Amino acid analysis: comparison and validation of gas chromatography with mass spectrometric detection and liquid chromatography with triple quadrupole mass spectrometric detection. Application in mouse embryonic fibrob

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MSc in Analytical Sciences

Amino acid analysis: comparison and validation of gas

chromatography with mass spectrometric detection and

liquid chromatography with triple quadrupole mass

spectrometric detection. Application in mouse

embryonic fibroblast NIH-3T3 cells.

Master Research Thesis

Alexandra Koukou

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MSc Chemistry

Analytical Sciences

Master Thesis

Amino acid analysis: comparison and validation of gas

chromatography with mass spectrometric detection and

liquid chromatography with triple quadrupole mass

spectrometric detection. Application in mouse embryonic

fibroblast NIH-3T3 cells.

by

Alexandra Koukou

June 2014

Supervisor:

dr H. Lingeman

Daily Supervisor:

P.

Krumpochova

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Abstract

The comprehension of metabolic profiling plays an important role in enhancement of knowledge about prognosis and diagnosis of disease. Amino acids are one of the mainly building blocks of cells among carbohydrates, lipids and nucleic acids. Therefore, the fluctuating blood concentration of amino acids may provide mechanistic insights into diseases. This thesis describes two methods for the determination of amino acids in standard solutions. One with gas chromatography (GC) coupled with mass spectrometric detection (MS) and one with liquid chromatography (LC) coupled with triple quadrupole (QQQ) mass spectrometric detection (MS). In both methods the sample pretreatment and derivatization steps were performed by Phenomenex EZ faast amino acid analysis kit for gas and liquid chromatographic analysis respectively and last 7 minutes per sample.

For the GC-MS method three different internal standards were tested; Norvaline, amino acids labeled with 13C and amino acids labeled with 13C/15N. The detection limit was ranging between 0.1 and 2.4 μM in the selected ion monitoring mode. The analysis time was approximately 7 minutes per sample.

For the LC-MS method no internal standard was used. The detection limit was 0.25 μM in the multiple reaction monitoring mode. The analysis time was approximately 25 minutes per sample.

Finally, the GC-MS method with amino acids labelled with 13C/15N was applied on mouse embryonic fibroblast NIH-3T3 cells in order to study the cancer mechanism.

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Acknowledgements

First and foremost, I would like to thank Petra Krumpochova, my daily supervisor, for made me feel so welcome from the first moment, for believing in me and for all the time she invested in my project.

Secondly, I want to thank Ben Bruyneel for being always next to me when I needed his help, answering the questions that I had.

Another important contribution in the attainment of my project was my cooperation with Azra Mujic-Delic, who providing me with biological samples.

Furthermore, I would like to thank Dr. Henk Lingeman for being my supervisor and for providing me with lot of useful ideas and feedback. I am also grateful about Prof. Wilfried Niessen and Prof. Manfred Wuhrer, who contributed with their own way in this project being always willing to share their knowledge.

Last but not least, big thanks to everyone in the group of Bioanalytical Chemistry of VU University. You listened to me and you advised me about both relevant and irrelevant subjects concerning my project.

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Abbreviations

3T3 : 3 day transfer, inoculum 3 x 105 cells

AA : Amino acids

AAA : a-Aminoadipic acid

ABA : a-Aminobutyric acid

aILE : allo-isoleucine

ALA : Alanine

ASN : Asparagine

ASP : Aspartic acid

ATP : Adenosine triphosphate

BGE : Background electrolyte

CAD : Charged aerosol detection gas

C-C : Cysteine-cysteine CDs : Cyclodextrins CE : Capillary electrophoresis CE : Collision energy CO2 : Carbon dioxide CoA : Coenzyme A

CTAB : Cetyltrimethylammonium bromide

CUR : Curtain gas

CXP : Collision cell exit potential

DMAB : 4-(N, N’-dimethylamino)-benzoic acid

DMAP : 4-dimethylaminopyridine

DMEM : Dulbecco’s Modified Eagle Medium

DP : Declustering potential

DTAB : Dodecyltrimethylammonium bromide

ECACC : European collection of cell cultures

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FIA : Flow injection analysis

FP : Focusing potential

GLN : Glutamine

GLU : Glutamic acid

GLY : Glycine

GS/MS : Gas chromatography coupled with spectrometric detection

H : Probe’s horizontal position

HCl : Hydrogen chloride

HGF : Heater gas flow

HIS : Histidine

HPLC : High performance liquid chromatography

HYP : Hydroxyproline

ILE : Isoleucine

IS : Ionspray voltage

L : Probe’s lateral position

LC/MS : Liquid chromatography coupled with spectrometric detection

LEU : Leucine

LLOL : Lower limit of Linearity

LOD : Limit of detection

LYS : Lysine

MET : Methionine

MRM : Multiple reaction monitoring

NEB : Nebulizer gas

OPA : ortho-Phthalaldehyde

ORN : Ornithine

PAS : p-aminosalicylic acid

PBS : Phosphate Buffered Saline

PFTBA : Perflurotributylanime

PHA : Phenylalanine hydroxylase

PHE : Phenylalanine

PMSF : Phenyl methyl sulfonyl flouride

PRO : Proline

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RSD % : Percentage relative standard deviation

SAR : Sarcosine

SDS : Sodium deoxycholate

SER : Serine

SIM : Selected ion monitoring

SPE : Solid phase extraction

TEM : Temperature THR : Threonine TRP : Tryptophan TYR : Tyrosine UV : Ultraviolet VAL : Valine

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1. Theoretical

1.1 Introduction

Over the past few decades numerous sample preparation, separation and detection techniques have been developed and have been applied in the bioanalytical field. The most challenging class of that field is the amino acids for two reasons. Firstly, amino acids have large differences in their chemical structures so their separation

becomes too difficult since they range from non-polar too highly polar amino acids. Secondly, amino acids play an important role in human metabolism and the metabolic profiling. Not only they are cell signaling

molecules, but they also have non-protein

functions.[1-2]In the body amino acids can be utilized as an energy source for major organs such as brain, liver, muscle and adipose tissue in most organisms. Catabolism of amino acids consists of two steps. In the first step the a-amino group is removed and converted into urea. In the second step the remaining carbon skeleton is converted into pyruvate, oxaloacetate, fumarate, succinyl CoA, a-keto-glutarate, acetyl CoA or acetoacetyl CoA which are further utilized as energy source in different metabolic

pathways. Amino acids as a source of energy are divided in two groups: ketogenic amino acids that can be degraded into acetyl CoA or acetoacetyl CoA and form ketone bodies; and glucogenic amino acids that can be degraded into pyruvate, oxaloacetate, fumarate, succinyl CoA or a-keto-glutarate and they are resulting to glucose formation. Of the twenty fundamental amino acids, tryptophan, phenylalanine, tyrosine and isoleucine are both ketogenic and glucogenic. Lysine and Leucine are only ketogenic and the rest are only glucogenic. [3-4] Tyrosine and tryptophan are used as precursors for the catecholamines (dopamine, norepinephrine and epinephrine) and the monoamine serotonin respectively. [5] Serine and

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alanine are utilized in the first step of ceramide de novo synthesis. [6] Glycine, Glutamine, Aspartic acid and Glutamic acid take a part in nucleotide catabolism. [4] Moreover, in clinical chemistry the increased amino acid levels in plasma are used as biomarkers for inborn errors of metabolism. For example, high concentration of phenylalanine indicates that the newborns suffer from phenylketonuria, a disease which characterized by a mutation in the gene phenylalanine hydroxylase (PHA), prevents the transmutation of phenylalanine to tyrosine and causes mental retardation. [7] It can be easily concluded that the quantitative analysis of amino acids is required for the understanding of chemical reactions that happened into human body and the prevention of human diseases.

