1. INTRODUCTION:

Artificial sweeteners (also called non-nutritive sweeteners) form an important class of food additives, which are commonly used in the food, beverages, confectionaries and pharmaceutical preparations. These sweeteners provide a taste of sweetness but with little or no intake of food energy. These are used by the diabetics and calorie conscious people¹. In India approximately 50.8 million of population is suffering from diabetics, which means in India itself large number of people would be consuming artificial sweeteners as a substitute of sucrose. The probability of encountering artificial sweeteners along with the various poisons in the toxicological analysis cannot be ruled out. There are large numbers of artificial sweeteners, but very few are approved to be used in foods, beverages and pharmaceutical preparations. In India Saccharin, Sucralose and Aspartame are permitted artificial sweeteners. Even though the cyclamate (Fig-1) is banned, it is still being used clandestinely. Saccharin, cyclamate and sucralose^2,3 are excreted largely unchanged. Aspartame is hydrolyzed to aspartic acid, phenylalanine and methanol and excreted².

Sucralose Aspartame

Cyclamate Saccharin

Fig-1: Structure of Artificial sweeteners

Thin layer chromatography (TLC) is commonly used as screening method for the forensic toxicological analysis of different samples like urine, stomach contents, blood and liver tissues etc. TLC is a presumptive screening method based on which we can selectively go for the other final confirmation methods. When TLC is used as a screening technique then the interference of other substances which are present along with analytes of interest in different biological matrices becomes an important consideration due to the possibility of false positive / negative results.

In this communication an attempt is made to study the interference of artificial sweeteners in the toxicological analysis of drugs and pesticides in different biological matrices by TLC.

2. EXPERIMENTAL:

2. 1 Material and Methods:

All the chemicals and solvents used were of analytical grade and purchased from Sd. Fine Chemicals Pvt Ltd (India). Pesticides and drugs standards were purchased from reputed sources. Saccharin was supplied by Kare Labs (India), sucralose was supplied by Virchows Labs (India), where as cyclamate, aspartame, phenylalanine and aspartic acid was purchased from Sigma-Aldrich (India). Precoated aluminum silica gel G F₂₅₄ TLC plates were purchased from Merck (Germany), where as twin trough TLC chambers (20 cm x 10 cm) were of Camag (Switzerland).

2. 2 Preparation of Visualization reagents

(i). Mercuric chloride-Diphenylcarbazone (HgCl₂-DPC):

(a) Dissolve 0. 1 g of DPC in 50 ml of ethanol.

(b) Dissolve 1 g of HgCl₂in 50 ml of ethanol.

Mix solutions (a) and (b) just before spraying.

(ii). Mercurous Nitrate: A saturated aqueous solution of Mercurous Nitrate.

(iii). Dragondorff reagent:

(a) Mix together 2 g of Bismuth sub nitrate, 25 ml of acetic acid and 100 ml of water.

(b) Dissolve 40 g of Potassium Iodide (KI) in 100 ml of water.

Mix together 10 ml of (a), 10 ml of (b), 20 ml of acetic acid and 100 ml of water.

(iv). Acidified Potassium permanganate: To 100 ml of 0. 5% aqueous solution ass 5 ml of 3% Sulphuric acid.

(v). Silver Nitrate (AgNO₃): 0. 1 M aqueous solution of AgNO_3.

(vi). Rhodamine B and Sodium Hydroxide (RHB-NaOH): Plates were sprayed with 0. 02% (w/v) solution of Rhodamine B in ethanol followed by saturated solution of Sodium Hydroxide in ethanol.

(vii). Diphenylamine-Zinc chloride (DPA-ZnCl₂): The spray comprises 0. 7% (w/v) of DPA and 0. 7% of ZnCl₂solution in acetone. After spraying plates were exposed to UV 254 nm radiation for 10 minutes and heated at 100oC until no further color change is observed.

2. 3 Sample Preparation:

For Drugs

Approximately 5 g of stomach tissues, its contents and 10 ml of urine were spiked separately with 10 mg of each of saccharin, sucralose, cyclamate, aspartame, and 10 mg each of methaqualone, paracetamol and phenobarbitone respectively.

For Pesticides

Approximate 5 g of stomach tissues and its contents were spiked with 1ml of 40% solutions of pesticide standards like phosphomidon, chlorpyriphos, malathion, methyl-parathion, endosulfan and propoxur and 10 mg of sweeteners like saccharin, sucralose, cyclamate and aspartame.

