Introduction

Solifenacin succinate (SOLS) is a urinary antispasmodic (anti-muscarinic class) drug. It acts as a direct antagonist at muscarinic acetylcholine receptors in cholinergically innervated organs. Its anti-cholinergic parasympatholytic action decreases the tonus of smooth muscle in the bladder, effectively reducing the number of required voids, urge incontinence episodes and urge severity and improving retention, facilitating increased volume per void [1].

Flavoxate HCl (FLXHC) is an antispasmodic drug that works by counteracting smooth muscle spasms of the urinary tract through directly acting on the muscle. It prevents spasms in the urinary tract and relieves painful or difficult urination, urinary urgency, excessive nighttime urination, pubic area pain, frequent urination and the inability to hold urine caused by urinary tract infections [2].

Tolterodine tartrate (TOLT) is an anti-muscarinic drug used in the treatment of urinary urge incontinence and other symptoms of an overactive bladder (Fig. 1) [3].

Fig. 1
figure 1

Chemical structures of SOLS, FLXHC and TOLT drugs

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Literature survey revealed that few chromatographic methods for SOLS [4,5,6,7,8,9,10,11,12,13,14,15], FLXHC [16,17,18,19] and TOLT [20,21,22,23,24,25,26,27], spectrophotometric methods for SOLS [28, 29], FLXHC [30, 31] and TOLT [32,33,34], potentiometric method for the determination of FLXHC [35, 36], spectrofluorimetric method for the determination of FLXHC [37] and voltammetric methods for the determination of FLXHC [38] and TOLT [39, 40] were reported for their determination in bulk, dosage forms and biological fluids.

Thermal decomposition measurements are used to study the physical and chemical changes that occur in the sample under temperature-controlled program. Therefore, one can explain a thermal analysis curve by relating the features of the property against temperature with possible chemical or physical events that have taken place in the system under observation. In thermogravimetric analysis (TG), the mass of sample was measured as a function of temperature at a predetermined rate where the sample may either lose mass to the atmosphere or gain mass by reaction with the atmosphere. In dynamic thermogravimetry (TG), the sample was subjected to continuous temperature changes under usually linear time. Meanwhile, in static thermogravimetric, the sample was maintained at a constant temperature for a period of time where any changes in mass are recorded. Derivative thermogravimetry (DrTG) can be obtained from TG, and it is used to know the decomposition steps of compounds.

Differential thermal analysis (DTA) is the measurement of the difference in temperature between a sample and a reference as heat applied to the system. It provides information on the chemical reaction, phase transformation and structure changes that occur in a sample during a heat-up or a cool-down cycle. The DTA measures the differences in energies released or absorbed and the changes in heat capacity of materials as a function of temperature. All materials behave in certain predictable ways when exposed to certain temperature.

Differential scanning calorimetry (DSC) is used to measure the temperature and heat flows associated with transition in materials as a function of time or temperature. These measurements provide qualitative and quantitative information about the physical and chemical changes that involve endothermic and exothermic processes or changes in heat capacity on DSC technique [41, 42].

The knowledge about drugs interaction mechanisms of drug with plasma proteins as a biomacromolecule was essential requirement to understand pharmacodynamics and pharmacokinetics of drugs [43]. As regards function of plasma proteins as drug carriers, there were a large number of researches about binding of drugs to them [44, 45]. As a result, plasma proteins interact with metal complexes in evaluating the worth drug candidate. Generally, proteins are known as the major targets for most of the drugs in organisms [46]. Their interaction with drug affected on the drug absorption, distribution and elimination in the circulatory system [47] and can prevent their rapid elimination from blood stream [48]. In this work, the behavior of SOLS, FLXHC and TOLT dugs was investigated using electron impact mass spectrometry at 70 eV and thermal analysis techniques such as TG, DrTG, DTA and DSC. These techniques were spirited in material characterization, and many authors have agreed these techniques to characterize the materials [49,50,51,52,53,54]. Therefore, the correct pathway of degradation (TA and MS) knowing this structural session of bonds can be used to predict the active sites of these drugs which responsible for their chemical reactivity. Correlation between the three techniques was discussed in order to throw more light on the degradation behavior of these drugs. Molecular docking of the three drugs under investigation was carried out using molecular docking studies to predict their binding modes toward E. coli bacterial RNA (4p20).

