1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation INTRODUCTION Skin whitening/lightening agents in cosmetics are often used to produce a more even skin tone, 1-3 The use of pharmaceutical active ingredients (AI) such as corticosteroids is prohibited in cosmetics due to the potential undesirable side effects that can occur. 1-5 Corticosteroids are highly effective drugs which are used to treat inflammatory skin conditions such as eczema and psoriasis. Topical preparations are usually in the form of creams, ointments, or gels. Long term use of corticosteroids can cause side effects including pustular psoriasis, permanent skin atrophy, and systemic effects such as hypertension, contact dermatitis, and diabetes. 4,5 The EU Cosmetics Regulation (EC 1223/2009) and US regulations, require that all cosmetic products be safe for use. 6,7 Regardless of the regulations against using corticosteroids in cosmetics, they can still be found in cosmetics marketed as skin lightening products, due to their effectiveness. 1-3 In this study, cosmetic products were obtained from online vendors. The samples were analyzed using a DART 8-11 source (IonSense, Saugus, MA, USA) coupled with Waters® ACQUITY QDa Mass Detector. DART is a desorption atmospheric pressure chemical ionization (APCI) method where a heated ionized gas (helium or nitrogen) is directed towards a target positioned between the DART source exit and the inlet tube leading to the ACQUITY QDa Detector’s ion block. Ions are generated in open air for analysis by mass detection. This mode of analysis typically allows samples be analyzed with little or no sample preparation. The analysis of cosmetics samples was accelerated and simplified in this study using the direct ionization method. SCREENING OF SKIN LIGHTENING PRODUCTS FOR THE CORTICOSTEROID CLOBETASOL PROPIONATE USING DIRECT ANALYSIS IN REAL TIME (DART) AND MASS DETECTION Marian Twohig 1 , Oliver Burt 2 and Chris Stumpf 1 1 Waters Corporation, 34 Maple Street, Milford, MA 01757 USA 2 Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK METHODS Instrumentation and software Ambient ionization was performed using the DART source with subsequent mass detection using the ACQUITY QDa. MassLynx Software was used for data acquisition and interpretation. The DART interface software was used to control the ionization settings and sampling speed. DART conditions Ionization mode: negative Temp.: 350 °C Sampling speed: 1.0 mm/sec Grid voltage: 350 V MS conditions MS system: ACQUITY QDa (Performance option) Ionization mode: negative Mass range: 100 to 600 Da Sampling rate: 5 Hz Sample analysis The standard compounds including clobetasol propionate, arbutin (a glycosylated hydroquinone), and four parabens (Figure 1) were dissolved in methanol and sequentially diluted to prepare the working solutions. Cosmetic samples including gel and cream formulations were obtained from internet vendors in the US. The cosmetic samples were transferred directly to the QuickStrip cards (Figure 2A) using a pipette tip for analysis with the DART-MS in negative ion mode, and helium gas heated to 350 °C. The sampling speed was set to 1 mm/s. Each sample on the card is analyzed consecutively as it traverses the DART-MS interface (Figure 2B). REFERENCES 1. Desmedt B, Courselle P, De Beer J O, Rogiers V, Deconinck E, De Paepe K. Illegal cosmetics on the EU market: a threat for human health? Arch. Toxicol. 88:17651766, 2014. 2. Desmedt B, Van Hoeck E, Rogiers V, Courselle P, De Beer JO, De Paepe K, Deconinck E. Characterisation of suspected illegal skin whitening cosmetics. J Pharm Biomed Anal. 90:8591, 2014. 3. Desmedt B, Courselle P, De Beer JO, Rogiers V, Grosber M, Deconinck E, De Paepe K. Overview of skin whitening agents with an insight into the illegal cosmetic market in Europe. JEADV, 30:943950, 2016. 4. Fiori J, Andrisane V. LC-MS method for the simultaneous determination of glucocorticoids in pharmaceutical formulations and counterfeit cosmetics products. J Pharm Biomed Anal. 91:185192, 2014. 5. Sik Nam Y, Kwon KI, Lee KB. (2011) Monitoring of clobetasol propionate and betamethasone dipropionate as undeclared steroids in cosmetic products manufactured in Korea. Forensic Sci Intl. 210:144148, 2011. 