in this study show a pseudo-induction period which is proportionai to the sodium hydroxide concentration added from the auto- burette, as shown in Fig. 3(b). Taking into

Table 1. Influence of other benzodiazepines on the kinetic determination of 0.8 &ml oxazepam

Tolerance Benzodiazevine added ratio

Flurazepam, chlotiazepam, loprazolam, lormetazepam, alprazolam

Diazepam* Nitrazepam, chlordi~epoxide, brazepam,

bromazevam

100 50

<I

*Maximum ratio assayed owing to its solubility.

account this behaviour, a concentration of 3.OM (20 ml of 7.5M sodium hydroxide in a final volume of 50 ml) was chosen for subsequent experiments. The concentration of 1-naphthol was varied over the range 1.38 x 10e4- 5.52 x 10e3M in order to investigate its effect on the reaction rate (Fig. 4), which increased with increase in the con~ntration of this reagent up to about 2.08 x 10e3M, above which it remained constant. A concentration of 3.47 x 10W3A4 (5 ml of 3.47 x IO-‘M I-naphthol in a final volume of 50 ml), which resulted in a maximal reproducibility and sensitivity, was selected.

The rate of addition of the coupling reagent, the initial volume of the sample solution and the stirring speed are the three instrumental vari- ables which exert an appreciable effect on the kinetic curves. The rate of addition of the alkaline l-naphthol solution was studied in the range 5-35 ml/min. The reaction rate remained virtually constant throughout this interval, although as with the effect of sodium hydroxide, a pseudo-induction period (the length of which was inversely proportional to the rate of addition) was also observed. Thus, a rate of 35 ml/min was selected for further experiments. The influence of the initial sample volume (between 60-100 ml) and the stirring speed (5&300 rpm) on the reaction rate was also examined. The values advocated under Procedure were chosen as optimal.

Kinetic determination of oxazepam

Quantification of the drug was achieved by plotting the reaction rate against its concen- tration. Linear correlation (I = 0.9995, n = 8) over the concentration range 0.15-3.65 pg/ml was obtained. A representative calibration graph for oxazepam provided the following linear regression equation:

Reaction rate = 0.023 + 0.216 [Oxazepam]

where the drug concentration is expressed in pg,/ml. The detection limit of the proposed kinetic procedure was determined by calculating the minimum concentration of drug that could be detected. A concentration of 40 ng/ml oxa- zepam gave rise to a response (reaction rate) that was three times the standard deviation of the reaction rate for 20 determintions on a sample containing a low analyte concen- tration2’ The precision (RSD = 1.26%) was confirmed by analysing 11 replicate samples each containing 1.0 ,ug/ml of oxazepam in the

Automatic kinetic determination of oxazepam 1179

Table 2. Analysis of synthetic mixtures of oxazepam and its hydrolysis product

Taken, rrnlrnl Oxazepam

Oxazepam Benzophenone

0.40 3.20 0.70 2.80 1.00 2.08 1 so 1.50 2.00 1.00 2.80 0.70 3.20 0.40

Found, mrlml

Error, %

Benzophenone

Found, Error, wnlml %

0.38 5.0 3.20 0.0 0.71 -1.4 2.78 0.7 0.96 4.0 2.03 1.5 1.46 2.6 1.50 0.0 2.02 -1.0 0.99 1.0 2.81 -0.3 0.68 2.8 3.15 1.6 0.40 0.0

reaction vessel. The sampling rate, as deter- mined for triplicate runs on the same sample, was about 60 samples/hr.

A study of interferences was made in order to determine the tolerance to other benzo- diazepines in the kinetic determination of 0.8 pg/ml of oxazepam. The tolerated limits for the foreign compounds were taken as the largest amounts yielding an error less than f 3% in the reaction rate measurements. As can be seen in Table 1, the proposed method is quite selective. The most serious interferences arise from other benzodiazepines or hydrolysis products of these including a primary amine group in their structures.

