Analysis of Biogenic Amines by GC/FID and GC/MS

Laura Nakovich

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

In

Chemistry

Dr. Harold M. McNair, Chair Dr. Paul R. Carlier Dr. Larry T. Taylor

July 16, 2003

Blacksburg, Virginia

Key Words: Gas Chromatography, Biogenic Amines, Propyl Chloroformate Derivatives, Mass Spectroscopy

Copyright 2003, Laura Nakovich

Analysis of Biogenic Amines by GC/FID and GC/MS

Laura Nakovich

(Abstract)

Biogenic amines (particularly histamine in the presence of putrescine and

cadaverine) can lead to scombroid food poisoning if levels become too high. Low levels

of these biogenic amines occur naturally, but high levels (FDA sets 50 ppm of histamine

in fish as the maximum allowable level) can lead to scombroid poisoning.

A number of analytical methods have been employed to analyze biogenic amines,

especially High Performance Liquid Chromatography (HPLC). Amines in general are

difficult to analyze by Gas Chromatography (GC) due to their lack of volatility and their

interaction with the GC column often leading to significant tailing and poor

reproducibility. GC methods could be simpler and cheaper than HPLC equipment in

some cases. Biogenic amines need to be derivatized before both GC and HPLC

analyses. The objective of this research was to develop a relatively fast, reproducible

method to derivatize and quantitate biogenic amines in fish at trace levels using GC/FID.

The derivatizing reagent used in the experiments was propyl chloroformate, useful for

aqueous samples. A method for GC/MS was also developed and used to confirm the

identity of six derivatized biogenic amines. To our knowledge no reference spectra for

these derivatives has been published.

It was concluded that best results are obtained using a Cold-On-Column (C.O.C.)

inlet with a short column (15 meters), thick film stationary phase (ZB-5, 1.00µm df), and

with recommendations to cut 40 cm from the inlet end of the column every 25 injections

when using C.O.C. Duplicate samples of Atlantic Salmon were analyzed on days 0, 3,

and 5. Levels of histamine were below 50 ppm for days 0 and 3, but day 5 showed

average levels of 160 pm (cadaverine), 1000 ppm (histamine), and 350 ppm (tyramine).

Good precision of six amine stardards at 50 ppm was shown: heptylamine 5.2%,

putrescine 5.6%, cadaverine 5.0%, histamine 9.9%, tyramine 5.1%, and spermidine 6.2%

RSD.

iv

Acknowledgements

First, I would like to thank God for giving me life and the opportunity not to take

even one day for granted. I would like to thank my research advisor, Dr. McNair, for

giving me the chance to work in a group and lab where I was given enough independence

to learn at my own pace. I thank you for believing in me when I was struggling. I would

like to thank my committee members Dr. Taylor and Dr. Carlier, for carefully reviewing

my thesis and giving me helpful suggestions. Also, I would like to thank many other

professors at Virginia Tech who have taught me so much and encouraged me along the

way, especially Dr. Hanson, Dr. Hasselman, and Dr. Wolfe.

Next I wish to thank all my family, but particularly my parents John and Joan

Nakovich and my brother John for their love and guidance and for never giving up on

me.

I am truly grateful for all the many friends I have made here at Virginia Tech who

have been such a blessing to me. I owe a tremendous thanks to Kari Urias and Dr.

Jennifer Smith; I would have perished long ago without you both. Also, I would like to

thank past and present members of the McNair group for your encouragement, especially

Iris Dadone, Amy Kinkennon, Kevin Schug, Brent Cunningham, Dr. Robert Boggeus,

and Jeff Cark.

Lastly, I would like to acknowledge and thank Phenomenex who supplied the GC

columns I used for this project.

v

Dedication

“Make voyages, attempt them. There’s nothing else.”

-Tennessee Williams

Dedicated to my parents John and Joan Nakovich and my brother John Nakovich.

Thank you for believing in me to help me see that if I work hard enough there are no

limits to what I can do, and for loving me enough to let me go.

vi

Table of Contents

Analysis of Biogenic Amines by GC/FID and GC/MS .................................................. ii

Acknowledgements .......................................................................................................... iv

Dedication .......................................................................................................................... v

Table of Contents ............................................................................................................. vi

Table of Figures.............................................................................................................. viii

Table of Structures ........................................................................................................... x

Table of Tables ................................................................................................................. xi

Chapter 1-Introduction and Background....................................................................... 1

GAS CHROMATOGRAPHY AND AMINE ANALYSIS.......................................................................... 2 DERIVATIZATION OF AMINES............................................................................................................ 3 CARBAMATE DERIVATIVES OF AMINES .......................................................................................... 6

Chapter 2-Experimental................................................................................................. 10

CHEMICALS USED .............................................................................................................................. 11 CHLOROFORMATE DERIVATIZATION OF AMINES ...................................................................... 12 CALIBRATION CURVES ...................................................................................................................... 14 ATLANTIC SALMON EXTRACTION AND DERIVATIZATION ........................................................ 16

Chapter 3-GC/MS Results of Standards....................................................................... 17

INTRODUCTION.................................................................................................................................... 18 AMINE STANDARD DERIVATIZED ................................................................................................... 18 HEPTYLAMINE DERIVATIVE ............................................................................................................ 19 PUTRESCINE DERIVATIVE ................................................................................................................ 21 CADAVERINE DERIVATIVE ............................................................................................................... 23 HISTAMINE DERIVATIVE .................................................................................................................. 26 TYRAMINE DERIVATIVE .................................................................................................................... 29 SPERMIDINE DERIVATIVE ................................................................................................................ 31 DERIVATIVE BLANKS ......................................................................................................................... 33

Chapter 4-GC/FID Results of Standards...................................................................... 36

CALIBRATION CURVES ...................................................................................................................... 37

Chapter 5-Salmon Analysis Results .............................................................................. 44

INTRODUCTION.................................................................................................................................... 45 SALMON RESULTS-DAY 1 ................................................................................................................. 45 SALMON RESULTS-DAY 3 ................................................................................................................. 46 SALMON RESULTS-DAY 5 ................................................................................................................. 48

vii

Chapter 6-Conclusions ................................................................................................... 52

GC/FID ANALYSIS............................................................................................................................. 53 GC/MS ANALYSIS .............................................................................................................................. 53 SALMON ANALYSIS ............................................................................................................................. 54 FUTURE APPLICATIONS .................................................................................................................... 54

Appendix.......................................................................................................................... 56

GC/FID DATA..................................................................................................................................... 57 GC/MS DATA ...................................................................................................................................... 59

References........................................................................................................................ 68

Vita ................................................................................................................................... 70

viii

Table of Figures FIGURE 1. GENERATION OF HISTAMINE, PUTRESCINE, AND CADAVERINE FROM THEIR CORRESPONDING

DECARBOXYLATED AMINO ACIDS. ......................................................................................................... 3 FIGURE 2. ACYLATION OF A SECONDARY AMINE BY AN ACYL CHLORIDE2...................................................... 4 FIGURE 3. FORMATION OF CARBAMATE DERIVATIVES FROM AMINES2. .......................................................... 7 FIGURE 4. FORMATION OF CARBAMATE DERIVATIVES FROM TERTIARY AMINES2. .......................................... 7 FIGURE 5. 50 PPM AMINE STANDARD DERIVATIZED (HEPTYLAMINE 6.51 MIN, PUTRESCINE 9.22 MIN,

CADAVERINE 9.70 MIN, HISTAMINE 9.94 MIN, TYRAMINE 10.63 MIN, SPERMIDINE 12.64 MIN)............ 19 FIGURE 6. DERIVATIZED HEPTYLAMINE, 6.51 MINUTES. .............................................................................. 20 FIGURE 7. HEPTYLAMINE DERIVATIVE GC/MS SPECTRA. ............................................................................ 20 FIGURE 8. DERIVATIZED PUTRESCINE, 9.22 MINUTES. ................................................................................. 22 FIGURE 9. PUTRESCINE DERIVATIVE GC/MS SPECTRA. ............................................................................... 22 FIGURE 10. DERIVATIZED CADAVERINE, 9.70 MINUTES. ............................................................................. 24 FIGURE 11. CADAVERINE DERIVATIVE GC/MS SPECTRA. ............................................................................ 24 FIGURE 12. DERIVATIZED HISTAMINE, AT 9.94 MINUTES............................................................................. 26 FIGURE 13. HISTAMINE DERIVATIVE GC/MS SPECTRA. ............................................................................... 27 FIGURE 14. DERIVATIZED TYRAMINE, 10.63 MINUTES. ............................................................................... 29 FIGURE 15. TYRAMINE DERIVATIVE GC/MS SPECTRA................................................................................. 30 FIGURE 16. DERIVATIZED SPERMIDINE, 12.64 MINUTES. ............................................................................. 31 FIGURE 17. SPERMIDINE DERIVATIVE GC/MS SPECTRA............................................................................... 32 FIGURE 18. DERIVATIVE BLANK RUN BY GC/MS, DILUTED 5:1. .................................................................. 34 FIGURE 19. DERIVATIVE BLANK RUN BY GC/MS, NOT DILUTED 5:1............................................................ 35 FIGURE 20. HEPTYLAMINE CALIBRATION CURVE ........................................................................................ 40 FIGURE 21. PUTRESCINE CALIBRATION CURVE............................................................................................ 40 FIGURE 22. CADAVERINE CALIBRATION CURVE .......................................................................................... 41 FIGURE 23. HISTAMINE CALIBRATION CURVE. ............................................................................................ 41 FIGURE 24. TYRAMINE CALIBRATION CURVE. ............................................................................................. 42 FIGURE 25. SPERMIDINE CALIBRATION CURVE............................................................................................ 43 FIGURE 26. DAY 1 SALMON SAMPLE A, RUN 3 OUT OF 5. ............................................................................ 45 FIGURE 27. DAY 1 SALMON SAMPLE B, RUN 3 OUT OF 5.............................................................................. 46 FIGURE 28. DAY 3 SALMON SAMPLE A, RUN 3 OUT OF 5; CADAVERINE 11.60, HISTAMINE 11.88, AND

TYRAMINE 12.59 MINUTES................................................................................................................... 46 FIGURE 29. DAY 3 SALMON SAMPLE B, RUN 3 OUT OF 5; CADAVERINE 11.61, HISTAMINE 11.88, AND

TYRAMINE 12.59 MINUTES................................................................................................................... 47 FIGURE 30. DAY 5 SALMON SAMPLE A, RUN 3 OUT OF 5; PUTRESCINE 10.96, CADAVERINE 11.47, HISTAMINE

11.73, AND TYRAMINE 12.45 MINUTES. ............................................................................................... 49 FIGURE 31. DAY 5 SALMON SAMPLE B, RUN 3 OUT OF 5; PUTRESCINE 10.96, CADAVERINE 11.47, HISTAMINE

