Applied Clinical Pharmacology and Toxicology (ISSN: 2577-0225)

Article / research article

"DLLME Extraction of Amino Acids from Blood Sample and its Profiling with Special References to Essential and Non-Essential Amino Acid by Thin Layer Chromatography."

S Sudhaker1*, RK Jain2, Mayurakshi Ray3, Sangita Saha3, Sohini Bose4

1Department of Toxicology, CFSL, Kolkata, India

2Department of Toxicology, CFSL, Guwahati, India

3Department of Biochemistry, West Bengal State University, Barasat, Kolkata, India

4Department of Forensic Science, Amity University, Noida, Delhi, India

*Corresponding author: S. Sudhaker, Department of Toxicology, CFSL, Kolkata, India. Tel: +91 3322841638; Fax: +91 3322849442; E-mail: smahatisudha@gmail.com

Received date: 11 April, 2017; Accepted date: 8 May, 2017; Published date: 9 May, 2017

Micro extraction technique, Dispersive Liquid-Liquid Micro Extraction (DLLME) is used for extracting amino acids from blood sample. Solubility of amino acids are checked in various solvent and tertiary mixture of mobile phase (1butanol: acetic acid: water) in ratio 8:2:2 is optimized and used for separating amino acid. Distinct different resolved Rf values were recorded for both blood samples and standard amino acids. By comparing the Rf values of the standard amino acids, phenyl alanine, valine and Aspartic acid were confirmed to be present in the blood samples. DLLME is a simple, rapid; environment friendly method is coupled with Thin Layer Chromatography (TLC). TLC is a simple chromatographic technique for separation, when combined with DLLME, the recovery and reproducibility of the method is found to be increased. Ninhydrin spray is used for chemically visualizing the amino acids. In this study better extraction efficiency that is 95% is achieved in comparison to conventional liquid-liquid micro extraction.

Keywords: Dispersive Liquid-Liquid Micro Extraction (DLLME); Thin Layer Chromatography (TLC); Non-Essential Amino Acid; Blood Sample;  Rf Values

Introduction

Amino acid is the building block of protein, which are organic compound containing amine and carboxyl functional group along with a side chain for specific each amino acid. There are 20 amino acid classified into two groups based on their requirement, among twenty-nine amino acids are essential amino acids which cannot be synthesized in the body and comes through food and nutritional supplement in few cases. There are eleven amino acids are non-essential which are synthesized in the body of an individual. When a person consumes any toxic or poisonous substances there occurs a change in metabolism of amino acids. Sometime it is observed that metabolism of amino acid come to a halt and many proteins are found to be denatured. Essential amino acids are used in various therapeutic purposes as nutritional supplement, when consumed in excess leads to poisoning. An attempt is made to identify the change in metabolism of amino acid and for profiling purpose; blood sample of those individuals and control samples were collected and analyzed. A simple thin layer Chromatography has been performed coupled with micro extraction technique DLLME. An identification factor in TLC is Retardation Factor or Rf Value. Rf value is defined as distance moved by the substance from origin divided by distance moved by the solvent from origin.

  1. In TLC the separation depends on several factor
  2. Solubility: The more soluble a compound the faster it will move up the plate.
  3. Attraction between the compound and silica: The more the compound interacts with silica the lesser it moves.
  4. Size of the compound: Larger the compound slower it moves it up to plate. The solubility of amino acid was checked and optimized mobile phase in TLC for better separation of amino acids. This method being a simple, rapid and inexpensive can be used in toxicology laboratory with minimum infrastructure.

Out of many techniques available for micro extraction, Dispersive Liquid-Liquid Micro Extraction has attracted the interest of forensic toxicology in recent year. It is the micro extraction procedure, used for extraction of amino acids from the post mortem blood samples. Blood samples are subjected for amino acids profiling by TLC. DLLME is simple, fast, inexpensive solvent micro extraction technique when compared with other methods and it consume a less amount of organic solvent. This DLMME extraction method is coupled with TLC is very much useful for rapid screening; the method is less time consuming and fast and thereby minimizing experiment time.

