Determination of Nicotine Extracted from Eggplant and Green Pepper by HPLC

Nicotine was separated from eggplant and green pepper seeds (Solanaceous) by High Performance Liquid Chromatography (HPLC).The concentration of nicotine in the eggplant extract (0.871-0.877 μg/ml) was determined by injecting standard material with 0.5 and 1.5 μg/ml, while the concentrations of nicotine in green pepper extract (0.613-0.618 μg/ml) was determined when the standard material was injected with 0.5 and 1.5 μg/ml. The qualitative chemical data was calculated from derivations of the standard material. Nicotine concentration was measured qualitatively in both extracts through the calibration curve and method of the standard addition. This technique has high accuracy and compatibility, bringing the proportion of relative recovery percentage of nicotine extracted from eggplant seeds to 95.880-103.104% and relative error of -4.119-3.104%. Relative recovery percentage of nicotine extracted from green pepper seeds was 97.284 103.518 % and relative error was -2.716-3.518%.


Introduction:
Nicotine alkaloids constitutes approximately 0.6-3.0 % of the dry weight of modern tobacco (1).It also occurs in edible plants of the Solanaceae family, which includes eggplant, potatoes, green pepper and tomatoes but under 200 nanograms per gram, dry weight (less than 0.0002%) (2).At high doses nicotine is associated with poisonings and is potentially lethal (3,4).Nicotine is linked to possible birth defects (5).During pregnancy, there are increased risks for type two diabetes, obesity, hypertension, dysfunction and infertility (4).Nicotine can be quantified in blood, plasma, or urine to confirm poisoning or to facilitate a forensic autopsy.Urinary or salivary concentrations are frequently measured for the purposes of pre-employment and health insurance medical screening programs (6).
There is effect of bacteria on some diseases which injure the Solanaceae family (7).Nicotine have a clear antibiotic effect on bacteria and fungi (8,9,10).Nicotine extracts can be utilized as alternative fungicides (11,12).
The chemical structure of nicotine (C 10 H 14 N 2 ), nicotine is crewed from two cycles (Pyridine, Pyrrolidine) (13)and is shown in fig. 1.

Figure 1. Chemical structure of nicotine
There are different ways to identify and quantify nicotine typically in blood and urine by RP-HPLC and GC-MS (14) or from plant matter HPLC (15).Separation of nicotine from cigarettes can be accomplished by TLC (16).
-Here, we separate and quantify nicotine from eggplant and green pepper seed extraction.
-We were able to obtain a quantitative estimation by HPLC and U.V. after preliminary qualitative analysis.

Materials and Methods:
Table 1 shows the chemical materials and their global origin which were used.The extraction of nicotine alkaloids was accomplished by the following protocol ( 17  Mix two solutions A+B and add distilled water until the volume reaches 100 ml .Take 1ml of the extract and add drops of the reagent.A white colour or off white residual is made when the alkaloid is present.Wagners' reagent is prepared as follows.Dissolve 2gm of potassium iodide in 20ml of distilled water.Add 1.3gm of I 2 .Mix well until dissolving.Add distilled water until volume of 100 ml is reached.Take 1ml of the extract and add drops of the reagent to the extract.A yellow color or sepia color residual is made which indicates the presence of an alkaloid.

The Standard Calibration Curve for Nicotine
Nicotine solution is prepared in concentrations of 5, 10, 15, 25, 35, 50 ppm from nicotine solution in100 ppm in 100 ml volume bottles.Absorption is measured in 254 nm an opposite blank solution of ethanol.

The Standard Addition Method (21)
The standard addition method was used for several concentrations in the analysis of nicotine extract and increasing volumes of the standard nicotine solution (100 ppm).The additives were added in Table 2 and absorption was measured at 254 nm.The method with the concentration is taken directly by the calibration curve.The separation occurred on liquid chromatography Shimadzu 10 AV-LC equipped with binary delivery pump model LC-10A shim adze, and the eluted peaks were monitored by UV-Vis 10 A-SPD spectrophotometer.

Table 2. Method of Standard Addition for Eggplant and Green Pepper Extract
The extract were centrifuged at 7,500 rpm for 15 min.The extract samples were re-suspended in 1.0 ml HPLC grade methanol by overtaxing.The mixture were passed through 2.5 um disposable filter, and stored at 4 o C for further analysis and then 100μl of the sample injected into HPLC system according the optimum condition.

Qualitative and Quantitative Analysis
The Preliminary Qualitative Screening on Nicotine extract eggplant and green pepper seeds and Table 1 shows the follwing screening results: Both reagents detected alkaloids, and specifically nicotine, in the eggplant and green pepper seeds (11).
The nicotine calibration curve was prepared as described in the materials and methods.Absorption was measured at 254 nm for concentrations of 5-50 ppm and the absorption values were measured against the concentration as show in Fig. 3.

