Atorvastatin (AT) and Ezetimibe (EZ) have high intra-subject variation, making bioequivalence investigation challenging. Herein, the current study has applied a novel clinical design, including a randomized, four-period, two-treatment, two sequences crossover open-label study design, to overcome the intra-subject variation for AT and EZ in bioequivalence investigation, post-oral administration of a single dose for 10/40 mg EZ and AT in forty healthy male adults, under fasting conditions, using a newly developed and validated bioanalytical method, which included a novel LLOQ of 0.04 ng/ml for EZ. Where the LC-MS/MS method has been validated for simultaneous determination of AT and EZ, including free EZ (unconjugated EZ) and total EZ (sum of free EZ and EZ-glucuronide).  AT with its labeled internal standards (IS; AT-D5) and EZ with its labeled IS EZ-D4 were extracted from plasma by protein direct precipitation using acetonitrile. The dynamic range was 0.4-60 ng/ml for AT, 0.04-6 ng/ml for free EZ, and 1-120 ng/ml for total EZ. The Cmax and AUC0-t of reference product for AT were 24.89, 124.20, free EZ 4.79, 59.53, and for total EZ were, 32.26, 243.97. All validation results were within the acceptance criteria. The outcome for investigated test product was bio-comparable to the reference product.

Atorvastatin (AT; Figure 1A) and Ezetimibe (EZ; Figure 1B) combo drug is used along with diet to control high cholesterol and triglycerides levels in the plasma, which helps prevent medical problems like a heart attack or stroke caused by clogged plasma vessels in patients at high risk of cardiovascular and cerebrovascular with atherosclerotic diseases (Ai et al., 2018; Zhan et al., 2018). It is also used for the treatment of homozygous familial hypercholesterolemia conditions. Furthermore, the combination use of EZ with AT helps to enhance the effect of lower doses and reduce the consequent side effects for homozygous familiar hyperlipidemia caused by statin (Hamilton-Craig et al., 2010). AT and EZ work in different mechanisms, where AT is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor “statin class,” acts as a lipid regulating drug which 

prevents the production of cholesterol in the body (Sirtori, 2014; Willey and Elkind, 2010). At the same time, EZ reduces cholesterol absorption from ingested foods (Prasad et al., 2013; Wang et al., 2013).

Pharmacokinetically, AT exhibits a dose-dependent and non-linear kinetics in human plasma post oral administration; it is very rapidly absorbed to raise its maximum concentration (Cmax) around 28 ng/ml, which reached within maximum time (Tmax) 1-2 hr with an AUC about 200 ng∙hr/ml, it just has approximately 15% bioavailability due to the high first-pass effect (Lins et al., 2003) which lead to high intra-subject variation (Hwang et al., 2020) as well as, EZ post oral administration is rapidly absorbed and extensively metabolized (>80%) to the pharmacologically active ezetimibe-glucuronide. Total EZ (sum of free EZ (unconjugated EZ) and EZ-glucuronide) concentrations reach a maximum of 1-2 hr (Kosoglou et al., 2005).

The bioequivalence studies for such variable intra-subject available drugs make clinical application challenging. They need highly sensitive analytical methods to detect the low levels of free EZ, where few studies have reported the bioequivalence of AT and EZ following two periods of crossover design (Abdelbary and Nebsen, 2013; FDA, 2011; Gowda et al., 2007).

To our knowledge, no reported method has applied a four-period crossover clinical design to overcome the intra-subject variation. Therefore, we designed for the first time in the current study a randomized, four-period, two-treatment, two sequences crossover clinical study to investigate the bioequivalence of AT and EZ simultaneously under fasting conditions. On the other hand, many bioanalytical methods for simultaneous determination of AT and EZ have been reported by different techniques, including LC-MS/MS (Abdelbary and Nebsen, 2013; El-Bagary et al., 2014; ELAWADY et al., 2021; Gowda et al., 2007), and the most sensitive reported a bioanalytical method for free EZ determination was not below 0.1 ng/ml for the lower limit of quantification (LLOQ) (Abdelbary and Nebsen, 2013). Herein, we developed and validated a new, more sensitive bioanalytical method, established upon 0.04 ng/ml as LLOQ to quantitate free EZ, including direct protein precipitation as a single extraction step for sample treatment which makes an easy, more economical, and rigid method.

AT calcium at purity = 92.1% with its labeled internal standard (AT-D5 purity = 86.1%) and EZ at purity = 99.9% with its labeled IS (EZ-D4 purity = 97.0%) were obtained from TRC. -Glucuronidase-Type HP-2 solution was obtained from Sigma. The collected plasma blank samples were obtained from donors at the Jordan center for pharmaceutical research (JCPR) clinical site. The LC-MS/MS-quality deionized water, acetonitrile, methanol, and acetic acid were purchased from Fisher, Germany; in addition to the other chemicals were all analytical grade.

Instrumentation

The Mass spectrometer was API 6500+, Applied Biosystems, MDS SCIEX, coupled to LC from Agilent 1200 series. Computer System of Windows 10 SP1 and Analyst 1.6.3 software for the data management system.

