Synthesis of a Unique Fluorescent Material to Print onto Medications for use in the Anti- Counterfeiting of Pharmaceuticals

Transcription

1 Synthesis of a Unique Fluorescent Material to Print onto Medications for use in the Anti- Counterfeiting of Pharmaceuticals Prepared by: Jamie Kern Faculty Advisors: Dr. Brian Logue Professor, Department of Chemistry and Biochemistry South Dakota State University Dr. Grant Crawford REU Program Director South Dakota School of Mines and Technology Dr. Alfred Boysen Professor, Department of Humanities South Dakota School of Mines and Technology Program Information: National Science Foundation Grant # Research Experience for Undergraduates Summer 2013 South Dakota State University 2201 Rotunda Lane Avera Science Center Brookings, South Dakota

2 Table of Contents Abstract Introduction Broader Impact Materials and Methods Reagents and Equipment Fluorescein Synthesis Printing of Fluorescein Inks onto Substrates Masking of Fluorescein s Color....7 Results and Discussion The Synthesis and Characterization of Fluorescein Printing of Fluorescein Inks onto Substrates Masking of Fluorescein s Color Conclusions References...24 Acknowledgments

3 Abstract Anti-counterfeiting of pharmaceuticals is a widely growing field due to the increased occurrence and sophistication of counterfeit medications. A fluorescent label that can be printed onto medications would provide a simple and efficient means by which an authentic pill may be distinguished from a counterfeit one. As a result, fluorescein inks have been used to print labels onto a variety of medications in order to combat counterfeiting. Fluorescein inks were found to be useful in printing labels onto enteric coated medications, particularly those with a yellow color. Further, it was found that inks could be mixed with the fluorescein to increase the covert nature of printing. The fluorescein-based fluorescent inks were used to print QR code labels onto medications to provide an additional level of security. 3

4 Introduction In today s society, counterfeiting of medications has become an issue of increasing significance. Though anti-counterfeiting technologies have become more sophisticated due to the advances of science and technology, counterfeiters have also become more clever due to these same advances. As a result, there is, and will likely always be, a need for the development of anti-counterfeiting techniques that are difficult for counterfeiters to replicate. This research focuses on synthesizing a fluorescent dye that will be printed onto medications to provide an additional level of authenticity. Flourescein is a well known redorange dye with several medical applications. Most commonly, fluorescein is used by optometrists for eye angiographies (Kaneshiro, 2012). Fluorescein fluoresces bright green when illuminated with long-wavelength (365 nm) ultraviolet light. Since it is already known to be biologically stable due to its current applications in the medical field, fluorescein is an ideal molecule to be used in the fluorescent labeling of medications. The fluorescein based ink was used to print quick response (QR) codes onto a variety of substrates. Broader Impact The World Health Organization estimates that less than 1% of medications in developed countries are counterfeit. Comparatively, between 10 and 30% of medications in developed countries are thought to be counterfeit due to a lack of regulations on medication distribution and production (Cabezas, 2010). Further, it is estimated that 30% of those counterfeit medications contain no active ingredients, with an even larger percentage containing an improper dosage of those ingredients (Dégardina, 2013). Consequently, the consumption of counterfeit medications 4

5 can be life-threatening. For example, forged tuberculosis and malaria drugs alone account for about 700,000 deaths worldwide per year (Harris, 2013). Currently, if a medication is suspected to be counterfeit, its physical characteristics, such as its appearance or mass, can be compared with known standards for the specific medication. Further, instrumental analyses using various types of spectroscopy or chromatography have been employed (Dégardina, 2013). Nonetheless, since developing countries are often the target of counterfeiters due to ease of access and fewer restrictions in place for the distribution of pharmaceuticals, sophisticated instruments may not be readily available for verifying the authenticity of medications. Additionally, the physical characteristics of a medication may be easy for sophisticated counterfeiters to mimic. Consequently, a simple, discrete, marker of identification that cannot be easily copied should be developed. Materials and Methods Reagents and Equipment Resorcinol and pthalic anhydride were purchased from Acros Organic (NJ). The sodium hydroxide (reagent grade) used was purchased from Fisher Scientific (Rochester, NY). The blue food coloring used was Great Value brand. Blue dextran was purchased from the Sigma Aldrich Chemical Company (St. Louis, MO). Concentrated sulfuric acid and methanol (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ). All samples were viewed under longwavelength (365 nm, 115 V, 60 Hz) ultraviolet light using a UVP lamp (Upland, CA). Fluorescein Synthesis Fluorescein was synthesized according to Figure 1 based on a similar procedure reported by Baeyer (1871). 5

6 Figure 1: The synthetic scheme for the production of fluorescein. In the hood, 1 g of resorcinol, 1 g of phthalic anhydride, and 4 ml of concentrated sulfuric acid were heated on a hot plate at about 200 o C until the solids melted (several minutes). As the solids began to melt, the material changed from white to a dark red color. The reaction mixture was then allowed to cool to room temperature for five minutes. Once the material had cooled, DI water (60 ml) was added. The solution was mixed for about five minutes using a glass stir rod, and the material at the bottom of the tube was scraped repeatedly to ensure adequate dissolution of the product. Then, NaOH (100 ml of a 1 M aqueous solution) was added and the solution was stirred. Subsequently, concentrated HCl was added 6