6. EU Regulation 1223/2009 [accessed on July 25th 2016]. 7. FDA: Hydroquinone Studies Under The National Toxicology Program (NTP). [accessed on July 25th 2016]. 8. Cody R, Laramee J, Durst H. Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions. Analytical Chem. 77, 8: 22972302, 2005. 9. Van Berkel GJ, Pasilis SP, Ovchinnikova O. Established and emerging atmospheric pressure surface sampling/ionization techniques for mass spectrometry. J Mass Spec. 43, 9:11631180, 2008. 10. Wells JM, Roth MJ, Keil AD, Grossenbacher JW, Justes DR, Patterson GE, and Barket DJ Jr. Implementation of DART and DESI Ionization on a Fieldable Mass Spectrometer, JASMS. 19, 10: 1419 1424, 2008. 11. Haunschmidt M, Buchberger W, Klampfl CK, Hertsens R. Identification and semi-quantitation of parabens and UV filters in cosmetic products by direct-analysis-in-real-time mass spectrometry and gas chromatography with mass spectrometric detection. Anal Methods. 3,99: 99104, 2011. 12. Ma Q, Bai H, Li W, Wang C, Li X, Cooks GR. Direct identification of prohibited substances in cosmetics and foodstuffs using ambient ionization on a miniature mass spectrometry system. Analytica Chimica Acta. 912: 6573, 2016. 13. Cabaleiro N, de la Calle I, Bendicho C, Lavilla I. An overview of sample preparation for the determination of parabens in cosmetics. Trends in Analytical Chemistry. 57: 3446, 2014. 14. Twohig M, Cooper J, Stumpf C. Identification of Skin Lightening Agents in Cosmetics using the ACQUITY Arc System with PDA, Mass Detection, and Empower Software. Waters Application Note no. 720005813en, November, 2016. RESULTS AND DISCUSSION Figure 3A shows the combined and subtracted spectra resulting from the analysis of a solvent standard of clobetasol propionate, a skin lightening cream (3B), and finally a skin lightening gel sample which did not declare the presence of clobetasol propionate on the label (3C). The packaging for the skin lightening cream sample listed the presence of clobetasol propionate at a level of 0.05% w/w. CONCLUSION The direct analysis of cosmetic samples using DART coupled with the ACQUITY QDa mass detector provides molecular ion information while allowing samples to be analyzed very rapidly and without the need for sample preparation or chromatographic method development. Clobetasol propionate is a highly effective drug that is widely used in dermatology; however its use requires a prescription from a licensed physician. The cosmetic skin lightening cream sample listed the presence of clobetasol propionate on the product information, however the product was sold without a prescription. In the skin lightening gel sample analyzed, the presence of clobetasol propionate was detected, but not declared on the label or the enclosed product information. Inaccurate or insufficient labeling of the cosmetics products increases the likelihood of adverse side effects, as cosmetics are usually used over long time periods with no medical supervision. In the current study the DART-MS has shown potential for the rapid screening of cosmetics samples suspected to contain clobetasol propionate. For precise and accurate quantitation, LC-UV/MS can be used on the screened samples that test positive for the presence of clobetasol propionate and other cosmetics additives. A B O O OH OH O H O H OH Arbutin C 12 H 16 O 7 Cl F O O H O O O CH 3 CH 3 C H 3 C H 3 H H Clobetasol Propionate C 25 H 32 ClFO 5 Methylparaben C 8 H 8 O 3 Ethylparaben C 9 H 10 O 3 Propylparaben C 10 H 12 O 3 Butlyparaben C 11 H 14 O 3 Figure 2. A. QuickStrip card used for sampling, B. automated multi- sample analysis Figure 1. Empirical formulas and structures for arbutin, clobetasol propionate and parabens that were analyzed in the study. Figure 3. A. Spectra from the direct analysis of clobetasol propionate standard; B. a skin lightening cream sample containing clobetasol propionate at 0.05% w/w; C. a skin lightening gel sample. In this example the y axis is normalized to the most intense peak in the spectrum. . Clobetasol propionate, with an [M-H] - ion corresponding to a mass-to-charge ratio (m/z) of 465 was observed in the