Quantification of Drugs and Pharmaceuticals Using N-Bromosuccinimide and Methyl

Introduction

Dobutamine hydrochloride, [Fig.1 (a)], is chemically as 4-(2-((1-methyl-3-(4-hydroxybenzene) propyl) amido) ethyl)-1, 2-di-hydroxybenzene hydrochloric salt, is an adrenalin receptor concussion medicine indicated obvious curative effect for coronary heart disease, acute myocardial infarction and expansionary cardiomyopathy.¹ The literature survey reveals that several analytical methods such as enzymatic catalytic spectrofluorimetry ², Spectrophotometry ^{3, 4, 5}, high performance liquid chromatography ^{6, 7} and flow-injection chemiluminescence method⁸ have been developed for determination of Dobutamine hydrochloride.

Lomefloxacin (Fig.  1  b)  chemically  known  as    (RS)-1-ethyl-6,8-difluoro-  7-(3- methylpiperazin-1-yl)-  4-oxo-quinoline-3-  carboxylic  acid  is  one  of  the  third  generation fluoroquinolones  with  some  specific  activity  in  upper    respiratory  tract  infections  and community  acquiredpneumonia.  It  is  also  used  in  meningitis,  osteomyclitis,  urinary  tract infections,  sexually  transmitted  diseases,  bacteraemia,  nosocomially  acquired  infections, gastrointestinal  infections  and  in  combination  with  other  agents  in  the  treatment  of tuberculosis.⁹ Because  of  its  physiological  significance  several  methods  have  been  developed  for  its quantitative  determination  viz.,Spectrofluorimetry ¹⁰,  Spectrophotometry ¹¹ and  HPLC .^12,13

Figure 1: Structures of the Drugs   Click here to View figure

 

Sildenafil citrate (SIL) is designated chemically as 1- h[3- (6,7-dihydro-1-methyl-7-oxo-3-propyl-1-Hpyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonylj-4-methylpiperazine citrate and has the structural formula shown in [Fig. 1(c)]. It is used in oral therapy for erectile dysfunction, is a selective inhibitor of cyclic guanosine mono phosphate (cGMP) specific phosphodiesterase type 5 (PDE5) .¹⁴ Some techniques have been developed for quantitative determination of SIL in pharmaceutical formulations. Some important ones are HPLC ^15,16,17, flow-injection analysis with multiple pulse amperometric detection ¹⁸, atomic emission spectrometry ¹⁹, spectrophotometry^20,21  and LC-MS. ²²

Ramipril chemically is (2S,3aS,6aS)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]-octahydrocyclopenta[b]pyrrole-2-carboxylic acid, is a highly lipophilic, long acting angiotensin converting enzyme (ACE) inhibitor, [Fig.1(d)]. It effectively reduces both supine alterations in the pulse rate. It is indicated for Hypertension and cardiac failure. ²³ Some methods for the analysis of ramipril are Spectrophotometry ^24,25,26, High-performance liquid chromatographic and chemometric based spectrophotometry ²⁷and simple colorimetry²⁸ have been employed.

Telmisartan or (2-(4-{[4-methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid or [Fig.1(e)] is a cardiovascular drug, indicated for hypertension.²⁹ Because of its physiological importance many physical and instrumental techniques have been developed for the quantification of TEL like HPLC^30-36, Spectrophotometry^37-40, LC-MS/MS ⁴¹ and HPTLC^{42, 43}.

Experimental

Instrument

All absorbance measurements were recorded on Shimadzu 140 double beam spectrophotometer as well as on Thermo Nicolet 100 & Elico double beam SL210 UV- Visible spectrophotometers using matched pair of Quartz cells of 10mm path length.

Materials and Reagents

All the reagents used were of analytical-reagent grade and distilled water was used throughout the investigation. NBS solution (0.01%) was prepared by dissolving N-bromosuccinimide (Himedia Laboratories pvt.Ltd, Mumbai) in water with the aid of heat and standardized. The solution was kept in an amber colored bottle and was diluted with distilled water appropriately to get 70 μg mL^–1 NBS for use in spectrophotometric method.

