Chemical Reagents and Derivatization Proceduresin Drug Analysis

Derivatization in conjunction with GC can be done :

in an off-line mode, often prior to the separation step (prederivatization),

to enhance the volatility, temperature stability, and/or detectability.

Prior to analysis by GC, compounds containing functional groups with active hydrogenssuch as COOH, OH, NH, and SH need to be protected.

Compounds with these functional groups tend to form intermolecular hydrogen bonds,reducing volatility. They are also thermally unstable and can interact with either fusedsilica or the stationary phase, causing peak broadening.

Common GC derivatives-alkylation

Common GC derivatives-acylation

Common GC derivatives-Silylation

Acylation reaction scheme

Derivatization of fluvoxamine with acetic anhydride

Decomposition of benzodiazepines during chromatographic analysis

Regroup an decomposition of temazpam

SPME/GC for Forensic Applications: Explosives,Fire Debris, and Drugs of Abuse

Fire Debris (Arson)A simple, inexpensive, rapid, and sensitive method for analyzing gasoline in fire debris, using

SPME for headspace sampling (capable of detecting ~0.1μL of petroleum product residue).

An accurate, simple, and rapid method for analyzing urine for methamphetamine and itsprincipal metabolite, amphetamine, using heated headspace SPME and capillary GC

Drugs of Abuse

Solid Phase microextraction (SPME)

Components of a Manual SPME Holder

Plain Hub

The O ring

Adjustableneedleguide/depthgauge

Plunger

PlungerretainingScrew

SPMEmanualholder

Septumpiercingneedle

Where fiber isexposed inheadspace/liquidsample

SPME fibers available• Fiber coating available:

– PDMS– PDMS/DVB– Polyacrylate– CAR/PDMS– CW/DVB– CW/TPR– StableFlex DVB/CAR/PDMS

• Different Phases available:– Non-bonded

• stable w/ some water-miscibleorganic solvents

• slight swelling may occur• NEVER use nonpolar organic

solvents

– Bonded• stable with ALL organic solvents• slight swelling possible w/

nonpolar solvents

– Partially Crosslinked• stable in most water-miscible

organic solvents• May be stable in some nonpolar

solvents, but slight swellingpossible

– Highly Crosslinked• Equivalent to the partially

crosslinked, but some bonding tocore has occurred in the past

• Different Phases available:– Non-bonded

• stable w/ some water-miscibleorganic solvents

• slight swelling may occur• NEVER use nonpolar organic

solvents

– Bonded• stable with ALL organic solvents• slight swelling possible w/

nonpolar solvents

– Partially Crosslinked• stable in most water-miscible

organic solvents• May be stable in some nonpolar

solvents, but slight swellingpossible

– Highly Crosslinked• Equivalent to the partially

crosslinked, but some bonding tocore has occurred in the past

Drugs of Abuse Confirmation by GC/MS

Drugs of Abuse Confirmation by GC/MS

Drugs of Abuse Confirmation by GC/MS

Headspace Gas Chromatography Method for DeterminingVolatile Compounds in Biological Fluids

5-mL blood collection tubes

Washed with distilled water, and dried in an oven at 105°C

After cooling to room temperature, 0.5 g NaCl and 0.1 g NaF were added to the tubes.

A 0.5-mL aliquot of sample and 0.5-mL of IS solution

Sealed with rubber caps, mixed, and placed in a 60°C water bath for 20 min (HS equilibrium)

A 150-μL gas aliquot was injected directly into GC (250-μL gas-tight syringe)

5-mL blood collection tubes

Washed with distilled water, and dried in an oven at 105°C

After cooling to room temperature, 0.5 g NaCl and 0.1 g NaF were added to the tubes.

A 0.5-mL aliquot of sample and 0.5-mL of IS solution

Sealed with rubber caps, mixed, and placed in a 60°C water bath for 20 min (HS equilibrium)

A 150-μL gas aliquot was injected directly into GC (250-μL gas-tight syringe)

Breath gas analysis

A method for gaining non-invasive information on the clinical state of an individual bymonitoring volatile organic compounds present in the exhaled breath.

Breath gas concentration can then be related to blood concentrations Containing more than 200 different volatile organic compounds (VOCs) VOCs are released within the human organism as a result of normal metabolic

activity or due to pathological disorders. VOCs enter the blood stream and are eventually metabolized or excreted via

exhalation, skin emission, urine, etc

Breath gas concentration can then be related to blood concentrations Containing more than 200 different volatile organic compounds (VOCs) VOCs are released within the human organism as a result of normal metabolic

activity or due to pathological disorders. VOCs enter the blood stream and are eventually metabolized or excreted via

exhalation, skin emission, urine, etc

Application of breath gas analysis

Asthma detection by exhaled nitric oxide Blood alcohol testing Lung cancer detection Diabetes detection Fructose malabsorption with hydrogen breath test Helicobacter pylori with urea breath test Diagnosis of bad breath Organ rejection

Asthma detection by exhaled nitric oxide Blood alcohol testing Lung cancer detection Diabetes detection Fructose malabsorption with hydrogen breath test Helicobacter pylori with urea breath test Diagnosis of bad breath Organ rejection

VOCs in breath identified as potential diagnosticmarkers of oxidative stress in various diseases

Blood VOCs

Breath analysis of VOCs offers a painless, simple and rapid way of assessing underlyingpathological conditions

There is a speculation that a significant proportion of these VOCs may be inhaled fromthe external environment.

By using the GC-MS technique, they found hexanal, 1-octen-3-ol and octane as possiblebiomarkers of liver cancer with good sensitivity and specificity in blood samples.By using the GC-MS technique, they found hexanal, 1-octen-3-ol and octane as possiblebiomarkers of liver cancer with good sensitivity and specificity in blood samples.

