2.1. Optimization of LC-MS/MS Conditions for SH045 and Internal Standard (IS)
To enable its application in preclinical in vivo studies, a sensitive method to quantify the new TRPC6 inhibitor SH045 in plasma was developed, covering optimization of high performance liquid chromatographic (HPLC) and tandem mass spectrometric (MS/MS) conditions as well as implementation of a suitable internal standard (IS).
Generally, due to the chemical structure of SH045 containing a hydroxyl group and carbamate functionality, the analyte was expected to get sufficiently ionized during electrospray ionization (ESI) in positive mode, further enhanced by formic acid (0.1%) which was superior to ammonium formate (2 mM) as an additive in the chromatographic solvent system.
Further, the chemical structure of the analyte also suggested that reversed-phase conditions are highly suitable for its chromatography. Hence, four different columns (A–D) were preselected from the wide spectrum of reversed-phase stationary phases available. In detail, the columns tested for gradient elution using acetonitrile with 0.1% formic acid were as follows: Column A—Luna C18(2) 100 Å (100 × 3.0 mm, 3 µm, Phenomenex, Aschaffenburg, Germany), column B —Luna C18 100 Å (50 × 2.1 mm, 5 µm, Phenomenex), column C—Zorbax Eclipse XDB-CN 80 Å (150 × 3.0 mm, 3.5 µm, Agilent Technologies, Waldbronn, Germany) and column D—Zorbax Eclipse XDB-C8 80 Å (100 × 3.0 mm, 3.5 µm, Agilent Technologies) (
Supplementary Figure S1 and Table S1). Of them, both Luna C18 columns (A and B) are known to cause retention of analytes mainly by hydrophobic interactions, whereas polar and ionic interactions are more pronounced characteristics of phases with shorter alkyl chains, such as the Zorbax Eclipse XDB-C8 column (D). Besides, another column with “extra dense bonding”, the Zorbax Eclipse XDB-CN column (C), was tested to check whether its further increased propensity to polar interactions is of advantage. Furthermore, to achieve run times not longer than 20 min, appropriate column dimensions, namely in terms of length, inner diameter and particle size, were chosen as stated. To enable measurements with an even shorter run time and therefore higher throughput, column B, having a length of 50 mm and an inner diameter of 2.1 mm, was also included in the survey.
The results of the comparison, which led to the decision to use the Zorbax Eclipse XDB-C8 column (D) for our study, are summarized in
Supplementary Table S1, while the related multiple reaction monitoring (MRM) chromatograms are shown in
Supplementary Figure S1. In brief, using columns A to D, gradient elution and injection of equal amounts of SH045, dissolved in pure solvent, the highest number of theoretical plates (
N) was calculated for the XBD-C8 column (D), which therefore exhibited the highest column efficiency. In contrast, the Luna C18 column (B, 50 × 2.1 mm) showed the highest value in terms of peak area, nearly twice as much as observed for column D. However, this superiority diminished almost completely when standard samples of SH045 prepared from mouse plasma were measured, which was of relevance for the planned pharmacokinetic study.
Asymmetry and tailing factors were calculated (
Supplementary Table S1). Most ideal for plasma samples appeared to be the tailing factor for the Luna C18 column (B). However, the chromatogram clearly showed an unfavorable peak tailing (
Supplementary Figure S1b) and therefore was not considered for further use. For the XBD-C8 column (D), a sufficient tailing factor was obtained consistent with an appropriate peak profile.
Taken together, among the columns tested, the Zorbax Eclipse XDB-C8 (column D) was revealed as the most suitable one and was finally chosen for the study. Using an optimized gradient method, SH045 eluted at 9.72 min during a total run time of 20 min.
Since no isotopically labelled derivative of SH045 was available to serve as IS, the suitability of three possible well-described drug candidates, namely dexamethasone, imipramine and primidone [
15], were examined together with the parent compound (+)-larixol.