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1.2 Historical Background

 Ion-exchange chromatography with postcolumn ninhydrin detection

In 1958, Moore, Stein and Spackman introduced an automatic amino acid analyzer based on ion exchange column chromatography after postcolumn ninhydrin derivatization.

A Li- or Na- based cation exchange system is used for complex or simpler physiological samples respectively. The proper pH, temperature and cation strength lead to the desirable separation. When ninhydrin reacts with the amino acid the reagent has purple color, shows an absorption at 570nm and the detection limit is around 10 pmol for the majority of them. In case of imino acids like proline the reagent has yellow color, shows an absorption at 440 nm and the detection limit is 50 pmol. The linearity range is obtained between 20-500 pmol. The analysis time ranges from 60 to 120 minutes.[8-9]

 High performance liquid chromatography with precolumn Ortho-phthalaldehyde

derivatization and fluorometric detection

Roth, in 1972, used strongly acidic cation exchange column followed by postcolumn oxidation with sodium hypochlorite. As precolumn derivatization used ortho-Phthalaldehyde (OPA) and thiol compound, such as N-acetyl-L-cysteine and 2-mercaptoethanol. Once again the proper pH and cation strength lead to the desirable separation. OPA reacts with primary amines in the presence of thiol compound and forms fluorescent isoindole products but it does not react with secondary amines, such as proline. In the last case, the reaction is accomplished because of the oxidation with sodium hypochlorite. The reagent that pass through the fluorometric detector has an excitation wavelength at 348 nm and an emission wavelength at 450nm. The detection limit is around 10 pmol and the linearity range is obtained between 10 pmol-10 nmol. The analysis time ranges from 40 to 70 minutes.[10]

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 Gas chromatography with ethyl chloroformate derivatization and flame ionization

detection

In 1990, Hušek introduced a method for the simultaneously analysis of 20 protein amino acids. Specifically, he combined the rapid gas chromatography (analysis time 10 minutes) with a short derivatization technique with chloroformate (20 minutes). He could even achieved shorter analysis time without sacrificing the resolution, using shorter and narrower bore capillaries. Ethyl chloroformate reacts with aqueous amino acid solutions and forms N (O, S)-ethoxycarbonyl ethyl esters. Masami, Yamamoto and Kiyama used isobutyl chloroformate instead of ethyl chloroformate but one extra step was necessary for the esterification of the carboxylic group.[1,11-12]

 Capillary zone electrophoresis with indirect absorbance detection

Kuhr and Yeung, in 1988, introduced a quite fast detection technique without derivatization step. Fluorophore or chromophore presence in the background electrolyte (BGE) was used for the indirect detection of the amino acids.[13] 18-20 amino acids were separated in 20-40 minutes using PAS and DMAB (10 mM) as a suitable background electrolytes, the pH in a range <10.3-11.2>; concentration of Mg2+ _{= 0.05 mM; DTAB= 0.25 mM or CTAB} 0.05mM.[14] However, with one single run lots amino acids cannot be efficiently separated from the baseline whereas Leu and Ile could not be separated at all. Seven years later, Lee and Lin tried to add cyclodextrins (CDs) to BGE, which contribute to better selectivity of CE. As a result, in 35 minutes they managed to separate all the twenty amino acids, except from Leu and Ile (pH 11 using; BGE PAS (10 mM); a-CD (20 mM)). Variants in the BGE can lead to better resolution but longer analysis time and the substitution of a-CD to β-CD can lead to Leu and Ile separation but inferior results.[15]

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1.3 A novel breakthrough approach

It can be agreed that amino acids are the “Achilles heel” on the separation analysis. During the last decades lot of techniques were introduced, but none of them could dominate. Among the techniques that are mentioned above, the first had low sensitivity, poor resolution, high costs and long analysis time. The second had the same drawbacks but better sensitivity. The third had a quite good analysis time but there were problems with the derivatization steps. And the last one did not require derivatization however the separation was not really efficiently. All these drawbacks can be overcome with the EZ-faast kit that Phenomenex introduced for simple and fast sample preparation (7 minutes) and simultaneously determination of 384 amino acids.

The procedure starts with a solid phase extraction as a clean-up step during which the amino acids and be extracted from the complex physiological fluids really fast. Afterwards, derivatization occurs, emulsify is formed and the upper organic phase is collected and analyzed on GC/MS or LC/MS system. In total, the sample preparation and analysis time for GC/MS is around 13 minutes and for LC/MS around 32 minutes.[16-17]

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1.4 Derivatization

Derivatization can be defined as a technique that modifies a compound, which is not suitable for the specific analytical procedure, to a product (derivative) with more suitable analytical properties as the original compound.[18]

The use of this technique can improve the following:

i. Suitability (e.g. solubility, polarity, volatility):

In high performance liquid chromatography the main problem is the existence of insoluble compounds in the mobile phase whereas in gas chromatography the existence of nonvolatile compounds.

ii. Efficiency (e.g. polarity):

In many cases compounds interact with each other’s or with the column, causing difficulties in the identification due to bad resolution and asymmetry of the peaks. iii. Detectability (e.g. atomicity):

The analysis of smaller amounts of material requires an extension in the range of their detectability.[19-21]

1.5 Types of Derivatization

There are three general types of derivatization that convert functional groups with active hydrogens (-OH, -COOH, -NH, -NH2, and -SH groups) to derivatized products that can be detected.

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 Silylation

The silylation mechanism involves the replacement of the active hydrogens by substituted silyl groups through a nucleophilic SN2 reaction.

where, X=halogen [21]

The most common reagents that are used for silylation derivatization are in the table below.[22]

Abbreviation Name Structure

BSA Bis-trimethylsilylacetamide BSTFA Bis-trimethylsilyltrifluoroacetamide MSTFA N-methyl-trimethylsilyltrifluoroacetamide TMCS Trimethylchlorosilane TMSI Trimethylsilylimidazole HMDS Hexamethyldisilzane

Table 1.5.1: Silylating Reagents.

Silylation is normally used in combination with gas chromatography to enhance the volatility of the analytes.

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 Alkylation ( Arylation )

The alkylation mechanism involves the replacement of the active hydrogens by aliphatic or aliphatic-aromatic groups. In case of acids, this procedure is known as esterification and contributes to better chromatograms.

RCOOH + PhCH2X → RCOOCH2Ph + HX

where, X=halogen or alkyl group R

The most common reagents that are used for alkylation derivatization are in the table below.[18]

PFBBr Pentafluorobenzyl bromide BF3 Boron trifluoride TBH Tetrabutylammonium hydroxide BB Benzylbromide DMF Dimethylformamide DAM Diazomethane

Table 1.5.2: Alkylating Reagents.

Alkylation is used in combination with gas chromatography to improve the volatility of the solutes as well as in liquid chromatography to improve the detectability on the separation efficiency. In the last case UV absorbing or mass spectrometry sensitive reagents are used.

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 Acylation

The acylation mechanism involves the replacement of the active hydrogens by acyl groups. Therefore, compounds with –OH,–NH and –SH can be converted to esters, amides and thioesters respectively.

where, R= acyl group[23]

The most common reagents that are used for acylation derivatization are in the table below.[18]

MBTFA N-methyl-bistrifluoroacetamide

PFPOH Pentafluoropropanol CF3CF2CH2OH

PFBCI Pentafluorobenzoyl Chloride

HFBI Heptafluorobutyrylimidazole

TFAA Trifluoroacetoic Anhydride CF3OCOCOCF3

Table 1.5.3: Acylating Reagents.

