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AMINO ACIDS

Amino Acids in Plasma/Urine LC-MS/MS Analysis Kit

In recent years metabolomics has facilitated gaining insight into discovering the potential disease diagnostic markers and observing fluctuations of certain metabolite levels in body fluids. Disease-related metabolites can be uncovered by following progress of the new methodologies which enable to identify a broad range of diagnostic biomarker candidates including amino acids. Owing to the vital roles of amino acids in metabolic regulations (protein synthesis, neurotransmission, hormone secretion, and immunity), they have become significant targets while taking snapshot of metabolic status. Therefore, elucidation of the circulating amino acids profile alterations by utilizing robust analysis method could shed light on not only in metabolic diseases but also in certain disorders such as autism, prostate cancer, Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, obesity, type 2 diabetes, liver cirrhosis, major depressive disorder, and cardiovascular disease.

Highlights

Quantitative analysis of underivatized 43 amino acids in 7.5 min. In need of determining allo-Isoleucine for MSUD, extra injection takes 11.0 min. (Leucine/Isoleucine/allo-Isoleucine).

One sample preparation for all amino acids-one vial for both abovementioned methods.

Just a few pipetting steps for the sample preparation; for plasma/serum-protein crash and shoot for urine-dilute and shoot.

Clinically important amino acids safeguarded by its own internal standard.

Consuming small volume of patient’s sample.

Long life span of HPLC column-up to 10000 injections.

Parameters

Alanine, Arginine, Aspartic acid, Asparagine, 2-Aminobutyric acid, Argininosuccinic acid, Anserine, 2-Aminoadipic acid, 3-Aminoisobutyric acid, β-Alanine, Ethanolamine, Phenylalanine, ortho-Phosphoserine, ortho-Phosphorylethanolamine, γ-Aminobutyric acid, Glutamic acid, Glycine, Glutamine, trans-4-Hydroxyproline, Homocitrulline, Homocystine, Histidine, 5-Hydroxylysine, Isoleucine, Carnosine, Lysine, Leucine, Allo-Isoleucine, Methionine, 3-Methylhistidine, 1- Methylhistidine, Norvaline, Ornithine, Proline, Cystine, Cystathionine, Sarcosine, Citrulline, Serine, Taurine, Tryptophan, Tyrosine, Threonine and Valine.

Matrix

Plasma/Urine

Sample Preparation

Plasma/Serum
  • Step 1
    Pipette 50 µL of serum/plasma patient’s sample (calibrator/control) into a glass centrifuge tube.
  • Step 2
    Add 50 µL of internal standard solution and vortex for 5 sec.
  • Step 3
    Add 700 µL of Reagent-1, vortex for 5 sec. then, centrifuge at 4000 rpm for 5 min.
  • Step 4
    Decant the clear supernatant into HPLC vial prior to injection.

Sample Preparation

Urine
  • Step 1
    Pipette 100 µL of urine sample into a glass centrifuge tube and dilute with 900 µL of HPLC-grade water.
  • Step 2
    Pipette 50 µL of diluted urine sample into a glass centrifuge tube.
  • Step 3
    Add 50 µL of internal standard solution and vortex for 5 sec.
  • Step 4
    Add 700 µL of Reagent-1, vortex for 5 sec. then, centrifuge at 4000 rpm for 5 min.
  • Step 5
    Decant the clear supernatant into HPLC vial prior to injection.

Chromatogram

METHOD PERFORMANCE

Analyte

LOQ (µmol/L) 6460 QQQ

Accuracy % 6460 QQQ

Repeatabilty % CV 6460  QQQ

Linearity R2 646 QQQ

1-Methylhistidine

0,2

85,3

2,94

0,9996

2-Aminoadipic acid

0,83

93,67

2,37

0,9992

2-Aminobutyric acid

0,22

93,67

4,12

0,9998

3-Aminoisobutyric acid

0,15

98,93

7,55

0,9995

3-Methylhistidine

0,3

99,87

4,19

0,9992

4-Aminobutyric acid

0,07

102,62

5,41

0,9994

5-Hydroxylysine

0,3

102,78

1,54

0,9988

Alanine

6,82

105,04

4,19

0,9988

Allo-isoleucine

0,22

98,3

1,65

0,9998

Anserine

0,21

106,86

4,13

0,9998

Arginine

0,15

103,88

3,98

0,9998

Argininosuccinic acid

0,62

93,04

2,97

0,9981

Asparagine

0,46

86,17

3,09

0,9997

Aspartic acid

1,06

91,19

3,44

0,9956

Beta-Alanine

1,0

91,84

9,11

0,999

Carnosine

0,12

96,08

3,97

0,9984

Citrulline

0,51

82,47

3,44

0,9977

Cystathionine

0,25

104,65

4,75

0,9997

Cystine

0,97

95,82

4,63

0,9998

Ethanolamine

0,56

104,25

5,67

0,9944

Glutamic acid

0,24

97,88

6,1

0,999

Glutamine

0,32

98,03

3,37

0,9999

Glycine

9,03

95,82

1,93

0,9984

Histidine

2,43

107

1,12

0,9999

Homocitrulline

0,89

104,4

3,71

0,9993

Homocystine

0,31

102,82

4,8

0,999

Isoleucine

0,22

110,92

1,39

0,9991

Leucine

0,2

92,22

4,85

0,9997

Lysine

0,09

113,46

5,12

0,9988

Methionine

0,41

87,42

1,88

0,9999

Norvaline

0,15

93,51

4,94

0,9988

Ornithine

1,39

93,34

3,54

0,9996

O-Phosphorylethanolamine

2,56

87

4,28

0,9976

O-Phosphoserine

0,12

83,96

4,49

0,9969

Phenylalanine

0,73

108,04

3,05

0,997

Proline

0,67

99,84

3,3

0,9998

Sarcosine

4,38

110,03

2,25

0,9964

Serine

0,89

99,26

4,58

0,997

Taurine

5,47

85,09

5,28

0,9956

Threonine

0,72

95,03

3,27

0,9999

trans-4-hydroxyproline

0,28

87,8

1,9

0,9914

Tryptophan

0,23

109,81

3,21

0,9998

Tyrosine

3,34

101,19

3,45

0,9991

Valine

5,11

98,65

6,08

0,9986
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