Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt

Corresponding Author: Y. M. issa, chemistry Department, Fac¬ulty of science, cairo University, Giza, Egypt. Tel: 00202-5676579; Fax: +5728843; E-mail: yousrymi@yahoo.com.

	ABSTRACT
	INTRODUCTION
	EXPERIMENTAL
	RESULTS AND DISCUSSION
	CONCLUSION
	REFERENCES

	ABSTRACT

  The interaction of diazepam with picric acid (I), 3, 5-dinitrobenzoic acid (II) and 2, 4-dinitrobenzoic acid (III) was found to be useful for its spectrophotometric determination. the quantitation was carried out at 475, 500, and 500 nm for the reaction with (I), (II) and (III), respectively. the effect of several variables on the coloring process was studied. the proposed methods have been applied successfully for the determina¬tion of diazepam in pure samples and in its pharmaceutical preparations with good accuracy and precision. the results were compared to those obtained by the pharmacopoeial methods. the linear ranges for obedi¬ence of beer’s law are up to 85.6, 180.2, and 128.6 µg/ml, ringbom ranges are 10.0-79.0, 15.2-177.8, 17.0-83.0 µg/ml, and rsD 0.048, 0.028, and 0.026% for reaction of diazepam with I, II, and III, respectively.

KEY WORDS:    diazepam; 2,4-dinitrobenzoic acid; 3,5-dinitrobenzoic acid; picric acid; spectrophotometric determination

	INTRODUCTION

  Diazepam (C16H13ClN2O) [7-chloro-1,3-dihydro-1- methyl-5-phenyl-2H-1,4-benzodiazpein], M. Wt. 284.75 [CAS (439-14-5)] is an important compound widely used therapeutically because of its relaxant, sedative, hypnotic and anticonvulsant properties.
  Several analytical procedures have been adapted for the assay of diazepam. They include non-aqueous titra¬timetry (1, 2), ultraviolet spectrophotometry (3-5), vis¬ible spectrophotometry (6-10), second order-derivative spectrophotometry (11, 12), fluorimetry (13, 14), high performance liquid chromatography HPLC (15-17), gas chromatography (18, 19), thin layer chromatography (20), polarography (21, 22), potentiometry (23, 24) and infrared assay (25). The official methods involve a non¬aqueous titration of diazepam by perchloric acid in acetic anhydride medium using Nile blue as indicator (2) and HPLC (17).
  Most of the old colorimetric methods involve hydrolysis of the benzodiazepine moiety to benzophenones, and thus lack specificity since this is the usual degradation pathway of benzodiazepines, and other needs solvent extraction be¬fore measurements. Therefore, a simple spectrophotomet¬ric method for determination of diazepam is needed. This is fulfilled in the present investigation by the application of Zimmermann reaction to the active methylene group adjacent to a carbonyl group in diazepam to produce high¬ly absorbing σ-complexes (26) upon reaction with picric acid (I), 3,5-dinitrobenzoic acid (II) and 2,4-dinitroben¬zoic acid (III), respectively. The present study describes the spectrophotometric determination of diazepam in pure samples and in its pharmaceutical preparations.

	EXPERIMENTAL

Apparatus

  Perkin Elmer Spectrophotometer model Lambda 1, Hanna instrument coductometer model HI8819N and Hanna pH meter model HI3313N were used for measuring absorbance, conductance and pH values, respectively.

Materials

  Diazepam [7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzo-diazpein] (M. Wt. = 284.75) was obtained from Memephis Co., Egypt and its purity was determined by the U.S. pharmacopoeial XX method (1). The pharma¬ceutical preparations (Farcozepam~, tablets 2 mg/tablet and Valepam~ ampoules, 10 mg/ampoule) were pur¬chased from the local market (Pharco Co., Egypt). All re¬agents were of analytical pure grade. They include sodium hydroxide, ethyl alcohol (99%), perchloric acid, acetic an¬hydride, Nile blue indicator, picric acid (I), 3,5-dinitroben¬zoic acid (II) and 2,4-dinitrobenzoic acid (III).

