**4. Monitoring**

In parallel, the authors have conducted a thorough monitoring campaign to modulate the range of the simulations and at the same time to check the usefulness of their outputs. We have to outline that the objective validation of our simulation program has taken place beforehand in other controlled experiments and test chambers [12–23].

To perform the evaluation, we installed data loggers in Saint Louis to monitor the dry bulb temperature, mean radiant temperature and relative humidity. The sensors were Onset models U12-012, UX 120-006 M and TMC6-HD, with data recorded every ten minutes. Temperature sensors had a measuring range from 0 to +100 ◦C and an accuracy of ±0.5 ◦C. The calibration before taking the measurements guaranteed the proper functionality of the setup.

Accumulative measures of light are not helpful from a spatial point of view, one-time measurements conducted simultaneously at a ground-floor grid work usually better. To that end, we have used a lux-meter PCE-170A with ISO calibration, a measuring range from 0 to 40,000 lux and an accuracy of ±0.3%. Of those, we present a comparison with simulated values for partly cloudy sky, which correspond well by not showing relevant discrepancies (Figures 12 and 13). The correlation coefficient of east-west and north-south main axis are 0.93 and 0.92, respectively.

The slight differences registered between simulated and measured data are presumably due to the event that on the days of performance of the experiments, as the sky was partly cloudy, some sunlight filtered from the southern fenestration increasing the levels on the north niche (bottom side in the graph). Besides, the main façade of the church faces east and through the upper choir perceptible intensities appear around midday but these were deliberately taken out of the simulation. The latter would explain minor augments of level near the entrance area to the east (left side of the figure).

**Figure 12.** Comparison of measured and simulated data for partly cloudy sky at midday on 21 December. 0 m corresponds to the central point of the church: (**a**) east-west axis, from entrance (left) to the altar (right). (**b**) south-north axis, from right to left respectively. Source: Almodovar-Melendo.

**Figure 13.** Extension of Figure 12, measurements effected at the ground floor in winter solstice at 12 h. Source: Cabeza-Laïnez.

We investigated other sky plus sun conditions with occasional registers, these showing the reliability of the simulations. As stated above, the model has been validated in previous campaigns of projects with straight parallelepiped shapes in laboratory-like conditions and for that reasons we are not in the position here to offer a full-range validation for such unusual architecture, as is this church.

In order to discuss the environmental performance of the building, measured daylighting values have been correlated to other environmental parameters such as temperature and relative humidity, relevant for the IEQ of the building [33,34]. Measurement data registered during the summer show that the lighting values are in a range of 200 to 400 lux, by all standards a steady level of lighting apt for the majority of visual tasks. On the other hand, we identified consistent temperatures nearing 25 ◦C in the floor level of the nave, at a time in which external shade temperature can reach 45 ◦C or even more on the north face of the cylindrical drum (Figure 14). This means that such adequate illumination is not attained by means of discomfort or excessive overheating, as is the case of so many contemporary buildings, fully glazed. Shading devices are generally not required in this kind of structure. However, we found photographs of late 1920s in which the tall windows were shaded from the inside with fabrics, a curiosity which may attest to the significant amount of light that impinges on the lower levels of the compound.

Theretofore, the property of the capacitive mass of this masonry and tile (ceramics) building is noticeable. Moreover, the low ratio of window to total surface area (under 10% as previously mentioned) averts excessive solar gains and contributes to obtain an optimal thermal performance. In this regard, we have to remind that daylight is the lighting source with the best light to heat ratio (up to 160 lumen/W).

The indoor relative humidity remained around 50% during the daily cycle though it could increase slightly at night.

Similar monitoring results were obtained by the authors at another baroque paradigm, the church of San Lorenzo in Turin (Italy) by Guarino Guarini (Figure 15). However, this temple is located almost 10 degrees more to the north than Saint Louis and markedly receives less solar radiation.

**Figure 14.** Temperature measured at the Church in summer to check overheating effects: (**a**) external temperature at the dome, (**b**) internal temperature at floor level near the altar. Notice that when external temperature in the north face of the drum reaches a scorching 50 ◦C, at the altar zone we measured a much cooler maximum of 26 ◦C. Source: authors.

**Figure 15.** Clear Sky monitoring in April at the famous church by C. G. Guarini. Turin. Lat. 45◦6 N. Source: Cabeza-Laïnez.