11.73, AND TYRAMINE 12.44 MINUTES. ............................................................................................... 49 FIGURE 32. RUN 3 OF 5, 100 PPM AMINE MIX (HEPTYLAMINE 8.30, PUTRESCINE 11.11, CADAVERINE 11.61,

HISTAMINE 11.88, TYRAMINE 12.59, SPERMIDINE 16.58 MINUTES). .................................................... 57 FIGURE 33. DERIVATIVE BLANK RUN WITH UNKNOWN PEAKS AT 13.42 AND 14.90 MINUTES. ..................... 57 FIGURE 34. RUN 3 OF 5, 50 PPM AMINE MIX (HEPTYLAMINE 8.29, PUTRESCINE 11.11, CADAVERINE 11.61,

HISTAMINE 11.87, TYRAMINE 12.59, SPERMIDINE 16.57 MINUTES). .................................................... 57 FIGURE 35. RUN 3 OF 5, 50 PPM AMINE MIX WITH HOT INLET 200�C (HEPTYLAMINE 8.86, PUTRESCINE 11.97,

CADAVERINE 12.53, HISTAMINE 12.85, TYRAMINE 13.89 MINUTES). ................................................... 58 FIGURE 36. RUN 3 OF 5, 20 PPM AMINE MIX (HEPTYLAMINE 8.28, PUTRESCINE 11.11, CADAVERINE 11.60,

HISTAMINE 11.88, TYRAMINE 12.59, SPERMIDINE 16.57 MINUTES). .................................................... 58 FIGURE 37. RUN 3 OF 5, 10 PPM AMINE MIX (8.293 HEPTYLAMINE, 11.11 PUTRESCINE, 11.61 CADAVERINE,

11.884 HISTAMINE, 12.601 TYRAMINE, 16.583 MINUTES SPERMIDINE). .............................................. 58 FIGURE 38. DAY 3 SALMON SAMPLE A, GC/MS RUN, CADAVERINE 9.67, HISTAMINE 9.91, AND TYRAMINE

10.60 MINTUES. ................................................................................................................................... 59

ix

FIGURE 39. DAY 3 SALMON SAMPLE A, GC/MS SPECTRA OF CADAVERINE 9.67 MINUTES.......................... 59 FIGURE 40. DAY 3 SALMON SAMPLE A, GC/MS SPECTRA OF HISTAMINE 9.91 MINUTES. ............................ 60 FIGURE 41. DAY 3 SALMON SAMPLE A, GC/MS SPECTRA OF TYRAMINE 10.60 MINTUES............................ 60 FIGURE 42. DAY 3 SALMON SAMPLE B, GC/MS RUN, CADAVERINE 9.68, HISTAMINE 9.91, TYRAMINE 10.60

MINUTES. ............................................................................................................................................. 61 FIGURE 43. DAY 3 SALMON SAMPLE B, GC/MS SPECTRA OF CADAVERINE 9.68 MINUTES. ......................... 61 FIGURE 44. DAY 3 SALMON SAMPLE B, GC/MS SPECTRA OF HISTAMINE 9.91MINUTES. ............................. 62 FIGURE 45. DAY 3 SALMON SAMPLE B, GC/MS SPECTRA OF TYRAMINE 10.60 MINUTES............................ 62 FIGURE 46. DAY 5 SALMON SAMPLE A, GC/MS RUN, PUTRESCINE 9.20, CADAVERINE 9.67, HISTAMINE 9.91,

TYRAMINE 10.60 MINUTES................................................................................................................... 63 FIGURE 47. DAY 5 SALMON SAMPLE A, GC/MS SPECTRA OF PUTRESCINE 9.20 MINUTES. .......................... 63 FIGURE 48. DAY 5 SALMON SAMPLE A, GC/MS SPECTRA OF CADAVERINE 9.70 MINUTES.......................... 64 FIGURE 49. DAY 5 SALMON SAMPLE A, GC/MS SPECTRA OF HISTAMINE 9.91 MINUTES. ............................ 64 FIGURE 50. DAY 5 SALMON SAMPLE A, GC/MS SPECTRA OF TYRAMINE 10.60 MINUTES............................ 65 FIGURE 51. DAY 5 SALMON SAMPLE B, GC/MS RUN, PUTRESCINE 9.20, CADAVERINE 9.68, HISTAMINE 9.92,

TYRAMINE 10.61 MINUTES................................................................................................................... 65 FIGURE 52. DAY 5 SALMON SAMPLE B, GC/MS SPECTRA OF PUTRESCINE 9.20 MINUTES. .......................... 66 FIGURE 53. DAY 5 SALMON SAMPLE B, GC/MS SPECTRA OF CADAVERINE 9.67 MINUTES. ......................... 66 FIGURE 54. DAY 5 SALMON SAMPLE B, GC/MS SPECTRA OF HISTAMINE 9.91 MINUTES. ............................ 67 FIGURE 55. DAY 5 SALMON SAMPLE B, GC/MS SPECTRA OF TYRAMINE 10.61 MINUTES............................ 67

x

Table of Structures STRUCTURE 1. N-METHYL-BIS[TRI-FLUOROACETAMIDE] (MBTFA)…………………………….4 STRUCUTRE 2. N,O-BIS(TRIMETHYLSILYL)TRIFLUOROACETAMIDE(BSTFA)………………....5 STRUCTURE 3. TRIMETHYLCHLOROSILANE (TMCS)………………………………………………6 STRUCTURE 4. N-METHYL-N-(TERT-BUTYLDIMETHYLSILYL)TRIFLUOROACETAMIDE (MTBSTFA)………………………………………………………………………………………………….6

xi

Table of Tables TABLE 1. SIX-AMINE STANDARD SOLUTION................................................................................................ 11 TABLE 2. STRUCTURES OF SIX AMINES......................................................................................................... 12 TABLE 3. GC CONDITIONS........................................................................................................................... 13 TABLE 4. GC/MS CONDITIONS .................................................................................................................... 14 TABLE 5. CONCENTRATION OF AMINE STANDARDS IN CALIBRATION CURVES ............................................ 15 TABLE 6. DERIVATIZED HEPTYLAMINE-PROPOSED FRAGMENT IONS .......................................................... 21 TABLE 7. DERIVATIZED PUTRESCINE-PROPOSED ION FRAGMENTS .............................................................. 23 TABLE 8. DERIVATIZED CADAVERINE-PROPOSED ION FRAGMENTS ............................................................ 25 TABLE 9. DERIVATIZED HISTAMINE-PROPOSED ION FRAGMENTS................................................................ 28 TABLE 10. DERIVATIZED TYRAMINE-PROPOSED ION FRAGMENTS .............................................................. 30 TABLE 11. DERIVATIZED SPERMIDINE-PROPOSED ION FRAGMENTS ............................................................ 33 TABLE 12. 100 PPM AMINE STANDARD........................................................................................................ 37 TABLE 13. 50 PPM AMINE STANDARD.......................................................................................................... 37 TABLE 14. 50 PPM AMINE STANDARD*, HOT INLET 200°C ......................................................................... 38 TABLE 15. 20 PPM AMINE STANDARD.......................................................................................................... 39 TABLE 16. 10 PPM AMINE STANDARD.......................................................................................................... 39 TABLE 17. DAY 3, SALMON SAMPLE A* ...................................................................................................... 47 TABLE 18. DAY 3, SALMON SAMPLE B*....................................................................................................... 48 TABLE 19. DAY 5, SALMON SAMPLE A* ...................................................................................................... 50 TABLE 20. DAY 5, SALMON SAMPLE B*....................................................................................................... 50 TABLE 21. DAY 5 SALMON-BIOGENIC AMINE LEVELS ................................................................................ 51

1

Chapter 1-Introduction and Background

2

Gas Chromatography and Amine Analysis

Gas chromatography (GC) has been one of the most versatile and widely

applicable techniques leading the field of analytical chemistry over the last forty years1.

The popularity of GC stems from its fast, simple, relatively inexpensive, and reproducible

nature1. As a method used to separate volatile and semi-volatile organic compounds, GC

can be utilized in numerous analyses including petrochemical1, environmental,

pharmaceutical, and food science, to name a few.

In GC the analyte is carried through the column by a mobile phase composed of

an inert gas such as helium or hydrogen1. As the vaporized analyte travels through the

column it interacts with the liquid stationary phase1. Depending on the analytes’

solubility for the stationary phase, they will separate and elute, from the column1. Upon

elution the analytes enter a detector, which produces an electrical signal1. This signal is

sent to a data system that generates an image, called a chromatogram, displaying the

analyte peaks1. Certain analytes, specifically nitrogen containing compounds (i.e.

aliphatic primary, secondary, and tertiary amines), can be difficult to detect using GC

because there is significant adsorption of the basic amines on the often acidic column as

well as decomposition of the analyte2. It would be advantageous to use GC in many

applications that analyze amine-containing compounds if a reproducible, reliable method

could be developed. An application of particular interest concerning amine analysis is

the detection and quantification of biogenic amines. Typically, the decarboxylation of

amino acids produces biogenic amines3 as shown in Figure 1.

3

H

CH

COOH

NH2

N

NCH2

H

NH2

CH2

CH2

N

N

CHHOOC

NH2(CH2)4 NH2 NH2 (CH2)5 NH2

HOOC

NH2(CH2)3CH NH2 (CH2)4 NH2NH2

CO2

CO2

CO2

L-histidine histamine

L-lysine cadaverine

putrescineL-ornithine

+

+

+

Figure 1. Generation of histamine, putrescine, and cadaverine from their corresponding decarboxylated amino acids.

The formation of biogenic amines can be facilitated by the production of bacteria4

in foods like cheese, wine, meat, and especially fish5,6. In fact, biogenic amines, namely

histamine, are used as markers for the degradation of fish7. The analysis of biogenic

amines is important because the degradation of fish can lead to Scombroid fish

poisoning8, if decomposition is not detected before the fish is sold in markets or prepared

in restaurants.

Derivatization of Amines

Although historically amines present difficulties in GC analysis, it is possible to

detect them if they are derivatized prior to investigation2. There are several advantages

4

of derivatizing amines in that it enables them to become volatile enough for GC analysis,

improves peak shape by reducing tailing, increases sensitivity and selectivity, and

enhances overall separation2. A number of methods can be employed to derivatize

amines such as acylation, silylation, and carbamate formation for example, as Kataoka

explains in a comprehensive review2.

Primary and secondary amines can be derivatized by acylation reactions2 where

an acyl group is added to the amine2. This can be achieved via the donation of an acyl

group, like an acid anhydride, acyl chloride, acyl imidazole, or an acyl amide2 (see Figure

2).

R

RNH CH3 C Cl

OR

RN C

O

CH3 HCl+ +

Figure 2. Acylation of a secondary amine by an acyl chloride2.

