GC/MS Lab, General Clinical Research Center at the University of Minnesota

GC/MS (Gas Chromatography/Mass Spectrometry) is a powerful analytical tool for separating and identifying volatile organic compounds. The capabilities of this integrated unit facilitate and support clinical research involving the use of stable isotopes. A gas chromatograph/mass spectrometer (GC/MS) has numerous applications for the clinical research laboratory. Virtually any metabolic process (involving carbohydrate, protein, or lipid) can be studied using stable isotope technology. The GCRC-GC/MS Core Laboratory at the University of Minnesota assists researchers by providing analysis of stable isotope enrichment in plasma and urine samples. The following services are provided:

• Assay Development
• Sample Purification
• Analyte Extraction
• Analyte Derivatization
• Analysis

For more information on these topics please scroll down. If you would like to know how the GCRC-GC/MS Core Laboratory can help you with your research needs please inquire to one of the below contacts.
 

Purification -  In the case of plasma this includes deprotienization of the samples. This is performed easily with a barium and zinc combination, concentrated ethanol, or  perchloric acid. Deprotienized plasma or urine samples may need further purification over ion exchange resin (see Pictures section). This purification step removes salts and differently charged species. We typically use 100-200 micron mesh resin of the hydrogen or formate forms.

Extraction - In certain methods a further purification step is necessary. The desired compound is extracted from the purification solvent by another, immisible solvent. This step further separates the desired and undesired species.

Derivatization - The desired analyte is then reacted with a specific reagent or combination of reagents to alter the structure of the analyte. This technique is employed for three main reasons: (1) to improve volatility and stability of the analyte, (2) to enhance chromatographic behavior and (3) to improve mass fragment delectability.

Analysis - The purified, derivatized samples can now be analyzed on the GC/MS system. This includes locating retention times if not known. Instrument parameters, such as temperature programs and detection windows, vary for each method. After the data acquisition calculations can be made. The atom percent excess of a stable isotope in a labeled species can be calculated by:

Where S is the ratio of the response (area under peak) of the labeled analyte to the unlabeled analyte in the sample and N is the same ratio in a natural abundance, or baseline, sample. The enrichment of label can also be extrapolated from a standard curve. In addition, relative or absolute concentrations of unlabeled or labeled species can be determined using the standard addition method and the calibration curve method.  

 
The ¹³C-Glucose enrichment in plasma was determined for CRC Protocols #482 and #508. The ¹³C label was located on the first carbon and is denoted by "*" in the above reaction. The mass fragment analyzed is m/z 161 (m/z160 for the unlabeled fragment) indicated by the dashed line. This derivatization method was also employed for d₂-Glucose labeled plasma samples.

For additional method information please email the GC/MS Core Lab.  

Amino Acids    Another group of commonly used tracers for metabolic analysis is the amino acids. We convert amino acids to their respective t-butyldimethylsilyl derivatives with acetonitrile and MTBSTFA +1% TBDMCS. Below are the reactions for three specific amino acids we have assayed.

The ¹³C-Phenylalanine, ¹⁵N-Phenylananine, ¹⁵N-Tyrosine, and d₄-Tyrosine enrichment in plasma were determined for CRC Protocol #545. As in the glucose reaction the location of the labels are denoted by "*" in the above reaction and the mass fragments analyzed are indicated by the dashed half brackets.

For additional method information please email the GC/MS Core Lab.  

Keto acids: KIC and KVAKeto acids, namely ketoisocaproic acid (KIC) and ketovaleric acid (KVA), are derivatized in a two step process. We first convert the keto acid to their respective quinoxalinol forms and then silylate the product creating the trimethylsilyl derivative of the quinoxalinol of a keto acid. The derivatization is performed using acidified o-Phenylenediamine and  BSTFA + 1% TMCS in pyridine.

 
The enrichment of ¹³C labeled KIC and ketovaleric acid KVA in plasma was determined for CRC Protocols #472 (closed) and  #518. As in the previous reactions, the location of the labels are denoted by "*" in the above reaction and the mass fragments analyzed are indicated by the dashed half brackets.

For additional method information please email the GC/MS Core Lab.  

Fatty AcidsTo analyze fatty acids we esterify the carboxyl group with methanolic hydrocholric acid. The below reaction is of a generic saturated fatty acid although all types can by derivatized and analyzed.

The ¹³C₄ and d₃₁ enrichment of palmitate (16:0) in plasma was analyzed with this method for CRC Protocol #640. The response of the molecular ion (M⁺) is used for calculations. A chromatograph of many different fatty acid methyl esters is show below (where the numbers of carbons and unsaturations are the first and second numbers of the peak label, respectively)

For additional method information please email the GC/MS Core Lab.  

Introduction to Gas Chromatography and Introduction to Mass Spectrometry from Virginia Tech Chemistry Department.

National Association for GCRC Core Laboratories.

University of Minnesota home page.

U of M GCRC home page.