Drug Determination: Sample Preparation and SPE

• Determination of drugs in biological fluids involves hyphenation techniques.
• Sample preparation involves steps to transform a sample for suitability in analysis.

Solid Phase Extraction (SPE) Importance

• It is vital for therapeutic efficacy, bioavailability, bioequivalence studies, and forensic toxicological analyses.
• Fluids and tissues are complex matrices, causing low selectivity and sensitivity.
• Sample procedures are needed for quantifying low dosages of active drug substances.

SPE Usage

• SPE simplifies complex sample matrices.
• It purifies compounds of interest.
• It reduces ion suppression in mass spectrometry applications.
• SPE can fractionate complex mixtures by classification.
• It concentrates analytes present at low levels.

Old Sample Preparation Procedures

• Extraction removes analytes and eliminates interfering compounds.
• Evaporation concentrates extracted analytes for analysis or re-constitution.
• Evaporation is the most time-consuming part of the analytical procedure.
• It can be the largest source of errors in the workflow.

Parameters

• Solvent use and costs are important.
• Extraction time impacts lab throughput.
• Recovery % is crucial from each extraction because low % yields poor analytical results.
• Relative Standard Deviation % (RSD) is a reproducibility measure with lower %RSDs yielding greater reproducibility.

Technique

• SPE is a cost-effective and versatile method for sample preparation.
• It's widely used in environmental and food labs.
• Prior to GC & GC/MS or LC & LC/MS analysis.
• Performed with inexpensive vacuum pumps, manifolds, and disposable cartridges or disks.

Solid Phase Extraction (SPE)

• This technique uses a solid adsorbent, in a cartridge or disk, to adsorb select species from solution.
• SPE isolates or cleans samples before analysis.

SPE Steps

• Bind and elute involves retaining analytes while matrix components do not.
• The analyte is washed and eluted with a strong solvent.
• Removal/trapping means the analyte is not retained and elutes, while the matrix components are retained.
• The captured analyte elution solvent can be further reduced in volume to concentrate the analyte prior to analysis.

Types of SPE

• Sorbent choice depends on the chemical properties of the species being separated.
• Understanding the sample matrix and analytes of interest is key for type selection.
• Ion exchange cartridges are selected if the analyte of interest is charged or ionizable.
• Types of SPE include reversed phase, normal phase, ion exchange (anion/cation), and mixed-mode phases.
• Mixed-mode phases have properties of more than one type of SPE material.

SPE Categories

• Adsorption
• Ion exchange
• Mixed Mode

SPE Type : Adsorption

• Normal. Analytes with low to high polarity/neutral and separates based on polarity with a non-polar sample matrix
• Least polar compounds elute first
• Reverse. Analytes with moderate to low polarity and separates based on hydrophobicity with an aqueous sample matrix.
• Most polar compounds elute first
• Cation. Analytes that are charged (+) or ionizable and separates based on charge in anAqueous sample matrix with low ionic strength.
• Weakly ionized compounds elute first
• Anion. Analytes that are charged (-) or ionizable and separates based on charge in an Aqueous sample matrix with low ionic strength.
• Weakly ionized compounds elute first
• Mixed-mode combines adsorption, hydrogen bonding, or ion-exchange properties in one cartridge.

Adsorption

• Reversed phase SPE techniques are suited to analytes with moderate to low polarity.
• Separates analytes based on hydrophobicity, with polar compounds eluting first.
• Employed in aqueous samples with reversed phase sorbents.
• Normal phase SPE is used when the analyte has low to high polarity, or is neutral.
• The cartridge contains silica and separation is polarity-based (least eluting first).
• Sample is in a non-aqueous matrix.
• Ion exchange SPE separates compounds based on charge.

Application

• The best way to separate 4-aminophenol and acetaminophen would be charge, using ion exchange.

Mixed Mode

• Mixed mode in Bind and elute SPE occurs when the analyte is retained when interferents will be rinsed
• Mixed Mode SPE is often used to retain one target analyte and reduce matrix contamination.
• Examples of mixed-mode SPE include reversed-phase with strong or weak cation/anion exchange.

SPE Cartridge

• Increases detector sensitivity and improves detection limits.
• Matrix Removal removes matrix and then elutes the sample into a solvent compatible with the analytical technique.
• Clean Up removes interferences prior to analysis.

