INTRODUCTION:

Loratadine (LOR) (fig.1), chosen for the current study, is a BCS class II drug (1), systematic (IUPAC) name is Ethyl4-(8-chloro-5,6-dihydro-11H benzo[5,6] cyclohepta [1,2-b]pyridin-11ylidene)-1-piperidinecarboxylate a long acting 2^nd generation tricyclic antihistamine. It is a piperidine derivative that antagonizes selectively histamine H₁ receptors. Loratadine is used in clinical practice to control symptoms of allergic rhinitis (2). Like the other 2nd generation antihistamines, it is claimed that loratadine has low sedation potential with no effect on cognitive and psychomotor performance.(3-5).

Many of analytical methods have been described for loratadine,as thin-layer chromatography(6) LC(7)liquid chromatography–tandem mass spectrometry method(8), liquid chromatography–electrospray mass spectrometry(9). non-linear second-order spectrophotometric data generated by a pH-gradient flow injection technique(10). And Capillary electrophoresis determination(11). The aim of this paper was the development of Spectrophotometric method for the identification and quantification of Loratadine in raw material and in tablets and syrups as pharmaceutical presentation.

The proposed method was based on the formation of charge transfer complexe between metol (N-methyl-p-hydroxy aniline) as π- acceptor and the studied drugs as n-donors in acetonitrile solvent.

Fig 1. Loratadine .Chemical formula C22H23Cl N2O2 Mol. Wt 382.8 gm Melting point 134-136 °C

EXPERIMENTAL:

Equipments

A (PG, England) uv-visible spectrophotometer with a 1 cm matched quartz cell were used for all absorbance measurements. pH meter (Hanna instrument pH 211, Italy).

Materials

All chemicals used were of analytical reagent grade unless otherwise stated.

Loratadine powder :

(Supplied by Philadelphia pharmaceutical, Jordan). And the stock solution (1000μg.ml^-1) was prepared with 521.7 mg of loratadine exactly weighed and dissolved in a 25 ml volumetric flask with acetonitrile. The commercial formulations used included Veegum, Clarityn, Methyl paraben as tablets, Propyl paraben, Raspberry flavor as syrups (supplied by SDI, Iraq). Stock dispersion of each suspending agent used were prepared.

Metol reagent solution (1×10^-2M):

Prepared by dissolving 0.1230 gm of N-methyl-p-hydroxy aniline in distilled water and made up to 100 ml in volumetric flask with the same solvent, and stored in dark bottle.

Procedure of pure drug :

Into a series of 25 ml calibrated flask, transfer an increased volumes of LOR of 500 ppm to cover the range of calibration graph 125-1500 μg of LOR in final volume 25 ml ,followed by 2 ml of metol (1×10^-2M).

The solutions are diluted to the mark with distilled water ..The color reach its maximum intensity and stability on standing for 50 min at room temperature then the absorbance is measured at 540nm against the reagent blank prepared in the same way butt contain no LOR..The color of the formed product is stable for 4h .For the optimization conditions and in all subsequent experiments a 1000 μg in final volume 25 ml was used (i.e. 40 ppm).

Procedure of pharmaceutical forms (tablets and syrups):

An accurately weighed amount of 10 powdered tablets or mixed content of 10 vials equivalent to 100 mg of the pure drug, was dissolved in 10 ml Acetonitrile and transferred into 100 ml calibrated flask and completed to the mark with distilled water. The flask with its contents was shaken well and filtered to obtain 1000 ppm, dilute 50 ml of this solution to 100 ml by distilled water to prepared 500 ppm of LOR. The measurement was carried out as described earlier under general procedure using suitable volume of last solution.

RESULTS AND DISCUSSION:

Metol reagent was utilized for the determination of LOR. The procedure depends on the formation of charge transfer complex upon the reaction of these reagents with LOR at acetonitrile medium. The reaction proceeds through the formation of a charge transferred colored product, which was measured spectrophotometrically.

The study and development of the method for the determination of LOR in bulk powder and pharmaceutical formulations, exploring its charge transfer reaction with metol reagent, was performed through optimization of the experimental conditions in order to achieve both maximum sensitivity and selectivity. This comprised investigation of the influence of the reagent concentration ,evaluation of the time required to complete the reaction , effect of temperature on the color intensity of the product and study and characterization of the reaction, which were carried out by the evaluation of the reaction stoichiometry and the verification of the proposed reaction mechanism.

