life science - shimadzu€¦ · life science • page 222 surface analysis of permanent wave...

Life Science

• 22Surface analysis of permanent wave processing hair using DART-MS

• Page 229Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromato-graph Mass Spectrometer)

PO-CON1454E

Surface analysis of permanent waveprocessing hair using DART-MS

ASMS 2014 MP 476

Shoji Takigami1, Erika Ikeda1, Yuta Takagi1,

Jun Watanabe2, Teruhisa Shiota3

1 Gunma University, Kiryu, Japan;

2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan;

3 AMR Inc., Meguro-ku, Tokyo, Japan

2

Surface analysis of permanent wave processing hair using DART-MS

IntroductionPermanent wave processing of hair is carried out at two processes as follows; (A) Reducing agent (permanent wave 1 agent) makes the bridge construction between the keratin protein molecular chains of hair, especially disul�de (S-S) bond of cystine residue cleaved to thiol (-SH) group and hear results a wave and curl.(B) Oxidizing agent (permanent wave 2 agent) makes -SH group oxidized to be reproduced S-S bond. As reducing agents used for permanent wave 1 agent, the thing of cosmetics approval, such as cysteamine hydrochloride and a butyrolactone thiol (brand name Spiera, other than quasi drugs, such as ammonium thioglycolate, acetyl cystein, and thiolactic acid, are used.

After hair is applying permanent wave processing and coloring repeatedly, the chemical structure of a keratin molecule and �ne structure in the hair have been damaged and it resulted as damage hair. It is thought that hair becomes dryness and twining if the cuticle which covers hair is damaged, so it is important to investigate the surface structure of hair and its chemical structure changing.DART (Direct Analysis in Real Time), a direct atmospheric pressure ionization source, is capable of analyzing samples directly with little or no sample preparation. Here, analysis of the ingredient which has deposited on the permanent wave processing hair surface was tried using this DART combined with a mass spectrometer.

The chemical state and property were investigated in the surface of the hair which repeated permanent wave processing with these reducing agents.

Figure 1 DART-OS ion source & LCMS-2020

High Speed Mass Spectrometer

Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec

TGA(thioglycolate)

CA(cysteamine hydrochloride)

BLT(butyrolactone thiol)

SH

O

HOO

O

SH

Fw 92 Fw 113 Fw 118

H2NSH

HCl

Wave ef�ciency is good in a weak alkaline (pH 8 - 9.5)

Wave ef�ciency is good in a weak alkaline (pH 8 - 9.5)

Wave ef�ciency is good in a weak acid (pH 6)

3


Methods and MaterialsThe Chinese virgin hair purchased from the market was washed with the 0.5% non-ionic surfactant containing saturated EDTA solution, and then it was considered as untreated hair sample. Permanent wave processing of hair was prepared as following; the 0.6M TGA solution and 0.6M CA solution which were adjusted to pH8.5 with aqueous ammonia and the 0.6M BLT solution adjusted to pH6.0 with arginine water, which were used as a reducing

agent. After hair sample was reduced for 15 minutes at 35°C using each solvent, it was carried out oxidation treatment at 35°C by being immersed in 8% sodium bromate solution (pH7.2) for 15 minutes. LCMS-2020 (Shimadzu) was coupled with DART-OS ion source (IonSense) and hear samples were held onto DART gas �ow directly, then their surface analyzed.

MS condition (LCMS-2020; Shimadzu Corporation)

Ionization : DART (Direct Analysis in Real Time)

Heater Temperature (DART) : 350°C

Measuring mode (MS) : Positive/Negative scanning simultaneously

Chinese Virgin Hair

0.5% Laureth - 9 solution - EDTA saturated 35°C 1h

Water washing and air drying

Untreated Permanent wave processing by agent 1 & 2 at 0.6M each

Analyzed by DART-MS

permanent wave 1 agent : TGA or CA (pH 8.5; aqueous ammonium) BLT (pH 6; arginine) 35°C 15min