With respect to acylation, the same statements can be made as for alkylation reaction. They are also used in combination with gas chromatography and liquid chromatography.

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The advantages and disadvantages of each type of derivatization are mentioned at the Table 1.5.4

Type of derivatization

Advantages Disadvantages

Silylation 1. Suitable for a wide range of compounds

2. Great variety of reagents 3. Easy preparation

1. Moisture sensitive reagents 2. Only aprotic organic solvents

can be used

Alkylation 1. Stable derivatives

2. Great variety of reagents 3. Reactions in a wide pH

range

1. Only suitable for amines and acidic hydroxyls

2. Toxic reagents

Acylation 1. Stable derivatives 2. Good sensitivity

3. Can activate carboxylic acids before the esterification

1. Difficult preparation

2. Dangerous, smelly and moisture sensitive reagents 3. By-products have to be

removed before analysis

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1.6 Derivatization modes

The reactions that were mentioned in the above paragraph can be performed in three different modes:[24-25]

 Pre-column Mode

The compounds are derivatized before the analytical separation, usually manually but can also be automated. After the derivatization the products are separated and detected.

The main advantages of pre-column derivatization are the freedom in choosing the most optimal reaction conditions and the limited reagent consumption. The latter enables the use of more expensive reagents and therefore improves the sensitivity because of the lower background levels. However, the directly mixing of the reagent with the sample and the resulting excess of derivatization reagent can be easily presented matrix effect.

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 Post-column Mode

The compounds are derivatized after the separation and before the detection with the aid of a second pump, so the reaction is fully automated. The automation is the main advantage of post-column derivatization since it making the method reproducible and suitable for quantitative analysis. Moreover, in comparison with pre-column derivatization there is less chance to appear matrix effects because the components of the sample are separated earlier and there is no directly mixing with the reagent. On the other hand, the improvement of selectivity is highly unlikely due to the necessity of high and constant amount of reagent. An important requirement on this mode is that the detection properties of the reagent have to be different compared with the detection properties of the derivative.

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 On-column Mode

The less frequently used approach is the On-column mode. In this case the derivatization is performed during separation. Particularly, the front-end of the capillary is used as a reaction chamber. The main advantage is the increase in speed since it can be a whole automated procedure. Moreover, the sample dilution is minimal, making this mode suitable for single-cell analysis. On the other hand, the number of suitable reactions and procedures is rather limited because the mixing of the analyte and the reagent plugs is obtained due to the difference of the electrophoretic mobilities.[26]

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1.7 Chloroformate derivatization

The method that is used for the purpose of this project is based on a chloroformate derivatization reagent, which can modify at the same time the amino and carboxylic groups of the amino acids forming highly stable derivatives. It is a pre-column mode derivatization, where alkylation occurs at the carboxylic group and acylation at the amino group.

The importance of this derivatization reagent can be concluded on the following:

 The esterification of the carboxylic group occurs simultaneously with the protection of the amino group. So, it is a single step reaction.

 The extraction step can be avoided since the reaction can be performed in an aqueous medium.

 The reaction can be done at room temperature and mild acidic or basic conditions, therefore no racemization phenomena can be occurred.

 The reagent is accessible, cheap and easy to handle.

 The reaction is extremely fast (approximately 60s) under continuously stirring.[27] Therefore, it can be concluded that multiple derivatization represents a simple, useful and effective technique for amino acid analysis and gave the “tinder” to search for new derivatization reagents. Another example of this kind of derivatization is the derivatization with diazomethane followed by acetic anhydride in the presence of 4-dimethylaminopyridine (DMAP) and pyridine lead to derivatives β-amino acids.[28]

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2.Experimental

2.1 Chemicals

Amino acids were purchased from the Sigma-Aldrich (Stock No.AA-S-18). The concentration of each amino acid is 2.5 μmoles per mL in 0.1 N HCl, except L-cystine, whose concentration is 1.25 μmoles per mL.

COMPONENT MOL. Wt. μMoles/mL

L-Alanine 89.09 2.50 Ammonium chloride 53.49 2.50 L-Arginine 174.2 2.50 L-Aspartic acid 133.1 2.50 L-Cystine 240.3 1.25 L-Glutamic acid 147.1 2.50 Glycine 75.07 2.50 L-Histidine 155.2 2.50 L-Isoleucine 131.2 2.50 L-Leucine 131.2 2.50 L-Lysine 146.2 2.50 L-Methionine 149.2 2.50 L-Phenylalanine 165.2 2.50 L-Proline 115.1 2.50 L-Serine 105.1 2.50 L-Threonine 119.1 2.50 L-Tyrosine 181.2 2.50 L-Valine 117.2 2.50

Table 2.1.1: Stock No. AA-S-18.

However, the above standard did not include three proteinogenic amino acids. For those three standard solutions were made, 2.5 μmoles per mL in water.

Component Supplier Mol. Wt. μMoles/mL

L-Glutamine 99% Sigma 146.14 2.50

L-Asparagine 98% Sigma 132.12 2.50

L-Tryptophan 98% Sigma/Bachem/99% Merck

204.23 2.50

Table 2.1.2: Proteinogenic amino acids.

Moreover, MilliQ water, methanol (Baker HPLC grade) and ammonium formate, formic acid ammonium salt (97% Aldrich/Sigma) were used for the HPLC experiments.

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2.2 Kit components

Kit components and materials are listed in Table 2.2.1 and Table 2.2.2.

Reagent Properties Ingredients Volume

Reagent 1 Internal Standard Solution

Norvaline 0.2mM N-propanol 10%

50 ml

Reagent 2 Washing Solution N-propanol 90 ml

Reagent 3A Eluting Medium Component I

Sodium Hydroxide 60 ml

Reagent 3B Eluting Medium Component II

N-propanol 40 ml

Reagent 4 Organic Solution I Chloroform 4 vials, 6 ml each

Reagent 5 Organic Solution II Isooctane 50 ml

Reagent 6 Re-dissolution Solvent Isooctane 80% Chloroform 20%

50 ml

SD 1,2 & 3[1]_{Amino Acid Standard}

Mixtures

Mix of amino acids 2 vials of each SD,2 ml each

Table 2.2.1: Reagents included at the EZ-faast kit.

Supplies Quantity

Sorbent tips in racks 4 x 96

Sample preparation vials 4 x 100

Microdispenser, 20-100 µL 1

Syringe, 0.6 mL 10

Syringe 1.5 mL 10

ZB-AAA 10m x 0.25mm Amino Acid Analysis GC Column

1

Autosampler vials with inserts 4 x 100

FocusLiners 5

User Manual 1

Table 2.2.2: Supplies included at the EZ-faast kit.

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SD1: 23 amino acids, 200 nmoles/mL each

AAA ASP GLY LEU PHE THR

ABA βAIB HIS LYS PRO TYR

aILE C-C HYP MET SAR VAL

ALA GLU ILE ORN SER

SD2: Complementary amino acids not stable in acidic solution, 200 nmoles/mL each

ASN GLN TRP

2.3 Additional equipment

Other required materials are listed in Table 2.3.1.