A stock solution of Methyl Orange (5×10^-4 M) was prepared by dissolving the dye (s. d. Fine Chem. Ltd., Mumbai) in water and filtered using glass wool. The dye solution was diluted to 50 μg mL^–1.

Hydrochloric acid (1 M): Concentrated hydrochloric acid (S.D. Fine Chem., Mumbai, India; sp. gr. 1.18) was diluted appropriately with water to get 1 M acid.

The pharmaceutical grade drugs were supplied by Arabindo pharmaceuticals and hetero drugs Pvt.Lmt Hyderabad. A stock standard solution of drugs were prepared by dissolving accurately weighed 10 mg of pure drug in water and diluting to 100 mL in a calibrated flask with water. The solution was diluted stepwise to get working concentrations.

Assay procedure

Aliquots containing 2-50 µg mL^-1 of drug were transferred into a series of 10 mL standard flasks using a micro burette. To this, 1 mL of NBS was followed by 1 mL of 1M HCl and contents were shaken well. After 30 minutes, 1 mL of Methyl Orange dye added to the content. Then contents were shaken well and diluted up to the mark. The absorbance of each solution was measured at 508 nm against the corresponding reagent blank.

Calibration curves were constructed for all the drugs by plotting the absorbance versus the concentration of drugs. The absorbance data was collected for six replicate experiments and absorbance to concentration ratio called the relative response was determined. The relative responses between 95% to 105% of average only are considered for construction of the Calibration curves [figure 2].

Figure 2: Calibration curves of the Drugs   Click here to View figure

 

Procdure Foe Assay of Pure Drug

Sample solutions of each drug in the beer’s law limits were chosen and recovery experiments were performed to check the accuracy and precision. The concentration chosen and recovery are tabulated in table 2. For this purpose standard deviation method also adapted.

Excellent recovery and %RSD being less than 2 speaks about the precision and accuracy of the method [Table 1].

Table 1: Determination of accuracy and precision of the methods on pure drug Samples

Drug	Taken (µg mL-1)	Found (µg mL-1)	ER (%)	Recovery (%)	RSD (%)	Proposed method Mean ± SD
DOB	5 8 11 14 17	4.96 7.98 11.01 14.02 16.93	0.8 0.25 0.09 0.14 0.41	99.20 99.75 100.09 100.14 99.59	0.386	99.90 ±0.38
LOM	5 10 15 20 25	5.01 9.96 14.92 20.01 25.02	0.20 0.40 0.53 0.05 0.08	100.20 99.60 99.47 100.05 100.08	0.323	99.96 ±0.319
SIL	4 6 10 16 20	3.98 6.01 9.94 16.02 19.95	0.50 0.17 0.60 0.13 0.25	99.50 100.17 99.40 100.13 99.75	0.353	99.82 ±0.349
RAM	2 4 6 10 12	2.01 3.95 6.01 9.96 11.97	0.50 1.25 0.17 0.40 0.25	100.50 98.75 100.16 99.60 99.75	0.662	99.81 ±0.71
TEL	6 10 14 18 20	5.89 9.92 13.91 17.88 19.89	1.80 0.80 0.64 0.67 0.55	98.17 99.20 99.36 99.33 99.45	0.529	98.91 ±0.531

 

Dobutamin Hydrochloride

Two Cardiject injection (50mg/4mL/injection)s of DOB were placed in a boiling tube and worked out to get  working standard solutions of 5µgmL^-1. Quantification was performed using 5,8,11,14&17 μg mL^-1 of Dobutamine hydrochloride.

Lomefloxacin Hydrochloride

Ten tablets of LOMEBACT were weighed accurately, and powdered. The powder equivalent to 50 mg was transferred into a 100 mL volumetric flask, containing a mixture of distilled water (10.0 mL) and    HCl  (2.0  mL).  The  flask  was  shaken  for  5  min  and  the  solution  was  filtered  using whatmann  No:  41  filter  paper  and  further  diluted  with  water  to  obtain  working  standard solution.

Sildenafil citrate

Four tablets of ALISIGA containing 25 mg each amounting about 100 mg of SIL was accurately weighed, dissolved in water and diluted to volume 100 mL calibrated flask. This solution was diluted stepwise to give a series of concentrations suitable for the construction of the calibration graph.