spectrum resulting from the analysis of a solvent standard (100 μg/mL). The isotopic pattern observed reflects the chlorine present in the chemical structure of clobetasol propionate (Figure 1) providing extra confirmation and increased confidence in the identification of the corticosteroid in both cosmetics samples. The same m/z and isotopic patterns were observed in both the gel and cream samples. The m/z 271 observed in the skin whitening gel sample indicated by the arrow in Figure 3C corresponds to the [M-H] - ion of arbutin which was listed as an ingredient on the product label. Arbutin is frequently used as a skin lightening agent in cosmetics, and currently, its use is not restricted. 2 m/z’s representative of parabens were observed in the samples. Parabens (Figure 1) are used as microbial inhibitors and are commonly found in cosmetic products. 11-13 Based on the observed m/z’s detected, the presence of methylparaben (m/z 151) and ethylparaben (m/z 165) was suspected in both samples, along with propylparaben (m/z 179) and butylparaben (m/z 193) in the lightening gel sample. Further analysis of the samples was carried out by Ultra HighPerformance Liquid Chromatography (UHPLC) with PDA and mass detection using the QDa mass detector. 14 UHPLC/PDA/MS Confirmation Instrumentation and software Separations were performed on the ACQUITY Arc System equipped with a 2998 Photodiode Array (PDA) Detector and the ACQUITY QDa Detector. Empower 3 Software was used for data acquisition and processing. Sample preparation The standard compounds were dissolved in methanol and sequentially diluted to prepare the working solutions. An aliquot (1g) of the cosmetics samples were weighed into 15 mL centrifuge tubes. Acetonitrile (5.0 mL) was added and the samples were shaken for 25 minutes. The samples were centrifuged at 3000 rpm for 10 minutes. An aliquot of the supernatant was syringe filtered using a 0.2 μm PVDF filter and placed in a vial in preparation for sample analysis. . LC conditions Column: CORTECS T3, 3.0 x 100 mm, 2.7 μm Solvent A: 0.1% formic acid in water Solvent B: Methanol Flow rate: 0.80 mL/min: Column temp.: 30 °C: Injection volume: 0.5 μL Gradient conditions: 0 min 0% B, 0.5 min 0% B, 2.2 min 2% B, 6.0 min 95% B, 8.0 min 99% B, 9.0 min 99% B, return to initial conditions. PDA detection: 210 to 400 nm MS conditions MS system : ACQUITY QDa (Performance option) Ionization mode: Negative ion electrospray (ESI) MS scan range: 100 to 600 m/z Sampling rate: 5 Hz Figure 4 (bottom) shows the PDA chromatogram resulting from the separation of the extracted skin lightening gel sample (Figure 2C). The sample analysis confirmed the presence of arbutin, the corticosteroid, clobetasol propionate as well as four parabens which are labeled using the Empower processing method. The t R of the peaks matched with those in the authentic standards (Figure 4 top). In addition the same m/z was obtained for the clobetasol propionate (inset), arbutin and parabens. Figure 4. ACQUITY Arc chromatogram resulting from the separation of a skin lightening gel sample (bottom) at 254 nm using a CORTECS T3 3.0 x 100 mm, 2.7-μm column. Standard compounds for t R matching are also shown (top), 100 μg/mL, 0.5 μL injection. Clobetasol propionate in the sample is indicated by the arrow and the corresponding ESI mass spectrum is shown inset. m/z 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 % 0 100 m/z 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 % 0 100 m/z 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 % 0 100 465 239 313 493 465 151 122 179 409 373 427 481 151 108 307 271 193 165 179 215 283 423 331 361 345 465 Skin lightening cream Skin lightening gel Clobetasol propionate standard Arbutin Parabens A B C Clobetasol Propionate 462 464 466 468 470 472 47 465 467 466 Arbutin Methyparaben Ethylparaben Propylparaben Butylparaben Clobetasol Propionate AU 0.00 0.06 0.12 0.18 0.24 Arbutin Methyparaben Ethylparaben Propylparaben Butylparaben Clobetasol Propionate AU 0.000 0.014 0.028 0.042 0.056 Minutes 0.00 1.80 3.60 5.40 7.20 9.00 Skin lightening gel sample 100 μg/mL standard 254 nm 465 466 467 m/z 0.00 462.50 465.00 467.50 470.00