Materials Supplier

1000µL - 200 µL pipette Gilson Pipetman Pipettes

200µL - 50 µL Gilson Pipetman Pipettes

100 µL - 20 µL Gilson Pipetman Pipettes

20 µL - 2 µL Gilson Pipetman Pipettes

Pipette tips Ultratip Greiner Bio-One

Vortex VWR International

Ultrasonic VWR International

4 µL Clear Vial, Screw Top Hole Cap PTFE/Silicone Septa

Supelco (Sigma Aldrich)

Container for proper waste disposal Afval Energie Bedrijf (Gemeente Amsterdam)

Septa Thermogreen LB-2 Septa for Shimadzu (conditioned) (Supelco/Sigma Aldrich)

Precolumn RP Phenomenex C18 4 x 2.00mm

Zorbax Eclipse XDB-C18, Rapid resolution HT, 4,6x50mm,1,8 micron Column

Agilent Technologies

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2.4 Storage and Stability

Reagents 2, 3a, 5 and 6 should be stored at room temperature. At the same conditions should be also stored the methanol and the mixture of ammonium formate in water and methanol. Whereas, the reagents 1, 3B and 4 should be stored at 4 o C. Last but not least, all the standards solutions should be stored at -20o C.

All the components have one year shelf life when they stored at recommended conditions.

2.5 Cleaning

A propanol: water mixture (1:2, v/v) should be used to flush the plastic syringes that are used for the SPE cleaning steps.

In addition, the Drummond Dialamatic Microdispenser should be cleaned with isopropanol: acetone mixture (1:1, v/v) at the end of the experiments.

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2.6 Preparation of Eluting Medium

The Eluting Medium should be prepared fresh each day before the beginning of the experiment. The volume is proportional of the number of samples. A combination of three parts of reagent 3A with two parts of reagent 3B consist the Eluting Medium, which should be stored at room temperature during the day and the remaining mixture should be discarded at the end of the day. The following table shows the necessary volume of each medium regard to the number of samples.

Number of Samples Reagent 3A (µL) Reagent 3B (µL)

2 300 200 4 600 400 7 900 600 12 1.5 1.0 14 1.8 1.2 19 2.4 1.6 24 3.0 2.0 29 3.6 2.4 34 4.2 2.8 39 4.8 3.2 44 5.4 3.6 49 6.0 4.0

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2.7 General Procedure for GC-MS experiments

Firstly, 100µL of sample was mixed with Reagent 1 in order to adjust pH and add internal standard -Norvaline. The mixture was slowly passed through sorbent tip and further on washed with Reagent 2. Thereafter, 200µL of freshly prepared Eluting medium was used to elute sample and sorbent from the tip. In the next step derivatization reagent (Reagent 4) was added to the mixture and the sample was vigorously vortexed. In the last step 100µL of Reagent 5 containing isooctane was added to sample in order to separate different phases after vortexing. Top organic phase was transferred to a fresh vial, evaporated under Nitrogen stream and re-suspended in Isooctane chloroform mixture.

2.8 General Procedure for LC-MS experiments

The procedure is identical with the previous except of re-dissolving step where the sample was re-suspended in 1:2 ammonium formate in water: ammonium formate in methanol instead of Reagent 6.

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2.9 Instruments Settings

 GC/MS

A QP2012 plus GC/MS (Shimadzu) is used with an automatic injector AOC-2Oi and an auto sampler AOC 2Os. The column was ZB-AAA 10m x 0.25mm Amino Acid Analysis GC Column.

For the GC helium is used as carrier gas at flow rate of 2ml/min, split injection ( 250 ° C ) and the oven temperature program was 30 ° C/ min from 110 ° C to 320 ° C.

The MS ion source temperature was 240 ° C, the interface 320 ° C and the scan range was 45-450 m/z. Finally, the MS was tuned and calibrated using Perflurotributylamine (PFTBA) every day.



LC/MS

A PE Sciex API 3000 (AB SCIEX) triple quadrupole mass spectrometer coupled to an Agilent 1100 Series LC system. The latter consist of a G1322A Degasser, a G1376A CapPump, a G1367A Wpals, a G1330B ALSTherm and a G13116A Colcom.

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2.10 Biological Samples

For the purpose of this experiment biological samples were performed by Azra Mujic-Delic. The procedure that she follows is describing below and it has three stages:

Cell culture:

Mouse embryonic fibroblast NIH-3T3 cells, obtained from European collection of cell cultures (ECACC), were cultured in the Dulbecco’s Modified Eagle Medium (DMEM) supplemented with a 10% bovine serum, penicillin (50 IU/ml) and streptomycin (50 mg/ml) in 5% CO2

atmosphere at 37 °C. Stable clones of NIH 3T3 expressing US28 receptor or empty vector were kept under a selective pressure of G418 (400 mg/ml) in the culture medium to ensure expression of US28 in all growing cells.

Determination of amino acid uptake and excretion:

Cells at the density of 75.000/well were plated in a 6 well dish and incubated in growth medium for 24hrs in 5% CO2 atmosphere at 37 °C. After 24hrs cell growth medium was

replaced with 0.5% bovine serum -containing medium to synchronize. After another 24hrs growth medium was exchanged for medium with 10% bovine serum. Since the change of medium supernatant and cell lysate were collected (time 0, 24, 48, 72 and 96 hours), amino acid analysis in the growth media and protein concentration determination was followed. In fact, 20L of extracellular media were used for further amino acid analysis.

Cell lysis and protein determination:

Cells were quickly washed with 0.5 ml of ice cold Phosphate Buffered Saline (PBS) and treated with 100 μl of Radioimmunoprecipitation assay (RIPA) lysis buffer (PBS containing Nonidet P-40 , 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS and 1mM PMSF

supplemented, 1 mM sodium orthovanadate with protease (Roche Applied Science,

Mannheim, Germany) inhibitor cocktails) on ice for 20 min. Cell extracts were clarified using centrifugation (18,000 rpm/10 min/4°C). Protein content was determined using BCA Protein Assay kit (Pierce).

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3. GC/MS Results

3.1 Full-SCAN GC/MS

In the first step sample containing standard amino acids was injected in order to collect data about the target ions, the reference ions and the retention time. Based on the obtained data, a selected ion monitoring method was created in order to increase method’s sensitivity.

Scheme 3.1.1: Chromatogram of standard derivatized amino acids using a MS SCAN mode. Range adjusted at 45-450 m/z.

C=200μM/L

Split Ratio=30

Retention Time (min)

Time

Abu

n

d

an

ce

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The target and the reference ions that are chosen for the determination of the amino acids are given at the Table 3.1.1. whereas, the retention time that is used for the selected ion

monitoring (SIM) method is listed in the Table 3.1.2.

Amino Acids Target Ions (m/z) Reference Ions (m/z)

Alanine 130.1 70.1 88.0 Glycine 116.1 56.1 74.1 Valine 116.1 72.1 55.1 Leucine 172.2 86.1 69.1 Isoleucine 130.1 101.1 - Threonine 101.1 74.0 56.1 Serine 60.1 146.1 74.1 Proline 70.1 156.1 114.1 Asparagine 69.1 155.1 113.1 Aspartic Acid 88.1 130.1 216.2 Methionine 61.1 101.1 56.1 Glutamic Acid 84.1 170.1 85.1 Phenylalanine 91.1 148.1 74.1 Glutamine 84.1 59.1 58.2 Lysine 170.2 84.1 128.1 Histidine 81.1 82.1 110.2 Tyrosine 107.1 164.1 74.0 Tryptophan 130.1 131.1 77.1

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Amino Acids Retention Time (min)

Alanine 1.103 Glycine 1.203 Valine 1.434 Leucine 1.616 Isoleucine 1.671 Threonine 1.879 Serine 1.920 Proline 1.992 Asparagine 2.095 Aspartic Acid 2.662 Methionine 2.687 Glutamic Acid 3.033 Phenylalanine 3.049 Glutamine 3.674 Lysine 4.369 Histidine 4.559 Tyrosine 4.848 Tryptophan 5.133

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3.2 Selected Ion Monitoring (SIM) for standards amino acids

With regard of the above data a SIM method for standards amino acids was set up. The method was split in 10 time periods, where each of the period monitors certain targeted ions and reference ions. This step is done in order to increase the sensitivity of the method.