7 dropwise to the solution until an orange precipitate formed. The mixture was then filtered using a Buchner funnel apparatus. The solid was allowed to dry overnight. Printing of Fluorescein Inks onto Substrates A 1.5 mm solution of fluorescein in 1:1 methanol:water was used to print onto medications at SDSMT using an Optomec M3D printer. The ink was allowed to dry onto the pill surface and the pill was then illuminated under long-wave ultraviolet light. Substrates included Equate 200 mg ibuprofen, Equate 325 mg aspirin, Quality Plus 500 mg acetaminophen, Motrin PM, Bayer 81 mg low dose aspirin, Major Pain Reliever Plus, and Equate 4 mg ChlorTabs. Masking of Fluorescein s Color Inks were prepared using blue dextran and blue food coloring dyes. Equate brand blue food coloring was diluted to a 20% solution in water. Two drops of the 20% blue food coloring solution were added per ml of the 1.5 mm fluorescein solution. The blue dextran-fluorescein ink was prepared using equal parts of the 1.5 mm fluorescein solution and a 10 mg/ml aqueous blue dextran solution. Results and Discussion The Synthesis and Characterization of Fluorescein Dark red fluorescein solid produced from the synthetic scheme outlined above is shown in Figure 2. In solution, fluorescein fluoresces bright green under long-wavelength ultraviolet light. This fluorescence was confirmed and is shown in Figure 3, with Figure 4 showing the excitation and emission spectra of fluorescein. The excitation-emission spectrum shows that fluorescein has a peak excitation at 448 nm and a peak emission at 519 nm. This is similar to 7

8 values reported by Bennet (2011) of 480 nm and 525 nm, respectively, with the deviation likely due to variations in the solvent identity or ph. The NMR of the fluorescein, Figure 5, further confirmed successful synthesis with all peaks corresponding to literature data (König, 2009) except for the peak at 3.4 ppm in the top spectrum, which was assigned to water of hydration. Figure 2: Red fluorescein precipitate. 8

9 Figure 3: Fluorescein dissolved in methanol under ambient light (top) and longwavelength ultraviolet light (bottom). 9

10 Intensity (cps) 900, , , , ,000 Excitation Emission 400, , , , Wavelength (nm) Figure 4: Excitation-emission spectra of fluorescein in water. 10

11 Figure 5: NMR of synthesized fluorescein (top) and from the literature (König, 2009) (bottom). Printing of Fluorescein Inks onto Substrates Fluorescein inks were used to print the QR code shown in Figure 6 onto a variety of medications. The QR code encodes for SDSM&T. Printing the fluorescein ink on Major Pain Reliever Plus resulted in slight dissolution of the pill (Figure 7). Therefore, the medication was considered incompatible with the current ink formulation and medications with enteric coatings were considered. The same ink formulation was printed on ibuprofen tablets. Fluorescein was highly covert on the tablets under ambient light, but showed excellent fluorescence when excited 11

12 by long wavelength UV light. In Figure 8, the QR code printed on the ibuprofen tablet is clearly visible under UV light, but essentially invisible to the naked eye under ambient conditions. Printing of this same QR code was attempted on other medications. The ink was fluorescent but not covert on the ChlorTabs (Figure 9). However, it was more discrete on the Bayer low-dose aspirin (Figure 10). This result, in combination with the result of the ibuprofen, suggests that the unmasked fluorescein ink is most suitable for pills that are enteric coated. If a covert ink is desired, the yellow color of the ink can be masked to varying degrees with either a yellow colored tablet or a tablet colored darker than the fluorescein ink. Figure 6: QR code printed onto medications. 12

13 Figure 7: A drop of a 1.5 mm fluorescein in methanol dried onto Major Pain Reliever Plus. 13

14 Figure 8: Fluorescein ink printed onto ibuprofen under ambient (top) and long wavelength ultraviolet light (bottom). 14

15 Figure 9: Fluorescein ink printed onto ChlorTabs under ambient light and long wavelength ultraviolet light. 15

16 Figure 10: Fluorescein ink printed onto low dose Bayer aspirin under ambient light and long wavelength ultraviolet light. 16