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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation

INTRODUCTION

Skin whitening/lightening agents in cosmetics are often

used to produce a more even skin tone,1-3

The use of

pharmaceutical active ingredients (AI) such as

corticosteroids is prohibited in cosmetics due to the

potential undesirable side effects that can occur.1-5

Corticosteroids are highly effective drugs which are used

to treat inflammatory skin conditions such as eczema

and psoriasis. Topical preparations are usually in the

form of creams, ointments, or gels. Long term use of

corticosteroids can cause side effects including pustular

psoriasis, permanent skin atrophy, and systemic effects

such as hypertension, contact dermatitis, and diabetes.4,5

The EU Cosmetics Regulation (EC 1223/2009) and US

regulations, require that all cosmetic products be safe for

use.6,7

Regardless of the regulations against using

corticosteroids in cosmetics, they can still be found in

cosmetics marketed as skin lightening products, due to

their effectiveness.1-3

In this study, cosmetic products were obtained from

online vendors. The samples were analyzed using a

DART 8-11

source (IonSense, Saugus, MA, USA) coupled

with Waters® ACQUITY QDa Mass Detector. DART is a

desorption atmospheric pressure chemical ionization

(APCI) method where a heated ionized gas (helium or

nitrogen) is directed towards a target positioned between

the DART source exit and the inlet tube leading to the

ACQUITY QDa Detector’s ion block. Ions are generated in

open air for analysis by mass detection. This mode of

analysis typically allows samples be analyzed with little

or no sample preparation.

The analysis of cosmetics samples was accelerated and

simplified in this study using the direct ionization

method.

SCREENING OF SKIN LIGHTENING PRODUCTS FOR THE CORTICOSTEROID CLOBETASOL PROPIONATE USING DIRECT ANALYSIS IN REAL TIME (DART) AND MASS DETECTION

Marian Twohig1, Oliver Burt2 and Chris Stumpf1

1Waters Corporation, 34 Maple Street, Milford, MA 01757 USA 2Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK

METHODS

Instrumentation and software

Ambient ionization was performed using the DART source with

subsequent mass detection using the ACQUITY QDa. MassLynx

Software was used for data acquisition and interpretation. The

DART interface software was used to control the ionization

settings and sampling speed.

DART conditions

Ionization mode: negative

Temp.: 350 °C

Sampling speed: 1.0 mm/sec

Grid voltage: 350 V

MS conditions

MS system: ACQUITY QDa (Performance option)

Ionization mode: negative

Mass range: 100 to 600 Da

Sampling rate: 5 Hz

Sample analysis

The standard compounds including clobetasol propionate, arbutin

(a glycosylated hydroquinone), and four parabens (Figure 1) were

dissolved in methanol and sequentially diluted to prepare the

working solutions.

Cosmetic samples including gel and cream formulations were

obtained from internet vendors in the US. The cosmetic samples

were transferred directly to the QuickStrip cards (Figure 2A) using

a pipette tip for analysis with the DART-MS in negative ion mode,

and helium gas heated to 350 °C. The sampling speed was set to

1 mm/s. Each sample on the card is analyzed consecutively as it

traverses the DART-MS interface (Figure 2B).

REFERENCES

1. Desmedt B, Courselle P, De Beer J O, Rogiers V, Deconinck E, De Paepe K. Illegal cosmetics on the EU market: a threat for human health? Arch. Toxicol. 88:1765–1766, 2014. 2. Desmedt B, Van Hoeck E, Rogiers V, Courselle P, De Beer JO, De Paepe K, Deconinck E. Characterisation of suspected illegal skin whitening cosmetics. J Pharm Biomed Anal. 90:85–91, 2014. 3. Desmedt B, Courselle P, De Beer JO, Rogiers V, Grosber M, Deconinck E, De Paepe K. Overview of skin whitening agents with an insight into the illegal cosmetic market in Europe. JEADV, 30:943–950, 2016. 4. Fiori J, Andrisane V. LC-MS method for the simultaneous determination of glucocorticoids in pharmaceutical formulations and counterfeit cosmetics products. J Pharm Biomed Anal. 91:185–192, 2014. 5. Sik Nam Y, Kwon KI, Lee KB. (2011) Monitoring of clobetasol propionate and betamethasone dipropionate as undeclared steroids in cosmetic products manufactured in Korea. Forensic Sci Intl. 210:144–148, 2011. 6. EU Regulation 1223/2009 [accessed on July 25th 2016]. 7. FDA: Hydroquinone Studies Under The National Toxicology Program (NTP). [accessed on July 25th 2016]. 8. Cody R, Laramee J, Durst H. Versatile New Ion Source for the Analysis of Materials in Open Air

under Ambient Conditions. Analytical Chem. 77, 8: 2297–2302, 2005. 9. Van Berkel GJ, Pasilis SP, Ovchinnikova O. Established and emerging atmospheric pressure

