INTRODUCTION:

Nicotine hydrogen tartrate (NHT) (Fig 1) is a pure crystalline salt of nicotine. Chemically it is (−)-1-Methyl-2-(3-pyridyl) pyrrolidine (+)-bitartrate salt. Nicotine is a potent parasympathomimetic alkaloid, it acts as a nicotinic acetylcholine receptor agonist. In smaller doses, its acts as a stimulant in mammals, while high amounts (30–60 mg) can be fatal. ^1-2

To date, all analytical methods described in literature for the determination of NHT involve potentiometric and spectrophotometric methods^3-4. The aim of the present study was to develop an accurate, precise, specific, reproducible and stability indicating HPLC method for the estimation NHT. The NHT was intentionally degraded by acid, base, oxide, dry heat, wet heat and UV light treatment to check the stability and to develop stability indicating assay method ^5-13. The developed analytical method was validated by means of precision, robustness, linearity and range. ¹⁴

Fig. 1: Structure of NHT

MATERIALS AND METHODS:

Chemicals and reagents:

NHT was supplied by J.L. Chaturvedi College of Pharmacy, Nagpur.

All the chemicals used for the study were of AR and HPLC grade. Millipore membrane filters (0.45μ) were used for filtration of mobile phase and working solutions. Double distilled and membrane filtered water was used throughout the experimental work.

HPLC instrumentation and conditions:

HPLC model Shimadzu SPD20-A equipped with ALC pump having variable wavelength UV- Visible detector with manual injector (20 µL). Each chromatogram was analyzed with LC Solutions software. Separations were achieved by using an Intersil C18 column (150 mm × 4.6 mm, 5 μm). The mobile phase consisted of a methanol: buffer in ratio 25:75 v/v was filtered through a 0.45 μm membrane filter to degas and pumped from the respective solvent reservoirs to the column at a flow rate of 1 ml/min. The run time was set at ten min.

FORCED DEGRADATION OF NICOTINE HYDROGEN TARTRATE:

Acid degradation:

Acid degradation of NHT was carried out in three different conditions.

1) Quantity of drug equivalent to 10 mg was dissolved in 20 ml of 0.5 M HCL and resulting solution refluxed 90 min at 80°C.

2) Quantity of drug equivalent to 10 mg was dissolved in 20 ml of 1 M HCL and resulting solution refluxed 5 h at 80°C.

3) Quantity of drug equivalent to 10 mg was dissolved in 20 ml of 5 M HCL and resulting solution refluxed 5 h at 80°C.

After cooling the resulting mixtures at room temperature, solutions was neutralized with NaOH and finally diluted with diluent (methanol: water in ratio 15:85 V/V) to obtain a final concentration of 100µg/ml.

Base degradation:

Alkaline degradation of NHT was carried out in three different conditions.

1) Quantity of drug equivalent to 10 mg was dissolved in 20 ml of 0.5 M NaOH and resulting solution refluxed 90 min at 80°C.

2) Quantity of drug equivalent to 10 mg was dissolved in 20 ml of 1 M NaOH and resulting solution refluxed 5 h at 80°C.

3) Quantity of drug equivalent to 10 mg was dissolved in 20 ml of 5 M NaOH and resulting solution refluxed 5 h at 80°C.

After cooling the resulting mixtures at room temperature, solutions was neutralized with HCL and finally diluted with diluent to obtain a final concentration of 100µg/ml.

Hydrogen peroxide degradation:

Accurately weigh quantity equivalent to 10 mg of NHT was dissolved in 20 ml of 30% hydrogen peroxide solution present in three different flasks and resulting solutions were then stored for 2 h, refluxed for 90 min and 2 h at 80°C, respectively. After cooling at room temperature, dilution was done with diluent to obtain a concentration of 100µg/ml.

Dry heat degradation:

The NHT was kept in oven at 60° C for 12 h, resulting sample was dissolved in diluent to obtain concentration 100 µg/ml to study dry heat degradation.

