DEVELOPMENT AND VALIDATION OF LC MS/MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF ΒETA SITOSTEROL, THYMOL AND OLEANOLIC ACID IN LEUCAS ASPERA LINN

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www.wjpr.net Vol 7, Issue 13, 2018. 896

DEVELOPMENT AND VALIDATION OF LC-MS/MS METHOD FOR

THE SIMULTANEOUS DETERMINATION OF ΒETA-SITOSTEROL,

THYMOL AND OLEANOLIC ACID IN

LEUCAS ASPERA

LINN

Prashant Hande1*, Ajitkumarbs1, Shailendra Rane2, Manish Hate2 and Jalil K. Shaikh3

1

Department of Gnird, Gurunanak Khalsa College, Matunga, Mumbai-400019, India.

2

Department of Chemistry, Ruia College, Matunga, Mumbai-400019, India.

3

Department of Ard, United State Pharmacopia, Hydrabad-500101, India.

ABSTRACT

A simple, precise and accurate LC-MS/MS method has been developed

for simultaneous determination of beta-sitosterol, thymol and oleanolic

acid in whole plant powder of Leucas aspera linn. MRM (Multiple

Reaction Monitoring) transitions 397.20>161.20, 151.10>109.10 and

455.40>455.55 were optimized on Shimadzu triple quadrupole mass

spectrometer instrument (Model: LCMS-8040) for quantification of

beta-sitosterol, thymol and oleanolic acid respectively.

Chromatographic method was developed on a Shimadzu shimpack-XR

C8 column (100mm x 2.0mm x 2µ) using gradient program. The

proposed method was validated for linearity, accuracy, precision, and

recovery, limit of detection (LOD) and limit of quantitation (LOQ). The validated

LC-MS/MS method can be used for a routine quality control analysis and simultaneous

quantitation of beta-sitosterol, thymol and oleanolic acid in Leucas aspera linn.

KEYWORDS: LC-MS/MS, Beta-sitosterol, Thymol, Oleanolic acid, Leucas aspera linn.,

Validation.

INTRODUCTION

Drugs used in medicine today are either obtained from nature or are of synthetic origin.

Natural drugs, obtained from plants and animals are called drugs of „biological origin‟ and

are produced in the living calls of plants or animals (Agrawal, and Paridhavi, 2007). Herbal

medicines are used for primary health care of humans with negligible side effects. Hence,

Volume 7, Issue 13, 896-904. Research Article ISSN 2277–7105

Article Received on 16 May 2018,

Revised on 05 June 2018, Accepted on 26 June 2018

DOI: 10.20959/wjpr201813-12813

*Corresponding Author Prashant Hande

Department of Gnird,

Gurunanak Khalsa College,

Matunga, Mumbai-400019,

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more usage and interest is due to promising activity and efficacy shown by many herbal

preparations in numerous clinical situations (Schuppan et.al., 1955). Although the traditional

remedies are advantageous, they have some limitations which include the lack of

standardization of raw materials and final products with non existence of criterion for quality

control. Now a day‟s new approaches for quality assessment cannot fulfill the requirements

of safety and efficacy of herbal medicines.(Chopra et.al., 1956; Ghosal et.al.,1973; Guha

et.al., 1996). Standardization of herbal preparations is based on phytochemical investigation

of one or more constituents as either active or marker compounds. Hence with the aid of

modern scientific methodology and techniques, the standardization of herbal constituents can

be carried out systematically and specifically which is the aim of this research work

(Agrawal, and Paridhavi, 2007). Recently the hyphenation of HPLC with tandem mass

spectrometry LC-MS/MS is widely used because of its better selectivity and sensitivity

(Montoro et.al., 2004; Sun et.al., 2005). Identification, separation and quantification of

bio-markers in complex samples can be performed in less time as well as reduced sample

preparation as compared to other analytical techniques. In this context, electrospray

ionizations (ESI) is soft ionization method and ESI-MS/MS has been shown to be a powerful

method for identification of variety of plants compounds(Rabaneda et.al,. 2003; Tolonen

et.al,. 2003).

Leucas aspera (wild) linn.(family-lamiceae) commonly called as „thumbai‟(Schuppan et.al,.

1955).It is found almost throughout India but majority in south part. In south, herbs are

available in the garden as well as near temples (Shome and Mehrotra, 1990). In leucas aspera

Linn., all parts of the plant possess useful therapeutic applications viz. anti-inflammatory,

antifungal, antioxidant, anti-insecticidal, and cytotoxic activities (Prajapati et.al,. 2010) and is

prescribed for the treatment of cobra venom poisoning(Mangathayaru et.al,. 2006). Many

reference reveal that the presence of triterpenoids in entire plants and nicotine contains arial

parts of leucas aspera Linn. (Kamat et.al,. 1994). whole plant reported to contain

beta-sitosterol(Jian et.al,. 1968; Mitra et.al,. 1992; Rai et.al, 2005), eugenol, citral, citronell,

linalool, thymol(Jian et.al,. 1968), oleanolic acid ursolic acid (Chaudhury et.al,. 1969).