Stock solutions

  5 × 10-2 M alcoholic solution of diazepam was prepared and standardized by titration in acetic anhydride medium with HClO4 dissolved in glacial acetic acid, using Nile blue hydrochloride as indicator (2). Further dilution were made to 3 × 10-3 M, 1.5 × 10-2 M and 5 × 10-3 M. Alcoholic solutions of the electron acceptors were prepared at con¬centrations of 3 × 10-3 M of (I), 1.5 × 10-2 M of (II) and 5 × 10-3 M of (III). The pH of the medium was adjusted using 5,7 and 10 M sodium hydroxide solutions.

Procedure

  A solution containing 10.0-79.0 (in case of reaction with I), 15.2-177.8 (in case of reaction with II) or 17.0-83.0 µg/ml (in case of reaction with III) of diazepam was trans¬ferred into 10 ml measuring flask. 0.7 ml of 3.0 × 10-3 M of I, 2 ml of 1.5 × 10-2 M of II or 0.6 ml of 5 × 10-3 M of III, respectively was then added, followed by the appropriate amount of 5, 7 or 10 M sodium hydroxide to give a final concentrations of 0.5, 2.5 or 4.0 M, respectively. The vol¬ume was then completed up to 10 ml with alcohol, shaked well and left for 50, 60 or 60 minutes, respectively, at room temperature for full color development. The formed com¬plexes remained stable for 1 day, 90 and 90 minutes for I, II and III, respectively. The absorbances were then mea¬sured at 475, 500 and 500 nm, respectively.

Application to pharmaceutical preparations

  Farcozepam~ tablets. The developed procedure was applied for the determination of diazepam in some dos¬age forms without prior separation. Thirty tablets of (Far¬cozepam 2 mg) were weighed accurately and powdered in an agate mortar. An amount corresponding to 20 mg of diazepam was transferred to a flask containing 30 ml of alcohol and the suspension was shaked with a mechanical shaker for 30 minutes, followed by treating for 1 minute in a bath subjected to the action of ultrasonic waves then fil¬tered, transferred to 50 ml measuring flask and diluted to the mark with ethyl alcohol. An aliquot was transferred to 10 ml measuring flask and treated as previously described. The concentrations of the drug were obtained from the calibration curve of diazepam and the recoveries, apply¬ing the new method, were calculated.
  Valepam~ ampoules. Solution of Valepam~ ampoule (10 mg/ampoule) was prepared by mixing the contents of 5 ampoules. Then 1.5 ml of this solution was diluted to 25 ml with ethyl alcohol in a measuring flask. An aliquot was transferred to 10 ml measuring flask and treated as previously described. The concentrations of the drug were obtained from the calibration curve of diazepam and the recoveries, applying the new method, were calculated.

RESULTS AND DISCUSSION

Formation of the complexes and determination of their stability constants

  The reaction of picric acid (I), 3, 5-dinitrobenzoic acid (II) and 2, 4-dinitrobenzoic acid (III) with active methy¬lene compounds in alkaline medium is known to proceed via the formation of ó-complexes (26). The complex is called Meisenheimer complex and the reaction is called Janovsky reaction. In the presence of excess I, II and III, the complex is oxidized to a coloured anion while the re¬agents are reduced to 2-amino-4,6-dinitrophenol, 3-ami¬no-5-nitrobenzoic acid and 2-amino-4-nitrobenzoic acid, respectively, under Zimmermann conditions (27).
  Diazepam was found to yield intensely red coloured products in case of reaction with I, II and III, whose max¬imum absorbances were found at 475, 500 and 500 nm, respectively, most probably due to formation of ó-com¬plexes between diazepam and I, II and III.
  The ratio of (diazepam:reagent) in the formed com¬plexes was determined by using the molar ratio method (28) and conductimetric titration (29). Application of mo¬lar ratio method indicates the formation of 1:1 in case of I, 1:1 and 1:2 in case of II, and 1:1 in case of III (diazepam: reagent) complexes, respectively. Application of conducti¬metric titration indicates the formation of 1:1 in case of I, 1:1 and 1:2 in case of II and 1:1 in case of III (diazepam: reagent) complexes, respectively.
  The stability constants of the complexes formed be¬tween diazepam and I, II or III, were calculated using Harvey and Manning method (30). The stability constants, ân , of the formed complexes were calculated applying molar ratio method by the aid of the following equation: where A is the absorbance at the drug concentration CD; Am is the absorbance at full color development; N is the stoichiometric ratio of the complex; CD is the concentra¬tion of drug.

