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Chuxin Chen Analytical problem: comparative analysis of arbutin and tranexamic acid in skin whitening products.

The research project was stimulated by the increasing tendency in consuming skin whitening products in the Asian beauty market. The research is important because a large number of Asian women use different kinds of whitening products to their skin every day. Sorts of anti-pigmentation compounds were added into the products, these products varies from prices and the effectiveness of whitening.
What I am going to make a comparison is the arbutin and tranexamic acid. Arbutin is widely used in the products with fair prices and the tranexamic acid is always preferred by the luxury products. The comparison will mainly focus on the whitening effects of the products with these two compounds and try to find out that the luxury component is not much better than the normal one. The hypothesis is tranexamic acid has better performance in skin whitening and more reliable to use externally.
The featured arbutin-added whitening product in the Asian beauty market is α-arbutin serials from DHC, Japan, and the tranexamic acid is mainly used by the sheiseido brand's whitening line. Thus the matrix of the analyte is whitening cream containing arbutin and whitening cream with tranexamic acid.
UV VIS ABSORPTION SPECTROMETRY
For arbutin, the maximum wavelength of the absorption is 227nm, the analyte was dissolved in the deionized water, the HPLC eluent was 40% methanol(v/v) in 0.02M phosphate buffer. however, the molar absorptivity cannot be found in various of articles, it is also difficult to figure out since no complete calibration curve was given.
Tranexamic acid: according to the research, the tranexamic acid compound is unable to be directly analyzed by the UV-Vis since no chromophore is in the molecule.(1) However, the derivatization is a better choice, through a high yield and sensitive reaction to the original analyte, make it more sensitively to be seen by the UV-Vis system.
Three most important steps of the experiment are:
1. Find out a suitable derivatizing agent, conduct the reaction with the target analyte. (the reaction should be as complete as possible, the evaluation of the irreversibility and the sentivity of are important.)
2. Keep the derivatized product from light source, acids, bases and oxidizing agent in case of the conversion of the analyte's interest.
3. The need for internal standards should be evaluated improvement of the method. (4)

(1) Nycz, Jacek E.; Malecki, Grzegorz; Morag, Monika; Nowak, Gerard; Ponikiewski, Lukasz; Kusz, Joachim; Switlicka, Anna. "Arbutin: Isolation, X-ray structure and computational studies". Journal of molecular structures vol.980. 2010, (0022-2860)
(2)Shih, Ying; Wu, Kuan-Lin; Sue, Jun-Wei; Kumar, Annamalai Senthil; Zen, Jyh-Myng. "Determination of tranexamic acid in cosmetic products by high-performance liquid chromatography coupled with barrel plating nickel electrode" Journal of Pharmaceutical and Biomedical Analysis. (2008) 0731-7085
(3) "Microdialysis sampling coupled to on-line high-performance liquid chromatography for determination of arbutin in whitening cosmetics." Journal of chromatography. B. Vol.829. 2005(1570-0232)
(4) Beverly Nickerson. "Sample Preparation of Pharmaceutical Dosage Forms: Challenges and Strategies for Sample Preparation and Extraction". page 333. ISBN:9781441996305
SIMILAR ANALYTICAL PROBLEMS
My research project is similar to Melissa Eubanks' silicones in cosmetics, since we both focus on specific compound that added in the cosmetics and skincare products, the difference is , she concerns the silicione washedovers to the environment and my issue more concern about the human health. Another similar problem is Abdihakim Abdullahi's mercury in skin supplies, that we both want to analyze compounds in the skin whitening products, that we may have similar approach to determine the target ingredient in products.
BLOG 6
Arbutin
CAS: 497-76-7
Can be purchased at sigma-aldrich company, the product number is A4256. The sample has a purity higher than 98% and the price is $104.50 for 10g.
Tranexamic acid
CAS: 1197-18-8
Can be purchased at sigma-aldrich company, the product number is 857653. The sample has a purity of 97% and the price is $41.50 for 10g