### **5. Discussion of the Results**

Due to the particular orientation of the church that defies the postulate of the longitudinal West-East axis from entrance to altar, which presided over European architecture until late Renaissance; the solar events like solstices and equinoxes are critical to define the performance of the central space under consideration. In fact, the fenestrated cylindrical drum allows the capture of solar radiation impinging from every solar position and suppresses the absolute necessity of offering a southern façade to achieve solar gains for natural heating and lighting, especially in winter, as was the case of Gothic architecture in England, France and Germany. The inner cylinder so to speak funnels direct solar radiation and due to its considerable height, it seldom permits the sunrays from reaching the floor level avoiding visual discomfort to the visitors.

In each case, we have investigated two situations: overcast conditions (isotropic but with hourly and monthly variation (Figure 16) and clear sky with sun (where orientation and hourly/monthly variations are mandatory). The first condition refers to conventional models for cold climates while the second condition is more innovative and typical of warmer regions but many simulation programs are unable to deal with it not to mention with the occurrence of curved surfaces (Figures 17–19). The daylighting levels have been calculated in a plane located 1 m above the floor, a position deemed for a sitting person.

Spectroscopy analyses were necessary to evaluate the reflective coefficients for this simulation. To this aim the authors conducted several on site tests. Such values tend to differ from those suggested by conventional colour charts, because ageing, candle fumes or inadequate refurbishments may have altered the original condition of the veneers.

**Figure 16.** Cloudy Sky at midday in winter. Source: Almodovar-Melendo and Cabeza-Laïnez.

One of the most unfavourable cases occurs in winter under cloudy sky condition. Even so, the average value is more than 80 lux (Figure 16) which is considered sufficient to perform basic tasks even with modern standards. The light field is rather homogeneous (from 120 to 80 lux) in the central nave and presents a marked axial symmetry appropriate for the liturgy and reinforcement of the design intentions. The minimum levels remain well over 50 lux.

In spring, the situation is much enhanced, as even in the mornings or afternoons the mean illuminance is about 150 lux and values lying in the range from 200 to 300 lux are easily reached (Figure 17). At noon (Figure 18) the average level is over 200 lux and a maximum of circa 1000 lux can be found in several points, the light is more varied but without abrupt contrasts in a soft progression from 200 to 500 lux towards the north niche where the sun impinges during the equinox.

The cloudy sky condition is slightly lower in its intensities but we have not presented that here since it is untypical of Seville in spring and autumn, its probability of occurrence is of 18% according to meteorological data; even so minimum values remain over 50 lux.

In summer, the mean illuminance lies in the range of 150 lux in early morning and late afternoon (Figure 19) and reaches 250 lux at noon.

If, following with the discussion on the simulations, we refer below to the vertical component of the radiation vector; the simulations show an interesting performance of light in the more likely event in Seville of enjoying a clear sky with direct sun (Figure 20). According to a TMY (typical meteorological year) of Seville provided in the software Energy Plus and derived from the Spanish Weather for Energy Calculations (SWEC), the probability of occurrence of non-clear skies in spring, autumn and summer combined is of 11%.

The illumination sectional vector keeps at an angle of around 45 degrees (which means it is not too steep), this clearly allows for distinct perception of artworks and wall decorations unlike Roman pantheon and derived churches like San Bernardo alle Terme (built under the dome of an ancient thermal bath).

**Figure 17.** Spring clear sky with sun. Probability of 74% (76% in autumn). The graph is symmetric at 15.15 h. Source: Almodovar-Melendo and Cabeza-Laïnez.

**Figure 18.** Spring clear sky with sun at midday, at some spots values of 1000 lux are registered. Source: Almodovar-Melendo and Cabeza-Laïnez.

**Figure 19.** Summer Clear Sky with sun. Symmetric with 8.45 h. Source: Almodovar-Melendo and Cabeza-Laïnez.

**Figure 20.** Daylight Section at the spring equinox with sun, midday. Values reach 300 lux at floor level. Source: Cabeza-Laïnez and Almodovar-Melendo.

Such character is less pronounced for the infrequent case of cloudy skies but can still be perceived and it has been checked against monitoring (Figure 21).

To sum up, the results examined from Figures 16–21 show that the daylighting field is balanced; with a range of around 100 lux in plan for cloudy sky in winter. In other sky conditions, values exceeding 300 lux can be amply found and over 200 lux are guaranteed in the central circle of the nave for a 56% of the total daytime. To clarify the results, we have compared in Figure 22 simulated data in Summer, Spring and Winter for clear sky with sun.

