CHLOROFORMATE DERIVATIZATION OF AMINES

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Chapter 2-Experimental

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.

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.

GC/FID Analysis

A Phenomenex ZB-5 (15m x 0.25mm id x 1.00 m d_f) 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.
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 d_f) 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 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.

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.

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.

Chapter 1-Introduction and Background
GAS CHROMATOGRAPHY AND AMINE ANALYSIS
DERIVATIZATION OF AMINES
CARBAMATE DERIVATIVES OF AMINES
Chapter 2-Experimental.
CHEMICALS USED.
CHLOROFORMATE DERIVATIZATION OF AMINES
CALIBRATION CURVES
ATLANTIC SALMON EXTRACTION AND DERIVATIZATION
Chapter 3-GC/MS Results of Standards
INTRODUCTION
AMINE STANDARD DERIVATIZED.
HEPTYLAMINE DERIVATIVE
PUTRESCINE DERIVATIVE
CADAVERINE DERIVATIVE
HISTAMINE DERIVATIVE
TYRAMINE DERIVATIVE
SPERMIDINE DERIVATIVE
DERIVATIVE BLANKS
Chapter 4-GC/FID Results of Standards
CALIBRATION CURVES.
Chapter 5-Salmon Analysis Results
INTRODUCTION.
SALMON RESULTS-DAY
SALMON RESULTS-DAY
SALMON RESULTS-DAY
Chapter 6-Conclusions
GC/FID ANALYSIS
GC/MS ANALYSIS
SALMON ANALYSIS
FUTURE APPLICATIONS
Appendix
References.
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