View image
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Blog 7
Blog 7
Atomic spectrometry is not useful to quantify the analytes , mass spectrometry may be used to determine the quantity of the analyte.
Analyte arbutin has a molecular weight of 272.25, the nominal molecular weight is 272.
Analyte tranexamic aicd has a molecular weight of 157.21, and the nominal molecular weight is 158.
Proper of ionization sources may be FD-TOF and ESI-trip Quad.
The mass spectrum(7) of tranexamic acid has its base peak at 122.7, and other peaks are at 95.1, 141.0 and 158.0. The spectrometry was LC-MS/MS, the spectrum was got by a sample which has a tranexamic acid concentration of 1E-4g/ml. The chromatohrapy was performed on surveyor liquid chromatography system, using a 5 micro hypurity C18 thermohypersil column (150*2.1mm i.d.) maintained at 20 oC.
The mass spectrum of arbutin was determined by a GC-MS spectrum(8), which is used very common in labs. The spectrum has its base peak at 73, however, the peak with is nominal molecular weight was not very high. The sample was prepared by diluted with DCM and injected 1micro liter in to the GC-MS system. The high purity helium was used as a carrier gas at 1ml/min flow rate. The oven temperature program was: from 120oC to 280oC in two minutes at a steady rate.
(7) Grassin Delyle, Stanislas. Clinica Chimica acta. 2010 Vol. 411 0009-8981
(8)Chisvert. A. Tanlata. 2010, Vol. 81. 0039-9140
blog 8
Sample preparation of arbutin for GC/MS spectrometer. (9)
1. Sample was weighed out in proper amount, (0.01-0.02g), was weighed in a 10-ml volumetric flask.
2. Sample was dissolved in approximately 5ml of DMF (an ultrasonic water bath was used to facilitate the sample solving process).
3. The solution was then diluted to mark with the DMF to dilute the sample.
4. The solution was filtered with a 0.45µm nylon membrane filter.
Sample preparation of tranexamic acid for LC/MS-MS.(10)
1. 10ml of the internal standard solution (0.5g/L) was made and added into the 100µL sample which was contained into a 1.5mL plastic tube.
2. 100µL of perchloric acid(2.5% w/w) were than added.
3. sample was then vortex mixed for 30s and centrifuged at 14,000rpm for 10min.
4. 100µL of the aqueous supernatant was decanted into another tube and 150µL of sodium hydroxide (0.1M) was added.
5. sample was vortex mixed for 10s, and transferred into injection vials for analysis.
References:
(9). Microdialysis sampling coupled to on-line high-performance liquid chromatography for determination of arbutin in whitening cosmetics. Journal of chromatography.B. 2005, vol.829, 1570-0232.
(10).Gaussin Delyle. S. , Chinica Chimica Acta., 2010. Vol. 411., 438-443
Blog 9
1. For my analytical problems, the gas chromatography, reverse phase chromatography, HILIC, affinity chromatography, chiral chromatography and size exclusion chromatography are suitable for analyze the compounds.
The Ion-Exchange chromatography is not suitable since there are no ions in my interested analytes.
2. I think the reverse phase chromatography is the most suitable technique to determine both analytes. Since both compounds are polar in structures, however, they each varied in the polarity substantially, the reversed phase chromatography is a good technique to separate.
3. The Nucleosil brand C18(11) (10µm,25*0.46cm) column was used for the separation, it is the medium density octadecyl , endcapped column, the structure of the stationary phase is "-(CH2)17-CH3". The PH stability of 20oC: 2-8, the number is 718966.
4. The mobile phase for the separation was acetonitrile/water (v:v 50:50), the PH of the mobile phase was adjusted to 2.6 by phosphoric acid (85%, w/w).(12)
5. The detector I am going to use is the UV-Vis detector, since both analytes are absorbed and emitted in the UV range.
References:
(11): http://www.mn-net.com/HPLCStart/NUCLEOSILphases/NUCLEOSILC18/tabid/6128/language/en-US/Default.aspx
(12): M. Saeed Arayne, Naima Sultana, Faiza Qureshi, Farhan Ahmed Siddiqui, Agha Zeeshan Mirza, Saima Sher Bahadur,Muhammad Hashim Zuberi., Chromatographia, 2009, Vol.70, 789-795

