1. Sergey Brevda Chem 4101 December 10, 2010
Levothyroxine Content in Medication Utilizing p-acceptors and Absorption Spectroscopy
Sergey Brevda
Chem 4101
December 10, 2010

2. Levothyroxine Sodium
Is a hormone released by the thyroid gland responsible for controlling human metabolic rate.
Deficiency in this hormone could lead to weight gain, cold intolerance, mental and physical retardation, and delayed reflexes.

3. Problem
Working as a pharmacy technician I encounter thyroid hormone deficient people everyday. It is not uncommon for them to compare generic and brand name levothyroxine medication. Their claim is that the brand name Synthroid® contains more T4 hormone than generic Levothyroxine manufactured by Mylan® Companies.
Hypothesis: Brand name Synthroid® and generic Levothyroxine have the same content of levothyroxine sodium which can be proven by capillary zone electrophoresis.

4. Possible Alternative Methods
Advantage
Disadvantage
HPLC-ICP-MS
Low limit of detection
Expensive and non-selective without sample prep
Capillary Zone Electrophoresis
Relatively low price, sample is recovered
Extensive sample preparation
Infared Spectroscopy
Very fast, matrix assisted IR has low sample preparation
The analysis is qualitative not quantitative
HPLC-Electrospray-Ion Trap MS
Low limit of detection, linear range from micrograms
Very expensive instrumentation, takes about 30 minutes for each run

5. Why Absorption Spectroscopy?
This technique is inexpensive. If Jenway 6305 spectrophotometer is used the entire setup is around 5, dollars.
Able to detect in the range of expected analyte content.
Has minimal sample preparation.
The trials can performed fast and with two injections by syringe.
Has minimal expected matrix interference.

6. Sample Preparation and Reaction
The samples (tablets of Levothyroxine and Synthroid of comparable strength) will simply be diluted to fit in the dynamic range 0.05 mmol to 0.25 mmol. This solution is then ultrasonicated and filtered through a membrane filter.
Chloranilic acid is than added as p-acceptor to take an electron from the n-donor: levothyroxine. The radical ion formed absorbs at 538nm.
The analyte does not absorb at that wavelength as shown on this complete absorption spectrum.

7. Prepared Solutions for Sampling
Both levothyroxine and chloranilic acid are dissolved to proper concentrations using ethanol.
Chloranilic concentration is set to be 1 mmol to be in sufficient quantity when analyte is introduced.
After each solution is prepared it is ultrasonicated as mentioned before. To the right is an example of a device responsible for that. Its task is to mix the components of the solution.

8. Instrumental Layout RC1,RC2: retention coils (65 cm) R: p-acceptor
B1,B2: automatic burettes
V1,V2: solenoid valves
W: Waste
RC1,RC2: retention coils (65 cm)
D: spectrophotometric detector
S: Sample or standard

9. Using The Instrumentation
Chloranilic acid is added to the first automatic burette (B1) and the first valve (V1) directs the solution towards the detector.
After this solution goes through almost the entire apparatus the second valve (V2) directs the current into waste (W).
The second valve (V2) allows the solution from the second automatic burette (B2) containing the sample to flow towards the second reaction coil (RC2) where the analyte reacts with chloranilic acid to form the chromogen.
The detector (D) measures the resulting absorbance and using the first valve (V1) the sample is directed towards waste (W).

10. Sample Results Measured Concentration
These results are done in the same way as proposed but for different brands producing artificial T4 and T3 (a similar hormone).
The equation for the dynamic range is : A=1045C with correlation coefficient of
Reproducibility was good with relative standard deviation lower than 4.6%.
The tests were ran at a speed of 26 per hour.

11. Conclusion
The optimal way to perform tests on levothyroxine containing medication with respect to amount of active ingredient in each table is through absorption spectroscopy after a chemical derivitization.
The resulting data is cheaply and quickly obtained. Furthermore, this data is still statistically relevant.
Based on previous work done to quantitatively measure levothyroxine levels in hypothyroidism medication made by different manufacturers the declared amount on the bottle is accurate.
In order to further the solution of this problem, studies involving actual patients exposed to Synthroid ® and Levothyroxine should be conducted.

12. References
C.N. Carducci, S.E. Lucangioli, VG. Rodriguez, G.C. Fernhdez Otero. “Application of extraction disks in dissolution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis.” Journal of Chromatography A, 730 (1996)
Cristina I.C. Silvestre, Joгo L.M. Santos, Joao L.F.C. Lima, Elias A.G. Zagatto. “Exploiting _-acceptors for the determination of thyroid hormones (T3 and T4) using a single interface flow system.” Talanta 79 (2009) 1177–1180.
Eric P. Windmaier, Hershel Raff Kevin Strang. “Vander’s Human Physiology.” Effects of Steroid and Thyroid Hormones. Colin H Wheatly; McGraw-Hill Companies: New York, 2008;11th edition, pp 425.
Nadia Soukhova, Offie P. Soldin, Steven J. Soldin. “Isotope dilution tandem mass spectrometric method for T4/T3.” Clinica Chimica Acta 343 (2004) 185–190.
Okabe, Nobuo O. Bulletin of the Chemical Society of Japan (1981):
Sasi S. Kannamkumarath, Rodolfo G. Wuilloud, Apryll Stalcup, Joseph A. Caruso, Himanshu Patel and Adel Sakr. “Determination of levothyroxine and its degradation products in pharmaceutical tablets by HPLC-UV-ICP-MS.” J. Anal. At. Spectrom. 2004, 19,
T. L. Jones-Lepp, D. A. Alvarez, J. D. Petty, J. N. Huckins. “Polar Organic Chemical Integrative Sampling and Liquid Chromatography–Electrospray/Ion-Trap Mass Spectrometry for Assessing Selected Prescription and Illicit Drugs in Treated Sewage Effluents.” Arch. Environ. Contam. Toxicol. 47 (2004) 427–439.