SIM for Standards

Start Time End Time Event Time

Amino Acids Ions

1.05 1.33 0.06 Ala-Gly 130.1,70.1,88 116.1,56.1,74.1

1.33 1.81 0.09 Val-IS-Leu-Ile 116.1,72.1,55.1 158.2 172.2,86.1,69.1 130.1,101.1

1.81 2.41 0.12 Thr-Ser-Pro-Asn 101.1,74,56.1 60.1,146.1,74.1 70.1,156.1,114.1 69.1,155.1,113.1

2.41 2.89 0.06 Asp-Met 88.1,130.1,216.2 61.1,101.1,56.1 2.89 3.4 0.06 Glu-Phe 84.1,170.1,85.1 91.1,148.1,74.1 3.40 4.05 0.05 Gln 84.1,59.1,58.2 4.05 4.49 0.05 Lys 170.2,84.1,128.1 4.49 4.73 0.05 His 81.1,82.1,110.2 4.73 5.02 0.05 Tyr 107.1,164.1,74 5.02 5.25 0.05 Trp 130.1,131.1,77.1

Table 3.2.1: SIM method for standard amino acids.

The below chromatogram is a representative one.

Scheme 3.2.1: Chromatogram of standard derivatized amino acids on SIM mode. Range adjusted at 45-450 m/z.

Retention Time

(min)

Ab

u

n

d

an

ce

(

a.u.)

C=0.01 μM/L

Split Ratio=30

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3.3 Repeatability (within a day)

AA/Concentration RSD% 0.1 μM 0.3 μM 0.6 μM 1.2 μM 2.4 μM 5 μM 10μM Alanine 5.4 5.5 4.4 3.6 0.6 1.1 0.5 Glycine 3.5 8.6 3.2 1.6 0.6 3.4 0.8 Valine 5.7 5.4 2.4 1.6 1.4 0.6 0.4 Leucine 9.2 2.0 2.7 3.2 1.0 1.7 0.6 Isoleucine 9.8 3.9 5.3 2.1 1.2 1.1 0.4 Threonine 13.5 10.6 9.4 4.2 3.0 4.2 2.9 Serine 14.3 8.9 7.1 4.2 1.6 3.5 3.7 Proline 4.1 2.2 3.6 3.1 0.3 0.6 0.8 Asparagine - - 11.9 5.6 4.2 5.4 0.7 Aspartic Acid - - 3.7 7.1 14.2 2.0 2.7 Methionine - - - 3.5 1.4 Glutamic Acid - - - 10.3 1.5 2.3 1.2 Phenylalanine - - 3.7 7.3 4.8 0.5 1.2 Glutamine - - - - 6.8 2.6 3.0 Lysine - 0.9 13.0 2.0 1.7 2.0 3.1 Histidine - - - - 9.5 6.6 6.7 Tyrosine - 3.3 3.3 3.5 3.5 2.9 1.7 Tryptophan - 11.1 6.0 4.6 3.5 3.1 2.6

Table 3.3.1: Relative standard deviation for all the amino acids in seven different concentrations.

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3.4 Intermediate Precision

AA/Concentration 10 μM 50μM

Recovery % Recovery %

Peak Area Retention Time Peak Area Retention Time

Alanine 99 100 88 100 Glycine 108 100 98 100 Valine 99 100 95 100 Leucine 106 100 104 100 Isoleucine 99 100 99 100 Threonine 114 100 103 100 Serine 119 100 96 100 Proline 94 100 91 100 Asparagine 116 100 90 100 Aspartic Acid 117 100 98 100 Methionine 115 100 99 100 Glutamic Acid 129 100 67 100 Phenylalanine 107 100 103 100 Glutamine 126 100 95 100 Lysine 136 100 97 100 Histidine 121 100 120 100 Tyrosine 119 100 108 100 Tryptophan 128 100 146 100

Table 3.4.1: The percentage recovery of peak area and retention time in two representative concentrations of all amino acids.

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3.5 Calibration Curves

For the purpose of this experiment six derivatized samples with standard amino acids are injected three times each at GC/MS. The concentration was chosen, had two magnitude range. Specifically, 0.1 μM, 1 μM, 5 μM, 10 μM, 25 μM and 50 μM. Norvaline was used as internal standard. The experiment was performed using as SIM method the one was described in paragraph 3.2 with split ratio 30 and injection volume 1 μL. In the next pages you will find a representative graph including calibration equation and correlation

coefficient. The rest are available in Appendix I. Moreover, below the graph you will find a table with the parameters of the calibration curve of each amino acid.

Alanine

Ar

ea

R

atio (

12

C

/N

or

va

line

)

Concentration (µM)

0 10 20 30 40 Conc. Ratio 0.0 1.0 2.0 3.0 4.0 5.0 A rea Ratio(x0.1) Y = 1.021928e-002X - 4.121476e-003 R^2 = 0.9989563 R = 0.999478

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Amino Acids

a

β

R

2

Alanine 1.021928e-002 - 4.121476e-003 0.9989563

Glycine 7.586305e-003 4.077981e-003 0.9996499

Valine 9.995392e-003 - 5.121873e-003 0.9992979

Leucine 1.086172e-002 - 2.920057e-003 0.9983499

Isoleucine 1.006918e-002 - 5.515763e-003 0.9983963

Threonine 6.919126e-003 - 1.38511e-003 0.9933719

Serine 3.08389e-003 -5.75994e-004 0.9875481

Proline 1.591607e-002 - 1.142593e-002 0.9978341

Asparagine 5.979105e-003 - 4.470555e-003 0.9905939

Aspartic Acid 3.890992e-003 - 3.216384e-003 0.9905092

Methionine 4.30262e-003 - 2.062193e-002 0.9728832

Glutamic Acid 3.640853e-003 - 4.719553e-003 0.9849679

Phenylalanine 8.686568e-003 - 2.76655e-003 0.9966394

Glutamine 1.253481e-003 - 5.236893e-003 0.9781025

Lysine 3.009924e-003 -3.043628e-003 0.9922608

Histidine 1.552537e-003 - 6.532646e-003 0.9862679

Tyrosine 1.154867e-002 - 2.634247e-002 0.9795034

Tryptophan 1.372151e-002 - 2.566159e-002 0.9802783

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3.6 Limit of Detection (LOD) and Linearity Range

Amino Acids

LOD (μM)

Linearity Range (μM)

Alanine 0.1 0.3-200 Glycine 0.1 0.3-200 Valine 0.1 0.1-200 Leucine 0.1 0.1-200 Isoleucine 0.1 0.1-200 Threonine 0.1 0.1-200 Serine 0.1 0.1-200 Proline 0.3 0.3-200 Asparagine 1.2 1.2-200 Aspartic Acid 0.6 0.6-200 Methionine 1.2 1.2-200 Glutamic Acid 1.2 1.2-200 Phenylalanine 0.6 0.6-200 Glutamine 2.4 2.4-200 Lysine 0.3 0.3-200 Histidine 2.0 2.0-200 Tyrosine 0.3 0.3-200 Tryptophan 0.3 0.3-200

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3.7 Amino acids labeled with

13

C

In order to correct the matrix effect and the sample degradation, 13C labeled standard amino acids were bought. During the validation procedure, 13C standards were added directly to the samples before adding Reagent 1. SCAN MS mode was used to identify the ion fragments of the label amino acids. All the spectra and chromatograms are available in Appendix II and Appendix III respectively.