Advantages of using acylation as a derivatization technique for amines are that the

reaction occurs readily with mild conditions, and not only amines but other active

substituents like phenols, thiols, and hydroxyls can become derivatized2. One

particularly beneficial acylating reagent is an acylimidazole called N-methyl-

bis(trifluoroacetamide) (MBTFA)2 shown in Strucutre 1.

C

O

C

O

CF3 N CF3

CH3

Structure 1. N-Methyl-bis[trifluoroacetamide]9(MBTFA).

5

When using MBTFA it is not necessary to remove acid by-products that may destroy the

column, as it is when using acid anhydrides and acyl chlorides in acylation reactions2.

Additionally, MBTFA can be used in conjunction with other derivatizing processes, such

as trimethylsilylation, to N-selectively acylate hydroxyamino molecules10.

A particularly useful reagent of choice for derivatizing amines by silylation is

N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)11 shown in Structure 2.

C

O

Si CH3CH3

CH3

CH3

CH3

CH3C

F

F

F

SiN

Structure 2. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)9.

As is true with other silylating reagents, BSTFA derivatizes hydroxyl and carboxyl

groups under moisture free conditions2. Primary amines are more readily derivatized

than secondary amines2. Trimethylchlorosilane (TMCS), as shown in Structure 3, is an

effective catalyst added to silylation reactions to facillitate the amino group

derivatization2. It should be emphasized that silylation reactions are most effective under

anhydrous reaction conditions, and therefore aqueous samples are not recommended for

such reactions2. It is possible that silyl groups may be added to either or both protons in a

primary amine upon derivatization so that mono- and/or di- N-trimethylsilyl (TMS)

derivatives may form2.

6

Si

CH3

CH3CH3

Cl

Structure 3. Trimethylchlorosilane (TMCS)9.

Another useful reagent for silylation reactions with amines is N-methyl-N-(tert-

butyldimethylsilyl)trifluoroacetamide (MTBSTFA)2 as shown in Structure 4. The

advantage of using MTBSTFA is that it generates derivatives that are orders of

magnitude more stable to hydrolysis than TMS derivatives2. This is attributed to the

protection of the silyl group from moisture by the bulky tert-butyl group of the

derivative2. Generally, t-BDMS derivatives are one to two orders of magnitude more

sensitive for GC analysis than corresponding TMS derivatives12.

CH33

C Si CN

O

CF3

CH3 CH3

CH3

Structure 4. N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)2.

Carbamate Derivatives of Amines

One of the most attractive methods used to derivatize amines for GC analysis is

through carbamate formation2. Primary, secondary, and tertiary amines as well as

phenol, thiol, and imidazole groups are derivatized with alkyl chloroformate reagents2.

7

In the alkyl chloroformate reaction, an alkyl group is added to the nitrogen of the amine

as shown in Figure 3. An advantage of preparing carbamate derivatives is that the

technique is not sensitive to moisture so aqueous samples can be derivatized, as

demonstrated by Ugland, et al.13 It is also possible to derivatize tertiary amines using

alkyl chloroformate derivatives (see Figure 4).

Figure 3. Formation of carbamate derivatives from amines2.

RNH

R'

RN

R'C

O

C

O

OR''

R''

R''O HClCl

Alkylchloroformate Carbamate

=methyl-, ethyl-, propyl-, butyl-, iso-butyl-, pentafluorobenzyl group

+ +

Amine

CH2

F

F

F F

F

O C

O

ClN

R

R

RN

R

RCH2

F

F

F F

F

O C

O

RCl+ +

Figure 4. Formation of carbamate derivatives from tertiary amines2.

The use of chloroformates as derivatizing reagents for amines has been

investigated for over thirty years. One of the earlier applications that utilized

chloroformate derivatization was demonstrated by Westley and Halpern14. In 1968 they

successfully derivatized asymmetric amino and hydroxyl groups with menthyl

chloroformate and analyzed them by GC14. The procedures used by Westley and Halpern

called for reaction times of over three hours14.

Chloroformate reagents can be especially useful in derivatizing problematic

tertiary amines2. A study conducted in 1976 focused on using

8

trichloroethylchloroformate to derivatize the tertiary amine pethidine, a pain reliever15.

In fact trichloroethylchloroformate was found to be a more effective derivatizing reagent

than pentafluorobenzyl chloroformate because it reacted three times faster with

pethidine16. The total derivatization time took around twenty minutes15. The detection

limits of pethidine in this method were 100 ppb using an electron capture detector15.

Further experiments have been conducted using various chloroformates in the

derivatization of tertiary amines. Pentafluorobenzyl chloroformate has been used to

derivatize tertiary amines as was mentioned above17. Specific applications where

derivatization of tertiary methyl amines is of significance are found in the analysis of

drugs containing methylamine17. Examples of these medications are imipramine and

trimipramine18, tri-cyclic antidepresants, and diphenhydramine and Recipavrin�,

antihistamines19. In 1978 the work of Sternson and Cooper showed that tertiary amino

compounds not containing the pyridine substituent group reacted well with chloroformate

reagents17. The time for the reaction was 50 minutes17. A gas chromatograph with an

electron capture detector was used for analysis, as well as a GC coupled with a mass

spectrometer (GC-MS) for further confirmation of the carbamate derivatives17.

Pentafluorobenzyl chloroformate is not effective for derivatizing dimethyl amine

alkane compounds containing a pyridine ring17. According to Sternson and Cooper there

may be a ring opening reaction involving the pyridine ring and the chloroformate reagent

such that derivatization of tertiary methylamines is hindered17. A brown tary mass is

what resulted in their experiment. It was determined that less than 10% of the

dimethylaminoalkane analyte was derivatized to the corresponding carbamate, and the

reaction was not reproducible17.

There are other chloroformate derivatization studies that have been performed

using amines as well. For example, in 1992 Hušek et al. were interested in determining

whether or not amines and aminoalcohols would react as carboxylic acids do when

derivatized with chloroformates20. In previous experiments, carboxylic acids had been

successfully derivatized into esters using chloroformates21. Hušek’s earlier work also

showed that it was possible to derivatize amino and hydroxy acids with chloroformates22.

Methylchloroformate (MCF) and ethylchloroformate (ECF) were the reagents of

choice for Hušek et al. in the amine and aminoalcohol experiments20. There were two

9

different methods employed in the studies20. The first used MCF or ECF to derivatize the

amines and chloroform as the organic liquid extractant20. The second method

incorporated a mixture of acetonitrile-water-methanol-pyridine before the addition of

either MCF or ECF and then lastly adding chloroform for the organic liquid extraction20.

It should also be noted that both methods used sodium bicarbonate in the derivatization of

amines and aminoalcohols, however in the second method the sodium bicarbonate was

added last while in the first method it was added first to the amine sample20.

As the results of the Hušek study indicated, both methods were successful in the

derivatization of the analytes of interest20. There were some differences in the derivatives

of spermine and spermidine in that reaction yields were acceptable with the use of only

method one20. Method two was the preferred technique for derivatization of the acidic

analytes20. An interesting note is that carboxylic acid groups next to alcoholic groups can

be esterified20. On side chains of the compounds of interest the primary amines react

more readily than the secondary amines, which require a longer reaction time20.

Esterification of the phenolic groups in the molecules was also shown to occur20. In

general, the reaction time for the derivatization was around ten minutes20.

The goal of the study conducted in this thesis was to develop a rapid,

reproducible, trace level quantitative method to derivatize and detect biogenic amines by

GC/FID and GC/MS.

10

Chapter 2-Experimental

11

Chemicals Used

The following biogenic amines were analyzed: heptylamine, putrescine,

cadaverine, histamine, tyramine, and spermidine. All standard amine mixtures were

prepared using HPLC grade water (Burdick & Jackson, Muskegon, MI) and a Nalgene

chemical-resistant propylene 50 mL volumetric flask. The purity of the standards and

their manufacturers are listed in Table 1.

Table 1. Six-Amine Standard Solution

Amine Purity Supplier

Heptylamine 99% Aldrich;

Milwaukee, WI

Putrescine 98% Sigma-Aldrich; St.

Louis, MO

Cadaverine 95% Aldrich;

Milwaukee, WI

Histamine 96% Aldrich;

Milwaukee, WI

Tyramine 99% Aldrich;

Milwaukee, WI

Spermidine 99% Aldrich;

Milwaukee, WI

The structures, molecular weight, boiling points, and pKa’s for the above amines

are shown in Table 2.

12

Table 2. Structures of Six Amines

Amine Molecular Weight (amu)

Boiling Point (�C)

Structure pKa123, pKa2

Heptylamine 115.22 154-156 NH2 10.67 Putrescine (1,4-butanediamine)

88.15 158-16023 NH2

NH2

9.35, 10.80

Cadaverine (1,5-pentanediamine)

102.18 178-180 NH2 NH2 10.05, 10.93

Histamine 111.15 167 N

NN H 2

H

6.04, 9.75

Tyramine 137.18 175-181 NH2

OH

9.74, 10.52

Spermidine 145.2523 128-130 NH2

NH

NH2 10.5124

Chloroformate Derivatization of Amines

Carbamate derivatives of the biogenic amines listed above were created using

propyl chloroformate as the derivatizing reagent. The derivatization procedure was based

on the methodology of Ugland et al13. All of the following procedures were preformed at

room temperature. To a 200�L aliquot of a standard amine solution (varying

concentrations), 200�L of a 1:4 mixture of chloroform (HPLC grade; Burdick & Jackson,

Muskegon, MI) and iso-octane (certified A.C.S.; Fisher Scientific Company, Fair Lawn,

NJ) was added to the plastic sample prep vial. To ensure that the solution remained at a

constant pH, 50�L of pH 12.2 K2CO3-KHCO3 buffer was also added to the mix. Finally,

1�L of propyl chloroformate (98% purity, Aldrich, St. Louis, MO) was added to the

solution. The solution was homogenized by a vortex mixer (Barnstead/Thermolyne,

Maxi Mix Plus; Dubuque, IA) for 1 minute. Next the sample vial was placed in an IEC

Spinette Centrifuge (Needham Heights, MA) for 5 minutes. Prior to GC analysis, 100µL

of the top organic layer was diluted by removing and depositing it into a glass sample

vial, containing 400µL of the chloroform/iso-octane solvent mixture. One microliter of

this solution was injected into the GC.

13

GC/FID Analysis

A Phenomenex ZB-5 (15m x 0.25mm id x 1.00�m df) capillary column

(Torrance, CA) was installed in an Hewlett Packard 6890 Gas Chromatograph (Little

Falls, DE) equipped with a flame ionization detector. The column was installed into a

Cold-On-Column (COC; Hewlett Packard, Palo Alto, CA) inlet suitable for a 0.25mm

I.D. column. This column was conditioned at 300�C overnight to ensure it was clean.

Blank runs were made before any samples were injected to further ensure that the system

was stable and uncontaminated. The GC conditions are listed in Table 3.

