Spectrophotometric and Spectrofluorimetric Methods for Simultaneous Estimation of Amlodipine and Metformin in Bulk and Synthetic Mixture.

 

Tejash H. Serasiya¹*, Satish Y. Gabhe² and Nurudin P. Jivani³

¹Department of Pharmaceutical Sciences, Jaipur National University, Jaipur - 302 025, India.

²C. U. Shah College of Pharmacy, S.N.D.T. Women’s University, Mumbai - 400 049, India.

³Smt. R. B.Patel Mahila Pharmacy College, Atkot - 360 040, India.

*Corresponding Author E-mail: tejashserasiya@rediffmail.com

ABSTRACT:

Purpose: the purpose of this article is to develop two simple, rapid, accurate and economical spectrophotometric methods and a sensitive spectrofluorimetric method for the simultaneous estimation of amlodipine (AD) and metformin (MT) in bulk and synthetic mixture.

Method: The Spectrophotometric determination involved simultaneous equations method that measured the absorbances of both the drugs at 238 nm (λ_max of metformin) and at 361 nm (λ_max of amlodipine) and Q- absorbance ratio method that measured the absorbance of both the drugs at 238 nm (λ_max of metformin) and at isoabsorptive point (256 nm). A spectrofluorimetric determination is based on the reaction between the drugs and 1-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl chloride) in presence of 1 M sodium carbonate (pH 10) to yield a highly fluorescent derivative that is measured at 450 nm and 520 nm after excitation at 345 nm for AD and MT, respectively. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized.

Results and discussions: Both spectrophotometry methods showed linearity over the range of 5-90 µg ml^-1 for AD and 1-10 µg ml^-1 for MT. The fluorescence intensity were linear over the range of 0.1-3.5 µg ml^-1 for AD and 0.05-1.3 µg ml^-1 for MT. Results of analyses were validated statistically and through recovery studies.

 

 

 

INTRODUCTION:

Hypertension is often present as part of the metabolic syndrome of insulin resistance. The prevalence of hypertension in type 2 diabetes has been reported to be 30–80%. In type I diabetes, the prevalence is up to 25% and hypertension is usually seen in association with nephropathy. Most diabetic complications occur in association with hypertension¹. People with both diabetes and hypertension have approximately twice the risk of cardiovascular disease as compare to non-diabetic people with hypertension. To improve outcome in hypertensive diabetic patients, Combination drug therapy is often required to control raised arterial pressure and blood glucose level².

 

Amlodipine (AD) (Fig. 1), 2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4-dihydropyri- dine, is a calcium channel blocking agent of the dihydropyridine derivative which is used in the treatment of hypertension and angina pectoris³. Metformin (MT) (Fig. 2), (N, N-dimethylimidodicarbonimidicdiamide hydrochloride) is a bigunide prescribed for the treatment of type II diabetes mellitus and is the drug of choice in obese patients. Thus, attempt to develop simultaneous estimation methods for AD and MT in bulk and synthetic mixture that may allow formulating single system and can reduce dosage frequency. Literature survey revealed that calcium channel blocking agent (AD) have beneficial action in hypertensive diabetes patient and reduce mortality rate in hypertensive diabetes patients by controlling blood pressure⁴. Due to important pharmacological responses of AD and MT in hypertensive diabetic patients, developments of simple yet sensitive and accurate methods for the determination of AD and MT are desirable. Detailed survey of literature revealed several chromatographic methods for determination of AD in pharmaceutical and biological fluids^5-11. Though these methods are sensitive enough, they are expensive and time consuming. Estimation of the AD has also been achieved by different spectrophotometric methds ^12-14. But the methods involve tedious extraction step or use of costly organic solvents.