The vertical situation of lighting also facilitates orientation and perception of the space and adds variety or spark in the disposition of liturgical spaces for Southern Spain. In a famous poem (by Angel Camacho), dating from 1733 the church of Saint Louis was compared, precisely for its lighting features, to the temples of Jerusalem and Zorobadel in ancient Babylon.

**Figure 21.** Daylight Section at the spring equinox in a cloudy situation. Maximum values of 200 lux on the ground. Source: Cabeza-Laïnez and Almodovar-Melendo.

**Figure 22.** Comparison of simulated data for a clear sky with sun at midday: (**a**) east-west axis, from entrance (left) to the altar (right). (**b**) south-north axis, from right to left respectively. Source: Almodovar-Melendo.

Coming back on the figures to describe this considerable success of religious architecture, the small proportion of window to total surface area (less than 10%) alongside with the low latitude of Seville (37◦22 N) may lead to such admirable results. We should notice that the values in this church are comparatively less than at its earlier baroque counterparts, which were mostly built in temperate north-central Italy and thus using wider fenestration in disregard of the heat.

Such could be the upfront explanation for the subdued illuminance levels attained on the inside of the Saint Louis temple. Especially if we compare them with some jewels of Roman Baroque (Figure 23), like Sant'Ivo by Borromini, the church at the site of ancient University of Rome and mainly Sant'Andrea by Bernini (Figure 24), an oval shaped masterpiece and not casually commissioned by the Jesuits as well.

So far, the analysis performed by the authors permit to trace a sure-handed line in the evolution of centralized temples pertaining to the baroque era. Such line is deeply chiselled in what we may call a proto-scientific artisanship of light within space.

**Figure 23.** Simulations at Sant'Ivo by Borromini in Rome. December at 9:55 am. Clear sky plus sun. Source: Almodovar-Melendo.

**Figure 24.** Simulations at Sant'Andrea by Bernini in Rome. Summer solstice. Source: Cabeza-Laïnez.

As we have clearly seen, sundry environmental and historical design strategies such as lighting, thermal comfort, et cetera, do intertwine in this analysis whilst at the same time they recall human and cultural nuances of architecture. For instance, the introduction of gilding and reflective specular veneers is critical in maintaining an acceptable visual perception with a restrained glazed surface. Such design features contribute to overcoming a situation perhaps created by an excessive seclusion from external climatic conditions.

Moreover, we confirm that the hypothesis by which the galleries of the nave were used as a place of study for the novices is plausible given the bright atmosphere that is achieved at the upper levels and the accuracy in which the educational and liturgical details of the interior of the dome cum drum are perceived from above.


**3.2 Targeted therapies**

Evolution of targeted therapies in thyroid cancer:

The increasing knowledge about the molecular alterations underlying thyroid cancer has greatly increased the interest in developing new drugs for targeted treatments in the last decade. The families of drugs that have primarily been investigated and most extensively studied for the treatment of thyroid cancer are tyrosine kinase inhibitors (TKIs). The first international clinical trial started in 2005 and explored the efficacy of motesanib diphosphate on 93 patients with progressive, locally advanced or metastatic, radioiodine refractory DTC. The median PFS was estimated to be 40 weeks [26]. The same drug was investigated for the treatment of locally advanced or metastatic, progressive, or symptomatic MTC in another single-arm phase 2 study which enrolled 91 patients. The median PFS was 48 weeks [27]. Despite these promising results, the drug was not FDA approved for these indications. Both studies used a single-arm design and were performed in a relatively small population of patients. Moreover, the lack of a placebo arm is another limitation. Soon after the motesanib

study, another, phase 2 study examining the effect of axitinib on 52 cases of locally advanced, unresectable, or metastatic MTC or RAI-R DTC was started. The median PFS and OS was 16.1 months and 27.2 months respectively. As in the previous studies, the single-arm design of this study makes the interpretation of the results rather difficult. Although the results appeared encouraging, no further studies have been planned using this drug [28]. These trials show that targeted therapies could lead to prolonged disease stabilization and objective response in patients with metastatic or recurrent thyroid cancers. Following these multiple other targeted agents soon entered clinical trials and there by a new era of treatment options emerged.