Water washing

Water washing

Britton - Robinson buffer (pH 4.6) 35°C 15min

Water washing

permanent wave 2 agent : 8% NaBrO3 solution (pH 7.2) 35°C 15min

Air drying

Repeat6 times

4


ResultAfter repeating operation of permanent wave processing 1-6 times using TGA (thioglycollic acid), CA (cysteamine), and BLT (Butyrolactonethiol), hair was immersed for 15 minutes at 35°C and with a �ush and air-drying, then permanent wave processing hair was prepared. In order to investigate the ingredient which has deposited on the permanent wave processing hair surface, DART-MS analysis

was performed. DART-MS analysis was conducted in order of #1 Untreated (woman hair), #2 control; ammonia treatment (pH 8.5), #3 0.6M thioglycolic acid (TGA) processing, #4 0.6M butyrolactone thiol (BLT) processing, #5 0.6M cysteamine hydrochloride (CA) processing and #6 control; arginine processing (pH 6).

In the DART mass spectra of #1 untreated and #6 control, many signals considered as triglyceride and diglyceride were detected in both positive and negative spectra obtained by DART-MS. In #3 0.6M thioglycolic acid (TGA) processing spectra, the signal in particular of TGA origin was not detected. In #4 BLT processing spectra (Figure 3), the signals considered to be oxidized BLT (3, 3'-dithiobis (tetrahydrofuran2-one), molecular weight 234) were detected at m/z 235 and 252 in the positive mode. The signal m/z 235 is equivalent to [M+H]+ and m/z 252, [M+NH4]+. In the negative mode, the signals, m/z 115,

231 were detected. They were considered the signal equivalent to [M-H]- and [2M-H]- of BLT oxide compound (C4H4O2S, molecular weight 116) in which two hydrogen atoms were removed from BLT. Carrying out permanent wave processing by BLT, it was found that the dimer of BLT accumulated on the cuticle surface.In #5 CA processing spectrum (Figure 5), the signal considered to be the dimer (Fw152) origin in which CA carried out S-S bond in the positive mode was detected at m/z 153. This is equivalent to [M+H]+.

Figure 2 TIC chromatogram of each sample analyzing with DART

0

25000000

50000000

75000000 2:TIC(+)

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

5000000

10000000

150000004:TIC(-)

#1 #2 #3 #4 #5 #6

Positive TIC m/z 30-2000

Negative TIC m/z 30-2000


5

Figure 3 DART-MS spectra of #4 BLT processingThe BLT-related signals were detected from the positive and the negative spectra.

Figure 4 DART-MS spectra of #5 CA processingThe CA-related signal was detected from the positive spectrum

100 200 300 400 500 600 700 800 900 1000 1100 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x10,000,000)

252

486282 368 424 516

100 200 300 400 500 600 700 800 900 1000 1100 m/z0.0

1.0

2.0

3.0

4.0

5.0Inten. (x100,000)

179

115

231

321347

411 501 579

235

Positive

Negative

[M+H]+[M+NH4]+

[M-H]-

[2M-H]-

100 200 300 400 500 600 700 800 900 1000 1100 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x1,000,000)

282124

391

252

468424 563

184600102 644 691 769 851 922

153

Positive

[2M+H]+


6

In order to indicate clearly the signals specifically detected in each sample, the extraction chromatograms (XIC) were shown (Figure 5). It turned out that BLT-related signals were detected only in #4 and the CA-related signal in #5. Moreover, although the signal intensity was weak, the signal at negative m/z 325 was detected from all samples. Negative m/z 325 is equivalent to [M-H]- of 18

methyl eicosanoic acid (18MEA, molecular weight 326). 18MEA is one of lipid components which protect a cuticle. There is no significant difference of this signal in the hair between treated hair and untreated hair. We would like to inquire so that intensity difference can be found out by further verifying the detection technique in the future.

