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Hello everyone!

In the previous lectures,

we have studied the commonly used pretreatment methods for biosamples.

With the continuous improvement of pharmaceutical analysis,

biosample pretreatment have been rapidly developed,

and many new techniques have emerged.

This topic will introduce three new technologies.

First, let's study the solid-phase microextraction.

The solid-phase microextraction,

SPME for short,

is a sample pretreatment technology developed in the 1990s

and has received great attention in the field of analytical chemistry.

As you can see from this picture,

the number of published papers increases rapidly.

SPME has many advantages:

for example, the operation is simple and fast;

no organic solvent is needed;

it integrates sampling, extraction,

and preconcentration with sample injection,

hence avoiding the errors caused by multi-step operations;

most interestingly,

it can be coupled to GC or LC to realize the automation.

The device of solid-phase microextraction is very simple,

and is similar to a chromatographic micro-injector.

It consists of a handle and an extraction head or fiber head.

The extraction head is a fused silica fiber

coated with an appropriate solid phase,

which could extract and concentrate the organic analyte.

The selection of the coating material

is based on 'like dissolves like' principle.

The polar compounds are extracted with polar coatings

(such as polyacrylate),

while the non-polar compounds with non-polar coatings

(such as polydimethylsiloxane).

There is a stainless-steel tube outside

to protect the fiber from being broken,

and the fiber head can be freely moved

inside the stainless-steel tube.

The thin stainless-steel tube could penetrate a rubber

or plastic gasket for sampling and injection.

Let's take a look at the two steps of SPME,

that is, extraction and desorption.

The first process is extraction.

Insert the extraction head into the sample vial,

and expose the extraction phase-coated fiber to the tested sample.

It can be divided into direct immersion SPME and headspace SPME.

In the direct immersion SPME,

the fiber is immersed directly in the sample solution

or gaseous sample to extract the target analyte.

But in the headspace SPME,

the fiber is parked above the sample solution for headspace extraction

without contacting with the sample matrix,

thus avoiding the interference from matrix.

The second step is desorption.

When SPME is coupled with GC,

thermal desorption is used to desorb the extracted analyte.

The extraction head is inserted in the GC injector,

and exposed to the heated carrier gas,

which continuously desorb the extracted analyte

for subsequent GC analysis.

When SPME is coupled with HPLC,

the fiber is inserted in a desorption tank,

the analytes would be desorbed from the fiber

by trace amount of solvent

and delivered to the column for HPLC analysis.

Then let's take a look at liquid-phase microextraction.

The liquid-phase micro-extraction,

LPME for short,

is based on the liquid-liquid extraction

and combines the advantages of liquid-liquid extraction

and solid-phase micro-extraction.

It is a fast and economic method with small amount of solvent

and a pretty simple device,

micro injector or polypropylene hollow fiber,

for extraction and injection.

Please take a look at the schematic diagram of the device:

a polypropylene hollow fiber is used to carry the organic solvent.

Through the immobilization in the porous hollow fiber,

the organic solvent can not only directly contact the given phase,

that is, the sample solution,

but also increase the contact area

between the organic solvent and the sample solution,

thereby facilitating the analyte mass transfer and diffusion.

Its separation principle is still based on the distribution

between the sample and small volume of organic solvent.

According to the properties of the solution in the hollow fiber,

liquid phase microextraction can be divided into two-phase

and three-phase systems,

of which the two-phase system is the most popular.

Let's look at a two-phase system,

also known as microporous membrane extraction,

in which the hollow fiber contains organic solvent.

According to the principle of "like dissolved like",

the analyte in the sample solution is extracted into the organic phase.

This technique has high sensitivity,

large enrichment ratio; simple and fast operation;

fewer solvents used,

and is an environmentally friendly technology.

Especially, the materials are widely available

without any need to use repeatedly,

which reduces the experimental cost compared to the SPME.

Therefore, this new type of extraction method

has become a very important sample pretreatment technique

in modern instrument analysis.

Finally, let's study the microdialysis technology.

Microdialysis is an in-vivo sampling technique

based on the principle of dialysis.

By combining the perfusion sampling with dialysis technique,

it can continuously sample the endogenous

or exogenous substances in the tissue fluid of the living body,

and can be coupled with various analysis method for online analysis.

Please take a look at the schematic diagram of the device.

The microdialysis probe is composed of a dialysis membrane,

inlet and outlet tubes.

The dialysis membrane is the heart of the device.

These membranes are chemically non-selective.

Small molecules passing through the membrane

are determined by the pore size of the membrane.

The microdialysis system is composed of a micro-perfusion pump,

a microdialysis probe, a sample collector,

connecting tubes and supporting equipment.

During the experiment,

the probe is buried in the target tissue (such as brain).

And an isotonic perfusate with similar composition of the tissue fluid

is perfused through the probe at a constant flow rate.

Please pay attention to this dynamic diagram.

The perfusate passes through the inlet tube,

probe and outlet tube in sequence.

When using a coaxial probe,

the perfusate flows out of the inner sleeve

and then enters the outer sleeve.

Due to the concentration gradient,

small molecular active substances in the tissue

diffuse across the dialysis membrane,

while cutting off the large molecules.

As you know,

most free drugs are small molecules

that can be taken out of the body by the perfusion fluid,

while the macromolecules like proteins and protein-bound drugs

cannot penetrate the membrane.

Therefore, the combination of microdialysis and analytical techniques

can determine the free drug concentration

in the extracellular fluid of various tissues straightly,

and is widely used in various fields

such as neuroscience and pharmacokinetics.

Compared with traditional sampling methods

(such as blood sampling or tissue homogenization),

the prominent advantage of microdialysis is that

it could perform online real-time in vivo sampling

without interfering the normal life process.

It can truly reflect the dynamic changes

of endogenous or exogenous substances in the organism

and greatly reduce animal numbers.

Moreover, the collected sample solution

does not contain any macromolecular compound,

thus the sample does not require complicated clean-up processing

and could be directly analyzed by HPLC,

high-performance capillary electrophoresis,

and LC-MS.

At last, the method could realize continuous online analysis.

That's all, thank you for watching.