Blog 13
Blog 13
Both of arbutin and tranexamic acid are electroactive, thus the capillary electrophoresis method was able to determine and quantify the analytes in samples.
Instrumentation: it is performed using a CHI 821b electrochemical workstation. The reference electrode is Ag/AgCl , the platinum wire is as the auxiliary electrode. The detector is the UV-Vis detector.
Sample prepration: sample and MnO2 powder (particle size≤5 µm) are prepared and make into an aqueous solution with deionized water. The standard stock solution (10 000 ppm in 0.1M H3PO4) is going to be diluted by H3PO4 in different volumes to make different standard sample solutions.
To quantify the analytes in samples, it is useful to detect the current of each samples since the concentration of analytes is proportional to the currents.
Reference:
(14)Jyh-Myng Zen, Hsueh-Hui Yang, Mei-Hsin Chiu, Chao-Hsun Yang, Ying Shin. Journal of AOAC International. Vol. 94, 2011, 985-990

Comments

Blog 13. It is unclear why you mention capillary electrophoresis. If they are using electrochemical detection, why mentioning UV detection. How would the analytes be identified? (-0.5 pt)

Hi, i like your blog, found it on yahoo. ive suffered with skin problems my whole life. And am always looking for good info. I'll be back, thankyou

Blog 9. Based on the molecular structures your compounds may be ionic at certain pH's. You may be able to use ion-exchange chromatography. You are not able to use size exclusion chromatography. (-0.2 pt).

Blog 10. Answers are correct.

Blog 11. Answers are correct.

Why do you use the verbs in past tense? You have not done the experiments. You are proposing to use these techniques?

For blogs 9 and 11. UV detection will work, but keep in mind that other species will also absorb in the UV. These other species may overlap with your analyte of interest. How would you address this issue?

Blog 11.
1. Suitable CE techniques are: Capillary Zone Electrophoresis (CZE) and Micellar Electrockinetic Chromatography (MEKC). The clEF is not suitable for my analysis, since my analytes are not amphiprotic species. And CGE is not suitable since my analytes are not macromolecules.
2. Among the techniques, CZE is my first choice, because the analytes will have different mobility in the column to separate into zone.
3. Conditions that would be used for the CZE: The CZE was performed in an uncoated fused-silica capillary. Sample were injected by the end of anode by pressure (3.45kpa) for 3 seconds and applied voltage 18kv. The CE was performed at 25 oC in phosphate buffer (125mM, Ph=3.00). detection was performed at cathode by UV absorption at 250nm.
4. The detector is UV-Vis, like the case in liquid chromatography ,both analytes are detectable under UV condition in range of 200-300nm, thus UV detector is efficient and easy to be used in this case.
Reference:
(13). Feng-Min Lin, Hwang-Shang Kou, Shou-Mei Wu, Su-Hwei Chen, Aij-Lie Kwan, Hsin-Lung Wu; Electrophoeresis; 2005. 26. 621-626

Blog 10.
1. The preferred technique for my project is the reversed phase liquid chromatography, the detector us UV-Vis.
2. Other projects using similar techniques are: Allison Blonski’s “Taurine in Energy drinks”; Ziyue Yi’s “Aflatoxin in the Oil”; Rajvi Mehta’s “DEHP Leaching from PVC into Contents of Medical Devices.”