At optimum conditions, the radical anion (absorbing species) was formed in the medium immediately after mixing of the reagent and showed maximum absorption at 540 nm in polar medium(fig.2). Thus, the wavelength was chosen for all further measurements in order to obtain highest sensitivity for the proposed methods.

Effect of the reagent concentration:

In Spectrophotometric analytical methods where maximum sensitivity is desired, the reagent concentration in solution is an important parameter to be studied, since the maximum conversion of the analyte into absorbing species depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. In order to achieve this objective, an experiment was performed when various concentration of metol solutions were added to fixed amount of the drug solution. A concentration of (1×10^-2 M) for LOR was found enough to develop the colour to its full intensity and give a minimum blank value and was considered to be optimum.

Effect of reaction time:

The optimum reaction time was determined by continuous monitoring of the absorbance at optimum wavelength of a solution containing 10 µg ml^-1 LOR plus 2.0 mL of (1×10^-2 M) reagent, at laboratory ambient temperature (25±2 °C). Stable absorbance values were observed from the beginning of the experiment up to 4 h. After this time, absorbance suffered a slight decrease. In view of these results, all spectral measurements were carried out after 5.0 min of mixing of the reagents and (25±2 °C)in order to make the method faster.

Effect of order of addition:

Drug–reagent–solvent was the favorable sequence of addition for complete color development and highest absorbance at the recommended wavelength.

Effect of temperature:

The effect of temperature on the colour intensity of the product was studied. In practice the same absorbance was obtained when the colour was developed at room temperature (25°C) but when the calibrated flask was placed in an ice-bath at (10°C) or in a water-bath at (40°C) a loss in colour intensity and stability were observed, it is therefore recommended that the colour reaction should be carried out at room temperature (25°C).

Analytical data:

Analytical data Employing the conditions described under procedure ,a linear calibration graph (Fig.3) for LOR and metol was obtained .The optical characteristics, such as Beer’s law, molar absorbability, correlation coefficient and other analytical data are summarized in Table (1). Under the optimum condition a linear calibration graphs was obtained over the concentration range of (10-50 μg.ml^-) for Loratadine. The limit of detections (signal/noise=3) were (0.24, 0.34 and 0.30 μg.ml^-1) and the correlation coefficients was 0.9997. The relative standard deviation of the method was better than 1.25%.

Accuracy and Precision:

The accuracy and precision of the method was determined at three different concentrations. Thee results shown in Table (2) indicate that satisfactory precision and accuracy could be obtained with the proposed method.

Stoichiometry of the reaction:

The stoichiometry of the reaction between Loratadine and metol was investigated using the molar ratio methodunder the optimized conditions. The results obtained (fig.4), show a 1:1 drug to reagent product was formed. In view of this result a reaction mechanism was proposed considering the transfer of free electron of the nitrogen atom present in one molecule of drug to the charge-deficient center of the reagent molecule.

Fig.2, (1:1)ratio of drug to reagent product

Fig. 3 . Stoichiometry of the reaction between LOR and metol

Mechanism of the Reaction:

The reaction between LOR and metol in the presence of acetonitrile yield a red product (λmax of 540 nm with a molar absorption coefficient of 2.824 ×10³ L.mole^-1.cm^-1), the reaction given in scheme 1 was postulated. The absorption spectrum of the colored product is given in Fig.5Under the reaction conditions, the complex may be regarded as a charge transfer complex type. The charge transfer may be presumed to be taking place involving electron transfer from the highest occupied (π)molecular orbital of pyridine cyclic of LOR to the lowest empty molecular orbital (π*) of metol. The addition of basic compounds that contain a lone pair of electrons, such as LOR, results in the formation of charge-transfer complexes of n–π type. This kind of complexes can be considered as an intermediate molecular-association compound that forms a corresponding radical anion in polar solvents. In this case, radical anions result from the total transfer of charge.

a b

Scheme 1: Proposed reaction mechanism for charge-transfer complexation of LOR (a) with Metol(b)

Analytical applications:

The proposed method was applied to the determination of Loratadine drugs in commercial tablets and syrups of pharmaceutical preparations. Good accuracy and precision were obtained for the studied drugs. The results obtained were given in (Table 1) which confirm the applicability of the method. Finally, the proposed method was compared successfully with the standard method.