Experimental

Materials

SOLS, FLXHC and TOLT drugs were obtained from Multipharma Pharmaceutical Company (Egypt), Unipharma Pharmaceutical Company (Egypt) and Pfizer Pharmaceutical Company (Egypt), respectively, their purity values were 100.3, 100.5 and 99.72% for SOLS, FLXHC and TOLT drugs, respectively.

Equipment and procedures

Thermal analyses

Thermogravimetric analysis, derivative thermogravimetry and differential thermal analysis measurements were taken by using simultaneous DTA-TG thermal analyzer apparatus (Shimadzu DTG-60H). The mass of samples was ranging from 4 to about 7 mg, using a platinum pan. Measurements were taken from ambient to 800 °C in dynamic nitrogen atmosphere with the flow rate of 30 mL min−1 and heating rate of 10 °C min−1.

DSC curves were obtained using Shimadzu DSC-50 cell. Approximately 3 mg of samples was weighed and placed in a sealed aluminum pan. An empty aluminum pan was used as a reference. The purity determination was performed in temperature range from 10 to 400 °C in nitrogen atmosphere with flow rate of 30 mL min−1 using heating rate of 10 °C min−1. DSC equipment was preliminary calibrated with standard of indium.

Mass spectrometry

The mass spectra of SOLS, FLXHC and TOLT drugs were recorded using Shimadzu GC–MS-Qp 1000 PX quadruple mass spectrometer with electron multiplier detector equipped with GC–MS data system. The sample was ionized by electron beam emitted from the filament, the generated ions being effectively introduced into the analyzer by the focusing and extractor lenses system. The MS was continuously scanned and the spectra obtained were stored. Electron ionization mass spectra were obtained at ionizing energy value of 70 eV, ionization current of 60 mA and vacuum of 10−6 Torr.

Molecular docking simulation

In order to find out the possible binding modes of the drugs under study against E. coli bacterial RNA (4p20), molecular docking studies were performed using Autodock MOA2008.10 software and it is rigid molecular docking software [55] and is an interactive molecular graphics program for calculating and displaying feasible docking modes of drugs and E. coli bacterial RNA (4p20) receptor. It necessitates the drugs and the receptor as input in PDB format. The structure of the drugs in PDB file format was created by Gaussian09 software. The crystal structure of the E. coli bacterial RNA (4p20) receptor was downloaded from the protein data bank (https://www.rcsb.org/pdb/home/home.do).

Results and discussion

Because of their importance as anti-cholinergic drugs, the chemistry and reactivity of SOLS, FLXHC and TOLT drugs have always been of great interest. Knowledge of thermal decomposition mechanism of SOLS, FLXHC and TOLT drugs was very important to understand the chemical processes that exact major fragmentation pathway in EI using conventional MS will be discussed.