SPE vs Liquid-Liquid Extraction (LLE)

• SPE is faster and decreases solvent use with out creating emulsions.
• SPE can be automated and requires less solvent, but there is more efficient extraction, but LLE has many phases that help with separation.

The Process of SPE Step

1. Conditioning is moisturizing of the solid phase with the matrix
2. Sample application which can occur when the sample is either pressed or drawn through solid phase
3. Washing and Elution
4. 4. Elution

Sorbent Substrates

• Types of SPE sorbents include Silica-Based, Polymeric, and Activated Carbon.
• Silica-Based Sorbents utilize silica substrate with bound functional groups, named for the functional group's character (e.g., C18).
• Polymeric Sorbents utilizes styrene-divinyl benzene copolymers modify to create custom ion exchangers .
• Activated Carbon contains high surface area ensures retention of multiple analytes in a challenging way.

Instruments

• The AutoTrace 280 SPE instrument by Thermo provides reliable automated SPE for determining organic pollutants.
• It saves time and reduces solvent use.

AutoTrace 280 SPE

• It reduces sample extraction cost.
• Improved productivity and analytical precision.

Hyphenated Separation Techniques

• This is a combination of two or more techniques to separate chemicals from solutions and subsequently detect them.
• It includes separation-separation, separation-identification, and identification-identification techniques.
• Separation techniques include SPE, GC,HPLC or CE
• Detection techniques include PDA, IR, MS, NMR or TLS

Hyphenated Techniques Advantages

• Fast and accurate analysis.
• Automation increases.
• Sample throughput expands.
• Reproducibility is better.
• Contamination is reduced
• Separation and quantification achieved at same time.

List of Hyphenated Techniques

• GC-MS
• LC-MS
• LC-NMR
• EC-MS
• CE-MS
• GC-IR
• LC-MS-MS
• LC-ESI-MALDI-TOF
• GC-MS-MS
• GC-NMR
• GC-AES
• ICP-MS
• ICP-AAS
• ICP-OES

Spectroscopy Hyphenation

• Ultraviolet/visible detection identifies/quantifies compounds thanks to conjugation systems.
• Infrared (IR) or FTIR detection is also used.
• NMR permits identifying and quantifying unknown compounds by chemical shifts.
• Mass spectrometry identifies/quantifies chemical compounds with mass-charge ratio spectral data.

Hyphenated GC

• In addition to hyphenations, it can also include automated online sample preparation systems before a GC such as:
• Static headspace
• Dynamic headspace
• Large volume injection
• Solid-Phase Micro Extraction.

GC-MS

• A GC-MS instrument is composed of a gas chromatograph and a mass spectrometer.
• It separates these chemical mixtures (gas chromatograph) and identifies or quantifiescomponents (MS detector).
• It has the most accurate and efficient tool for analyzing volatile organic samples.
• It is used to analyze a urine sample for Tetrahydrocannabinol,(THC)
• THC appears as a GC peak, and gives a molecular ion at 314, its molecular weight.

GC-MS Applications

• Quantitation of pollutants in drinking and wastewater.
• Drug quantitation in metabolites and urine for pharmacological and forensic purposes.
• Identification of unknown organic compounds.
• Synthetic chemistry and reactions.
• Drug analysis + pesticide and Herbicide Detection

LC-MS

• LC-MS detects compounds from polyaromatic to peptide and proteins.
• Can identify and measure the purity of compounds
• Is used for determination of pesticides, herbicides and organic pollutants in environmental monitoring.
• Proteome samples can be analyzed with this technique.

GC-IR

• GC-IR technique is hyphenation of gas chromatography and Infrared spectroscopy.
• It can be both very sensitive, very expensive. and sample recovery because IR is non-destructive.
• GC does it through its separation, with IR performs performing the identification.
• Effluent from GC is directly forwarded into the heated pipe of IR at atmospheric pressure.
• Infrared red spectroscopy identifies the compound by identifying the functional groups.

GC-IR Applications:

• Pharmaceutical + Industrial
• DNA Analysis of blood samples, other fluids.

GC-AES/GC-OES

• Combination of gas chromatography with atomic emission spectroscopy.
• Atomic emission spectroscopy is one of the elemental analysis techniques
• GC performs the components and with the help of AES the elemental of components is performed.
• Elemental composition of every peak separated by GC is determined.