Scheme 3.7.1: Chromatogram of 13C labeled derivatized amino acids using a SCAN MS mode. Range adjusted at 45-450 m/z.

Retention Time (min)

Ab

u

n

d

an

ce

(

a.u.)

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3.8 Selected Ion Monitoring (SIM) for amino acids labeled with

13

C

With regard on the data that were collected from the spectra a new SIM method about label amino acids was set up.

SIM for 12_C/13_C-14_N Start Time End Time Event Time

12_C 13_C 12_C 13_C 12_C 13_C 12_C 13_C

1.05 1.33 0.06 Ala-Gly 130.1 132.1 116.1 117.1 102 104

1.33 1.81 0.09 Val-IS-Leu-Ile 116.1 120.1 158.2 172.2 177.2 130.1 135.1

1.81 2.41 0.12 Thr-Ser-Pro-Asn 101.1 103 60.1 62 70.1 74.1 69.1 72 2.41 2.89 0.06 Asp-Met 88.1 90 61.1 69 101.1 104 114.1 116 2.89 3.4 0.06 Glu-Phe 84.1 88 91.1 98 3.40 4.05 0.05 Gln 84.1 88 4.05 4.49 0.05 Lys 170.2 175.2 4.49 4.73 0.05 His 81.1 85 4.73 5.02 0.05 Tyr 107.1 114.1 5.02 5.25 0.05 Trp 130.1 139.1

Table 3.8.1: SIM method for labeled 13C amino acids.

Scheme 3.8.1: Chromatogram of labeled derivatized amino acids on SIM mode. Range adjusted at 45-450 m/z.

Retention Time (min)

Abu

n

d

an

ce

(

a.

u

.)

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3.9 Amino acids labeled with

13

C-

15

N

Furthermore, amino acids labeled with 13C and 15N were added as internal standard. As a result, instead of 100 μL of sample, 98 μL of sample and 2 μL of label were added. The rest of the procedure followed precisely.

At the begging, SCAN MS mode was used to identify the ion fragments of the label amino acids. The spectra and chromatograms are available in Appendix IV and Appendix V respectively.

Scheme 3.9.1: Chromatogram of 13C-15N labeled derivatized amino acids using SCAN MS mode. Range adjusted at 45-450 m/z.

Retention Time (min)

Abu

n

d

an

ce

(

a.

u

.)

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3.10 Selected Ion Monitoring (SIM) for amino acids labeled with

13

_C-

15

_N

With regard on the data that were collected from the spectra a new SIM method about label amino acids was set up

SIM for 12_C/13_C-15_N Start Time End Time Event Time

12_C 13_C 12_C 13_C 12_C 13_C 12_C 13_C

1.05 1.33 0.06 Ala-Gly 130.1 132.1 116.1 117.1 102 104

1.33 1.81 0.09 Val-IS-Leu-Ile 116.1 120.1 158.2 172.2 177.2 130.1 135.1

1.81 2.41 0.12 Thr-Ser-Pro-Asn 101.1 103 60.1 62 70.1 74.1 69.1 72 2.41 2.89 0.06 Asp-Met 88.1 90 61.1 69 101.1 104 114.1 116 2.89 3.4 0.06 Glu-Phe 84.1 88 91.1 98 3.40 4.05 0.05 Gln 84.1 88 4.05 4.49 0.05 Lys 170.2 175.2 4.49 4.73 0.05 His 81.1 85 4.73 5.02 0.05 Tyr 107.1 114.1 5.02 5.25 0.05 Trp 130.1 139.1

Table 3.10.1: SIM method for labeled 13C-15N amino acids.

Scheme 3.10.1: Chromatogram of labeled derivatized amino acids on SIM mode. Range adjusted at 45-450 m/z.

Retention Time (min)

Abu

n

d

an

ce

(a.

u

.)

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3.11 Biological Samples

Amino acid analysis in biological samples is considered as an important laboratory test for the diagnosis of cancer mechanism. Specifically, it is considered that there are some amino acids, whose difference in concentration implies cancer. But firstly, a brief reference to Warburg effect is necessary.

Scheme 3.11.1: Warburg Effect.

Tumor Cells

+/-O

₂

Glucose

Pyruvate

Lactate

95%

Aerobic

Glycolysis

(4ATP)

Warburg Effect

O

₂ Mitochondrial

CO

₂

5%

Normal

Cells

-O

₂

+O

₂

Glucose

Pyruvate

Lactate

Anaerobic

Glycolysis

(2ATP)

O

₂ Mitochondrial

CO

₂

Oxidative

Phosphorylation

(36ATP)

Lactate

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Normal cells obtain ATP as a source of energy through the mitochondrial oxidative phosphorylation in the presence of oxygen instead of glycolysis, due to the higher yield of ATP. In addition, the presence of oxygen results in the inhibition of glycolysis. On the other hand, tumor cells can activate glycolysis even in the presence of adequate oxygen levels causing the Warburg effect. [29]

The metabolic phenomenon where cancer cells rely on aerobic glycolysis instead of oxidative phosphorylation, was described for the first time by Otto Warburg over 80 years ago and it was named after him. Aerobic glycolysis is an inefficient way to generate ATP producing only 2 molecules of ATP per molecule glucose whereas oxidative phosphorylation generates 32 ATP molecules per molecule glucose. The question immediately arises as to why cancer cells would switch to less productive form of energy production. One hypothesis describes increased aerobic glycolysis as an adaptation to hypoxic environment. Others argue that increased glycolysis should facilitate uptake and incorporation of nutrients into the biomass. Although, the exact mechanisms underlying the Warburg effect are unclear, the importance of increased glycolysis in cancer cells has been experimentally demonstrated.

Consequently, it is accepted that glycolysis provides tumor cells with glucose to make ATP intermediates such as nonessential amino acids, which serve as building blocks for tumor cells. Specifically, glutamine is essential for cancer cell growth due to nutritional value as carbon and nitrogen source. Moreover, it provided acid resistance. Therefore, the reduced concentration implies Warburg effect. The reverse is happening to alanine, where the increased concentration implies Warburg effect because pyruvate is converting to alanine. [30]

In our experiment we compared amino acids uptake and release in extracellular medium of two cell lines, that differ in expression of viral receptor US28. Expression of such a receptor was described to activate multiple signaling pathways with consequent cancer phenotype.

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Scheme 3.11.2: Glutamine concentration in the extracellular cell growth media in time. Red bars show control cells MOCK, blue bars show cells US-28 expressing the viral receptor.

Glutamine is a source of energy side to a glucose and it was described in literature that certain cancer lines are utilizing more glutamine compare to glucose. It was even described that glutamine uptake is life depending and its depletion from growing media leads to a cell death. Alanine usually serves in the cell metabolism as a sink for nitrogen in the presence of flux of glutamine. Based on the huge uptake of glutamine, we assume that excretion of alanine would take a place.

Scheme 3.11.3: Alanine excreting growth to extracellular growth media in time. Red bars show control cells MOCK, blue bars show cells US-28 expressing the viral receptor.

0 50 100 150 200 250 medium 0 24 48 72 96 C on cen tr at ion ( μ M)

Time points (hours)

Glutamine

MOCK US-28 0 10 20 30 40 50 60 medium 0 24 48 72 96 C on cen tr at ion ( μ M)

Time points (hours)

Alanine

MOCK US-28

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The last graph represent isoleucine’s concentration in the extracellular growth media of two different cell lines collected in different time points. The reduced concentration of isoleucine shows that is utilized in very small amounts, therefore is not directly used as a source of energy but only for the protein synthesis.