Methods and Materials

All the reagents were of analytical grade and standard reference material were procured from sigma Aldrich pvt ltd. Silica gel 60F-254 pre-coated TLC aluminum plates (20×20 cm, 0.25mm layer thickness) were procured from Merck, Germany. Acetonitrile, acetone, methanol, butanol, acetic acid, Millipore water were used in the extraction and thin layer chromatography method as a mobile phase was procured from Merck and qualigens. The standard solutions of each amino acid were prepared in Millipore water in 1ppm to 10 ppm range for LOD and LOQ measurement. Mini centrifuge (Tommy) is used for centrifugation. Cyclomixer (Remi) is used for mixing the sample.

Extraction of Blood Sample

A new simple micro extraction technique was developed for determination of amino acid from the blood samples and control samples. The amino acids were extracted by DLLME based on separation of blood plasma and deposition of the cell extracts.

Procedure

A mixture of 1ml blood sample, 1ml of methanol is added and shaken well. Then 1ml of Acetonitrile is added to the tube.

Then the blood sample was centrifuged at 3000 rpm for 5 to 6 minutes. After centrifugation pellets that is the cell extracts are found to be settle at the bottom, and the supernatant is found floating at the top of the reaction mixture. After that supernatant is separated and air dried and reconstituted with methanol and spotted on the TLC plates along with the amino acid standard.

Thin Layer Chromatography (TLC) Procedure

We used Aluminum support pre-coated silica coated TLC plates 20*20 cm. To activate the silica, plates are heated at 1100c on the TLC activator (camag) for 30 minutes. Then the chamber is washed and rinsed with the newly prepared solvent that is (1 butanol, acetic acid, dist, water) in the ratio of 8:2:2 that is 4:1:1. Then the chamber is saturation with mobile phase and it is set to equilibrate with mobile phase for 30 minutes. Then on a TLC plates, 5µl of the extracted samples and standard amino acids were loaded equidistantly using capillary tubes and the plate is then kept inside the chamber and allow running for 10 cm. And when the solvent front reaches 10cm the plates are taken out and allowed to air dry at room temperature. Then the newly prepared .2% ninhydrin spray reagent is sprayed on the TLC plates in the spraying chamber TLC (Fume wood), Camag and the wet plate is air dried in a room temperature and slightly warmed in a TLC Activator, Camag and the spots appears on the plate and Rf values were recorded .

Results and Discussion

The Rf values of the amino acid standards were measured and the extracted samples amino acids Rf were also measured. The amino acids phenyl alanine, Valine and Aspartic acid were found to be more clear in the samples extracted blood samples and matches with the standard amino acid by their unique Rf value confirms the presence in the blood. This method is very rapid and sensitive and the LOD and LOQ were found to be 1-5mg and 5-10mg respectively was tabled in (Table 3).

Other amino acids were not detected because those amino acids were extracted in the form of different metabolites. According to the Rf values mentioned in the (Table 1), Rf value of phenyl alanine .63 is found to be as that of the spot 6 of sample 1 mentioned in (Table 2). In the same way the RF values of the valine and aspartic acid are .53 and .21 respectively are found to be same as that of the fourth spot of the sample 1, second spot of the same sample mentioned in (Table 4).

Table 4: The solubility of amino acids were checked in various solvents and the mobile phase that is the tertiary mixture of solvent (1 butanol: acetic acid: water) in the ratio of 8:2:2 were tried and optimized for separation of amino acids by the thin layer chromatography coupled with DLLME. DLLME is a very advanced process compared to the liquid-liquid extraction because DLLME is an environment friendly, less time taking and uses very little amount of solvent. The known amounts of amino acids were spotted with known loading capacity and 98% repeatability and 95% recovery were achieved in this method. The excessive intake of nine essential amino acids provided through nutritional supplements or diet leads to poisoning only two essential amino acid such as phenyl alanine and valine and non-essential amino acid, Aspartic acid could be extracted from the blood sample from the above mentioned procedure. In this study control blood samples from donors who are healthy human beings were also collected and subjected for extraction and identification. This method can be adopted in any forensic science laboratory for extraction and identification of amino acids.

