Determination of cyclamate in urine by derivatized gas chromatography-mass spectrometry.

AIM: It is important in toxicological/drug screening work to rule out the possible interfering analytes, to eliminate the false positive or negative results. In this paper, we describe a simple, selective, and sensitive derivatized GC-MS method for the determination of cyclohexylsulfamic acid (cyclamate) in urine.
MATERIALS AND METHODS: Elite- 5MS capillary column was used for the separation of analytes and detection using GC-MS. The analysis was carried out in selected ion monitoring mode (SIM) in the range of 26 to 200 using m/z values of 57, 30, 55, 41, 44, 67, 82, 98, and 39. RESULTS AND DISCUSSION: The method is based on the conversion of cyclamate into nitroso derivative of cyclamate followed by its gas chromatography-mass spectrometry determination. The limit of detection, limit of quantitation, and linearity range of the proposed method were found to be 0.2 μg/ ml, 0.7 μg/ml, and 1-15 μg/ml, respectively. The recovery of the present method is in the range of 88-94%.
CONCLUSION: The proposed method can be applied for detection and quantification of cyclamate in urine.

Urine testing has become the most frequently performed type of analysis in drug abuse screening and forensic toxicology.[1] Urine is a high-volume specimen, which is easily obtainable through a medically non-invasive process in non-fatal cases and in post mortem samples. Under normal conditions, it is sterile and contains high concentrations of water-soluble metabolites, which are markers for therapeutic drugs/abused drugs and their metabolites. Sometimes, people may use several methods to defeat the detection of drug either by substituting the urine sample itself or by consuming/adding the substances that can mask the original banned or prohibited drug in urine. The possibility of interference by artificial sweeteners in therapeutic/drug abuse monitoring in urine cannot be overruled. Therefore, there is a need of sensitive analytical methods which are able to detect these interfering substances unequivocally.

Artificial sweeteners play an important role in our society not only for diabetic patients but also for people using low-calorie foods and for people who want to lower the costs of foods. Cyclohexylsulfamic acid (Cyclamate) [Figure 1] is used extensively in many diets, medical and food products as an artificial non-nutrition sweetener. It is a white, odorless crystalline powder. In dilute solution, it is about 30 times as sweet as sucrose.[23] Several studies show that larger part of cyclamate injected orally gets excreted unchanged,[45] whereas very little amount of ingested cyclamate is metabolized to cyclohexylamine. Studies show that the conversion of cyclamates to cyclohexylamine is not through human metabolism but is by the action of intestinal flora.[6] As major part of ingested cyclamate is excreted unchanged in the urine, there is a possibility of its interference and may give false-positive results. Schutz et al. reported such kind of possible false-positive detection of amphetamine due to the presence of cyclamate.[7]

Figure 1

Structure of cyclamate

Several analytical methods like paper electrophoretic method[8] and paper chromatography[9] high-performance liquid chromatography with pre-column derivatization[10] have been reported by several researchers for determination of cyclamate in urine. Paper chromatographic and paper electrophoretic methods are not so sensitive, whereas HPLC method with pre-column derivatization requires lengthy derivatization procedure and need a special cleanup procedure to remove the excess reagent. However, gas chromatography (GC) coupled with mass spectrometer (MS) is found to be more rapid and sensitive; cyclamate has to be derivatized prior to analysis.

In the work reported herein, we determined the cyclamate as nitroso derivative by GC-MS, using toluene as internal standard.

Experimental

Materials

Cyclamate was purchased from Sigma-Aldrich (India). Sodium nitrite, sulfuric acid, sodium chloride, toluene, and n-hexane were of analytical reagent grade and purchased from S d Fine chem. (India). Water used for preparation of reagents was double distilled prepared in the laboratory.

Equipment

GC was performed using a Perkin-Elmer Clarus 600S GC-MS system controlled by Turbomass 5.3.0 software, equipped with an electron-impact (EI) ion source (electron energy, 70 eV) and an electron multiplier detector capable of recording ions from m/z 26 to m/z 200. The capillary column was a 15 m × 0.25 mm i.d. fused-silica column coated with a 0.1 μm film of Elite-5MS. The injector temperature was kept at 200°C, whereas inline and source temperature was kept at 150°C. The column oven temperature was initially maintained at 60°C for 2 minutes then programmed @ 12°C/min to 150°C which was further increased @ 35°C/min to 250°C which was held for 2 minutes. Solvent delay of initial 2 minutes was maintained for each chromatographic run.

Standard preparation

0.1 mg/ml stock solution of cyclamate was prepared with double distilled water. The stock solution was further diluted to give five different working standard solutions in linearity range (3 to 15 μg/ml).

Sample preparation

Three human volunteers aged between 27-45 years were orally given 100 mg 100 mL–1 aqueous solution. And, after different interval of time, approximately 20 ml of urine was collected from each volunteer. 10 ml of urine sample was centrifuged and the supernatant layer was used for the experiment.

Derivatization

Five milliliter of each supernatant obtained for standard and urine was taken in a screw-capped test tube, to that 2 ml of (0.5% sodium nitrite) and 10% H2SO4 was added into the test tube and covered with screw cap and kept on a water bath at 30°C for 10 minutes. The resultant derivative was extracted using 20 × 3 ml of n-Hexane with 100 mg of sodium chloride.

The hexane extract was evaporated to dryness and reconstituted in 100 μl of n-hexane and to that 100 μl of 1% toluene (1 ml toluene made up to 100 ml with n-hexane) was added as internal standard. 2 μl of each standard and sample was injected into the GC-MS system.

Method validation

Preliminary validation of the method was performed by checking the linearity, precision, recovery, detection, quantification limits, and repeatability.

