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Standardization of Phytopharmaceuticals: Qualitative Evaluation and Quantification of Carbohydrates in Medicinal Plants using TLC, Matrix Free MELDI-TOF-MS and GC-MS.

Byline: Muhammad Nasimullah Qureshi, Guenther Stecher and Guenther Karl Bonn

: Summary: Pharmaceutical industry requires reliable and less time consuming procedures for the quality control analysis of raw materials and finished products. The study was focused on thin layer chromatography (TLC), gas chromatography (GC) and matrix free material enhanced laser desorption ionization time of flight mass spectrometry (mf-MELDI-MS). Standards and microwave assisted water extracts from Achillea milleffolium (herb), Althaea officinalis (roots), Equisetum arvense (herb), Juglans regia (leaves), Matricaria chamomella (flowers) and Taraxacum officinale (herb) were the samples used for this study.

The presence of mono- (fructose and glucose) and disaccharide (sucrose) in all the plants under study was proved through the TLC analysis. These results were confirmed by the Mf-MELDI-MS experiments. Gas chromatography coupled to mass spectrometry (GC-MS) confirmed the data obtained via TLC and mf-MELDI-MS, delivering additionally quantified values, but needed a long time. A carbohydrate of higher oligomerization degree could not be found. Among the six plants, glucose was found in highest concentration in Taraxacum officinale. Fructose is present in appreciable quantity in Matricaria chamomella while Althaea officinalis has the highest amount of sucrose among the plants under study.

Key words: Herbal extracts, Carbohydrates, Thin layer chromatography, mf-MELDI-MS, GC-MS.

Introduction

Standardization of phytopharmaceuticals makes it important to evaluate the chemical profile including the analysis of primary and secondary metabolites [1-7]. Accurate identification and quantification of carbohydrates in plants being the third bio-informative macromolecule [8] is important in research and development as well for the determination of the quality of food, fruits and for process control during the production [1, 4]. They are important for controlling immunological recognition, in defense against pathogens, protein folding and placement [8].

A number of methods for the determination of sugars in plants have been published. These analytical approaches can be classified into two main groups. These are: procedures for the analysis of analytes without separation like enzymatic, reduction, and electrochemical methods, polarimetry; and separation methods such as chromatography and electrophoresis [4, 9]. Among the chromatographic methods gas chromatography is the mostly used method for these determinations. The determination of carbohydrates through matrix free material

enhanced laser desorption ionization mass spectrometry (mf-MELDI-MS) is a relatively new approach [4, 10-12]. Benefits of the method generally are: less time consuming sample preparation, ease of handling, and fast analysis. The study focuses on the qualitative and quantitative analysis of carbohydrates in different plant extracts employing thin layer chromatography (TLC), gas chromatography (GC) and mf-MELDI-MS.

TLC is a low cost, simple and the most often used analytical method for the qualitative or semi- quantitative assessment of carbohydrates from plants. A number of samples can be analyzed simultaneously. Mf-MELDI-TOF-MS was used for the qualitative analysis of carbohydrates in plants. It does not need any derivatization before analysis and analysis can be performed within a short period of time. Gas chromatography (GC) coupled to MS detection was chosen for quantification of the carbohydrates in plant extracts as it is the most widely used and is the most sensitive technique through which carbohydrates below concentration of nano moles, can be identified. Furthermore,

identification of carbohydrates can be performed not only on the basis of retention time but the produced fragmentation pattern can be compared with the standard spectra from the spectral library [13]. Prior to GC-MS analysis, carbohydrates have to be converted into volatile and stable derivatives, i.e. acetate or trimethylsilyl (TMS) derivatives [4, 14-26]. Generally, silylation is the most common method for the derivatization of organic compounds containing active hydrogen atoms. This produces less polar, high volatile, thermally and catalytically stabile derivatives [27]. BSTFA was used for the production of silylated analytes without oxime preparation [4].

Results and Discussion

TLC, mf-MELDI-MS have been used for the qualitative investigations while quantification of carbohydrates in medicinal plant extracts were performed using GC hyphenated with MS.