HPLC Conditions

Chromatographic conditions consisted of a mobile phase of 0.03

LC-MS/MS analysis

The optimized tandem MS parameters exhibited a high quantitative detection efficiency. The molecular ion for AT and EZ were detected with their daughter fragment upon +MRM scan Mode at the mass transition of m/z 559.1 à 466.15 and 408.5à271.1, respectively. The optimized chromatographic conditions were also efficient enough to separate AT (RT 2.75 min) and EZ (RT 0.68 min for total and free EZ) from the plasma matrix with an excellent quantitative peak as seen in Figure 2B for AT LLOQ and Figure 2D for free EZ LLOQ, where AT and free EZ with their ISs eluted within the total run time of 3.2 min.

Figure 2:

Specificity and carryover

The extraction method from human plasma was specific to quantitate AT, total EZ, and free EZ over IS, and no endogenous peaks were observed through validation and routine analysis as seen from chromatograms in Figure 2A for AT blank and 2C for total EZ blank, where a chromatogram for the extracted plasma’s blank sample shows no any prominent peak compared to the LLOQ in Figure 2B and 2D, respectively.

Furthermore, the carryover test exhibited an efficient washing system for the injection port. All injected subsequent blank samples to the high concentration didn’t contain any AT, EZ, or IS residual peaks.

Standard calibration curve and linearity

The peak area ratio of each AT, free EZ, and total EZ to IS in human plasma was linear over the selected dynamic range of 0.400 to 60.000, 0.040 to 6.000, and 1.000 to 120.000 ng/ml, respectively. Where the R2 for all curves were ≥ 0.999, and the mean (n=10) corresponding calibration function is y = 0.00152 (±4.7) x + 0.00097, y = 0.01045 (±3.2) + 0.00165, y = 0.00014 (±2.9) + 0.00091, respectively.

Within- and between-run sensitivity (LLOQ), accuracy, and precision

The within- and between-run accuracy and precisions for analysis of AT and free EZ and total EZ in plasma (spiked QC including LLOQ) were all within the acceptance criteria, as shown in Table 1.

Table 1:

Recovery and matrix effect

The recovery values obtained from the protein direct extraction procedure were all above 92% in TA and fee EZ plasma extraction as presented in table 2, while the recovery values obtained from the liquid-liquid extraction procedure that followed in total EZ extraction from plasma were above 19%, which validated properly and passed all validation sections successfully. summarizes the complete recovery for AT, EZ, and IS obtained by directly comparing the peak areas of extracted QC samples with unprocessed spiked post-extraction from human plasma.

Table 2:

The effect (matrix factor; (MF)) of extracted plasma matrix on AT, free EZ, and total EZ were all below 12%, as well as the IS- normalized MF was less than 4%, as examined through 6 different plasma sources at both QC low and high and given by the mean of IS-normalized MF.

Stability

AT, free EZ, and total EZ stability were examined out- and within the plasma matrix in the short and long-term using both QC low and high, and all results for each corresponding stability condition were above 91%.

Clinical application

During clinical application, there were no clinically relevant abnormalities at physical examination, all findings were normal for all participant volunteers, and there were no safety concerns during the study. For AT, free EZ, and total EZ, no significant difference was found between the Cmax and AUC0-t of the plasma for either treatment. The concentration-time profile for analysis of AT, free EZ, and total EZ in human plasma post oral administration of the test drug product AT+EZ (40+10) mg/ tab compared to the reference product under fasting conditions is presented in Figure 3 for fourth participant volunteers derived from the two sequences in the comparative form of test (T) vs. reference (R) drug products.

Figure 3:

Table 3 summarizes the statistical analysis for major pharmacokinetic (PK) parameters for the test product of AT, free EZ, and total EZ compared to the corresponding reference product.

Table 3:

Based on the pk parameters of Cmax and AUC0-t derived from measurements of AT, free EZ, and total EZ in human plasma for each drug product, the test product was bioequivalent with the reference product of Ezetrol® (EZ tablet 10mg/tablet) and one film-coated tablet of Lipitor® (AT film-coated tablet 40mg/tablet).

The described method for determination of AT, free EZ, and total EZ in human plasma by tandem MS was successfully validated and used to estimate clinical bioequivalence of EZ + AT (10+40) mg/tab that coadministration as one tablet of Ezetrol® (EZ tablet 10mg/tablet) and one film-coated tablet of Lipitor® (AT film-coated tablet 40mg/tablet), which were bioequivalent related to Cmax and AUC0-t of AT, free EZ, and total EZ.

Ahmed Abu-awwad: Writing original draft – review & editing; Data curation.

Khaled W. Omari; Investigation, Review & editing.

Basel Arafat: Methodology; Data curation; Formal analysis; Resources.

Eyad Mallah: Software; Supervision.

Mona Bustami: Formal analysis, Software.

Eleni Loukeri: Funding acquisition.

Tawfiq Arafate: Project administration, Methodology, Validation, Investigation, Resources.

Acknowledgments

The authors are grateful to the Jordan center for pharmaceutical research (JCPR) represented by its great team.

Conflicts Of Interest

The authors report no financial or other conflicts of interest in this work.

Funding

There is no funding to report.

Ethical Approval

Ethical approval was obtained and kept for this study.

Data Availability

All data generated and analyzed are included in this article.