Table 1:Analytical data obtained from proposed method.

PARAMETER	VALUE
Beer’s Low limits(μg.ml^-1)	2–40
Molar absorbativity(lit.mole^-1.cm)	2.431 × 10⁴
Sandell’s sensitivity(μg.cm^-2)	0.01162
Slope(b)	0.0729
Intercept(a)	0.0121
Correlation coefficient(R2)	0.9991
λmax (nm)	540
R.S.D(%)	1.037

Table2: accuracy and precision of the proposed method

Amount of LOR μg.ml^-1		Recovery %	RSD %
Present	Found	Recovery %	RSD %
30.00	30.28	101.39	1.90
40.00	40.28	100.93	0.48
80.00	80.40	98.99	0.25

General procedure for the determination of LOR drug:

The flow manifold is show in Fig. 5 a two channel manifold were used for the (FI) Spectrophotometric determination of drug. Four channel peristaltic pump [Pergen CH-8152, USA] minipuls (3) peristaltic pump was employed to transport the carrier stream. Local injector valve was used for injection of the drug sample. Flexible vinyl tubing of 0.8 mm internal diameter was used for the peristaltic pump. The reaction coil (RC) was made from glass with an internal diameter of 0.5 mm. In Fig. 6, the channel 1 was used to transport metol and channel 2 to introduce acetonitrile. The drug sample was injected through the injection valve into the resulting stream of the mixture of 2.033 x 10^-2 M metol with acetonitrile and were propelled by the peristaltic pump with an individual flow rate of (1.2,1.5, 1.5) ml.min^-1and The absorbance of the red colored species was measured at λ max 540 nm against the reagent blank prepared similarly omitting the drug.

Fig.4: Manifod of FIA system for determiation of FIA

Where: IV: injection valve. R.C: reaction coil. SX: drugs sample (LOR). P: peristaltic pump. D: detector. W: waste.

The LOR drug reacted with metol in the presence of acetonitrile to form an intense red product that can be measured at 540nm . Fig.5 showed the spectrum of the drug and product. It was found that the sensitivity of the color products depends on the reaction conditions and were optimized as follow.

Effect of the metol concentration:

The effect of various concentration of metol was investigated. A concentration of ( 100µL) gave the highest responce and were used for further experiments. The results obtained are shown in Table.3.

Table3: Effect of the metol concentration

Volume of metol (2. x 10^-2 M)

5ml

10 ml

20 ml

50 ml

75 ml

100 ml

Peak height

(cm)

3.4

4.8

7.2

8.4

9.2

10.2

Effect of flow rate:

Flow rate is an essential parameter in FIA. The results obtained showed that a flow rate of (2.5 ml.min^-1) gave the highest response for LOR as shown in Table.4 and was used in all subsequent experiments.

Table4: Flow rate of a reagent and carrier

Time (min)	Flow rate (ml/min)	Time of response (sec)	Peak height (cm)
2.30	2	20	6.4
2.00	2.5	16	7.8
1.40	3.0	12	7.4
1.30	3.3	10	7.2
1.20	3.8	6	6.6
50	6.0	2	4.8

Effect of reaction coil length:

The coil length is an essential parameter that affected on the sensitivity of the color reaction product and was investigated in the range of (50-200) cm. The results obtained showed that a coil length of (50cm) gave the highest absorbance for LOR as shown in Table .5 and were used in all subsequent experiments.

Table 5: The effect of reaction coil length on the product

Length of coil (cm)	Absorbance (mV)
50	482	486	488
100	426	427	426
150	436	436	448
200	440	436	432

Effect of injected sample volume:

The effect of sample volume was investigated by injection of a volume of difference length of sample loop. The results obtained showed that an injection sample of (50 µl) gave the best absorbance for LOR drug as shown in Table.6 and were used in the general recommended procedure.