In brief, to enable MS detection for quick examination, their enhanced product ion (EPI) spectra were recorded. Based on the most intense signals of fragment ions, MRM methods were set up. Using them, the four potential IS were found to eluate at 4.64, 4.64, 1.96 and 9.46 min respectively, under the aforementioned optimized LC conditions for SH045. From this series, (+)-larixol (9.46 min) was preselected because of its retention time close to that of SH045 (9.72 min), while maintaining peak separation during the applied chromatographic method and so avoiding possible cross-talk during MS detection.
Furthermore, (+)-larixol and its congener SH045, both belonging to the class of labdane diterpenes, were anticipated to show similar behavior during sample preparation due to their structural similarity. Hence, having confirmed that (+)-larixol had no influence on the extraction of SH045 from plasma nor it was detectable in plasma after administration of SH045, it was finally chosen as IS for method development and application (
Figure 1).
In order to quantify SH045 with the aid of (+)-larixol as IS, MS detection methods for both compounds were optimized using MRM in positive ion mode with ESI. In brief, solutions of the analytes (100 ng/mL) in water/acetonitrile (ACN) 1:1 (
v/
v) containing 0.1% formic acid were infused by a syringe pump, while an automated sample optimization procedure was performed. For SH045 and IS, the most intense MRM transitions were
m/
z 364.3→
m/
z 151.2,
m/
z 364.3 →
m/
z 271.3 and
m/
z 364.3 →
m/
z 289.3, as well as
m/
z 307.2 →
m/
z 271.2,
m/
z 307.2 →
m/
z 220.9,
m/
z 307.2 →
m/
z 265.9 and
m/
z 307.2 →
m/
z 151.2, respectively. Of these, the latter showed highest signal intensities and were selected for detection. When recording the EPI spectrum for SH045 (
Figure 1a), the product ion of
m/
z 289.3 was not the most intensive, however, it was used for quantitation since it provided the most intensive signal for detection by MRM. The optimized MRM conditions applied for detection of SH045 and IS throughout the study are specified in
Table 1.
In addition, a rationale for the fragmentation pattern observed in the EPI spectrum of SH045 (
Figure 1a) is given in
Figure 1d. Starting from the parent ion [M+H]
+ at
m/
z 364.3, the
N-methylcarbamate moiety was cleaved (-
m/
z 75), followed by an elimination of water (-
m/
z 18), which in turn resulted from the alkyl hydroxyl group in the molecule. Consequently, the latter was observed for (+)-larixol as well (
Figure 1b), and the formation of a fragment ion at
m/
z 271.3 was similarly detected. Further, cleavage of the bicyclic ring system finally resulted in the main fragment ion of
m/
z 151.2 for both SH045 and (+)-larixol.
2.5. Concentration-Time Profile of SH045 in Plasma
The validated LC-MS/MS method was successfully applied in a first exploratory kinetic study in mice after administration of a single dose of SH045 (20 mg/kg body weight, i.p.,
n = 6) (
Figure 3). Further, data received were used to calculate pharmacokinetic parameters based on a non-compartmental analysis (
Table 6).
SH045 was rapidly absorbed into the vascular system with a maximum plasma concentration (Cmax) of 756 ± 85 ng/mL at the time to maximum concentration (Tmax) of 30 min. The area under the plasma concentration-time curve for 6 h (AUC0–6) was calculated with 1410 ng/mL × h, whereas AUC from time zero to infinity (AUC0–∞) was 1473 ng/mL × h. This reflects an extrapolated AUC from the last measured time point to infinity, AUCextra, of only 4.3%. This low amount corresponds to a nearly complete systemic drug exposure covered by the non-compartment modeling and suggestive for adequate computing of the elimination function.
The calculated half-life, T1/2, of SH045 was 1.3 h, however the mean residence time (MRT) was slightly longer than the T1/2.
A semilogarithmic plot of mean drug concentrations in plasma versus time indicates that the drug elimination follows a first-order kinetics (
Figure 3). The volume of distribution at steady state (V
ss) of 25 L is very similar to the volume of distribution during the terminal phase (V
z) of 25.5 L, which indicates a preferential extravascular appearance of SH045 (
Table 6).