surface sampling/ionization techniques for mass spectrometry. J Mass Spec. 43, 9:1163–1180, 2008. 10. Wells JM, Roth MJ, Keil AD, Grossenbacher JW, Justes DR, Patterson GE, and Barket DJ Jr. Implementation of DART and DESI Ionization on a Fieldable Mass Spectrometer, JASMS. 19, 10: 1419–1424, 2008. 11. Haunschmidt M, Buchberger W, Klampfl CK, Hertsens R. Identification and semi-quantitation of parabens and UV filters in cosmetic products by direct-analysis-in-real-time mass spectrometry

and gas chromatography with mass spectrometric detection. Anal Methods. 3,99: 99–104, 2011. 12. Ma Q, Bai H, Li W, Wang C, Li X, Cooks GR. Direct identification of prohibited substances in

cosmetics and foodstuffs using ambient ionization on a miniature mass spectrometry system. Analytica Chimica Acta. 912: 65–73, 2016.

13. Cabaleiro N, de la Calle I, Bendicho C, Lavilla I. An overview of sample preparation for the determination of parabens in cosmetics. Trends in Analytical Chemistry. 57: 34–46, 2014.

14. Twohig M, Cooper J, Stumpf C. Identification of Skin Lightening Agents in Cosmetics using the ACQUITY Arc System with PDA, Mass Detection, and Empower Software. Waters Application Note no. 720005813en, November, 2016.

RESULTS AND DISCUSSION

Figure 3A shows the combined and subtracted spectra resulting from the analysis of a solvent standard of clobetasol propionate, a skin

lightening cream (3B), and finally a skin lightening gel sample which did not declare the presence of clobetasol propionate on the label

(3C).

The packaging for the skin lightening cream sample listed the presence of clobetasol propionate at a level of 0.05% w/w.

CONCLUSION

The direct analysis of cosmetic samples using DART coupled

with the ACQUITY QDa mass detector provides molecular ion

information while allowing samples to be analyzed very rapidly

and without the need for sample preparation or chromatographic

method development.

Clobetasol propionate is a highly effective drug that is widely

used in dermatology; however its use requires a prescription

from a licensed physician.

The cosmetic skin lightening cream sample listed the presence of

clobetasol propionate on the product information, however the

product was sold without a prescription.

In the skin lightening gel sample analyzed, the presence of

clobetasol propionate was detected, but not declared on the label

or the enclosed product information.

Inaccurate or insufficient labeling of the cosmetics products

increases the likelihood of adverse side effects, as cosmetics are

usually used over long time periods with no medical supervision.

In the current study the DART-MS has shown potential for the

rapid screening of cosmetics samples suspected to contain

clobetasol propionate.

For precise and accurate quantitation, LC-UV/MS can be used

on the screened samples that test positive for the presence of

clobetasol propionate and other cosmetics additives.

A

B

O O

OH

OH

OH

OH

OH

ArbutinC12H16O7

Cl

F

O

OH

O

O

O

CH3

CH3CH3

CH3

HH

ClobetasolPropionateC25H32ClFO5

MethylparabenC8H8O3

EthylparabenC9H10O3

PropylparabenC10H12O3

ButlyparabenC11H14O3

Figure 2. A. QuickStrip card used for sampling, B. automated multi-

sample analysis

Figure 1. Empirical formulas and structures for arbutin, clobetasol propionate and parabens that were analyzed in the study.

Figure 3. A. Spectra from the direct analysis of clobetasol propionate standard; B. a skin lightening cream sample containing clobetasol

propionate at 0.05% w/w; C. a skin lightening gel sample. In this example the y axis is normalized to the most intense peak in the spectrum.

.

Clobetasol propionate, with an [M-H]- ion corresponding to a mass-to-charge ratio (m/z) of 465 was observed in the spectrum resulting

from the analysis of a solvent standard (100 μg/mL).

The isotopic pattern observed reflects the chlorine present in the chemical structure of clobetasol propionate (Figure 1) providing extra

confirmation and increased confidence in the identification of the corticosteroid in both cosmetics samples. The same m/z and isotopic

patterns were observed in both the gel and cream samples.

The m/z 271 observed in the skin whitening gel sample indicated by the arrow in Figure 3C corresponds to the [M-H]- ion of arbutin which

was listed as an ingredient on the product label. Arbutin is frequently used as a skin lightening agent in cosmetics, and currently, its use

is not restricted.2

m/z’s representative of parabens were observed in the samples. Parabens (Figure 1) are used as microbial inhibitors and are commonly

found in cosmetic products.11-13

Based on the observed m/z’s detected, the presence of methylparaben (m/z 151) and ethylparaben (m/z

165) was suspected in both samples, along with propylparaben (m/z 179) and butylparaben (m/z 193) in the lightening gel sample.