Scheme 3.11.3: Isoleucine excreting growth to extracellular growth media in time. Red bars show control cells MOCK, blue bars show cells US-28 expressing the viral receptor.

To sum up, taking the above under consideration only glutamine is an important source of energy. The rest amino acids, like isoleucine, show a small reduction on their concentration at cells expressing US-28 receptor because they are utilized by the protein synthesis. For that reason the rest graphs will not be provided.

Based on this analysis we do not see any significant differences in uptake or excretion amino acids and we could conclude that metabolism of these cells does not differ. However, for real conclusion more metabolites analysis would have to be performed.

0 10 20 30 40 50 60 70 medium 0 24 48 72 96 C on cen tr at ion ( μ M)

Time points (hours)

Isoleucine

MOCK US-28

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4. LC/MS Results

4.1 Manual Tuning for Probe and Turbo Ionspray Gas

The first step on validation of a method using LC/MS is the tuning.

The initial conditions of probe’s horizontal position (H), probe’s lateral position (L) and heater gas flow (HGF) were:

 H=15

 L=-5

 HGF=6500

The values were chosen and measured for the optimization as well as the resulting graphs are shown particularly in Appendix VI.

For the optimum conditions was chosen “The Golden Section” between the maximum intensity of the peak and the less possible gas consumption:

 L= -5

 H=3

 HGF=5000

4.2 Flow injection analysis (FIA)

Except of the probe and the turbo ionspray gas there are other parameters that should optimized and they are related to each amino acid separately.

The EZfaast Kit has already provided us with a manual that gives us representatives values for all the compound-dependent parameters and source dependent parameters as well.

However, it has to be checked if those parameters are suitable for the instrument that we are using. Specifically, the compound-dependent parameters that need to be optimized are the Declustering Potential (DP), the Focusing Potential (FP), the Entrance Potential (EP), the Collision Cell Exit Potential (CXP) and the Collision Energy (CE). The Nebulizer Gas (NEB),

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the Curtain Gas (CUR), the Temperature (TEM) and the Ionspray Voltage (IS) are the source-dependent parameters that have to be checked.

Two amino acids and the internal standard were checked and the given values were verified. The data of those measurements can be found in Appendix VII. The below table sums up the values of the compound-dependent parameters for each amino acid. The source-dependent parameters will be given in the paragraph 4.4.

Amino Acids DP FP CE CXP EP Glycine 21 130 13 8 10 Asparagine 60 250 15 10 10 Leucine 16 110 21 10 10 Glutamine 26 150 21 10 10 Threonine 40 200 17 10 10 Serine 25 140 17 8 10 Alanine 30 210 27 4 10 Methionine 21 140 17 12 10 Proline 26 160 19 10 10 Lysine 36 200 17 8 10 Aspartic Acid 30 110 19 14 10 Histidine 63 190 33 12 10 Norvaline 21 130 17 10 10 Glutamic Acid 26 150 21 10 10 Tryptophan 60 350 21 10 10 Tyrosine 60 300 43 8 10 Isoleucine 40 200 17 10 10 Phenylalanine 40 200 17 14 10

Table 4.2.1: Compound-dependent parameters based on EZ-faast manual.

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4.3 Full-SCAN LC/MS

In the first step sample containing standard amino acids was injected in order to check the retention time and the precise mass of parents and products ions. In following tables are listed all the masses. In Appendix VIII you will find the spectra that verify the choice of those masses. In addition, table 4.3.3 shows the retention time of its amino acid in time order.

Parent Ions

Amino acids m/z (manual) m/z (after scanning)

Glutamine 275.3 275.6 Serine 234.3 234.3 Asparagine 243.3 243.3 Leucine 260.2 260.4 Glycine 204.1 204.5 Threonine 248.3 248.7 Alanine 218.3 218.6 Proline 244.3 244.6 Methionine 278.3 278.4 Aspartic Acid 304.3 304.6 Histidine 370.2 370.3 Valine 246.3 246.6 Glutamic Acid 318.3 318.6 Isoleucine 260.3 260.5 Phenylalanine 294.3 294.5 Tyrosine 396.2 396.7 Lysine 361.2 361.7 Tryptophan 333.3 333.8

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Product Ions

Amino acids m/z (manual) m/z (after scanning)

Glutamine 172.1 172.5 Serine 146 146.2 Asparagine 157.2 157.2 Leucine 172.1 172.3 Glycine 144 144.2 Threonine 160.2 160.2 Alanine 130.1 130.2 Proline 156.2 156.3 Methionine 190.1 190.5 Aspartic Acid 216.2 216.5 Histidine 196.3 196.5 Valine 158.1 158.3 Glutamic Acid 172.1 172.4 Isoleucine 172.1 172.6 Phenylalanine 206.1 206.4 Tyrosine 136.2 136.4 Lysine 301.3 301.6 Tryptophan 245.1 245.6

Table 4.3.2: Mass of product ions based on manual and after scanning.

Amino Acids Retention Time

Glutamine 1.25 Serine 1.40 Asparagine 1.44 Leucine 1.52 Glycine 1.58 Threonine 1.64 Alanine 2.23 Proline 2.83 Methionine 2.97 Aspartic Acid 3.38 Histidine 3.43 Lysine 3.48 Norvaline 3.57 Glutamic Acid 3.71 Tryptophan 3.95 Phenylalanine 4.68 Isoleucine 4.78 Tyrosine 6.78

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4.4 Multiple Reaction Monitoring (MRM)

An MRM method was developed for the determination of the amino acids. This method was divided in three time periods, each of them has a group of amino acids with their parent and product ions that are allowed to pass through the triple quadrupole. The amino acids were grouped on periods based on the time order of their appearance on the chromatogram and the source-dependent parameters. The compound-dependent parameters can be defined for each amino acid separately whereas the source-dependent parameters only for periods resulting on compromises.

Period Start Time End Time Amino Acids Q1 Mass Q3 Mass

1st 0.000 1.995 Glycine 204.5 144.3 Asparagine 243.4 115.0 Leucine 260.3 172.3 Glutamine 275.6 172.3 Threonine 248.6 160.5 Serine 234.2 146.2 2nd 0.1995 4.300 Alanine 218.6 88.4 Methionine 278.4 190.4 Proline 244.5 156.2 Lysine 361.6 301.6 Aspartic Acid 304.5 216.5 Histidine 370.3 196.4 Norvaline 246.4 158.3 Glutamic Acid 318.6 172.3 Tryptophan 333.7 245.5 3rd 4.300 7.305 Tyrosine 396.6 136.4 Isoleucine 260.4 172.3 Phenylalanine 294.4 260.3

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The compound-dependent parameters have already given at table 4.2.1 whereas the source-dependent parameters of each period are given below.

Period Nebulizer (NEB) Curtain Gas (CUR) CAD Gas (CAD) Ionspray Voltage (IS) Temperature (TEM) Entrance Potential (EP) 1st ₁₂ ₁₂ ₁₀ ₁₆₀₀ ₄₇₅ ₁₀ 2nd ₁₂ ₁₂ ₁₀ ₁₆₀₀ ₄₇₅ ₁₀ 3rd ₁₀ ₁₀ ₁₀ ₁₆₀₀ ₄₇₅ ₁₀

Table 4.4.1: Source-dependent parameters based on EZ-faast manual.

The following chromatograms indicate the distribution of the amino acids during the periods.