Table 3. GC Conditions

Temperature Program 50�C (1 min hold) to 280�C (5 min hold) @20�C/min

Carrier Gas Helium-59cm/sec Injection Volume 1�L,manual injection

Inlet Setting Oven Tracking Detector FID at 250�C

One microliter of the derivatized amine standard was injected into the GC manually for

five replicate injections. It should also be noted that standard solutions of each individual

amine were analyzed by GC/FID and GC/MS to confirm the identities of the peaks in the

mix based on retention times and spectra. The percent Relative Standard Deviation

(%RSD) was calculated using peak areas for all replicate injections.

GC/MS Analysis

An Agilent HP5-MS (15m x 0.25mm id x 0.25µm df) column was installed in an

HP-6890 GC coupled with an HP-5973 Mass Selective Detector. Before the column was

connected to the MSD it was conditioned overnight at 300°C. A 50 ppm derivatized

amine standard was injected into the GC/MS for analysis and confirmation of the identity

of the derivatives. The conditions used are specified in Table 4. The GC/MS system was

14

not equipped with a cold-on-column inlet so a hot inlet was used with a 2 mm internal

diameter, straight tube liner not containing glass wool.

Table 4. GC/MS Conditions

Temperature Program 50�C (1 min hold) to 280�C (5 min hold) @20�C/min

Carrier Gas Helium-59cm/sec Injection Volume 1�L,manual injection

Injection Port Temperature 200°C Split Ratio 30:1

Transfer Line 280�C

Calibration Curves

A standard amine mix of 2000 ppm was prepared in a 50 mL volumetric Nalgene

flask. The 2000 ppm solution was diluted using serial dilution to produce the following

concentrations: 500, 250, 100, and 50 ppm. Each of these solutions was derivatized and

diluted as specified earlier. Exact concentrations of each component in the standard mix

are shown in Table 5. Then the derivatives were analyzed by GC/FID with the conditions

described in Table 3. The RSD was calculated and calibration curves were constructed

(see Chapter 4) based on these trace concentrations run five times each.

15

Table 5. Concentration of Amine Standards in Calibration Curves

Amine Level 1 (ppm) Level 2 (ppm) Level 3 (ppm) Level 4 (ppm)

Heptylamine 78 39 16 7.8

Putrescine 88 44 18 8.8

Cadaverine 87 44 18 8.7

Histamine 91 45 18 9.1

Tyramine 93 47 19 9.3

Spermidine 93 46 19 9.3

16

Atlantic Salmon Extraction and Derivatization

A fillet of fresh Atlantic Salmon was purchased from Kroger for a five day study.

After purchase it was immediately brought into the lab and analyzed as descrbed below.

The method is based on part in work done by Antoine et al25. It should be noted that the

salmon was analyzed in duplicate. Fifteen grams of the salmon were weighed on a

Mettler AE 260 Delta Range Scale (Columbus, OH). The fish was then chopped up into

very small fragments with a knife and placed into a plastic Nalgene bottle, which

contained 50 mL of methanol (Burdick and Jackson, Muskegon, MI) and 50 mL of HPLC

grade water. The solution in this bottle was homogenized by vortex mixing for two

minutes. The salmon mix was then placed into a 45°C water bath for 45 minutes. Next

the extract was cooled to 30°C in a cold water bath. Upon cooling, a portion of the mix

was centrifuged in plastic tubes for 20 minutes. Then 200 µL of the supernate was used

for derivatization by the same propyl chloroformate derivatization method used on the

standard amines, however the sample was not diluted 1:5 after derivatization. Lastly, the

derivatized salmon extracts were analyzed by the GC/FID method five times and one

time by the GC/MS method. The entire salmon extraction and derivatization process was

repeated in duplicate on days three and five. After the initial salmon analysis on day one

and until day five, the salmon fillet was stored at 4°C. The resulting data from the

salmon study is included and analyzed in Chapter 5-Salmon Analysis Results.

17

Chapter 3-GC/MS Results of Standards

18

Introduction

It was necessary to perfom GC/MS analysis on the biogenic amines once they had

been derivatized to confirm that the derivatives actually existed. It was not sufficient to

use only single standards of each derivatized biogenic amine and retention times for

confirmation of peak identity because it would not have ensured that the peak was

actually the propyl chloroformate derivative and not a contaminant or degradation

product. To my knowledge there is no spectra libraray containing the propyl

chloroformate derivatives of the biogenic amines examined in this study. Therefore, it

was important to perform a comprehensive analysis of the derivatized amine ion

fragments.

Amine Standard Derivatized

The chromatogram of the 50 ppm derivatized amine standard is shown in Figure 5. There are eleven major peaks that appear, as well as a few minor ones that

should be examined. Analysis of individual peaks is discussed in depth in this chapter.

19

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

320000

Time-->

Abundance

TIC: 13MAY00.D

1.82

2.71

6.51

8.27

9.22 9.70

9.94

10.63

11.28

12.06

12.64

Figure 5. 50 ppm Amine Standard Derivatized (heptylamine 6.51 min, putrescine

9.22 min, cadaverine 9.70 min, histamine 9.94 min, tyramine 10.63 min, spermidine

12.64 min).

Heptylamine Derivative

The chromatogram above has been blown up to show the heptylamine derivative

peak at 6.51 minutes (see Figure 6). The peak shape is gaussian with no evidence of

fronting or tailing. There is a small peak preceding the heptylamine peak, which may be

an impurity.

20

6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.100

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

320000

Time-->

Abundance

TIC: 13MAY00.D 6.51

Figure 6. Derivatized heptylamine, 6.51 minutes.

Upon examination of the heptylamine GC/MS spectra it is apparent that

heptylamine did in fact become derivatized. As shown in Figure 7 there are several main

ions to consider.

20 40 60 80 1001201401601802002202402602803003203403603804004200

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

32000

34000

36000

38000

m/z-->

Abundance

Average of 6.498 to 6.522 min.: 13MAY00.D (-)116

43

15874

20199

140 356177 281257 402223 327240

Figure 7. Heptylamine derivative GC/MS spectra.

21

The fragments and ions for the heptylamine derivative are listed in Table 6.

Table 6. Derivatized Heptylamine-Proposed Fragment Ions

Fragment Ion (molecular

weight)

Suggested Structure

Molecular Ion 201

CH26HN C

O

OCH2CH2CH3CH3

+.

Derivative

Fragment Ion

160

CH26HN C

O

CH3 OH

H.. +

Derivative

Fragment Ion

104

NC

O

H

OCH2CH2CH3

H

H

.

.+

Derivatizing

Reagent Fragment

Ion

43 CH3CHCH3+

Putrescine Derivative

The putrescine peak at 9.22 minutes is shown in Figure 8.

22

180000

160000

120000

100000

9.00 9.05 9.10 9.15 9.20 9.25 9.30 9.350

20000

40000

60000

80000

140000

Time-->

Abundance

TIC: 13MAY00.D

9.22

Figure 8. Derivatized Putrescine, 9.22 minutes.

There is minimal tailing and no fronting on the analyte peak. There does not appear to be

evidence of degradation products. The spectra for putrescine contains fragments as proof

that the derivative was formed (see Figure 9).

2 0 4 0 6 0 8 01 0 01 2 01 4 01 6 01 8 02 0 02 2 02 4 02 6 02 8 03 0 03 2 03 4 03 6 03 8 04 0 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0

4 0 0 0

4 5 0 0

5 0 0 0

5 5 0 0

6 0 0 0

6 5 0 0

7 0 0 0

7 5 0 0

8 0 0 0

8 5 0 0

9 0 0 0

9 5 0 0

1 0 0 0 0

1 0 5 0 0

1 1 0 0 0

1 1 5 0 0

1 2 0 0 0

1 2 5 0 0

m /z -->

A b u n d a n ce

A ve ra g e o f 9 .2 1 6 to 9 .2 2 6 m in .: 1 3 M A Y 0 0 .D (-)1 56

1 1 6

4 3

7 0

9 8 2 0 1

1 7 3

2 6 0

1 3 9 3 4 12 8 1 3 1 32 37 3 8 6 4 15

Figure 9. Putrescine derivative GC/MS spectra.

23

The fragments of the putrescine derivative are located in Table 7. It is clear that the

molecular ion is present. The remaining derivative fragments listed are further evidence

that the parent compound was broken apart in the GC/MS analysis.

Table 7. Derivatized Putrescine-Proposed Ion Fragments

Fragment Ion

(molecular

weight)

Suggested Structure

Molecular

Ion

260

C

O

OCH2CH2CH3CH3 CH2

4NH NHC

O

OCH2CH2

+.

Derivative

Fragment

Ion

201

C

O

OCH2CH2CH3CH2

4NH NHCO

+

Derivative

Fragment

Ion

173

C

O

OCH2CH2CH3CH2

4NH NH

.

.+

Derivative

Fragment

Ion

116

C

O

CH3CH2CH2O NH CH2+

Derivatizing

Reagent

Fragment

Ion

43 CH3CHCH3+

Cadaverine Derivative

The enlarged in cadaverine peak from Figure 5 is shown in Figure 10. There is no

fronting and only slight tailing on the analyte peak. Also, there do not appear to be any

24

degradation or decomposition product peaks from cadaverine. As the GC/MS spectra in

Figure 11 shows there are several ions of interest that confirm the prescence of

derivatized cadaverine.

180000

160000

120000

100000

9.50 9.55 9.60 9.65 9.70 9.75 9.80 9.850

20000

40000

60000

80000

140000

Time-->

Abundance

TIC: 13MAY00.D

9.70

Figure 10. Derivatized Cadaverine, 9.70 minutes.

20 40 60 80 100120140160180200220240260280300320340360380400420

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

m/z-->

Abundance

Average of 9.694 to 9.706 min.: 13MAY00.D (-)116

43

84

170 215

18714362274

329 405355297249

Figure 11. Cadaverine derivative GC/MS spectra.

25

Table 8 shows the fragment structures of the cadaverine derivative GC/MS

spectra.

Table 8. Derivatized Cadaverine-Proposed Ion Fragments

Fragment Ion

(molecular

weight)

Suggested Structure

Molecular

Ion

274 O

OCH2CH2CH3CH2 5

NH CCH3 NHC

O

OCH2CH2

+.

Derivative

Fragment

Ion

215 O

CH2 5NH CCH3 NHC

O

OCH2CH2 +

.

.

Derivative

Fragment

Ion

143

CH2CH3 NHC

O

OCH2CH2 CHCH2+

Derivative

Fragment

Ion

116

CH2CH3 C

O

OCH2CH2 NH+

Derivatizing

Reagent

Rragment

Ion

43 CH3CHCH3+

26

Histamine Derivative

The histamine peak from Figure 5 is shown in Figure 12. The peak shape is good

with no fronting or tailing.