Table 6 :Volume of Sample injected (µl)

Volume of Sample injected (µl)	1	5	10	20	50
Peak height (cm)	1.9	3.4	6.2	9.5	10.5

Calibration graph for the determination of LOR drug by FIA system:

Under the optimum condition a linear calibration graph. Fig.6was obtained over the concentration range of (10-70 μg.ml^-1) for LOR. The limit of detections (signal/noise=3) were (10μg.ml^-1). The correlation coefficients was 0.9991. The relative standard deviation of the method was better than 1.47% .

Fig .5:Calibration graph for the determination of LOR drug by FIA system

Table 6: The application of the proposed method for the determination of Loratadine in pharmaceutical preparations.

Sample	Amount of drugs Taken μg.ml-1	RSD%*	Recovery %
Sample	Amount of drugs Taken μg.ml-1	RSD%*	Proposed method	Standard method
Veegum	20	0.84	100.90	100
Clarityn	20	0.60	100.20
methyl paraben	20	0.86	99.16
propyl paraben	20	0.43	100.40
raspberry flavor	20	0.68	99.40

* average of three determination.

Fig .6: Spectrum of Lor and Metol for the determination of LOR drug by FIA system:

Under the optimum condition a linear calibration graph. Fig.7

Fig. 7: Calibration graph of Loratadine drugs

CONCLUSIONS:

The reported method is simple, rapid and sensitive. It has the advantages of a wide range of determination and high accuracy. The complex formed is stable for at least 4 h, thus permitting quantitative analysis to be carried out with good reproducibility.

REFERENCES:

1. M.Z. Khan, D. Rausl, R. Zanoski, S. Zidar, J.H. Mikulci´ c, L. Krizmani´ c, M. Eskinja, B. Mildner, Z. Knezevi´ c, Classification of loratadine based on the biopharmaceutics drug classification concept and possible in vitro–in vivo correlation, Biol.Pharm. Bull. 27 (2004) 1630–1635.

2. Lourance DR,Benett PN and Brown MJ . Histamine antihistamines and allergies .In Clinical pharmacology, ninth ed.2003:p 553-556)

3. Shamsi Z,Kimber S,Hindmarch I.An investigation into the effects of cetrizine oncognitive functions and psychomotor performance in healthy volunteers.Eur. J.Pharmacol. 2001;56(12):865-71.

4. Hindmarch JS,Meadows.The acute and subchronic effects of levocetrizine,loratadine,, cetrizine, promethazine and placebo on cognitive function ,psychomotorperformance and wheal and flare.Curr.Med.Res.Opin.2001;17(4):241-55.

5. Hindmarch JS, Shamsi Z.The effects of single and repeated administration of ebastine on cognitive and psychomotor performance in comparison to tripolidine and placebo in healthy volunteers.Ccurr.Med.Res.Opin.2001;17(4):274-81).

6. G. Popović, M. Čakar, and D. Agbaba Simultaneous determination of Loratadine and preservatives in syrups by thin-layer Chromatography(Acta Chromatographic, No. 19, 2007 Serbia ).

7. F.J. Rupe´rez, H. Ferna´ndez, C. Barbas.Determination of loratadine and related impurities .Journal of Pharmaceutical and Biomedical Analysis .29 (2002) 35–41 LC, Spain .

8. A.D. de Jager, D. Badenhorst, T. Scanes, H.K.L. Hundt, K.J. Swart, A.F. Hundt. Sensitive liquid chromatography–tandem mass spectrometry method for the determination of loratadine and its major active metabolite descarboethoxyloratadine in human plasma. Journal of Chromatography A, 914 (2001) 37–43 F.C.W. Sutherland, , South Africa.

9. Jianguo Sun ,Guangji Wang ,Wei Wang, Shuai Zhao, Yi Gu, Jinwen Zhang, Mingwen Huang, Fen Shao, Hao Li, Qi Zhang. Haitang Xie Journal of Pharmaceutical and Biomedical Analysis 2005, Pages 217–224 . China .

10. Culzoni MJ, Goicoechea HC. Anal Bioanal Chem. 2007 Dec;389(7-8):2217-25. Epub 2007, Argentina.

11. H. Ferna´ndez, F.J. Rupe´rez, C. Barbas , Spain Journal of Pharmaceutical and Biomedical Analysis 31 (2003) 499/506).

Received on 29.01.2013 Modified on 07.02.2013

Asian J. Research Chem. 6(3): March 2013; Page 221-225