8/11/2019 Acid Tioglicolic

1/4

8/11/2019 Acid Tioglicolic

2/4

510 N. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 509512

Tri-sodium phosphate dodecahydrate (Na3PO412H2O, 98%) was

bought from Beijing Chemical Reagent Co. (Beijing, China). Dode-

cyltrimethylammonium bromide (DTAB, 99%) was purchased from

J.K. Scientific Ltd. (Beijing, China). Tetradecyl trimethyl ammonium

chloride (TTAC, >98%) was obtained from Tokyo Chemical Industry

Co. Ltd. (Tokyo, Japan). Cetyltrimethylammonium bromide (CTAB,

99%)and calcium thioglycolate trihydrate (98%)were purchased

from SigmaAldrich, (St. Louis, MO, USA). Eighty-five samples

including fifty permanent wavings, 16 hair straighteners and 19

depilatory creams were bought from the local market.

2.2. Apparatus and conditions

All CE experiments were performed on a P/ACE MDQ sys-

tem (Beckman Instruments, Fullerton, CA, USA) equipped with

an auto-sampler and a photodiode array (PDA) detector. Data

acquisition and treatment was controlled using 32 Karat soft-

ware, version 8.0 (Beckman Coulter, Brea, CA, USA). The running

buffer was 300mmol L1 Na3PO4 (without pH adjustment) con-

taining 0.5 mmolL1 CTAB. A 1:10 dilution of the running buffer

was used as the sample buffer. An uncoated fused-silica capillary

(Yongnian Ruifeng Sepu Peijian Plant, Heibei Province, China) of

50mi.d.

40.2cm (375m o.d., effective length to the detectionwindow was 30 cm) was used. The new capillary was conditioned

by successively flushing with 1 mol L1 NaOH at 20 psi for 20min,

ultrapure water for 5min, and the running buffer for 5min. To

ensure the repeatability, before each subsequent run, the capillary

was rinsed with 1 mol L1 NaOH, ultrapure water, and the running

buffer for 3, 2, and 2 min, respectively. The separation was car-

ried out at a constant voltage of5 kV (current about 124A).

The detection wavelength was set at 236 nm. The sample was

introduced into the capillary by hydrodynamic injection at 0.5 psi

for 10 s. All electrophoresis runs were performed at a temperature

of 25 C.

2.3. Preparation of calibration curve

A TGAstandardstock solution (10mg mL1) was prepared daily

by accurately weighing 20 mg of calcium thioglycolate trihydrate

into a 1.5 mL of sample vial. 1 mL of water was added and then

vortex-mixed. A series of standard solutions at the final concentra-

tions of 0.006, 0.025, 0.125, 0.250, 0.500 and 1.000 mg mL1 were

made and kept in dark to avoid possible photodissociations[16].

2.4. Sample pretreatment

An appropriate amount of samples were weighed into a 15 mL

centrifuge tube with a cap and then diluted with sample buffer

to the mark of 10 mL. The solutions were thoroughly vortexed to

make the samples homogeneous. The mixedwater-based solutions

could be injected directly, while the mixed oil-based samples mustbe centrifuged at 9000 rpm for 10 min and then the resultant clear

supernatant was directly injected.

3. Results and discussion

3.1. Optimization of CE conditions

3.1.1. Selection of the running buffer

TGA is a weak organic acid with two pKa values, pKa1 = 4.32and

pKa2 = 10.20 which comes from the dissociation of its carboxyl and

thiol groups, respectively [17]. Asa ruleof thumb,thepH oftherun-

ning buffer should be adjusted to at least two units below the pKa1or above the pKa2 of TGA to ensure its complete ionization[18].

Phosphate, with low UV absorption and wide pH buffer capacity

Table 1

Comparison of the results of the TGA in 16 cosmetic samples determined by CE,

HPLC and IC.

Sample CE HPLC IC

Content (%, w/v) Content (%, w/v) Content (%, w/v)

1 / /

2 / /

3 0.7

4 / / /

5 / /

6 4.1 4.0 4.3

7 1.3

8 1.1

9 8.7 8.4 8.2

10 8.1 7.7 8.0

11a 2.7 2.6 2.8

12a 3.4 3.3 2.9

13a 2.3 2.4 2.3

14a 3.7 4.0 3.7

15a 2.7 2.7 2.6

16a 3.4 3.4 3.4

/, Not detected; , cannot be quantified due to interferences.a Oil-based sample.