Analytical instruments

Breath analysis can be done with various forms of mass spectrometry, but thereare also simpler methods for specific purposes, such as the Halimeter and thebreathalyzer.

Gas chromatography-mass spectrometry GC-MS

Proton transfer reaction mass spectrometry PTR-MS and PTR-TOF

Selected ion flow tube mass spectrometry SIFT-MS

Ion mobility spectrometry IMS

Fourier transform infrared spectroscopy FTIR

Laser spectrometry Spectroscopy

Chemical sensors resp. Electronic nose

Breath analysis can be done with various forms of mass spectrometry, but thereare also simpler methods for specific purposes, such as the Halimeter and thebreathalyzer.

Gas chromatography-mass spectrometry GC-MS

Proton transfer reaction mass spectrometry PTR-MS and PTR-TOF

Selected ion flow tube mass spectrometry SIFT-MS

Ion mobility spectrometry IMS

Fourier transform infrared spectroscopy FTIR

Laser spectrometry Spectroscopy

Chemical sensors resp. Electronic nose

Metabolome analysis

Applied to biomarker detection and disease diagnosis in medical studies

Changes in metabolite levels reflect the functional status of a cell because alterations in

their levels occur downstream of DNA, RNA, and protein.

Owing to the rapid development of mass spectrometry analytical techniques

metabolome analysis is becoming an important experimental method in life

sciences including the medical field.

Metabolomic panel has been evaluated as potential candidate biomarkers.

Metabolomic study for diagnostic model of oesophageal and Colorectal cancer using GC-MS

Applied to biomarker detection and disease diagnosis in medical studies

Changes in metabolite levels reflect the functional status of a cell because alterations in

their levels occur downstream of DNA, RNA, and protein.

Owing to the rapid development of mass spectrometry analytical techniques

metabolome analysis is becoming an important experimental method in life

sciences including the medical field.

Metabolomic panel has been evaluated as potential candidate biomarkers.

Metabolomic study for diagnostic model of oesophageal and Colorectal cancer using GC-MS

Drug analysis in biological specimens

Several drugs abuse may be involved, including alcohol, amphetamines, cannabis,cocaine, opiates and prescription drugs, such as benzodiazepines.

Drug analysis in urine, blood, hair, oral fluid (saliva), nail clippings, tissue (adiposeand brain) and breath is used to determine the presence or absence of specifiedparent drugs or their metabolites.

The drug are usually present in these alternative speciments at low concentrationsand the amount of specimen available for analysis is small. In this regard, the usaeof highly sensitive technique is necessary.

Several drugs abuse may be involved, including alcohol, amphetamines, cannabis,cocaine, opiates and prescription drugs, such as benzodiazepines.

Drug analysis in urine, blood, hair, oral fluid (saliva), nail clippings, tissue (adiposeand brain) and breath is used to determine the presence or absence of specifiedparent drugs or their metabolites.

The drug are usually present in these alternative speciments at low concentrationsand the amount of specimen available for analysis is small. In this regard, the usaeof highly sensitive technique is necessary.

Example of a 200-ng/ml, 0.1-μl injection of TFA derivatives of amphetamine anddeuterated internal standard with 50:1 split; analysis run time slightly more than 1 min

Scheme of the analytical method for acidicpharmaceuticals and phenolic antiseptics

Scheme of the analytical method for estrogens

The developed SPE procedure was now optimized for biological matrices suchas plasma, blood, brain tissue and hair samples, as the extraction of ADsfrom these matrices is of interest in the field of clinical toxicology (plasma)and forensic toxicology (blood, brain, hair).

Optimization of the SPE procedure for extraction ofantidepressants (Ads) from biological matrices

For plasma and blood, the developed SPE method had to be optimized due totheir protein content.

Sample preparation scheme

Spectra and fragmentation patterns ofheptafluorobutyrylated fluoxetine

Rapid Determination of Cyanides inBiological Material by HS-GC/MS

HCN is liberated during an incubation step for 60 min at 60 °C by concentratedphosphoric acid from the matrix in a headspace vial

Subsequently transformed to cyanogen chloride Cl-CN by reaction with chloramine T.

The ions m/e = 61 and 63 for Cl-CN are monitored by SIM/MS and 1-BuOH is usedas internal standard (m/e = 56, 31 and 41).

Cocaine, amphetamines (including ecstasy), and opiates can be accuratelydetected for one month, given a short 1.3 cm segment of hair.

Detection of opiates (morphine, codeine, and6MAM) in human hair

Rapid determination of amino acids in neonatal bloodDerivatization with isobutyl chloroformate followed by solid-phase microextractionand gas chromatography/mass spectrometry

SPME-GC-MS TIC chromatograms for MSUD-positiveblood (a) and a control blood (b) samples were derivatized

Determination of Estradiol in Blood by GC/MS/MS

This applications note illustrates the selectivity and sensitivity advantages of anion trap GC/MS/MS approach to enhance selectivity for the hormone estradiol(estrogen) in blood.

Sample Preparation. Estradiol standards were derivatized with BSTFA (with 1%TMCS as catalyst).

MS/MS Method Development.dissociation of the isolated parent ion at m/z 416 to characteristic product ions atm/z 285 and 326.

This applications note illustrates the selectivity and sensitivity advantages of anion trap GC/MS/MS approach to enhance selectivity for the hormone estradiol(estrogen) in blood.

Sample Preparation. Estradiol standards were derivatized with BSTFA (with 1%TMCS as catalyst).

MS/MS Method Development.dissociation of the isolated parent ion at m/z 416 to characteristic product ions atm/z 285 and 326.

Automated method development for estradiol-di-TMS

Optimal CID voltage is 85 V