Acknowledgements

The authors are gratefull to the Director, CFSL, Kolkata and the chief forensic scientist for providing all facilities to carryout the work.

 

Serial No.

 

Name of the amino acids

 

  1.  

 

  1.  
  1.  
  •  
  1.  
  1.  
  1.  

Aspartic acid

  1.  
  1.  
 

 

Table 3: LOD and LOQ value.

 

 

Name of Amino Acid

 

Classification

 

Spraying reagent

 

Rf value

Alanine

Non- essential

Ninhydrin

.33

Aspartic acid

Non-essential

Ninhydrin

.21

Glutamic acid

Non-essential

Ninhydrin

.32

Ornithine Mono hydrochloride

Non-essential

Ninhydrin

.10

Serine

Non-essential

Ninhydrin

.23

Glycine

Non-essential

Ninhydrin

.20

Proline

Non-essential

Ninhydrin

.30

D-L nor leucine

Proteinogenic

Ninhydrin

.65

D-L amino butyric acid

-

Ninhydrin

.42

Arginine

Non-essential

Ninhydrin

.15

Threonine

Essential

Ninhydrin

.27

Tryptophan

Essential

Ninhydrin

.70

Valine

Essential

Ninhydrin

.53

Lysine

Essential

Ninhydrin

.08

Histidine

Essential

Ninhydrin

.11

Isoleucine

Essential

Ninhydrin

.65

Leucine

Essential

Ninhydrin

.10

Methionine

Essential

Ninhydrin

.55

Phenyl alanine

Essential

Ninhydrin

.63

 

 

Table 1: Rf values of standard amino acid.

 

 

  •  

 

Distance travel by solvent

 

  1. value

Spot1

6.7 cm

.04

Spot2

6.7cm

.15

Spot3

6.7cm

.23

Spot4

6.7cm

.32

Spot5

6.7cm

.50

Spot6

6.7cm

.59

  •  

 

 

Spot1

6.7 cm

.04

Spot2

6.7 cm

.16

Spot3

6.7 cm

.25

Spot4

6.7 cm

.33

Spot5

6.7 cm

.49

Spot

6.7 cm

.59

  •  

Distance travel by solvent

  1. value

Spot1

7.5cm

.08

Spot2

7.5cm

.22

Spot3

7.5cm

.29

Spot4

7.5cm

.37

Spot5

7.5cm

.56

Spot6

7.5cm

.63

  •  

 

 

Spot1

7.5cm

.07

Spot2

7.5cm

.22

Spot3

7.5cm

.30

Spot4

7.5cm

.37

Spot5

7.5cm

.57

Spot6

7.5cm

.66

 

 

Table 2: Rf value of unknown blood sample.

 

 

  •  

 

Distance travel by solvent

 

  1. value

Spot1

6cm

.08

Spot2

6cm

.21

Spot3

6cm

.26

Spot3

6cm

.35

Spot4

6cm

.53

Spot5

6cm

.65

  •  

 

 

Spot1

6cm

.08

Spot2

6cm

.16

Spot3

6cm

.25

Spot4

6cm

.33

Spot5

6cm

.51

Spot6

6cm

.65

 

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Citation: Sudhaker S, Jain RK, Ray M, Saha S, Bose S (2017) DLLME Extraction of Amino Acids from Blood Sample and its Profiling with Special References to Essential and Non-Essential Amino Acid by Thin Layer Chromatography. Appl Clin Pharmacol Toxicol 2017: ACPT-103.

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