9.75 9.80 9.85 9.90 9.95 10.00 10.05 10.10 10.15 10.20

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

32000

34000

36000

38000

40000

42000

44000

46000

48000

Time-->

Abundance

TIC: 13MAY00.D

9.94

Figure 12. Derivatized Histamine, at 9.94 minutes.

The GC/MS spectra (Figure 13) from the histamine derivative peak is proof that

histamine was derivatized. shows the histamine derivative fragments. Histamine is one

of the more difficult analytes to derivatize because it contains primary, secondary, and

tertiary amines. It is interesting to note that the spectra (Figure 13) provides evidence to

confirm that two of the three nitrogens in histamine were derivatized with the propyl

chloroformate reagent.

27

20 40 60 80 1001201401601802002202402602803003203403603804000

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

m/z-->

Abundance

Average of 9.928 to 9.943 min.: 13MAY00.D (-)180

82

43

135

224 403197

341110283 325153 38725365 302 371

Figure 13. Histamine derivative GC/MS spectra.

28

Table 9. Derivatized Histamine-Proposed Ion Fragments

Fragment Ion (molecular weight) Suggested Structure

Molecular Ion 283

N

N

C

O

NH

C

O

OCH2CH2CH3

OCH2CH2CH3

+.

Derivative Fragment Ion 197 N

N

C

O

NH

H

OCH2CH2CH3

+.

Derivative Fragment Ion 180

N

NCH CH2

C

O

OCH2CH2CH3

+.

Derivative Fragment Ion 168

N

NCH3

C

O

OCH2CH2CH3

+.

Derivatizing Reagent

Fragment Ion

43 CH3CHCH3+

29

Tyramine Derivative

The tyramine derivative peak from Figure 5 is shown (Figure 14). There is a

small amount of tailing on the peak and no fronting.

1 0 .4 01 0 .4 51 0 .5 01 0 .5 51 0 .6 01 0 .6 51 0 .7 01 0 .7 51 0 .8 01 0 .8 51 0 .9 00

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

3 0 0 0 0

3 5 0 0 0

4 0 0 0 0

4 5 0 0 0

5 0 0 0 0

5 5 0 0 0

6 0 0 0 0

6 5 0 0 0

7 0 0 0 0

7 5 0 0 0

8 0 0 0 0

8 5 0 0 0

9 0 0 0 0

9 5 0 0 0

T im e -->

A b u n d a n ce

T IC : 1 3 M A Y 0 0 .D

1 0 .6 3

Figure 14. Derivatized Tyramine, 10.63 minutes.

The following GC/MS spectra is sufficient proof to show that tyramine was

derivatized (Figure 15). Further analysis of the spectra (Table 10) shows the various

fragments of the tyramine derivative.

30

20 40 60 80 100120140160180200220240260280300320340360380400

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

m/z-->

Abundance

Average of 10.621 to 10.635 min.: 13MAY00.D (-)120

43

162

77206

104 145 309 331253223180 37128061 400

Figure 15. Tyramine derivative GC/MS spectra.

Table 10. Derivatized Tyramine-Proposed Ion Fragments

Fragment Ion (molecular weight) Suggested Structure

Molecular Ion 223

OCH2CH2CH3

H

C

O

NH

O

+.

Derivative Fragment Ion 120

HO

CH2 CH

+.

Derivative Fragment Ion 107

OH

CH2+

Derivatizing Reagent

Fragment

43 CH3CHCH3+

31

For tyramine only the amine, and not the hydroxyl group, was derivatized.

Spermidine Derivative

The spermidine peak from Figure 5 is shown zoomed in below in Figure 16. The

peak has good shape with no fronting or tailing.

12.45 12.50 12.55 12.60 12.65 12.70 12.75 12.80

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

Time-->

Abundance

TIC: 13MAY00.D

12.64

Figure 16. Derivatized Spermidine, 12.64 minutes.

The GC/MS spectra for the spermidine peak contains significant information as proof that

spermidine was derivatized (Figure 17). The derivative fragments from the spectra are

listed in Table 11.

32

20 40 60 80 100120140160180200220240260280300320340360380400420

0

500

1000

1500

2000

2500

3000

m/z-->

Abundance

Average of 12.627 to 12.644 min.: 13MAY00.D (-)156

213

43

70 130

25998 284173

316 403196 342242 377

Figure 17. Spermidine derivative GC/MS spectra.

33

Table 11. Derivatized Spermidine-Proposed Ion Fragments

Fragments Ion

(molecular

weight)

Suggested Structure

Molecular

Ion

403

C

O

C

O

OCH2CH2CH3CH24

N CH23

NH

OCH2CH2CH3C

O

CH3CH2CH2O NH

+.

Derivative

Fragment

Ion

316

C

O C

O

OCH2CH2CH3

CH24

N CH23

NHCH3CH2CH2O NH+

Derivative

Fragment

Ion

259 C

O

NCH3CH2CH2O NH CH23

C

O

OCH2CH2CH3

CH2

+

Derivative

Fragment

Ion

130 CH2CH2 N

HOCH2CH2CH3C

O

+

Derivatizing

Reagent

Fragment

Ion

43 CH3CHCH3+

Derivative Blanks

There are several peaks that appear in Figure 5 (retention times 1.82, 2.71, 8.27

minutes) that are not amine derivatives or degradation products. These peaks, as well as

34

a few smaller ones, appear in Figure 18 of a derivative blank that was run after it was

diluted 5:1 in the organic solvent mix iso-octane/chloroform. The blank included the

same amounts of reagents and water, but not the amines. The derivative blank was

prepared the same as all other derivatives. The peak at 1.87 minutes is a xylene that

shows up in most runs and the peak at 2.72 minutes is identified as propyl carbonate.

The remaining peaks in Figure 18 are a variety of siloxane peaks most likely from the

column or septa that did not show up regularly in other analyses.

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.000

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

Time-->

Abundance

TIC: 05310302.D 1.87

2.72

4.11

5.33

6.43

7.41

8.27 9.03 9.73 10.51

Figure 18. Derivative blank run by GC/MS, diluted 5:1.

One additional derivative blank was run that had been treated in the same way as

the derivative above, except that is was not diluted 5:1. The resulting chromatogram is

shown in Figure 19. There are several other peaks that appear in Figure 5, which can be

explained by this blank derivative run (retention times 1.82, 2.71, 11.28, and 12.06

minutes). These peaks are not amine derivatives or degradation products, however they

do appear consistently in almost all derivatives run by either GC/MS or GC/FID (results

in Chapter 4-GC/FID Results of Standards).

35

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

Time-->

Abundance

TIC: 05310301.D

1.84

2.71

11.29

12.06

Figure 19. Derivative blank run by GC/MS, not diluted 5:1.

36

Chapter 4-GC/FID Results of Standards

37

Calibration Curves

A calibration curve was constructed using the following concentrations: 100, 50,

20, and 10 ppm. The results of the 100 ppm amine standard are listed in Table 12.

Table 12. 100 ppm Amine Standard

Data File 77.7ppm Hep 8.30min 87.7ppm Put 11.11min 87.3ppm Cad 11.61 min 90.7ppm His 11.88 min 93.3ppm Tyr 12.59 min 92.5ppm Spe 16.58 min06240301.d 818.7 317.1 427.5 82.1 100.7 285.606240302.d 883.9 344.8 463.0 85.8 109.7 313.406240303.d 943.3 371.0 496.7 89.3 117.4 337.006240304.d 963.5 377.8 505.8 87.4 119.8 346.706240305.d 1027.8 403.6 539.8 90.5 127.8 371.3Average 927.4 362.9 486.6 87.0 115.1 330.8Std Dev 79.6 33.1 42.9 3.3 10.3 32.7%RSD 8.6 9.1 8.8 3.8 9.0 9.9

The % RSD for the amines is good at less than 10%. The resolution and peak shapes are good with slight fronting on heptylamine, putrescine, and cadaverine (Appendix, Figure 32). There are two peaks at 13.41 and 14.89 minutes that are unknown, but they appear consistently in the runs and are also in the derivative blank run (Appendix, Figure 33). The signal to noise ratios for the 100 ppm standard are all equal to or greater

than 20:1. Table 13 lists the results of the 50 ppm standard amine mix. The % RSD’s are

still below 10% for all of the amines and even below 7% for all amines except histamine.

Table 13. 50 ppm Amine Standard

Data File 38.9ppm Hep 8.30 min 43.9ppm Put 11.11 min 43.7ppm Cad 11.61 min 45.4ppm His 11.88 min 46.7ppm Tyr 12.59 min 46.3ppm Spe 16.58 min06250324.d 400.8 234.0 304.9 69.7 73.8 199.306250325.d 390.6 228.1 296.8 62.8 69.5 195.006250326.d 360.1 208.5 274.4 56.7 66.4 177.006250327.d 362.7 210.9 276.8 60.5 66.0 177.006250328.d 360.2 208.2 274.6 54.2 65.5 175.7Average 374.9 217.9 285.5 60.8 68.2 184.8Std Dev 19.4 12.2 14.3 6.0 3.5 11.4%RSD 5.2 5.6 5.0 9.9 5.1 6.2

38

The peak shapes and resolution are good (see Appendix, Figure 34). There does not

appear to be any fronting or tailing. At 50 ppm the signal to noise ratios for the standard

amines are all equal to or greater than 20:1.

A set of data was run at 50 ppm using the same GC/FID conditions as listed in

Table 3 except that there was no oven tracking because the inlet used was not COC, but a

hot inlet at 200°C. The liner used was a 2 mm inner diameter, straight tube, with no glass

wool. The results of the study are listed in Table 14 and a representative chromatogram

is shown in the Appendix, Figure 35. The spermidine peak is not detected in this set of

runs and the tyramine peak shape is very broad, tailing, and there appears to be a doublet.

Overall, the %RSD is at least 9% higher than the 50 ppm sample run COC. Also, the

peak areas using the COC inlet are an order of magnitude higher than they are for the hot

inlet. The poor results of the hot inlet study may be attributed to thermal degradation of

the carbamate derivatives in the inlet. It would have been very difficult and perhaps

impossible to use a hot inlet for quantitative analysis with the amine derivatives and still

achieve the same low limits of detection seen here (10ppm). Therefore, COC was

employed as the inlet of choice for the quantitative portion of the analysis.