Thin Layer Chromatography of Carbohydrates (TLC)

TLCs have been developed for the identification of mono- and oligosaccharides in plants under study. Their presence was confirmed in comparison to standards developed parallel to plant extracts. Fructose, glucose, sucrose were detected in plant extracts. Table-1 show results obtained from TLC analysis.

Matrix free Material Enhanced Laser Desorption Ionization Mass Spectrometric Analysis of Carbohydrates (mf-MELDI-MS)

Fig. 1 shows the mf-MELDI mass spectra of all the six plant extracts under study. A number of signals are produced in the mass range of m/z 20 to750. Labeled signals represent carbohydrates present in the plant extracts. Carbohydrates produce sodium and potassium adducts signals in MELDI-MS [4, 10]. Monosaccharides (glucose and fructose) and disaccharide (sucrose) were detected in all the extracts. Besides carbohydrates, several unknown signals marked with asterisks were detected which

show the presence of other metabolites but their nature is not clear.

Gas ChromatographyMass Spectrometry ofCarbohydrates (GC-MS)

Quantification of sugars was performed using GC coupled to MS. Derivatization of the target analytes were performed according to the method optimized by M. N. Qureshi et al 2011 [4]. Qualitative results from TLC, MELDI and GC-MS analyses identified monosaccharide (fructose and glucose) and disaccharide (sucrose) in plants. Calibration curves for glucose, fructose and sucrose were obtained from the data produced by analyzing standard solutions of various concentrations through GC-MS. Concentrations of the working standard solutions, regression equations and the R2 valuesobtained for the standards are as below

Fructose: conc. (0.5 mg/ml 4 mg/ml)y 0.0461x + 0.0092 R2 0.9915

Glucose: conc.(0.5 mg/ml 5 mg/ml)y 0.104x + 0.0322 R2 0.9905

Sucrose: conc.(0.5 mg/ml 4 mg/ml)y 0.1936x - 0.0332 R2 0.9932

Using these equations, quantification of the three carbohydrates fructose, glucose and sucrose were performed in six plants. GC chromatograms obtained from these analyses have been shown in Fig.2.

Table-2 shows quantification results of carbohydrates in six plants. Different carbohydrates are present in different plants in different concentration. Among the six plants Taraxacum officinale (herb) has the highest amount of glucose. Fructose is present in appreciable quantity in Matricaria chamomella (flowers) while Althaea officinalis (roots) has the highest amount of sucrose among the plants under study.

Table-1: Results from TLC analysis of plant extracts.

Standards###Achillea###Althaea###Equisetum###Juglans###Matricaria###Taraxacum

###Fructose###+++###+++###+++###+++###+++###+++

###Glucose###+++###+++###+++###+++###+++###+++

###Sucrose###+++###+++###+++###+++###+++###+++

Experimental

Chemicals and Reagents

N,O-bis-(trimethylsilyl)trifluoroacet- amide (BSTFA), phenyl-AY-D-glucopyranoside (98%), imidazole, dichloromethane, glucose (99.5%), fructose ( 99%), sucrose (99%) were purchased from Fluka (Sigma-Aldrich GmbH, Germany). Acetic acid, acetone, aniline, n-butanol, diphenylamine, ethanol, phosphoric acid, pyridine and silica gel TLC plates 20 cm A- 20 cm were obtained from Merck AG (Darmstadt, Germany).

Nitrogen and helium gas were purchased from Messer Austria GmbH. All these chemicals and reagents were of analytical grade and used without further purification. Water purified by a Nano Pure-unit (Branstead, Boston, MA, USA) was used. Plant materials were provided by Bionorica Phytoneering Company Germany.

Experimental procedures, used for the extraction, TLC, mf-MELDI-MS and GC-MS analyses, are similar to those employed by M. N. Qureshi et al 2011 [4].

Table-2: Quantification of carbohydrates in different plant extracts through GC-MS.

Plant Name###Fructose (mg/g)###Glucose (mg/g)###Sucrose (mg/g)

###Achillea###56.143###27.537###33.696

###Althaea###3.862###0.000###290.912

Equisetum###108.985###46.630###66.566

###Juglans###113.838###68.590###45.353

Matricaria###134.045###68.842###41.857

Extraction

High-performance microwave digestion system (MLS 1200 MEGA, Leutkirch, Germany) was employed for the extraction. Powdered plant material (1 g) was extracted in double distilled water (10 ml) for 200 seconds at 150 Watts. Extracts were

centrifuged at 14.6A-1000 g (Centrifuge 5415D, Eppendorf AG Hamburg, Germany).