(pKa1 = 2.12, pKa2 = 7.20 and pKa3 = 12.36), is a popular buffer sys-tem for CE. Considering that TGA salts at pH 7.09.5 or 12.7 and

TGA esters at pH 6.09.5 are usually used in cosmetic formulations

[6]and only phosphate can provide a good buffer capacity at pH

higher than (pKa2 (of TGA)+ 2), phosphate was chosen as a priority

running buffer for the subsequent optimizations.

3.1.2. Optimization of the concentration of Na3PO4and the pH of

the separation buffer

Although the analysis of only a single analyte TGA in cosmetic

samples was studied in this work, however, in such a case, the

researchers were oftendismayedby the interferences coming from

the sample matrix. In the present work, a real sample 12 (listed in

Table 1)was used to optimize the following conditions of the CE

method.The concentration of Na3PO4 played a key role for the sep-

aration of TGA. Keeping the concentration of CTAB (0.5 mmol/L)

unchanged, the concentration of Na3PO4(without pH adjustment)

increasing from 100mmol L1 to350mmol L1, not only decreased

the EOF, but also suppressed the adsorption of TGA to the capillary

wall. As shown in Fig.1, the detection sensitivity was enhancedand

the separation efficiency was increased. However, high concentra-

tions of phosphate would produce large amount of Joule heat due

to its inherently high conductivity,which could make theTGA peak

broadened. As a result, 300mmol L1 Na3PO4was chosen.

The pH of running buffer is another keyfactor for the separation

of TGA from the cosmetic sample matrix. It controls both the TGA

charge state and the level of EOF. Since the running buffer with pH

close to the pKa3value of H3PO4(12.36) can provide better buffer-ing capacity [19], the effectof the running buffer at pH 12.35,12.60

(without pH adjustment) and 12.85 were investigated while the

concentration of 300 mmolL1 Na3PO4 was kept constant. It was

found that under all the three pH values, the TGA could be baseline

separated from the interfering peaks coming from sample matrix.

Consideringthat therunningbuffer at pH 12.60could be easilypre-

paredand hada high buffercapacity,300 mmol L1 Na3PO4 without

pH adjustment was chosen for the following experiments.

3.1.3. The selection of the EOF-reversing agent and its

concentration

Samples are normally injected at the anode and detected at the

cathode. At pH > 7, the EOF is sufficient to ensure most species

to move from the anode to the cathode in the order of cations,

8/11/2019 Acid Tioglicolic

3/4

N. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 509512 511

Fig. 1. Effect of Na3PO4 concentration on separation. Na3 PO4 concentration

(mmol L1 ): (a) 100, (b) 200, (c) 300, and (d) 350. (1) TGA and (2) unknown peak.

Running buffer: x mmol L1 Na3PO4 and 0.5mmolL1 CTAB. Uncoated capillary:40.2 cm50m, i.d.(30 cm to thedetector). Injectiontime: 10 s. Injectionpressure:

0.5 psi. Separation voltage: 5 kV, detection: UV 236 nm, temperature: 25 C.

neutrals and anions. However, the negatively charged TGA

migrated against the EOF and moved to the anode under the opti-

mized running buffer pH 12.60, which made it difficult to migrate

out of the capillary in a reasonable time (within 30min). To ensure

that the TGA could rapidly move to the detection window, an

EOF-reversing agent was needed[20].Long-chain alkyl trimethyl

quaternary salts are frequently used as EOF modifiers for anionic

analysis. In this study,threeEOF modifiers,namely DTAB, TTAC and

CTAB were investigated. The migration time of TGA was reduced

alongwiththe increasedlength of thealkylchain as shownin Fig.2.

Itmight be related to the surfactant structures, thelonger thechain,the stronger the adsorption and the ability to control EOF[21].As

a result, CTAB was selected.

Once EOF is reversed and reaches a stable value, the EOF no

longer depends on the CTAB concentration when its concentration

is 0.1mmolL1 which corresponds to 10% of its standard critical

micelle concentration. On this occasion, the surface of capillary is

a dynamic structure represented by the bilayer assembly instead

Fig.2. Effectof theEOF-reversingagenttypes onmigration time. (a)CTAB,(b) TTAC

and (c) DTAB. (1) TGA, other CE conditions were the same as in Fig. 1.