Table 14. 50 ppm Amine Standard*, Hot Inlet 200°C

Data File 38.9ppm Hep 8.86 min 43.9ppm Put 11.97 min 43.7ppm Cad 12.52 min 45.4ppm His 12.85 min 46.7ppm Tyr 13.94 min 46.3ppm Spe 16.58 min06290303.d 16.6 5.9 7.8 NQ 12.5 ND06290304.d 18.6 6.7 8.6 NQ 7.3 ND06290305.d 14.2 5.0 6.8 NQ 7.0 ND06290306.d 22.3 7.8 9.8 NQ 6.3 ND06290307.d 14.2 4.5 6.1 NQ 5.4 NDAverage 17.2 6.0 7.8 7.7Std Dev 3.4 1.3 1.5 2.8%RSD 19.8 22.1 18.7 36.1

*NQ-not quantitatable, ND-not detectable

The 20 ppm standard amine mix results are shown in Table 15. The %RSD for the amines is good in the range from 2.4% to 5.3%. The peak

shapes and resolution (see Appendix, Figure 36) are still good with signal to noise ratios

greater than or equal to 13:1.

39

Table 15. 20 ppm Amine Standard

Data File 15.5ppm Hep 8.29min 17.5ppm Put 11.11min 17.5ppm Cad 11.61min 18.1ppm His 11.87min 18.7ppm Tyr 12.59min 18.5ppm Spe 16.57min06250301.d 158.7 146.2 179.8 45.9 44.1 126.706250302.d 149.5 138.2 170.6 40.9 43.1 121.606250303.d 150.2 139.1 171.8 44.1 44.9 122.606250304.d 157.5 144.7 179.1 41.6 46.3 129.106250305.d 150.8 140.8 172.2 40.7 44.3 124.5Average 153.3 141.8 174.7 42.6 44.5 124.9Std Dev 4.4 3.5 4.4 2.3 1.2 3.1%RSD 2.9 2.5 2.5 5.3 2.6 2.4

The last set of data for the calibration curve is the 10 ppm standard, which is shown in

Table 16. The %RSD is consistantly good between 3.2% and 5.8% at the 10 ppm level.

The peaks are gausssian and there are some extra peaks and drifting baseline toward the

end of the run that are amplified at this low concentration (see Appendix, Figure 37).

Quantitation is still possible at this level based on a signal to noise ratio of 12:1 or better

for all standard amines.

Table 16. 10 ppm Amine Standard

Data File 7.8ppm Hep 8.29min 8.8ppm Put 11.12min 8.7ppm Cad 11.62min 9.1ppm His 11.89min 9.3ppm Tyr 12.61min 9.3ppm Spe 16.61min06250306.d 70.4 79.1 96.9 28.0 30.6 64.106250307.d 67.9 76.2 95.0 25.4 30.1 61.806250308.d 62.8 70.3 90.6 28.8 28.1 57.006250309.d 68.7 77.3 97.2 27.8 29.9 62.006250310.d 68.8 77.7 99.5 29.8 30.1 64.0Average 67.7 76.1 95.8 28.0 29.8 61.8Std Dev 2.9 3.4 3.3 1.6 1.0 2.9%RSD 4.3 4.5 3.5 5.8 3.2 4.7

The calibration curve for heptylamine is shown in Figure 20. The linearity is good with a

correlation coefficient of R2 = 0.9911.

40

Calibration Curve for Heptylamine

y = 12.259x - 47.918R2 = 0.9911

-200.00

0.00

200.00

400.00

600.00

800.00

1000.00

0 20 40 60 80 1

Concentration (ppm)

Peak

Are

a (p

A)

00

Figure 20. Heptylamine Calibration Curve

Below is the calibration curve for putrescine (Figure 21). The linearity is good with a

correlation coefficient of R2 = 0.9973.

Calibration Curve for Putrescine

y = 3.4524x + 63.398R2 = 0.9856

0.0050.00

100.00150.00200.00250.00300.00350.00400.00

0 20 40 60 80 1

Concentration (ppm)

Peak

Are

a (p

A)

00

Figure 21. Putrescine Calibration Curve.

41

Figure 22 shows the calibration curve for cadaverine. The correlation coefficient

is very good at R2 = 0.9918.

Calibration Curve for Cadaverine

y = 4.7845x + 72.619R2 = 0.9918

0.00100.00200.00300.00400.00500.00600.00

0 20 40 60 80 1

Concentration (ppm)

Peak

Are

a (p

A)

00

Figure 22. Cadaverine Calibration Curve

The calibration curve for histamine is shown in Figure 23. The linearity is good

with a correlation coefficient of R2 = 0.9746.

Calibration Curve for Histamine

y = 0.6852x + 26.628R2 = 0.9746

0.0020.0040.0060.0080.00

100.00

0 20 40 60 80 1

Concentration (ppm)

Peak

Are

a (p

A)

00

Figure 23. Histamine Calibration Curve.

42

Figure 24 shows the calibration curve for tyramine. The correlation coefficient is

good at R2 = 0.9962.

Calibration Curve for Tyramine

y = 0.9877x + 22.922R2 = 0.9962

0.0020.0040.0060.0080.00

100.00120.00140.00

0 20 40 60 80 100

Concentration (ppm)

Peak

Are

a (p

A)

Figure 24. Tyramine Calibration Curve.

The last calibration curve is for spermidine, and it is shown in Figure 25. The

linearity is good with an R2 = 0.9836.

43

Calibration Curve for Spermidine

y = 3.0528x + 48.422R2 = 0.9836

0.0050.00

100.00150.00200.00250.00300.00350.00400.00

0 20 40 60 80 1

Concentration (ppm)

Peak

Are

a (p

A)

00

Figure 25. Spermidine Calibration Curve.

44

Chapter 5-Salmon Analysis Results

45

Introduction

The guidance level that the FDA has set for the limit of histamine in fish is 50

ppm26. It has been shown that people are more likely to become sick from scombroid

toxicity with levels of 200 ppm or higher, however if there is 50 ppm histamine in one

part of the fish there may be 500 ppm in another part of the fish so the lower limit is used

as the acceptable standard27. It is significant to also consider the presence of other

biogenic amines such as putrescine and cadaverine because it is believed that they

contribute to the toxicity of histamine28. The results of the salmon study, including levels

of biogenic amines detected, are presented in this chapter.

Salmon Results-Day 1

After the fresh salmon was extracted, derivatized, and run by GC/FID it was

determined that no biogenic amines were present in either sample replicate 1 (A) or

replicate 2 (B). The quantitation limit was based on the lowest level of the calibration

curve at 10 pmm, with limits of detection estimated to be around 2-3 ppm for GC/FID.

The resulting chromatograms are shown in Figure 26 and Figure 27.

min0 2 4 6 8 10 12 14 16

pA

0

200

400

600

800

1000

1200

1400

FID1 B, (LAN\06250314.D)

4.3

50

4.4

12

4.9

63

6.3

35

12

.28

6

Are

a: 16

5.999

13

.45

2

14

.90

8

15

.344

Figure 26. Day 1 Salmon sample A, run 3 out of 5.

46

Figure 27. Day 1 Salmon sample B, run 3 out of 5.

There are several small, unknown peaks in the chromatograms, but they do not

correspond to the biogenic amines that were run in the standards. GC/MS runs did not

show evidence of biogenic amine derivatives for either sample A or B for day one.

Salmon Results-Day 3

After the salmon samples for day three were extracted, derivatized and run by GC/FID there were some trace levels of biogenic amines found, namely cadaverine, histamine, and tyramine. These GC/FID runs are shown in Figure 28 and Figure 29.

min0 2 4 6 8 10 12 14 16

pA

0

200

400

600

800

1000

1200

FID1 B, (LAN\06250320.D)

4.3

48

4.4

11

6.3

32

13

.67

3

min0 2 4 6 8 10 12 14 16

pA

0

200

400

600

800

1000

1200

FID1 B, (LAN\06270303.D)

4.3

43

4.4

06

6.3

31

11

.60

2 1

1.8

75

12

.28

6 1

2.5

87

Figure 28. Day 3 Salmon sample A, run 3 out of 5; cadaverine 11.60, histamine

11.88, and tyramine 12.59 minutes.

47

min0 2 4 6 8 10 12 14 16

pA

0

50

100

150

200

FID1 B, (LAN\06270309.D)

4.3

48

4.4

10

6.3

09

10

.58

9

11

.60

7 1

1.8

78

12

.59

3

13

.67

2

15

.35

4

Figure 29. Day 3 Salmon sample B, run 3 out of 5; cadaverine 11.61, histamine

11.88, and tyramine 12.59 minutes.

Levels of histamine were too low to quantitate based on a signal to noise ratio less than 10:1. Cadaverine had a signal to noise ratio greater than 10:1, and based on extrapolation of the calibration curve the average level of cadaverine was found to be 13 ppm (0.62 mg cadaverine/100 mg salmon, 6.2ppm for sample A and 2.0 mg cadaverine/100 mg salmon, 20 ppm for sample B). The percent RSD is very high (73%) at these low levels of cadaverine so the reproducibility of the data is not as good. Tyramine had a signal to noise ratio equal to 20:1 for sample A and 11:1 for sample B and average peak areas did fall within the calibration curve. For sample A there was 5.4 mg tyramine/100 mg salmon (54 ppm) and for sample B there was 6.2 mg tyramine/100 mg salmon (62 ppm). The average, therefore, was 58 ppm tyramine with standard deviation of 6 ppm and %RSD of 9.7%. The data for the replicate injections of samples A and B are listed below in Table 17 and Table 18, respectively.

Table 17. Day 3, Salmon sample A*

Data File Hep 8.29min Put 11.12min Cad 11.62min His 11.89min Tyr 12.61min Spe 16.61min06270301.d ND ND 68.1 NQ NQ ND06270302.d ND ND 104.5 NQ 41.1 ND06270303.d ND ND 82.1 NQ 32.7 ND06270304.d ND ND 106.1 NQ 42.4 ND06270305.d ND ND 93.3 NQ 38.7 NDAverage 90.8 38.7Std Dev 16.0 4.3%RSD 17.6 11.1

*ND-not detectable, NQ-not quantitatable.

48

Table 18. Day 3, Salmon sample B*

Data File Hep 8.29min Put 11.12min Cad 11.62min His 11.89min Tyr 12.61min Spe 16.61min06270307.d ND ND 88.2 NQ NQ ND06270308.d ND ND 103.2 NQ 40.5 ND06270309.d ND ND 113.1 NQ 43.9 ND06270310.d ND ND 102.7 NQ 41.0 ND06270311.d ND ND 96.8 NQ 40.1 NDAverage 100.8 41.4Std Dev 9.2 1.7%RSD 9.1 4.2

*ND-not detectable, NQ-not quantitatable.

GC/MS analysis was done for both samples A and B of day 3 to confirm the

prescence of cadaverine, histamine, and tyramine. These chromatograms and spectra are

displayed in the Appendix; Figure 38 through Figure 45.

Salmon Results-Day 5

On the last day of the study four biogenic amines were detected after extraction,

derivatization, and GC/FID and GC/MS analysis. Putrescine, cadaverine, histamine, and

tyramine were identified. Putrescine was detected with a signal to noise ratio of 11:1 for

sample A and 20:1 for sample B, however based on the peak areas being too low the

putrescine was not quantifiable. Cadaverine, histamine, and tyramine were quantitatable.