#### *3.2.1 Multikinase inhibitors*

Various multikinase inhibitors has proven to be an effective treatment option for metastatic and recurrent Thyroid Cancers, given the activity of the PI3K/Akt/mTOR and MAPK signaling pathways in this disease. Currently, there are four drugs, all oral multikinase inhibitors, approved in the treatment of differentiated and medullary thyroid cancer. Sorafenib is a multikinase inhibitor that targets the VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF. Lenvatinib is another kinase inhibitor that targets VEGFR1–3, fibroblast growth factor receptors (FGFR) 1–4, PDGFR*α*, RET, and c-Kit signaling pathways. Vandetanib selectively targets RET, VEGFR, and EGFR signaling and cabozantinib is a kinase inhibitor of RET, VEGFR2, and MET. All these kinase inhibitors are antiangiogenic, but they also have additional targets that may contribute to their efficacy. The response rates of the above agents vary from 30 to 50% in various trials.

Sorafenib and lenvatinib are FDA approved for advanced radioiodine refractory and poorly differentiated thyroid cancers and vandetanib and cabozantinib are approved for recurrent or metastatic medullary thyroid cancers.

Role of multikinase inhibitors in differentiated thyroid cancer:

Two randomized placebo-controlled phase III clinical trials that led to US Food and Drug Administration (FDA) approval of TKIs for treatment of progressive RAIrefractory DTC are the DECISION and SELECT trials.

Sorafenib was approved by the FDA in 2013 on the basis of DECISION trial in locally advanced or metastatic radioiodine refractory differentiated thyroid cancer [29]. DECISION, a double-blind, placebo-controlled, phase 3 trial trial reported a significant progression-free survival (PFS) benefit of 5 months with sorafenib. Median progression-free survival in the sorafenib group was 10·8 months vs. 5·8 months in the placebo group (p < 0·0001). However, the overall response rate [ORR] was 12% in the trial. Sorafenib did not improve overall survival (OS), although PFS was longer with sorafenib. The SELECT trial, a phase 3 randomized double-blind trial investigated lenvatinib versus placebo in patients with progressive iodine-refractory DTC which showed a significant improvement in PFS of 14 months with lenvatinib compared with placebo. The median PFS was 18.3 months in the lenvatinib group and 3.6 months in the placebo group (P < 0.001) and lenvatinib had an ORR of 65% [30].

Cabozantinib has shown activity in patients with radioiodine-refractory DTC who have been previously treated with lenvatinib, sorafenib, or both TKIs in COSMIC -311, a doubleblind, placebo-controlled, phase 3 trial [31]. The trial showed significant improvement in progression-free survival for cabozantinib over placebo (5·7 versus 1·9 months, p < 0·0001) and an ORR of 15%.Based on the above trial results, FDA approved cabozantinib for the treatment of locally advanced or metastatic radioactive iodine–refractory differentiated thyroid cancer that has progressed after prior VEGF-targeted therapy in 2021.

Role of multikinase inhibitors in medullary thyroid cancer:

Vandetanib was approved for the treatment of locally advanced or metastatic medullary thyroid cancer based on the ZETA trial, a randomized double blind phase 3 trial that demonstrated a PFS benefit of 11 months (p < 0.0001) with vandetanib compared with placebo in patients with MTC. At a median follow up of 24 months, vandetanib demonstrated an ORR of 45% [32]. One major limitation in the design of the study was that there was no requirement for disease progression prior to enrolment in the trial. Because of the above limitation, patients with indolent disease could have been part of this clinical trial.

In the EXAM trial a double-blind, phase III trial, which investigated cabozantinib versus placebo in locally advanced or metastatic medullary thyroid cancer, patients were required to have disease progression at the time of study entry. At a median follow-up of 13.9 months, the trial showed that the placebo arm had a much shorter PFS of 4 months compared with 11 months for those in the cabozantinib arm (p < .001), with an ORR of 28% [33]. The below table summarises the four approved kinase inhibitors used in thyroid cancers (**Table 1**).