Thermal analysis decomposition of drugs

Thermogravimetry is a kind of technology to measure the change in the mass of the samples with the temperature [56,57,58,59]. Figure 2 shows the thermal analyses data (TG, DrTG and DTA) for SOLS, FLXHC and TOLT drugs under the goal of this article. The TG data showed two main mass losses for SOLS and TOLT drugs and four main mass losses for FLXHC drug. The first stage occurred at 161–280 °C, 215–254 °C and 195–295 °C as a result of an estimated mass losses of 26.53, 8.51 and 91.78% which may be accounted for the loss of C7H12NO, HCl and C23H30NO7 molecules (calcd. mass loss = 26.22, 8.53 and 90.96%) for SOLS, FLXHC and TOLT drugs, respectively. The second stage occurred at 280–800 °C, 254–307 °C and 295–650 °C as a result of 72.88, 27.043 and 9.14% estimated mass losses which may be due to the loss of C20H20NO5, C9H8 and C3H7 molecules (calcd. mass loss = 73.67, 27.11 and 9.041%) for SOLS, FLXHC and TOLT, respectively. For FLCHC, the third stage occurred at 307–365 °C due to loss amount to 54.47% may be assigned to the loss of C13H13O4 molecule (calcd. mass loss = 54.44%). The fourth stage occurred at 365–650 °C as a result of 9.87% estimated mass losses which may be due to the loss of C2H4N molecule (calcd. mass loss = 9.81%). The thermal degradation patterns of these drugs are shown in Schemes 1, 2 and 3. These mass losses appeared as endothermic and exothermic peaks as given by DTA data. The first mass loss appeared at 152, 236 and 210 °C as endothermic peaks due to melting point of SOLS, FLXHC and TOLT drugs, respectively. Thus, these drugs melt with decomposition. The second mass loss appeared in DTA as endothermic peaks at 255 and 338 °C for SOLS and at 300 and 238 °C for FLXHC and TOLT, respectively. There were also exothermic peaks at 364, 401, 444 and 510 °C for SOLS and at 281, 313, 366, 451, 488 and 535 °C for TOLT. The third and fourth mass losses appeared in DTA as endothermic peak at 331 °C and exothermic peak at 511 °C for FLXHC.

Fig. 2
figure 2figure 2

TG, DrTG and DTA curves of SOLS, FLXHC and TOLT drugs

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Scheme 1
scheme 1

Thermal decomposition pattern of SOLS drug

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Scheme 2
scheme 2

Thermal decomposition pattern of FLXHC drug

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Scheme 3
scheme 3

Thermal decomposition pattern of TOLT drug

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The thermal analysis results showed that FLXHC, SOLS and TOLT drugs started to decompose at 215, 195 and 161 °C, respectively. Therefore, the order of drugs under investigation was FLXHC > TOLT > SOLS according to their stability.

Mass spectral fragmentation

Electron ionization (EI) mass spectra of SOLS, FLXHC and TOLT drugs at 70 eV were recorded and investigated (Fig. 3). Schemes 4, 5 and 6 show the mass fragmentation patterns of the studied drugs, the loss of C7H12NO molecule to produce C20H20NO5 (m/z = 355, R.I. = 32%) for SOLS and the loss of HCl to produce C24H25NO4 (m/z = 392, R.I. = 53%) and C9H8 to produce C15H17NO4 (m/z = 275, R.I. = 42%) for FLHXC which agree with the results of thermal analysis of these drugs. The important ions are represented in Table 1.

Fig. 3
figure 3

Mass spectra of SOLS, FLXHC and TOLT drugs

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Scheme 4
scheme 4

The mass fragmentation pathways of the principle fragment ions of SOLS

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Scheme 5
scheme 5

The mass fragmentation pathways of the principle fragment ions of FLXHC

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Scheme 6
scheme 6

The mass fragmentation pathways of the principle fragment TOLT

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Table 1 Relative intensity (R.I) and possible fragments for important ions produced from SOLS, FLXHC and TOLT drugs
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Determination of purity of SOLS, FLXHC and TOLT drugs

For the determination of purity of drugs, DSC technique was utilized. It was based on the assumption that the impurities will lower the melting point of a pure substance. The melting transition of pure substance (100% crystalline) should be infinitely sharp, but impurities or defects in the crystal structure will broaden the melting range and lower the melting point [60]. In a system that contains impurities, Van’t Hoff equation approximately held and allowed the purity value to be calculated as follows: Tf = T0 − [(R T 20  × /ΔH f)· 1/F], where T f is the melting temperature of the sample, T 0 is the melting point of a pure substance in Kelvin (K), R is the gas constant, ΔH f is the heat of fusion, F is fraction of sample melted at T f and x is mole fraction of impurities in the original sample. Figure 4 shows the DSC curves of SOLS, FLXHC and TOLT drugs. It was obvious that very strong and sharp endothermic peaks appeared at 147.5, 236.2 and 213.70 °C which may be attributed to the melting of SOLS, FLXHC and TOLT drugs, respectively. Table 2 shows melting point values and degree of purity of SOLS, FLXHC and TOLT which agree with those of the reported methods.