Fig. 3. Effectof samplebuffer on separation:(a) pure water as thesample medium,

(b) one-tenth dilution of the running buffer as the sample buffer, and (c) running

buffer as the sample buffer. (1) TGA and (2) unknown peak. Other CE conditions

were the same as inFig. 1.

of the solid backbone of silica[21].In order to further verify this

observation,CTAB concentrations at 0.2, 0.5 and1.0 mmol L1 were

studied, respectively, with no differences between the separation

efficiency and the migration time observed. Thus, CTAB with con-

centration of 0.5 mmolL1 was chosen because the EOF is stable at

this concentration[14,21].

3.1.4. Choice of sample buffer

For efficient sample stacking, the conductivity of the sample

buffer should be relatively lower than that of the running buffer.

In this study, pure water, one-tenth dilution of the running buffer

and the running buffer were tried as the sample buffer ( Fig. 3).As

expected, one-tenth dilution of the running buffer showedthe best

result.

3.2. CE method validation

3.2.1. Linearity, limit of detection, limit of quantitation, precision

and recovery

The corrected peak areas (Ac) versus the concentrations (,

mgmL1) of TGA showed a good linear relationship (Ac=50.33

+ 40.174) with a correlation coefficient (r) of 0.9998. The limit

of detection (S/N=3) and limit of quantitation (S/N= 10) were

0.002mgmL1 and 0.006 mg mL1, respectively.

Sample 12 was prepared seven times within a day to evaluate

the intra-day precision of the CE method. Samples were accurately

weighed (0.1g for each) and treated as described in Section2.4.To

evaluate the inter-day precision of the CE method, sample 12 waspretreated in triplicate each day on seven consecutive days. Both

the intra- and inter-day precisions of the method were 1.4% and

the average content of TGA in sample 12 was 3.4%.

The average recoveries at the three spiked levels (0.125, 0.250

and 0.500mgmL1) were 96.9%, 102.3% and 94.0% with relative

standard derivations 2.1%, 3.9% and 2.2%, respectively.

3.2.2. Comparison of CE, HPLC and IC results

Sixteen samples of hair-treatment products and depilatory

creams were analyzed by CE, HPLC and IC, respectively. The

results are listed in Table 1. For most of the samples, the

results of the TGA assayed by CE were in good agreement

with the values obtained by HPLC and IC. However, for the

HPLC method, six out of 16 cosmetic samples could not be

8/11/2019 Acid Tioglicolic

4/4

512 N. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 509512

Fig. 4. Chromatogram and electropherogram of the sample 3. (A) Chromatogram, (B) electropherogram. (1) TGA and (2) interfering peak. Other CE conditions were the

same as inFig. 2. HPLC conditions: Mobile phase: acetonitrile and 0.01mol L1 potassium dihydrogen phosphate solution (pH 2.5) with volume ratio of 10:90. Flow rate:

1.0mLmin1. Detection wavelength: 215nm. Column temperature: 30 C. The sample injected was 20 L.

quantified due to the sample matrix interferences. The chro-

matogram and electropherogram of the sample 3 are shown in

Fig. 4. For the IC method, the TGA could not be quantified due

to the serious interferences suffering from the sample matrix

insamples 1,2, 3,5, 7 and8. Allthedatademonstrated the reliability

and practicality of the new CE method.

3.3. Real sample analysis

Sixty-nine commercially available samples, including forty per-

manent wavings, sixteen hair straighteners and thirteen depilatory

creams were successfully analyzed. None of these samples con-

tained TGA exceed the permitted level.

4. Conclusions

The developed CE method demonstrated the straightforward

preparation, robustness, and accuracy for TGA analysis, and has

been recommended for routine quality control analysis of cosmet-

ics.

Acknowledgement

The project for training high-level medical technical personnel

in health system in Beijing City is gratefully acknowledged.

References

[1] Y.F.Zabashta,A.V. Kasprova, S.P.Senchurov, Y.E.Grabovskii, The location of thethioglycolic acid molecules in intrafibrillar unordered areas of the human hairkeratin structure, Int. J. Cosmet. Sci. 34 (2012) 223225.

[2] K. Suzuta, S. Ogawa, Y. Takeda, K. Kaneyama, K. Arai, Intermolecular disul-fide cross-linked structural change induced by permanent wave treatment ofhuman hair with thioglycolic acid, J. Cosmet. Sci. 63 (2012) 177196.