Photochemical degradation:

Quantity of NHT equivalent to 10 mg was transferred to 100 ml of volumetric flask and diluted to a mark with diluent. Resulting solution was then exposed to 254 nm for 24 h.

Water hydrolysis:

Accurately weigh quantity of NHT was dissolved in 15 ml of water and then refluxed for 4 h at 80°C, after cooling at room temperature dilution was done with diluent to obtain a concentration of 100 µg/ml.

Development of HPLC method:

HPLC studies were carried out on all the reaction solutions individually and on a mixture of the solutions in which decomposition was observed. The separations were achieved by using, methanol: buffer (buffer solution was prepared by dissolving 2.72 g of KH₂PO₄ and 0.18 g n-Heptane salt in 1000 ml of double distilled water) in a ratio 25:75 v/v as the mobile phase. It was filtered through 0.45 µ membrane filter and degassed before use. The working concentration 100 µg/ml was used for all degradation samples. The elution was carried out at the flow rate of 1ml /min. Detection was carried out at 260 nm at ambient temperature. A typical chromatogram of NHT was shown in (Fig. 2).

The peak area, retention time (Rt), capacity factor, theoretical plate, and peak symmetry were calculated for replicate estimation of same concentration of drug for five times. The values obtained demonstrated the suitability of the system for the analysis of the NHT. System suitability parameters fall within 2 % coefficient of variation (% RSD) during routine performance of the method. The summary of the system suitability results were showed in the (Table 1).

Table No 1: System suitability parameters of NHT

Injection	Retention time (min)	Tailing	No. of theoretical plates	Peak area
1	6.922	1.12	7930	379170
2	6.928	1.12	7908	378470
3	6.938	1.12	7851	379117
4	6.924	1.13	7926	378671
5	6.927	1.12	7939	378495
Mean	6.93	1.12	7910.80	378784.60
Std. Dev.	0.0062	0.0045	35.2803	337.1829
% RSD	0.0892	0.3986	0.4460	0.0890

Table No 2: Stress study of NHT

Stress conditions	Duration (Time)	Temp.	Peak area (Standard)	Peak area* (sample)	% Purity*	±SD	% RSD
0.5 M HCl	90 min	80°C	364880	354330.00	98.99	0.4636	0.4683
1 M HCl	5h	80°C	379320	373239.33	98.98	0.6995	0.7066
5 M HCl	5h	80°C	379320	387948.67	99.29	1.0517	1.0592
0.5 M NaOH	90 min	80°C	364880	389590.00	98.28	0.8771	0.8924
1 M NaOH	5h	80°C	379320	374247.33	99.34	0.7398	0.7447
5 M NaOH	5h	80°C	379320	388936.00	99.30	0.3424	0.3448
30% H₂O₂	2 h	-	379320	264001.67	73.50	0.8510	1.1578
30% H₂O₂	90 min	80°C	364880	189206.67	85.14	1.1050	1.2978
30% H₂O₂	2h	80°C	379320	264001.67	73.50	0.8510	1.1578
Dry heat	12 h	60°C	379320	345449.33	99.59	1.0979	1.1025
Photolytic at λ -254 nm	30h	Room temp	379320	345580	98.10	1.2590	1.2707
Water hydrolysis	4 h	80°C	379320	352689.67	98.83	0.7779	0.7871

*Results are mean of three replicates

Table No 3: Linearity study for NHT

Sr.No.	Concentration (µg/ml) () (µg/ml)	Peak area*
1	20	84088
2	40	147763
3	60	231594
4	80	313114
5	100	389742
6	120	478277
7	140	559436

*Results are mean of three replicates

VALIDATION PARAMETERS:

Linearity:

Accurately weigh quantity of drug equivalent 100 mg of NHT was transferred to 100 ml of volumetric flask and diluted with diluent to a mark. (Stock solution)

Stock solution was father diluted to get the concentration of 20, 40, 60, 80 100, 120 and 140 µg/mL. Equal volume of each solution was injected individually and peaks were recorded and graph was plotted as concentration verses area response of NHT.