Presence of alkaloids in leucas aspera linn. are more significant from the chemotaxonomic

point of view. Beta sitosterol, thymol and oleanolic acid are all belongs to the secondary

metabolites which are bio active phyto compounds(Ali et.al,. 2007). Beta-sitosterol, thymol

and oleanolic acid are reported to help in curing the cholesterol absorption,

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1992; Best et.al,. 1956), antimutagenic effect (Mezzoug et.al,. 2007), antitumor(Andersen,.

2006).

According to the literature survey no method has been developed for simultaneous estimation

of these three bio-markers by LC-MS/MS. Hence, the principle of the study was to develop a

simple, economic, rapid, precise standardized and validated method for simultaneous

quantitative estimation of beta-sitosterol, thymol and oleanolic acid from the herbs and herbal

formulation.

MATERIALS AND METHODS

Plant material and sample preparation

Whole plant material was collected from Kerala district and herbarium of leucas aspera linn.

was prepared and authenticated from MS university (Vadodara). The plants collected were

washed under running tap water. The plant kept for drying in oven at temperature 40±2⁰C.

The dried plant material was used for further studies. 250 mg of whole plant material of

leucas aspera linn. was extracted with 10 mL of methanol. The mixture was vortexed for 5

min and put for overnight extraction. Extract was filtered through 0.2micron syringe filter

and then subjected to LC-MS/MS analysis.

Chemicals and standard solutions Preparation

All the chemicals used in the experiments were of LCMS grade. Standard beta-sitosterol

(98.0% purity), standard thymol (98.0% purity) and standard oleanolic acid (98.0% purity)

were procured from sigma Aldrich chemie (steinheim Germany). The stock solutions of beta-

sitosterol, thymol and oleanolic acid (1mg/mL) were prepared separately in methanol. From

this individual stocks, mix working stock solution containing 10ug/mL of each standards

were prepared in methanol. This working stock solution was used for preparation of different

calibration standards.

Instrumentation and Chromatographic Conditions

Chromatographic development was performed on Shimadzu Nexera, UHPLC (Ultra High

Performance Liquid Chromatograph) system with LC-30AD pumps, SIL-30A autosampler

and CTO-20AC as column oven. LABsolutions software (version 5.80) was used for

operating the instrument. Shimadzu LCMS-8040 model (Triple Quadrupole Mass

Spectrometer) was used for optimization of MRM transitions for beta-sitosterol, thymol and

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Analysis was performed on shimadzu, shimapack-XR, C8 column (100mm x2.0mm, 2.2 μm).

The mobile phase comprising of A: 0.1% formic acid and B: methanol was filtered through a

0.2 μm membrane filter (Millipore) and degassed by sonication. Gradient method was

[image:4.595.151.442.313.455.2]

developed for chromatographic separation which is given in table1.

Table 1.

Time % Mobile phase A % Mobile phase B

0.00 55 65

2.00 20. 95

6.00 20 95

6.00 55 65

8.00 STOP STOP

RESULTS AND DISCUSSION

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 (x10,000)

3:OLEANOIC ACID 455.40>455.55(-) CE: 11.0 2:THYMOL 151.10>109.10(+) CE: -11.0 1:BETA-SITOSTEROL 397.20>161.20(+) CE: -21.0

O L E A NO IC A CI D T HY M O

L _BET

A -S IT O S T E RO L

Figure1. Overlay mass chromatograms of beta-sitosterol, thymol and oleanolic acid

standard mixture.

1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 (x1,000) 3:455.40>455.55(-)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

-0.25 0.00 0.25 0.50 0.75 1.00 1.25

1.50(x1,000)1:397.20>161.20(+)

B E T A -S IT O S T E RO L

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0(x100,000)_{1:397.20>161.20(+)}

B E T A -S IT O S T E RO L

0 250 500 750 Conc.

0.0 0.5 1.0 1.5 2.0 2.5 Area(x1,000,000)

1 2 3 4 5 6

7

8

9

[image:5.595.84.511.76.234.2]

Beta-sitosterol_1000 ng mL-1 Linearity_ (1-1000 ng mL-1)

Figure 2: Beta-sitosterol mass chromatograms and linearity graph.

1.0 2.0 3.0 4.0 5.0 6.0 7.0

1.0 1.5 2.0 2.5 3.0 3.5 (x1,000) 2:151.10>109.10(+)

1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 (x1,000) 2:151.10>109.10(+) T HY M O L

Thymol_blank run Thymol_1 ng mL-1

1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 (x100,000) 2:151.10>109.10(+) T HY M O L

0 250 500 750 Conc.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Area(x1,000,000)

2 3 4 5 6

7

8

9

Thymol_1000 ng mL-1 Linearity_ (1-1000 ng mL-1)

[image:5.595.80.517.270.639.2]

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1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 (x1,000) 3:455.40>455.55(-)

1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 (x1,000) 3:455.40>455.55(-) O L E A NO IC A CI D

Oleanolic acid blank run Oleanolic acid _1 ng mL-1

1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 (x1,000,000) 3:455.40>455.55(-) O L E A NO IC A CI D

0 250 500 750 Conc.