The representative chromatograms for the GC/FID runs are shown in

Figure 30 and Figure 31.

49

Figure 30. Day 5 Salmon sample A, run 3 out of 5; putrescine 10.96, cadaverine

11.47, histamine 11.73, and tyramine 12.45 minutes.

min0 2 4 6 8 10 12 14 16

pA

0

25

50

75

100

125

150

175

FID1 B, (LAN\06290315.D)

4.2

22

4.2

83

9.1

37

10

.44

9

10

.96

4

11

.46

8 1

1.7

34

12

.11

0 1

2.4

45

12

.94

2

13

.44

5

min0 2 4 6 8 10 12 14 16

pA

0

25

50

75

100

125

150

175

200

FID1 B, (LAN\06290322.D)

4.2

26

4.2

87

5.9

86

9.1

39

10

.45

0

10

.96

3

Are

a: 307

.156

11

.46

7 1

1.7

32

12

.10

9

12

.44

2

12

.94

2

Figure 31. Day 5 Salmon sample B, run 3 out of 5; putrescine 10.96, cadaverine

11.47, histamine 11.73, and tyramine 12.44 minutes.

The peaks shapes are good overall except for a small shoulder on the cadaverine peak. The signal to noise ratios for cadaverine, histamine, and tyramine are all at least 75:1 for both samples A and B. In Table 19 and Table 21 the %RSD’s for the analyte peak areas for day five samples A and

B, respectively. All the %RSD’s are good at less than 10%.

50

Table 19. Day 5, Salmon sample A* Data File Hep 8.15min Put 10.96min Cad 11.46min His 11.72min Tyr 12.44min Spe 16.10min06290313.d ND 40.6 298.9 303.9 121.7 ND06290314.d ND 42.0 307.3 310.3 122.2 ND06290315.d ND 43.9 327.6 324.2 129.1 ND06290316.d ND 41.1 307.4 302.6 121.4 ND06290317.d ND 43.8 323.0 318.3 127.1 NDAverage 42.3 312.8 311.9 124.3Std Dev 1.5 12.0 9.3 3.6%RSD 3.6 3.8 3.0 2.9

*ND-not detectable, NQ-not quantitatable.

Table 20. Day 5, Salmon sample B*

Data File Hep 8.15min Put 10.96min Cad 11.46min His 11.72min Tyr 12.44min Spe 16.10min06290319.d ND 53.2 339.0 223.1 149.3 ND06290320.d ND 46.8 297.1 198.5 130.4 ND06290322.d ND 49.7 317.9 209.4 138.0 ND06290323.d ND 48.8 310.8 207.5 135.4 ND06290324.d ND 41.3 262.3 177.4 115.5 NDAverage 48.0 305.4 203.2 133.7Std Dev 4.4 28.5 16.9 12.3%RSD 9.1 9.3 8.3 9.2

*ND-not detectable, NQ-not quantitatable.

The quantitative data for the day five salmon analysis is listed in Table 21. The

%RSD is good for cadaverine and tyramine at less than 6%. Histamine is a bit higher at

33.7%. The histamine level is extremely high and is out of range of the calibration curve

so that may contribute to the reproducibility inconsistencies. It is clear from this data that

after five days of salmon stored at 4°C, it is not fit to be prepared for human

consumption, based on the levels of biogenic amines, without significant risk of

scombroid poisoning.

51

Table 21. Day 5 Salmon-Biogenic Amine Levels

Analyte

Peak

Sample A

Concentration

(ppm)

Sample B

Concentration

(ppm)

Average

Concentration

(ppm)

Standard

Deviation

(ppm)

%RSD

Cadaverine 168 162 165 4 2.6

Histamine 1390 855 1123 379 33.7

Tyramine 342 372 357 22 5.9

GC/MS analysis was performed on the salmon samples from day five. These

chromatograms and spectra can be found in the Appendix; Figure 46 through Figure 55.

52

Chapter 6-Conclusions

53

GC/FID Analysis

In summary, the propyl chloroformate derivatization method employed for the

biogenic amines heptylamine, putrescine, cadaverine, histamine, tyramine, and

spermidine, was successful for trace level quantitation. The limits of quantitation are

below 10 ppm, where the error bars on the calibration curves are the largest, however

Relative Standard Deviation is still low and acceptable at less than 5% at the 10 ppm

level for all six amines. The signal to noise ratios at 10 ppm are 12:1 for both histamine

and tyramine, therefore limits of detection are estimated to be 2-3 ppm.

The advantages of using this method are that it is a rapid derivatization method

taking less than ten minutes and it is not moisture sensitive. Extraction and derivatization

from urine and aqueous matrices are possible. The chromatography is very reproducible

(~4% RSD) at trace levels (10 ppm). It should be noted that it is best to use a column 15

meters or perhaps shorter, as opposed to a 30 meter column. Additionally, a thicker

stationary phase film such as 1.00 µm df will lengthen the life of the column. It is also

recommended that the column be cut at the inlet end 40 cm every 25 runs so that

maximum sensitivity and reproducibility are achieved. It also may be useful to

incorporate an internal standard into the method to increase limits of quantitation and

improve reproducibility.

GC/MS Analysis

The GC/MS method used to confirm the identities of the biogenic amine

derivatives proved to be successful. This method is not, however, recommended for

quantitation using a hot split inlet, due to the thermal degradation of the carbamate

derivatives. For multiple injections using the hot inlet in the GC/MS method, the

reproducibility is not sufficient for good, quantitative analysis. Although for a quick,

qualitative confirmation using the hot split inlet, with GC/MS, works well.

54

Salmon Analysis

The extraction and derivatization process for biogenic amines from the salmon is

a relatively fast and reproducible method. The extraction time may be optimized further

depending on the type of fish to be analyzed. Fish samples can be troublesome due to the

complex matrices, especially those high in fish oils. This method works well on fish

samples largely due to the methanol that is 50% of the liquid extraction media. Salmon

extraction attempts were made using only water, and it was found that derivatization of

the supernate after centrifuging the extract was not possible. This is most likely due to

the matrix effects of the fish oils that caused the derivative to form an insoluble clump of

white matter. It is believed that the methanol is useful to break up the fish oils so that the

derivatization can proceed.

It is not known how addition of methanol to the homogeneous salmon mix affects

the efficiency of the derivatization. It is possible that trace levels of water exist in the

organic layer of the derivative, due to the solubility of methanol in chloroform and the

solubility of water in methanol. This may slightly decrease the overall efficiency in the

derivatization of aqueous biogenic amine samples. For the derivatization of the salmon

extracts clearly two distinct layers were formed, the top being the organic and the bottom

the aqueous layer. In between the two layers was a thin, white, insoluble membrane most

likely composed of some residual fat from the salmon, although it did not appear to

interfere with the analysis.

Based on the salmon study conducted in this thesis, and considering the 50 ppm

histamine safety level set by the FDA26, it is recommended not to use raw salmon in food

preparation after three days, even if it is stored in the refrigerator.

Future Applications

The methods developed in this thesis were applied to fish samples, but may also

be applied to other biogenic amine containing samples, especially in aqueous media. The

derivatization method used here was based on Ugland’s work13, which incorporated

derivatization of urinary amines such as amphetamine and methamphetamine, using

55

propyl chloroformate as one of the more successful derivatizing reagents in the trials.

Ugland also experimented successfully with solid phase microextraction (SPME) directly

in the amine/derivatizing solution where the derivatized amines partition to the SPME

fiber13. A quantitative technique similar to this may be possible for biogenic amine

derivatives in aqueous solutions, especially with the availability of an automated

headspace system to achieve a more rapid analysis. It can be hypothesized that even

greater sensitivity may be obtained with the use of SPME in the derivatization process.

56

Appendix

57

GC/FID Data

pA

500

Figure 32. Run 3 of 5, 100 ppm amine mix (heptylamine 8.30, putrescine 11.11,

cadaverine 11.61, histamine 11.88, tyramine 12.59, spermidine 16.58 minutes).

Figure 33. Derivative blank run with unknown peaks at 13.42 and 14.90 minutes.

min0 2 4 6 8 10 12 14 160

100

200

300

400

FID1 B, (LAN\06240303.D)

4.3

51

8.2

95

10.

921

11.

113 1

1.61

4 1

1.87

7

12.

592

13.

409

14.

888

16.

575

min0 2 4 6 8 10 12 14 16

pA

0

25

50

75

100

125

150

175

200

FID1 B, (LAN\06240307.D)

4.3

49

7.1

15

12.

395

13.

424

13.

676

14.

908

min0 2 4 6 8 10 12 14 16

pA

0

50

100

150

200

250

FID1 B, (LAN\06250326.D)

4.3

47

8.2

94

11.

110

11.

608

11.

874

12.

592

16.

568

Figure 34. Run 3 of 5, 50 ppm amine mix (heptylamine 8.29, putrescine 11.11,

cadaverine 11.61, histamine 11.87, tyramine 12.59, spermidine 16.57 minutes).

58

Figure 35. Run 3 of 5, 50 ppm amine mix with hot inlet 200�C (heptylamine 8.86,

putrescine 11.97, cadaverine 12.53, histamine 12.85, tyramine 13.89 minutes).

Figure 36. Run 3 of 5, 20 ppm amine mix (heptylamine 8.28, putrescine 11.11,

cadaverine 11.60, histamine 11.88, tyramine 12.59, spermidine 16.57 minutes).

min0 2 4 6 8 10 12 14 16

pA

4

6

8

10

12

14

16

18

20

FID1 B, (LAN\06290306.D)

8.8

62

11.

972

12.

525

12.

851

Area

: 6.31

32

13.

890

min0 2 4 6 8 10 12 14 16

pA

20

40

60

80

100

FID1 B, (LAN\06250303.D)

4.3

48

8.2

89

11.

110

11.

609

11.

880

12.

592

13.

431

16.

573

min0 2 4 6 8 10 12 14 16

pA

10

20

30

40

50

60

FID1 B, (LAN\06250308.D)

4.3

48

8.2

93

11.

115

11.

615

11.

884

12.

601

16.

583

Figure 37. Run 3 of 5, 10 ppm amine mix (8.293 heptylamine, 11.11 putrescine,

11.61 cadaverine, 11.884 histamine, 12.601 tyramine, 16.583 minutes spermidine).

59

GC/MS Data

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

Time-->

Abundance

TIC: 06270312.D

2.28

2.66

9.67

9.91

10.60

Figure 38. Day 3 Salmon sample A, GC/MS run, cadaverine 9.67, histamine 9.91,

and tyramine 10.60 mintues.

20 40 60 80 1001201401601802002202402602803003203403603804000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

m/z-->

Abundance

Average of 9.669 to 9.679 min.: 06270312.D (-)116

43

84

170

215100

143 18768

274252 342233 309 403373

Figure 39. Day 3 Salmon sample A, GC/MS spectra of cadaverine 9.67 minutes.