Further analysis of the samples was carried out by Ultra HighPerformance Liquid Chromatography (UHPLC) with PDA and mass

detection using the QDa mass detector.14

UHPLC/PDA/MS Confirmation Instrumentation and software Separations were performed on the ACQUITY Arc System equipped with a 2998 Photodiode Array (PDA) Detector and the ACQUITY QDa Detector. Empower 3 Software was used for data acquisition and processing. Sample preparation The standard compounds were dissolved in methanol and sequentially diluted to prepare the working solutions. An aliquot (1g) of the cosmetics samples were weighed into 15 mL centrifuge tubes. Acetonitrile (5.0 mL) was added and the samples were shaken for 25 minutes. The samples were centrifuged at 3000 rpm for 10 minutes. An aliquot of the supernatant was syringe filtered using a 0.2 µm PVDF filter and placed in a vial in preparation for sample analysis. . LC conditions Column: CORTECS T3, 3.0 x 100 mm, 2.7 μm Solvent A: 0.1% formic acid in water Solvent B: Methanol Flow rate: 0.80 mL/min: Column temp.: 30 °C: Injection volume: 0.5 μL Gradient conditions: 0 min 0% B, 0.5 min 0% B, 2.2 min 2% B, 6.0 min 95% B, 8.0 min 99% B, 9.0 min 99% B, return to initial conditions. PDA detection: 210 to 400 nm MS conditions MS system : ACQUITY QDa (Performance option) Ionization mode: Negative ion electrospray (ESI) MS scan range: 100 to 600 m/z Sampling rate: 5 Hz Figure 4 (bottom) shows the PDA chromatogram resulting from the separation of the extracted skin lightening gel sample (Figure 2C). The sample analysis confirmed the presence of arbutin, the corticosteroid, clobetasol propionate as well as four parabens which are labeled using the Empower processing method. The tR of the peaks matched with those in the authentic standards (Figure 4 top). In addition the same m/z was obtained for the clobetasol propionate (inset), arbutin and parabens.

Figure 4. ACQUITY Arc chromatogram resulting from the separation of

a skin lightening gel sample (bottom) at 254 nm using a CORTECS T3

3.0 x 100 mm, 2.7-μm column. Standard compounds for tR matching are

also shown (top), 100 μg/mL, 0.5 μL injection. Clobetasol propionate in

the sample is indicated by the arrow and the corresponding ESI mass

spectrum is shown inset.

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

%

0

100465

239 313 493

465

151

122 140

179

409373 427 481

151

143

108

307

271193

165179

215 283 423331 361345 465

Skin lightening cream

Skin lightening gel

Clobetasol propionate standard

Arbutin

Parabens

A

B

CClobetasolPropionate

m/z438 440 442 444 446 448 450 452 454 456 458 460 462 464 466 468 470 472 474 476 478 480 482 484 486 488 490 492 494 496

%

0

100

m/z438 440 442 444 446 448 450 452 454 456 458 460 462 464 466 468 470 472 474 476 478 480 482 484 486 488 490 492 494 496

%

0

100

m/z438 440 442 444 446 448 450 452 454 456 458 460 462 464 466 468 470 472 474 476 478 480 482 484 486 488 490 492 494 496

%

0

100465

467

465

467

465

448

451

467

466

Arb

uti

n

Meth

yp

ara

ben

Eth

ylp

ara

ben

Pro

pylp

ara

ben

Bu

tylp

ara

ben

Clo

beta

so

l P

rop

ion

ate

AU

0.00

0.06

0.12

0.18

0.24

Arb

uti

n

Meth

yp

ara

ben

Eth

ylp

ara

ben

Pro

pylp

ara

ben

Bu

tylp

ara

ben

Clo

beta

so

l P

rop

ion

ate

AU

0.000

0.014

0.028

0.042

0.056

Minutes

0.00 1.80 3.60 5.40 7.20 9.00

Skin lightening gel sample

100 µg/mL standard254 nm

465

466

467

Inte

ns

ity

0.0

7000.0

14000.0

21000.0

28000.0

m/z

460.00 462.50 465.00 467.50 470.00