Choose, Activation, Loading, and Shower of SPE Cartridge

1. Choose an SPE cartridge or filter

First of all, according to the physical and chemical properties of the analyte and the sample matrix, the stationary phase with a strong retention capacity of the analyte should be selected. If the analyte is negatively charged, use anion exchange packing, otherwise use cation exchange packing. If it is a neutral analyte, it can be extracted with reverse-phase packing.

The size and specifications of the SPE cartridge or filter should be determined by the concentration of the analyte in the sample. For in vivo samples with a lower concentration, generally use as few stationary phase fillers as possible to extract larger volumes of samples.

2. Activation

Before extraction, flush the small column with a solvent filled with a small column or flush the filter with 5-10ml of solvent. Generally use methanol and other water first.

Flush the packing with a soluble organic solvent, because methanol can wet the surface of the adsorbent and penetrate into the non-polar silica gel bonding phase, making silica gel easier.

Wet with water, then add water or buffer to rinse. Before loading the sample, the SPE packing should be kept moist. If the packing is dry, the sample retention value will be reduced; and the drying degree of each cartridge will be different, which will affect the reproducibility of the recovery rate.

C18A Reversed Phase SPE Cartridges

3. Loading

Generally, the following measures can be taken:
(1)Adjust with 0.1mol/L acid or alkali to make pH<3 or pH>9, centrifuge to take the supernatant for extraction;
(2)Precipitate the protein with methanol, acetonitrile, etc., and take the supernatant, Dilute with water or buffer and extract;
(3)Precipitate protein with acid or inorganic salt and take supernatant, adjust pH to extract;
(4) Add water and buffer after 15 minutes of sonication, and extract supernatant. The drug concentration in the urine sample is high. Dilute it with water or buffer before adding the sample. If necessary, acid and alkali hydrolysis reactions can be used to destroy the combination of drug and protein and then extract. The flow rate should be controlled to 1ml/min, the fast flow rate is not conducive to the combination of the test object and the fixed.

4. Shower

The cleaning solvent of reversed-phase SPE is mostly water or buffer solution. A small amount of organic solvent, inorganic salt, or pH can be added to the cleaning solution. The cleaning solution added to the small column should not exceed the volume of a small column, while the SPE membrane is 5 to 10ml.

5. Elution test substance

An elution solvent with a weak ionic strength of 5 to 10 ml but capable of washing the analyte should be selected. If higher sensitivity is required, the eluent can be evaporated and the residue can be reconstituted with the mobile phase before injection. After elution of the in vivo sample contains more water, the freeze-drying method can be used.

SPE packing with weak retention capacity can be used to wash the analyte with a small volume and weak eluent, and then use a more polar HPLC analysis column such as the C18 column to analyze the eluate. If the analyte can be ionized, the pH value can be adjusted to suppress sample ionization to enhance the retention of the analyte in the reversed-phase SPE packing, adjust the pH value during ionization to make it ionized and elute with a weaker solvent, collect After the eluent, the pH value is adjusted to achieve the best separation effect in HPLC analysis.

During the elution process, the flow rate should be slowed down, and two small-volume elutions should be used instead of one large-volume elution. The recovery rate is higher.