60

20 40 60 80 100120140160180200220240260280300320340360380400

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

m/z-->

Abundance

Average of 9.906 to 9.920 min.: 06270312.D (-)180

81

43

224

138105 281 355327163 389303253203 408

Figure 40. Day 3 Salmon sample A, GC/MS spectra of histamine 9.91 minutes.

20 40 60 80 1001201401601802002202402602803003203403603804000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

m/z-->

Abundance

Average of 10.596 to 10.605 min.: 06270312.D (-)120

43

162

91

206 309223 25062 179 329145 283 381355 415

Figure 41. Day 3 Salmon sample A, GC/MS spectra of tyramine 10.60 mintues.

61

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.000

50000

100000

150000

200000

250000

300000

350000

400000

450000

Time-->

Abundance

TIC: 06270313.D

2.66 4.58

9.68

9.91

10.60

12.21

12.98

Figure 42. Day 3 Salmon sample B, GC/MS run, cadaverine 9.68, histamine 9.91,

tyramine 10.60 minutes.

20 40 60 80 1001201401601802002202402602803003203403603804000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

m/z-->

Abundance

Average of 9.672 to 9.682 min.: 06270313.D (-)116

43

84

170 215

10068 143 187

274252 360 405377327

Figure 43. Day 3 Salmon sample B, GC/MS spectra of cadaverine 9.68 minutes.

62

20 40 60 80 1001201401601802002202402602803003203403603804000

200

400

600

800

1000

1200

1400

1600

1800

2000

m/z-->

Abundance

Average of 9.908 to 9.918 min.: 06270313.D (-)180

81

43

138224

111 19765 283 355157 265241 376 405332

Figure 44. Day 3 Salmon sample B, GC/MS spectra of histamine 9.91minutes.

20 40 60 80 100120140160180200220240260280300320340360380400420

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

9500

10000

10500

m/z-->

Abundance

Average of 10.598 to 10.610 min.: 06270313.D (-)120

43

162

77206

309223103 281144 250 328 415180 356 391

Figure 45. Day 3 Salmon sample B, GC/MS spectra of tyramine 10.60 minutes.

63

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

Time-->

Abundance

TIC: 06290303.D

1.77

2.27

2.66

2.71 9.20

9.68 9.92

10.61

12.04 12.21

12.99

Figure 46. Day 5 Salmon sample A, GC/MS run, putrescine 9.20, cadaverine 9.67,

histamine 9.91, tyramine 10.60 minutes.

2 0 4 0 6 0 8 01 0 01 2 01 4 01 6 01 8 02 0 02 2 02 4 02 6 02 8 03 0 03 2 03 4 03 6 03 8 04 0 00

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0

1 1 0 0

1 2 0 0

1 3 0 0

1 4 0 0

1 5 0 0

1 6 0 0

m /z -->

A b u n d a n ce

A ve ra g e o f 9 .1 9 3 to 9 .2 0 8 m in .: 0 6 2 9 0 3 0 3 .D (-)1 1 6

1 5 6

4 3

7 0

2 0 1

9 8

1 7 3

2 6 0 3 0 5 4 1 51 3 9

Figure 47. Day 5 Salmon sample A, GC/MS spectra of putrescine 9.20 minutes.

64

20 40 60 80 1001201401601802002202402602803003203403603804000

1000

2000

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4000

5000

6000

7000

8000

9000

10000

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16000

m/z-->

Abundance

Average of 9.669 to 9.688 min.: 06290303.D (-)116

43

84

170215

14318762

274255 298 341 415376

Figure 48. Day 5 Salmon sample A, GC/MS spectra of cadaverine 9.70 minutes.

2 0 4 0 6 0 8 01 0 01 2 01 4 01 6 01 8 02 0 02 2 02 4 02 6 02 8 03 0 03 2 03 4 03 6 03 8 04 0 04 2 00

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0

1 4 0 0 0

1 5 0 0 0

1 6 0 0 0

1 7 0 0 0

1 8 0 0 0

1 9 0 0 0

2 0 0 0 0

2 1 0 0 0

2 2 0 0 0

2 3 0 0 0

2 4 0 0 0

2 5 0 0 0

m /z -->

A b u n d a n ce

A v e ra g e o f 9 .9 0 8 to 9 .9 2 2 m in .: 0 6 2 9 0 3 0 3 .D (-)1 8 0

8 1

4 3

1 3 8 2 2 4

1 1 0 2 8 3

6 0 3 2 71 9 7 4 1 72 4 91 5 5 4 0 1

Figure 49. Day 5 Salmon sample A, GC/MS spectra of histamine 9.91 minutes.

65

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000

20000

21000

22000

m/z-->

Abundance

Average of 10.600 to 10.612 min.: 06290303.D (-)120

43

162

91

206223 30962 250145 327179 389279

Figure 50. Day 5 Salmon sample A, GC/MS spectra of tyramine 10.60 minutes.

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.000

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

320000

Time-->

Abundance

TIC: 06290304.D

1.78

2.28

2.66

2.71

7.37

9.20

9.68

9.92

10.61

Figure 51. Day 5 Salmon sample B, GC/MS run, putrescine 9.20, cadaverine 9.68,

histamine 9.92, tyramine 10.61 minutes.

66

40 60 80 100 120 140 160 180 200 220 240 260

0

200

400

600

800

1000

1200

1400

1600

m/z-->

Abundance

Average of 9.196 to 9.206 min.: 06290303.D (-)116

156

7043

20130

102 129

88 17356145

191 260

Figure 52. Day 5 Salmon sample B, GC/MS spectra of putrescine 9.20 minutes.

2 0 4 0 6 0 8 0 1 0 01 2 01 4 01 6 01 8 02 0 02 2 02 4 02 6 02 8 03 0 03 2 03 4 03 6 03 8 00

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0

1 4 0 0 0

1 5 0 0 0

1 6 0 0 0

1 7 0 0 0

1 8 0 0 0

1 9 0 0 0

2 0 0 0 0

2 1 0 0 0

2 2 0 0 0

2 3 0 0 0

2 4 0 0 0

m /z -->

A b u n d a n c e

A ve ra g e o f 9 .6 7 3 to 9 .6 8 3 m in .: 0 6 2 9 0 3 0 4 .D (-)1 1 6

4 3

8 4

1 7 02 1 5

1 0 01 4 36 8 1 8 7

2 7 4

2 5 5 3 8 7

Figure 53. Day 5 Salmon sample B, GC/MS spectra of cadaverine 9.67 minutes.

67

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1000

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3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

m/z-->

Abundance

Average of 9.910 to 9.922 min.: 06290304.D (-)180

81

43

138 224

110 28360 207158 415255 315

Figure 54. Day 5 Salmon sample B, GC/MS spectra of histamine 9.91 minutes.

20 40 60 80 1001201401601802002202402602803003203403603804000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000

m/z-->

Abundance

Average of 10.600 to 10.612 min.: 06290304.D (-)120

43

162

77206 309223103 250145 341267 40760 377

Figure 55. Day 5 Salmon sample B, GC/MS spectra of tyramine 10.61 minutes.

68

References

1McNair, H.M.; Miller, J.M. Basic Gas Chromatography, John Wiley & Sons: New

York, 1998. 2Kataoka, H.J. Chromatogr. 1996, 733, 19. 3Rice, S.L.; Koehler, P.E. J. Milk Food Technol. 1976, 39, 166. 4Yen, G.C.; Hsieh, C.L. J. Food Sci. 1991, 56, 158. 5Maga, J.A. Food Sci. Nutr. 1978, 10, 373. 6Coutts, R.T.; Baker, G.B.; Pasutto, F.H. Adv.Drug Res., 1986, 15, 169. 7Mietz, J.L. J. Assoc.Off. Anal. Chem., 1978, 61, 139. 8Arnold, S.H.; Brown, W.D. Adv. Food Res., 1978, 24, 114. 9Sullivan, J.E. J. Chromatogr. Sci., 1977, 15, 196. 10Donike, M. J. Chromatogr., 1973, 78, 273. 11Drouett-Coassolo, C.; Aubert, C.; Coassolo, P.; Cano, J. J. Chromatogr., 1989, 487,

295. 12Ibanez, M.A.D. J. Chromatogr., 1989, 488, 237. 13Ugland, H.G.; Krogh, M.; Rasmussen, K.E. J. Chromatogr., 1997, 701, 29. 14Westley, J.W.; Halpern, B. J. Org. Chem., 1968, 33, 3978. 15Hartvig, P.; Karlsson, K.E.; Johansson, L.; Lindberg, C. J. Chromatogr., 1976, 121,

235. 16Hartvig, P.; Ahnfelt, N.O.; Karlsson, K.E. Acta Pharm. Suecica, 1976, 13, 181. 17Sternson, L.A.; Cooper, A.D. J. Chromatogr., 1978, 150, 257. 18Hartvig, P.; Handl, W.; Vessman, J.; Svahn, C.M. Anal. Chem., 1976, 48, 390. 19Hartvig, P. Anal. Lett., 1974, 7, 223. 20Hušek, P.; Huang, Z.H.,; Sweeley, C.C. Anal. Chim. Acta, 1992, 259, 185. 21Hušek, P.; Rijks, J.A.;p Leclercq, P.A.; Cramers, C.A. J. High Resolut. Chromatogr.,

1990, 13, 663. 22Hušek, P. J. Chromatogr., 1991, 547, 307. 23Merck Index 12th Ed., Merck & Co., Inc., 1996, Whitehouse Station, NJ. 8140, 8889. 24Zotou, A.; Loukou, Z.; Soufleros, E.; Stratis, I. Chromatographia, 2003, 57, 429.

69 25Antoine, F.R.; Wei, C.; Otwell, S.; Sims, C.A.; Littell, R.C.; Hogle, A.D.; Marshall,

M.R. J. Agric. Food Chem., 2002, 50, 4754. 26Anonymous. 1996. Fish and fisheries products hazards and controls guide, 1st ed. U.S.

Food and Drug Administration, Office of Seafood, Washington, D.C. 27Gingerich, T.M.; Lorca, T.; Flick, G.; Pierson, M.D.; McNair, H.M. J. Food Prot.,

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70

Vita Laura Nakovich was born to John and Joan Nakovich on November 13, 1977 in

Silver Spring, MD. She grew up in Herndon, VA with her parents and brother John.

After high school she attended Virginia Tech where she received a Bachelor’s of Science

degree in Biochemistry in May of 2000. She did undergraduate research in Dr. McNair’s

lab for two semesters and one summer. In the fall of 2000 she began work on her

Master’s degree in Analytical Chemistry under the advisorship of Dr. Harold McNair.

She will finish her Master’s degree in September of 2003 and plans to pursue an industry

career in the field of analytical chemistry.