Precision:

Precision of analytical method was ascertained by replicate estimation of same concentrations of drug for five times. The results are expressed as SD and % RSD of series of measurements.

LOQ and LOD:

LOQ and LOD value represent the sensitivity of the proposed analytical method. LOD and LOQ of the drugs were calculated using the following equations.

LOD = 3.3 x [σ]/S

LOQ = 10 x [σ]/S

Where σ = the standard deviation of the response and S = the slope of the calibration curve (S) (Table 4).

Robustness:

The robustness of the assay method was established by introducing small changes in the HPLC condition i.e. flow rate (0.8, 1 and 1.2 ml/min). Robustness of the method was studied using five replicates at a concentration level of 100 µg /ml.

Table No 4: Robustness study of NHT

Sr. No.	Flow rate (ml/min)
Sr. No.	0.8	1	1.2
1	390342.00	365668.00	268491.00
2	394437.00	361602.00	270662.00
3	387396.00	363972.00	266363.00
4	390852.00	368629.00	271252.00
5	394304.00	364527.00	266946.00
Mean	391466.20	364879.60	268742.80
±S.D.	2961.6869	2567.6011	2175.7042
% RSD	0.7566	0.7037	0.8096

Results are mean of three replicates

RESULT AND DISCUSSION:

No stability indicating method is available in the official compendia using HPLC for analyzing NHT till now. The present proposed method was used for the complete separation of the analytes in less than 10 min and the method can be successfully applicable to perform long-term and accelerate stability studies of NHT. The representative chromatogram obtained for NHT is shown in Fig. 1. The theoretical plates were 7910 (more than 2000) and tailing was1.12 (less than 2) for the NHT peak (Table 1). The method permits detection and quantitation of NHT in the presence of its degradation products. It was subjected to stress conditions as per ICH guidelines. The chromatograph of NHT degradation in acidic and basic condition showed no additional peak and there is no change in peak area, so it was found to be stable in acidic (Fig. 3-5) and basic (Fig. 6-8) conditions. The chromatogram of the hydrogen peroxide (30 %) degraded sample for NHT show additional peaks as follows, sample stored for 2 h show additional peaks at retention time 4.187 and 4.887 min, reflux for 90min at 80°C showed additional peaks at Rt 4.153 min and 5.507 min and reflux for 2 h at 80°C showed additional peaks at Rt 4.327, 4.733 and 6.95 min. (Fig. 9-11) Additional obtained indicating that NHT undergoes degradation under hydrogen peroxide condition.

Fig. 2: Chromatogram of NHT

Fig.3:Chromatograph of NHT subjected to acid (0.5 M) degradation at 80°C for 90 min

Fig. 4: Chromatograph of NHT subjected to acid (1M) degradation at 80°C for 5 h

Fig.5: Chromatograph of NHT subjected to acid (5M) degradation at 80°C for 5 h

Fig.6: Chromatograph of NHT subjected to base (0.5M) degradation at 80°C for 90min

Fig.7: Chromatograph of NHT subjected to base (1M) degradation at 80°C for 5 h reflux

Fig.8: Chromatograph of NHT subjected to base (5M) degradation at 80°C for 5 h.

Fig.9: Chromatograph of NHT subjected to H₂O₂ (30%) degradation at 80°C for 90 min

Fig.10: Chromatograph of NHT subjected to H₂O₂ (30%) degradation for 2 h

Fig.11: Chromatograph of NHT subjected to H₂O₂ (30%) degradation at 80°C for 2h

Fig.12: Chromatograph of NHT subjected to dry heat degradation at 60 ºC for 12 h

Fig.13: Chromatograph of NHT subjected to photo degradation at 254 nm for 30 h

Fig.14: Chromatograph of NHT subjected to water hydrolysis at 80°C for 4 h

It was found to be stable under dry heat (Fig. 12) and photolytic conditions (Fig. 13). Chromatograph of water hydrolysis shows no additional peak (Fig. 14).The drug can be analyzed in the presence of different degradation products by using isocratic conditions and mobile phase containing methanol: buffer in ratio 25:75 v/v (Table 2).