0.0 1.0 2.0 3.0 4.0 5.0 6.0Area(x1,000,000)

1 2 3 5 6

7

8

9

[image:6.595.74.519.75.413.2]

Oleanolic acid _1000 ng mL-1 Linearity_ (1-1000 ng mL-1)

Figure 4: Oleanolic acid mass chromatograms and linearity graph.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.00 0.25 0.50 0.75 1.00 1.25(x100,000)

3:OLEANOIC ACID 455.40>455.55(-) CE: 11.0 2:THYMOL 151.10>109.10(+) CE: -11.0

1:BETA-SITOSTEROL 397.20>161.20(+) CE: -21.0

O L E A NO IC A CI D B E T A -S IT O S T E RO L

Figure 5: Plant extracts chromatograms.

Method validation summary

Limit of Detection (LOD) and Limits of Quantitation (LOQ)

The signal-to-noise ratio of 3:1 and 10:1 was used to establish LOD and LOQ, respectively.

The LOD and LOQ of beta-sitosterol was 0.3 ng mL-1 and 1.0 ng mL-1, thymol was 1.0 ng

[image:6.595.135.453.446.610.2]

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www.wjpr.net Vol 7, Issue 13, 2018. 902 Linearity

A good linearity was achieved in the concentration ranges of 1 ng mL-1 to 1000 ng mL-1 for

beta-sitosterol, thymol and oleanolic acid. The regression equations and correlation

coefficient for the reference were y= 2672.97x-336.406, R2 = 0.9952 for beta-sitosterol and y

= 3497.87x+835.334, R2 = 0.9985 for thymol and y = 5146.21x+230.593, R2 = 0.9914 for

oleanolic acid.

The experiment was performed three times and the mean was used for the calculations. The

data was analyzed by linear regression least squares fitting. The statistical data obtained is

given in Table 2.

Table 2.

Parameters Beta-sitosterol Thymol Oleanolic acid

Linearity range[ng mL-1] 1ng mL-1-1000 ng mL-1

Slope [m]1 2672.97 3497.87 5146.21

Intercept [c]1 -336.406 835.334 230.593

Correlation Coefficient [R2] 0.995 0.998 0.991

LOD [ng mL-1] 0.3 1 0.1

LOQ [ng mL-1] 1 3 0.3

Intraday precision(n=5 COV) 0.81 0.77 0.63

Interday precision (n=5 COV) 0.48 0.59 0.75

1

of the equation y = mx + c, where y is peak area, m is the slope, x is the concentration, and c

is the intercept.

Recovery

Three replicates at 50 ng mL-1, 100 ng mL-1, and 200 ng mL-1 concentration for the

beta-sitosterol, thymol and oleanolic acid were prepared for recovery determination. The mean

recovery for beta-sitosterol, thymol and oleanolic acid were 70%, 101% and 89%

respectively.

Assay

The developed HPLC method was used for simultaneous determination of beta-sitosterol,

thymol and oleanolic acid from whole plant powder of leucas aspera linn. The sample

working solution (10 μL) was injected and the area of both beta-sitosterol, thymol and

oleanolic acid peak was measured. From the calibration curve, the amount of beta-sitosterol,

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time of beta- sitosterol, thymol and oleanolic acid in sample solution was 5.28, 2.02 and

3.383 respectively.

The mean assay value of beta- sitosterol was found to be 1.580 ug per 250 mg of plant

powder with % RSD as 1.136 and mean assay value of oleanolic acid was found to be 0.427

ug per 250 mg of plant powder with % RSD as 1.458. Thymol was found to be below

detection level in plant extract.

Precision and Accuracy

The intra-day and inter-day precision was used to study the variability of the method. The %

RSD for intra-day and inter-day precision for beta-sitosterol were 0.81 and 0.48%,

respectively, Thymol were 0.77 and 0.59% respectively and oleanolic acid were 0.63 and

0.75% respectively.

CONCLUSION

The application of a simple, rapid and accurate LC-MS/MS method for the simultaneous

quantitation of beta- sitosterol, thymol and oleanolic acid in whole plant powder of Leucas

aspera linn. The method was validated to track the active principles in the complex mixture

of herbal ingredients. The method could be extended for the marker-based standardization of

other herbal product containing beta- sitosterol, thymol and oleanolic acid.

The method was found to be simple, precise, accurate, specific and sensitive and can be used

for routine quality control of herbal raw materials and for the quantification of these

compounds in plant materials.

ACHKNOWLEGMENT: Special thanks to Shimadzu Analytical (India) Pvt. Ltd. for

providing their facility to complete the work.

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