Expression of U-II in NZWR on High Fat Diet after iliofemoral balloon angioplasty

  One day post-angioplasty the naïve artery demon¬strated moderate UII-IR in both the endothelial and me¬dial SMC cells (Fig 2A). UII-IR in the injured artery one day post-angioplasty was localized predominantly to the sub-intima (Fig 2B), which was similarly noted in animals on normal chow diet one day post-angioplasty. Expect¬edly, the endothelium was absent in these arteries.
  In concordance with observations in arterial segments of animals on normal chow diet, animals on high fat diet exhibited elevated UII expression in both naïve and in¬jured arteries seven days post-angioplasty. Naïve arteries demonstrated moderate to strong UII-IR in the tunica me¬dia, while the endothelium demonstrated relatively strong UII-IR (Fig 2C). The injured arteries on the other hand exhibited strongest UII-IR in the neointima, with lesser IR in the media (Fig 2D). These latter arteries also demon¬strated re-endothelialization with strong IR.
  At 14 days post angioplasty, naïve arteries exhibited moderate to strong UII-IR in both the endothelium and in the media (Fig 2E). Contralateral injured arteries exhibited substantial intimal thickening with abundant UII-IR in foam cells in the abluminal side of the neointima (Fig 2F). The tunica media also exhibited relatively strong UII-IR in these arteries.
  At 28 days post-angioplasty, Naïve arteries exhibited moderate endothelial and weak medial IR (Fig 2G). A rel¬atively small neointima was observed in this artery dem¬onstrating the atherogenic potential of the high fat diet in these animals. Similarly to injured vessels, the neointima of this naïve artery demonstrated strong UII-IR (Fig 2H). The contralateral injured arteries exhibited very large in¬timal thickenings with very strong IR in the endothelial, neointimal and medial layers (Fig 2I). Consistently, stron¬gest UII-IR was noted in the outer neointima in associa¬tion with foam cells.
  The values of stability constants of the formed com¬plexes are depicted in Table 1. It was found that the se¬quence of increasing stability of the complexes is I < III < II.

