The Specific Use Method of SPE Cartridge

1. Select the SPE cartridge or filter membrane

Firstly, according to the physical and chemical properties of the object to be measured, and the substrate of the sample, the fixed phase with strong retention ability should be selected. If the object to be tested has a negative charge, anion exchange packing can be used; otherwise, cation exchange packing can be used. If it is a neutral substance to be measured, it can be extracted with reverse phase packing. The size and specification of the SPE cartridge or membrane should depend on the concentration of the sample to be measured. For in vivo samples with low concentration, less fixed phase filler should be used to extract larger volume samples.

Diol SPE Cartridges

2. The activation

Before extraction, rinse the cartridge with a solvent filled with the cartridge or rinse the filter membrane with 5 ~ 10ml solvent. Generally, methanol and other water can be used first.
The soluble organic solvent rinses the filler because methanol wets the adsorbent surface and penetrates the non-polar silica gel bonding phase, making the silica gel easier.
Wet with water, then rinse with water or buffer. Before sample addition, SPE packing should be kept moist. If the packing is dry, the sample retention value will be reduced. However, the different drying degree of each cartridge will affect the reproducibility of the recovery rate.

3. On the sample

Generally, the following measures can be taken:

(1) using 0.1mol/L acid or base regulation, pH<3 or pH>9. Centrifugal extraction of upper liquid;

(2) the protein was precipitated with methanol, acetonitrile, etc., then the supernatant was taken and diluted with water or buffer solution for extraction;

(3) the supernatant was taken after the protein was precipitated with acid or inorganic salt, and the pH value was adjusted for extraction;

(4) after ultrasound for 15min, add water and buffer solution, and extract with supernatant. The drug concentration in the urine sample is relatively high. Before adding the sample, dilute with water or buffer solution. When necessary, acid and alkali hydrolysis reaction can be used to destroy the combination of drug and protein, and then extract. The flow rate should be controlled to 1ml/min, and the fast flow rate is not conducive to the combination of the object to be measured and fixation.

4. The elution

The cleaning solvent of reversed-phase SPE is mostly water or buffer solution. A small amount of organic solvent, inorganic salt, or pH value can be added into the cleaning solution. The cleaning fluid added to the cartridge should not exceed the volume of one cartridge, and the SPE filter membrane is 5 ~ 10ml.

5. Elute the object under test

5 ~ 10ml eluent with weak ionic strength but capable of washing the substance to be measured should be selected. If higher sensitivity is required, the eluent can be dried before the mobile phase recombined residue is injected. In vivo samples, after elution contains more water, can choose the freeze-drying method. The SPE filler with weak retention ability can be washed with small volume and weak eluent, and then the eluent can be analyzed by the HPLC cartridge with strong polarities, such as C18 SPE cartridge.

If the substance to be measured can be ionized, the pH value can be adjusted to inhibit the sample ionization, so as to enhance the retention of the substance to be measured in the reverse SPE filler. During elution, the pH value can be adjusted to ionize the substance and elute it with a weak solvent. After collecting the eluent, the pH value can be adjusted to achieve the best separation effect in the HPLC analysis. In the process of elution, the flow rate should be slowed down, two small volume elution instead of a large volume elution, the recovery rate is higher.