 

Figure 1: Structure formula of amlodipine besylate

Figure 2: Structure formula of metformin hydrochloride

 

Similarly, literature survey for MT revealed various methods based on chromatographic and spectrophotometric for its determination in pharmaceutical and biological fluids^15-24. Most of these are time consuming; involve expensive instrumentation or the use of excess organic solvents and highly skilled human resources. On the other hand, Spectrophotometry and spectrofluorometry are still the technique of choice since it is simple yet sensitive, economical and rapid. Literature survey revealed that no analytical method is yet reported for the simultaneous estimation of AD and MT in the bulk and synthetic mixture. Therefore, it was thought worthwhile to develop simple, accurate, rapid and economical spectrophotometry and a sensitive spectrofluorimetry method for the simultaneous estimation of AD and MT in synthetic mixture.

 

The present paper describes two spectrophotometric methods and a spetcrofluorimetric method based on its reaction with 1-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl chloride) to form a highly fluorescent product for simultaneous determination of AD and MT in synthetic mixture. The proposed methods are sensitive, simple, fast, efficient, and were successfully used to determine the AD and MT in synthetic mixture.

 

MATERIALS AND METHODS:

Apparatus:

The spectrofluorimetric measurements were recorded using ARF-1501 Shimadzu Spectrofluorometer, equipped with Xenon arc lamp. Spectral and absorbance measurements were made on Heliosα V4.60, serial no. 132206, with matched pair quartz cells corresponding to 10 mm path length. Sartorius CP224S analytical balance was used for weighing the samples, an ultrasonicator (Frontline FS4) was used for proper dissolution of samples. All glassware used (in each preparation) were soaked overnight in a mixture of chromic acid and sulphuric acid, rinsed thoroughly with double distilled water and dried in hot air oven.

 

Reagents and Solutions:

All chemicals used were of analytical grade and solvents were of spectroscopic grade.

§  Amlodipine besylate and Metformin hydrochloride were kindly provided by Khandelwal lab. Pvt. Ltd., Uttaranchal.

§  1-Dimethyl aminonaphthalene-5-sulphonyl chloride (Dansyl chloride), purchased from Sigma Aldrech. A stock solution containing 0.1% w/v of dansyl chloride was freshly prepared in acetone.

§  Sodium carbonate (Qualigen Fine Chemicals, Mumbai), 1M aqueous solution (pH 10).

§  Methanol used was of analytical grade and obtained from Qualigen Fine Chemicals, Mumbai.

 

Standard drugs solution:

Stock solutions of Amlodipine besylate and Metformin hydrochloride were prepared separately by dissolving the pure compounds in methanol to obtain each of 100 µg ml^-1 for spectrophotometric determination. For spectrofluorimetric determination, Stock solutions containing 100 µg ml^-1 of Amlodipine besylate and Metformin hydrochloride were prepared separately by dissolving 10 mg of (each) pure drug in 100 ml of solvent mixture (Acetone:1M sodium carbonate, 30:70) and the mixture was sonicated till dissolved. The volume was completed to the mark with same solvent mixture (Acetone:1M sodium carbonate, 30:70).

 

Synthetic drug mixture:

The synthetic mixture containing AD and MT were prepared in the ratio of 1:2 with common excipients, which are used in the tablet formulations, and sonicated for 20 min. These solutions were filtered through whatman filter paper No. 41 and diluted to obtain the concentrations 100 µg ml^-1 of AD and 200 µg ml^-1 of MT for simultaneous determination.

 

Figure 3: Overlain emission spectra of Amlodipine and metformin in optimum condition

 

 

Figure 4: Overlain spectra of Amlodipine and Metformin

 

 

SPECTROPHOTOMETRIC METHODS:

Determination of absorbance maxima:

The standard drug solutions were appropriately diluted and scanned separately in the range of 200 to 400 nm against methanol as a blank. Maximum absorbance was obtained at 238 nm and 361 nm for MT and AD, respectively. Iso-absorptive point was found at 256 nm (Fig. 4). Test solutions were prepared by appropriate dilution of standard stock solutions such that resulting solution contain concentration range 5-90 µg ml^-1 for AD and 1-10 µg ml^-1 for MT. Absorbance of each solution was measured at the three wavelengths 238 nm, 256 nm and 361 nm. Molar absorptivity for both the compounds was calculated at three wavelengths 238 nm (λ_max of metformin), 256 nm (iso-absorptive point) and 361 nm (λ_max of amlodipine). MT did not show any absorbance at 361 nm, hence its absorptivity was taken as zero in the calculations.