Limits of detection and determination

Limits of detection (LOD) and limits of quantification (LOQ) of the method were determined by injecting standard serial dilutions (made from stock solution of 0.1 mg/ml of progressively decreasing concentrations run on GC-MS. The LOQ and LOD were the concentrations for which signal-to-noise ratio (S/N) was 10:1 and 3:1, respectively.

Linearity

For linearity checking, Stock solution (0.1 mg/ml) was further diluted to give the final concentration of 0.1 μg/ml. And, these solutions were injected into the GC-MS system and linear curve was drawn by taking the resultant's peak areas and concentration.

Precision

The precision of the method was evaluated on the basis of analyzing the urine samples spiked with three different concentrations (4 μg/ml, 8 μg/ml, and 12 μg/ml) of cyclamate in the linearity range for repeating three times.

Recovery/accuracy

The accuracy of the method was expressed as the percentage recovery of cyclamate. Recovery studies were carried out by standard addition method where three different concentrations (4 μg/ml, 8 μg/ml, and 12 μg/ml) of cyclamate spiked in urine samples. Recovery studies were carried out on pure samples also.

Repeatability

The consistency of the results for the samples was checked by repeating the experiment six times per day (intraday) and consecutively for 3 days (interday). The standard deviation of the repeated recovery values was calculated.

Robustness

Robustness is a measure of a method's immunity to small but deliberate variations in the conditions used. Injector temperature, source and interface temperatures were deliberately changed and the effects were monitored.

Results and Discussion

Cyclamate is a white, odorless artificial sweetener, which is acidic in nature. Some of the researchers have carried out pre-column derivatization followed by separation and UV detection. However, the methods are tedious and time consuming. It was also analyzed by electrophoretic method which is also a time-consuming process. Always there is a need of method which leads to high sensitivity combined with the possibility of achieving efficient separations of complex mixtures. GC-MS seems more rapid, accurate, and sensitive method, even though it needs to be derivatized. In this paper, we used a modified GC method[11] for the analysis of cyclamate in urine.

As we discussed earlier, cyclamate should be derivatized prior to analysis by GC. Several researchers used silylation method for the analysis, but it is also a cumbersome and time-consuming procedure. However, method described by Yan et al.[11] was based upon the formation of nitroso derivative of cyclamate formed upon the reaction of cyclamate in presence of sodium nitrite and sulfuric acid. We tried to give the hypothetically plausible reaction mechanism as shown in Scheme 1. Method described by Yan et al.[11] was carried out at 4°C. But upon performing these reactions at different temperature conditions, it was found that by increasing the reaction temperature we can reduce the reaction time. And, it was found that when we performed the same reaction at 30°C on water bath, the reaction time was reduced to 10 minutes, which in turn gives high output in short span of time which also reduces the analysis time.

Scheme 1

Showing the plausible reaction for conversion of cyclamate into cyclamate-nitroso derivative

Upon completion of reaction, sodium chloride was added to the reaction mixture prior to the extraction of cyclamate derivative. Sodium chloride was added to increase the ionic strength of derivative which is easily extractable in the organic solvents. Several organic solvents [Table 1] were tried for the extraction of cyclamate derivative. It was found that hexane gives the maximum yield of cyclamate derivative when used as extraction solvent.

Table 1

Showing the recovery of cyclamate derivative in different solvents

Gas chromatography

GC method proposed by Yan et al.[11] was based upon the chromatographic separation of cyclamate derivative and its determination by electron capture detector (ECD) in fruit juices. We have adopted the Yan et al.[11] method for determining cyclamate in urine by using a mass selective detector instead of ECD. As mass spectrometry is the more sensitive and specific method, in the proposed method, we detected cyclamate derivative using modified GC-MS method. We performed the analysis in selected ion monitoring mode (SIM) in the range of 26 to 200 using m/z values of 57, 30, 55, 41, 44, 67, 82, 98, and 39. The structure of nitroso derivative of cyclamate shows a molecular weight of 129 from its structure and this has been confirmed by the mass spectrum which showed the presence of peaks at different m/z values which has been formed after the fragmentation [Figure 2] of the nitroso derivative of cyclamate and we also tried to give the plausible fragmentation mechanism as shown in Scheme 2 The proposed chromatographic conditions gives well-resolved peak of cyclamate in urine samples [Figure 3].

Figure 2

Mass spectrum obtained for nitroso derivative of cyclamate

Scheme 2

Showing the plausible fragmentation pattern of nitroso derivative of cyclamate

Figure 3

Showing the chromatogram obtained for standard and urine samples

The limit of detection and limit of quantitation of the proposed method was found to be 0.2 μg/ml and 0.7 μg/ml, respectively. The method was found to be linear in the concentration range of 1-15 μg/ml [Figure 4] and also the regression value (R2) was found to be 0.9997 [Table 2]. The recovery of cyclamate in urine samples by proposed method was found to be in the range of 88 to 95% with the precision in terms of standard deviation (SD) value of <1.63 [Table 3]. The intraday and interday reproducibility values in terms of standard deviation were found to be less than 2.1 and 2.9, respectively [Tables 4 and 5]. The proposed method is selective and five times more sensitive compare to Yan et al.[11] method.

Figure 4

Showing the calibration curve obtained for cyclamate (y = 4415.5x-488.76) with the regression coefficient of 0.9997

Table 2

Showing the values of LOD, LOQ, linearity, and regression value

Table 3

Showing recovery and precision data in urine samples

Table 4

Showing intraday reproducibility data

Table 5

Showing interday reproducibility data

Conclusion

The proposed method can be adopted by forensic science laboratories and drug abuse monitoring laboratories. It will be useful for ruling out the presence of cyclamate in urine samples tested for the screening of different kind of drugs and poisons, which in turn help in eliminating the false-positive and false-negative results.