Ramipril

To determine the content of Ramipril in pharmaceutical preparations, 20 tablets of Corpil (lable claim: 5 mg/tablet) were weighed and finely powdered. A portion of the powder equivalent to 50mg. Ramipril was stirred with 50 mL doubly distilled water and let stand for 10 minutes. The residue was filtered on Whatmann No.42 filter paper and wash with doubly distilled water. This solution was further diluted as necessary to complete the analysis concentration solutions for assay.

Telmisartan

Four tablets (Teli: 20mg/tablet) were weighed and powdered. Accurately weighed quantity of tablet powder equivalent to about 25 mg of telmisartan was transferred into 50 mL volumetric flask, added 25 mL of acetonitrile and shaken for ten minutes, the volume was then adjusted to mark with acetonitrile and mixed, the solution was filtered through Whatmann filter paper No.42 and the filtrate was then appropriately diluted with acetonitrile to get a final concentration of 6 μg mL^-1 of Telmisarta

Results and Discussion

Each method developed for quantification of drugs has been validated in terms of precision, accuracy, Limit of detection, Limit of quantification, Linearity, Selectivity and Ruggedness. The Beer’s law limits, Slope, Intercept, Correlation coefficient, Sandell’s sensitivity and Regression equations for each drug are tabulated in [table 2]. To assess the precision each experiment was repeated at least 6 times and accuracy is estimated in terms of percent recovery and percent RSD. Excellent percent recovery and RSD being less than 2 for each drug demonstrates accuracy and precision of the methods.

Table 2: Analytical and regression parameters of Spectrophotometric method

Parameter	DOB	LOM	SIL	RAM	TEL
λmax (nm)	508	508	508	508	508
Beer’s law limits (µg mL-1)	5-35	5-50	4-28	2-14	6-42
Molar absorptivity (L moL-1 cm-1)	8.5 x104	1.2×106	1.9×104	2.5×104	5.2×104
Sandell sensitivity* (µg cm-2)	0.035	0.167	0.036	0.013	0.067
Limit of detection (µg mL-1)	0.623	1.37	0.885	0.166	0.286
Limit of quantification (µg mL-1)	1.931	4.138	2.681	0.503	0.866
Regression equation (Y**=a+bX)	0.031+0.053X	0.031+0.006X	0.004+0.028X	0.069+0.081X	0.02+0.016X
Intercept, (a)	0.031	0.031	0.004	0.069	0.02
Slope, (b)	0.053	0.006	0.028	0.081	0.016
Correlation coefficient, (r2)	0.993	0.993	0.997	0.995	0.991
Standard deviation of intercept (Sa)	0.010	0.002	0.008	0.005	0.0014
Standard deviation of slope (Sb)	0.002	0.001	0.001	0.002	0.00133

 

Effect of acid concentration

To study the effect of acid concentration, different types of acids were examined (H₂SO₄, HCl, and H₃PO₄and CH₃COOH) to achieve maximum yield of Redox reaction. The results indicated that the hydrochloric acid was the preferable acid with NBS as oxidant. The reaction was performed in a series of 10 mL volumetric flask containing 8.0 μgmL^-1 0f the cited drugs, different volumes (0.5–2.5 mL) of 1M HCl and 1 mL of NBS (0.01%) were added. After 5.0 min of heating time at 60 ± 2°C in a water bath, the solution was cooled for about 3.0 min, 1 mL of Methyl Orange dye were added, then complete to 10 mL total volume with water. It was found that the maximum absorbance was obtained at 1 mL of 1M HCl. Above this volume, the absorbance decreased. Therefore, a volume of 1 mL of 1M HCl was used for all measurements.

Effect of heating time

In order to obtain the highest and most stable absorbance, the effect of heating time on the oxidation re-action of drugs were catalyzed by heating in a water bath at 60 ± 2°C for the periods ranging for 5-10 min. the time required to complete the reaction and maximum absorbance was obtained after 5.0 min of heating. After oxidation process, the solution must be cooled at least for 3.0 min before addition of dye.