 

Method I (Simultaneous equation method):

In the simultaneous equation method concentrations of MT and AD in the synthetic mixture were found out by solving following equations;

 

C_m= (A₂a_a1-A₁a_a2) / (a_m2a_a1-a_m1a_r2)                                     (1)

C_a= (A₁a_m2- A₂a_m1) / (a_m2a_a1-a_m1a_a2)                                    (2)

 

Where; C_m & C_a are the concentration of MT and AD in the sample solution, A₁ & A₂ are absorbance of the sample solution at 238 nm and 361 nm, respectively, a_m1 & a_m2 are molar absorptivity of MT at 238 nm and 361 nm, respectively and a_a1 & a_a2 are molar absorptivity of AD at 238 nm and 361 nm, respectively.

Method II (Q-absorbance ratio method):

In the Q-absorbance ratio method concentration of MT and AD in the sample solutions were calculated using equations

 

C_m2 = (Q_o- Q_a/Q_m- Q_a) × A₃/a_m3                                                                  (3)

C_a2 = A₃/a_a3- C_m2,                                                                (4)

 

Where, A₁ and A₃ are absorbance of sample solution at 238 nm and 256 nm; and a_m3 and a_a3 are molar absorptivity of MT and AD at 256 nm; a_m1 and a_r1 are molar absorptivity of MT and AD at 238 nm. Q_o = A₁/A₃, Q_m = a_m1/a_m3 and Q_a = a_a1/a_a3.

 

SPECTROFLUORIMETRIC METHOD:

Binary mixture solutions containing AD and MT were prepared by transferring different aliquots (0.1–3.5 ml) of 10 µg ml^-1 AD solution and (0.05–1.3 ml) of 10 µg ml^-1 MT solution into series of 10 ml calibrated volumetric flasks. To each flask volume was adjusted to 5 ml by adding solvent mixture (Acetone:1M sodium carbonate, 30:70). 0.8 ± 0.1 ml of 0.1 % w/v dansyl chloride solution was added. The content was mixed well after stoppering the flasks and let stand for 45 min, and then completed to the mark with acetone. The reaction mixture was allowed to stand for 10 min. The final concentrations were in the ranges 0.05-1.3 µg ml^-1 for MT and 0.1-3.5 µg ml^-1 for AD. The fluorescence intensity of the reaction product was measured at 450 nm and 520 nm after excitation at 345 nm for determination of AD and MT, respectively.

 

 

Table 1: Quantitative parameters of spectrophotometric methods

Parameter	238 nm		361 nm	256 nm
	AD	MT	AD	AD	MT
Beer’s limit, µg m-1	5-90	1-10	5-90	5-90	1-10
Molar absorptivity, l mol-1cm-1	19464.49	17430.42	6805.2	7029.7	2050.5
Sandell’s Sensitivity, µg cm-2	0.0513	0.0573	0.146	0.142	0.487
LOD, µg ml-1	0.28	0.12	0.27	0.14	0.17
LOQ, µg m-1	0.87	0.36	0.83	0.44	0.52
Regression equation*, Y= mX + C	Y=0.033X+0.036	Y=0.104X+0.001	Y=0.012X-0.014	Y=0.013X-0.015	Y=0.011x+ 0.004
Correlation coefficient, r2	0.998	1.0	0.999	0.998	0.998

^* six independent analysis; LOD is limit of detection; LOQ is limit of quanification

 

 

RESULTS AND DISCUSSION:

