Detection and Characterization of Simvastatin and its Metabolites in Rat Tissues and Biological Fluids Using the MALDI High-Resolution Mass Spectrometry Approach


Animal, materials and reagents

Sprague-Dawley rats were obtained from the College of Pharmacy animal facility. All animal experiments were conducted in accordance with the standards set forth in the guidelines for the use and care of experimental animals of the National Institute of Health and the Animal Experiments Oversight Committee. The study was validated and approved by the King Saud University Animal Ethics Committee (No. KSU-SE-19-73). The study was conducted in accordance with ARRIVE guidelines. SV was obtained from Sigma-Aldrich, Kenilworth, NJ, USA. Methylcellulose (viscosity: 400 cP) was obtained from Sigma-Aldrich, Saint Louis, MO, USA, distilled/deionized water (Milli-Q Advantage A10) from Merck Millipore Frankfurter Strasse 250 Darmstadt Germany, rotor stator homogenizer (sirial number: SNTH21832; 5,000 to 35,000 rpm) from Omni International, Kennesaw, GA, USA, liquid nitrogen (Air Liquid, Riyadh, SA), methanol (purity HPLC-99, 9%) from Sigma-Aldrich, Saint Louis, MO, USA, metabolic cages, oral gavage tubes, syringes, surgical tools, death pestle, beakers/glass tubes/glass vials and d other equipment (c. College of Pharmacy.

In vivo experimental procedure

Six rats were randomized into two groups, with 3 in each group (1 for control). Simvastatin was administered orally by gastric gavage at a dose of 100 mg/kg body weight (diluted in 0.5% methylcellulose). Physiological saline solution was administered orally to the control group (average weight 205 ± 15 g). The animals were placed in metabolic cages for the collection of urine and feces. Rats were sacrificed by cervical dislocation 1 h after drug administration, and blood and tissue samples (brain, heart, kidney, lung, and liver) were collected. Tissues from one group of rats were disrupted by homogenization by applying shear force, while tissues from the other group were disrupted by pulverization (grinding). The ruptured cells were then extracted, centrifuged and reconstituted before being directed to mass data analysis using MALDI Orbitrap MS.

Rat models

Preparation of vehicle SV for oral administration in rats

The vehicle for SV was water + 0.9% NaCl + 0.5% methylcellulose. Vehicle SV (viscous solution) was prepared by boiling 100 mL of distilled water in a 500 mL conical flask. Then 1g of methylcellulose was added slowly over about 2-3 minutes with vortexing. The mixture was cooled to 30-40°C with stirring, then 100 ml of a freshly prepared 1.8% NaCl solution was added and stirred overnight at 4°C.

Oral gavage and collection of biological samples (group I)

Label, weigh and dose the rats (in duplicate): The tails of the rats were labeled with different colors of dyes. Rats were weighed separately and readings were recorded (Table S-1). Depending on the weight of each rat, the amount of simvastatin (100 mg/kg) to be dissolved in the recommended volumes of vehicle (water + 0.9% NaCl + 0.5% methylcellulose; 5 ml/kg was used) was calculated based on “Oral Dosage (Gavage) in SOP Adult Mice and Rats”, “Recommended Dose Volumes for Common Laboratory Animals” and “Administration of Substances to Laboratory Animals: Routes of Administration and Factors to consider “38. Rats were dosed separately and placed in metabolic cages at 30 min intervals to ensure sufficient time for sacrifice and collection of biological samples from individual rats 1 h after drug administration (Table S-2) . Samples of blood, urine, tissues (brain, heart, kidney, lung and liver) and intestinal contents (feces) were collected and placed at -80°C prior to sample preparation, except for the blood, which was placed at 4°C.

Oral gavage and collection of biological samples (group II)

Label, weigh and dose rats (in duplicate): The same processes were performed for group II as for group I (Tables S-3, S-4).

Preparation of biological samples using different cell disruption methods

Homogenization of biological samples taken from group I rats

Collected blood was allowed to clot by incubating undisturbed at room temperature for 30 min. The clot was removed by centrifugation at 1000–2000 × g for 10 min in a refrigerated centrifuge. The supernatant (serum) was transferred to a clean Eppendorf tube using a Pasteur pipette. Tissue samples (brain, heart, kidney, lung and liver) were removed from the -80°C freezer and thawed. The organs were cut into small pieces and distributed in different glass tubes (to ensure better homogenization). Extraction of SV and its metabolites from each tissue was performed by adding 1-2 ml of a methanol-water mixture (9:1, v/v), depending on the amount of tissue in each tube of glass (plastic tubes were avoided through the use of an extraction solvent). Further extraction was performed by homogenizing all samples using a rotor-stator homogenizer at 25,000 rpm for 20 s. Supernatants were pooled (combined extracts are from different parts of the same organ of the same organism) and collected after centrifugation at 3000 × g for 5 min and stored at 4 °C. Four milliliters of the aforementioned methanol-water mixture (9 : 1, v/v) were added to 202 mg of rat feces, and 4 ml of pure methanol was added to 1 ml of serum and 0.5 ml of urine to extract SV and its metabolites (for each rat ). All samples were then homogenized separately and centrifuged at 3000 × g for 5 min. The supernatants were evaporated to dryness using a stream of nitrogen and dissolved in 300 μl of an acetonitrile-water mixture (50:50, v/v). Rat faecal samples were diluted 1000 times before analysis.

