3.2.5. Desorption Time

Effect of time on desorption of analytes from sorbent is presented on Figure 8. Only slight differences of desorption kinetics between investigated compounds were noted. The amount of eluted analytes increased up to 4 min and then a plateau was observed.

**Figure 8.** Effect of desorption time on percentage of eluted analytes.

### *3.3. Application of Si-PANI Sorbent for Pretreatment of Plant Material*

Based on the conducted experiments, the optimal conditions for d-SPE of triterpenic acids were as follows: Si-PANI (+) impregnated with water, 3 min of extraction time, 2 mL of methanol water mixture (1:1, *v*/*v*) as washing solution, 4 min of desorption time and methanolic solution of ammonia as elution solvent. To verify the utility of the procedure, methanolic extracts from *Viscum album* L. and *Ocimum basilicum* L. were purified with the use of the above conditions. Moreover, MSPD was also conducted to assess the application potential of Si-PANI for isolation of triterpenic acids directly from raw plant material. The conditions for MSPD were established based on optimized d-SPE procedure and were as follows: Si-PANI (+) impregnated with water, 3 min of grinding time, 2 mL of methanol water mixture (1:1, *v*/*v*) as washing solution, and methanolic solution of ammonia as elution solvent.

The amount of triterpenic acids isolated using both techniques was determined by HPLC method. The validation parameters are summarized in Table 1 and the results of quantification are presented in Table 2.

**Table 1.** Validation parameters for determination of triterpenic acids (*n* = 5).


**Table 2.** The content of investigated analytes obtained with the use of various extraction methods (mg analytes/g of dried material ± SD).


As can be seen, the differences between a determined amount of investigated compounds in raw and d-SPE purified extracts were relatively low (5.1 and 5.9% for BA and OA, respectively in *Viscum album* and 7.5 and 9.2% for OA and UA, respectively in *Ocimum basilicum*). Moreover, the significant reduction of the accompanying matrix comparing to raw extracts was observed in three-dimensional (3D) HPLC chromatograms (Figure 9) and this proved the utility of the proposed method for sample pre-treatment of plant extracts.

**Figure 9.** 3D HPLC chromatograms of investigated plant extracts obtained with the use of various extraction methods; I-*Viscum album* L. and II-*Ocimum basilicum* L.

Pre clean-up of the sample is an especially essential step before chromatographic analysis. It allows us to extend the column longevity because it prevents the clogging of inter-grain spaces and pores that may led to the reduction of an active surface of stationary phase and decreasing of chromatographic system efficacy (lower theoretical plate number, resolution and peak symmetry). It also prevents the excessive increase of pressure in the chromatographic system.

The reduction of the matrix was also observed for MSPD; however, the differences of quantified analytes in raw and MSPD extract were significant (in the range of 36–42%); thus, despite the simplicity, and relatively low time-consumption, this technique may be considered only for preliminary screening studies.

In our previous study [18], two commercially available sorbents were applied for SPE of triterpenic acids; however, they were less favorable due to weak sorption (octadecyl silica) or difficult elution (aminopropyl silica). The cost of Si-PANI is slightly higher than bare silica; however, it is lower than its other modifications and Si-PANI may be an alternative for these types of sorbents.