Absorbance spectra of AD showed two λ maximum 240nm and 361nm while MT showed maximum absorbance at 238nm. The overlain absorbance spectra (Fig. 4) showed one iso-absorptive point at 256 nm. This wavelength was used in Q-absorbance ratio method for simultaneous estimation of AD and MT while λ maximum 238nm and 361nm were selected for simultaneous estimation of MT and AD by simultaneous equation method. Both spectrophotometric methods showed linearity over concentration range of 5-90 µg ml^-1 for AD and 1-10 µg ml^-1 for MT. LOD and LOQ revealed that both spectrophotometric methods are quite sensitive as shown in table 1. These methods were further validated through % recovery study. The % recoveries obtained were 99.72±0.09 to 101±0.05 for AD and 99.6±0.09 to 100.13±0.14 for MT by simultaneous equation method and 99.6±0.04 to 100.66±0.06 for AD and 99.72±0.11 to 100.1±0.09 for MT by Q-absorption ratio method. No interference due to the excipients was observed. The proposed validated methods were thus successfully applied in the determination of AD and MT in bulk and synthetic mixture.

 

Dansyl chloride (DNS-Cl) (1-dimethylaminonaphthalene-5-sulphonyl chloride) was first introduced by Weber for the preparation of fluorescent conjugates of albumin, and has frequently been used as a fluorescent reagent for peptides and proteins^25,26,27. It is found that primary and secondary amines, imidazoles and phenols react quantitatively with dansyl chloride under suitable conditions to produce the corresponding sulphonamides or phenolic esters^28,29. Dansyl chloride was used for the determination of some primary, secondary amines, imidazoles and phenols^30-33. In the present spectrofluorimetric method (Table-2), AD and MT were found to react with Dansyl chloride at pH 10.0 forming a highly fluorescent derivative with λ maximum emission at 450 nm and 520 nm after excitation at 345 nm (Fig. 3).

 

Optimization of reaction conditions:

Many experimental parameters may affect the development of the reaction product and its stability. Such factors were changed individually while the others were kept constant. The factors include pH, concentration of the dansyl chloride, temperature, reaction time and dilution time.

 

Table 2: Quantitative parameters of spectroflourimetric methods

Parameter	AD	MT
λex, nm	345 nm	345 nm
λem, nm	520 nm	450 nm
Linearity range, µg ml-1	0.1- 3.5	0.05 – 1.3
LOD, µg ml-1	0.021	0.012
LOQ, µg ml-1	0.065	0.036
Regression equation*, Y=mX + C	y = 147.7x + 3.802	y = 445.0x + 9.619
Correlation coefficient, r2	0.999	0.999

^* Six independent analysis

 

Effect of pH:

The influence of pH on the fluorescence intensity of the reaction product was studied. Maximum fluorescence intensity was obtained upon using mixture of acetone and 1M Sodium carbonate solution. The pH of the reaction mixture was found to be 10. However, under the proposed chosen conditions and wavelengths used, there was no interference arisen from any dansyl hydroxide formed, as indicated by the low fluorescence intensity of the reagent.

 

Effect of Concentration of Dansyl chloride:

The influence of the concentration of dansyl chloride was studied using different volumes of 0.1% w/v of the dansyl chloride solution. It was found that, the reaction of dansyl chloride with drugs started upon addition 0.1 ml of 0.1% w/v dansyl chloride solution in the presence of sodium carbonate (pH 10.0). Flourescence intensity was found to be increased with increasing the volume up to 0.7 ml and then remains constant up to 1 ml. Therefore, 0.8±0.1 ml of 0.1% w/v of dansyl chloride solution was chosen as the optimal volume of the reagent (Fig. 5).

 

Figure 5: Effect of volume of dansyl chloride (0.1 % w/v) on the fluorescence intensity of the reaction product at pH 10.0.