17 Masking of Fluorescein s Color Another approach to covert/semi-covert printing was tested: printing a visible design on the pharmaceutical tablet with embedded fluorescent molecules where the fluorescence has special properties. To implement this approach, fluorescein molecules were added to blue colored inks and printed on pharmaceuticals. Visually blue inks by themselves should not produce visually yellow/green fluorescence. Therefore, the presence of yellow/green fluorescence from long wavelength UV light is unusual and could be used as an anti-counterfeiting strategy. To test this approach, blue dextran and blue food coloring inks with embedded fluorescein were printed onto acetaminophen and Motrin substrates (Figures 11-14). On the Motrin tablets, the inks are particularly fluorescent and the fluorescence on the acetaminophen is very difficult to discern. This could be explained by variations in coatings between the acetaminophen and Motrin. The ink on the Motrin pills shows no sign of fluorescent properties under ambient light, but is highly fluorescent under long-wave ultraviolet light. Consequently, it was shown that blue dyes are useful in improving the covertness of fluorescein based inks. 17

18 Figure 11: Blue dextran fluorescein solution printed onto acetaminophen under ambient light (top) and long wavelength ultraviolet light (bottom). 18

19 Figure 12: Blue dextran fluorescein solution printed onto Motrin under ambient light (top) and long wavelength ultraviolet light (bottom). 19

20 Figure 13: Blue food coloring fluorescein solution printed onto acetaminophen under ambient light (top) and long wavelength ultraviolet light (bottom). 20

21 Figure 14: Blue food coloring fluorescein solution printed onto Motrin under ambient light (top) and long wavelength ultraviolet light (bottom). 21

22 Conclusions Summary Fluorescein was synthesized and used to make a fluorescent ink. The NMR and fluorescence spectra of the synthesized fluorescein were compared with that from the literature in order to confirm the product. Subsequently, the ink was used to print labels onto medications. It was determined that fluorescein labels are most suitable for medications that are enteric coated because the compound is commonly dissolved in aqueous solvent. Damage to the medication can result if the labels are printed onto pills that are not safety coated, as was seen with the Major Pain Reliever Plus. Additionally, fluorescein inks should be printed onto medications that are either yellow in color, or that are darker in color than the ink, for optimum covertness. For those medications that are lighter in color, blue dextran and blue food coloring were used as masking agents for the original ink. These blue inks could be printed onto lighter colored medications without it being immediately known that the fluorescein is present. Future Work Printing of the fluorescent label should be tried with various types of medications, including those with gel capsules, in order to determine which medications this label will be suitable for. The color of a medication s surface, as well as its chemical components, appears to affect the fluorescence and covertness of this ink. In order to make these fluorescent labels more covert, additional investigations should be made into the masking of fluorescein on the surface of the medication. For example, microencapsulation of the ink by a polymer that degrades under ultraviolet light could be investigated. This would allow for the fluorescein to be undetectable until the medication is 22

23 illuminated under ultraviolet light by the physician, pharmacist, or consumer. At this point, the polymer capsule would degrade, releasing the fluorescein solution and resulting in fluorescence under long-wave ultraviolet light. Similar to the microcapsule idea, a UV degrading quencher of fluorescein should also be investigated. If a molecule was added to the fluorescein solution that could quench the fluorescence, it may improve the covertness of the ink. Then, when illuminated under long-wave ultraviolet light, the quencher would degrade, resulting in fluorescence occurring. For an additional level of security, on-off fluorescence of fluorescein on medications could also be investigated. Abebe and Sinn have created a fluorescein based chemosensor for the detection of copper that has on-off fluorescence characteristics (2011). Their research could be used as the basis for an investigation into on-off fluorescence of fluorescein under other conditions, such as certain wavelengths of light. In this way, illumination with a specific wavelength of light could result in fluorescein fluorescing, while another wavelength could lead to the quenching of the fluorescence. For those medications for which the fluorescein ink is not compatible, for example, those that are not enteric coated, other types of fluorescent inks should be investigated. 23

24 References Abebe, F. A., & Sinn, E. (2011). Fluorescein-based fluorescent and colorimetric chemosensors for copper in aqueous media. Tetrahedron Letters, 52, Baeyer, A. (1871). Berichte der deutschen chemischen gesellschaft.4, 555. Bennet, T. J. (2011). Fluorescein fundamentals. Retrieved, 2013, from page=fa. Cabezas, M. (2010). Counterfeit medicines as global treat. Pharmaceuticals Policy and Law, 12, 179. Dégardina, K., Roggoa, Y., & Margotb, P. (2013). Understanding and fighting the medicine anticounterfeit market. Journal of Pharmaceutical and Biomedical Analysis. Harris, J. (2013). Fake drugs kill over 700,000 people every year. Retrieved, 2013, from Kaneshiro, N. K., Lusby, F. W. & Zieve, D. (2012). MedlinePlus: Fluorescein angiography. Retrieved, 2013, from medlineplus/ency/article/ htm. König, B. (2009). Sustainability in the organic chemistry lab course. Retrieved, 2013, from 24

25 Acknowledgments This work was made possible by the National Science Foundation REU Security Printing and Anti-Counterfeiting Site EEC Additionally, thanks to Dr. Brian Logue for his advice and guidance throughout this experience and to all of the chemistry and engineering faculty at the School of Mines and Technology, especially Dr. Alfred Boysen and Dr. Jon Kellar, and University of South Dakota for their information and direction. 25