2. 4 Extraction Procedure:

Extraction of Drugs from Urine

To 10 ml of urine sufficient phosphoric acid or tartaric acid was added to adjust the pH to 3. The urine was extracted with two 30 ml portion of diethyl ether, evaporated to dryness⁴. To the aqueous solution left after the above extraction, sufficient dilute ammonia solution was added to adjust the pH to 8 and extracted with two 30 ml portions of chloroform. The combined extracts were washed with water and filtered. A little tartaric acid was added to prevent the loss of volatile bases and evaporated to dryness.

Extraction of Drugs from Stomach contents

Any fragments of capsules or tablets or powdery material was removed and suspended in water for extraction. If the sample contained much food residue or mucus, stable emulsions are produced during the extraction; in such a case the following pre-treatment was necessary.

An excess of solid ammonium sulphate was added together with a few drops of 10% phosphoric acid and was heated while stirring. The filtrate was then extracted by the method described above for urine.

Residue obtained after above drug extraction procedure⁴ was reconstituted in 0. 1 ml of methanol

Extraction of pesticides from biological matrices

Solid or semi-solid samples were homogenized with an equal mass of water. Food stuffs, stomach contents or stomach tissues were homogenized with water, treated with sat-CaCl₂ solution, allowed to stand overnight and filtered. An aliquot of the filtered sample or homogenate was shaken for 5 min with an equal volume of a mixture of hexane: toluene (1:1 v/v). After layers separation the organic phase was retained. The aqueous fraction was adjusted to pH 2 by the addition of 1 M sulphuric acid and then extracted with an equal volume of diethyl ether. The aqueous fraction was retained for later examination. The Hexane-Toluene and ethereal fractions were combined and then evaporated to dryness. The residue thus obtained was reconstituted in 0. 1 ml of hexane or toluene.

2. 5 Standard solution:

Standard solutions of sucralose (1mg/ml), saccharin, and the drugs (methaqualone, paracetamol, phenacetin and phenobarbitone) were prepared in methanol. Solutions (10% v/v) of pesticides (phosphomidon, chlorpyriphos, malathion, methyl-parathion, endosulfan and propoxur) prepared in hexane. Standard solutions (1mg/ml) of aspartame, aspartic acid and phenylalanine were prepared in water. Cyclamate solution (1mg/ml) was made in acetonotrile.

2. 6 Chromatography:

Residues obtained after above extraction procedures were reconstituted in suitable solvents (methanol for drugs and hexane or toluene for pesticides) and spotted 10 uL using micro syringe along with standards solutions on TLC plates (distance from lower edge 15 mm, distance from the left side 15 mm, distance between bands 10 mm and solvent front 90 mm). Plates were developed and visualized by methods described for analysis of drugs and pesticides as per the procedure described previously⁵ and it is also described in tables (2-5).

3. RESULTS AND DISCUSSION:

Artificial sweeteners like saccharin, sucralose and cyclamate are slightly acidic in nature and nearly 80-98% of them are excreted unchanged, where as aspartame which is slightly neutral gets hydrolyzed to aspartic acid, phenylalanine and methanol (Fig-2). TLC methods which are generally used for toxicological screening were applied to check the interference of these artificial sweeteners and /or their metabolites.

When the pesticide extraction method was applied it was observed that artificial sweeteners were extracted in the second (ethereal) extract of the sample (Table-1). As in the method under reference⁴ it is mentioned that after hexane and ethereal extract two extracts are to be combined and evaporated to dryness, then there is a probability that artificial sweeteners which were extracted in ethereal extract would interfere in the screening of pesticides. By taking only hexane extract in which normally most of the pesticides get extracted, the interference of artificial sweeteners can be avoided as they would not get extracted in the hexane. The hexane and ethereal extract were combined and applied to TLC plate and developed by standard methods. The spots were visualized under UV light at 254 nm. It was observed that only saccharin spot showed slight movement with low R_f value and in no way interfered with pesticides spots (Table-2). And when we applied the visualization reagent for detection of pesticides then it was found that all four artificial sweeteners and hydrolyzed products of aspartame did not show any response to the visualization reagents used (Table-3). Thus the artificial sweeteners even though present in the sample did not interfere in the screening procedure for pesticides.