Table 3: Results of recovery study

Drug	Amount, µg ml-1	Spectroflourimetry		Method Ib		Method IIc
	Taken + Added	Found±SDa	% RSDa	Found±SDa	% RSDa	Found±SDa	% RSDa
AD	5 + 0	5.02±0.05	0.99	5.05±0.05	0.99	4.98±0.04	0.8
	5 + 4	9.03±0.08	0.88	9.02±0.07	0.77	9.06±0.06	0.66
	5 + 5	10.01±0.07	0.69	9.99±0.08	0.8	10.03±0.09	0.89
	5 + 6	10.95±0.09	0.82	10.97±0.09	0.82	11.01±0.08	0.72
MT	10 + 0	10.02±0.06	0.59	9.96±0.09	0.9	10.01±0.09	0.89
	10 + 8	17.97±0.14	0.77	18.02±0.13	0.72	17.95±0.11	0.61
	10 + 10	20.04±0.12	0.59	19.98±0.16	0.8	19.98±0.17	0.85
	10 + 12	22.01±0.16	0.72	22.03±0.14	0.63	22.02±0.19	0.86

^a average of six independent analysis; ^bSimultaneous equation method; ^cQ-absorbance ratio method; SD is standard deviation; %RSD is percentage relative standard deviation.

 

Table 4: Results of precision and ruggedness

Parameter	Spectroflourimetry		Method Ia		Method IIb
	AD	MT	AD	MT	AD	MT
Precision, % RSD
Intra-day, n=3	0.16 - 0.98	0.21 – 1.05	0.19-1.04	0.14-1.12	0.23-1.17	0.25-1.26
Inter-day, n=6	0.21 – 1.05	0.11 – 1.24	0.17-1.17	0.28-1.35	0.19-1.28	0.27-1.32
Repeatability, n=6	0.28	0.32	0.42	0.29	0.34	0.41
Ruggedness, % RSD
Analyst I, n=6	0.36	0.29	0.26	0.51	0.38	0.32
Analyst II, n=6	0.28	0.25	0.14	0.36	0.25	0.17

^aSimultaneous equation method; ^bQ-absorbance ratio method

 

Effect of Temperature:

Same derivatization procedure was carried out at different temperature higher than the room temperature. Results showed there was no any significance changed in fluorescence intensity with increasing temperature.

 

Effect of Reaction Time:

Different time intervals were tested to ascertain the time after which the solution attains its highest fluorescence intensity. It was found that after 40 min, the reaction product reaches the highest fluorescence intensity (Fig. 6) and remains stable at room temperature for 20 min.

 

Figure 6: Effect of reaction time on the fluorescence intensity of the reaction product with dansyl chloride at pH 10.0.

 

Effect of Diluting Solvent:

Different solvents were tried to dilute the reaction mixture throughout the study. It was observed that acetone gave the highest fluorescence intensity. Dilution with water or 1M sodium carbonate, or the mixture (acetone: 1M sodium carbonate, 30:70) produces no fluorescence. Using of acetonitrile and isobutylmethylketone produce relatively low fluorescence intensity. The increase of fluorescence intensity obtained with acetone due to lowering the fluorescence of the blank reagent (Fig. 7).

 

Figure 7: Effect of diluting solvent on fluorescence intensity of reaction product.

 

Figure 8: Effect of dilution time on the fluorescence intensity of reaction product with dansyl chloride at pH 10.0.

Effect of Dilution Time:

Dilution times were tested to ascertain the time after which the solution attains its highest fluorescence. It was found that dilution with acetone after 10 min, the reaction product reached its highest fluorescence intensity (Fig. 8).

 

Validation of Proposed Methods:

The accuracy and validity of the proposed methods were ascertained by performing recovery studies via standard addition technique. Pre analyzed drug mixture was spiked with pure AD and MT at three different levels and the total amount of drugs were determined by the proposed methods. The recoveries of the pure drugs added were quantitative (Table 3), and revealed that the common additives, such as talc, starch, hydroxyl propyl methyl cellulose, magnesium stearate and calcium dihydrogen orthophosphate, and experiments did not interfere in the determination. Precision of the proposed methods was studied as intra-day, inter-day and repeatability (Table 4). Repeatability was performed by analyzing same concentration of drugs for six times. The results showed the best approval of the proposed methods in routine use.  Ruggedness of the proposed method was determined by analysis of aliquots from homogenous slot by different analysts using similar operational and environmental conditions.