Blog 9
1. For my analytical problems, the gas chromatography, reverse phase chromatography, HILIC, affinity chromatography, chiral chromatography and size exclusion chromatography are suitable for analyze the compounds.
The Ion-Exchange chromatography is not suitable since there are no ions in my interested analytes.
2. I think the reverse phase chromatography is the most suitable technique to determine both analytes. Since both compounds are polar in structures, however, they each varied in the polarity substantially, the reversed phase chromatography is a good technique to separate.
3. The Nucleosil brand C18(11) (10µm,25*0.46cm) column was used for the separation, it is the medium density octadecyl , endcapped column, the structure of the stationary phase is “-(CH2)17-CH3”. The PH stability of 20oC: 2-8, the number is 718966.
4. The mobile phase for the separation was acetonitrile/water (v:v 50:50), the PH of the mobile phase was adjusted to 2.6 by phosphoric acid (85%, w/w).(12)
5. The detector I am going to use is the UV-Vis detector, since both analytes are absorbed and emitted in the UV range.
References:
(11): http://www.mn-net.com/HPLCStart/NUCLEOSILphases/NUCLEOSILC18/tabid/6128/language/en-US/Default.aspx
(12): M. Saeed Arayne, Naima Sultana, Faiza Qureshi, Farhan Ahmed Siddiqui, Agha Zeeshan Mirza, Saima Sher Bahadur,Muhammad Hashim Zuberi., Chromatographia, 2009, Vol.70, 789-795

The samples are not arbutin or tranexamic acid. Your samples are likely skin formulations. You cannot use 'past verb tense' because you are proposing an extraction procedure. (-0.1 pt)

If you use a different preparation procedure for each compound, your comparative analysis may not be possible because the extraction procedure will be different. It may be better to use the same extraction procedure or figure out a way to account for recovery

Blog 8
Sample preparation of arbutin for GC/MS spectrometer. (9)
1. Sample was weighed out in proper amount, (0.01-0.02g), was weighed in a 10-ml volumetric flask.
2. Sample was dissolved in approximately 5ml of DMF (an ultrasonic water bath was used to facilitate the sample solving process).
3. The solution was then diluted to mark with the DMF to dilute the sample.
4. The solution was filtered with a 0.45µm nylon membrane filter.
Sample preparation of tranexamic acid for LC/MS-MS.(10)
1. 10ml of the internal standard solution (0.5g/L) was made and added into the 100µL sample which was contained into a 1.5mL plastic tube.
2. 100µL of perchloric acid(2.5% w/w) were than added.
3. sample was then vortex mixed for 30s and centrifuged at 14,000rpm for 10min.
4. 100µL of the aqueous supernatant was decanted into another tube and 150µL of sodium hydroxide (0.1M) was added.
5. sample was vortex mixed for 10s, and transferred into injection vials for analysis.
References:
(9). Microdialysis sampling coupled to on-line high-performance liquid chromatography for determination of arbutin in whitening cosmetics. Journal of chromatography.B. 2005, vol.829, 1570-0232.
(10).Gaussin Delyle. S. , Chinica Chimica Acta., 2010. Vol. 411., 438-443

Blog 6. Good answers.
Blog 7. There are not ionization sources: FD-TOF and ESI-trip Quad. They are an ionization source and a mass analyzer. GC-MS and HPLC-MS/MS are hyphenated techniques. Example of mass specta? (-0.5 pt).

Blog 7
Atomic spectrometry is not useful to quantify the analytes , mass spectrometry may be used to determine the quantity of the analyte.
Analyte arbutin has a molecular weight of 272.25, the nominal molecular weight is 272.
Analyte tranexamic aicd has a molecular weight of 157.21, and the nominal molecular weight is 158.
Proper of ionization sources may be FD-TOF and ESI-trip Quad.
The mass spectrum(7) of tranexamic acid has its base peak at 122.7, and other peaks are at 95.1, 141.0 and 158.0. The spectrometry was LC-MS/MS, the spectrum was got by a sample which has a tranexamic acid concentration of 1E-4g/ml. The chromatohrapy was performed on surveyor liquid chromatography system, using a 5 micro hypurity C18 thermohypersil column (150*2.1mm i.d.) maintained at 20 oC.
The mass spectrum of arbutin was determined by a GC-MS spectrum(8), which is used very common in labs. The spectrum has its base peak at 73, however, the peak with is nominal molecular weight was not very high. The sample was prepared by diluted with DCM and injected 1micro liter in to the GC-MS system. The high purity helium was used as a carrier gas at 1ml/min flow rate. The oven temperature program was: from 120oC to 280oC in two minutes at a steady rate.
(7) Grassin Delyle, Stanislas. Clinica Chimica acta. 2010 Vol. 411 0009-8981
(8)Chisvert. A. Tanlata. 2010, Vol. 81. 0039-9140

