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Rapid development of quantitativemethod for determination of plantgrowth regulators and streptomycinin fruits using LC/MS/MS

ASMS 2016 WP 234

Anant Lohar, Shailendra Rane, Ashutosh Shelar,

Purushottam Sutar, Shailesh Damale, Deepti Bhandarkar,

Rashi Kochhar, Ajit Datar, Jitendra Kelkar

and Pratap Rasam

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

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

Mumbai-400059, Maharashtra, India.

2

Rapid development of quantitative method for determination of plant growth regulators and streptomycin in fruits using LC/MS/MS

IntroductionPlant growth regulators (PGRs) and antibiotics are commonly used by farmers for enhancing growth of crops and to protect against infections respectively. However, extensive usages of these compounds is known to have adverse effect on human health[1],[2]. Each standard has different linearity range as shown in Table 2. It is challenging to quickly develop multi-analyte quantitation method for determination of varied compounds simultaneously[3]. One interesting approach can be Method Scouting, which allows user to conduct all

method development exercises automatically. This results in swift development of an optimum method which will suit all analytes. Highly sensitive quantitative method was developed quickly using Nexera Method Scouting system for determination of eight PGRs and streptomycin (shown in Figure 1) on LCMS-8040 (shown in Figure 2), a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan.

Figure 1. Structures of PGRs and antibiotics

Chlormequat chloride Paraquat dichloride Diquat dibromide

Streptomycin 6 Benzyl adenine Mepiquat chloride

Ethephon 4 Chlorphenoxyacetic acid(4 CPA)

Gibberellic acid

The standards of PGRs and streptomycin procured from Sigma Aldrich were used for analysis. Mixed standard stock of PGRs and streptomycin was prepared in the water : methanol (1:1 v/v). Further this stock was serially diluted to prepare calibration levels ranging from 0.1 ng/mL to 1000 ng/mL in water : methanol (1:1 v/v).

Sample preparation

Method of analysis

Preparation of aqueous calibration levels

3


In a typical method scouting procedure, data was collected using a number of mobile phase and column combinations. Analyzers switching manually between these combinations are limited by the number of work hours in a single day, making it impossible to perform method scouting ef�ciently. The Nexera Method Scouting System is capable of automatically investigating up to 96 combinations of mobile phases and columns, without such time restrictions, thereby signi�cantly improving method development productivity.For primary method scouting, different mobile phases were checked e.g. 0.1 % formic acid in water, 5 mM ammonium acetate, 10 mM ammonium acetate etc. as aqueous phases and methanol, acetonitrile, 0.1 % formic acid in acetonitrile etc. as organic phases. Columns like C18 and HILIC of different makes and dimensions were

tested (shown in Figure 3). Various gradient programs were also tried. Automatic batch �le was created using Method Scouting software and data were acquired by LabSolutions software (Shimadzu Corporation, Japan).Solvent blending is a powerful tool in order to save the labor and time required for preparing mobile phases. For example, a wide analysis range can be performed with varied concentrations of the buffer and organic solvent by just assigning aqueous solutions to pump A and organic solutions to pump B. When chromatographic elution patterns are investigated with various compositions of the solvents, using an automated solvent blending eliminates wastage of solvent and reduce labors.By using solvent blending function, formic acid concentration in 20 mM ammonium acetate was optimized.

LC/MS/MS analysis

Figure 2. LCMS-8040 triple quadrupole mass spectrometer

Matrix matched standards were prepared in fresh watermelon juice purchased from local market. In 50 mL centrifuge tube, 10 mL of watermelon juice sample was taken. To this sample, 10 mL of acidi�ed methanol was added and vortexed for few minutes. Solution was then centrifuged at 5000 rpm for 5 min. Supernatant was

collected and �ltered through 0.2 micron �lter. This �ltered extract was used as a diluent for matrix matched calibration levels.The matrix matched calibration levels were prepared in a same way as aqueous standards.