 

Application in Synthetic Mixture:

The proposed methods were evaluated in the assay of synthetic mixtures. Table 3 shows the results obtained by the application of the proposed models on the synthetic samples. Six replicate determinations were carried out on each experiment. These results confirm satisfactory to the drug content and indicate the high precision and accuracy of the proposed methods when applied to synthetic mixture.

 

CONCLUSION:

The proposed spectrophotometry and spectrofluorimetry determination are suitable techniques for the reliable analysis of combination of MT and AD in bulk and their mixtures. The most striking features of the proposed methods are their simplicity and speed, which render them suitable for routine analysis in control laboratories. In addition, Spectrofluorimetric method possesses the advantage of high sensitivity, which may be an incentive to other workers to consider it for the biological fluids.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Dr. C. N. Patel, principal, Shri Sarvajanik Pharmacy College, Mehsana, for providing the research facilities. Thanks are also extended to M/s. Khandelwal lab. Pvt. Ltd., India for providing the gift samples of pure amlodipine besylate and metformin hydrochloride.

 

REFERENCES:

1.       Michael DF. Diabetes and Hypertension. Diab Met Rev 1987; 3: 463–524.

2.       Bakris GL, Williams M, Dworkin L. Preserving renal function in adults with hypertension and diabetes: a consensus approach. National Kidney Foundation Hypertension and Diabetes Executive Committees Working Group. Am J Kidney Dis 2000;36: 646-661.

3.       Haria M, Wagstaff AJ. Amlodipine: A reappraisal of its pharmacological properties and therapeutic use in cardiovascular disease. Drugs 1995; 50:560-586.

4.       Mattew RW. Diabetes and Hypertension: How long should you go and with which drugs. Am J Hypertens 2001;14:17S-26S.

5.       Yeung PK, Mosher SJ, Pollack PT. Liquid chromatography assay for amlodipine: Chemical stability and pharmacokinetics in rabbits. J Pharm Biomed Anal 1991;9: 565-568.

6.       Josefsson M, Zackrisson AL, Norlander B. Sensitive high-performance liquid chromatographic analysis of amlodipine in human plasma with amperometric detection and a single-step solid-phase sample preparation. J Chromatogr B: Biomed Appl 1995; 672: 310-17.

7.       Pandya KK, Gandhi TP, Modi IA. Detection and determination of total amlodipine by high-performance thin-layer chromatography: a useful technique for pharmacokinetic studies. J  Chromatogr. B: Biomed  Appl 1995; 667: 315-26

8.       Agrekar AP, Powar SG. Simultaneous determination of atenolol and amlodipine in tablets by high-performance thin-layer chromatography. J Pharm Biomed  Anal 2000; 21: 1137-48

9.       Bresford AP, Marcrac PV, Stopher DA. Analysis of amlodipine in human plasma by gas chromatography. J  Chromatogr 1987; 420: 178-87

10.     Yasuda T,  Tanaka M, Iba K. Quantitative Determination of Amlodipine in Serum by Liquid Chromatography with Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry. J Mass Spectrom 1996;31: 879-88

11.     Mohamed YE, Naglaa ME, Bahia AM. Fluorimetric determination of amidarone, amlodipine and propafenone.  Bull Fac Pharm 1998; 36: 1-8.

12.     Sridhar k, Sastry CSP, Reddy MN, Sankar DG. Spectrophotometric determination of amlodipine besylate in pure forms and tablets.  Anal Lett 1997; 30: 121-30.

13.     Rahman, N,  Azmi SNH. Spectrophotometric Determination of Amlodipine Besylate by Charge-Transfer Complex Formation with p-Chloranilic Acid. IL Farmaco 2001;  56: 731-39.