Figure 5 XIC chromatorgam of each sample analyzing with DART

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

0

500000

1000000 2:152.85(+)

0

5000000

10000000 2:234.70(+)

0

5000000

100000002:251.75(+)

0

250000

4:114.95(-)

0

500000

4:230.90(-)

0

500000

1000000

1500000 2:123.85(+)

0

50000

1000004:325.15(-)

Positive XIC m/z 153


Negative XIC m/z 231


#1


#2 #3 #4 #5 #6




For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

© Shimadzu Corporation, 2014

First Edition: June, 2014

www.shimadzu.com/an/

ConclusionsBy direct analysis of the hair by DART-MS, the chemical structure change in the surfaces of hair, such as permanent wave processing, was able to be observed.

PO-CON1469E

Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

ASMS 2014 TP761

Sanket Chiplunkar, Prashant Hase, Dheeraj Handique,

Ankush Bhone, Durvesh Sawant, Ajit Datar,

Jitendra Kelkar, Pratap Rasam

Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh

Chambers, Makwana Road, Marol, Andheri (E),

Mumbai-400059, Maharashtra, India.

2


IntroductionCosmetics, fragrances and toiletries (Figure 1) are used safely by millions of people worldwide. Although many people have no problems, irritant and allergic reactions may occur. Irritant and allergic skin reactions are the types of contact dermatitis. Essential oils present in fragrance contain some natural and synthetic compounds, which may cause allergic reactions to the end user after application. There are 26 potential allergens listed by

European Directive (EU) 2003/15/EC and International Fragrance Association (IFRA)[1] labeled on cosmetics. Shimadzu MDGC-GCMS technology facilitates the identi�cation and quanti�cation of these allergens to comply with the threshold limits of 100 ppm for rinse-off products.Co-eluting peaks were resolved completely with the help of MDGC-GCMS heart-cut technique.

Figure 1. Cosmetics, fragrances and toiletries

Method of Analysis

Shampoo samples were collected from local market. Standard solutions of 23 allergens were procured from ACCU Standard and dilutions were carried out in Ethanol/Acetonitrile to yield 1000 ppm concentration. Further dilutions were made in methanol.MDGC-GCMS technique was effectively used to minimize matrix effect. Co-eluting peaks were resolved with heart-cut technique using two columns of different

polarities. In MDGC-GCMS, 1st instrument was GC-2010 Plus equipped with FID as a detector and 2nd instrument was GCMS-QP2010 Ultra with MS as a detector. Columns in both the instruments were connected with Deans switch. Allergens in shampoo samples were determined by using this technique. For sample preparation, following methodology was adopted.

Extraction of allergens from shampoo sample

Part method validation was carried out by performing system precision, sample precision, linearity and recovery study. For validation, solutions of different concentrations

were prepared using 40 ppm (actual concentration) standard stock solution mixture of allergens.

1) Blank Solution : 10 mL of methanol was transferred in 20 mL centrifuge tube and vortexed for 5 minutes. The mixture was then centrifuged for 5 minutes at 3000 rpm. This solution was filtered through 0.2 µm nylon syringe filter. Initial 2 mL was discarded and remaining filtrate was collected.

2) Sample Solution : 1 g of shampoo sample was weighed in 10 mL volumetric flask and diluted up to the mark with methanol. Above mixture was transferred in 20 mL centrifuge tube. Further processing was done as mentioned in blank solution.

3) Spike Sample Solution : For recovery study, 1 g of sample was spiked with different volumes of standard stock solution. The above procedure was repeated for preparing different concentration levels of allergens in samples. These spiked samples were treated as mentioned in sample solution.

3


MDGC-GCMS Analytical ConditionsThe instrument con�guration used is shown in Figure 2. Samples were analyzed using Multi-Dimensional GC/GCMS as per the conditions given below.

