Cancer Metabolite Profiling by GCxGC J.-M. D. Dimandja Spelman College

Cancer Research Strategies Curative Preventive

Bio-Metabolic Profiling Impulse Response Biological System First developed by Roger Williams and his associates in the 1940s (using paper chromatography) Examined over 200,000 samples from a variety of subjects (alcoholics, schizophrenics, etc.) and produced suggestive evidence of characteristic metabolic patterns associated with each of the groups. Williams, R.J., et al. “Individual Metabolic Patterns and Human Disease: An exploratory Study Utilizing Predominantly Paper Chromatographic Methods. U. Texas Publication No (1951)

Carcinogenesis and Metabolic Profiling  Carcinogenesis is known to affect a complex network of metabolic interrelationships, leading to significant changes in concentration of a large number of body fluids.

Organic Acids  Organic acids are well established as important biochemical indicators of abnormal metabolism created by various diseases.

Goals of Metabolic Profiling  Characterization of normal and pathologic states  Studies of drug metabolism  Human developmental studies Horning, E.C., Horning, M.G. “Metabolic Profiles: Gas-Phase Methods for Analysis of Metabolites. Clin. Chem. 17, 802 (1971)

Challenges of Metabolic Profiling  Number of samples to analyze (for proper statistical treatment of the data)  Sample complexity  Sample preparation  Sample separation  Data processing

Starting Point

Sample Preparation Methoximation pH 13 SPE Acidification pH 2 SPE Silylation GC/MS K.-R. Kim et al. “Gas Chromatographic Profiling and Pattern Recognition Analysis of Urinary Organic Acids from Uterine Myoma Patients and Cervical Cancer Patients. J. Chrom. B, 712 (1998), Hours

Sample Separation

Data Processing

Challenges of the Kim Method Sample Preparation Sample Separation Data Processing

Goal of our Laboratory Sample Preparation Sample Separation Data Processing Impact: Higher Throughput

Sample Preparation

pH Reduction

Acidified Sample

Dichloromethane Addition

Liquid/Liquid Extraction

Liquid-Liquid Extraction

Filtration GCxGC Silylation in Pyridine Aqueous removal Sodium Sulfate Drying 2 Hours

GCxGC Instrumental Conditions  Pegasus 4D (LECO Corporation, St. Joseph, MI)  Sample Inlet  1 uL injection, splitless  Helium carrier gas (1 mL/min)  1st Dimension Column  30 m Rtx-1 column (Restek Corporation, Bellefonte, PA), 0.25 mm i.d., 0.25 um film  40°C to 280°C (5°C/min), 12 min hold at 280°C  Modulator  4-second modulation  80 msec release time  70°C to 310°C (5°C/min), 12 min hold at 310°C  2nd Dimension Column  1.5 m Rtx-50 column, 0.25 mm i.d., 0.25 um film  45°C to 285°C (5°C/min), 12 min hold at 285°C  TOF MS  100 scans/sec acquisition ( amu)  Ion source temperature: 200°C  Transfer Line temperature: 285°C  FID  Detector temperature: 285°C

GCxGC Peak Apex Plot

Data Processing  “Extract” a pattern out of a profile  Compare profiles  Similarity indexing

Urinary Sample Comparisons  Similarity Index scale  Similar to library searching in Mass Spectrometry  Calculation of residual error  1st dimension retention  2nd dimension retention  Normalized relative peak area

Urinary Profile (Non-Diseased State) Hippuric acid trans-Aconitic acid 2-Hydroxybutyric acid Pyruvic acid Citric acid

Urinary Profile (Diseased State) Hippuric acid trans-Aconitic acid 2-Hydroxybutyric acid Pyruvic acid Citric acid

Profile Comparison Similarity Index: 628

Conventional Chromatography Sample Separation Sample Inlet Sample Detection

Alphabetography Letter Sorter Name mixture Alphabet Signal Profile

Stephen Reichenbach e n t c h i S e p e h R abcdefghijklmnopqrstuvwxyz en b a c h

abcdefghijklmnopqrstuvwxyz

abcdefghijklmnopqrstuvwxyz

Similarity Matching Table for Steve Reichenbach

Similarity Matching Table for John Dimandja

First Name Last Name

John Seeley John Dimandja 1D similarity Index: 654 2D first name similarity Index: D second name similarity index: 654

Citric Citramalic Malic Monocarboxylic Acids Dicarboxylic Acids Tricarboxylic Acids Aromatics

Multi Level Similarity Indexing

Conclusions  Multidimensional profiling method has been demonstrated for selected organic acid standards; more standards need to be added to improve the screening process.  GCxGC allows for high-throughput sample preparation strategies  GCxGC-MS is indispensable for method development; GCxGC-FID can be used for routine analysis  Advanced chemometric methods need to be further developed for effective data processing

Acknowledgements  LECO Corporation  Restek Corporation  NSF/SEI Grant  Ms. Deanna J. Scott  Ms. Isioma Enwerem  Ms. Courtnea Rainey