Preparation of matrix matched calibration levels

4


Column : Grace Alltima HP HILIC (50 mm L x 2.1 mm I.D.; 3 µm)

Mobile phase : A: 20 mM ammonium acetate containing 0.05 % formic acid in water

B: 0.1 % formic acid in acetonitrile

Gradient program (B%) : 0.0-1.0 min → 80 (%); 1.0-2.5 min → 80-57 (%);

2.5-4.0 min → 5 (%); 4.0-4.1 min → 5 (%);

4.1-5.0 min → 80 (%); 5.0-12.0 min → 80 (%)

Flow rate : 0.8 mL/min

Oven temperature : 35 °C

Injection volume : 20 µL

MS interface : Electro Spray Ionization (ESI)

Nitrogen gas �ow : Nebulizing gas 3 L/min; Drying gas 15 L/min

MS temperature : Desolvation line 300 °C; Heating block 400 °C

Table 1. Optimized LC/MS/MS conditions for PGRs and antibiotic analysis

Figure 3. Method scouting software

Figure 4. Solvent blending

5


The preliminary data (Figures 5A to 5D) showed that the combination of 20 mM ammonium acetate containing 0.15 % formic acid and 0.1% formic acid in acetonitrile with HILIC column gives good peak shapes and separation. This mobile phase composition was further optimized with solvent blending software (as shown in Figure 4).It was observed that the formic acid concentration in aqueous phase regulates the peak shapes. Therefore, automated batch �le was created for optimization of formic acid concentration by using solvent blending

software and 20 mM ammonium acetate containing 0.05 % formic acid showed maximum response compared to all other combinations especially for streptomycin (shown in Figure 6).The LC/MS/MS method was developed using method scouting for simultaneous quantitation of PGRs and streptomycin. MRM transitions used for these compounds are given in Table 2. Linearity studies were carried out using external standard calibration method and results of linearity studies for both aqueous and matrix matched standards are tabulated in Table 2.

LC/MS/MS analysis results of PGRs and streptomycin

Results

Figure 5A. A=20 mM ammonium acetate 0.1% formic acid B= 0.15% formic acid in acetonitrile

Figure 5B. A= 0.1 % formic acid in water and B= methanol

Figure 5C. A= 10 mM ammonium formate and B= 0.1 % formic acid in methanol

Figure 5D. A=10 mM ammonium acetate and B= 0.1 % formic acid in methanol

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5(x10,000)

12:Ethephon TIC(-)(20.00)11:GA TIC(-)10:1-NA TIC(-)9:4-CPA TIC(-)8:2,4 D TIC(-)(20.00)7:6-BA TIC(+)6:CCC TIC(+)5:Diquat TIC(+)(20.00)4:Mepiquate TIC(+)3:Paraquat TIC(+)2:Azhadirectin TIC(+)1:Streptomycin 2 TIC(+)

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0 (x1,000,000)

12:Ethephon TIC(-)11:GA TIC(-)10:1-NA TIC(-)9:4-CPA TIC(-)8:2,4 D TIC(-)7:6-BA TIC(+)6:CCC TIC(+)5:Diquat TIC(+)(50.00)4:Mepiquate TIC(+)3:Paraquat TIC(+)(100.00)2:Azhadirectin TIC(+)1:Streptomycin 2 TIC(+)

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0(x100,000)

12:Ethephon TIC(-)(20.00)11:GA TIC(-)10:1-NA TIC(-)(50.00)9:4-CPA TIC(-)8:2,4 D TIC(-)7:6-BA TIC(+)(0.50)6:CCC TIC(+)5:Diquat TIC(+)4:Mepiquate TIC(+)3:Paraquat TIC(+)(50.00)2:Azhadirectin TIC(+)(50.00)1:Streptomycin 2 TIC(+)(100.00)

0.0 2.5 5.0 7.5 10.0 min

0.00

0.25

0.50

0.75

1.00

1.25(x10,000,000)

12:Ethephon TIC(-)(50.00)11:GA TIC(-)(50.00)9:4-CPA TIC(-)(100.00)8:2,4 D TIC(-)(100.00)7:6-BA TIC(+)6:CCC TIC(+)(0.50)5:Diquat TIC(+)(20.00)4:Mepiquate TIC(+)3:Paraquat TIC(+)2:Azhadirectin TIC(+)(100.00)1:Streptomycin 2 TIC(+)(100.00)

6


Figure 6. Optimization of formic acid concentration by solvent blending

The matrix matched calibration levels were prepared and injected in triplicate. The calibration curve of PGRs and streptomycin are shown in Figure 7 to Figure 15 and the correlation coef�cient of > 0.99 was observed for all the compounds.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