Fig. 4
figure 4

DSC curves of SOLS, FLXHC and TOLT drugs

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Table 2 Melting point and degree of purity of SOLS, FLXHC and TOLT drugs
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Molecular docking studies

The method of molecule simulation is an efficient way to predict the correct binding mode and the interaction region. Targeting the minor groove of DNA through binding to a small molecule has long been considered as an important tool in molecular recognition of a specific DNA sequence. It was confirmed that the major factor affecting on the distribution, metabolism, the free concentration and the elimination rate of drugs was the binding affinity of drugs to E. coli bacterial RNA (4p20). Moreover, the half-life of drugs depends on their E. coli bacterial RNA (4p20) binding that affected on the dosage of drugs. Therefore, the study of interaction between E. coli bacterial RNA (4p20) and SOLS, FLXHC and TOLT drugs was essential to investigate the pharmacokinetics and availability of drugs in tissues. The study of interaction of SOLS, FLXHC and TLOT molecules and E. coli bacterial RNA (4p20) can express carcinogens’ carcinogenic properties, contributing to further understanding of the molecular mechanisms of drug, as well as the drug–DNA interactions in rational drug design. The big and small grooves are the mainly binding sites of DNA. The interaction of binding mode can be non-covalent binding, covalent binding and long-range assembly. The first step is to explore the interaction of the binding model of the series of compounds and DNA molecule. Then, the binding energy must be calculated.

Molecular docking studies for the investigated drugs have been performed using MOE 2008.10. software [55] to predict their binding modes toward E. coli bacterial RNA (4p20) [61,62,63]. Figure 5 shows the binding models of SOLS, FLXHC and TLOT to the bacterial RNA. The 2D interaction maps (Fig. 6) showed the different modes of interactions between each drug and the bacterial RNA (Table 3).

Fig. 5
figure 5

Binding models of a SOLS, b FLXHC and c TOLT to the bacterial RNA

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Fig. 6
figure 6

2D interaction maps of a SOLS, b FLXHC and c TOLT to the bacterial RNA

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Table 3 Molecular docking of SOLS, FLXHC and TLOT drugs with E. coli bacterial RNA (4p20)
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The O14 and six-membered ring of SOLS compound were the active sites and bound with O2P(Adenosine31, B) and N4(Cytosine32, B) active sites of E. coli bacterial RNA (4p20) via hydrogen bond and π-interaction, and the binding energy was found to be −3.6 and −1.5 kcal mol−1, respectively. The minimum binding energies of the FLXHC compound with DNA were −3.1, −1.1 and −0.7 kcal mol−1. There are a H-bond and π-interaction between N4 (Cytosine8, A), N4 (Cytosine32, B) and N4 (Cytosine10, A) of E. coli bacterial RNA (4p20) and O27 (pyrone ring), N48 (piperidine ring) and six-membered ring of FLXHC compound, respectively. The minimum energy docked pose revealed that the TLOT compound fitted into the curve contour of the targeted DNA in the groove. Moreover, –C=O group (O32, carbonyl group) and N34 (quinuclidine) of the TLOT compound acted as strong H-bond donors or acceptors and were engaged in hydrogen-bonding interactions with N4 (Cytosine30, B) and N6 (Adenosine29, B) in the DNA. The binding energy was found to be −1.3 and −1.2 kcal mol−1, respectively.

Conclusions

This study provided further insights into application of experimental TA and MS techniques on SOLS, FLXHC and TOLT drugs. From the application of these techniques, it was concluded that there were some similarities in TA and MS fragmentation pathways for SOLS and FLXHC drugs but there was difference in TA and MS fragmentation pathway for TOLT drug. DSC technique can be used to determine purity of drugs giving compatible results with the results obtained using the reported methods. This work revealed the importance of the thermal analysis and DSC techniques for the quality control of SOLS, FLXHC and TOLT drugs. The way of interaction between the active a sites in the drugs with E. coli bacteria was studied and confirmed using molecular docking simulation program.