Scheme 4.4.1: Chromatogram of first period’s amino acids. GLN SER ASN LEU GLY THR

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Scheme 4.4.2: Chromatogram of second period’s amino acids.

Scheme 4.4.3: Chromatogram of third period’s amino acids. ALA PRO MET HIS ASP GLU VAL TYR ILE PHE

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The gradient program that was used to accomplish the necessary equilibrium, avoid the retention shifting and the contamination of the column is describing below.

Time (min) Flow (ml/min) A% (10mM ammonium formate in water) B% (10nM ammonium formate in methanol) 0 0.6 32 68 13 0.6 17 83 13.01 0.6 5 95 18 0.6 5 95 18.0 0.6 32 68 25 0.6 32 68

Table 4.4.2: The gradient program according to time.

Scheme 4.4.4: The fluctuations of the mobile phase.

60 65 70 75 80 85 90 95 100 0 5 10 15 20 25 30 B% G ra d ie n t Time (min)

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4.5 Calibration Curves

For the purpose of this experiment nine derivatized samples with standard amino acids are injected three times each at LC/MS. The concentration was chosen, had three magnitude range. Specifically, 0.25 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 10 μM, 25 μM, 50 μM and 100 μM. Norvaline was used as internal standard. The experiment was performed using the MRM method that was described in the above paragraph. In the next pages you will find a

representative graph including calibration equation and correlation coefficient. The rest are available at Appendix IX. Moreover, below the graph you will find a table with the

parameters of the calibration curve of each amino acid.

Glycine

y = 51726x + 93576 R² = 0.9984 0.00E+00 1.00E+06 2.00E+06 3.00E+06 4.00E+06 5.00E+06 6.00E+06 0 20 40 60 80 100 120 P e a k A rea Concentration (μM)

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Amino Acids

a

β

R

2 Asparagine 128055 - 8810.4 0.9996 Leucine 505704 133475 0.9951 Glutamine 193378 74903 0.9846 Threonine ₃₀₉₆₂ ₅₅₂₁₂ _0.9979 Serine 83401 264814 0.9944 Alanine 20124 - 61986 0.9732 Methionine 217566 - 499397 0.9941 Proline 1E+06 210795 0.9918 Lysine 183816 705.56 0.9991 Aspartic Acid 247631 74509 0.9995 Histidine 992948 253136 0.9983 Glutamic Acid 320678 18095 0.9999 Tryptophan 16366 - 1337.6 0.9985 Tyrosine 238980 4586 0.9999 Isoleucine 1E+06 69562 0.9935 Tyrosine 1E+06 25739 0.9994

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4.6 Relative Standard Deviation (RSD %)

In order to check the precision and the repeatability of the method the relative standard deviation was calculated for 8 different concentrations.

Relative Standard Deviation of Peak Area

Amino Acids

Concentration

0,25μM 0,5 μM

1 μM

3 μM

5 μM 10 μM 50 μM 100 μM

Glycine 3.42 2.01 6.04 21.44 4.32 6.61 8.84 6.57 Asparagine 5.32 6.93 8.84 22.76 4.23 7.45 6.45 4.99 Leucine 3.94 0.61 16.14 25.37 9.31 11.69 7.16 4.53 Glutamine 4.95 4.99 5.65 20.83 5.61 5.63 8.14 4.60 Threonine 3.94 0.52 5.14 23.01 5.11 11.00 7,83 5.73 Serine 0.73 1.37 13.77 25.64 4.36 7.09 7.49 7.01 Alanine 8.44 3.09 10.52 18.80 3.65 2.35 9.40 3.27 Methionine 11.49 19.60 1.21 21.09 3.61 1.05 10.16 1.52 Proline 1.31 2.59 3.31 19.64 2.08 1.30 7.05 2.32 Lysine 5.53 1.54 4.97 22.87 1.51 5.91 7.56 2.97 Aspartic Acid 4.52 2.56 4.48 21.23 1.15 2.51 7.85 3.28 Histidine 0.54 6.16 4.53 22.56 0.81 1.32 9.53 4.60 Glutamic Acid 4.89 3.36 3.68 22.27 4.42 5.63 5.92 3.94 Tryptophan 8.49 11.06 9.90 23.49 9.09 13.66 1.18 4.84 Tyrosine 4.04 0.80 9.81 26.39 2.24 7.28 1.44 3.45 Isoleucine 4.26 2.24 3.22 19.12 2.87 1.16 7.19 2.50 Phenylalanine 2.81 5.09 3.66 20.97 3.04 1.60 9.86 2.39

(61)

P a g e 60 | 132

4.7 Limit of Detection (LOD) and Linearity Range

Amino Acids

LOD (μM)

LLOL(μM)

Linearity Range

(μM)

Glycine 0.25 0.25 0.25-100 Asparagine 0.25 0.25 0.25-10 Leucine 0.25 0.25 0.25-10 Glutamine 0.25 0.25 0.25-10 Threonine 0.25 0.25 0.25-100 Serine 0.25 0.25 0.25-100 Alanine 0.25 3 0.25-100 Methionine 0.25 1 0.25-100 Proline 0.25 0.25 0.25-5 Lysine 0.25 0.25 0.25-10 Aspartic Acid 0.25 0.25 0.25-10 Histidine 0.25 0.25 0.25-10 Glutamic Acid 0.25 0.25 0.25-10 Tryptophan 0.25 0.25 0.25-10 Tyrosine 0.25 0.25 0.25-10 Isoleucine 0.25 0.25 0.25-5 Phenylalanine 0.25 0.25 0.25-10

Amino acid analysis: comparison and validation of gas chromatography with mass spectrometric detection and liquid chromatography with triple quadrupole mass spectrometric detection. Application in mouse embryonic fibrob

MSc in Analytical Sciences

Amino acid analysis: comparison and validation of gas

chromatography with mass spectrometric detection and

liquid chromatography with triple quadrupole mass

spectrometric detection. Application in mouse

embryonic fibroblast NIH-3T3 cells.

Master Research Thesis

Alexandra Koukou

MSc Chemistry

Analytical Sciences

Master Thesis

Amino acid analysis: comparison and validation of gas

chromatography with mass spectrometric detection and

liquid chromatography with triple quadrupole mass

spectrometric detection. Application in mouse embryonic

fibroblast NIH-3T3 cells.

by

Alexandra Koukou

June 2014

Supervisor:

dr H. Lingeman

Daily Supervisor:

P.

Abstract

Acknowledgements

Contents

Abbreviations

1. Theoretical

1.1 Introduction

1.2 Historical Background

1.3 A novel breakthrough approach

1.4 Derivatization

1.5 Types of Derivatization

1.6 Derivatization modes

1.7 Chloroformate derivatization

2.Experimental

2.1 Chemicals

2.2 Kit components

2.3 Additional equipment

2.4 Storage and Stability

2.5 Cleaning

2.6 Preparation of Eluting Medium

2.7 General Procedure for GC-MS experiments

2.8 General Procedure for LC-MS experiments

2.9 Instruments Settings

 GC/MS

LC/MS

2.10 Biological Samples

Cell culture:

Determination of amino acid uptake and excretion:

Cell lysis and protein determination:

3. GC/MS Results

3.1 Full-SCAN GC/MS

C=200μM/L

Split Ratio=30

Retention Time (min)

Time

Abu

n

d

an

ce

3.2 Selected Ion Monitoring (SIM) for standards amino acids

Retention Time

(min)

Ab

u

n

d

an

ce

(

a.u.)

C=0.01 μM/L

Split Ratio=30

3.3 Repeatability (within a day)

3.4 Intermediate Precision

3.5 Calibration Curves

Alanine

_C-

_N