Table 1. Method validation parameters

Figure 2. Multi-Dimensional GC/GCMS System by Shimadzu

Figure 3. Schematic diagram of multi-Deans switch in MDGC-GCMS

Parameter Concentration

System Precision

Sample Precision

Linearity

Accuracy / Recovery

10 ppm

10 % in Methanol

2.5, 5, 7.5, 10, 15 (ppm)

5, 10, 15 (ppm)

4


MDGC-GCMS analytical parametersChromatographic parameters (1st GC : GC-2010 Plus)

• Column : Stabilwax (30 m L x 0.25 mm I.D.; 0.25 μm)

• Injection Mode : Split

• Split Ratio : 5.0

• Carrier Gas : Helium

• Column Flow : 2.27 mL/min

• Detector : FID

• APC Pressure : 200 kPa (For switching)

• Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min)

50.0 0.00

15.00 100.0 0.00

5.00 240.0 43.67

Chromatographic parameters (2nd GCMS : GCMS-QP2010 Ultra)

• Column : Rxi-1ms (30 m L x 0.25 mm I.D.; 0.25 μm)

• Detector : Mass spectrometer

• Ion Source Temp. : 200 ºC

• Interface Temp. : 240 ºC

• Ionization Mode : EI

• Event Time : 0.30 sec

• Mode : SIM and SCAN

• Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min)

80.0 13.00

3.00 180.0 0.00

10.00 260.0 20.67

• Total Program Time : 75.00 min

Results

MDGC-GCMS technique was used to avoid matrix interference from sample. Using multi-Deans switch and heart-cut technique (Figure 3), co-eluted components from the 1st column were transferred to the 2nd column with different polarity.

Sample analysis using MDGC-GCMS

5


Figure 5. Chromatogram with 1st column (FID)

Figure 4. Chromatogram of spiked sample solution before switching

Table 2. Summary of results for precision on GC and GCMS

Figure 6. SIM chromatogram with 2nd column (MS)

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 min

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

uV (x100,000) Chromatogram

Sam

ple

- 1

Lim

onen

e

Lina

lool

Met

hyl h

eptin

e ca

rbon

ate

Sam

ple

- 2

Sam

ple

- 3 Citr

al -

1

Citr

al -

2 Citr

onel

lol

Ger

anio

lBe

nzyl

Alc

ohol

Hyd

roxy

-citr

onel

lal

Cin

nam

al

Euge

nol

Am

yl c

inna

mal

Ani

syl a

lcoh

olC

inna

myl

alc

ohol

Fern

esol

- 1

Isoe

ugen

olFe

rnes

ol -

2Fe

rnes

ol -

2H

exyl

cin

nam

ald

ehyd

e

Cou

mar

in

Am

ylci

n na

myl

alc

ohol

Sam

ple

- 5

Benz

yl b

enzo

ate

Sam

ple

- 6

Benz

yl s

alic

ylat

e

Benz

yl C

inna

mat

e

12.0 13.0 14.0 15.0 16.0 17.0 min

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

uV (x10,000) Chromatogram

Met

hyl h

eptin

e ca

rbon

ate

Sam

ple

- 2

Sam

ple

- 3

Citr

al -

1

Citr

al -

2

Citr

onel

lol

Ger

anio

l Benz

yl A

lcoh

ol

25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 min

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0uV (x10,000) Chromatogram

Am

yl c

inna

mal

Ani

syl a

lcoh

olC

inna

myl

alc

ohol

Fern

esol

- 1

Isoe

ugen

ol Fern

esol

- 2

Fern

esol

- 2

Hex

yl c

inna

m a

ldeh

yde

26.5 27.0 27.5 28.0 min0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50uV (x10,000) Chromatogram

26.2

56

26.4

91

28.1

05

Target compound - Isoeugenol

27.0 27.5 28.0 28.5 29.0 29.5-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

(x100,000)

134.00 (100.00)115.00 (100.00)92.00 (100.00)137.00 (100.00)109.00 (100.00)138.00 (100.00)103.00 (100.00)149.00 (100.00)164.00 (100.00)

Target compound - Isoeugenol

Summary of results

Result

% RSD for area (n=6) < 2.0

% RSD for area (n=6) < 2.0

Concentration

10 ppm

Unknown

Sample name

23 Allergens mixture

Shampoo

Type of sample

Standard

Cosmetic

Sr. No.