We also observed that these four artificial sweeteners got extracted in the acidic extract of the urine, stomach contents and tissue samples spiked with drugs. So there is no need of checking of interference of these sweeteners with the basic drugs because they were not extracted in basic extract. When we observed the plates of acidic drugs along with artificial sweeteners under UV 254 nm it shows that saccharin spot moved negligibly giving small R_f value and phenylalanine (hydrolyzed product of aspartame) spots moved considerably giving higher R_f value (Table-4), where as other sweeteners didn’t responds to these solvent systems.

The interference of saccharin and phenylalanine could be avoided by spraying with reagents applied for visualization of acidic and neutral drugs, because they would not respond to these visualization reagents. However only the cyclamate could be visualized with one of the reagents (Table-5) but it was at the point of origin only. Thus with the combination of response to visualization reagents and R_f values it would be possible to discern between the artificial sweeteners and other substances unequivocally.

Aspartic acid Phenylalanine Methanol Aspartame

Fig-2: Hydrolysis products of Aspartame

Table-1 Shows the data on extraction of artificial sweeteners in different extracts.

Compound Name	Drug extraction method		Pesticide extraction method
Compound Name	Acidic extract	Basic extract	Hexane extract	Ether extract
Saccharin	Positive	Negative	Negative	Positive
Sucralose	Positive	Negative	Negative	Positive
Cyclamate	Positive	Negative	Negative	Positive
Aspartame	Positive*	Negative	Negative	Positive

*. Aspartame got hydrolyzed into Aspartic acid and Phenylalanine

Table-2 Shows the HR_fvalues (Observed under UV 254 nm) of artificial sweeteners for different solvent systems used for Pesticides analysis.

Compound Name	HR_f(Observed under UV 254 nm)
Compound Name	Hexane : Acetone (80 : 20 v/v)	Toluene : Acetone (95 : 5 v/v)	Chloroform : Acetone (90 : 10 v/v)	Chloroform
Saccharin	-	3	5	2
Sucralose	-	-	-	-
Cyclamate	-	-	-	-
Aspartame	-	-	-	-
Phenylalanine	-	-	-	-
Aspartic acid	-	-	-	-
Phosphomidon	7	15	43	7
Chlorpyriphos	64	54	64	28
Malathion	30	39	58	39
Methyl-Parathion	31	58	66	33
Endosulfan	40	49	57	95
Propoxur	20	11	60	13

Table-3 Shows the Response of artificial sweeteners to different visualization reagent used for visualization of Pesticides.

Compound Name	Visualization reagent
Compound Name	Silver Nitrate	Rhodamine B- Sodium hydroxide	Diphenylamine-Zinc Chloride
Saccharin	-	-	-
Sucralose	-	-	-
Cyclamate	-	-	-
Aspartame	-	-	-
Phenylalanine	-	-	-
Aspartic acid	-	-	-
Phosphomidon	+	+	+
Chlorpyriphos	+	+	+
Malathion	+	+	+
Methyl-Parathion	+	+	+
Endosulfan	+	+	+
Propoxur	+	+	+

Table-4 Shows the HR_fvalues (Observed under UV 254 nm) of artificial sweeteners compounds for different solvent systems used for acidic drugs analysis.

Compound Name	HR_f(Observed under UV 254 nm)
Compound Name	Chloroform : Acetone (80 : 20 v/v)	Ethylacetate : Methanol : Ammonia (85 : 10 : 5 v/v)	Ethylacetate
Saccharin	4	15	6
Sucralose	-	-	-
Cyclamate	-	-	-
Phenylalanine	29	65	-
Aspartic acid	-	-	-
Paracetamol	7	57	43
Phenacetin	29	65	52
Methaqualone	56	70	62
Phenobarbitone	27	71	70

Table-5 Shows the Response of artificial sweeteners to different visualization reagent used for visualization of drugs

Compound Name	Visualization reagent
Compound Name	Mercuric chloride- Diphenylcarbazone	Mercurous Nitrate	Dragondorff reagent	Acidified Potassium permanganate
Saccharin	-	-	-	-
Sucralose	-	-	-	-
Cyclamate	+*	-	-	-
Phenylalanine	-	-	-	-
Aspartic acid	-	-	-	-
Paracetamol	-	-	-	+
Phenacetin	-	-	-	+
Methaqualone	-	-	+	_
Phenobarbitone	+	+	-	-

*. Spot was not moved.