Effect of oxidant concentration

When a study on the effect of NBS on color development was performed, it was observed that in both cases the absorbance increased with increase in the volume of NBS.  It reached maximum when 1 mL of 70 µg mL^-1 NBS   solution was added to a total volume of 10 mL for drugs solutions. The color intensity decreased above the upper limits. Therefore, 1 mL of 70 µg mL^-1 NBS   was used for all measurements.

Effect of dye concentration

In order to ascertain the linear relationship between the volume of added NBS and the decrease in absorbance of Methyl Orange dye, experiments were performed using 1 mL of 1M HClwith varying volumes of NBS. The decrease in absorbance was found to be linear up to the 1 mL of NBS with optimum volume 1.0 mL of Methyl Orange dye for fixed concentration drug solution. The color was found to be stable up to 24 hours.

Application To Formulations

The proposed methods were applied to the determination of drugs in tablets. The results in [Table 3] showed that the methods are successful for the determination of drugs and that the recipients in the dosage forms do not interfere.

Table 3: Results of assay of tablets by the proposed methods and statistical evaluation and recovery experiments by standard addition method.

Pharmaceuticals/ tablets/ injection	Drug in tablet(µg mL-1)	Drug added(µg mL-1)	Total found(µg mL-1)	ER%	Recovery %	RSD%	Reference method mean±SD	Proposed method±SD
DOB (CARDIJECT)	4.06.010.0 15.0 20.0	1.01.50.0 5.0 5.0	4.967.4710.01 19.98 25.0	0.800.130.10 0.10 0.00	99.2099.6100.1 99.9 100	0.366	99.75±0.425	99.80±0.365
LOM (LOMEBACT)	5.09.015.0 20.0 30.0	0.51.00.0 5.0 0.0	5.4810.0115.0 24.99 30.0	0.360.100.0 0.04 0.0	99.64100.1100 99.96 100	0.176	99.97± 0.203	100.0±0.176
SIL (ALISIGA)	3.06.011.0 20.0 22.0	1.02.01.0 0.00 2.0	3.967.9511.98 20.0 23.96	1.00.630.17 0.0 0.17	99.099.3899.83 100 99.83	0.412	100.01±0.402	99.61±0.411
RAM (CORPIL)	1.03.56.0 9.0 12.5	1.00.50.0 1.0 1.5	1.984.06.0 9.98 13.97	1.00.00.0 0.20 0.21	99.0100.0100 99.8 99.79	0.415	99.95±0.396	100.01±0.414
TEL (TELI)	5.011.018.0 20.0 25.0	1.001.000.00 5.0 3.0	5.9612.0217.97 24.98 28.01	0.670.170.17 0.08 0.04	99.33100.1799.83 99.92 100.04	0.322	100.0±0.298	99.92±0.322

 

Statistical analysis of the results using Student’s t-test for accuracy and F-test for precision revealed no significant difference between the proposed methods and the literature method at the 95 % confidence level with respect to accuracy and precision [Table 4].

Table 4: Student’s t-test and f-test values for Pharmaceutical analysis

Pharmaceuticals/tablets/injection	DOB	LOM	SIL	RAM	TEL
Student’st-test	0.262(3.182)	0.246(3.182)	0.038(3.182)	0.077(3.182)	0.134(3.182)
f-test	0.738(4.75)	0.752(4.75)	1.045(4.75)	1.093(4.75)	1.168(4.75)

 

Recovery experiment was performed via standard addition technique to ascertain the accuracy and validity of the proposed methods. To a fixed and known amount / concentration of drug in tablet powder, pure drug was added at three levels (50, 100 and 150 % of the level present in the tablet) and the total was found by the proposed methods. Each experiment was repeated six times and the percent recovery of pure drugs added was within the permissible limits showing the absence of interference by the inactive ingredients in the assay.

Conclusion

This is simple, rapid, and cost-effective methods for the determination of drugs have been developed and validated. The proposed method is more sensitive and the methods depends on the use of simple and cheap chemicals and techniques but provide sensitivity comparable to that achieved by sophisticated and expensive technique like HPLC. Thus, they can be used as alternatives for rapid and routine determination of bulk sample and tablets.

 Aknowledgement

The authors are thankful to the Head, Department of chemistry, University College of Science, Osmania University, Hyderabad, Telangana-500 007 (INDIA). One of the authors (KS) is thankful to UGC-JRF.

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