Cryogenic crushing/pulverization of organs harvested from Group II rats

Tissue samples (brain, heart, kidney, lung and liver) were removed from the -80°C freezer prior to spraying each sample. Liquid nitrogen was poured into a mortar to pre-cool the mortar-pestle assembly. The sample previously frozen at -80°C was made brittle with liquid nitrogen before or during grinding. The frozen tissues were ground into a homogeneous powder. Each powdered tissue sample was transferred to a 15 ml Falcon tube containing 4-5 ml of the extraction solvent consisting of methanol-water (9:1, v/v), depending on the amount of powder in each tube , and well mixed . Samples were then centrifuged at 16,000 × g for 10 min. Supernatants were collected and stored at 4°C. The sample preparation method for biological fluids and feces was the same as the procedure used for Group I.

Mass Spectrometry (MS) Analysis


Mass spectra were measured using an ultra-high resolution MALDI-LTQ Orbitrap MS platform (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nitrogen UV laser (337 nm, 60 Hz) with a beam diameter of about 80 μm × 100 μm. The LTQ Orbitrap instrument operated in positive ion and negative ion modes over a normal mass range (m/z 100–1000). The tuning parameters were individually optimized for the matrices used in the present study. The number of laser shots and power were determined based on tests performed with automatic gain control on a small area of ​​the tested tissue slide (target value was set at 5.105). The SV parent compound was used to determine the parent spectrum which serves as a control spectrum to tune the MS conditions. The MS parameters optimized for SV were as follows: the analysis was carried out in FTMS with a positive/negative mode, ASF/AGC was maintained; plate movement has been set to monitor CPS mode; laser energy was applied from 5 to 25 µJ; and the scan range was set to 100–1000 m/z. Data were analyzed using Thermo Xcalibar Qual Browser software, version 3.1.

LC/MS ion trap

LC-MS/MS measurements were performed using a model 6320 ion trap (Agilent Technologies, USA) equipped with an electrospray ionization (ESI) source. Electrospray ionization was performed at room temperature in positive/negative mode. Voltage was maintained at 4.5 kV, nebulizer pressure was 60 psi, dry gas was 12 l/m, dry bulb was 350°C, trap drive level was 100%, and the capillary temperature was 325°C. The scan range has been set from 100 to 1000 Daltons. The column used was an Eclipse plus C18 (150 × 4.6 mm, 5 microns). LC separation was performed using a mobile phase composed of ammonium acetate buffer (pH=4) in water (solvent A) and acetonitrile (v/v) (solvent B). Gradient chromatography (30 min run) was performed with an ammonium acetate buffer (pH=4) in a water/acetonitrile mixture as the mobile phase at a flow rate of 0.3 mL/min. The program started with 70% mobile phase A, then the amount of mobile phase B was increased by 30-70% in 30 min. The vials containing the samples to be analyzed by HPLC were placed in the autosampler integrated into the HPLC machine, and 1 µl of a particular sample was injected into the HPLC system connected to the Agilent ion trap.

Selection and optimization of the MALDI matrix

The optimization of the different matrices, 2,5-DHB, 1,5-DAN, 9-AA and CHCA, selected for MALDI was carried out with SV and the extract of rat liver tissues (where most of the SV metabolites should be detected). The four matrix solutions (2,5-DHB, 1,5-DAN, 9-AA and CHCA) were prepared by dissolving each in H2O/ACN (v/v, 50:50). 2,5-DHB (10 mg), 1,5-DAN (1 mg), 9-AA (1 mg), CHCA (10 mg) were separately dissolved in 500 µL (H2O/CAN), respectively, to obtain a saturated supernatant. The solutions were centrifuged to collect the supernatant and obtain saturated matrices. The selection of appropriate matrices was made based on the strongest MS signal obtained from the analysis of SV and liver extracts.

Methods for depositing samples on the MALDI plate

The suitability of different methods of loading samples onto a MALDI plate, namely the dried droplet method (mixing the matrices and the sample in an Eppendorf tube, pouring the sample into a 96-well MALDI plate, then co-crystallization of sample and template during solvent evaporation), plate mixing method (templates were poured into a plated 96-well MALDI plate and sample was then added before the template dried and was mixed using a pipette; the mixture was then co-crystallized during solvent evaporation) and the sandwich method (the matrices were poured into a 96-well MALDI plate, dried, the sample was then added on top of the dry matrix and another layer of each matrix added), was compared for standard SV and extracts from tissue samples (i.e. liver) for determine the method that obtained the signal the p more intense for SV in positive and negative ion modes. A micromolar solution of SV and an in vivo reconstituted sample (in 1 ml) were used for the analysis. To taste vs. matrix were used at ratios of 1:1, 1:5, 1:10, 1:25 and 1:50 with three analytical replicates.


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