[3] D. Oikawa, W. Takeuchi, S. Murata, K. Takahashi, Y. Sekine, Measurement ofconcentrationsof thioglycolicacid, dithiodiglycolicacid andammoniain indoorair of a beauty salon, J. Occup. Health 54 (2013) 370375.

[4] Z.Wang, X.Ren, D.Wang, Y.Guan, L.Xia, Effectsof thioglycolicacid onpartheno-genetic activation ofxenopusoocytes, PLoS ONE 6 (2011) e16220.

[5] L. Xia, S. Hou, X. Ren, Z. Wang, Effects of thioglycolic acid on in vivo oocytesmaturation in mice, PLoS ONE 6 (2011) e23996.

[6] T.Zhao, HygienicStandardforCosmeticsHealth, MilitaryMedicalPress,Beijing,China, 2007, pp. 239241.

[7] Z. Zhong, D. Du, C. Liang, J. Yao, Study on analysis of thioglycolic acid in cos-metics by ion chromatography, Wei Sheng Yan Jiu 33 (2004) 491493.

[8] V. Cavrini, V. Andrisano, R. Gatti, G. Scapini, HPLC determination of thiogly-colic acid and other aliphatic thiols in cosmetic formulations using ethacrynicacid as precolumn derivatization reagent, Int. J. Cosmet. Sci. 12 (1990)141150.

[9] J.M. Zen, H.H. Yang, M.H. Chiu, Y.J. Chen, Y. Shih, Determination of thioglycolicacid in hair-waving products by disposable electrochemical sensor coupledwithhigh-performanceliquidchromatography,J. AOACInt. 92 (2009)574579.

[10] Methods for Determination of Thioglycolic Acid in Cosmetics, Documents ofChinas Food and Drug Administration, Accessory Four, 2012, pp. 1719.

[11] L. Wang, Z. Chen, Determination of thioglycolic acid and cysteine in hair-

treatment products at ceramic carbon composite electrodes, Electroanalysis9 (1997) 12941297.[12] N. Bansal, A kinetic method for the determination of organosulfur compounds

by inhibition: determinationof cysteine,2,3-dimercaptopropanol and thiogly-colic acid, Transition Met. Chem. 35 (2010) 483490.

[13] M. Sierra-Rodero,J.M. Fernndez-Romero, A. Gmez-Hens, Photometric deter-mination of thioglycolic acid in cosmetics by using a stopped-flow reverseflow-injection system and the formation of gold nanoparticles, Microchem. J.97 (2011) 243248.

[14] E.A. Dutra, M.I.R.M. Santoro, G.A. Micke, M.F.M. Tavares, E.R.M. Kedor-Hackmann,Determination of-hydroxyacidsin cosmeticproductsby capillaryelectrophoresis, J. Pharm. Biomed. Anal. 40 (2006) 242248.

[15] P. Wang, X. Ding, Y. Li, Y. Yang, Simultaneous determination of eleven preser-vatives in cosmetics by micellar electrokineticchromatography, J. AOAC Int.95(2012) 10691073.

[16] A.R. Attar, D.E. Blumling, K.J. Knappenberger, Photodissociation of thioglycolicacid studied by femtosecond time-resolvedtransientabsorption spectroscopy,

J. Chem. Phys. 134 (2011) 024514.[17] P. Ke, X. Sun, Preparation of CdS self-assembled monolayers and interfacial

sensing for DNA, Chin. J. Huaqiao Univ. (Nat. Sci.) 32 (1) (2011) 4852.[18] Beckman Coulter, Introduction to Capillary Electrophoresis, Beckman Instru-

ment Inc., 1996, pp. 18.[19] K. Altria, Buffer preparation hints, tipsand common errors,LCGC AsiaPacific

10 (2) (2007) 2529.[20] J.E. Melanson, N.E. Baryla, C.A. Lucy, Dynamic capillary coatings for electroos-

motic flow control in capillary electrophoresis, Trends Anal. Chem. 20 (2001)365374.

[21] M.F.M. Tavares, R. Colombara, S. Massaro, Modified electroosmotic flow bycationic surfactant additives in capillary electrophoresis: evaluation of elec-trolyte systems for anion analysis, J. Chromatogr. A 772 (1997) 171178.