Studies corrected for blog 4:
(1). Randomly find 100 (or more) volunteers to test the effectiveness of the products, I decide to test the skin area after ears since that part were less likely to be exposed under the UV and the test results may be well kept. Each of the testers will have the arbutin added cream after the left ear and the tranexamic added cream after the right ear, compare the brightness of the skintone after 2 months’ period.
(2). Kept check the duration of the whitening effects once a month after they stop using the cream, decide which product has given a better and durable result.
(3). Test the side effects of each product if they were absorbed overdoze by skin and into the blood.

Blog 4 - A & B should be part of your literature search and will help you define the studies. C & D does not help to test your hypothesis. (-1 pt).

Blog 5 - Answers are ok. Consider that the molecular structure of arbutin also includes a sugar moiety that can be derivatized.

Blog 5
For the analyte of arbutin, there were no resources directly introduce the fluorescence property of the compound, however, according to the Table 15-1 of textbook, I assume that the arbutin compound, (2R,3S,4S,5R,6S)-2-Hydroxymethyl-6- (4-hydroxyphenoxy)oxane-3,4,5-triol, is a fluorescent and will have an approximately wavelength of fluorescence at 285-365nm, this hypothesis is based on the hydroxyl group and ether structure.
For the tranexamic acid, since it is not a fluorescent, a derivatization was carried out by reacting with 4-chloro-7-nitrobenzofurazan, the derivatization has a maximum excitation at 462nm and a maximum emission at 520nm.(5) The solvent is water and methonal.
For my analytes, the spectrofluorometer is a good instrument to detect since both emission and excitation were able to detect at one time.
Reference:
(5) Huertas-Perez. “Simple, rapid, and sensitive liquid chromatography-fluorescence method for the quantification of tranexamic acid in blood” Journal of chromatography. Vol. 1157. 2007 (0021-9673).

Hypothesis: the tranexamic acid is more effective, stable and healthy to body as an whitening ingredient than the arbutin.
1. Studies: (A) Identify the theories of how each analyte works effectively to bright skintone. (B) Identify the side effects of each analyte as a whitening ingredient, the probability to result an irritation, how they work to sensitive skins and what will happen if use overdoses of each product. (C) Identify the stability of each analyte, which is more likely to oxidized in normal temperature, and which is more light sensitive. (D) Search for relative clinic data to find support that the tranexamic acid is more effective.
2. Since the exact analytes’ level of each whitening cream was not reported by the manufacturer, I only found the relative effective level of each analyte. For arbutin, it is reported that the limiting safe concentration is 7%, and the 5% is the concentration at which arbutin works at its most function. (1)
For tranexamic acid, the limiting safe concentration is 3%, and it is always used at a concentration of 0.5%.(2)
Online references:
(1) http://baike.baidu.com/view/136184.htm
http://baike.baidu.com/view/1283092.htm

I found the posting for BLOG 3. However, the answers to "what studies are similar?" and "what studies are different" are missing (-0.5 pt).

This problem is similar to my analytical problem in the sense that we both took an approach on cosmetics and will most likely be dealing with the same type of matrixes, however my approach is on a toxin found in some creams while Chuxin's is a comparative analysis between two different compounds to see which is more effective.

BLOG 2- If molar absorbtivity was not found, how would you calculate it? (-0.15 pt).

Posting in Melissa's and/or Abdihakim's entry? (- 0.15 pt)

BLoG 3? (-1 pt).

Please revise grammar carefully before posting. Let me know if you want to talk about it.
What is your hypothesis? (-0.2 pt)
What is the matrix? (-0.2 pt)