#### **Table 1.**

*Four approved kinase inhibitors based on their phase 3outcomes.*

#### *3.2.2 Selective inhibitors*

#### *<IP_ADDRESS> BRAF and/or MEK inhibitors*

Role of BRAF and/or MEK inhibitors in Anaplastic thyroid cancer:

BRAF inhibitors alone, or in combination with MEK inhibitors, have been extensively studied in *BRAF* mutated thyroid cancer. Dabrafenib (BRAF inhibitor) and trametinib (MEK1/2inhibitor) was FDA approved for BRAFV600E mutated ATC based on the safety and efficacy data in a phase II, open-label BRF117019 trial in which 16 patients with BRAF V600E mutant ATC were enrolled [34]. At a median follow-up of 47 weeks, the ORR was 69%. Seven of the 16 patients had continued response to therapy. Median overall survival and progression free survival were not reached in the study. The common adverse effects observed in the study were fatigue, pyrexia, and nausea.

The outcomes of a series of six initially unresectable *BRAF* V600E mutated ATC patients who received neoadjuvant dabrafenib and trametinib followed by a R1 or R0 surgical resection was reported by Wang et al. All six patients continued treatment with dabrafenib and trametinib after surgery. All six patients could undergo complete surgical resection. Analysis of the surgical specimen revealed 0–5% viability in five patients, whereas it was 50% viable in one patient. OS at 6 months and 1 year was 100% and 83%, respectively and loco-regional control rate was 100% in the series [35]. In a large single-institution cohort study at MD Anderson Cancer Center on 479 patients with ATC treated from 2000 to 2019, there were 20 patients treated with BRAF directed neoadjuvant targeted therapy followed by surgery. The 1-year overall survival was 94% in this group [36].

Role of BRAF and/or MEK inhibitors in differentiated thyroid cancer:

BRAF inhibitiors (vemurafenib or dabrafenib) alone or in combination with MEK inhibitors are effective for differentiated thyroid cancers. Vemurafenib was tested in 51 patients with BRAF V600E mutated PTC in a non-randomized, open-label phase II study. 10 of 26 patients who were VEGFR tyrosine kinase inhibitor-naive had partial response (PR) rates of 38.5%, and a majority had at least stable disease (SD) (57.5%). Median PFS was 18.2 months and Median OS was not reached [37]. In another phase II, randomized study, patients with BRAFV600E mutated PTC were randomized to dabrafenib monotherapy or dabrafenib with trametinib. 10 of 26 patients, who received dabrafenib monotherapy, had RECIST defined partial response, while nine of 27 in the combination arm had a partial response of the disease. A total of 50% and 54% in monotherapy and combination, respectively, had at least 20% decrease in target lesions. Median PFS for dabrafinib and trametinib combination was 15.1 months vs. 11.4 months for dabrafenib alone [38].

Mammalian target of rapamycin (mTOR) inhibitors:

The PI3K/Akt/mTOR pathway is downstream of RAS and activation of this pathway mostly occurs in advanced thyroid cancers. Everolimus and temsirolimus are drugs that are inhibitors of the mTOR pathway. These drugs have been studied in several phase 2 clinical trials of all thyroid cancer subtypes.

50 patients (33 DTC, including 13 HCTC; 10 MTC; and 7 ATC) were enrolled in the most recently published everolimus trial by Hanna *et al.* Disease progression within previous 6 months was an inclusion criterion for enrollment to the trial. Six percent of patients in the trial achieved partial response and 74% of patients experienced stable disease. The median PFS was 12.5 months for the entire cohort of patients in the trial. The median PFS in DTC and MTC was 12.9 and 13.1 months, respectively [39].

*Systemic Therapy in Thyroid Cancer DOI: http://dx.doi.org/10.5772/intechopen.106462*

The combination of everolimus with other targeted agents has also been tried. In a trial combining everolimus and sorafenib, 55% of patients achieved partial response, which is higher than single-agent sorafenib reported in literature [40].

Similarly, when a combination of sorafenib and temsirolimus was studied in follicular-derived thyroid cancer, partial response was seen in 22% patients [10]. Adverse effects of m-TOR inhibitors include mucositis, anorexia, pancytopenia, hyperglycemia, liver function test abnormalities, and rarely pneumonitis.