Method is highly specific for the determination of NHR as

· The blank solution (Mobile phase) should not show any interference at the retention time corresponding to the peak of NHT and its impurities.

· The blank solution of peroxide degradation should not show any interference at the retention time corresponding to the peak of NHT and its impurities.

The method was validated for parameters like precision, linearity, LOD, LOQ, and robustness. Linearity was studied over the concentration range 20–140 µg/mL (Table 3) and the regression equation was found to be y= 4009.3x – 5886.6 with correlation coefficient of 0.9997, indicating that method is accurate. The LOQ was found to be 0. 358 µg/mL and LOD was found to be 0.118 mg/mL. The %RSD of precision (Table 1) and robustness studies (Table 4) was found to be less than 2.0%, indicating that the method is precise and robust.

CONCLUSION:

A specific stability indicating HPLC method was developed for the quantitative determination of NHT and it was validated with respect to various analytical parameters. Statistical data of various analytical parameters proves that method is suitable for routine analysis and long term and short term stability study of NHT with good linearity, robustness and precision. Developed method can be used for the quantitative determination of NHT with simultaneous separation of their degraded products. The mobile phase, solvent mixture and degradation product under same condition as that of analytical conditions of NHT does not show any interference to analyte peak, peak purity is satisfactory. Thus the method is rightfully used in quality control laboratory for release of production batches and stability study.

REFERENCE:

1. Neil MJO, Heckelman PE, Koch CB, Roman KJ, Kenny C M, D’Arecca MR. The Merck Index, An Encyclopedia of chemicals drugs and biological. Merck Research Laboratories, NJ, USA, 2006; p 1128.

2. http://www.sigmaaldrich.com/catalog/product/sigma/n5260?lang=en&region=IN

3. Chauhan SS, Lin S, Madan PL. Preparation and evaluation of nicotine hydrogen tartrate fast dissolving films for smoking cessation. Asian J Pharm 2012; 7 (3): 181-192.

4. Willits CO, Swan Margaret L, Connelly JA, Brice BA. Spectrophotometric determination of nicotine. Anal Chem 1950; 22: 430–433.

5. Bakshi M, Singh S. Development of validated stability-indicating assay methods-critical review. J Pharm Biomed Anal 2002; 28:1011–1040.

6. Singh S, Bakshi M. Guidance on Conduct of Stress Tests to Determine Inherent Stability of Drugs, Pharm Tech On-Line 2000; April: 1-14.

7. Kumarn BSP, Annapurna MM, Pavani S. Development and validation of a stability indicating RP-HPLC method for the determination of Rufinamide. J Pharm Ana 2013; 3(1): 66–70.

8. Ngwa G. Forced Degradation as an Integral Part of HPLC Stability-Indicating Method Development. Drug Delivery Technology. June 2010, 10 (5): XX.

9. Shah DA, Bhatt KK, Mehta RS, Baldania SL, Gandhi TR. Stability indicating RP-HPLC estimation of nebivolol hydrochloride in pharmaceutical formulations Indian J Pharm Sci 2008; 70: 591-595.

10. Dangi M, Chaudhari D, Sinker M, Racha V, Damle M et al. Stability indicating HPTLC method for estimation of nebivolol hydrochloride and amlodipine besylate in combination. Eur asian J Anal Chem 2010; 5(2): 161-169.

11. Sherikar O, Mehta P. Comprehensive assessment of degradation behaviour of aspirin and atorvastatin singly and in combination by using a validated RP-HPLC method. Sci Pharm 2013; 81: 195–210.

12. ICH Q1A (R2). Stability Testing of New Drug Substances and Products. Geneva: International Conference on Harmonization; 2003.

13. ICH Q2 (R1). Validation of Analytical Procedures: Text and Methodology. Geneva: International Conference on Harmonization; 2005.

Received on 30.07.2014 Modified on 15.08.2014

Asian J. Research Chem. 7(9): September 2014; Page 787-794