The acid-induced degradation product of oxazepam, 2-amine-5chlorobenzophenone, is obviously a major interferent for the proposed method; however, its perturbation can be avoided by simultaneously determining ox- azepam and its hydrolysis product using one hydrolysed and another unhydrolysed sample aliquot. Laboratory-prepared mixtures of ox- azepam and its degradation product were thus assayed. Table 2 lists the results obtained; the analysis of oxazepam/benzophenone mixtures was feasible over the weight ratio range from 1: 8 to 8: 1 (maximum relative error, rfi 5%). These results are quite satisfactory and confirm that the proposed method allows the resolution

of this mixture at lower degrees of hydrolysis of oxazepam in the sample (about to 10%). The average of the results from 11 samples each containing 1.0 pg/ml oxazepam and hydrolysis product yielded an overall relative standard deviation of 1.5% for the method.

Applications

In order to check the applicability of the proposed method we used it to analyse for oxazepam in pharmaceutical and urine samples. The pharmaceutical samples were treated as described above, and in order to detect potential matrix effects from other components, the re- coveries of oxazepam added to these samples were also determined. Table 3 shows the results obtained in the determination of oxazepam in such samples, which are consistent with the nominal values of this drug given by the manu- facturers (the recoveries ranged from 93.1 to 100.7%, with an average of 96.5%). These results show the good performance of the pro- posed method for the kinetic determination of oxazepam.

The analysis for oxazepam in urine samples is of great clinical significance since this drug is the main urinary metabolite of several benzo- diazepines such as chlordiazepoxide, diazepam, halazepam, medazepam, etc.2’ Therefore, its determination in this type of sample can be used

Table 3. Kinetic determination of oxazepam in dosage forms by the CAR technique

Oxazepam, mg Stated by Found by Mean recovery,

Sample manufacturer proposed method* %

Adumbram 10 9.2kO.l 100.7 Aplakil 20 18.9 f 0.6 94.7 Buscopaxt 9.7*0.1 93.1 SuxidinaJ :.!I 4.86 & 0.07 98.2 Novo-aerofil§ 5.0 4.75 f 0.08 95.9

*Average of four determinations. Co-occurring substances include tbutilescopol- amine bromide (10 mg); loxazepam as succinate acid, aspergillus oryzae (4200 units), metochlopramide hydrochloride (5 mg) and dimeticone (10 mg); §metochlopramide hyrochloride (5 mg) and dimeticone (10 mg).

Table 4. Recovery of oxazepam added to urine samples

Added, Found, Mean recovery, unlml rcalml %

2.00 2.05, 1.85 97.5 5.00 4.83, 4.91 98.0

10.00 9.35, 9.86 96.0 15.00 14.45, 14.15 95.3 18.00 17.05, 17.45 95.8

as a bulk analysis of these drugs or for the quantitative analysis of a given drug when only one benzodiazepine is present. Different urine samples were analysed by pretreating them as described under Experimental. The samples were spiked with 2-18 pg/ml oxazepam (Table 4) and the average recovery found, 96.5 f 1.2%, shows the reliability of the pro- posed kinetic method for the determination of this drug in this type of sample. Finally, it is worth noting that this method can be used to determine smaller amounts of oxazepam in urine samples if these are used entirely for this analysis after the pretreatment. Thus, if 50 ml of urine are treated and then diluted to 60 ml in the reaction vessel to determine its oxazepam content by the proposed CAR method, the minimum amount of oxazepam that can be determined in this case is about 50 ng/ml, taking into account the detection limit of the proposed kinetic method.

CONCLUSIONS

Oxazepam was assayed in dosage forms and urine samples by using the continuous-addition- of-reagent technique. The method used for this purpose is simple, rapid, precise and more sensi- tive than the corresponding equilibrium ap preach in which the determination is feasible over the range 2-160 pg/ml.‘O*” The present method can be applied to quality control testing and drug stability monitoring as a good alterna-

1180 MANUEL CARMONA et al.

tive to other methods available for the determi- nation of this drug.

Acknowfedgemenr-The authors are grateful to the CYCIT (Project PB87-0821) for financial support granted for the realization of this work.

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