Determination of diazepam

  The formation of the above mentioned complexes was utilized for the spectrophotometric determination of diaz¬epam in pure form, by measuring the absorbances of the formed complexes with I, II and III at 475, 500 and 500 nm, respectively. Table 2 summarizes the different param¬eters of this determinations, i.e. the wavelength of maxi¬mum absorption (λmax), molar absorptivity (ε), specific ab¬sorptivity (a), Sandell's sensitivity ($), range of obedience of Beer's law, Ringbom range and the statisticals of the calibration curve. The results shown in Table 2 reveal that Beer's law is obeyed up to 85.6, 180.2 and 128.6 µg/ml of diazepam in case of determination using I, II and III, respectively; with detection limits of 10.0, 15.2 and 17.0 µg/ml, respectively.
  The applicability of the proposed methods was tested, also, for the determination of diazepam in pharmaceutical preparations. It was found that the proposed methods can be applied successfully for the determination of diazepam both in pure form and in pharmaceutical preparations. Ta¬ble 3 summarizes the results of such determinations. The mg taken of the drug [as determined by the official method (2)] is shown versus the mg found by the proposed meth¬ods. The good recoveries obtained (98.9-101.3%) indicates the accuracy of these methods. Also, the precisions of the methods were tested by calculating the relative standard deviations for different determinations. The results shown in Table 3 indicate low values of the relative standard de¬viation, which taken as an evidence for the precision of the present methods.
  In order to assess the accuracy and precision of the present methods, the mean values obtained by the pro¬posed methods were compared with each other using t¬test, and the variances were compared with those of the official one (2) using F-test. The obtained t-values range from 0.48 to 1.92, which are lower than the tabulated value at 99% confidence level and 12 degrees of freedom (3.06). The obtained F-values range from 1.72 to 4.75, which are lower than the tabulated value at 99% confi¬dence level and 6, 5 degrees of freedom for the official and proposed methods, respectively (5.95). This means that there is no significant difference in accuracy of the proposed methods. Also, the proposed methods are of comparable precision with the official ones at 99% confi¬dence level as shown by the calculated "t" and "F" values shown in Tables 3 and 4.
  The pharmacopoeial methods (2, 17) for determina¬tion of diazepam in the raw material, tablets, and injec¬tion depends on non-aqueous titration with 0.1 N perchlo ric acid in acetic anhydride medium, using 1% solution of Nile blue hydrochloride in glacial acetic acid as indicator to yellowish-green end point (2) or HPLC (17). The mini¬mum quantities determined by these methods are 1 mg/ ml or 10 mg/ml in case of USP (17) or British pharma copoeia (2), respectively; as well as the technique needs certain precautions to keep anhydrous medium. The pro¬posed methods are used for the determination of much lower concentration (10 µg/ml), as well as it has a high reproducibility.
  The effect of interference of different cations and an¬ions on the absorbances of the formed complex was stud¬ied and it was found that in case of complexes of diazepam with picric acid, 3,5-dinitrobenzoic acid and 2,4-dinitro¬benzoic acid, up to 20 folds of Na+, Mg2+, K+, Zn2+ Pb2+, PO43-, Cl-, I-, Br-, S2O32-, SO42-, oxalate, citrate, tartrate, salicylate, acetate, nitroprusside, gluconate, pyroborate, hy¬drogen tartrate, metvanadate, sucrose, lactose, dextrose,glutamine, glycine and L-aspragine do not interfere. On the other hand, Ba2+, Ca2+, Fe2+, Fe3+, Hg2+, CN-, NO2 -, SCN, MnO4-, Cr2O7 2-, and EDTA interfere.
  Various spectrophotometric methods have been used for determination of diazepam. The method proposed by Sadeghi (6) is based on the reaction of diazepam with bro¬mocresol green at pH 3.5, extracting the colored product into chloroform and measuring the absorbance of chloroform layer at 410 nm. Although this method is sensitive (Beer’s law is obeyed within the range 2-60 µg/ml), but it needs tedious and time consuming extraction procedure. The methods proposed by Popovici depends on the reac¬tion of diazepam with picric in benzene (7) or chloroform (8) medium to form a colored product which is measured at 400 nm. Beer’s law is obeyed within the range 20-300 µg/ml. The minimum quantity determined by this method is 20 µg/ml, as well as the benzene and chloroform sol¬vents are expensive and have carcinogenic effect. Another spectrophotometric technique (9) is based on extraction of diazepam from hydrochloric acid medium into dichlo¬romethane (CH2Cl2) as a colored ion pair complex with orange II. Beer’s law is obeyed from 0.6-10 µg/ml, the molar absorptivity (ε) value is 1.15 × 104. This method is very sensitive, but in needs, also, prior extraction with or¬ganic solvent. The spectrophotometric method based on extraction of diazepam from aqueous solution at pH 1.2 into chloroform as a colored complex with Alizarin violet 3B or Alizarin brilliant violet R which is measured at 560 nm (10), has also the disadvantage of prior extraction with a harmful organic solvent. Beer’s law was obeyed from 4-16 µg/ml.
  Comparing the proposed methods with the published ones reveals that the new methods are simple, need no pri¬or separation steps and can be applied for determination of very low concentrations (10 µg/ml) with corresponding coefficient of variation ranges from 2.6-4.8% (n=7). Also, the reagents used are common and available. In conclu¬sion, the new methods are comparable in accuracy and precision with the published ones.

  Table 1 shows clinical characteristics of the subjects. There were significant differences in body mass index (BMI), SBP and DBP between EH and NT subjects. Among male subjects, there were significant differences in pulse rate, total cholesterol, smoking habit, uric acid and alcohol consumption between EH and NT subjects. There were no significant differences in creatinine or HDL-cholesterol between EH and NT subjects.

	CONCLUSION

  In conclusion, the proposed procedures are simple, inexpensive, and more sensitive than the official method. The developed procedures can be applied to the determi¬nation of diazepam in some dosage forms without prior separation. The developed procedure, being simple and rapid, can be recommended for routine analysis in drug quality control laboratories. Recovery experiments were carried out for the drug in its respective formulation. The excellent recoveries indicate the absence of interference from frequently encountered excipients or additives.

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