Figure 3. The Standard Curve of Nicotine area of sample area of standard
The accuracy and compatibility of the standard nicotine was tested by the relative recovery percentage and percentage relative error calculated.
The method was accurate and compatible with recovery of 97.370 -103.108% and error of -2.629-3.108% of nicotine as shown in Table 4.The method of standard additions was used to analyze nicotine extracted from eggplant seeds as shown in the work method.Absorption was measured at 254 nm for concentrations of 0-40 ppm and the absorption values of concentration were calculated as in Fig. 4.

Figure 4. Standard curved additions of nicotine extract from eggplant seeds
The accuracy and compatibility of the standard additions of nicotine extracted from the eggplant seeds were tested by calculating recovery percentage and of 95.880-103.104% and error of -4.119-3.105% as shown in Table 5.The method of standard additions was used to analyze nicotine extracted from green pepper seeds as shown in the methods above.Absorption is measured at 254nm for concentrations 0-40 ppm and the absorption values concentration are calculated as show in Fig. 5.The accuracy and compatibility of the standard additions of nicotine extracted from the green pepper seeds were tested by calculating recovery percentage and relative error percentage.
The method was found to be highly accurate and compatible, with 97.284 -103.518% and error of -2.716-3.518%as shown in

HPLC
The analysis results by HPLC of nicotine extracted from eggplant seeds refers to the appearance of many peaks.One of them was identical to the standard compound at 0.5 and 1.5 μg/ml from the standard.Its concentration was 0.871-877 μg/ml, as seen in Tables (7,8) and Figs.(6,7,8).The HPLC results from green pepper seeds refers to the appearance of many peaks.One of them is identical to the standard compound.Its concentration was 0.613-0.618μg/ml, as seen in Tables (7,9) and Figs.(6,7,9).

Figure 9. HPLC readout of green pepper seed extract
In figs.8 and 9 the peak doesn't appear under base line, we give direction to the decatur, so as not to record the pack on 5000 UV.Any area more than 5000 UV appears as a retention time and area.As long as retention time of material identical for standard compound was detected the sample.It is also quantities calculated as the area.Through tables (8,9) and figures (7,8), it was concluded that nicotine appeared in eggplant and green pepper seed extracts.It can be concluded that Solanaceous plants contain nicotine and it can be determined by HPLC (12).

): 1 - 3 -
Weight 10 gm of dried powdered seeds of both eggplant (Solanum melongena) and green pepper (Capsicum annuum).2-Add 200ml ethanol in soxhlet to the powdered seeds.Concentrate the extraction by rotary evaporation at 40 o C for 6 hours.4-Dissolve dried substance in 5 ml ethanol and add 30 ml of sulfuric acid to the extraction.5-Evaporate ethanol using rotary evaporator to get rid of acid solution and add ammonium hydroxide 10% to until the solution reaches a pH of =9.6-Put solution in separation funnel adding chloroform, and concentrate in rotary evaporator.7 -Dry extraction at 50 o C. 8-Add 5 ml of 10% ammonia solution and 5 ml 95% methanol to 5 gm of dried extraction.9-Put extraction in shaker water bath in 60 o C for 15 mints.Cool and filter using Watman no.11 filter paper.10-Concentrate the filtrates in a vacuum rotary evaporator until it reaches 1 ml(18).Fig.(2) explain the steps of extraction method.

Figure 2 .
Figure 2. Shows the extraction of nicotine from the seeds of eggplant and green pepper Preliminary Qualitative Screening (19, 20) reagent for Alkaloids Mayers' reagent is prepared by using solution A+ B as follows.Solution (A) is prepared as follows Dissolve 1.4 gm from mercuric chloride in 60 ml of distilled water.Solution (B) is prepared as follows Dissolve 5gm of potassium iodide in 10 ml of distilled water.

Quantitative
Determination of Nicotine in Eggplant and Green Pepper by HPLCThe main compound is separated on FLC (22) (Fast Liquid Chromatographic) column under the following conditions: Column: Phenomenex C-18,3μm Particle size (50 x 4.5mm I.D) column.The mobile phase is: 0.01M potassium phosphate buffer pH 3.2: methanol HPLC grade (80:30,V/V)with a flow rate 1.2 ml/min.Detection was of UV light at 254 nm with an injection volume of 100 μl.The unknown concentrations were calculated according to the following law:

Figure 5 .
Figure 5.Standard curved additions of nicotine extract from green pepper seeds