1

2


6

Figure 7. Linearity graph for linalool

Table 3. Linearity by GC

For the quantitation studies, the shampoo sample was spiked with allergens standard to achieve 5, 10 and 15 ppm concentrations. Recovery studies were performed on 13 allergens, having co-elution or matrix interference, using heart-cut technique. The quantitation of these allergens was carried out using 2nd detector (MS) in SIM mode.

In below recovery study, some allergens had recovery value out side the acceptance limit (70-130 %). Optimization can be done by means of change in sample clean up procedure and filtration study.

Quantitation of allergens in shampoo sample

Name of allergen

Linalool

Methyl heptine carbonate

Citronellol

Geraniol

Hydroxy citronellal

Cinnamal

Amyl Cinnamal

Coumarin

Amylcin namyl alcohol

Benzyl benzoate

Sr. No.

1

2

3

4

5

6

7

8

9

10

Linearity (R2)

0.9945

0.9949

0.9965

0.9962

0.9973

0.9959

0.9976

0.9971

0.9983

0.9979 0.0 2.5 5.0 7.5 10.0 12.5 Conc.0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Area (x10,000)

Figure 8. Linearity graph for benzyl cinnamate

Table 4. Linearity by GCMS

Name of allergen

Limonene

Benzyl alcohol

Citral - 1

Citral - 2

Eugenol

Anisyl alcohol

Cinnamyl alcohol

Isoeugenol

Farnesol - 1

Farnesol - 2

Hexyl cinnam aldehyde

Benzyl salicylate

Benzyl cinnamate

Sr. No.

1

2

3

4

5

6

7

8

9

10

11

12

13

Linearity (R2)

0.9945

0.9871

0.9889

0.9902

0.9894

0.9916

0.9937

0.9902

0.9919

0.9929

0.9932

0.9853

0.9927

0.0 2.5 5.0 7.5 10.0 12.5 Conc.0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Area(x10,000)

For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

© Shimadzu Corporation, 2014

First Edition: June, 2014

www.shimadzu.com/an/


Conclusion• MDGC-GCMS method was developed for quantitation of allergens present in cosmetics. Part method validation was

performed as per ICH guidelines.[2] Results obtained for reproducibility, linearity and recovery studies were well within acceptable limits.

• Simultaneous SCAN/SIM and high-speed scan rate 20,000 u/sec are the characteristic features of GCMS-QP2010 Ultra, which enables quantitation of allergens at very low concentration level.

• Matrix effect from cosmetics was selectively eliminated using MDGC-GCMS with multi-Deans switching unit and heart-cut technique.

• MDGC-GCMS was found to be very useful technique for simultaneous identi�cation and quantitation of components from complex matrix.

Reference[1] IFRA guidelines (International Fragrance Association), GC/MS Quanti�cation of potential fragrance allergens, Version 2,

(2006), 6.[2] ICH guidelines, Validation of Analytical Procedures: Text And Methodology Q2(R1), Version 4, (2005).

Figure 9. Overlay SIM chromatogram of unspiked and spiked sample

Table 5. Quantitation of allergens – Recovery Study

Name of allergen

Limonene

Benzyl alcohol

Citral - 1

Citral - 2

Eugenol

Anisyl alcohol

Cinnamyl alcohol

Isoeugenol

Farnesol - 1

Farnesol - 2

Hexyl cinnam aldehyde

Benzyl salicylate

Benzyl cinnamate

Sr. No.

1

2

3

4

5

6

7

8

9

10

11

12

13

Level -15 ppm

127

114

101

97

96

94

98

103

83

84

121

63

66

Level -210 ppm

% Recovery

126

114

106

103

105

105

106

108

95

95

122

47

61

Level -315 ppm

129

123

114

112

116

116

115

118

107

106

130

32

5625.0 27.5 30.0 32.5

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

(x1,000)

Farnesol-1

min

Farnesol-2

Spiked

Unspiked

m/z : 69.00

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