Fluorescence Spectrometric Determination of Drugs Containing α-Methylene Sulfone/Sulfonamide Functional Groups Using N 1-Methylnicotinamide Chloride as a Fluorogenic Agent

A simple spectrofluorometric method has been developed, adapted, and validated for the quantitative estimation of drugs containing α-methylene sulfone/sulfonamide functional groups using N 1-methylnicotinamide chloride (NMNCl) as fluorogenic agent. The proposed method has been applied successfully to the determination of methyl sulfonyl methane (MSM) (1), tinidazole (2), rofecoxib (3), and nimesulide (4) in pure forms, laboratory-prepared mixtures, pharmaceutical dosage forms, spiked human plasma samples, and in volunteer's blood. The method showed linearity over concentration ranging from 1 to 150 μg/mL, 10 to 1000 ng/mL, 1 to 1800 ng/mL, and 30 to 2100 ng/mL for standard solutions of 1, 2, 3, and 4, respectively, and over concentration ranging from 5 to 150 μg/mL, 10 to 1000 ng/mL, 10 to 1700 ng/mL, and 30 to 2350 ng/mL in spiked human plasma samples of 1, 2, 3, and 4, respectively. The method showed good accuracy, specificity, and precision in both laboratory-prepared mixtures and in spiked human plasma samples. The proposed method is simple, does not need sophisticated instruments, and is suitable for quality control application, bioavailability, and bioequivalency studies. Besides, its detection limits are comparable to other sophisticated chromatographic methods.

Methyl sulfonyl methane (MSM) (1) is a relatively new dietary supplement form of sulfur that is found in our living tissues. MSM supports healthy connective tissues like tendons, ligaments, and muscle. Thus, it is important in conditions such as arthritis, muscle pains, and bursitis. MSM should be considered an integral part of any health care practice because of its physiological action, indirect importance, and current/future uses [3][4][5].
In this report, MSM was used as a model compound and was found to react successfully with NMNCl quantitatively yielding a fluorophore. Because there are marketed dosage forms containing MSM, it was decided to apply the developed method for its analysis in its marketed dietary supplement dosage forms in view of the rather elaborate and costly methodologies reported for its analysis in the available literature. The reported methods include Fourier-transform infrared (FT-IR) spectrometry [6], Raman spectroscopy [7], CIMS [8], GC [9], GC with flame ionization detector [10], GC with flame photometric detection [11], capillary 2 International Journal of Analytical Chemistry GC [12], GC-MS for estimating volatile sulfur compounds [13,14], solid-phase microextraction-GC-MS [15], dynamic headspace-GC-MS [16], reversed phase HPLC followed by headspace GC-MS [17], and direct thermal desorption prior to GC-MS application [18]. Tinidazole (2) is a synthetic antiprotozoal agent. Survey of the available literature reveals several methods for the analysis of 2 active pharmaceutical ingredient (API) and pharmaceutical formulations in the presence of possible impurities and related substances [19,20]. These methods include fluorometry involving the reduction of its nitro group and subsequent measurement of its emission intensity at 420 nm [21], UV [22], electrochemical method [23], HPTLC [24,25], LC/MS/MS [26], HPLC [27], and GLC [28]. Further, 2 was analyzed in biological fluids containing its major metabolite using HPLC-MS [29].
Rofecoxib (3) is a nonsteroidal anti-inflammatory drug (NSAID) introduced in 1998 with lots of fanfare claiming it a selective COX-2 inhibitor with minimal side effects, and 3 was withdrawn in September, 2004 because of safety concerns regarding its untoward cardiovascular side effects leading to several deaths [30,31].
Nimesulide (4) is a sulfonanilide analogue clinically used anti-inflammatory agent; it is not related to conventional NSAIDs, which usually present a carboxyl or hydroxyl functional group [38]. Several methods were described for the determination of 4 in the presence of its compendia-related substances [39] as well as in the presence of its metabolites [40]. These methods include spectrophotometric ones as near-infrared [41], UV techniques [42], fluorometry using egg phosphatidylcholine liposomes [43], and colorimetric methods [44]. Electrochemical methods involve flow amperometry [45] and adsorptive stripping voltammetry [46]. Separation methods involve HPLC [47,48] and HPLC-MS/MS [49].
Nakamura and Tamura described the application of the reaction of N 1 -methylnicotinamide chloride (NMNCl) to the analysis of various compounds containing α-methylene carbonyl groups [50]. Nakamura and Tamura also made qualitative tests on the reaction mechanism and established the cyclized α-adduct fluorophore [50]. This reaction was not tested before for compounds containing α-methylene groups adjacent to other functional groups.
All previous publications based on the fluorophore produced by reaction with NMNCl described the utility of this reagent to react with drugs containing active methylene α to carbonyl functional groups. This paper formulates our continuous effort to extend the utility of NMNCl to determine different classes of drugs containing active methylene α to various groups such as cyclic ketone, (ketamine, griseofulvin, and levonorgestrel) sulfoxide (PPIs), and sulfone/sulfonamide (1-4) and even to groups that would produce active methylene upon hydration, for example, methyne group, such as levonorgestrel and ethinyl estradiol (unpublished results). This paper describes the application and validation of the reaction of NMNCl with some drugs containing α-methylene sulfone groups.