(x10,000)

1:Streptomycin 2 TIC(+) 1_PGR_HILIC-G_0.1%FA in 20mMAA_0.1%FA in ACN_5_95_005.lcd1:Streptomycin 2 TIC(+) 2_PGR_HILIC-G_0.2%FA in 20mMAA_0.1%FA in ACN_5_95_007.lcd1:Streptomycin 2 TIC(+) 3_PGR_HILIC-G_0.15%FA in 20mMAA_0.1%FA in ACN_5_95_009.lcd1:Streptomycin 2 TIC(+) 1_PGR_HILIC-G_0.05%FA in 20mMAA_0.1%FA in ACN_5_95_005.lcd

Figure 7. Streptomycin Figure 8. Paraquat Figure 9. Mepiquat

0 250 500 750 Conc.

0.0

1.0

2.0

3.0

Area (x100,000)

8 9 10

11

12

13

14

0 100 200 Conc.

0.0

0.5

1.0

1.5

2.0Area (x1,000,000)

5 6 7

8 9

10

11

0.0 1.0 2.0 3.0 Conc.

0.0

0.5

1.0

1.5Area (x10,000,000)

1 2

3

4

R2= 0.9947 R2= 0.9941 R2= 0.9982

7


0 250 500 750 Conc.

0.0

1.0

2.0

Area (x1,000)

9 10

11

12

13

14

Figure 10. Diquat Figure 11. Chlormequat Figure 12. 6 Benzyl adenine 0 250 Conc.

0.0

0.5

1.0

Area (x10,000,000)

5 6 7 8

9 10

11

12

0 250 500 Conc.

0.0

0.5

1.0

Area (x1,000,000)

5 6 7 8 9 10

11

12

13

0.0 25.0 50.0 Conc.

0.0

0.5

1.0

Area (x1,000,000)

1 2

3

4

5

0 250 500 750 Conc.

0.0

2.5

5.0

7.5

Area (x10,000)

7 8 9 10

11

12

13

14

Figure 13. Ethephon

0 250 500 750 Conc.

0.0

0.5

1.0

Area (x10,000)

10

11

12

13

14

Figure 14. Gibberellic acid Figure 15. 4 CPA

R2= 0.9927 R2= 0.9954 R2= 0.9942

R2= 0.9952 R2= 0.9911 R2= 0.9967

Table 2. Result table of aqueous and matrix matched calibration standards

Streptomycin

Paraquat

Diquat

6 Benzyl adenine

Mepiquat

Chlormequat

Gibberellic acid

Ethephon

4 CPA

Name of molecule

582.00>176.05

186.00>171.05

183.90>128.05

225.80>91.00

114.00>98.15

122.10>58.15

345.05>239.10

143.00>106.90

184.70>127.05

MRM transition

Positive

Positive

Positive

Positive

Positive

Positive

Negative

Negative

Negative

Polarity

50-1000

5-75

5-100

5-750

5-250

5-500

100-1000

50-1000

100-1000

Linearity rangein ppb

0.9947

0.9941

0.9927

0.9972

0.9982

0.9958

0.9992

0.9948

0.9977

Aqueousstandard

0.9983

0.9941

0.9927

0.9942

0.9954

0.9954

0.9911

0.9952

0.9967

Matrix matchedstandards

Sr.No.

1

2

3

4

5

6

7

8

9

Correlation coef�cient (r2)

© Shimadzu Corporation, 2016

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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.

First Edition: June, 2016

www.shimadzu.com/an/


Conclusion• The Nexera Method Scouting System enhances method development ef�ciency. • An automated solvent blending system can provide the same level of accuracy as manual premixing. • With the help of method scouting and solvent blending techniques, it was very easy to develop a quantitative

multi-residue analytical method for different class of compounds.

Disclaimer : The product and applications in this poster are intended for Research Use Only (RUO). Not for use in diagnostic procedures.

References[1] Sorensen MT, Danielsen V. International journal of Androl, (2006), 129-33.[2] Ana Coste, Laurian Vlase. Plant Cell Tiss Organ Cult 106, (2011), 279-288.[3] X. Esperza, E. Moyano,. Journal of Chromatography A, Volume 1216, (2009), 4402-4406.

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