Thin Layer Chromatography (TLC)

Standard carbohydrate solutions (1.0 mg/ml) and plant extracts were applied on the pre-activated (at 100 C for 30 min) silica plate (1-2 L) through an automated sample applicator (ATS 4, Camag, Berlin, Germany).

TLC was developed using mobile phase n- butanol:acetic acid:H2O (8:3:2) and was dried at 60C. Visualization of the bands was achieved bydrying the TLC plate at 120 C for 1 hour after reacting the plate with a solution consisting of 5 ml of acetone, 0.1 ml of aniline, 0.1 g of diphenylamine and 0.75 ml of phosphoric acid (85%). Identification was achieved by comparing the Rf values and colour of the extracts with bands resulting from the carbohydrates standards.

Matrix Free Material Enhanced Laser DesorptionIonization Mass Spectrometry (mf-MELDI-MS) Sample Preparation for MELDI-MS Analysis10 mg of synthesized MELDI material [10] was suspended in methanol (1 ml) of and sonicated for 3 min. A thin layer of the suspension was made on a stainless steel target and dried at room temperature. Extract (0.5-1 L) was placed on the layer and dried with a blow of nitrogen gas.

Instrumentation

Analysis was performed using MALDI mass spectrometer (Ultraflex MALDI TOF/TOF, Bruker Daltonics, Bremen, Germany) in the positive ionization mode. Desorption was obtained by using a Nitrogen laser (337 nm) was used for the desorption of analytes and laser energy was adjusted as per requirement. 20 and 18.6 kV voltage was impressed on the ion source 1 and 2 respectively. 1601 V was used for the detection and for parameter control during recording Flex Control (version 2.0, Bruker Daltonics) was used. Data was evaluated using Flex Analysis (version 2.0, Bruker Daltonics). 100-500 laser shots were used to obtain each spectrum.

Gas ChromatographyMass Spectrometry (GC-MS) Derivatization100 L standard solution (1 mg/ml) / sample were combined with 50 L internal standard solution

(2.5 g/100 ml phenyl-AY-D-glucopyranoside in 50% EtOH) and 150 L imidazole buffer (pH 7). The mixture was thoroughly mixed and dried. 0.5 ml of a mixture consisting of N,O-bis(trimethylsilyl)trifluoro acetamide (BSTFA) and pyridine (2:8) were added to the dried sample and mixed at 1400 rpm for 7 min at30 C. Silylation was performed under microwave radiation for 4 min at 180 Watts. After centrifugation1 L of derivatized sample was injected into the GC-MS for analysis.

Chromatography of TMS Derivatives

A GC-MS from Agilent Technologies equipped with an auto-sampler (CTC Analytics, Switzerland) was used. A constant flow rate of 1 ml/min of carrier gas (Helium) was used. Separation of the analytes was performed on a DB-1ms capillary column (60 m A- 250 m A- 0.250 m). Injector was operated in a splitless mode at 60 C. The temperature at the injection was maintained at 60 C for two min. The temperature was raised to 310 C in1.44 min and held at 310 C for 5 min. The columntemperature program started at 60 C for one min and rose to 200 C in 5.60 min. Then temperature was elevated to 220 C in 10 min, to 280 C in further 15 min and to 285 C in 5 min. The temperature was hold at 285 C for 30 min and then was brought to310 C in 2.50 min. It was kept at this temperaturefor 10 min. Start conditions were achieved within2.50 min. Total elution time was 83.60 min. MSscanning was performed from m/z 40 to 800.

Acknowledgement

Higher Education Commission (HEC) of Pakistan is acknowledged for providing scholarship during the course of work.

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Title Annotation:thin layer chromatography; matrix free material enhanced laser desorption ionization time of flight mass spectrometry; and gas chromatography-mass spectrometry
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Apr 30, 2014
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