Results and Discussion
When 1, 2, 3, and 4 (for chemical structures and plausible pathway of the reaction, cf. Figure 1) were allowed to react with NMNCl under the optimal conditions specified for each, strong fluorescent products were obtained. The optimal wavelengths of excitation and emission of the reaction product were determined using synchronous wavelength search and listed in Table 1.
Different variables affecting the reaction between the chosen drugs and NMNCl, including sodium hydroxide concentration and volume, volume and concentration of the added NMNCl, and pH values, were studied to optimize the reaction conditions to give maximum fluorescence intensity (Figures 2, 3, and 4).
These results have revealed a good and dynamic linearity ranges of the proposed method with different drugs. The good linearity of these relations was indicated by the corresponding regression equations shown in Tables 2 and  3 for standard solutions and spiked human plasma samples, respectively.

Accuracy.
The accuracy of the proposed method was studied according to the ICH topic Q2B (R1) [51], by preparing spiked human plasma samples containing various concentrations, lying within the linearity range of each drug, and analyzing them using the proposed method. The results, expressed as % recovery ± S.D., are shown in Table 4 for spiked human plasma samples.

Precision.
The precision of the method was judged by performing intraday and interday triplicate analyses of different concentrations covering the linearity range of each International Journal of Analytical Chemistry  Table 5 for spiked human plasma samples.

Specificity.
To study the specificity of the proposed method, three synthetic mixtures of 1, 2, and 3 and two synthetic mixtures of 4 were prepared to contain the possible interfering substances used during pharmaceutical formulations. These mixtures were analyzed using the proposed method and the results, were expressed as % recovery ± S.D., and were as follows: 99.8% ± 3.0 for 1, 100.3% ± 2.8 for 2, 99.6% ± 3.5 for 3, and 100.7% ± 1.7 for 4.   2.6. Assay of Pharmaceutical Preparations. All the pharmaceutical preparations available in the local market for each drug were analyzed using the proposed method. The results, expressed as % recovery ± S.D., are illustrated in Table 6.

Determination of 2 and 4 in Volunteer's Blood.
The success in the application of the highly sensitive proposed procedure for the determination of 2 and 4, in spiked human plasma samples with good accuracy and precision, encouraged the investigator to study its application for monitoring the drug level in the blood of a volunteer receiving 2 or 4 therapy. The level of 2 and 4 was monitored in the blood of volunteers, and their concentrations were found to be 48 μg/mL and 35 μg/mL, respectively, that lie in the therapeutic levels of 2 (47.7 ± 7.5 μg/mL) and 4 (38 ± 10.6 μg/mL).

Conclusion
The proposed method makes use of the high sensitivity and specificity of the fluorometric analysis to reach low limits of detection and quantitation for all the studied drugs in standard solutions, synthetic mixtures, pharmaceutical preparations, spiked human plasma samples, and patient's or volunteer's blood. The method is simple; it gives results comparable to those obtained by other techniques that require elaborate instrumentation and time-consuming sample preparation procedure. The method showed good accuracy and precision suitable for quality assurance and could be recommended for    International Journal of Analytical Chemistry   Nimesulide (4): Sulide 50 mg and 100 mg tablets (Alkan Pharma), Nimalox 100 mg tablets (Sigma) and sulidan 100 mg tablets (Modern Pharmaceutical Co. (MPC)).

Stock Standard Solutions of Drugs.
Stock standard solutions were prepared in distilled water for 1, methanol for 3, and ethanol for 2 and 4 to contain 10 mg/mL, 10 mg/mL, 0.2 mg/mL, and 0.25 mg/mL for 1, 2, 3, and 4, respectively.

Serial Standard Solutions of Drugs.
Aliquots of the stock solution were diluted quantitatively with the same solvent to obtain serial standard solutions in concentration ranging from 0.1 to 15 mg/mL, 0.1 to 10 μg/mL, 0.01 to 20 μg/mL and 0.1 to 250 μg/mL for 1, 2, 3, and 4, respectively. Each synthetic mixture containing 1, 2, 3, or 4 was extracted with 100 mL of distilled water for 1, methanol for 2, or ethanol for 3 and 4, filtered, and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain serial dilutions in concentrations ranging from 0.1 to 25 mg/mL, 0.1 to 10 μg/mL, 0.01 to 20 μg/mL and 0.1 to 25 μg/mL, for 1, 2, 3, and 4, respectively.

Assay Solutions of Drugs in Their Pharmaceutical Preparations.
Twenty tablets were finely powdered, a quantity of the powder, equivalent to one tablet of 1-4, was transferred with the aid of several portions of distilled water for 1, methanol for 3 or ethanol for 2, 4 to a 100 mL volumetric flask and the volume was completed with the same solvent. The resulting solution was filtered and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain 100 μg/mL, 6 μg/mL, 5 μg/mL and 6 μg/mL solutions, for 1, 2, 3 and 4, respectively.  4.10. General Fluorometric Procedure. One milliliter of each drug standard solutions, assay solutions of synthetic mixtures, assay solutions of pharmaceutical preparations, assay solutions of plasma samples, or the assay solution of the volunteer's plasma was transferred to 10.0 mL screw-capped test tube. Solutions of sodium hydroxide and NMNCl were added. The mixture was cooled (in ice) for the indicated time, then the pH was adjusted using formic acid and heated for the indicated time and then was cooled in ice for 5 minutes (optimum NaOH concentration and volume, volume and concentration of added NMNCl, reaction pH values) and cooling and heating times are indicated in Table 1. The mixture was transferred to 10.0 mL volumetric flask, and the resulting solution was completed using distilled water. In case of 4, the pH of the reaction product was adjusted to 10.0 before completing to volume with distilled water. The intensity of the resulting fluorescence was measured at the optimal wavelengths indicated in Table 1. The fluorometric measurements were performed against reagent blank experiments. Concentrations of the drugs were calculated from the corresponding calibration graphs prepared simultaneously.