14.     Arayne MS, Sultana N, Zuberi MH. Development and validation of RP-HPLC method for the analysis of metformin. J Pharm Sci 2006; 19: 231-39.

15.     Vasudevan M, Ravi J, Suresh B, ION-pair liquid chromatography technique for the estimation of metformin in its multicomponent dosage forms. J Pharm Biomed Anal 2001; 25: 77-87.

16.     Kolte BL, Raut BB, Deo AA, Simultaneous High-Performance Liquid Chromatographic Determination of Pioglitazone and Metformin in Pharmaceutical-Dosage Form. J  Chromtogr  Sci 2004; 42: 27-36.

17.     Zarghi SM, Foroutan SM, Shafaati A, Khoddam A. Rapid determination of metformin in human plasma using ion-pair HPLC. J Pharm Biomed Anal 2003; 31: 197-207

18.     Zhang M, Moore GA, Lever M. Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk. J Chrom B 2002;766: 175-82.

19.     Tache F, David V, Farca A. HPLC-DAD determination of Metformin in human plasma using derivatization with p-nitrobenzoyl chloride in a biphasic system. Microchemical J 2001;68: 13-21.

20.     El-Khateeb SZ,  Assaad HN, El-Bardicy MG, Ahmad AS. Determination of metformin hydrochloride in tablets by nuclear magnetic resonance spectrometry. Anal Chim Acta 1988; 208: 321-30.

21.     Hassan SM, Mahmoud WH, Elmosallamy MA. Determination of metformin in pharmaceutical preparations using potentiometry, spectrofluorimetry and UV–visible spectrophotometry. Anal Chim Acta  1999; 378: 299-312.

22.     Habib IH, Kamel MS. Near infra-red reflectance spectroscopic determination of metformin in tablets. Talanta 2003; 60: 185-196.

23.     Ashour S, Kabbani R. Direct spectrophotometric determination of metformin hydrochloride in pure form and in drug formulations. Anal Lett 2003;36: 361-372.

24.     Seiler N. Use of the Dansyl Reaction in Biochemical Analysis. Methods Biochem Anal 1972; 18: 259-264.

25.     Rosmus J, Deyl Z. Chromatographic methods in the analysis of protein structure: The methods for identification of N-terminal amino acids in peptides and proteins. J Chromatogr  1972; 70: 221-332.

26.     Seiler N, Wiechmann M. Progress in Thin Layer Chromatography and Related Methods, Vol. 1. Ann Arbor-Humphrey Science Publishers, Ann Arbor MI 1970; 94.

27.     Boulton AA. The automated analysis of absorbent and fluor- escent substances separated on paper strips. Methods Biochem Anal 1968;16: 327-39.

28.     Penzes LP, Oertel GW. Determination of steroids by densitometry of derivatives II. Direct fluorometry of dansyl estrogens. J Chromatogr. 1970; 51: 325-37.

29.     Gray WR, Hartley BS. The structure of a chymotryptic peptide from pseudomonas cytochrome c-551. Biochem J 1963; 89: 59-67.

30.     Ayad MM. Spectrofluorimetric microdetermination of imidazoline derivatives using 1-dimethylaminonaphthaline-5- sulphonylchloride. Analyst 1984;109: 431-42.

31.     Frei-Hausler M, Frei RW. An investigation of fluorigenic labelling of chlorophenols with dansyl chloride. J Chromatogr 1973; 84: 214-222.

32.     Dennis JG, Charles BR, Steele SC, Donald R, Devanter V. Determination of polymyxin E1 in rat plasma by high-performance liquid chromatography. J. Chromatogr. B. 2003;789: 365-374.

33.     Houdier S, Perrier S, Defrancq E. A new fluorescent probe for sensitive detection of carbonyl compounds: sensitivity improvement and application to environmental water samples. Anal Chim Acta  2000;412: 221-230.

 

 

 

 

Received on 18.12.2010        Modified on 11.01.2011

Accepted on 27.01.2011        © AJRC All right reserved