#### *<IP_ADDRESS> Selective RET inhibitors*

Selpercatinib is an oral selective RET kinase inhibitor for RET mutated MTC and RET fusion-positive thyroid cancers. The safety and efficacy of selpercatinib (160 mg twice daily) in patients with RET-mutant MTC was evaluated in the phase I/II LIBRETTO-001 trial. The ORR was 69% among 55 patients previously treated with TKIs including cabozantinib and vandetinib. Among 88 TKI naïve subjects, ORR was 73% and ORR was 62% in a cohort of patients with RET-fusion positive thyroid cancer [41]. Selpercatinib is well tolerated with very few adverse effects reported like fatigue, diarrhea, constipation, dry mouth, nausea, and dyspnea. Pralsetinib, is a second potent RET-inhibitor with activity in RET-fusion positive MTC. ARROW is a phase I/II trial of pralsetinib for RET-mutated cancers. Among 13 RET- fusion positive thyroid cancer patients enrolled in the trial, overall response rates were 91% and all patients had at least stable disease. For RET-mutated treatment naïve MTC patients, overall response rates were 74%, while overall response rates of around 60% were reported for previously treated patients [42]. Pralsetinib is also well tolerated like selpercatinib, with constipation, elevated liver enzymes, hypertension, fatigue, and peripheral oedema being the most common side effects.

#### *<IP_ADDRESS> Other selective inhibitors*

The existence of gene fusions in *NTRK*, *ALK*, and *ROS1* in a subgroup of patients with PTC, PDTC, and ATC has added to the understanding of the genetic basis of thyroid cancer and such patients may have more aggressive disease. Larotrectinib is a selective pan-NTRK (A, B, and C) inhibitor. Larotrectinib has been studied in cancer patients that harbor NTRK fusions, including in those with NTRK fusion–positive DTC and ATC [43]. In a study of 28 patients with TRK fusion–positive iodine-refractory DTC, ORR was 75% [44]. Overall, larotrectinib was well tolerated and the most common adverse effects reported were fatigue, nausea, vomiting, abnormal liver function tests and dizziness.

Entrectinib is a potent inhibitor of NTRK like larotrectinib, but it also targets ROS1 and ALK [45]. NCT02568267, an open-label, multicenter, global, phase 2 basket study of entrectinib is enrolling patients with thyroid tumors (including PTC) harboring gene rearrangements in *NTRK*, *ROS1*, or *ALK*.

There is one case report reported in literature of an ATC patient with an *ALK* rearrangement, who has been successfully treated with crizotinib (an ALK inhibitor) after failing standard therapy [46]. An open-label study of ceritinib, an ALK inhibitor, recruiting ATC patients (NCT02289144) is currently ongoing.

#### **3.3 Immunotherapy**

Immuno-oncology is another area that is gaining momentum in advanced thyroid cancer, including immune checkpoint blockade. Inhibition of programmed cell death

#### **Table 2.**

*Ongoing clinical trials of immuno-oncology treatments for thyroid cancer.*

protein (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and other proteins in the checkpoint cascade, is currently being investigated as new molecular targets in patients with advanced thyroid cancers (**Table 2**). Updated guidelines that incorporate testing for immuno-oncology markers may need to be developed depending on the results of these trials.


**3. Differential Scanning Calorimetry (DSC) Experiments**

DSC experiments are performed under air atmosphere to diesel, Jatropha oil methyl ester (JOME), and its B-20 blend at 10°C/min in Universal TA instruments with alumina pan. Performing experiments on liquid samples is difficult due to evaporation and non-stability of complex during heating. Hermetically sealed pans *CFD Combustion Simulations and Experiments on the Blended Biodiesel Two-Phase Engine Flows DOI: http://dx.doi.org/10.5772/intechopen.102088*

**Figure 9.** *Mesh Convergence study.*

are used in experiments with universal cramper. Two types of calibration are performed on instrument: (i) Initially with T-Zero (Temperature) calibration; and (ii) Enthalpy calibration. Initially Experiments are performed without the samples to get baseline further with known material (sapphire or Al2O3) heat of fusion is calculated by heating up to its melting point. Heat of fusion value is compared with theoretical estimates.


*2.5. HPLC Analysis*

All analysis were performed on an Agilent Series 1260 (Agilent Technologies, La Jolla, CA, USA) system, equipped with an autosampler, a quaternary pump, a diode-array detector and a column compartment, controlled by Agilent1260 LC software. Separation was achieved on a ZORBAX SB-C18 analytical column (4.6 × 250 mm, 5 μm, USA) [47]. The mobile phase was 0.05% H3PO4 aqueous solution and acetonitrile, and the ratio was 95:5. The detection wavelength was 213 nm, the mobile phase flow rate was 1 mL/min and the injected sample volume was 10 μL. The temperature remained at 25 ◦C.

#### *2.6. Static Adsorption*